BIOCHIMIE, 1978, 60, 979-987.

A n t i t u m o u r drugs

T. A. CONNORS.

with latent activity.

MRC Toxicology Unit, Medical Research Council Laboratories, Woodmanslerne Road, Carshalton, Surrey SM5 4EF.

The concept of l a ten t ly act ive drugs, that is chemica l s w h i c h are not themselves act ive but whi.ch requ i re convers ion in vioo to the i r act ive form, was wel l es tab l i shed before chemothe ra py was cons ide red to be a feasible form of cancer t rea tment . Perhaps , for th is reason, the synthes is of l a ten t ly act ive agents or p r o d r u g s r ep resen ted one of the ear l ies t a t tempts to design more spe- cific an t i cance r agents. Some of the ea r ly w o r k in th is f ield has been desc r ibed by Sel igma et al [1], Evere t t and Ross [2], I sh ida te el al [3], Ar- nold et al [4] and Hebborn and Daniel l i [51.

The a iky la t ing agenis p roved to be p a r t i c u l a r l y useful in the design of l a ten t ly act ive chemica l s s ince member s of the series range w i d e l y in the i r tox ic i ty to mammals , some agents having an I,D~o in rodents of 0.1 m g / K g or less whi l e others have LDso values in excess of 5,000 mg/~Kg. Fu r the r - more smal l changes in chemica l s t ructure , such as might occur by metabol i sm in vivo, could lead to the fo rma t ion of metabol i l es some tens or thou- sands of t imes more toxic than the pa ren t com- pound. The methyl ester of p h o s p h o r a m i d e mus-

Walker cells in vitro.

O O II . . 0 - M -" ~ ~OCH3

M P~ NH 2 "~ NH 2

i D9o I Dgo

2 ~g/ml lOOOpg/ml

Fro. l. - - Cytotoxicity of phosphoramide mustard and its methyl ester.

t a rd for ins tance (figure 1) is quite non- toxic to cells in cul ture hav ing an 'LD~o of a round 100 ~g/ ml for W a l k e r tumour cells w h i c h are very sen- si t ive to the ac t ion of a lky la t ing agents. The ester

is also quite non- toxic to animals . The free acid, however , ki l ls W a l k e r turnout cells at a round 2 ~g/ml. If a type of tumour cell exis ted w h i c h had an enzyme capab le of h y d r o l y s i n g the methy l ester then the ester w o u l d be a h igh ly specif ic agent for this type of tumour , s ince its low toxi- c i ty in vivo impl ies that there is ins igni f icant con- vers ion to the free ac id in normal cells.

F igure 2 shows a few examples nvhere metabo- l i sm of a lky la t ing agents in vivo produces der iva- t ives w h i c h are quite different in the i r cytotoxi - city. Azo mus ta rds are genera l ly quite non- toxic but some may be r educed to the c o r r e s p o n d i n g p - p h e n y l e n e d i a m i n e s w h i c h m a y be severa l thou- sand fold more toxic than the pa ren t azo com- pound. On the other hand methyl -d i -2-ch loroe thy l - amine is deme thy la t ed in vivo to d i -2-chloroethyl - amine w h i c h is ve ry much less toxic. S imi lar ly , a number of n i t rogen mus ta rds are monodech lo ro - e thy la ted by mic rosomal enzymes, the resu l tan t monofunc t iona l agents i nva r i a b ly being less toxic (and wi thou t an t i tumour act ivi ty) .

Whi le the tox ic i ty of an a lky la t ing agent may be inf luenced by such factors as its pa r t i t i on coeffi- cient and its p h a r m a c o k i n e t i c p roper t i e s , for those a lky la t ing agents w h i c h are not metabo l i sed in vivo there is a b r o a d co r re l a t ion be tween alky- la t ing ac t iv i ty and tox ic i ty as shown in table I. The monofunc t iona l analogue in the table has poor a lky la t ing ac t iv i ty and low toxic i ty . As the bas ic i ty of the n i t rogen atom is inc reased by sub- s t i tu t ion wi th e lec t ron re leas ing groups so both the a lkyla t ing ac t iv i ty and tox ic i ty increase [6]. Such a cor re la t ion is to be expec ted s ince the cy- to toxic i ty of a l~yla t ing agents is due to a lky la t ion of cer ta in ce l lu lar macromolecules . If a chemica l has only low a lkyla t ing ac t iv i ty then it is quite l ikely, if its b io logica l ha l f life is reasonably short , that a large p r o p o r t i o n of the drug may be excre ted unchanged. Compared wi th an a lkyla- l ing agent w i th h igh reac t iv i ty there wi l l b~ cor- r e spond ing ly less in t r ace l lu l a r a lky la t ion and hence less toxic i ty .

980 T. A. Connors .

The aim in the design of a latently active drug is the synthesis of a molecule with very low alkylating ability but of such a structure that it may be converted in . i vo to a highly reactive spe-

other requirements has meant that in the past ma- ny well designed chemicals have been synthesi- sed but have not been tested under the correct conditions. In the first case the ,difference in toxi-

(C, CH2 C H 2 ) 2 N - ~ N = N - - ~

R R

D (CI'CH2 CHa)aN-~NH2 R

ACTIVATED

CHa 'N/CH2 CH2 CI

\CH2 CHa CI

N/CH2 CH2 CI R.

\CH2 CH2 C I Fro. 2.---Activation or deactivation

in vivo.

NH/CH2 CH 2 CI

\CH 2 CH 2 CI LESS ACTIVE

m R'NH' CH2 CH2 CI DEACTIVATED

of nitrogen mustards by n~etabolism

TABLEAU I.

Alkylating Toxicity Compound activity (LD.~ o,

(K'so X t 0 3 ) ,~mole/kg)

N/C H~ 4.9 3000

~ \CH2CH2CI

CH2CH~C1

H O O C ~ N / 4.6 915

\CH2CH2C1

CH~CH~CI

~CH2CH~CI 13.0 367

/CH2CH2CI

HO--~ ~r--- N 48.6 74 \CH:CH:Cl

~ _N/CH:CH~'Br 123.0 39

\CH_,CH~Br

cies by a known metabolic pathway. However, a number of other condit ions must be fulfilled be- fore a latently active agent will have a selective ant i tumour effect. The failure to recognise these

BIOCHIMIE, 1978, 60, n ° 9.

city between the latent drug and its active mcta- bolite should be high, at least ten and preferably a hundred or thousand fold. Secondly the model system (usually a tumour bearing animal) used to

A n t i t u m o u r d r u g s w i t h la t en t ac t i v i t y . 981

test the hypothesis must be shown to conta in the act ivat ing enzyme in the tunmur which must also be absent or in much lower concent ra t ion in all normal host tissues. ~ e latent ly active drug should also be shown to be a substrate for the en- zyme unde r physiological condit ions. Final ly, the alkylat ing half life of the active metaboli te should be very short so that cytotoxic alkylat ions are confined main ly to the tissue, that is the tumour ,

].

Gluco ~ M

i 1

. o

Fro. 3. - - Metabolism of aniline mustard in rodents. Formation of glueuronide in liver followed by hydro- lysis of the glueuronide in a tumour.

wh ich activates the agent. Once an alkylat ing agent has been demonstra ted to be highly speci- fic by virtue of its selective activation in a model tumour, then its c l inical tr ial would be in those human cancers wh ich have the same biochemical propert ies of the model system.

In fact very few latent ly active alkylat ing agents have been synthesised which meet all these con- d i t i ons Where good an t i tumour selectivity is achieved by metabolism in vivo it is usually the result of a complicated series of b iochemical pa thways of act ivat ion and deactivation. In the case of an i l ine mustard, for example, w h i c h is highly selective against a mouse plasma cell tu- mour, metabolism is required both in the l iver and in the tumour to obtain selectivity [7]. Figu- re 3 shows that the relat ively non-toxic ani l ine mustard (LDs0 about 250 mg/Kg) is conver ted in the l iver by hydroxyla t ion and conjugat ion to the less toxic g lucuronide which is the major plasma metabolite. In the plasma cell tumour, the glucu- ronide is hydrolysed to the aglycone which is highly toxic (LDs0 about 2-3 mg/Kg) because the tumour, unl ike all other tissues, has very high ~-glucuronidase activity at physiological pH. In this case most of the condi t ions for selectivity have been fulfilled wi th the result that ani l ine mustard is usual ly very effective against tumours wi th high levels of :~-glucuronidase [8].

It is of interest that many years before the dis- covery of the mechanism of act ion of ani l ine mus- tard, it had been proposed that latent ly active al- kylat ing agents might be more selective if their act ivat ion requi red more than one metabolic stage [9]. Dalton and Hebborn [10] prepared a series of N,N-bis(2-chloroethyl) -N'-glycyl-p-phenylenedi- amines wh ich were to be activated by two conse- cutive stages (fig. 4). In the first stage an acylase is required to conver t the paren t compound to a g lycinamide derivat ive which can then be acted upon by an aminopept idase to release a highly reactive and cytotoxic p -pheny lened iamine mus- tard.

Cyclophosphamide is one of the best and the safest of alkylat ing agents in the cl inical chemo- therapy of cancer, although it undoubted ly owes its activity to one or more metabolites. Based on reports that many tumours had high levels of phosphamidase [11, !2], F r i edman and Seligman [13] synthesised the phosphate nmstard (fig. 5) which might, in the presence of phosphamidase enzymes, be converted to the su lphur mustard de- rivative. Some t ime later, Arnold et al [4], wor- king on s imi lar lines, synthesised many hun- dreds of phosphorous conta in ing compounds one of wh ich was cyclophosphamide. However, it soon becaine clear that lhe activity of cyclophos- phamide was not due to hydrolys is by phospha- midase enzymes to nor-HN2 but was the result of a p re l imina ry act ivat ion step which occured pre- dominan t ly in the l iver E14]. From exper iments of

BIOCHIMIE, 1978, 60, n ° 9.

982 T. A. Connors .

the type shown in table II [1,5], the impor tance of microsomal metabol ism can be demonstrated. It can be seen that the concent ra t ion of cyelophos- phamide or its metaboli tes required to cause re- gression of the WaIker ca rc inoma in vivo is far

and quant i ta t ive ly assessed for their eytoxi,city [16]. The method is shown in figure 7. Two of the most toxic metaboli tes separated in this way were subsequent ly shown to be two isomers of 4-hy- d roxycyc lophosphamide [171. A number of other

M~-NHCOCH~NHCOCH ~ (ACyt.++) -~ M - ~ N H C O C H 2 N H , .

(A mlnopep,,da,e) "~ M--~NH2 M = --N(CH:CH~CIh

FIG. 4. - - La ten t l y active n i trogen m u s t a rd requir ing t, wo en- z y m a t i c stages for activation.

less than that requi red to kill the same cells in vitro. The in vioo si tuat ion can be s imulated in vitro by the addi t ion of l iver microsomes and an

TABLE II.

Cone. of Endoxan to kill Walker tumour 90 per cent cells

In vivo ca 20 "(/ml In vitro 800 T/ml In vitro Jr- microsomes 20 -[/ml

NADPH generat ing system. In s imilar experi- ments [161 it has been shown thai small numbers of Walker turnout cells growing in vitro are ex-

0 H

O--P--OCH~_ CH2SCH:CH.,CI

) HOCH.,CH2SCH2CH2C1 Fro. 5 . - Latentl.o active mustard designed to be

activated by enzymatic hydrolysis of the phosphate group.

t remely sensit ive to low concent ra t ions of cyclo- phosphamide in the presence of microsomes (figure 6). Based on the sensi t ivi ty of these tumour ceils a small scale cytotoxici ty test was developed whereby the metaboli tes of cyclophosphamide could be separated by th in layer chromatography

BIOCHIMIE, 1978, 60, n ° 9.

metaboli tes of cyclophosphamide that had pre- viously been identif ied were the major u r i na r y metaboli te carboxycyelophosphamide [18] and a minor u r i n a r y metaboli te 4 -ke tocydophosphamide

MICROSOMAL ACTIVATION OF ENDOXAN IN CULTURE

, o I 70 ~

Untreated I O 60 controls (X)

- - 5 0 x Microsomcs

alone ~ 4o

d s o pglml

20 Endoxon

,0

I I I I O 20 40 60 80

HOURS Fro. 6. - - Activation of Endoxan (cyclophosphami-

de) in vitro by microsomes to products cytotoxic iv rapidly proliferating Walker turnout cells.

[19]. Evidence for the existence of a r ing opened form of 4-hydroxycyclophosphamide had also been obtained E20! and acrolein and phosphora-

A n t i t u m o u r drugs w i t h la ten t ac t iv i ty . 983

mi(te mustard had been identif ied after microso- m-~l metabolism of cycIophosphamide in vitro [211. This complex metabolic pa thway led at least two groups [17, 22] to propose the scheme of figure 8 as the mechanism for the selective action of cyclophosphamide. The drug is metabolised

23, 24]. However, 4-hydroxycyclophosphamide may also break down spontaneously by a 6-elimi- nat ion react ion to form acrolein and phosphora- mide mustard [17]. The proposal is that whi le the major i ty of these act ivat ions and detoxifications take place in the l iver a cer ta in amount of the

CYTOTOX1CITY TEST FOR DRUG METAE~OLITES SEPARATED i~Y TLC

Trace of radio- ~ act ive label Controls

Th in [ ' ~1 ~1 i ~ 1 ~ B i a n k silica layer plate Uncha ncjed, labelled

dru 9 from T LC plate

:.llll 1 1 Silica scraped off plate and weighed Equal wleigits °f e°ch into sterile tubes 1 I J 1

~ : : t ¢ " i:grO~:~ U Supernatant

Radioactivity mea su red

Serial dilutions Celt suspensions~

Incubated lh at 37°C Cells arc counted every 24 h ~,

ml

Grown in 96-welt plastic plate (12 wells per treatment)

Fro. 7. - - Method for determining the cytoxicity, of metabolites of cgclophosphamide prodzzced by metabolism by mzcrosomes in vitro and separated by thin layer chromatography.

Centrifuged at 2009, 2 rain

Suspended in fresh medium

pr imar i ly by liver microsomes to its 4-hydroxy derivat ive wh ich is in equi l ib r ium wi th its tau- tomeric r ing opened form. Both tautomers are en- zyme substrates and may be conver ted to 4-keto- cyclophosphamide and carboxy.cyclophosphamide both of ~vhich are non toxic me,tabolites [20, 22,

4-hydroxyder ivat ive wil l pass into the blood stream and enter both normal cells w h i c h are sen- sitive to alkyJating agents and whose death cau- ses toxici ty (e.g. bone mar row cells) and tumour cells. In tumour cells the 4-hydroxy compound may break down to form the toxic phosphoramide

BIOCHIMIE, 1978, 60, n ° 9.

984 T. A . Connors .

mustard while in normal cells some of the com- pound may be converted to the non-toxic .excre- tory products . Thus, al though both normal cells and tumour cells wil l be alkylated by phosphora- mide mustard being produced in the l iver and serum by l~-elimination, there wil l be less overall a ikylat ion of normal cells if they can effectively detoxify any 4-hydroxycyclophosphamide that reaches them. In m a n y animal tumour systems cyclophosphamide is often about twice as selec- tive (judged by t 'herapeutic index) as other ni tro-

That such compounds are still selective an t icancer agents is due to the fact that the l iver wi th its low mitol ic index is resistant to aIkylat ing agents which act p r imar i ly on cells in cycle. According to this theory there should be a greater alkylat ion of the macromolecules of turnout cells than of those normal cells of the host wh ich are part icu- lar ly sensitive to alkylat ing agents.

Brock and Hohorst I2:5] consider the interac- tio'n of 4-hydroxycyclophosph~mide wi tb thiols

00--CH 2 " / \ .

M-P\ ? (CH3}2

NH--CH 2 / O . O - - C H 2

\ / NH--CH

I OH

EN'ZY~;TIC / /

00--C.H2 "I )clc M'P\ NH ~C

I! 0 NON TOXIC

M=N (CH2CH2CI)2

00--CH2 i i / \

"~ M'I" C{CH3) 2 -" \ /

NH 2 CHO

ENZ p- ELIMINATION

V-C c;c O o- MP MP\

N\H2 ~H NH2 TOXIC

Fro. 8. - - Path~ways of metabolism of cyclophosphamide in vivo.

gen `mustards such as chlorambuci l . The increase in therapeu.Uc index may be a measure by wh ich normal sensit ive cells of the host (but not tumour cells) can deactivate any 4-hydroxycyclophospha- mide to which they are exposed.

Brock and Hohorst [25] oppose this work ing hypothesis on the grounds that ii assumes that tu- mour cells should conta in a higher level of the activated metaboli te (phosphoramide mustard) than the liver. This is of course not an essential requiremen{ for the hypothesis. In the major i ty of studies on the d is t r ibut ion of alkylat ing agents in tumour bear ing animals, the concen t ra t inn of drug is al~vays found to be higher in the l iver with its good blood supply than in the tumour.

BIOCHIMIE, 1978, 60, n o 9.

to be in some way responsible for the high selec- t ivi ty s.een 'with .cyclophosphamide. The 4-hy- droxy derivative is a reactive N-methylo,1 and ~vvill react =wilh many na tura l ly occur r ing amines and thiols. 4-(SR),-cyclophosphamide complexes 'may reform 4-hydroxycyclophosphamide and this ~vill undoubted ly have an effect on the pharmacokine- tics of release of the cytotoxic metabolite, phos- phoramide mustard [2~]. ,Whether this react ion in some u n k n o w n way contr ibutes to the high selec- t ivi ty of cyclophosphamide remains to be investi- gated.

Certainly two lines of research support the scheme out l ined in figure 8. Cox et al [24] have shown that the cytosol of normal tissues can pro-

Antitumour drugs with latent activity. 985

CH 3 I 0--CH2 0 0~CH ?//1 6~ l/1 6 \

M-P~ H 2 M-P2 3 t, \3 I.~/CH2 NH--CH NH~CH2

M = N (CH2CH2CI) 2

?H 3 ?H3 O0 --CH 00--CH '

M g CH~ ---~M. H~ ? O--CH 2 / \

'4" M'P\ ~H 2 Further NH--C Oxidation / I I u At C-4 HO H3 OH 0 Not

~H3 ~Ha ?/O--C\H2 00--CH 0.0--CH

I \ I \ M.P ?H 2 M.Ip/~ CH2 ....~ M.~:~ CH 2

N H:/C3~o 0 ; H~H?(O NH2 CO'OH

C~=CH.CO.CH3P ~/0 "~CH3.CH=CH.CH O M'P'XNH 2

/ O ~ CH 2 M" P \

N H -- CH2/CH2"

/ / 0 / O ~ CHa II

M . P \ /CHa - NH--CH

I ENZYMAT/ OH

FIG. 9. - - Pathways of metabolism o[ S-methyl and 6-methglcyclophos- phamide in vioo.

? / O - - CH2 • M-P \ /CH 2

NH 2 CHO

ENZYMAT>/ ICHEMICAL

?__O--CH2. / \ ? . ,O--CH21 ~/ C ~p~O- • M" ~-H2 M "1- ACROLEIN MP\NH C/c"2 \N.2 C60H N.2

II O NON TOXIC TOXIC

Fxc. 10. Pathways of metabolism of 5,5-dimethyh'gclophosphamide in vivo.

BIOCHIMIE, 1978, 60, n ° 9.

986 T. A. Connors .

tect Walker cells in vitro against cyclophospha- mide act ivated by microsomes. The assumption is that the 4-hydroxy der ivat ive is deact ivated to some extent by enzymes in the cytosol and no longer breaks down completely to phosphoramide mustard. Extracts of tumour cells had no such pro tec t ive effect.

Stronger ev idence comes f rom pred ic t ions ma- de, on the basis of the scheme of figure 8, of the probable an t i tumour act ivi ty of methyl der ivat i - ves of cyc lophosphamide . Figure 9 sho~vs that 6 - m e t h y l c y d o p h o s p h a m i d e can be metabol ised si- mi la r ly to cyc lophosphanf ide and can also under- go #-el iminat ion [27]. P red ic tab ly this der iva t ive should have s imilar proper t ies to cyc lophospha- mide. The 4-methyl der ivat ive howeve r may be hydroxy la t ed by microsomes but fur ther oxida- t ion at C4 is then not possible. Thus there is no oppor tun i ty for normal sensi t ive cells to detoxify the hyd r oxy der iva t ive and it avill break down in both normal cells and turnout cells to phosphora- mide mustard. The h igh select ivi ty of cyclophos- phami de should thus be lost. 5 ,5-dimethylcyclo- phosphamide , figure 10, may be hydroxy la t ed and fur ther metabol ised to the co r respond ing 4-keto and ca rboxyamide derivat ives. However , there is no possibi l i ty of 8-el iminat ion to form the act ive metaboli te. Table III shows that all these predic-

TABLE III.

((carboxy-)) Phosphor- LD,~o/I D.o Substituent and (~ keto- )~ amide PC6 turnout

derivatives mustard

- - -~- -J- 93 6-methyl -~- -~- 92 4-methyl - - + 54 5,5-dimelhyl- + - - c~ 0

t ions have subsequent ly been conf i rmed by tests on nfice bear ing a plasma cell turnout [28]. Cyclo- phosphamide has a high therapeut ic index of around 90 w h i c h is at least twice the value of most ni t rogen mustards tested against the turnout. The 6-methyl analogue wh ich is metabol ised simi- lar ly to the paren t compound has an ident ica l therapeut ic index. The 5,5-methyl analogue w h i c h cannot form the act ive metaboli te, phosphoramide nmstard has no an t i tumour act ivi ty. The results wi th the 4-methyl analogue are of pa r t i cu la r in- terest s ince it has only about half the therapeut ic index of cyc lophosphamide . Since in this case it

BIOCHIMIE, 1978, 60, n ° 9.

is the fur ther metabol ism of the 4-hydroxy deriva- t ive that is b locked then the h igh select ivi ty af cyc lophosphamide may be assumed to be depen- dent on the fur ther metabol ism of the 4-hydroxy der ivat ive and presumably on the compara t ive di f ferences of this detoxif icat ion pa thway in nor- mal and tumour cells.

Metabolism of alkylat ing agents can thus be seen to play an impor tan t role in de te rmin ing the i r an t i tumour selectivity. ~f we had a greater knowledge of the differences in b iochemis t ry , es- pec ia l ly drug metabol is ing proper t ies , of tumours and normal tissues the avay 'would be open to the design of agents w i th high an t icancer specificity. Unfor tuna te ly the s i tuat ion has changed lit t le sin- ce Harpe r [29] stated ¢ The development of com- parative b iochemis try of various tissues has lag- ged behind the ingenuity of the synthet ic chemist , so that although in m a n y cases the gun could be loaded w i t h suitable ammuni t ion , we lack infor- mat ion about the target ~.

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Antitnmonr drugs with latent actioity. 987

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B1OCH1MIE, 1978, 60, n ° 9.