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(CANCER RESEARCH 29, 2435-2442, December 1969]
The Need for Additional Alkylating Agents and
Antimetabolites
Charles Heidelberger1
McArdle Laboratory for Cancer Research, University of Wisconsin,
Madison, Wisconsin 53706
The title that was assigned to me is a clear invitation
topontificate. Instead, I will share with you a few modest andnot
very original thoughts and speculations. These are basedon a large
literature and a small experience of tilling the soilamong certain
obscure and unnatural pyrimidines. This engagement with the
rationality of antimetabolites followed anignoble exodus from the
quagmire surrounding an even moreobscure alkylating agent. Thus, my
bias is already exposed.
It is not my purpose to cover the enormous literature inthese
two fields of alkylating agents and antimetabolites.Rather, I shall
refer to reviews whenever possible. Nor do Iintend to explore
systematically structure-activity relationships of every series of
compounds, detailed screening results,comparative toxicology,
clinical pharmacology, andmechanisms of action. I will try merely
to focus on certainfacts, trends, and promising leads that happen
to interest meand suggest points of departure for future synthetic
chemicalforays. I shall emphasize drug design at the expense of
ignoringthe exciting advances in cell-cycle kinetics and clinical
combination chemotherapy that have been emphasized elsewherein this
Symposium. I shall forbear from intoning the customary litany of
our ignorance of the essential molecular lesions ofmalignancy and
intend to proceed with what is known. But Ihad better proceed.
ALKYLATING AGENTS
The alkylating agents as a group are among the clinicallymost
useful compounds in cancer chemotherapy. Their chemistry and
attempts to design agents showing selectivity ofaction have been
reviewed by Ross (46). Hirschberg (24) hasmade a very comprehensive
compilation of the earlier information about their chemotherapeutic
properties, and this hasbeen expanded and brought up to date by
Ochoa andHirschberg (39). Various aspects of the mechanism of
actionof alkylating agents in many systems have been reviewed
byBrookes and Lawley (7), Lawley (32), Wheeler (57, 58), andWarwick
(56). Oliverio and Zubrod (40) have discussed theirclinical
pharmacology.
The alkylating agents with chemotherapeutic activity
arechemically reactive and at least difunctional. The structures
ofa few of the important compounds are shown in Chart 1.Compound I
is sulfur mustard, the original prototype, andII-VIII are members
of the nitrogen mustard (HN2) family,
CH3N'l
0
0>C ...^i-'N
f X
^
XCH2CH2CI
XCH2CH2CI
I,CH2CH2CI
HN CHSNVCH2CH2CI XCH2CH2CI
E HI
CICH,CH2X
XCH2CH2CI
^rrH
m
oCICH2CH2NH C- NCH2CH2CI
NO
•yirr
CH2-CH2
CH2-CH2
X
/CH2-0XMCH2P-lN
CH,-N'H3ZECH2-CH2XCH2CH2CI1XSCH2CH2CI
CH2-N^N^Nsl
i^
IS.
0 0
CH^SOCH2CH2CH2CH2OSCH2
0 0
'American Cancer Society Professor of Oncology.
Chart 1. I, sulfur mustard; II, noi-HN2; III, nitrogen mustard,
HN2;IV, Nitromin; V, phenylalanine mustard (Sarcolyán, Melphalan);
VI,uracil mustard; VII, cyclophosphamide (Cytoxan, Endoxan);
VIII,l,3-bis(2-chloroethyl)-l-nitrosourea, BCNU; IX,
triethylenemelamine(TEM); X, Thio-TEPA; XI, Myleran.
with their characteristic 0-chloroethyl groups. Two examplesof
ethyleneimines (aziridines) are IX and X, and the
remainingimportant class of methane sulfonates is illustrated by
Myleran(XI). Literally thousands of these compounds have
beensynthesized, in which the reactive 0-chloroethyl,
ethylene-imine, or methane sulfonate groups have been hung
ontoinnumerable "carriers," which some mystics endow with
remarkable biologic specificity. Thus, we have
phenylalaninemustard (V, Sarcolysin, Melphalan), uracil mustard
(VI),antimalarial mustards, steroidal mustards, aromatic
mustards,quinonoid mustards, etc.
Do these "carriers" in fact impart any biologic specificity
to
the reactive groups that they embrace? In order to answer
thisquestion it is necessary to have reliable comparative data
onthe activities of a large number of compounds. Fortunately,
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CharlesHeidelberger
these data have been provided by a truly heroic andmonumental
study by Schmidt et al. (50). These investigatorsstudied 51
ß-chloroethylamines, 25 aziridines, 39 methanesulfonates, and 12
nonalkylating compounds for their toxicityto mice, rats, dogs, and
monkeys and their activities against 18transplanted rat tumors and
7 transplanted mouse tumors.This tour de force was published in a
monograph of 1528pages (50).
It has been alleged by many that the proof of
thechemotherapeutic specificity of the "carrier" rests on the"fact"
that the L-phenylalanine mustard has greater tumor-
inhibitory effectiveness than the D isomer or the DL mixture(V).
Let us examine the most reliable information on thissubject as
presented by Schmidt et al. (50). Their data on therat tumors are
given in Table 1. At the bottom of the table isthe mean for 10
tumors of the ratio of the LDi0/ED90 (orED60), the therapeutic
index, for the L isomer over the Disomer, which is 1.05 ±0.19.
Thus, there is no difference in thetherapeutic index of the two
isomers although, admittedly, theL isomer is more toxic and is more
active against the tumorsthan the D isomer. On the other hand, when
the ratio of the Lisomer to the DL mixture is taken for 16 tumors,
it is 0.76 ±0.08. This finding that the DL mixture has a
bettertherapeutic index than the L isomer resists
rationalexplanation. However, the same comparison in mouse
tumors(Table 2) shows that the L isomer is better than the D and
DL,but not strikingly so. If the ratios of all the rat and mouse
tumorscombined are taken, the mean L/DL ratio of the
therapeuticindex for 22 tumors is 0.92 ±0.09, and the L/D ratio
for 16tumors is 1.20 ±0.15. Neither of these ratios is
significantlydifferent from 1. In addition to demonstrating
biologicvariability, these data show clearly that if these optical
isomersrepresent the proof for the chemotherapeutic specificity of
the"carrier," then the case is lost, even though there are
differences in overall toxicity. Schmidt et al. (50)
haveconcluded that "with possibly two exceptions, both in the
Table 1
Tumor L DL L/DI/7 D L/D6
Table 2
Walker 256, CH,subcutaneousWalker256, CH,pulmonaryWalker256,
CH,ascitesWalker256, CB,subcutaneousWalker256,
CB,pulmonaryWalker256, SK,subcutaneousWalker256,
SK,pulmonaryYoshida
asciteshepatomaYoshidaascitessarcomaDunningIRC-741
leukemia,subcutaneousDunningIRC-741 leukemia,ascitesLymphoma
8,subcutaneousAdenocarcinomaR-35,subcutaneousAdenocarcinomaR-35,pulmonaryMurphy-Sturm
lymphosarcoma,subcutaneousNovikoff
hepatoma,
ascites178338.5331220332812.5112.51583121101012445425.5142.515.51.131.001.060.410.330.331.000.450.611.001.461.000.620.401.000.46102121101415310112.51.701.570.410.331.422.200.670.801.001.000.46
TumorSarcoma
180,subcutaneousAdenocarcinoma755,subcutaneousL1210,
subcutaneousL1210,ascitesEhrlichascitesAdenocarcinoma
SAH-I-I,
subcutaneousL1.52.5.1.04.04.53.5DL1.02.51.02.53.02.0L/DLa
D11!111.5.0.0.6.5.71.02.51.02.51.7L/Dè11112.5.0.0.6.6
Effects of optical isomers of phenylalanine mustard
ontransplantable rat tumors (50). LDi 0 'I DC,,,or LD10/EDoo-
°L/DL:mean of 16 tumors, 0.76 ±0.08.bL/D: mean of 10 tumors,
1.05 ±0.19.
Effects of optical isomers of phenylalanine mustard on
transplantablerat tumors (50). LDi0/ED90 orLD10/ED60.
°L/DL,mean of 6 tumors, 1.38 ±0.11., mean of 5 tumors, 1.54
±0.26.
methane sulfonate series, there was no indication that
specificclasses of alkylating agents or members within a class
wereendowed with unique antitumor activity. Apart from
theseexceptions, cyclophosphamide (VII) and the isomerie
phenylalanine mustards (V) were the outstanding representatives
invirtually every test system examined whether the neoplasmwas
highly sensitive or relatively insensitive to
alkylatingagents."
Let us now examine cyclophosphamide (VII, Cytoxan,Endoxan) a
little more closely. This compound "was designed
in accordance with the principle of transversion of an
inactivetransport form to an active form at special sites in the
body"
(5). It was thought that tumors are rich in phosphoamidases,and
hence that the P-N bond would be selectively cleaved inthe tumor to
activate the drug. However, it was soon foundthat cyclophosphamide
was not active against the growth oftumor cells in culture but was
metabolized to an active form inthe liver (15). Hence, the
properties of the compound were asdesigned, except that the
activation takes place in the liverrather than in the tumor. Since
that time a great deal of workhas been done, particularly by Brock
(5) and Friedman (17) toelucidate the nature of this activation
process. It was originallybelieved that after a series of
hydrolyses, nor-HN2 (II) wasliberated and was the therapeutically
active compound.However, this does not seem to be so, and the
present state ofthis exceptionally complicated situation appears to
be that themetabolism of cyclophosphamide in vivo does not parallel
itschemical behavior. Considerably more work is needed beforean
understanding is reached of the almost unique tumor-inhibitory
activity of cyclophosphamide.
Another compound of particular interest is
1,3-bis(2-chlo-roethyl)-l-nitrosourea (BCNU) (VIII), which is
unusual in thatit crosses the blood-brain barrier and is active
againstintracerebral L1210 leukemia (49). Although it
containsß-chloroethyl groups, there is some doubt as to whether it
isreally an alkylating agent, since it reacts to only a slight
extentwith 4-(p-nitrobenzyl)-pyridine (59). It inhibits DNA andRNA
synthesis in LI210 leukemia cells in vivo and in vitro(60), and its
effect on a DNA polymerase system iscomparable to that of
/3-chloroethylisocyanate ; it appears toreact with the enzyme
rather than with the primer (61).
It is clear that there is very little relationship between
thedistribution, excretion, and metabolism of the alkylatingagents
and their mechanism of action (39,40). Although theyare often
considered to be "radiomimetic" compounds, this
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Additional Alkylating Agents and Antimetabolites
analogy is probably overdone (56). Studies on the mechanismof
resistance of experimental tumors to alkylating agents havebeen
critically reviewed by Wheeler (58). The main fact is thatthere is
considerable cross-resistance among these structurallydiverse
compounds, but Wheeler (58) cautions against assuming from that
fact that the alkylating agents act by a commonmechanism; he
considered altered permeability as an important factor in
resistance, and the repair of alkylated DNA (seebelow) was unknown
at that time.
What then is the mechanism of action of the alkylating
agents,if, indeed, a single mechanism exists? It appears from
anoverwhelming body of data that the primary action of
thesecompounds is a direct attack on DNA. The chemistry of thishas
been worked out in a brilliant series of investigations byBrookes
and Lawley, which has been reviewed by Lawley (32).They showed that
sulfur mustard (I) exerts a nucleophilicattack on the N-7 of
guanine residues in the DNA, whichlabilizes the glycosidic bond and
causes the release of guaninefrom the DNA, which can thus give rise
to mutations. Theyalso showed that sulfur mustard could react with
two guaninesto produce a cross-linking, which they postulate to be
theprimary cytotoxic action, and which would prevent DNAreplication
(7). They have demonstrated with sensitive andresistant strains of
bacteria that in the latter there is an activerepair mechanism that
excises the cross-linked guanines fromthe DNA (33). They also found
in bacteria that similarcross-linking was obtained with
triethylenemelamine (TEM)(IX) and butadiene diepoxide, and produced
additional evidence that the cross-linking is interstrand (34).
Crathorn andRoberts extended this work to mammalian cells (HeLa)
andhave demonstrated that with sulfur mustard there is a
directrelationship between lethality and attack on DNA; they
alsofound repair-excision of alkylated guanines from the DNA(11).
Ruddon has reported that DNA reacted with HN2 (HI)has a decreased
template activity for RNA polymerase, and toa lesser extent for DNA
polymerase (48), which is opposite tothe inhibitory effects of
nucleic acid biosynthesis found inintact cells.
However, the view that the primary chemotherapeutic effectof
alkylating agents results from their attack on DNA is
notuniversally accepted, largely upon the basis of work done inmice
with tumors. Golder et al. (19) found that the extent ofalkylation
by HN2 was very slight and that there was someevidence of
crosslinking DNA to protein. Wheeler and Alexander (59) studied the
in vivo alkylation of sensitive andresistant tumors implanted
bilaterally into the same mouse,and found no correlation between
the extent of alkylation ofthe DNA and chemotherapeutic effect.
However, it is evidentto all those who are concerned with the
binding of carcinogensto DNA that there is considerable specificity
in binding, and infact Doskocil and Sormova (13) have found that
not allguanines in DNA are equally reactive to sulfur mustard.
Untilthe nature of this specificity is understood, the mechanism
ofaction of the alkylating agents will remain somewhat obscure.
What then is the need for additional alkylating agents?Schmidt
et al. (50) have stated, "In one sense the lack of
specificity of the various alkylating agents in both
thetherapeutic activity and toxicity spheres is disappointing.
Onthe surface at least, it offers little hope that any agent of
this
chemical type will be found which will affect some neoplasmsin a
unique way ...." Therefore, I feel that continuation of a
vast effort of synthesis aimed at the random attachment
ofalkylating groups to a fanciful collection of "carriers" can
no
longer be justified. However, there appear to me to be
threeareas in which specific and rational syntheses could be
justifiedat this time: (a) preparation of possible metabolites
ofcyclophosphamide and its derivatives, (b) synthesis of
furtherderivatives of BCNU, and (c) once we have a much
moresophisticated knowledge of the parameters regulating
thespecificity of the attack on DNA, this information might beput
to use in the area of drug design.
ANTIMETABOLITES
Let us herewith define a metabolite as some naturallyoccurring
compound produced during metabolism and anantimetabolite as a
compound related structurally to themetabolite which prevents its
further utilization by competingwith it for an enzyme. This
definition and field came out ofthe pioneering efforts of D. D.
Woods (64), who in 1940recognized the metabolite-antimetabolite
relationship ofp-aminobenzoic acid and sulfanilamide. The role of
thesecompounds in pharmacology and biochemistry was explainedwhen
the structure and function of folie acid were elucidated.Reviews of
various aspects of cancer chemotherapy withantimetabolites of many
sorts have been provided by Stock(53), Langen (30), Baker (2), and
Timmis (54).
Antifolics
The antifolics in cancer chemotherapy have three distinctions:
(a) they were the first compounds to be effective againstacute
leukemia in children; (b) they cure a high percentage offemale
patients with choriocarcinoma; and (c) they are nowwhere the action
is.
Folie acid (XII) is reduced to tetrahydrofolate by theenzyme
dihydrofoÃ-ate reducÃ-ase,which is powerfully inhibitedby
antifolates such as Amethopterin (XIII,
Methotrexate).Tetrahydrofolate reacts with formaldehyde (or other
compounds at the same oxidation level) to give isomerie formyl
orméthylènetetrahydrofolates, which are the coenzymes of
allone-carbon metabolism. Consequently, these coenzymes arerequired
for two steps of purine synthesis and for thymidylatesynthetase. A
great deal of work has been done on themechanism of resistance to
antifolics, and suffice it to say thatthis is not fully understood
at present. Although Amethopterin is a very powerful inhibitor of
dihydrofolate reductase,there is still a need to obtain more
selective and specificcompounds.
A simple modification, by the addition of a méthylènegroupto
give homofolic acid (XIV), has been studied by Friedkin etal. (43).
They have found some antimalarial activity with thiscompound and
have further demonstrated that tetrahydro-homofolates are
inhibitors of thymidylate synthetase. Veryrecently, Hutchison (27)
has reported that a series ofquinazoline analogs, of which XV is
the most active, exertgreater inhibitions of bacteria and tumor
cell growth than doesAmethopterin. Obviously, further work along
these two linesis indicated.
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CharlesHeidelberger
A Vc-NHCHCH2CH2COOH
COOH
xn
^-C-NHCHCH2CH2COOH
COOHXIII
_ O~VcONHCHCH2CH2COOH
COOH
k ^-C-NHCHCH2COOH
COOHzz
impermeable to the compound (3). Hopefully, this difficultymay
not be insurmountable. If so, we can look forward toinhibitors of
dihydrofolate reductase with unsurpassedspecificity to cancer
tissue. Here, then, is a supreme exampleof carrying out enzymatic
structure activity studies combinedwith skillful and imaginative
chemistry.
These latter studies of Hitchings (25) and Baker (3) gave riseto
the third category of distinction listed above.
NH OH OH
XVIII
SH
XVII
Chart 2. XII, folie acid; XIII, amethopterin (Methotrexate);
XIV,homofolic acid; XV, quinazoline analog; XVI,
pyrimethamine(Daraprim); XVII, Bakei and Meyer's analog.
A classical study of dihydrofolate reducÃ-ase inhibition
byanother series of inhibitors, the 2,4-diaminopyrimidines,
illustrated by XVI, Daraprim, an active antimateria!, has been
carriedout by Hitchings (25). He has studied the kinetics of
interactionof a series of these compounds with dihydrofolate
reductasesisolated from bacteria and various protozoa and has
foundstriking differences. This makes it possible to achieve
aremarkable specificity in the chemotherapy of infectiousdisease by
taking advantage of these species differences inenzymes.
Baker has for many years been working on "active-site-directed
irreversible inhibitors" of various enzymes, including
dihydrofolate reducÃ-ase. This productive research has
beendescribed in his book (2) and in innumerable papers since.
Bystudying the kinetics and nature of the inhibition of thisenzyme
with a large number of structurally related compounds, he has
"mapped" the region of the active site in terms
of ionic and hydrophobic regions. As the near-culmination ofthis
approach, Baker and Meyer (3) have recently reportedthat compound
XVII has the fantastic specificity of being apowerful irreversible
inhibitor of the enzyme isolated fromLI210 cells, but not from
mouse liver, spleen, and intestine!This finding has remarkable
connotations as to the nature ofthe carcinogenic change, but it
cannot yet be interpreted.Surely, this compound must be the "magic
bullet" that we
have all been searching for, with apparent specificity
forirreversible inhibition of only the tumor enzyme. Unfortunately,
however, this compound is inactive in vitro atinhibiting the growth
of LI210 cells which are presumably
SH ' 0
k CH3
xn XXtt XXTT1NHo NH2 NH2 ,oc>
HO
XXVI" ~ "~" CHNHCH2CH=CC >!>
HO OH
Chart 3. XVIII, adenine; XIX, guanine; XX, 8-azaguanine;
XXI,6-mercaptopurine; XXII, thioguanine; XXIII,
dimethyltriazenoirnid-azole carboxamide; XXIV, psicofuranine; XXV,
arabinosyladenine;XXVI, Cordycepin; XXVII, R = H, Tubercidin, R =
CM, Toyocamycin,R = CONH2, Sangivamycin; XXVIII, Formycin; XXIX,
isopentenyl-adenosine.
Furine Ant ¡metabolites
Various aspects relating to these compounds have beenreviewed by
Brockman (6), Cohen (10), Fox (16), andHeidelberger (23). The
naturally occurring purine bases,adenine (XVIII) and guanine (XIX),
have a large number ofanalogs related to them in various ways. A
number of theserepresent isosteric replacements in the imidazole
ring: 8-azaguanine (XX), Tubercidin (XXVII), and Formycin
(XXVIII).Two very important purine base analogs are
6-mercaptopurine(XXI) and thioguanine (XXII), which are effective
in cancerchemotherapy. Their nucleosides are known and have
beenstudied extensively, and it is of great interest that
thea-anomer of 2'-deoxythioguanosine, in contrast to all others
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Additional Alkylating Agents and Antimetabolites
studied, is biologically active and is incorporated into
DNA(36). It has also been found that
6-methylmercaptopurineribonucleoside has quite a different
mechanism of action than6-mercaptopurine (6-MP) and its
ribonucleoside (4), and thatthe periodate oxidation product of its
ribonucleoside inhibitstumor growth and has other interesting
biologic actions (28).
Another interesting compound is
4(5X3,3-dimethyl-triazene)-imidazole-5(4)-carboxamide (XXIII),
which is ananalog of an intermediate in purine biosynthesis and
hasconsiderable clinical activity against melonomas (51).
Itsbiochemical mode of action is not yet known (to this author),but
it represents an important new series of compounds that isworthy of
further chemical and biochemical exploration.
Arabinosyladenine (XXV) has considerable activity againstsome
tumors, and studies of its mechanism of action have beencarried out
(cf. 10, 23). Several purine nucleoside antibioticswith interesting
structures and tumor-inhibiting activity havebeen isolated,
characterized, and studied. These includeCordycepin (XXVI),
Psicofuranine (XXIV), Tubercidin, Toyo-camycin, Sangivamycin
(XXVII), and Formycin (XXVIII)(cf. 16). This latter compound is of
particular interest because anumber of its biologic properties have
been explained by Wardand Reich (55) as being due to the fact that,
in polynucleotideform, its individual residues exist in the syn,
rather than in thecommon anti, conformation. This poses interesting
possibilities for other C-nucleosides.
Isopentenyl adenosine (XXIX) was isolated from transfer RNAand
its structure determined by Hall et al. (20). It was foundto be a
powerful cytokinin in plants and also had inhibitoryactivity
against tumor cells and possibly against acute leukemiain children.
Because of this interesting activity, a number ofrelated compounds
have been synthesized and tested byFleysher et al. (14).
Although this presentation has been necessarily brief
andsuperficial, it is clear that there are a number of
interestingstructural modifications of purine nucleosides with
biologicactivity. There are also several other different types
ofstructural modifications that I have not listed here.
Therefore,it is quite clear that further synthetic work with purine
andpurine nucleoside analogs should be biologically, and
probablychemotherapeutically, rewarding.
Pyrimidine Antimetabolites
Some general reviews of this subject are those of Stock
(53),Langen (30), Baker (2), Timmis (54), and Brockman (6).
The pyrimidine bases that occur in the nucleic acids areuracil
(XXX), thymine (XXXI), and cytosine (XXXII). Theonly pyrimidine
antimetabolite that is chemotherapeuticallyactive as the free base
is 5-fluorouracil (FU) (XXXIII), whichwas originally synthesized on
the basis of a very definiterationale. With all other pyrimidine
analogs, the nucleosidesare required for biologic activity. A
derivative more active thanFU is 5-fluoro-2'-deoxyuridine (FUDR)
(XXXIV); and trifluo-
rothymidine (XXXV), in addition to being a potent
tumorinhibitor, is also the most active compound known to
inhibitthe replication of DNA viruses. These compounds have
beenthoroughly reviewed (22, 23). 5-Iodo-2 '-deoxyuridine
(IUDR),
(XXXVI) is clinically active against herpes simplex
keratitis
and is incorporated into DNA instead of thymine, but it hasbeen
disappointing in cancer chemotherapy (cf. 23). Aninteresting
approach to increasing the chemotherapeutic effectof a close
relative, 5-iodo-2'-deoxycytidine (ICDR), was made
by Woodman (63), who complexed the 5 -phosphate of ICDRwith
various polycations and found that it was incorporatedinto mouse
tumor DNA in vivo to a considerably greaterextent than was the
nucleoside. Such an approach may findapplication to other
nucleotide analogs.
Chart 4. XXX, uracU; XXXI, thymine, XXXII, cytosine;
XXXIII,5-fluorouracil; XXXIV, 5-fluoro-2'-deoxyuridine, FUDR;
XXXV,trifluorothymidine; XXXVI, 5-iodo-2'-deoxyuridine, IUDR;
XXXVII,
arabinosyl cytosine; XXXVIII, 6-azauridine; XXXIX, 5-azauridine;
XL,3-deazacytidine; XLI, S'-fluorothymidine; XLII, Showdomycin.
Arabinosylcytosine (ara-C) (XXXVII) is a compound that isvery
active against tumors, human acute leukemias, and DNAviruses (cf.
10, 23). Its mechanism of action is also underintensive study. It
is rapidly deactivated by deamination toarabinosyluracil (ara-U),
and interesting research has beencarried out to find inhibitors of
this deaminase in the hope ofincreasing the chemotherapeutic
effects of ara-C (8).
Isosteric replacements have also been fruitful with pyrimidine
nucleosides. 6-Azauridine (XXXVIII) and 5-azauridine(XXXIX) have
shown moderate activity against varioustumors, and this field has
been reviewed by Skoda (52). It is ofinterest that although
trifluorothymidine and 6-azathymidine
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CharlesHeidelberger
have biologic activity, a compound,
5-trifluoromethyl-6-aza-2'-deoxyundine, that combines both
substitutions, was
biologically inert (12). Another compound with an
isostericreplacement that has activity against bacteria and tumor
cellsin culture is 3-deazacytidine (XL) as very recently reported
byRobins et al. (45). In our laboratory we are
synthesizingcomparable compounds in the deoxyribonucleoside series,
aswell as other pyrimidine nucleoside analogs with
isostericreplacements. Langen and Kowollik (31) have recently
prepared 5 '-fluorothymidine (XLI), which is active against
thy-
midylate kinase, and hence is a nucleotide, rather than
anucleoside, analog.
A compound that appears superficially to resemble apyrimidine
nucleoside in the antibiotic Showdomycin (XLII)(cf. 16). However,
biochemical work by Roy-Burman et al.(47) indicates that it does
not act as a pyrimidine analog, butrather it inhibits various
enzymes as a consequence of thealkylating properties of the
maleiimide moiety.
As in the case of the purine antimetabolites, the
pyrimidinenucleosides are continuing to provide interesting new
pharmacologically active compounds, including many that are
notspecifically mentioned here. Further chemical efforts in
thisfield seem to be fully justified.
Poly imdeot ¡des
There is now abundant evidence that mammalian cells cantake up
intact polynucleotides. This has been reviewed forDNA by Ledoux
(35), and Click has shown that DNAobtained from mouse thymus can
actually inhibit the growthof L1210 tumors in vivo (18). In our own
studies, we madeoligonucleotides of FUDR-5'-phosphate (FUDRP) in
1961
(44), but these did not show any greater activity than FUDRat
inhibiting the incorporation of formate into the DNAthymine of
Ehrlich ascites cells in vitro.
An important recent discovery by Park and Baron (41)
H2 N C CH2CH2ÇH COOH
NH,
XL-Ill
HgH-C-CH2CH COOH
NH2
TTCV
HgN-C-NHOH
yo/ir
CH,CH-CH CHO3 i i
OH OH
NEN-CH2CO CH2CH2CH-COOH
NH2
XLIV
N5NCH2CO CH2CH COOHNH2
XLVI
CH3C-CHO CH2-CH-CHO
O OH Ã’H
XLVIM XLIX
demonstrated that double-stranded poly-inosinic:poly-cytidylic
acid preparation (poly-IC) is capable of preventingor inhibiting
viral replication in vitro and in vivo by stimulating interferon
production. Very recently, Levy et al. (37) andHoman et al. (26)
have reported that poly-IC inhibits thegrowth of several tumors in
mice, and they have done somepharamacologic studies with the
polymer. This opens up anexciting new field of macromolecular
chemotherapy. Particu-lary when more is known about the mechanisms
of theseeffects, it appears possible that major advances in
specificcancer chemotherapy may be in the offing. In these days
ofwild speculations about genetic engineering, it may not betotally
inappropriate to prophesy that one day cancer may bereversed by
appropriate polynucleotide chemistry [for areview of the chemistry
of polynucleotides, see Michelson etal. (38)]. For example, if as
proposed by Pitot andHeidelberger (42), carcinogenesis is the
result of theperpetuated deletion of a represser, then if the
messengerRNA that codes for this represser were synthesized, it
couldcause the revision of that cancer to normal. Impossible?
Whoknows?
Other Miscellaneous Antimetabolites
Again, the listing of these compounds will not be complete,but
are selected arbitrarily according to my o ».ncurrentinterests.
Glutamine (XLIII) is an important biosyntheticintermediate, and
diazo-oxo-norleucine (DON, XLIV) is itsantimetabolite. In view of
the interesting therapeutic effects ofL-asparaginase,
Handschumacher et al. (21) designed ananalogous inhibitor of the
substrate asparagine (XLV), namelydiazo-oxo-nor-L-valine (XLVI),
which they showed to haveenzymatic specificity. Such an approach
may be applied toother amino acids, should other enzymes of amino
acidmetabolism be found with tumor-inhibitory activities.
Hydroxyurea (XLVII) has been shown at high concentrationsto be a
reversible inhibitor of DNA synthesis (65), and it hassome cancer
chemotherapeutic activity which has stimulatedthe synthesis of
related analogs. It has been shown by Krakoffet al. (29) to be an
inhibitor of ribonucleoside diphosphatereducÃ-ase.
Lastly, I would like to consider three aldehydes. Veryrecently
Apple and Greenberg (1) reported that the combination of two
metabolites in minor branches of the 3-carbonstage of glycolysis,
2-oxopropanal (XLVIII) and 2,3-dihy-droxypropanal (XLIX), cured
some mice with transplantedtumors. It has been demonstrated by
Ciaranfi et al. (9) thatL-eryrfiro-2,3-dihydroxybutyraldehyde, an
inhibitor of protein synthesis and not of glycolysis, has
considerableantitumor activity. Thus, it appears that more
synthesis ofaldehydes may be in order.
CONCLUSIONS
Chart 5. XLIII,glutamine; XLIV, diazo-oxo-norleucine (DON);
XLV,asparagine; XLVI, diazo-oxo-norvaline; XLVII, hydroxyurea;
XLVIII,2-oxopropanal; XLIX, 2,3-dihydroxypropanal; L,
L-erythro-2,3-duiy-droxybutyraldehy de.
What is the need for additional alkylating agents
andantimetabolites? In my opinion there is no need for
additionalalkylating agents dreamed up ad hoc, but possibly there
is aneed for compounds specifically designed relative to the
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Additional Alkylating Agents and Antimetabolites
interest in the metabolism and modes of action of
cyclophos-phamide and BCNU. I hope that I have made a convincing
casefor the need for the design and preparation of
additionalantimetabolites. The limits for these should only be
theimagination and vision of the investigator and his
chemicalskill.
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1969;29:2435-2442. Cancer Res Charles Heidelberger The Need for
Additional Alkylating Agents and Antimetabolites
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