The Mechanism of Action of Alkylating Agents
G. P. WARWICK
(Chester Beatty Research In3tiiute, Institute of Cancer Research: Royal Cancer Hospital,
@
Fuiham Road;
S.W. 3, England)
SUMMARY
The term “alkylating agent― has been defined and a detailed discussion made of the
mechanisms
(Sni
and
Sn@) by which
they
interact
with
nucleophilic
centers.
The
various nucleophiles likely to be encountered in vivo are discussed in terms of their
relative reactivities toward alkylating agents.
With regard to Sn@ reactors it was possible to group them into four sections on the
basis of their chemical reactivity and the “spread―
in their affinity toward different
nucleophiles. The groups comprised (a) the epoxides, ethyleneimines,
and $-lactones;
(b) the primary alkyl methanesulfonates;
(c) primary alkyl halides; and (d) a-halo
acids and ketones.
An attempt
has been made to correlate
the reactivity
of the Sn@ and Sni reactors
with their known pharmacological properties and the likelihood of their reaction with
the various cellular constituents. In particular, the effects of the alkylating agents on
nuclear material and cell division, the blood components, male fertility, and the im
mune
action
nucleic
The
and as
response
tion have
No attempt
the literature
have been discussed
in detail,
and the chemistry
involved
in the inter
of the alkylating agents with compounds of biological importance such as the
acids, proteins, and peptides has been critically examined.
importance and limitations of the alkylating agents as chemothenapeutic
drugs
tools for examining the basic mechanisms involved in carcinogenesis and muta
been considered
in the light
of recent
findings.
will be made to review the whole of
relevant to the mode of action of
in the elucidation of fundamental
mechanisms of
action.
alkylating
agents, since this has been done many
The term “alkylating agent― in its widest sense
times in the past, and more recently by Ross (7@) denotes those compounds capable of replacing a
and by Wheeler (87). Because of the growing com
hydrogen atom in another molecule by an alkyl
plexity of the field, it might be instructive to ex
radical, and this of course involves electrophilic
amine the chemical reactivities and alkylating po
attack by the alkylating agent so that the defini
tentials of some of the different classes of alkylat
tion must be extended to include those reactions
ing agents in relation to what is known about their
involving addition of the radical to a molecule con
pharmacological
properties. The different types of taming an atom in a lower valency state—for ex
compound which can function as alkylating agents
ample, formation of a sulfonium compound from a
or electrophilic
reagents include alkyl halides,
sulfide. The alkylating agents exhibit a diversity of
alkyl methanesulfonates,
alkyl sulfates, alkyl phos
pharmacological
properties including the capacity
phates, halogenomethyl
ethers, @2-chloroethyl sul
to interfere with mitosis, to cause mutations, and
fides, @-ch1oroethylamines, epoxides,
@-lactones, to initiate and promote malignant tumors; some
ethyleneimines
and ethyleneimides,
diazoalkanes,
of them have a stimulatory action on the nervous
activated
ethylenic compounds, halogenomethyl
system, and others are powerful vesicants or
ketones and esters, methylolamines,
etc., and, al
lachrymators.
When attempting to find a parallel
though only a few of these classes are useful as ism between the pharmacological
effects elicited
palliatives in clinical therapy, all are capable of re
by the alkylating agents and their chemical reac
acting with at least some nucleophilic centers with
tions at particular cellular sites, it is important to
the result that even inactive compounds can help
consider, among other variables, the particular
1315
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1316
Cancer
mechanism of alkylation involved, since this will
largely determine the chemical groups which will
be attacked
inside the cell and their position
within the cell. Almost every review which has
been written on the alkylating agents has included
at least some description of the mechanisms in
volved in unimolecular and bimolecular reactions,
and the justification for indulging in this again in
detail is inherent in the problem with which we are
concerned. The alkylating agents, unlike many
other drugs used in medicine, produce their effects
by binding covalently with cell constituents,
and
@
R@
—x ___
@
R+Y
®
CHART
9
1.—Mechanism
• 9
R+X
RY
of first-order
nucleophilic
substitu
tion Sn 1 reaction.
R1
Ogston (68) in the case of mustard
gas (see later)
CHART
2.—Ethyleneimmonium
phatic nitrogen
their
pendent
V
ion
@cH2
formed
from
an
yields a carbonium
to some
extent
concentration,
and
is the
of binding,
although
such'as
amount
in the
reac
au
TABLE
on factors
gas. Mustard
ion which is sta
tive form, then in a particular system the relative
amount of any group reacting is proportional
to
mustard.
sites and extents
1963
bilized in the form of a threo-membered
ring, and
this accounts for its greaten capacity to discrimi
nate between anions. This stability is even greater
in the case of the immonium ion formed from au
phatic
@-chloroethylamines due to the greater
basicity of nitrogen than sulfur (Chart
@).
Each nucleophile (canbonium ion, immonium
ion, etc.) has a competition factor which was meas
ured by Ogston in the case of mustard gas by
observing the extent of reaction in aqueous solu
tion with various anions (Table 1). In the case of
compounds such as thiols, amines, and acids, ac
count must be taken of the amount in the reactive
form at a particular pH in considering likelihood
of reaction with an alkylating agent. If one no
members that alkylating agents pick out centers
of high electron density, thiols and acids will be
most reactive when ionized, in contrast to amines
which become unreactive when protonated. If the
competition factor for a particular group is F, c is
its
:(@2@ZC1
Vol. p23, September
Research
1
COMPETITION FACTORS FOR
MUSTARD G@s (63)
de
solubil
ity, will be determined mainly by their electro
philic (or alkylating) potential; and this involves
consideration
of ease of electron displacement,
resonance energies, bond strengths, steric factors,
etc.
There are two generally accepted basic mecha
nisms of alkylation, first-order nucleophilic sub
stitution
(Sni) and second-order
nucleophilic
substitution
(Sn@), although care should be taken
in making too rigid a classification. In the former,
the rate-controlling
stage in the reaction is ioniza
tion to form a solvated carbonium ion, followed
by a rapid combination with a solvent molecule or
new nucleophilic reagent such as an anion or sul
fide (Chart 1). Since the driving force of the reac
tion is a displacement of electrons away from R,
then reactions of this type will be facilitated by the
presence of electron-repelling
substituents (such as
methyl) in R. The carbonium ion, being an ex
tremely unstable and hence reactive species, will
be to some extent indiscriminate in its combination
with nucleophilic centers. The more unstable car
bonium ions, such as those derived from i-propyl
methanesulfonate,
will tend to react rapidly with
solvating water molecules, whereas in some cases
more discrimination is possible as demonstrated by
SubstanceF
(Competi.
.
tion
Cysteine ethyl ester
Methylamine
Phosphate
Pyridine
Chloride
Acetate
Formate
1 .SX1O'
3.9X10'
75
54
21
10
3
factor)pHThiosulfate
7
13
8
7
7
7
7
Nitrate
0.2
7
Glucose2.7X104
08
7
Fl c. However, Alexander (1) has drawn attention
to
the
fact
that
apparent
competition
factors
of
groups in macromolecules
may be increased by
adsorption of the agent onto the macromolecule,
thereby increasing effective concentration.
Ross
(74) assessed the reactivity
ethylarylamines
drolysis
in aqueous
of a number of chloro
by measuring
acetone
their rates of hy
and found that electron
releasing substituents
in the benzyne ring such as
p-methyl greatly increased the rate of hydrolysis,
whereas it was retarded by p-nitro-, p-phenylazo-,
or other electron-attracting
substituents.
The other essential features of Sni reactions
are (a) that the rate of any particular reaction will
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1963 American Association for Cancer Research.
WARwICK—Mechanism
of Action
be essentially independent of both the concentra
tion and the nature of the other reagent. Thus, an
ionized
thiol,
a powerful
nucleophile
because
of
the electron distribution
on the sulfur atom, will
react at the same rate as, e.g., an amine or hy
droxide ion, less powerful nucleophiles, and (b)
that no reaction will take place until primary
ionization has occurred. Both these factors are in
contrast
to what
is observed
in Sn@ reactions
and
are probably very significant when comparing the
pharmacological
properties of the two classes of
reactant.
Mustard gas (dichlorodiethyl
sulfide) and the
aromatic nitrogen mustards are the Sni reactors
with which we will be concerned in this discussion.
Mustard gas is an extremely reactive compound
(half-life in water about 3 mm. at 37°C.) because
of the presence and position of the electron-releas
ing sulfur atom relative to the chlorine atoms
which facilitates formation of a carbonium ion
through the unstable ring system shown. The
carbonium ion (which may exist in the form of a
threo-membered
ring) can discriminate
between
nucleophiles to some extent, but it is far too reac
tive and, hence, toxic to be of any use in therapy
(Chart 3).
The aromatic nitrogen mustards were developed
because
they
retained
the
capacity
to
react
by
ci@—@
ci
acH,cH,—@-cI@ —k cicii@cii@s
cii,cij,
CHART
3.—Mechanism
of ionization
of mustard
gas
Y@RL@—*Y_R + Xe
CHART 4.—Mechanism
Agents
1317
sition state is high compared with the initial state.
Bond strength
is obviously
important,
since,
whereas electron affinity increases in the order
I < Br < Cl < F, replacement by an alkylating
agent increases in the reverse order, I being most
easily expelled.
Compounds
such as primary
alkyl halides,
methanesulfonates,
and phosphates are not par
ticularly powerful reactors, their rate of reaction
with water and other nucleophiles being usually
relatively low. Thus, whereas mustard gas has a
half-life of 3 mm. in water at 87°C., ethyl meth
anesulfonate
has a half-life of 36.3 hours and
that of methyl bromide is 116 hours (75). The no
action rate increases considerably in the presence
of centers of high electron density, especially
TABLE
2
RELATiVE REACT1VITIES OF NUCLEOPRILES
TOWARD METHYL BROMIDE (84)
Nucleophilek/kwNucleophilek/kwH,O
CH,C00
Pyridine
}IPOi
500
4000
Thiosulfate1.3X10' 4 .0X10
6300
0H1
16000HS
an
Snl mechanism;
but the electron-withdrawing
capacity of the aromatic ring greatly reduced the
rate of carbonium ion formation so that corn
pounds could be synthesized which could reach
distant sites in the organism before reacting with
tissue constituents.
/1
of Alkylating
of Sn2 reaction
In second-order nucleophiic substitution a car
bonium ion is not formed, but a transition com
plex is formed involving both reactants, and hence
the rate of reaction is dependent upon both con
centrations
(Chart 4). In this case the reagent
helps in the breaking of the R-X bond, and this
will occur most readily if the energies of the Y-R
and R-X bonds in the transition state are high, if
the electron affinity of X is high compared with
that of Y, and if the resonance energy of the tran
anions. Thus, the ability of Y to replace X in R-X
by the Sn@ mechanism can be assessed by compar
ing reaction rate with V as compared with water.
This has been done by Swain for methyl bromide
(84).
The major nucleophiic
centers present inside
cells are ionized thiol groups, ionized carboxylic
acids, ionized phosphates, and unionized amines,
and, ignoring accessibility and concentration
fac
tors, reaction would proceed most readily in the
order thiol > amine > phosphate > carboxyl.
Compounds such as a-halogenocarboxylic
acids
and ketones are highly reactive Sn@ reagents
toward
certain
tron-attracting
(Chart
nucleophules
capacity
5) . However,
by
virtue
of the
of the carbonyl
elec
group
for reasons to be discussed
later, its reactivity toward weak nucleophiles such
as the carboxyl ion or the phosphate ion is low. In
this context a significant factor in considering
Sn@ reactors is the variation in relative reactivity
of a particular reagent toward different nucleo
philic centers. For example,
rate constant (thiosulfate)
rate constant (acetate)
is @05for ethyl methanesulfonate,
4380 for methyl
bromide, and 16,600 for the iodoacetate ion (75,
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1963 American Association for Cancer Research.
1318
Cancer Research
p.
@).Thus, it is obvious that the “spread―
in no
activity
toward
different
nucleophilic
centers
varies considerably from reagent to reagent and is
of fundamental
importance
in determining
the
reactions which a particular
drug will undergo
in vivo and hence its pharmacological properties.
The factors which determine whether an agent
will react by an Snl or an Sn@ mechanism are
obviously complex, and it is dangerous to be too
rigid in this type of classification, since even sol
vent changes can sometimes lead to a modification
in mechanism. There occur also those compounds
such as the epoxides and ethyleneimines which, by
virtue
of their
strained
three-membered
ring
struc
ture, fall into a different category from most other
Sn@ reactors, as will be considered later. In the
case of alkyl halides and alkyl methanesulfonates,
the rate and mechanism of reaction depend on
@
Vol. @Z3,September
1963
halides leads to a further drop in Sn@ rate, but
now the Snl mechanism becomes of importance,
and the two mechanisms operate simultaneously
and at a comparable rate (37) . The reason for
this is that the extra methyl group facilitates pni
mary ionization of the halide ion (Sni) but retards
the Sn@process for electronic reasons and because
it hinders the approach of the nucleophile and sub
sequent formation of the transition complex (pni
many stenic effect) . The presence of three methyl
groups as in tert-butyl halides causes a complete
changeover to the Sni mechanism, and the rate of
hydrolysis becomes faster than the Sn@ hydrolysis
of methyl halides under the same conditions (61).
A change in mechanism can also be observed
sometimes if an attacking nucleophile is replaced
by one of less nucleophilic potential under the
same experimental
conditions. At first the effect
will be to decrease the velocity of the Sn@ process,
but with even weaker nucleophilic reagents a point
e
‘@2
will be reached in which the reagent is no longer
x + IcH,—@c-oH—@x--cH,cooH+ I
able to assist in the breaking of the R-X bond
CHART 5.—Mechanism
of reaction
of iodoacetic
acid
which must, therefore,
occur unaided by the
reagent, and the mechanism will become Sni
probably coupled with a decreased reaction rate
cHa
CH@
I
S
(39). For example, in the substitution of the tn
@H35020
(@H2@,0S0PI3methylsulfonium
CH,S0,O•CCH3
.
coso,cH3
ion by various anions it was
found that the specific rates were OH(743),
CHART 6.—Dimethylmyleran
and Myleran
PhO—(13.4), CO@(7.4), Br(7.8),
Cl(7.3),
ously indicating an Sn@ mechanism for the first
whether the reactant is primary, secondary, or ten
tiary. In some cases, such as in the reaction of two anions and an Snl mechanism at a lesser rate
for the others. A situation similar to this probably
alkyl bromides with pynidine, the rate of reac
occurs in the case of a-haloketones and acids, mdi
tion to form an ethylene derivative is in the order
eating their tremendous
potential to react with
tent. > sec. > prim., whereas in the simultaneous
powerful
nucleophiles
such as ionized
thiols
by an
reaction to form quaternary
salts the order is no
Sn@
process
and
their
relative
unreactivity
toward
versed. The differences in the reaction rates of
weaken nucleophiles such as ionized acid groups
primary,
secondary,
and tertiary
alkyl compounds
(hence the misnomer “specific
thiol reactors―).
are generally attributed
to the inductive effect.
The earlier data concerning the alkyl halides is
The replacement of a hydrogen atom by a methyl
probably of fundamental
importance in consider
group is acid-weakening—i.e.,
it induces a nega
ing
the
biological
properties
of the various agents.
tive charge on the atom on which substitution has
For example, the greater reactivity
of methyl
occurred. It therefore increases the ease of ioniza
as compared with
tion of a halide ion from the molecule. This ease halides (or methanesulfonates),
of separation can be more than compensated for the corresponding ethyl derivatives, probably ex
plains the greater toxicity of the former in vivo.
by the smaller attraction of the reacting base. Con
One surprising finding is the fact that dimethyl
sequently, the primary halides require a stronger
myleran (@,5-dimethanesulfonyloxyhexane)
is so
basic reagent than the tert-halides, but this order
similar
to
Myleran
(1,4-dimethanesulfonyloxybu
is reversed when the reagent acts as an ionizing
tane) in its pharmacological
properties (see later)
rather than a basic reagent (61).
(Chart 6). Unlike Myleran, it will tend toward an
In general methyl halides react almost exclu
Sni mechanism in its reactions, forming a very Un
sively by an Sn@ process, but introduction of an
stable
carbonium ion extremely reactive toward
other methyl (electron-releasing)
substituent (giv
its
own
solvating water in aqueous media and
ing ethyl) leads to a reduction in the rate of reac
hence unreactive toward other nucleophiles. How
tion by the Sn@ mechanism, the factor usually be
ever, the possibility of its reacting by an Sn@
ing about 10, as in the reaction with amines. Intro
duction of another methyl group as in iso-propyl
mechanism may become more important in non
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1963 American Association for Cancer Research.
WARwICK—Mechanism
of Action
aqueous media, and the implications of this will be
considered later.
Striking differences in pharmacological
effects
have been found on the one hand between alkylat
ing agents which react essentially by an Sni
mechanism, and many of those which react by the
Sn@ mechanism, between monofunctional
and bi
functional agents of both types, and between mdi
vidual members of the Sn@ type. The differences
observed between the various Sn@ reactors are
probably explicable on the basis of the foregoing
discussion, and from the point of view of chemical
reactivity and “spread―
in affinity toward differ
ent nucleophiles the Sn@ reactors can be broadly
classified into four groups:
a) the epoxides, ethyleneimines, and fl-lactones;
b)
the
Myleran
c)
primary
alkyl
methanesulfonates
(e.g.,
and ethyl methanesulfonate);
primary
alkyl
halides
(e.g.,
of Alkylating
anticipate epoxides and ethyleneimines
to react
readily with a range of nucleophiles in a somewhat
similar way to the mustards. They have in fact
been shown to react readily with thiols, amines,
and acids (73), and this ease of reaction with di
verse nucleophiles is reflected in their biological
properties,
since
end-products
chromosome
poietic
system)
very
and
ketones
of bond-making
in
many
similar
to the
respects
mustards
from the epoxides in their potential
because no strained ring is present
mechanism will mainly operate in
making will be more important than
ing, so that both the concentration
ing reagent
and its nucleophilicity
although
(35).
of reactivity,
and the Sn@
which bond
bond-break
of an attack
will be of prime
it should be mentioned
that
dibromobutane);
®
a-haloacids
produce
Group (b), the primary methanesulfonates, differ
importance,
(e.g.,
iodoacetic
acid and co-bromoacetophenone).
Group (a) .—Compnises compounds which bear
a resemblance
in structure
to the ethyleneim
monium ions which are derived from aliphatic
nitrogen mustards and which react by an Sn@
mechanism with nucleophiles (see earlier) (Chart
7).
Epoxide reactions can be acid-catalyzed,
but
this will probably be of little significance in vivo.
The presence of a strained three-membered
ring
in these compounds makes them somewhat unlike
the other Sn@Zreactors in the finer details of their
reaction kinetics. In a normal Sn@ process, in
which an entering nucleophile causes the expulsion
of a weaker nucleophile such as in the reaction of
an ionized thiol with methyl iodide, the process of
bond-making is considered a more important dniv
ing force of the reaction than that of bond-break
ing. In the transition state of such a reaction the
process
they
(inhibition
of mitosis, mutation,
breaks, cancer, effects on the hemo
and,
d)
1319
Agents
(new -S-C-bond)
will al
ready have progressed to a considerable extent. If
as mentioned earlier the process of bond-breaking
(-C-halogen) is the more important driving force
of the reaction, then the reaction kinetics tend
toward unimolecular and the rate of ionization be
comes the rate-determining
step. There are obvi
ously gradations between the two extreme mecha
nisms as already mentioned, and the epoxides of
biological interest fall into a class in which, while
second-order kinetics are followed, the process of
bond-breaking
has progressed to a considerable
extent in the transition complex because of the
strained
three-membered
ring structure.
Since
bond-breaking
is a more important driving force
than bond-making
in such reactions, one would
0®
R—CH—CH2
+ X + H —@
RCH--CH,X
@1
CHART
7.—Mechanism
6H
of epoxide
reactions
the true Sn@ tendency will be greater in the
methyl esters than the ethyl. Because of their rela
tive unreactivity
is found a greater
compared
with epoxides, there
“spread―in their affinity toward
nucleophiles,
and their reactivity
toward thiol
groups will be far greater than toward amines on
acids. In this connection Brookes and Lawley (@1)
have found that both ethyl methanesulfonate
and
Myleran have much less reactivity toward guanine
in DNA than the mustards and epoxides, whereas
Roberts and Warwick (65, 66) have demonstrated
that almost all the urinary reactivity
excreted
after injection of these compounds could be ac
counted for by reaction with the powerfully
nucleophilic thiol group in vivo. In the case of
ethyl methanesulfonate
much of the C'4 in the
drug was exhaled as C―02which could have been
derived either by hydrolysis of esters formed by
reaction
with carboxyl
groups on phosphate
groups. In the case of Myleran, much less of the
drug was metabolized in this way. The capabilities
of an alkylating agent to react with various nucleo
philes is reflected faithfully in the biological and
pharmacological
effects which it elicits, leading in
the case of these two agents to effects much less
potent than were observed with the mustards,
epoxides, etc. Thus Myleran is much less toxic
than mustard gas, possesses none of its vesicant
properties,
has only moderate activity against
transplanted tumors, causes far fewer chromosome
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13@20
Cancer Research
Vol.
@3,September
1963
“breaks―
(33), and, although it decreases the num
ber of circulating granulocytes,
it has far less ef
action is a contributor
feet
various agents leading to different biological ef
fects. There is no proof yet which alternative is
correct, and yet certain vital experiments, which
remain to be done, may make the situation clearer.
The past literature is full of examples implicating
the importance of thiol reactions in all sorts of bio
logical phenomena (10), and yet a really critical
assessment of some of these claims can leave little
doubt that they were based on naïve or over
simplified assumptions leading to a very confused
situation. The potential importance of thiol neac
tion remains (58), and yet more research needs to
be done to discard outmoded ideas and to bring
the whole field into line with modern concepts.
Speculative “paperchemistry― leads us nowhere,
except possibly in circles, and there is no sub
stitute for doing the vital experiments which can
finally clear up these points. More will be said on
-SR reaction later.
Group (d) comprises those agents, the a-halo
acids and ketones, which in their reaction mecha
nism lie in the other extreme from undoubted Sni
reactors.
With these potent Sn@ reactors the
process of bond-making
far outweighs in im
on the
mustards
peripheral
lymphocyte
count
than
the
and epoxides (30) ; and its effects on the
immune
response
and
on
male
fertility
in
rats
again differs fundamentally
from the mustards
and ethyleneimines
(see later).
Group (c), comprising the primary alkyl halides,
represents,
with
the
exception
of the
methyl
corn
pounds, agents even more unneactive than the pni
mary alkyl methanesulfonates.
Differences in the
nucleophilicity
of the Br and CH3SO2O anions
coupled with differences in the C-Br and C-
OSO2CH3bond strengths and a weak capacity
to undergo
Sn@ reactions
makes
the
halides
(e.g.,
ethyl bromide and dibromobutane)
even poorer
candidates than the methanesulfonates
as far as
diversity of reactions in vivo are concerned. Thus a
decreased reactivity toward all nucleophiles is oh
served, so that even fewer biological effects would
be anticipated;
and this is so, since 80 mg. of di
bromobutane
can be administered
safely to a
300-gm. rat compared with 4 mg. of Myleran, and
this compound produces none of the pharmaco
logical effects observed with Mylenan. The in
creased
discussed
toxicity
reactivity
of methyl
compounds
earlier, and this is reflected
of methyl
bromide
compared
has
been
in the high
with
ethyl
bromide.
Roberts and Warwick showed that dibromo
butane reacted with cysteine in t@itroin a manner
similar to that of Myleran leading to a sulfonium
ion (66). Since dibromobutane
lacks the pharmaco
logical properties of Myleran, this evidence could
be used to discount the importance of such a reac
tion in vivo and, indeed, the importance of thiol
reaction in general. However, such reasoning is
probably not valid, because it does not take ac
count of the different thiol groups in the cell, their
different reactivity, or the difference in alkylating
potential between Myleran and dibromobutane.
The major thiol reaction in vivo will be with gluta
thione, which will probably have little permanent
effect on cellular function ; reaction with thiol pro
teins or enzymes might have greater significance,
and there is no evidence yet that dibromobutane
will react with these in vivo. The site of thiol reac
tion inside the cell is probably very important,
since it is now almost
agent will combine
certain
that every
extensively
alkylating
with thiol groups
and that the site at which
it occurs and its extent differ with those of the
portance
the
process
of bond-breaking,
and
so the
nature of the attacking nucleophile is vitally im
portant,
leading to extreme reactivity
toward
powerful
nucleophiles
such
as ionized
thiol
groups
and a very weak level of reactivity toward less
nucleophilic centers. Thus as mentioned earlier
k(thiosulfate)
k(acetate)
is 16,600, as compared with 9205for ethyl methane
sulfonate.
Probably many resonance forms contribute to
the transition state (920), but the peculiar reactiv
ity of compounds of this type is a direct result of
the presence of the powerful electron-attracting
carbonyl group. Because of this, compounds of this
type
were
once
regarded
as
specific
thiol
reactors,
and all their effects from enzyme inhibition to gen
eral toxicity in vivo were ascribed on the basis of
-511 reaction, despite warning by Michaelis and
Schubert (59) that amine reaction could also oc
cur. In this connection Anson and Stanley (5) later
showed that, although tobacco mosaic virus was
not inactivated
by p-chionomercunibenzoate
(a
in vivo, and yet compounds such as the mustards,
powerful
rnethanesulfonates,
halides, and a-haloketones
and acids manifest very different effects on cells.
There are two conclusions to be drawn from this:
acetarnide and iodoacetic acid, implicating reac
tion at another site. More recently, Stark, Stein,
and Moore (83) have shown that the inactivation
of nibonuclease is due to reaction with the ring
nitrogen of histidine. This reaction with tertiary
(a) that thiol reaction in no case contributes
pharmacological
end-effects,
to
or (b) that thiol re
511 reactor),
it was
inactivated
by
iodo
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1963 American Association for Cancer Research.
WARWICK—Mechanism
of Action
amines is of interest, since Roberts and Warwick
(67) have shown that bromoacetic
acid reacts
readily with the punine portion of deoxyguanylic
acid and that a low level of reaction with the
guanine of DNA also occurred. It remains to be
seen how significant such reactions are in tiivo,
however, because of the high competition by other
groups such as thiol in the cytoplasm. In this con
nection, the same authors have shown (67) that,
after incubating
Krebs ascites cells with C'4bromoacetic acid, there is a steady uptake of label
and the ratio
specific activity
specific
activity
(protein)
(DNA)
was approximately
2@; and mthas yet to be estab
lished whether any DNA reaction other than that
associated with residual DNA protein has oc
curred. It is significant that in the case of mustard
gas and Myleran the ratio was nearer 1 (@1).
From the foregoing discussion it is probably true
to say that, with some exceptions, the different
types of alkylating agent are potentially capable
of reacting with all nucleophilic centers and that
the pharmacological
properties
of a particular
agent will depend on (a) ease of penetration, (b) to
some extent mechanism of alkylation,
and (c)
“spread―
in capacity to react with different nucleo
philes. The mustards, for example, probably pro
duce such diverse effects because they can easily
penetrate
cells, their chance of reaching most
parts
of the
cell
is high
because
of their
unreactive
nature until they are ionized (a relatively slow
process), and the carbonium ions once formed are
reactive toward most nucleophiles,
leading to
some reaction at most parts of the cell. The van
ous Sn@ reactors fall into different categories, and
compounds such as the epoxides and ethylenei
mines produce effects similar to the mustards be
cause of their relatively low reactivity which en
ables them to reach most parts of the cell un
altered, coupled with a satisfactory gradient in the
“spread―
of their affinity toward different nucleo
philes.
Some of the biological and pharmacological
properties of the alkylating agents will now be dis
cussed in the light of their chemical reactivities.
The time will surely come when the barriers which
divide chemistry from biology must crumble and
they will merge into one conceptual framework.
If the static concepts of chemistry could be made
more dynamic, and the dynamic aspects of biology
more static, then the meeting point would seem
much nearer.
of Alkylating
THE
EFFECT
13f21
Agents
OF ALKYLATING
AGENTS
ON
Nucj@n MATERIAL ANDCELL DIVISION
When it was discovered that in many respects
the bifunctional
chloroethylamines
and chioro
ethylsulfides produced cytological and pharmaco
logical effects (e.g., cell death, chromosome ab
normalities,
degenerative
changes in the bone
marrow and testis, suppression of the immune no
sponse) similar to those produced by ionizing radia
tion, the term “radiomimetic―was applied to them
(@8). Although certain of the alkylating agents
produce end-results
which appear superfluously
like those produced by radiation, detailed investi
gation has often revealed significant differences
between them, and the term in many respects leads
to confusion in the minds of chemists and biolo
gists alike. Loveless and Revell (56) set forth their
precise biological criteria upon which chemical
substances were to be classified as radiomimetic,
and this involved restriction “tothose substances
which can be shown unequivocally to react upon
the resting cell in such a way as to produce an
alteration of the genetic material which is revealed
by the appearance of chromosome breakage and
rearrangement
in subsequent divisions.― Certain
compounds
which caused pyknosis, and which
Dustin had referred to as radiomimetic, were not
covered by this definition. Others have preferred
to retain the original broad definition, but since
this time so many alkylating agents have been
discovered to mimic superfluously the effects of
radiation in, say, one respect only, that the term
ceases to have great significance. Ethyl methane
sulfonate, for example, shows none of the properties
of radiation except that it is a powerful mutagen
(53). Mylenan, possibly atypical in some respects,
nevertheless has a profound destructive effect on
granulocyte production (3@), causes certain chro
mosomal abnormalities
(45), depresses the im
mune response (1@) (in a manner very similar to
radiation) and causes infertility in rats (48) (also
in a manner reminiscent of radiation) ; but, unlike
certain mustards, it has only a moderate inhibit
ing effect on the transplanted
Walker rat car
cinoma. Thus these compounds would seem to be
“radiomimetic,― partially
“radiomimetic,― or
“nonradiomimetic,― depending on the particular
biological phenomenon
being considered. Koller
(46) has compared the effectsof several alkylating
agents with those of radiation
and concludes
“thatin view of the dissimilarities, it would be an
error to infer a similarity of the mode of action of
alkylating agents and x-nays, basing the inference
on the similarity of some end products.―
Of the various types of alkylating agent which
have been tested for tumor growth-inhibiting
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1963 American Association for Cancer Research.
13@2
Cancer Research
properties, those which have emerged as the most
active include the Sni reactors such as the aro
matic nitrogen mustards and the Sn@ reactors of
the HN@ type, epoxides, ethyleneirnines.
Other
Sn@ reactors such as the aliphatic methanesulfo
nates (Myleran)
show a low order of activity
against the transplanted
Walker tumor, whereas
those Sn@ reactors such as the a-halo acids and
ketones have been found to be inactive while
possessing
a high
order
of general
toxicity.
The
other factor of importance in determining efficacy
in this respect is functionality,
and it is now well
known that bifunctionality
leads to a profound
increase in capacity to interfere with mitosis, pro
vided the reactive centers are spaced at an opti
mum distance apart. Thus in the chemical sense
greatest
activity
is
associated
with
undoubted
Snl reactors or those Sn@ reactors which, as dis
cussed earlier, show some tendency toward the
Sni mechanism in the bond-breaking
and bond
making process and which have a reasonable no
activity toward most nucleophiles. Biesele et al.
(18), in discussing the chemistry of alkylating
agents, postulated that the most active compounds
in causing chromosomal
aberrations
were those
which possessed or could be transformed into un
stable threo-membered
rings. In a comment on this
postulate,
Loveless
and
Ross
(57)
drew
attention
to the “radiomimetic―properties of 1,4-dimeth
anesulfoxyloxybutane
(Myleran)
in inhibiting
the growth of Sarcoma 180 and in its effect on the
blood and bone marrow, and the capacity of
dimethylsulfate
to produce chromosome breakage
in plant material. Since both these compounds are
incapable of forming three-membered
ring sys
tems, the postulate of Biesele et at. was obviously
an oversimplification,
and Loveless and Ross (57)
argued
that
capacity
to form
a carbonium
ion was
a more general feature of “radiomimetic―chemi
cals. Actually, it is doubtful whether epoxides,
ethyleneimines, and methanesulfonates
do, in fact,
form true carbonium ions under conditions likely
to be met with in the organism, and it is difficult
therefore to accept this generalization
as it is
worded. As indicated earlier the term “nadiomi
metic― is often applied far too indiscriminately
to
sometimes ill-defined biological phenomena, and
it would seem more profitable and less misleading
to define carefully the biological effects manifested
by each alkylating agent rather than to refer to
some property as “nadiomimetic―because it may
vaguely
correspond
to
a particular
property
of
radiation.
Any apparent similarity between the
biological properties of radiation and the alkylat
ing agents undoubtedly
results from the capacity
of each entity to modify cellular structure and
Vol. 23, September
1963
function by direct combination with cellular com
ponents such as nuclear and cytoplasmic material.
The most “radiomimetic―chemicals will be those
capable of penetrating deep into the cell and of no
acting with a diversity of cellular sites, whereas
less reactive chemicals or those which alkylate by
a different basic mechanism may possess far fewer
“radiomimetic―properties.
Tumor inhibition with the alkylating agents no
sults from their cytotoxic action, which probably
is the result of a complex series of changes varying
in fundamental mechanism with different types of
alkylating agent, and even from cell type to cell
type. Lethal cytotoxic action is usually of two
types called interphase death and mitotic death.
Interphase death occurs without cells' going into
division or attempting to go into division. Alkylat
ing agents vary in their capacity to kill cells at this
stage, and individual cell types vary in their sus
ceptibility to this type of death. Thus fully dif
ferentiated
lymphocytes
tend to be extremely
sensitive to the action of mustards (but not to
Myleran), whereas most other differentiated cells
do not. The mechanism of interphase death is
probably complex and results from damage to
many cellular sites.
In mitotic death the primary damage is thought
to occur in the “nestingstage,― and chromosome
aberrations and other effects become cytologically
apparent at the time of division. Many changes
occur, such as clumping and bridging of chromo
somes, and some of the effects are cumulative dur
ing successive cell divisions leading eventually to
nonviable cells. The action is not restricted to
malignant
cells,
but
extends
to proliferating
of all types (35).
Various differences in detail of action
been reported to exist between the mustards
HN@) and the methanesulfonates
cells
have
(e.g.,
(e.g., dimethyl
myleran) . Thus in comparing their actions on
lymphoma cells in culture, Alexander (3) found
that HN@ immediately
arrested
cell division,
whereas dimethylmyleran
behaved like x-rays,
and at least one division occurred before cell pro
liferation stopped. Koller (45) has compared in
detail the cytological effects produced by various
alkylating agents and again found differences in
their modes of action and also differences in no
sponse to varying doses of alkylating agent. He
found that in a series of dimethanesulfonyloxy
esters, the greatest diversity of effects on cells of
the Walker tumor was shown by Myleran. He also
found that it could produce suppression of mitosis,
and “radiomimetic― and cytotoxic
effects, al
though these probably differed in detail from those
observed with the mustards. Alexander reported
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1963 American Association for Cancer Research.
\\ARWICK—Mechafl?$m
of Action
of Alkylating
Agents
13@23
(3) that, after all treatments, there was an increase poiesis was prolonged, cytological abnormalities
in volume of cultured lymphoma cells; with HN@ were not seen (cf. 45) at the doses used, and mitosis
the cell volume doubled, and then there was de
proceeded normally. They explain these different
generation,
whereas with x-rays and dimethyl
effects on the basis of the capacity of the mustards
myleran a wide spectrum of cell sizes was obtained,
to attack ruthlessly nuclear material in the resting
including “giants,―
although nuclear granulation
stage and to lyse fully differentiated lymphocytes,
similar to that seen with HN@ was not observable
whereas they consider that Myleran prolongs the
after @4hours.
intermitotic interval in all proliferating cells inre
Alexander' has also found that iodoacetic acid
spective of the stage in the mitotic cycle at which
produces different effects from either the mustards
they are exposed. Differences in normal inter
or the methanesulfonates
on lymphoma cells. It
mitotic levels which vary with different cell types
seems to have little effect on cell division, but
could explain the effects. Lajtha (47) has mdi
when a critical dose level is reached the cells cated that erythropoietic
cells normally divide
break up. This would be consistent with the no about twice as often as granulopoietic
cells, and
activity
of this compound
mentioned
earlier, re
delay in each series would explain why depletion
sulting in its major reactions occurring in the cy
of erythropoiesis occurs more rapidly than that of
cells. The authors do not consider
toplasm (67). The need for bifunctionality
and a granulopoietic
certain critical level of nuclear reaction (whether
that Myleran has any selectivity for particular
this be with nucleic acid, protein, or cofactor) seem cell types and “thatthe apparent resistance of
essential for “radiomimetic―cell death. “Cyto lymphoid tissue to Myleran may depend as much
plasmic death― may be a factor of importance in on the ratio of mitotic frequency to life-plan as to
considering mustard action on fully differentiated
the lack of damage to mature lymphocytes.―
lymphocytes and indeed in the inhibition of tumor
In their effects on the circulating gnanulocytes
growth
in general.
Koller
(45) considers
that many
and lymphocytes,
Elson has found (30) that the
factors must be considered when assessing the
epoxides and ethyleneimines produce effects simi
causes of cell death in a dynamic population and
lan to those produced by the mustards—not sun
that “theability of the alkylating agents to pro
pnising in view of the similarity in reaction kinetics
duce nadiomimetic injuries may not be the most
discussed earlier—whereas he found the effects
important property that determines the effective
produced by the carcinogens 4-aminostilbene
and
ness of these drugs in the therapy of cancer.―
the polycyclic hydrocarbons to be more like those
of Myleran, a result difficult to explain at present
EFFECTS
ON THE BLOOD
but possibly of great significance. Ethylmethane
sulfonate (half-Myleran)
was not active.
Elson (30) showed that the effects of whole
As
in
the
case
of
tumor
growth-inhibition,
bi
body x-radiation
on the number of circulating
platelets, lymphocytes,
and neutrophils could be functional alkylating agents of all series were much
essentially reproduced by the administration
of more efficient than the corresponding monofunc
tional derivatives, and in the Mylenan series opti
suitable combinations of Chlorambucil
(a bifunc
mum activity was found when n = 4 or 5 (31).
tional mustard) and Myleran, and he concluded
Potent Sn@ reactors such as a-halo acids and
therefore that each of these chemicals was only
have not been tested as ex
partially “radiomimetic.―Elson, Galton, and Till ketones apparently
tensively as the foregoing compounds,
whereas
(3@) consider
that Myleran
appears
to act on the
alkyl
halides
such
as
dibromubutane
(group
[c]
resting stage and prolongs the intermitotic
inter
val, whereas mustards appear to act mainly on Sn@2reactors) possess no detectable activity (note
cells undergoing mitosis and possibly during the lower chemical reactivity).
latter part of the DNA synthetic period. They also
EFFECTS
ON MALE FERTILITY
exert a particularly destructive action on mature
lymphocytes which Mylenan does not. They found
Very interesting results which may contribute
that the effects of Chlorambucil appeared rapidly,
to an elucidation of the mode of action of x-rays
were transient,
and were accompanied
by con
and the alkylating agents have been found by com
spicuous mitotic abnormalities.
Recovery pro
paring the effects of these agents on the process of
ceeded rapidly except in the lymphoid tissue, and
spermatogenesis
in rats and mice. The process fol
there was a transient overcompensation
in the
lows the path spermatogonia —@
spenmatocytes —+
granulocyte
series. Myleran, on the other hand,
spenmatids —+spermatozoa in epididymis, and it
produced gradual effects, depression of hemo
has been found that different agents can affect dif
ferent stages in the process. Once again it tran
I p@
Alexander,
personal
communication.
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1963 American Association for Cancer Research.
18@4
Cancer
Vol. @23,September
Research
1963
spires that Myleran most closely follows the effects
of radiation.
Intrapenitoneal
injection into mice of aiiphatic
mustards produced destructive
changes in the
@
was found that irradiation
and Myleran sup
pressed antibody production maximally if given
about @—4
days before the antigen, whereas nitno
gen mustard and TEM did so when given about
testis, and recovery was observed 3—4weeks later
days after the antigen, and antibody production
(48), whereas in rats potency returned after @—4could be suppressed for some weeks in each case,
months (34) . It was of interest, however, that two
following a single injection given at the appropni
aromatic nitrogen mustards, Melphelan and Chlo
ate time interval. Once antibody production
is
rambucil, did not interfere with the fertility of under way, however, these agents have no further
male rats (4@, 44) at doses which were adequate
inhibiting effect. Berenbaum tentatively
explains
to cause transient leukopenia in the peripheral
these relative effects by assuming that immuno
white count (@9) and inhibit tumor growth (13).
logically competent cells are produced from pnimi
Jackson (43) concludes, therefore, that capacity to tive stem cells which give rise to more differenti
inhibit tumor growth or to interfere with the bone
ated cells which can produce antibody. He en
marrow
does
not
imply
capacity
to damage
visages early cell types to be sensitive to the action
of x-nays and Mylenan, but not to TEM or the
germinal epithelium. This, of course, may have
been due to poor penetration rather than to a dif
mustards, whereas the later, more differentiated
forms are sensitive to the latter agents, but not to
ferent mode of action.
2,@,6-Triethyleneimino-1,3,5-triazine (TEM), the former. Other alkylating agents have unfortu
which was placed with the epoxides as far as nately not yet been tested, but the foregoing no
sults will be discussed later in the light of known
chemical reactivity is concerned, produced effects
indicative
of
selective
interference
with
certain
reactions
stages of spermatogenesis
(19), mainly, probably,
with spermatocytes
(11, 4@), since the infertility
was found at 4 weeks and recovery was rapid after
a single dose of 0.@mg/kg. Five daily doses of 0.@
mg/kg, however, caused sterility for several weeks,
and early and late stages of spermatogenesis
were
affected, and probably mature sperm in the epi
didymis were also attacked (as with the mustards).
The relative insusceptibility
of spermatogenia
is
in contrast in that they are the germinal tissue
most sensitive to radiation (6@, 79) and to My
leran.
After one dose of Myleran (11 mg/kg) the testis
histology remained normal until the 8th week,
when sterility rapidly developed (@6, 4@), indicat
ing an effect on an early stage of spermatogenesis.
The other germinal cells present at the time of
treatment
appeared
to develop normally
for
about 45 days, resulting in a systematic depletion
of spermatogonia,
spermatocytes,
spermatids, and
spermatozoa,
in that sequence (4@). Increased
doses still did not affect other spermatogenic cells
which continued to proliferate.
Dimethylmyleran
produced similar effects at a
lower dose level (44). This apparent specificity of
action of Myleran is probably in some way related
to its chemical reactivity. The fact that the effects
which it produces are similar to those produced by
radiation and unlike those produced by the mus
tards and TEM is probably highly significant.
EFFECTS
Different
sponse,
ON THE
IMMUNE
RESPONSE
(1@)
effects were found on the immune no
depending
on
the
agent
tested.
Thus,
it
of these
agents
in vivo.
CHEMICAL INTERACTIONS WITH
CELL
CONSTITUENTS
General conaiderations.—In discussing the basic
chemistry of the alkylating agents it was found
logical to separate these compounds into various
types or groups depending on whether they were
essentially Sni reactors on Sn@ reactors, and fun
ther subdivision was possible when a more detailed
assessment was made. In considering some of the
pharmacological
properties
of the alkylating
agents, it was found that, where comparative
assessments have been made, in a broad sense
these faithfully reflected the type of alkylating
agent involved. Thus, one can distinguish between
the pharmacological
properties manifested by the
mustards, the epoxides and ethyleneimines on the
one hand, and the alkyl methanesulfonates
on the
other, in considering their pharmacological effects.
Although fewer biological studies have been car
ned out on the alkyl halides and the a-haloko
tones and acids, it is evident that again these differ
from one another and from the above compounds.
Since these complex processes involving cells
and alteration of cellular structure and function
almost
certainly
involve
direct
chemical
combina
tion with cellular constituents, and since the level
of any particular
combination
will depend on
many
variables,
the
most
important
of
which
is
probably chemical reactivity, then an examination
of cellular components
after treatment,
or of
altered biochemical
integrity
after treatment,
should help to pin-point
the lesions involved.
This type of reasoning, valid on misleading, has
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1963 American Association for Cancer Research.
WARwICK—Mechanism
of Action
guided most workers studying the mechanism of
action of alkylating agents for many years. The
general opinion seems to be that, in the case of
compounds
such
as the
carcinogenic
hydrocarbons,
of Alkylating
13@2@5
Agents
cellular constituents,
the nature of this binding in
some cases, and that in vitro studies can often help
to elucidate tantalizing
in vivo problems. The
progress
made
so far,
much
as it seems
when
one
the carcinogenic azo dyes, etc., we must keep an
open mind whether these agents cause their effects
by actually binding to cellular constituents
on
whether they are doing something much more
subtle—such as finding their way between macro
molecular strands and exerting their effects by es
sentially physical means ; however, in the case of
the alkylating agents, cellular binding is of prime
importance.
To digress a little, there is good reason for be
lieving that the carcinogenic hydrocarbons
and
azo dyes do, in fact, need optimum structural re
assesses it against the almost complete void which
existed 10 years ago, is still extremely limited; and,
although we are in a position to make some intelli
gent guesses as to the mechanism by which the
alkylating agents exert some of their effects (e.g.,
their cytotoxic action), it would be unwise to make
any dogmatic assessments at this stage, no matter
how justifiable they may seem. However, the sun
face has been scratched, and doubtless the task of
a reviewer at the end of another 10 years will be a
comparatively
reassuring one.
quirements
(7) which
and
particular
may enable
them
Metabolism of ethyl methanesulfonale and My
electron
distributions
leran.—The
to alter
cellular
pharmacologically
active alkylating agent did in
fact alkylate nucleophilic centers in vivo was oh
tamed when Roberts and Warwick (65) demon
strated the presence of N-acetyl-S-ethylcysteine
and related compounds in the urine of rats which
had received C'4-ethyl methanesulfonate
by intra
penitoneal injection. The bulk of the radioactivity
which appeared during the first few hours was
probably derived from reaction with glutathione
known to be present in high concentration
in
mammalian cells and with which ethyl methane
sulfonate reacted readily in vitro. That which ap
peared later probably came from ethylated pro
tein. Because of the related structure
of 1,4dimethanesulfoxyloxybutane
(Mylenan) and its
enchanced pharmacological
properties, it was con
sidered of interest to examine the urinary products
excreted after its injection. Because of the bifunc
tional nature of the drug it was considered that
some type of cross-linked product might be formed
func
tion by some kind of physical means; but there is
growing evidence that compounds of this type can
in fact bind firmly to cellular constituents, both to
proteins and to nucleic acids. To quote but two
examples, Heidelberger (36) has shown dimethyl
benzanthracene
to combine both with protein and
DNA in vivo, whereas the Millers (60) and others
have shown dye binding to liver proteins following
administration
of carcinogenic azo dyes, and, more
recently, Roberts and Warwick (68) have shown
the combination
of a metabolite
of dimethyl
aminoazobenzene
with nucleic acids in vitro.
These findings tend if anything to cloud the com
plicated situation even further; they may be mis
leading, but if direct covalent combination
with
cellular constituents is proved to be a potent force
in producing all the pharmacological
properties of
the alkylating agents, then these findings might
be of the utmost importance in elucidating at least
some of the phases of the complex carcinogenic
process with these and other agents.
In the case of the alkylating agents where one
compound
can be cytotoxic, carcinogenic,
and
mutagenic, the correlation between chemical reac
tions at the cellular level with any of these mani
festations becomes exceedingly difficult. However,
attempts have been made to study the types of
chemical reactions which some of these reagents
can undergo inside cells, and in the main the no
sults obtained have indicated a close parallelism
between the types of reactions observed in vitro
using purified chemicals and those observed in
vivo, and it has also been found that levels of bind
ing to various constituents
were as one might
have anticipated on the basis of the reactivity of
the drug concerned. These studies are probably
important
if only to show that definite covalent
binding can occur between alkylating drugs and
first
clear-cut
demonstration
that
a
in vivo. However, the major urinary metabolite
was found to be 3-hydroxytetrahydrothiophene
1,1-dioxide (66), which was probably derived from
a sulfonium compound formed by the interaction
of both alkylating arms with one thiol group. This
was followed by the removal of tetrahydrothio
phene and its further metabolism to the hydroxy
sulfone. The reaction is of interest, because it in
volves not only modification of an existing thiol
group but its later removal from the amino acid
moiety in the form of tetnahydrothiophene.
In the
case of protein reaction, this could lead to the pro
duction of a new sequence of amino acids by the
replacement
of an existing molecule of bound
cysteine by a molecule of bound lanthionine
or
bound aminoacrylic acid (69). Bound lanthionine
would result if the sulfonium compound was at
tacked by another molecule of ionized glutathione,
whereas bound aminoacrylic acid would result by
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Cancer Research
13@?26
hydrolytic decomposition (Chant 8). The dehydro
peptide or dehydroprotein
might undergo addition
of a molecule of glutathione or other thiol forming
bound lanthionine.
It is extremely difficult to assess the importance
of these reactions when considering the mechanism
of action of Myleran. It has been stated (@1) that
the low level of reaction of Mylenan with nucleic
acids in vitro and in vivo at therapeutically
effec
tive doses makes these centers seem unlikely vital
points of attack in this case. It is possibly relevant
that, in the series of dimethanesulfonyloxy
esters,
those having four or five methylene groups are the
most active in depressing granulocyte count and
are also those which could undergo the ring closure
CH2—CH2o
@
012
I
CH2@―
NHR
.—@ I
G@
::s
CH2—Q1@
,NHR
+
Gs—cIl,—cH
e
r2_cI@4H_c@
i@
@COR'
NHR
—*
cii@—cH@
@
@0R'
CH,—cH2
I
S+@H@zC
cH2—cH2
,NHR
Vol. p23, September
1963
one glutamic acid unit by valine in a polypeptide
chain of 300 units. However, these ideas are specu
lative, and more data regarding the extent of pro
tein alteration by Myleran in vivo and the func
tions which these proteins play in cellular integrity
are needed. The related compound,
dimethyl
myleran, which in many respects is similar to My
leran in its pharmacological properties, should be a
useful tool in elucidating some of these problems,
since
if reactivity
toward
anions
is low and
for
steric reasons ring closure and dethiolation are not
possible in this case, then the relevance of these
changes to the mode of action of Myleran would be
questionable. Work on this aspect of the problem is
in progress, with highly tritiated dimethylmyleran.
In connection with the mode of action of Myleran,
Timmis (85), on the basis of its similarity in
action to thioguanine, has suggested that it might
combine with nucleic acids in vivo or with nucleo
tides to form a type of antimetabolite
which could
act by lengthening intermitotic
intervals rather
than directly killing cells. He suggested that
Myleran might first interact with homocysteine to
form a compound capable of alkylating
purine
bases in a similar manner to adenosyl-methionine.
Metabolism of aromatic nitrogen mustards and
mustard gas.—Itwas considered of interest in view
\ coR
S of the ring closure reaction of Myleran to deter
mine whether similar reactions occurred in vitro
CHART 8.—Reactions
of the sulfonium
ion formed
from
and in vivo in the case of the bifunctional mus
Myleran and a cysteinyl moiety with glutathione
and with
tards. Reaction with thiol groups in vivo seemed
hydroxide ion.
probable, and in the case of the bifunctional com
pounds ring closure also seemed probable in view
(_@ N'2
G ---@ I\-N@
CH2IG of the optimum spacing of the reactive centers,
\=J
@cH2
—
cii@
\@=/
‘Qi2@CH2-SG since six-membered rings are normally particularly
stable.
@.s
G
Under in vitro conditions with the use of bis
chloroethylaniline,
bis-chloroethyl-p-anisidine,
or
CHART 9.—Reaction of an aromatic nitrogen mustard with
glutathione to form a cross-linked product.
mustard gas, and either cysteine or glutathione,
evidence was obtained
that intermediate
sul
and dethiolation depicted earlier. Also in this con
fonium compounds were in fact formed (70), but
nection one should recall the many similarities
their instability was such that isolation proved im
which exist between the pharmacological
effects
possible. In the presence of excess cysteine or
produced by Mylenan and ionizing radiations.
glutathione
the only products isolated were the
The change in amino acid sequence in a protein
cross-linked cysteinyl or cross-linked bound cys
or peptide chain constitutes
a type of chemical
teinyl compounds,
in contrast to the situation
mutation induced without the intermediate inter
found with Myleran. Thus, in this case dethiola
vention of DNA or RNA. If certain proteins,
tion did not occur. Assuming the formation of an
either in the nucleus or the cytoplasm, act as sup
intermediate
sulfonium ion, the reaction sequence
pressons or cell-regulators, then one could envisage
followed in the case of glutathione
and aniline
such a change having possibly far reaching effects,
mustard would be as shown in Chart 9. The sec
and here may be recalled the drastic effects of the
ondary
reaction
of the sulfonium
ion with gluta
change of one amino acid unit in a protein in rela
thione was obviously so fast that hydrolytic cleav
tion to the production of sickle cell anemia (38).
age to 4-phenylthiazan
was not possible. The inter
esting feature of these reactions is, of course, the
The difference between the normal hemoglobin
and that in the mutant cell is the replacement of point of attack on the sulfonium compound by the
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1963 American Association for Cancer Research.
WAnwI@—Mechanism
of Action
glutathione molecule, leading to ring opening, to
form cross-linked products instead of dethiolation
and ring release as in the case of Myleran.
On the basis of these observations, cross-linking
of protein strands in vivo following treatment with
reactive bifunctional mustards seems likely (71),
and because of the chemical properties of the
mustards discussed earlier, restriction as to the
cellular sites involved would seem less likely
than with powerful Sn@ reactors such as iodoace
tate. Thus, the mustards because of their unre
active nature prior to ionization would be more
capable of deeper penetration
into cells and,
hence, reaction with a greater range of thiols than
the Sn@reactors of the iodoacetate type. The argu
ments used in the past to dismiss the importance
of thiol reaction in determining
any of the im
portant pharmacological
properties of the alkylat
ing agents are for these reasons not valid (74, 8@).
The range of so-called specific -SH reactors tested
for their capacity to inhibit tumor growth, etc., has
been limited; the compounds
have often been
monofunctional,
when it is known that bifunc
tionality is a requisite for high antitumor activity;
and most of the bifunctional agents tested have
not had their reactive centers spaced an optimum
distance apart (5@). In addition, account was
obviously not taken of the probability that, be
cause of the inherent chemical reactivity of such
compounds, they would probably never reach cel
lular sites accessible to agents such as the mus
tards, epoxides, ethyleneimines,
and, to a lesser
extent, the aliphatic methanesulfonates.
Now that
it is known that both the mustards and the au
phatic methanesulfonates
are capable of extensive
thiol reaction with -SH groups in vivo, coupled
with the knowledge that the mustards can cross
link, while Myleran can bring about a unique type
of dethiolation and at cellular sites probably in
accessible to -SH reactors of the iodoacetate type,
it will be necessary to reconsider the possible nelo
vance of particular types of -SH reaction to the
mode of action of the alkylating agents. Greater
knowledge of the site and extent of reaction is no
quired and, in particular, whether thiol reaction in
the nucleus plays any role in inhibition of cell
division or in those processes leading to ma
lignancy. If in the future alkylation of proteins or
peptides and, in particular,
alkylation of those
bearing reactive thiol groups is shown to have no
part to play in determining any of the important
pharmacological propertiesof the alkylatingagents,
the experiments leading to such conclusions will
be mainly those which now remain to be done,
since those which have been done can lead us to
no definite conclusion. The writer is not a sup
of Alkylating
Agents
13@27
porter of any theory of mode of action whose
foundations
nest on particular reactions at par
ticular cellular sites, which excludes other possi
bilities, but he wishes to draw attention to certain
oversimplifications
in reasoning which have led to
premature
conclusions regarding -511 reaction,
where in fact a great deal of work remains to be
done. It may transpire that much of the reasoning
used in the past to explain the mode of action of
alkylating agents has its foundation in ovensimpli
fication, and although theories to explain certain
pharmacological
events are often useful in leading
to further experimentation,
they can be a drag on
rapid progress if their exponents and others regard
them as an end in themselves.
Reaction of aikylating agents with nucleic acids.—
Since the importance of the nucleic acids in cellu
lar metabolism
was established,
many workers
have been attracted to the idea that all the im
pontant biological properties
of the alkylating
agents can be explained on the basis of reaction
with nucleic acids, particularly
DNA. Since the
alkylating agents cause gross mitotic abnormali
ties and can effect gene mutations, this hypothesis
has always had many followers. Nobody would
doubt that the alkylating agents lead to an altena
tion in the structure and function of nuclear DNA
and consequently in cellular. integrity, but there is
evidence that some of these alterations might be
mediated by indirect effects at many sites, such as
those indicated earlier when thiol reaction was
being considered.
The effects of the alkylating agents on DNA,
RNA, and protein synthesis have been recently
well reviewed by Wheeler (87). It transpires that
the mustards are capable of inhibiting DNA syn
thesis to a greater extent than RNA synthesis,
whereas the methanesulfonates
such as Myleran
have little effect on either—results which may help
to clarify the apparent differences in phanmaco
logical properties of the two classes of compound.
Considering the popularity of the concept of di
rect action on DNA it seems incredible that the
actual loci of the reactions should be overlooked
for so long. Despite the work of Young and Camp
bell (90), who first drew attention to the reactivity
of the guanine moiety, and of Wheeler, Morrow,
and Skipper (88, 89), who pointed out that
quaternization
of a ring nitrogen atom was likely
in the reaction of certain punines with alkylating
agents, attention continued to be directed toward
reaction with ionized phosphate groups as the most
probable points of reaction in the nucleotides (@,
74). Although estenification of phosphate groups
seems probable in some cases it is a difficult neac
tion to demonstrate because of the lability of the
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13@8
Cancer Research
Vol. 23, September
1963
products formed and because differences in the
liberation of acid, interpreted
as being due to
esterification, also occur following quaternization
of the ring nitrogen atoms. It remained for Lawley
and Wallick (50), possibly stimulated by the re
marks of Skipper (80), to demonstrate
that di
methylsulfate
reacted with guanylic acid to form
7-methylguanine,
and it was further shown by
Lawley (49) that the product formed from deoxy
guanylic acid and dimethylsulfate
was much more
labile and that liberation of 7-methylguanine
oc
at pH 7, and in this case breakdown probably oc
curs with the formation of an aldehydopynimi
dine derivative by ring opening (Chart 11).
In the case of reaction with DNA, the apuninic
acid formed after removal of the alkylated guanine
will be relatively unstable and could lead to main
chain breakdown.
These reactions may be of fundamental
im
portance in explaining some of the phanmacologi
cal effects of the alkylating in mechanistic chemi
cal terms. Thus it was postulated
(55) that the
curred
greater
appreciably
at
pH
7.@ (37° C.).
Thus,
a
mechanism was established by which alkylation
of guanine moieties of DNA could effect changes
0H(cH3),S04
oH?@'
@IcH
HZN'@N'@@I@
H@N@%
R
R- raos.
@
à @p@sta
CHART 10.—R.eaction of deoxyguanylic
acid with dimethyl sulfate.
acid or guanylic
0HcH3
H,N
R
CHART
11.—Breakdown
form an aldehydropyrimidine
of
an
alkylated
guanylic
acid
to
derivative.
due to quaternization,
elimination of the alkyl
ated guanine moieties at physiological pH, and
possibly fission of the polymer chain.
The reaction can be depicted as in Chart 10,
when R = nibophosphate or deoxynibophosphate,
and the product will have another form contnibut
ing to the resonance hybrid in which the positive
charge resides on the alkylated nitrogen atom.
Where R = deoxynibophosphate,
breakdown can
probably occur in two ways, leading in the one
case to the 7-methylguanine
and a carbonium ion
R@which will react with a molecule of water leading
to the liberation of acid and an apuninic acid, or
breakdown could occur at the other nitrogen atom
leading to a regeneration of the original deoxygua
nylic acid and liberation of CH@. Alkylated (e.g.,
methylated)
guanylic acids are much more stable
efficiency
of certain
bifunctional
alkylating
agents in killing cells and interfering with mitosis
might be due to their capacity to cross-link parts
of DNA, or DNA to protein, or protein to protein.
We have seen how proteins might be cross-linked
(71), some evidence is available for linking of
nucleic acid to protein (77), and Alexander, Cou
sens, and Stacey (4) obtained evidence of cross
linking of DNA in nucleoprotein
with nitrogen
mustards.
This they interpreted
as being solely
due to reaction with phosphate group, but in the
light of the findings of Brookes and Lawley (@1)
that bis-guanine products are formed after reac
tion of various alkylating
agents with nucleic
acids, they have had to reconsider their interpreta
tion, and it is the belief of Lett, Parkins, and Alex
ander (51) that in some cases the alkyl groups of
phosphate esters can transalkylate
to the 7-posi
tion of guanine accounting
for the apparently
higher reactivity of guanine when in DNA than
when it appears in nucleosides (75, p. 79). The
main evidence which they present for this comes
from spectral data, the U.V. changes which ac
company base alkylation being delayed in some
cases as if two reactions were occurring; and they
also interpret changes in “coiling―
as being due to
estenification. Whatever the true interpretation,
the fact remains that parts of DNA can be cross
linked in vivo, and DNA will be more or less in
activated and degraded depending on the extent of
reaction. Some of the cytotoxic effects of, say, the
mustards
will be explicable on this basis, but
whether the whole complex process of lysis of fully
differentiated
lymphocytes,
for example, can be
explained entirely by such mechanisms remains to
be tested.
Brookes and Lawley (@1, @)have studied the
extent of combination of various alkylating agents
with DNA and have found a simple rule to
apply—the more reactive the compound and the
greaten its tendency to react with all nucleophiles,
the greater the extent of reaction with the guanine
moiety. Thus the mustards reacted extensively,
whereas the methanesulfonates,
halides, and sul
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1963 American Association for Cancer Research.
WARWICK—Mechanism
fates reacted much less extensively
of Action
and more slow
ly. These findings again reflect the differences in
pharmacological
properties displayed by the van
ous compounds. Thus the mustards, highly reac
tive toward most nucleophiles, able to penetrate
deep into cells, able to cross-link nucleic acids and
probably proteins, are able to create havoc among
cells, causing effects from inhibition of cell division
to complete cellular disruption and lysis in con
trast to the less reactive reagents such as Myleran
which would be expected to react very little with
DNA at therapeutic doses and unable to cross-link
proteins through their thiol groups (see earlier).
The greater efficiency of mustards in causing cell
death probably
results from their heightened
capacity to react with all nucleophiles within the
cell, not merely to their greater reactivity toward
DNA.
The low level of reaction of Myleran with
nucleic acids in vivo leads one to seek for other
explanations for its pharmacological effects. There
is no scope in this discussion for considering other
implications of DNA reaction such as mutagenesis
and the difficulties which have been found in inter
preting some of the biological phenomena
en
countered—for example, the “uniquenessof ethyl
ation― (@3, 51, 53) ; but let it suffice to say that,
although
difficulties have been encountered
in
interpreting
mutation
esterification
put forward
(f), a working hypotheth has been
by Lawley and Brookes (%4) which
seems
to fit most
of the
in
the
facts
light
and
of
which
phosphate
can
prob..
ably be tested.
MUTATION
AND CANCER
Although bifunctionality
in alkylating agents is
a normal requisite for high killing action against
cells, this is not the case with regard to muta
genesis and carcinogenesis, since in these respects
monofunctional
agents are often as active or more
active than their bifunctional counterparts,
and
this is in line with the concept that cross-linking is
lethal. Some evidence exists that the physiological
state of the cell influences the occurrence
of
‘spontaneous' or induced chromosomal abnormal
ities (15—17) in some cases; Loveless (54) has
strongly supported the hypothesis that mutation
in the case of the alkylating agents involves direct
attack on nucleic acid. The fact that monofunc
tional alkylating agents can be both mutagenic
and carcinogenic coild be used as evidence to
support the mutational theory of cancer. On the
other hand, one should not ignore appealing al
ternatives
which involve much more extensive
alteration of cellular integrity and alteration in
of Al/cylating
Agents
13@9
delicately balanced cellular homeostasis. There is
much evidence to support an alteration in the in
tegrity
of mitochondria
and the endoplasmic
reticulum during administration
of carcinogens (8,
9), a consequence of the alteration of lipo-protein
membranes which constitute the structural
sup
port of the organized multi-enzyme
complexes.
Thus 3'-DAB (dimethylaminoazobenzene)
has a
profound
effect on microsomal
swelling when
fed for 4 weeks (6), in contrast to the inactive
@-methyl DAB, and the former compound also
produces a significant decrease in the mitochon
drial population in the cell (64, 78). During the
early stages of carcinogenesis with any agent the
cell population is being continuously modified, and
many cells die as a result of excessive interference
with their cytoplasmic
and nuclear elements.
Those extensively modified cells which survive will
be those which have been able to adapt themselves
to the unfavorable conditions; but with many of
their complex feedback controls and fine structure
damaged they will be unable to respond to the
normal stimuli of intercellular dependence and will
cease to obey the stringent rules of organismal
organization
(81). The great complexity of the
intracellular
effects produced by the alkylating
carcinogens, ranging from slight alterations in cell
structure to cell death, makes the problem of sort
ing out the chain of events leading to malignancy
one of the utmost difficulty. The relatively long
time lapse between treatment with the carcinogen
and the emergence of malignant tumors is prob
ably related to the multitude of changes occurring
in the cell population
during the ‘adaptive'
period. The oncogenic viruses are probably more
specific in their effect and faster acting because of
their information
content and may eventually
prove to be better tools for elucidating the chain
of events leading to malignancy than the chemical
carcinogens which in many ways have not lived
up to earlier expectations.
CONCLUSION
No attempt has been made to discuss the actual
biochemical lesions such as effects on cellular syn
thetic mechanisms which result from the action of
the alkylating agents. Effects on glycolysis, en
zymes, and so on have been well reviewed by
Wheeler (87). Rather, an attempt has been made
to correlate the basic alkylating capacities of the
various compounds
with gross biological end
products, and some attempt has been made to
classify the various agents into groups or classes
as far as possible.
Some conclusions can be reached regarding
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1330
Cancer
Research
Vol. @23,September
1963
these comparisons, the main one being that the
facile explanations for their mode of action. We
most reactive compounds of the Snl or pseudo
now have some idea of the chemical sites with
which a few of them interact intracellularly,
but
Sni type with a satisfactory “spread―
in their re
activity toward all nucleophiles are those which
from a very mechanical organic chemical view
manifest the most diverse pharmacological
effects.
point. Much more work now needs to be directed
It must also be relatively obvious from the fore
to the study of their effects in relation to cell popu
going discussion that none of the studies carried
lation dynamics and their effects in relation to
out on the mechanism of action of alkylating
intercellular dependence and to dedifferentiation,
agents has given any information as to how better
since it is in these dynamic aspects that the
drugs with a greater selectivity of action might
explanation of the carcinogenic process lies.
be designed, although suggestions have been made
Wheeler (87) indicates at the conclusion to his
as to why certain drugs are more efficacious than
review that there is a need “to
pinpoint the critical
others. This statement is depressing, and yet the
site of alkylation and the real cause of anticancer
obvious must be faced—and this is that the
activity.― Assuming that there is a “criticalsite of
alkylating agents so far designed are fan too crude
alkylation,― which is debatable,
one wonders
in their killing properties to be of any permanent
whether knowing it would help in designing better
use in cancer therapy. Of course one must always
chemotherapeutic
agents. We now have drugs
leave room for the unexpected breakthrough which
which will attack almost every cellular site, and
may take the form of a drug with a special “car it is difficult to envisage a new type of alkylating
rier― group (14) or a drug possessing “latent― agent which has not yet been tried.
activity, and yet attempts which have been made
More research on the effects of combinations of
so far to make this kind of breakthrough
have
drugs on tumors might be worth while—there
proved somewhat disappointing.
Thus, for ex
would seem to be endless possibilities to exploit
ample, Ross and Warwick (76, 86) made a series
and the recent work of Dean and Alexander (f27)
of mustard derivatives of azobenzene which would
demonstrating the potentiating
effect of iodoacet
remain chemically unreactive until reduction of amide on the destructive
effect of radiation on
the azo linkage occurred in vivo, and it was hoped
cultured lymphoma cells and bacteria is encourag
that some selectivity of action might be achieved.
ing. In this connection, Roberts and Warwick' ob
However, the compounds are relatively toxic, and
served the effect of pretreatment
of rats with a
one (40) has undergone clinical trial with disap
large dose of ethyl methanesulfonate
prior to injec
pointing results. Yet more of this type of research
tion with Myleran on the peripheral blood count.
It was hoped that ethyl methanesulfonate, while
is needed.
Compounds possessing all the various types of inactive itself against granulocytes, might potenti
chemical reactivity
have been tried, and no ate the effects of Mylenan, since it is itself a thiol
obvious solution to the problem of what to try next
reactor. Preliminary
results have proved disap
has been forthcoming. Thus, compounds such as pointing, but in some respects rats are not ideal
Myleran, Chiorambucil, etc., which are now used
test subjects from the point of view of following
as palliatives in the treatment of chronic leukemia
minor variations in peripheral blood count due to
and Hodgkin's disease, have certainly played a large background fluctuations, and the experiment
will probably be repeated in some other way.
part in relieving distressing symptoms and have
Bearing in mind the chemical, as well as the bio
enabled certain patients to remain active when
otherwise they may have been bedridden; and in logical, properties of the various agents, some
interesting combinations which have not yet been
this sense the efforts which have gone into
tried might come to light.
planning them and synthesizing them has prob
At this Institute further attempts are being
ably been justifiable.
However, whether many
more of these compounds should be made is a made to exploit differences between normal and
tumor tissue in several ways which have been do
debatable point.
scnibed at length by Ross (7@2),and the attempts
The alkylating agents have stimulated much re
being made by Connors and Elson (@25)to lower
search on fundamental
problems concerning cell
general toxicity while maintaining antitumor ac
metabolism,
mutagenesis,
cancinogenesis, and so
tivity by prior treatment with certain compounds
on, and it is probably in these fundamental ways
such as thiols, etc., is worthy of comment. Some of
that they have proved most useful. However,
these studies assume different pH levels between
their very number, the diversity of pharmacologi
tumor tissue and normal tissue, and results are
cal effects which they manifest, and the number of
cellular sites with which they interact preclude
2 Unpublished
data.
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1963 American Association for Cancer Research.
WARWICK—Mechanism
of Action
awaited with interest. Studies on the mechanisms
of resistance are urgently required, but this sub
ject is to be covered by Dr. Wheeler.
Some fresh approaches are needed to lift cancer
chemotherapy
from the somewhat unsatisfactory
situation in which it finds itself, but most of all is
needed a really critical assessment from all points
of view of the situation as it stands at the moment.
of Alkylating
some Alteration
and Tumour
Inhibition
by Nitrogen
Mustards: The Hypothesis of Cross-Linking
Alkylation.
Nature, 166:1112—13,1950.
19. BocK, M., and JACKSON, H. The Action of TEM on the
Fertility of Male Rats. Brit. J. Pharmacol., 12: 1—7,1957.
20. BRANCH, G. E. K., and C@vzx, M. The Theory of Organic
Chemistry, p. 439. New York: Prentice-Hall,
Inc., 1947.
21. BROOKES,P., and LAWLET, P. D. The Reaction of Mono
and Di-Functional
Alkylating Agents with Nucleic Acids.
Biochem. J., 80:496—503, 1961.
22.
REFERENCES
1. ALEXANDER,P. The Reaction of Carcinogens with Macro
molecules. Adv. Cancer Res., 2: 1—72,
1954.
2.
. Comparisonof the ChangesProducedby Ionizing
Radiations and by the Alkylating Agents: Evidence for a
Similar Mechanism at the Molecular Level. Ann. N.Y.
Acad. Sci., 68:1225—1337, 1958.
3.
. Comparison of the Action of Ionizing Radiations
and Biological Alkylating Agents (Nitrogen Mustards) at
the Cellular and Sub-Cellular Level. Sci. Rep. Inst. Super.
Sanita, 1:537—50,1961.
4. ALEXANDER,P. ; COUSENS,S. F. ; and STACEY,K. A. Drug
Resistance
in Microorganisms.
Ciba Foundation
Sym
posium, p. 294. London: Churchill, 1957.
5. ANSON, M. L., and STANLEY, W. M. Some Effects of
Iodine and Other Reagents on the Structure and Activity
of Tobacco Mosaic Virus. J. Gen. Physiol., 24:679-90,
1941.
6. ARCOS, J. C., and ARCOS, M. Fine Structural Alterations in
Cell Particles during Chemical Carcinogenesis.
1. The In
fluence of Feeding of Aminoazo Dyes on the Swelling and
Solubilization
of Rat-Liver
Microsomes.
Biochim. Bio
phys. Acta, 28:9—20, 1958.
7.
. Molecular Geometry and Mechanism of Action of
Chemical Carcinogens.
Fortsch.
Arzneimittelforschung,
4:407—581,
1962.
8. ARCOS, J. C. ; GRIFFITH, G. W. ; and CUNNINGHAM, R. W.
Studies on the Swelling of Rat Liver Mitochondria
in
Relation to Tumour Incidence during Feeding of Aminoazo
Dyes. Experientia, 15:316—17, 1959.
9.
. Fine Structural Alterations in Cell Particles during
Chemical Carcinogenesis.
J. Biophys. Biochem. Cytol.,
7:49-60, 1960.
10. BARRON, E. S. G. Thiol Groups of Biological Importance.
Adv. Enzymol., 11:201—66, 1951.
11. BATEMAN, A. J., and JACKSON,H. Mutagenic Action of
TEM. Ann. Rep. Brit. Emp. Cancer Campaign, p. 473,
1958.
12. BERENBAUM, M. C. The Effect of Cytotoxic Agents on the
1331
Agents
. The Alkylationof Guanosineand GuanylicAcid.
J. Chem. Soc., pp. 3923—28,1961.
23.
. Ann. Rep. Brit. Empire Cancer Campaign, pp.
8—13,1960.
24.
. Ionization of DNA Bases or Base Analogues as a
Possible Explanation of Mutagenesis, with Special Refer
ence to 5-Bromodeoxyuridine.
1962.
J. Mol. BioL, 4:216-19,
25. CONNORS,T. A., and ETaON, L. A. Reduction of the
Toxicity of Alkylating Agents in Rats by Thiol Pretreat
ment. Biochem. Pharmacol., 11:722—32, 1962.
26. CRAIG, A. W.; Fox, B. W.; and JACKSON, H. Sensitivity of
the Spermatogenic Process in the Rat to Radiomimetic
Drugs
and X-Rays.
Nature,
181:353—54,
1958.
27. DEAN, C. J., and ALEXANDER, P. Sensitization of Radio
Resistant Bacteria to X-Rays by Iodoacetamide.
Nature,
196:1324—26, 1963.
28. DUSTIN, P. Some New Aspects of Mitotic Poisoning.
Nature,
159:79497,1947.
29. ELSON, L. A. Comparison of the Physiological Response to
Radiation
and Radioniimetic
Chemicals.
Patterns
of
Blood Response. In: G. C. DE HEVESY (ed.), Adv. in
Radiobiology,
pp. 372—75. Edinburgh:
Oliver & Boyd,
1957.
30.
. Hematological
Effects of the Alkylating Agents.
Ann. N.Y. Acad. Sci., 68:826—33, 1958.
31.
. The Effects of Myleran and Homologous Com
pounds on the Blood. Biochem. Pharmacol., 1:39—47, 1958.
32. ELSON, L. A.; GALTON,D. A. G.; and TIu@, M. The Action
of Chlorambucil and Busuiphan (Myleran) on the Haemo
poietic Organs of the Rat. Brit. J. Haematol., 4:355—74,
1958.
33. FARMY, 0. G., and FAHMY,M. J. Cytogenetic Analysis of
the Action of Carcinogens
and Tumour
Inhibitors
in
Drosophila Melanogaster. IX. The Cell-Stage Response of
the Male Germ Line to the Mesyloxy Esters. J. Genetics,
46:361—72,
1961.
34. GOLDECK,H., and HAGENAR, H. Der Stickstoff host Em
fuss auf die Fertilitat
und Spermiogenese
der Labora
toriumsratte. Z. gus. exp. Med., 117:67—80,1951.
35. HADDOW, A. The Chemical and Genetic Mechanisms
of
Carcinogenesis.
II. Biologic Ailcylating Agents. In: F.
Biochem. Pharmacol., 11:29—44, 1962.
HOMBURGER (ed.), The Physiopathology
of Cancer, pp.
BERGEL,F. and STOCK,J. A. Cytotoxic a-Amino Acids and
602—85,2d ed. New York: Hoeber, 1959.
Peptides. Ann. Rep. Brit. Emp. Cancer Campaign, p. 6,
36. HEIDELBERGER,C. ; and DAVENPORT, G. R. Local Func
1953.
tional Components of Carcinogenesis. Acta Internat. Unio
BERGEL, F.; STocic, J. A.; and WADE, R. Peptides and
contra Cancrum, 17:55—62, 1961.
Macromolecules
as Carriers of Cytotoxic
Groups. In:
Biological Approaches to Cancer Chemotherapy,
pp. 125— 37. HUGHES, E. D.; INGOLD, C. K.; and SHAPIRO, U. G.
Mechanism of Substitution at a Saturated Carbon Atom.
34, London: Academic Press, 1961.
Part VI. Hydrolysis of Iso-Propyl Bromide. J. Chem. Soc.,
BIESELE, J. J. Induced Imitations of Mitotic Anomalies.
pp. 225—36,1936.
Ann. N.Y. Acad. Sci., 71: 1054—67,1958.
38. Hu@r, J. A., and INGRAM, V. M. The Chemical Effects of
. Studies on Mitosis in Purine Treated Tissue Cul
Gene Mutations in Some Abnormal Human Haemoglobins.
tures. In: S. C. PALAY (ed.), Frontiers in Cytology, pp.
Symposium on Protein Structure,
pp. 148—51. London:
84—112. New Haven:
Yale Univ. Press, 1958.
Methuen & Co., Ltd., 1958.
. Mitotic Poisons and the Cancer Problem. New
York: ELsevier, 1958.
39. INGOLD, C. K. Structure
and Mechanism
of Organic
BIESELE, J. J.; PHILIPS, F. S.; TrnEasdu,
J. B.; BmtcHE
Chemistry, p. 335. Ithaca: Cornell Univ. Press; and Lon
Production of Antibody to T.A.B. Vaccine in the Mouse.
13.
14.
15.
16.
17.
18.
NAL,
J.
H. ; BUCKLEY,
S. M.;
and
STOCK,
C.
C.
Chromo
don: Messrs. G. Bell & Sons, Ltd., 1953.
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1963 American Association for Cancer Research.
133@
Cancer Research
Vol. @23,September
1963
40. ISRAEL8, L. G. ; RITZMANN, S. Laboratory
and Clinical
Studies on a New Azo-Mustard,
4-Di-2―-chloroethyl
amino-2-methyl-2'-carboxyazobenzene(CB1414).
Acta Un.
62. OAKBERG, E. F. Sensitivity and Time of Degeneration of
Spermatogenic
Cells Irradiated
in Various Stages of
Maturation
in the Mouse. Rad. Res., 2:369—91, 1955.
63. OGSTON, A. G. The Replacement
Reactions of $-@‘41. JACKSON, H. Side Effects of Some Tumour Inhibiting
Dichiorodiethyl
Suiphide and of Some Analogues in Aque
Drugs. J. Soc. Chem. md., p. 949, 1957.
ous Solution:
The Isolation
of $-chloro-$'-hydroxy
42.
. The Effects of Radiomimetic Chemicals on the
Diethyl Sulphide. Trans. Faraday Soc., 44:45—50, 1948.
Fertility
of Male Rats. J. Fac. Radiol.,
9:217—20, 1958.
64. PRICE, J. M.; Mn@Erc, E. C.; MILLER, J. A.; and WEBER,
43.
. AntifertilitySubstances.Pharm.Rev., 11:135—72,
G. M. Studies on the Intracellular Composition of Livers
1959.
from Rats Fed Various Aminoazo Dyes. II. 3'-Methyl,
44. JACKSON,
H.; Ciwo, A.W.; and Fox,B. W.Comparative
2'-Methyl
and 2-Methyl-4-dimethylaminoazobenzene;
3Effects of Certain Alkylating Agents on the Proliferation of
Methyl-4-monomethylaminoazobenzene
and 4'-Fluoro-4Normal and Tumour Tissues. 1. Spermatogenesis
and
dimethylaminoazobenzene.
Cancer Res., 10: 18—27,1950.
Tumour Growth. Acta Unio. Internat.
contra Cancrum,
65. ROBERTS, J. J., and WARWICK, G. P. Studies of the Mode
16:673—75,
1960.
of Action of Tumour Growth Inhibiting Alkylating Agents.
45. Kou@zm, P. C. Comparative
Effects of Alkylating Agents
I. The Fate of Ethyl Methanesulphonate
(“HalfMy
on Cellular Morphology. Ann. N.Y. Acad. Sci., 68:783—
leran―) in the Rat. Biochem. Pharmacol., 1:60—75, 1958.
801,1958.
66.
. Studies of the Mode of Action of Tumor Growth
46.
. Comparison of the Biological Effects of X-Rays
Inhibiting Alkylating Agents. III. The formation of Tetra
and Radiomimetic
Chemical Agents. Proc. Third Aus
hydrothiophene-1,1-dioxide
from 1,4-Dimethanesulpho
tralasian Conference on Radiobiology.
Radiobiol, pp. 218—
nyloxybutane
(Myleran), S-@9-L-Alanyltetrahydrothiophe
86, 1960.
niummesylate, Tetrahydrothiophene, and Tetrahydrothio
47. LAJTHA, L. G. Bone Marrow Cell Metabolism. Physiol.
phene-1,1-dioxide,
in the Rat,
Rabbit
and Mouse.
Rev., 37:50-05, 1957.
Biochem. Pharmacol., 6:217—27, 1961.
48. LANDING, B. H.; Goum@, A.; and Nos, H. A. Testicular
67.
. The Reaction of Some Compounds of Biological
mt.Cancer.
16:665—71,
1960.
Lesions
in Mice
Following
Parenteral
Administration
Interest with Nucleic Acids. Biochem. J., 84: 119 r@,1962.
of
Nitrogen Mustards. Cancer, 2: 1075—82,1949.
49. LAwLET, P. D. The Hydrolysis of Methylated Deoxy
guanylic Acid at pH 7 To Yield 7-Methylguanine.
Proc.
Chem. Soc., pp. 290—91,1957.
50. LAWLEY, P. D., and WAu@icu, C. A. The Action of
Alkylating Agents on Deoxyribonucleic
Acid and Guanylic
Acid. Chem. & lad., p. 633, 1957.
51. Ls'rr, J. T.; PARKINS,G. M.; and ALEXANDER,P. Physico
chemical Changes Produced in DNA after Alkylation.
Arch. Biochem. Biophys., 97:80—93, 1962.
68.
52. LoVELESS,
A. QualitativeAspects of the Chemistryand
71.
Biology of Radiomimetic (Mutagenic) Substances. Nature,
167:338—42,
1951.
53.
. The Influence of Radiomimetic Substances on De
oxyribonucleic Acid Synthesis Studied in E8cherichia coli
Phage Systems. III. Mutation of T2 Bacteriophage
as a
Consequence of Alkylation in Vitro: The Uniqueness of
Ethylation.
Proc. Roy. Soc., s.B., 150:497—508, 1959.
54.
. The Involvement
of Deoxyribonucleic
Acids in
Chemical Mutagenesis.
Biochem. Pharmacol.,
4:29—33,
1960.
Carcinogenesis.
2-Chloroethylarylamines. Biochem. Pharmacol. (in press).
72.
73.
74.
75.
76.
61. Nou@m@,C. R., and DnisMous, R. The Reaction of Alkyl
Bromides with Pyridine. J. Am. Chem. Soc., 54:1025-84,
1932.
. Studies of the Mode of Action of Tumour Growth
Inhibiting Alkylating Agents. VI. The Metabolism of Bis
@9-Chloroethyl Sulphide (Mustard Gas) and Related Corn
pounds. Biochem. Pharmacol. (in press).
Ross, W. C. J. Biological Alkylating Agents. London:
Butterworths,
1962.
. The Chemistry of Cytotoxic Alkylating Agents.
Adv. Cancer Res., 1:397—449, 1953.
. in Vitro Reactions of Biological Alkylating Agents.
Ann. N.Y. Acad. Sci., 68:669—81, 1958.
. Biological Alkylating
Agents, p. 25. London:
Butterworths,
1962.
Ross, W. C. J., and WARWICK, G. P. AryI-2-halogeno
alkylamines. Part XVIII. The Rates of Reduction of Sub
stituted 4-Di-(2-chloroethyl)arninoazobenzenes by Stan
nous Chloride, Hydrazine,
and the Xanthine
Oxidase
Xanthine System. J. Chern. Soc., pp. 1724—32,1956.
77. RUTMAN, R. J. ; STEELE, W. J. ; and PRICE, C. C. Observa
tions on the in Vise Alkylation of Ehrlich Tumour Cell
78.
79.
J. Nail. Cancer Inst., 15:
1571—90,
1955.
of Hy
acids in Vitro.
197:87—88, 1963.
. The Mode of Action of Alkylating Agents. II.
Studies of the Metabolism of Myleran. The Reaction of
Myleran with Some Naturally Occurring Thiols in Vitro.
Biochem. Pharmacol., 6:205—16, 1961.
70.
. Studies of the Mode of Action of Tumour Growth
Inhibiting Alkylating Agents. IV. In Vitro Reactions of
60. Mni@ica,
E. C., and Mu@su,J. A.BiochemicalInvestiga
tions on Hepatic
Reaction
with Nucleic
Nature,
Alkylation. Nature, 168: 1113—14,
1950.
58. M@zi.&, D. Biochemistry
of the Dividing CelL Ann. Rev.
Biochem., 30:669—88, 1961.
59. Mxcn.&zus, L., and SCHUBERT, M. P. The Reaction of
lodoacetic
Acid on Mercaptans
and Amines. J. BioL
Chem., 106:331—41, 1934.
Liver Carcinogenesis:
69.
55. LOVELESS,
A.; GOLDACRE,
R. J.; and Ross, W. C. J. Mode
of Production of Chromosome Abnormalities by the Nitro
gen Mustards: the Possible Role of Cross-Linking. Nature,
163:667—69,
1949.
56. LovELEss, A., and REVEL, S. New Evidence on the Mode
of Action of Mitotic Poisons. Nature, 184:938—44, 1949.
57. LovELEss, A., and Ross, W. C. J. Note Following Biesele,
J. J. St at. Chromosome Alteration and Tumour Inhibition
by Nitrogen Mustards: the Hypothesis of Cross-Linking
. Azo-Dye
droxymethylaminoazobenzene
80.
81.
DNA by Nitrogen Mustards.
Biochem. Biophys. Res.
Comm., 4:278—82, 1961.
SCHNEIDER, W. C. Intracellular
Distribution
of Succiic
Dehydrogenase,
Cytochrome
Oxidase, Adenosinetriphos
phatase and Phosphorus Compounds in Normal Rat Liver
and in Rat Hepatornas. Cancer Res., 6:685, 1946.
SHAVER, S. L. X-Irradiation
Injury and Repair in the
Germinal Epitheium
of Male Rats. I. Injury and Repair
in Adult Rats. Am. J. Anat., 92:391-432, 1953.
SKIPPER, H. E. Discussion of the Biological Effects of
Ailcylating Agents. Ann. N.Y. Acad. Sci., 68:808, 1958.
SMITHERS, D. W. Cancer: An Attack on Cytologism.
Lancet, pp. 493—99,1962.
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1963 American Association for Cancer Research.
W@wic@x—Mechanism
of Action of Alkylating
82. STACEY, K. A.; COBB, M.; Cousm@s, S. F.; and ALzw@
DEE, P. The
Reactions
of the “Radiomimetic― Alkylating
Agents with Macromolecules
in Vitro. Ann. N.Y. Acad.
Sci.,
68:682—701,
1958.
83. ST@urn,G. R.; STm1@@,
W. H.; and MooRz, S. Relations be
tween the Conformation of Ribonuclease and Its Reactiv
ity toward Iodoacetate. J. Biol. Chem., 236:436—42, 1961.
84. SWAIN,
C. C. Abstr. 13thNail. Org.Chem.Symposium,
Am. Chem. Soc., p. 65, 1953.
85. Tmssxs, G. M. The Action of Antimetabolites
and Bio
logical A&ylating Agents on the Synthesis of Deoxyribo
nucleic Acid, and a Possible Relation between the Mech
anisms of Action. Biochem. Pharmacol., 4:49—56, 1960.
86. W@tuwicx, G. P. Relative Rates of Reduction of a Series of
Azo Compounds. J. Soc. DyersColourists,
75:291—99, 1959.
87. WHEmms,
1333
Agents
G. P. Studies
Related
to the Mechanisms
of
Action of Cytotoxic Alkylating Agent@ Cancer Res., 22:
651—88,1962.
88. WHEELER, G. P.; Moimow, J. S.; and S@xppm@,H. E.
Studies with Mustards. L Ion Exchange Chromatography
of Reaction Mixtures of Purines and Pyrimidines
with
Nitrogen Mustard. Arch. Biochem. Biophys., 57:124—32,
1955.
89.
. Studies with Mustards.
II. Paper Chromatog
raphy and Radioautography
of Reaction
Mixtures
of
Various Compounds
with S's-Sulphur
Mustard.
ibid.,
pp.
133—39.
90. YOUNG,
E. G., and [email protected]@,
IL B. The Reactionof
fl-fl'-Dichlorodiethyl
Sulphide
Res., 25B:37—48, 1947.
with
Proteins.
Canad.
J.
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1963 American Association for Cancer Research.
The Mechanism of Action of Alkylating Agents
G. P. Warwick
Cancer Res 1963;23:1315-1333.
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