A05 001

Cancer Chemotherapy 1
Cancer Chemotherapy
Bernhard Kutscher, ASTA Medica AG, Frankfurt am Main, Federal Republic of Germany
Gregory A. Curt, National Cancer Institute, Bethesda, Maryland 20205, United States
Carmen J. Allegra, National Cancer Institute, Bethesda, Maryland 20205, United States
Robert L. Fine, National Cancer Institute, Bethesda, Maryland 20205, United States
Hamza Mujagic, National Cancer Institute, Bethesda, Maryland 20205, United States
Grace Chao Yeh, National Cancer Institute, Bethesda, Maryland 20205, United States
Bruce A. Chabner, National Cancer Institute, Bethesda, Maryland 20205, United States
1. Introduction . . . . . . . . . . . . . . 2 3.8. Nitrosoureas . . . . . . . . . . . . . . 17

2. Antimetabolites . . . . . . . . . . . . 3 3.8.1. Mechanism of Action . . . . . . . . . 18
2.1. Methotrexate . . . . . . . . . . . . . 3 3.8.2. Mechanisms of Resistance . . . . . . 18
2.1.1. Mechanism of Action and Mecha- 3.8.3. Analogs . . . . . . . . . . . . . . . . . 18
nisms of Resistance . . . . . . . . . . 3 3.9. Procarbazine . . . . . . . . . . . . . . 19
2.1.2. Analogs . . . . . . . . . . . . . . . . . 5 3.9.1. Mechanism of Action . . . . . . . . . 19
2.2. Fluoropyrimidines . . . . . . . . . . 6 3.9.2. Mechanisms of Resistance . . . . . . 20
2.2.1. Mechanism of Action . . . . . . . . . 6 3.10. Dacarbazine . . . . . . . . . . . . . . 20
2.2.2. Mechanisms of Resistance . . . . . . 7 3.11. Hexamethylmelamine . . . . . . . . 20
2.2.3. Other Fluoropyrimidines . . . . . . . 7 3.12. Mitomycin-C . . . . . . . . . . . . . 20
2.3. 5-Azacytidine . . . . . . . . . . . . . 8 4. Anthracyclines . . . . . . . . . . . . 21
2.3.1. Mechanism of Action . . . . . . . . . 8 4.1. Mechanism of Action . . . . . . . . 22
2.3.2. Mechanism of Resistance . . . . . . 8 4.2. Mechanism of Resistance . . . . . 23
2.3.3. New Analogs . . . . . . . . . . . . . . 8 4.3. Analogs . . . . . . . . . . . . . . . . . 23
2.4. Cytosine Arabinoside (Ara-C) . . 9 5. Intercalating Anthracenes and
2.4.1. Mechanisms of Resistance . . . . . . 9 Analogs . . . . . . . . . . . . . . . . . 24
2.4.2. New Analogs . . . . . . . . . . . . . . 10 5.1. Mitoxantrone . . . . . . . . . . . . . 24
2.5. Deoxycytidine and Analogs . . . . 10 5.2. Analogs . . . . . . . . . . . . . . . . . 24
2.6. 2-Halopurines and Analogs . . . . 11 6. Antitumor Antibiotics Other than
2.7. 6-Mercaptopurine and Anthracyclines . . . . . . . . . . . . 25
6-Thioguanine . . . . . . . . . . . . . 12 6.1. Actinomycin D . . . . . . . . . . . . 25
2.7.1. Mechanism of Action . . . . . . . . . 12 6.2. Bleomycin . . . . . . . . . . . . . . . 25
2.7.2. Mechanism of Resistance . . . . . . 13 6.2.1. Analogs . . . . . . . . . . . . . . . . . 26
2.7.3. New Analogs . . . . . . . . . . . . . . 14 6.2.2. Mechanism of Action . . . . . . . . . 26
3. Alkylating Agents . . . . . . . . . . 14 6.3. DNA Interactive Natural Products 26
3.1. Nitrogen Mustard . . . . . . . . . . 14 7. Antitubulin Agents . . . . . . . . . . 28
3.1.1. Mechanism of Action . . . . . . . . . 14 7.1. Vinca Alkaloids . . . . . . . . . . . . 28
3.1.2. Mechanisms of Drug Resistance . . 15 7.1.1. Vincristine and Vinblastine . . . . . 28
3.2. Melphalan . . . . . . . . . . . . . . . 15 7.1.2. Vindesine . . . . . . . . . . . . . . . . 29
3.2.1. Mechanism of Action . . . . . . . . . 15 7.1.3. Vinorelbine . . . . . . . . . . . . . . . 29
3.2.2. Mechanism of Resistance . . . . . . 15 7.2. Podophyllotoxin and Its Deriva-
3.3. Cyclophosphamide . . . . . . . . . . 16 tives . . . . . . . . . . . . . . . . . . . . 30
3.3.1. Mechanism of Action . . . . . . . . . 16 7.3. Camptothecin and Analogs . . . . 30
3.3.2. Mechanism of Resistance . . . . . . 16 7.4. Taxoids . . . . . . . . . . . . . . . . . 31
3.4. Chlorambucil . . . . . . . . . . . . . 16 7.5. Epothilone A and B . . . . . . . . . 33
3.5. Thio-TEPA . . . . . . . . . . . . . . . 16 8. Heavy-Metal Complexes . . . . . . 33
3.6. Ifosfamide . . . . . . . . . . . . . . . 17 8.1. cis-Platinum . . . . . . . . . . . . . . 33
3.7. Estramustine . . . . . . . . . . . . . 17 8.1.1. Mechanism of Action . . . . . . . . . 34
c 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

10.1002/14356007.a05 001
2 Cancer Chemotherapy
8.1.2. Mechanisms of Resistance . . . . . . 34 9.4. LHRH Analogs . . . . . . . . . . . . 41

8.2. Carboplatin . . . . . . . . . . . . . . 35 9.4.1. LHRH Agonists . . . . . . . . . . . . 41
8.3. Analogs . . . . . . . . . . . . . . . . . 35 9.4.1.1. Leuprorelin Acetate . . . . . . . . . . 42
9. Hormonally Active Anticancer 9.4.1.2. Goserelin . . . . . . . . . . . . . . . . 43
Drugs/Antihormones . . . . . . . . 36 9.4.2. LHRH Antagonists . . . . . . . . . . 43
9.1. Antiestrogens . . . . . . . . . . . . . 36 9.4.2.1. Receptor Assays . . . . . . . . . . . . 44
9.1.1. Antagonists . . . . . . . . . . . . . . . 36
9.4.2.2. Peptidomimetics . . . . . . . . . . . . 44
9.1.2. Tamoxifen, Toremifene . . . . . . . . 36
10. Signal Transduction Inhibitors . . 45
9.1.3. Analogs . . . . . . . . . . . . . . . . . 37
9.2. Aromatase Inhibitors . . . . . . . . 38 10.1. Enzyme Inhibitors . . . . . . . . . . 45
9.3. Antiandrogens . . . . . . . . . . . . . 39 10.2. Phospholipid – Based Antineo-
9.3.1. Flutamide . . . . . . . . . . . . . . . . 39 plastics . . . . . . . . . . . . . . . . . . 45
9.3.2. Nilutamide . . . . . . . . . . . . . . . 40 11. Economic Aspects . . . . . . . . . . 46
9.3.3. Bicalutamide . . . . . . . . . . . . . . 40 12. References . . . . . . . . . . . . . . . 46
1. Introduction $ 100 × 109 per year in treatment expenses and

lost wages.
Malignant tumors represent one of the most Increase in the incidence of cancer, mostly
common human diseases worldwide. Based on as a result of an aging population, is the driv-
an estimation made in the United States, cancer ing growth in the marketplace. However, new
will become the leading cause of death in the medicines like hormone-analogs have lead to de-
year 2000 [1]. crease in severity of the side effects of cancer
Unfortunately, the subset of human cancer therapy and have spurred wider use. According
types that are amenable to curative treatment still to a review [11] of ca. 90 approved anticancer
is rather small. Although there is a tremendous drugs, more than 60 % are of natural origin
progress in understanding the molecular events or modeled on natural products parents. Cancer
that lead to malignancy and many agents are chemotherapeutics can be grouped according to
known that effectively kill cancer cells, progress their pharmacological and mechanistic profiles
in development of clinically innovative drugs into
that can cure humans is slow [2], [3].
Antimetabolites
The heterogeneity of malignant tumors with
Alkylating agents
respect to their genetics, biology, and biochem-
DNA-intercalating (agents) antibiotics
istry as well as primary or treatment-induced
Mitose inhibitors
resistance to therapy hamper curative treatment
Signaltransduction-inhibitors
[4], [5].
Searching for antineoplastic agents with im- Two primary events in cell proliferation are
proved selectivity to malignant cells remains the DNA replication and cell division [12], [13]. The
central task for drug discovery and development cell cycle has been divided into four sequen-
[5], [6]. tial phases (G0 , G1 , S, M) and the cytostatics
According to a survey published in 1997 vary in the way they interfere with the cell cy-
more then 315 drugs are under development in cle. Phasespecific agents/drugs, e.g., those in-
the United States for the treatment of cancer. teracting in G1 , S- or M-phase, are the mitose-
This figure includes 42 drugs for treatment of inhibitors vincristine or vinblastine or the an-
lung cancer, 58 for breast cancer [8], [9], 60 for timetabolites methotrexate or cytarabine, attack-
treatment of skin tumors (60), 36 for prostate ing in the S-phase. Alkylating drugs such as cy-
cancer, and 35 for colon cancer. clophosphamide, cisplatin, or carboplatin inhibit
In 1997 more than 1500 Americans are ex- and damage the cell in all phases and are thus
pected to die of cancer each day and more than a phase unspecific. In general cells in the resting
million new cases will be diagnosed with over- phase (G0 ) are insensitive.
all medical costs of $ 35 × 109 . The total dis- Approximately 70 % of patients diagnosed as
ease costs are estimated to sum up to more than having cancer have metastatic disease, i.e., dis-
ease that has spread beyond the primary site at

the time of diagnosis [15]. However, the steady
progress made in the treatment of cancer with
drugs has contributed to the curing of an in-
creasing proportion of patients with metastatic
disease. The greatest change and improvements
in cancer treatment have occurred because of Methotrexate
the discovery and clinical development of drugs
and the demonstration that metastatic cancer can Most importantly, methotrexate can inhibit
be cured by these agents. Drugs, such as cy- dihydrofolate reductase (DHFR, K i = 10−11 M),
toxan, adriamycin, vincristine, cis-platinum, and a key enzyme for the maintenance of biologi-
bleomycin, all developed since 1960, are now cally active intracellular reduced folate pools.
regularly used by physicians to treat patients The folate-requiring reactions utilize reduced
who would have been considered incurable a folates, and all reactions except that catalyzed
short time ago. by thymidylate synthase maintain folates in a
This article details the pharmacology and reduced state during carbon transfer. Thymidy-
clinical use of the major classes of anticancer late synthase, which catalyzes the methylation
agents, including (1) antimetabolites, (2) alky- of deoxyuridylate to thymidylate (required for
lating agents, (3) anthracyclines and analogs, DNA synthesis), requires the transfer of a car-
(4) other antitumor antibiotics, (5) antitubulin bon group from the folate cofactor N 5−10 -
agents, (6) platinum complexes, (7) antihor- methylene tetrahydrofolate with resultant oxi-
mones, and (8) signaltransduction-inhibitors. In dation of the folate to dihydrofolic acid. Ox-
each case, special emphasis is given to the idized folates must be reduced to the tetra-
progress made in developing clinically useful hydro form by DHFR to be useful for intra-
drugs and analogs that retain antitumor activity cellular metabolism. Inhibition of DHFR fol-
while decreasing host toxicity. lowing methotrexate exposure ultimately leads
to depletion of intracellular reduced folates.
Cessation of first thymidylate and then purine
nucleotide synthesis occurs as an indirect ef-
2. Antimetabolites
fect of methotrexate on reduced-folate levels.
Methotrexate metabolites (polyglutamates) may
2.1. Methotrexate also have direct inhibitory effects on folate-
requiring enzymes, e.g., thymidylate synthase
Folic acid analogs comprise a class of antineo- [18], and these effects may be important in in-
plastic agents of which methotrexate has gained ducing cytotoxicity.
the most widespread clinical use. These agents
were the first to produce impressive remissions
in acute leukemia [16] and cures in choriocarci- 2.1.1. Mechanism of Action and
noma in women [17]. Mechanisms of Resistance
Reduced folates (tetrahydrofolates) are the
biologically active form of folates required as Transport. At concentrations less than
cosubstrates in one-carbon transfer reactions. 10 µM, methotrexate and reduced folates en-
Included in these reactions are several impor- ter cells via an energy-dependent, temperature-
tant enzymatic steps in the de novo synthesis of sensitive carrier mechanism [19]. The affinity
purines and pyrimidines. for this carrier has been variously reported to
Methotrexate (NSC-740) [59-05-2], N-(4- fall between 1 and 10 µM for tumor cell lines
{(2,4-diamino-6-pteridinyl)methyl]methylami- [19–21] and to be 87 µM for normal intestinal
no}benzoyl)-l-glutamic acid, C20 H22 N8 O5 , M r epithelial cells [22]. These differences in effi-
454.46, is a 2,4-diamino, N 10 -methyl analog of ciency of transport may account for some of the
folic acid that is capable of inhibiting certain selectivity of methotrexate for neoplastic cells.
folate-requiring reactions. In addition to this carrier-mediated transport sys-
tem, there exists a second, low-affinity trans-
port mechanism that is poorly understood but duration of binding to dihydrofolate reductase
appears to play a role in the transport of drug are directly related to the length of the polyglut-
when concentrations exceed 20 µM [21], [23]. amate tail [29].
Methotrexate and reduced folates do not com-
pete for uptake by this process, and it may rep- Interaction with Dihydrofolate Reductase.
resent a means for drug entry in cells resistant to The binding of methotrexate to dihydrofolate re-
low doses of drug by virtue of defective trans- ductase has been extensively investigated by X-
port by the high-affinity mechanism. In some ray crystallographic and amino acid sequencing
models, the sensitivity of a cell to methotrex- studies [30–34]. Methotrexate binds in a stoi-
ate can be directly correlated with efficiency of chiometric fashion to a hydrophobic pocket in
drug transport, i.e., sensitive cells have a greater the target enzyme DHFR [35]. The binding affin-
capacity for drug transport and longer intracel- ity depends on multiple factors, including pH,
lular retention of drug when compared to meth- salt concentration, and NADPH concentration,
otrexate-resistant cells [24]. Because decreased and has been reported to be ca. 10 pM [36]. In
membrane transport may play a role in clinical the cell, methotrexate is a reversible inhibitor ca-
drug resistance, a number of analogs with high pable of being displaced by high concentrations
lipid solubility have been developed that can cir- of substrate. Thus, free intracellular drug in ex-
cumvent a transport deficit. Methotrexate esters, cess of the cellular dihydrofolate reductase bind-
diaminopyrimidines, and triazenates have been ing capacity is required to maintain complete
synthesized and used with success against ex- inhibition of the enzyme and thereby produce
perimental cell lines with defective methotrex- and maintain a state of reduced-folate depletion.
ate transport. If an excess of intracellular drug is not main-
tained, the intracellular reduced-folate pool re-
Intracellular Metabolism. Once inside the covers through enzymatic reduction of oxidized
cell, naturally occurring folates may be metabo- folates and cellular metabolism resumes.
lized to polyglutamates; that is, additional glu- Cellular resistance to methotrexate has been
tamyl moieties are added to the terminal gluta- most commonly associated with an increase in
mate present on the parent compound. This pro- dihydrofolate reductase activity. In general, the
cess allows for selective intracellular retention of amplified enzyme is identical to the native pro-
the polyglutamated forms and an increased affin- tein in its affinity for methotrexate; however, al-
ity for certain folate-requiring enzymes, such as tered methotrexate affinity has been reported to
thymidylate synthase, and for enzymes required correlate with sensitivity to methotrexate in a se-
for the de novo production of purine nucleotides ries of murine leukemias [36]. Increased reduc-
[25]. Like the naturally occurring folates, in- tase activity and resistance to methotrexate has
tracellular methotrexate is also polyglutamated also been demonstrated in a number of cell lines
with the addition of from one to four additional made resistant in vitro by stepwise increases
glutamyl residues [26]. This process has been in drug concentration [37], [38], and in human
demonstrated in a variety of tissues, including tumor samples from clinically resistant tumors
human breast cancer cell lines [26], normal hu- [39]. The increased enzyme levels can be cor-
man liver [27], and murine leukemia cells [28]. related to gene amplification that may take the
The polyglutamates of methotrexate are selec- form of small new pieces of chromosomal ma-
tively retained by the cells and appear to have terial, called double minutes, or of large chro-
an enhanced inhibitory potential for certain en- mosomes, referred to as homogeneously stain-
zymes [18]. The inhibitory capacity of metho- ing regions (HSRs). The former variety of am-
trexate polyglutamates for dihydrofolate reduc- plification imparts relatively unstable resistance,
tase appears to be somewhat greater than that of which requires the ongoing selective pressure of
the parent drug. The selective retention of meth- drug presence to be maintained [40], whereas the
otrexate polyglutamates may be critical for the HSRs represent a more durable form of amplifi-
delayed cytotoxicity exhibited by cells capable cation and thus resistance. Several investigators
of polyglutamate synthesis. In vitro experiments [41–43] have successfully transvected amplified
using MCF-7 human breast cancer cells have reductase genes into normal hematopoietic cells,
demonstrated that the intracellular retention and allowing greater marrow resistance to metho-
trexate, an important dose-limiting toxicity of ther lipophilic DHFR-inhibitors are trimetrex-

the drug. ate [82952-645] [47], and edatrexate [48] which
are clinically studied and have shown activity,
Determinants of Cytotoxicity. Methotrex- e.g., in non-small cell lung cancer. Thymidylate
ate is an S-phase-specific agent whose cytotoxic synthase (TS) is the rate-limiting enzyme in the
effects are determined by drug concentration and anabolism of thymidine resulting in the incorpo-
duration of cell exposure. These effects may be ration into DNA. Raltitrexed (company codes:
altered by the cellular milieu. The toxic effects ZN-1694, D-1694, ICI-D1694) [112887-68-0]
of methotrexate can be completely reversed by [49], is currently under clinical investigation
exogenous administration of the end products with response rates in colon and breast cancers
(purines and thymidine) whose de novo synthe- of up to 30 % [50], [51]. Myelosuppression
sis is inhibited by methotrexate treatment. Also, seems to be the predominant dose limiting toxi-
the synthesis of these products may resume if city.
an exogenous source of reduced folates is pro-
vided. These data provide the rationale for the
treatment of patients with high-dose methotrex-
ate and subsequent administration of a reduced
folate in the form of leucovorin calcium (N 5 -
formyl tetrahydrofolic acid) as “rescue.” The re-
versal of methotrexate cytotoxicity by reduced Piritrexim
folates is a competitive process. The reasons

for the competitive nature of this relationship
are unclear but may be the result of a shared
membrane transport system. In addition, meth-
Raltitrexed
otrexate may have direct inhibitory effects on
enzymes other than dihydrofolate reductase that
require competitive levels of the folate cosub-
strate to overcome the inhibition.
2.1.2. Analogs Trimetrexate
Many new analogs to methotrexate have been

developed in an effort to circumvent the cellular
resistance that occurs with prolonged meth-
otrexate exposure. As mentioned, drugs with AG 331
increased lipid solubility have been success-
ful in treating transport-resistant cells in vitro
[44]. The lipophilic derivative metoprine and
variations of the 10-deazaaminopterin series are
the most interesting additions. Of the 10-deaza
series, an ethyl sub-stitution at the 10-position
Edatrexate
imparts a marked increase in cytotoxicity when
compared to methotrexate [45]. The analog pos-
sesses an improved membrane transport ability Crystallographic data and computer-assisted
while retaining a high affinity for dihydrofolate drug design led to the development of thymidy-
reductase. A new antineoplastic agent is pir- late synthase (TS) inhibitors of the type of
itrexim isothionate [79483-69-5], 6-[(2,5-di- AG 331 [52].
methoxyphenyl)methyl]-5-methylpyrido[2,3- Finally, CB 3717, a potent inhibitor of
d]pyrimidine-2,4-diamine mono-2-hydroxy- thymidylate synthase, is toxic for cell lines with
ethanesulfonate, C17 H19 N5 O2 [46], that in- altered dihydrofolate reductase; and homofo-
hibits dihydrofolate reductase (DHFR). Fur- late, a de novo purine inhibitor requiring di-
hydrofolate reductase for activation, is effective 5-FdUMP. In the presence of methylene tetra-
in reductase-amplified lines [44]. hydrofolate, 5-FdUMP forms a stable ternary
complex with thymidylate synthetase (TS), in-
hibiting this critical enzyme to cause “thymine-
2.2. Fluoropyrimidines less death.” As expected, cytotoxicity is pre-
vented in the presence of exogenous thymi-
5-Fluorouracil (NSC-19893) [51-21-8], 5- dine in these cells with intact salvage path-
fluoro-2,4-(1H, 3H)-pyrimidinedione, 5-FU, ways. Inhibition of TS has long been consid-
C4 H3 FN2 O2 , M r 130.08, is a fluorinated ered the principal mechanism of 5-FU cytotoxi-
pyrimidine whose structural formula resembles city, but it has also been demonstrated that 5-FU
thymine; the hydrogen in the 5-position of the can be converted to 5-FUMP, either by orotic
naturally occurring pyrimidine being replaced acid phosphoribosyl-transferase (OPRTase) in
by fluorine. The synthesis of 5-FU in 1957 [15] the presence of phosphoribosyl pyrophosphate
represents the first successful effort in the ra- (PRPP), or by stepwise conversion to the ri-
tional design of anticancer drugs [53], and was bonucleotide 5-FUR (by uridine phosphorylase)
predicated on the earlier observation that malig- followed by formation of 5-FUMP by uridine
nant cells selectively utilized uracil (and possi- kinase. This intermediate can be converted to
bly toxic uracil analogs) in vivo [16], [54]. 5-FdUMP by ribonucleotide reductase to in-
hibit TS. Alternatively, 5-FUMP can be phos-
phorylated to 5-FUTP, which may be fraudu-
lently incorporated into RNA to induce cytotox-
icity. In a number of tumor models, loss of clono-
genic capacity is directly correlated with the ex-
tent of incorporation of 5-FUTP into RNA [55],
[56]. This RNA-specific toxicity is not reversed
by thymidine. However, the precise mechanism
of RNA-induced cell kill is speculative. The
5-Fluorouracil 5-FUDR
most consistent structural effect of 5-FU expo-
sure is impaired processing of ribosomal RNA
Since the original synthesis of 5-FU [57]. This concept is not supported by current ev-
and its nucleoside 5-FUDR (NSC-27640) idence, however, as neither the synthesis nor the
[50-91-9], 2 -deoxy-5-fluorouridine, floxuri- translation of messenger RNA (mRNA) appears
dine, C9 H11 FN2 O5 , M r 246.21, much has been affected by documented 5-FUTP incorporation
learned about the mechanism of action of the into mRNA. Using human colon carcinoma
fluoropyrimidines. These drugs are useful in the cells propagated in vitro, neither quantitative
treatment of a wide range of human malignan- nor qualitative differences in the translational
cies. products (polypeptides) of 5-FUTP-containing
mRNA could be demonstrated [58]. However,
small nuclear RNA species responsible for exon
2.2.1. Mechanism of Action recognition during RNA splicing do contain sig-
nificant quantities of uridylic acid [59]. Specific
Both 5-FU and 5-FUDR are prodrugs that re- substitution of 5-FUTP into this RNA fraction
quire intracellular metabolism to their respec- may be critical for 5-FU toxicity.
tive nucleotides for cytotoxicity. The pathways As shown in Figure 1, 5-FdUMP can be fur-
for fluoropyrimidine activation are shown in Fig- ther phosphorylated to 5-FdUTP, which can be
ure 1. Each drug is enzymatically activated by incorporated into tumor cell DNA [60], [61].
different routes to FdUMP, FUMP, or FUTP, and This mechanism of drug action has been par-
each of these fluorinated nucleosides has differ- ticularly difficult to appreciate, since the fraud-
ent mechanisms of cytotoxicity. ulent base is quickly excised from DNA by
Thymidine phosphorylase converts 5-FU to the enzymes uracil-DNA glycosylase and dUTP
the deoxyribonucleotide 5-FUDR, which is then nucleotidohydrolase. Thus, when tumor cells
phosphorylated by thymidine kinase to yield are incubated at low (0.1 µM) concentrations
Figure 1. Pathways for fluoropyrimidine activation
of 5-FUDR, single-strand DNA shifts to lower elevated levels of target protein represent the
molecular mass species, suggesting excision of end result of specific gene amplification (as has
the fluorinated base [62]. However, the actual been documented for methotrexate resistance)
presence of 5-FdUTP in cellular DNA cannot remains to be determined.
be detected until tumor cells are exposed to
higher drug concentrations. Apparently, the im-
portance of 5-FU incorporation into DNA varies 2.2.3. Other Fluoropyrimidines
from tumor to tumor, with human promyelocytic Attempts to develop fluoropyrimidines with im-
leukemia cells incorporating nearly 100-fold proved therapeutic indexes have resulted in the
more 5-FdUTP into DNA than mouse leukemia synthesis and clinical trial of so-called masked
cells [63]. The importance of this pathway both fluoropyrimidines. The masked fluoropyrimi-
to tumor cell cytotoxicity and therapeutic index dine 5 -deoxy-5-fluorouridine is a nontoxic pro-
remains to be elucidated. drug that is converted to 5-FU by pyrimidine
nucleoside phosphorylase. Because this enzyme
may be present to a greater degree in some tu-
2.2.2. Mechanisms of Resistance mor cells than in normal human bone marrow,
an improved therapeutic index can be demon-
Tumor cells selected for in vitro resistance may strated in vitro using breast, sarcoma, leukemia,
demonstrate a deletion of critical drug-activating and colon carcinoma cells [75]. Since 5 -deoxy-
enzymes, including uridine kinase [64], orotic 5-fluorouridine requires conversion to 5-FU and
acid phosphoribosyltransferase [65], [66], and is in itself nontoxic, tumor cells that lack phos-
uridine phosphorylase [67]. Methylene tetra- phorylase activity are resistant to the masked
hydrofolate is required for 5-FdUMP inhibition compound, while remaining cross-sensitive to
of TS, and decreased availability of intracellu- 5-FU [76].
lar folates has been involved as a mechanism of Ftorafur is a second masked fluoropyrimidine
5-FU resistance [68]. that is less myelosuppressive than 5-FU. How-
In addition, alterations in the target enzyme ever, the drug has a higher incidence of gastroin-
TS can result in drug resistance. Resistant cells testinal and nervous system toxicity, which is
have been described with altered thymidylate probably due to organ-specific localization of
synthetase having decreased affinity for FdUMP activating enzymes [77]. In early clinical stud-
[69–71]. In addition, increased specific activity ies, ftorafur has shown antitumor activity in pa-
of TS has been reported in drug-resistant fibrob- tients with 5-FU refractory colorectal and breast
lasts [72] and tumor cells [73], [74]. Whether cancer [78].
2.3. 5-Azacytidine [87]; methylation appears to inhibit gene expres-

sion. Thus the globin gene is hypomethylated
5-Azacytidine (NSC-102816) [320-67-2], in bone marrow as compared to other tissues
4-amino-1-β-d-ribofuranosyl-1,3,5-triazine- [88–91]. The DNA containing even low levels
2(1H)-one, 5-azacitidine, C8 H12 N4 O5 , M r of 5-azacytidine is a potent inhibitor of the en-
244.21, is a pyrimidine analog that was first zyme responsible for cytosine methylation, i.e.,
isolated as a fermentation product from Strep- DNA cytosine methyltransferase [92], [93]. The
toverticillium cultures [79] and was chemically enzyme inhibition is disproportionately great
synthesized in Czechoslovakia in 1964 [80]. compared with the small amount of incorpo-
Structurally, 5-azacytidine differs from cytidine rated fraudulent base, and appears to result
by the substitution of nitrogen in the 5 position from formation of a stable complex between 5-
of the pyrimidine nucleus. azacytidine residues and the methyltransferase,
similar to the complex formed with thymidy-
late synthase and FdUMP [94]. Thus, in vitro
treatment with 5-azacytidine can induce DNA
hypomethylation and differentiation of murine
cells [95], [96]. More recently these observa-
tions have been extended to clinical medicine.
Azacytidine treatment in a patient with severe β-
thalassemia could stimulate gamma-globin syn-
thesis by inducing hypomethylation and expres-
5-Azacytidine sion of the gamma-globin gene [97].
After conversion to a triphosphate, azacyti-
dine also competes with CTP for incorporation
2.3.1. Mechanism of Action into RNA [98] and inhibits maturation of ribo-
somal and transfer RNA [99]. This causes dis-
5-Azacytidine shares the facilitated transport assembly of polyribosomes [100] and interferes
system for cytidine for entry into cells [81] and with protein synthesis [101].
must be phosphorylated to exert cytotoxic ef-
fects. Conversion to the monophosphate is cat-
alyzed by the enzyme uridine – cytidine kinase 2.3.2. Mechanism of Resistance
[82], and this is likely the rate-limiting step for
drug activation [83]. 5-Azacytidine monophos- Cellular resistance to 5-azacytidine may be
phate inhibits the enzyme orotidylate decar- the result of either decreased drug activa-
boxylase and interferes with de novo pyrimidine tion or possibly increased drug degradation.
biosynthesis [84]. Subsequent metabolism of the Drug metabolism by cytidine kinase appears
monophosphate to azacytidine di- and triphos- to be rate-limiting in drug activation; dele-
phate, catalyzed by cytidine monophosphate ki- tion of this enzyme has been reported in 5-
nase and nucleoside diphosphate kinase, occurs azacytidineresistant cells in vitro [81], [102].
rapidly and does not appear to be a rate-limiting Cytidine deaminase degrades 5-azacytidine to 5-
step in drug activation [81]. azauridine; however, the role of this enzyme in
The diphosphate of azacytidine is a substrate drug resistance remains uncertain. For example,
for ribonucleotide reductase and dAzaCTP (de- drug toxicity may be dependent on deamination;
oxyribonucleotide triphosphate of azacytidine) bacteria incapable of forming 5-azauridine from
for DNA polymerase, allowing direct incorpo- 5-azacytidine are resistant to the drug [103].
ration of drug into DNA [85]. This pathway
may be critical for cytotoxicity because active
DNA synthesis correlates with drug sensitivity 2.3.3. New Analogs
in vitro [86]. In addition, incorporation of 5-
azacytidine into DNA may affect gene expres- In clinical trials 5-azacytidine has demonstrated
sion. Mammalian DNA contains ca. 5 % of in- consistent antileukemic activity, inducing com-
corporated cytosine methylated in the 5 position plete remissions in a significant number of
heavily pretreated patients with acute myeloge- the triphosphate ara-CTP, which is responsible
nous leukemia [104]. Acute dose-limiting toxi- for inhibition of DNA synthesis.
cities associated with bolus drug administration
(severe gastrointestinal symptoms, fever, life-
threatening hypotension) may be ameliorated
by administering the drug via constant intra-
venous infusion [105]. However, 5-azacytidine
is chemically unstable, undergoing ring open-
ing at the 5,6-imino double bond to form N-
formylamidinoribofuranosylguanylurea, which
further decomposes to ribofuranosylurea [106].
The halflife of this decomposition is 4 h, com- Cytosine arabinoside
plicating the task of strict dosage control of pro-
The precise mechanism by which ara-CTP
longed infusions.
inhibits DNA synthesis remains uncertain. Ara-
To circumvent the problem of aque-
CTP does inhibit both DNA polymerase α [110]
ous instability, the hydrolytically sus-
and β [111]. The former is essential for DNA
ceptible 5,6-imino bond of 5-azacytidine
synthesis, the latter for DNA repair. Ara-CTP
was reduced to produce dihydro-5-
can be directly incorporated into DNA as well,
azacytidine (NSC-264880) [62402-31-7],
and this pathway correlates strongly with cy-
4-amino-5,6-dihydro-1-β-d-ribofuranosyl-
totoxicity. The extent of drug incorporation
1,3,5-triazine-2(1H)one · monohydrochloride,
into DNA is proportional to cell kill in both
C8 H14 N4 O5 · HCl, M r 282.7 [107]. This com-
acute myelocytic and promyelocytic leukemia
pound has completed phase I trials in the 1980s
cells [112]; incorporation and cytotoxicity can
with preliminary evidence of activity in lym-
be modulated by thymidine [113]. Also ara-C-
phoma; in addition it exhibits the unusual dose-
substituted DNA is unstable under conditions of
limiting toxicity of chest pain at the maximally
alkaline elution, suggesting drug-induced strand
tolerated dose [108].
fragility and breakage [114]. Ara-CTP incor-
poration also directly blocks strand elongation
[115] and causes premature strand termination
[116]. This results in accumulation of DNA
peaks of small sizes, suggesting that preexisting
DNA may be nicked following exposure to ara-C
[117]. A further effect of ara-C exposure is inhi-
bition of DNA repair, as determined by alkaline
elution studies in L 1210 cells [118]. As might
Dihydro-5-azacytidine be expected, cells pretreated with ara-C are more
sensitive to ionizing radiation, suggesting poten-
tiation of radiation damage by inhibition of DNA
repair [119], [120–123].
2.4. Cytosine Arabinoside (Ara-C)
Ara-C (NSC-63878) [147-94-4], 4-amino-1-β-
d-arabinofuranosyl-2(1H)-pyrimidone, cytara- 2.4.1. Mechanisms of Resistance
bine, cytosine arabinoside, C9 H13 N3 O5 , M r
243.22, an antimetabolite that is a structural ana- A number of mechanisms of resistance to ara-C
log of cytidine, differs from the physiologic nu- have been described. Ara-C itself is a prodrug
cleoside by the epimeric configuration of the β- that is metabolized first to ara-CMP by deoxy-
trans-hydroxyl group at the 2 position of the cytidine kinase, then to ara-CDP by pyrimidine
sugar. The drug is transported into cells by a nucleoside monophosphate kinase, and finally
carrier-mediated process with shared affinity for to ara-CTP by nucleoside diphosphate kinase.
deoxycytidine [109]. Once ara-C has entered the Resistant cell lines lack the initial, rate-limiting
cell, cytotoxicity is dependent on formation of activating enzyme [115], [124].
Since the 1980s, HPLC has been used to high levels of cytidine deaminase because the
separate and semipurify deoxycytidine kinase, catabolism product, FIA-uracil, is more toxic
pyrimidine nucleoside monophosphate kinase, than the parent compound [138].
and pyrimidine nucleoside diphosphate kinase
from cell extracts [125]. In murine leukemia
cells selected for ara-C resistance, deoxycyti- 2.5. Deoxycytidine and Analogs
dine kinase was found in lower specific activity,
and similar results have been reported for other The pyrimidine antimetabolite gemcitabine (LY-
systems [126]. 188011, dFdC) [095058-81-4], 2’-deoxy-2’,2’-
In addition, expansion of the intracellular difluorocytidine, C9 H11 F2 N3 O4 is an analog of
pool of the physiologic substrate deoxycytosine deoxycytidine and a result of a program initi-
triphosphate can inhibit ara-C activation by feed- ated at Lilly Research to synthesize fluorinated
back inhibition of the initial activating enzyme d-ribose and fluorinated nucleosides [139–142].
[127]. Moreover, increased dCTP pools may The difluorinated analog of cytarabine, gemc-
also compete directly with ara-CTP for DNA itabine was identified as novel antimetabolite
polymerase. Experiments have shown a relation- with a broad spectrum of antitumor activity.
ship between duration of leukemia remission Trade names: Gemicitabine monohydro-
and the ability of tumor cells obtained from pa- chloride [122111-03-9] is on the market as
tients to form and retain ara-CTP in vitro [128], Gemzar in the indication of palliative treatment
but the mechanisms underlying this relationship of locally advanced or metastatic non-small cell
remain to be established. lung cancer.
Although some workers have reported that in- Mechanism of Action. Gemcitabine shows
creased drug catabolism by cytidine deaminase good activity against human leukemic cell lines,
may underlie both de novo and acquired drug re- a number of murine solid tumors, and human
sistance in patients with leukemia [129], a def- tumor xenografts [143–146]. Gemcitabine was
inite clinical correlation between response and significantly more cytotoxic than cytarabine in
levels of this enzyme has not been established in Chinese hamster ovary cells. The major cellular
several studies [130–132]. metabolite is the 5’-triphosphate of gemcitabine.
The cytotoxicity was competitively reversed by
deoxycytidine, suggesting that the biological
2.4.2. New Analogs activity required phosphorylation by deoxycyti-
dine kinase [145].
Several ara-C analogs have been rationally de- Tumor-bearing mice were treated with ei-
signed with the goal of overcoming specific ther gemcitabine or cytarabine (20 mg/kg). DNA
mechanisms of resistance. Compounds with synthesis reached 1 % of control levels upon
lipophilic side chains are relatively resistant administration of gemcitabine. The greater
to inactivation by cytidine deaminase. Another accumulation of gemcitabine-5’-triphosphate
analog with lipophilic modifications in the side compared with cytarabine-5’-triphosphate may
chain is enocitabine (NSC-239336, BHAC) cause greater cytotoxicity and therapeutic activ-
[055726-47-1] [133], [134]. N 4 -Behenoyl-ara- ity [146].
C (BHAC) has undergone clinical trial in pa-
tients with acute leukemia [135]. Despite its
lipophilicity, the drug does not enter the cere-
brospinal fluid and concentrates in bone mar-
row and red blood cells. N 4 -Palmitoyl-ara-
C can be administered orally and appears to
be more active than the parent compound in
preclinical models [136]. Although developed
as an antiviral agent, 2 -fluoro-5-iodo-1-β-d-
Gemcitabine
arabinofuranosylcytosine (FIAC) has significant
antitumor activity [137]. Interestingly, this com- Further gemcitabine may enhance its own cy-
pound may be relatively cytotoxic for cells with totoxic effects by self-potentiation mechanisms
that act on, e. g., deoxycytidine monophosphate behaves more like an analog of deoxycytidine
deaminase, deoxycytidine kinase or on DNA- than adenine or deoxyadenine as indicated by
synthesis [147], [148]. reports [155] demonstrating that the presence of
fluorine in the 2-position of the adenine ring al-
ters its function as a substrate for deaminase and
2.6. 2-Halopurines and Analogs nucleoside kinases. This results in differences
in biological activity and metabolism. Halo-
Cladribine (2-CdA, RWJ-26251) 004291-
genation does not simply block deamination,
63-8, is a purine deoxyribonucleoside with re-
but also influences the enzyme that carries out
markable antileukemic activity. It represents a
the phosphorylation, as a result cytotoxicity is
significant advance over existing therapy be-
increased [156]. Fludarabine phosphate may se-
cause it is given as a single 7-day continuous
lectively inhibit the incorporation of thymidine
treatment, thus minimizing the side effects ob-
and uridine into the DNA molecule by inhibiting
served with multiple treatments. Remission rates
both ribonucleotide reductase [157] and DNA
of up to 89 % lasting for up to 25 months were ob-
polymerase [158]. The maximum tolerated dose
served in clinical trials in patients with hairy cell
(MTD) in heavily pretreated patients with ad-
leukemia [149–152]. The antimetabolite used
vanced malignancy/solid tumors on the daily
for first line treatment of hairy cell leukemia
regimen was about 15 mg/m2 . Granulocytope-
[153].
nia and thrombocytopenia were dose-limiting
Trade name: Leustatin.
[159–161].
Fludarabine phosphate (NSC-312887, 2-F-
ara-AMP) [075607-67-9] [154], 2-fluoro-9-
(5-O-phosphono-β-d-arabino-furanosyl)-9H- Pentostatin. The cytotoxic and im-
purin-6-amine, C10 H13 FN5 O7 P, M r 365.21, munosuppressant pentostatin (NSC-218321,
another cytotoxic purine antimetabolite, acts CI-825, PD-81565, YK-176, 2-deoxyco-
via inhibition of DNA synthesis. The product formycin, 2’-dCF) [063677-95-2], (R)-3-(2-
is used for treatment of patients with chronic deoxy-β-d-erythro-pentofuranosyl)-3,6,7,8-
lymphocytic leukemia. tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol,
Trade names: Fludara (Berlex), Benefluor C11 H16 H4 O4 , M r 268.13, mp 220 – 225 ◦ C, can
(Schering AG). be isolated from the fermentation broth of Strep-
tomyces antibioticus NRRL 3238 [162], [163]
or Aspergillus nidulanus Y 176-2 or Emericella
[164]. The adenosine nucleoside analog pento-
statin, is the most potent inhibitor of adenosine
deaminase, which is an important and ubiquitous
cellular enzyme. The inhibition of this enzyme
leads to accumulation of dATP which inhibits
ribonucleotide reductase and thus DNA synthe-
sis. Pentostatin was launched for treatment of
Cladribine
hairy-cell leukemia refractory to α-interferon.
Trade names: Nipent (Parke-Davis, Lederle),
Coforin (Katetsuken).
Fludarabine
Mechanism of Action. Fludarabine and its Pentostatin

soluble derivatives interfere with phos-
phorylation, e.g., in L 1210 cells. Fludarabine
Mechanism of Action. The highest activity of 6-Mercaptopurine is used for maintenance

adenosine deaminase is found in lymphoid tis- therapy of acute lymphocytic and acute myel-
sue as well as in the malignant cells of acute ogenous leukemia, and 6-TG is used primarily
lymphoblastic leukemia [165]. Since pentostatin for remission induction in acute myelogenous
is the most potent inhibitor of adenosine deam- leukemia. Although 6-MP and 6-TG have an
inase it was expected to possess antitumor important role in chemotherapy of leukemia pa-
properties against certain malignancies, espe- tients, the purine analogs produce low-response
cially acute leukemias, chronic myelogenous rates in patients with solid tumors, lymphomas,
leukemia, and lymphomas. Surprisingly, when and chronic lymphocytic leukemia.
tested against murine tumor models and also
against various tumor cell cultures no antitumor
effect was found [166]. However, in a phase I 2.7.1. Mechanism of Action
clinical trial the compound was found to produce
a drop in lymphoblast count and showed antitu- 6-Mercaptopurine has been used as an anti-
mor activity in acute leukemia and lymphoma. neoplastic and immunosuppressive agent for
The toxicity observed consisted mostly of CNS decades, but the precise mechanism by which it
side effects including nausea, hepatic and re- exerts its cytotoxic effects has not yet been estab-
nal dysfunction. In combination with vidarabin lished. Both 6-MP and 6-TG must be converted
(Ara-A) (→ Chemotherapeutics, Chap. 4.6.1.) to their thiol nucleotide form, which is the active
superiority to monotherapy was demonstrated cytotoxic moiety. The conversion is catalyzed
in various studies [167], [168]. Durable com- by hypoxanthine guanine phosphoribosyltrans-
plete remission was observed after pentostatin ferase (HGPRT) [179–181] and the reaction is
treatment in patients with hairycell leukemia re- dependent on the phosphoribosylpyrophosphate
sistant to α-interferon [169–174]. (PRPP) level in the cells [182–186]. 6-Thiouric
acid (6-TU) is the major catabolic product of
6-MP. The rapid conversion of 6-MP to 6-TU
2.7. 6-Mercaptopurine and by xanthine oxidase [187], [188] in leukemic
6-Thioguanine cells may be a possible mechanism of 6-MP re-
sistance [182], [189], [190]. The antineoplastic
The purine antimetabolites, 6-mercaptopurine effect of 6-TG is similar to that of 6-MP. In its
(NSC-755) [50-44-2], 1,7-dihydro-6H-purine- nucleotide form, 6-TG inhibits de novo purine
6-thione, 6-MP, C5 H4 N4 S, M r 152.19; and biosynthesis and purine interconversions [191–
6-thioguanine (NSC-63878) [154-42-7], 2- 195].
amino-1,7-dihydro-6H-purine-6-thione, 6-TG, The cytotoxicity of 6-MP and 6-TG has been
C5 H5 N5 S, M r 167.21, have been used for linked to (1) the interference with de novo purine
several decades in the treatment of leukemia biosynthesis and purine interconversions, (2) the
and certain other neoplastic diseases. The 6- inhibition of in vitro RNA synthesis, and (3) the
thiopurine analog, 6-MP, was first synthesized in incorporation into DNA during S phase, result-
1952 [175]; subsequently the same workers syn- ing in a deformation of the DNA.
thesized 6-TG in 1955 [176]. The clinical evalu- The interference with de novo purine
ation of 6-MP in the treatment of acute leukemia biosynthesis by 6-MP is regulated by 6-
and chronic myelocytic leukemia was carefully MP nucleotides. These nucleotides inhibit
studied in 1953 [177]. Shortly thereafter, the 2- the enzyme 5-phosphoribosylpyrophosphate
amino analog of 6-MP, 6-TG, was submitted for amidotransferase that catalyzes the initial reac-
clinical evaluation as an antitumor agent [178]. tion in the purine biosynthetic pathway [196].
The 6-MP nucleotides also inhibit conver-
sion of inosine monophosphate (IMP) to ade-
nine monophosphate (AMP) and to xanthine
monophosphate (XMP), and limit the avail-
ability of XMP to form guanine monophos-
R = H, 6-Mercaptopurine
R = NH2 , Thioguanine
phate (GMP), thereby interfering with the sup-
ply of purine precursors for nucleic acid synthe- treated with allopurinol. Furthermore, allopuri-
sis [181]. In the 1980s two new findings pertain- nol increased the plasma level of 6-MP in rab-
ing to the cytotoxicity mechanism in tumor cells bits [207]. The data suggest that inhibition of 6-
have been reported. Studies of human lymphoma MP catabolism by allopurinol may contribute to
revealed that 6-MP was a potent inhibitor of cel- a greater availability of 6-MP to tissues. Stud-
lular RNA synthesis and that 6-thioITP inhib- ies of the effect of allopurinol on the kinetics
ited both the RNA polymerase I and RNA poly- of oral and intravenous 6-MP in Rhesus mon-
merase II activities of these cells [197]. These keys and in humans demonstrated that allopuri-
data suggested that direct inhibition of the en- nol pretreatment resulted in a nearly 400 % in-
zymes mediating transcription by 6-thio-IMP crease in peak plasma concentration of 6-MP
may be one of the mechanisms for the cytotoxic in monkeys and a 500 % increase in humans,
action of 6-MP in human tumor cells. Using 6- but only when 6-MP was administered orally
TG as a cytotoxic agent resulted in severe chro- [208]. Allopurinol pretreatment had no effect on
mosome damage in wild-type CHO cells [198]. the kinetics of intravenously administered 6-MP.
Gross unilateral chromatid damage resulted, and This difference is due to the action of allopurinol
the unilateral nature of this damage was proba- on liver or intestinal xanthine oxidase and in-
bly due to malfunction of 6-TG-containing DNA hibition of first-pass metabolism of oral 6-MP.
as a replication template. This finding may explain the low and variable
plasma levels of mercaptopurine in patients with
acute lymphoblastic leukemia treated with oral
2.7.2. Mechanism of Resistance 6-MP [209]. Although these studies emphasize
the catabolic pathway for purines, all the purine
Several mechanisms of resistance to these agents and pyrimidine metabolic enzymes may be im-
have been described in experimental tumors and portant to the bioavailability and activation of
relate to the pathways of antimetabolite activa- antimetabolites.
tion and degradation. A decreased HGPRT ac- To clarify the mechanisms of resistance
tivity in tumor cells diminishes antimetabolite to thiopurines and potential drug interactions
activation, and this resistance pathway has been in tumor cells, studies have focused on the
reported by several workers [199–202]. How- thiopurine-resistant cell lines deficient in HG-
ever, the HGPRT-regulated mechanism as a ba- PRT (L 1210) and the regulation of PRPP for-
sis for drug resistance in human leukemic cells mation in thiopurine-resistant cell lines.
is relatively uncommon [203]. The resistance of The thiopurines are inactive in the base form
6-MP is related to an increase in alkaline phos- and must be converted to their respective nu-
phatase in sarcoma cells [204]. Increased alka- cleotides. This activation step requires PRPP as
line phosphatase, a membrane-bound enzyme the cofactor and HGPRT as the enzyme to cat-
that converts the active mononucleotide to 6- alyze the conversion to nucleotide. A major bio-
thioinosine and inorganic phosphate, has also chemical effect of methotrexate is the suppres-
been reported in human leukemia patients re- sion of purine biosynthesis and expansion of the
sistant to drug treatment [205], [206]. Another PRPP pool. Studies of the cytotoxic and bio-
enzyme in the degradation pathway of 6-MP that chemical interaction of methotrexate and 6-TG
needs to be considered for drug efficacy and in L 1210 mouse leukemia cells demonstrated
bioavailability is xanthine oxidase, which is re- that methotrexate can markedly enhance 6-TG
sponsible for converting 6-MP to 8-OH-6-MP activity [210]. Preexposure of cells to metho-
and subsequently to thiouric acid, which is ex- trexate resulted in a large increase in cytotoxic
creted through the urine. potency of 6-TG, whereas simultaneous expo-
To understand further the bioavailability and sure caused an antagonism of 6-TG cytotoxic ac-
pharmacokinetics of thiopurines, the effect of al- tivity. Although PRPP pools were not measured
lopurinol on 6-MP catabolism was studied. Al- quantitatively, the effect of methotrexate preex-
lopurinol, an analog of hypoxanthine, enhances posure was to increase PRPP pools, enhance the
the therapeutic efficacy of 6-MP by inhibiting activation of 6-TG to 6-TG monophosphate, and
xanthine oxidase. The urinary excretion of 6- thereby increase its incorporation into RNA.
MP metabolites is markedly reduced in patients
2.7.3. New Analogs the SN 1 reaction, a highly reactive intermedi-

ate forms initially and quickly reacts with a nu-
Alkyl disulfide derivatives have been used as cleophile to produce an alkylated product. This
masked compounds of 6-mercaptopurines. The reaction follows first-order kinetics because the
antitumor effect of these derivatives has been rate-limiting step is formation of the intermedi-
measured by their ability to decrease the degra- ate. The SN 2 reaction is a second-order reaction
dation of 6-MP monophosphate. Of the seven and thus is dependent on the concentration of
6-alkyl disulfide derivatives of 6-MP and 6-TG both the alkylating agent and its target nucle-
in L 1210 leukemia cells tested, decyl deriva- ophile [216]. In general, alkylating agents that
tives of both 6-MP and 6-TG were the most ef- react by an SN 1 mechanism, such as nitrogen
fective with therapeutic ratios as high as 50 and mustard, are less selective in their reactions than
48, while those of parent compounds were 6.2 SN 2 agents, but this rule is not always true. Se-
and 5.0, respectively [211]. Partial circumven- lectivity also depends upon membrane perme-
tion of thiopurine resistance may have resulted ability, charge, and reactivity of the drug.
from cellular uptake of intact acylated bis(6-MP-
9-β-d-ribofuranoside-5 )pyrophosphate deriva-
tives in 6-MP-resistant human cell lines deficient 3.1. Nitrogen Mustard
in HGPRT (L 1210) [212].
The 6-MP-resistant sublines of P 388 and 3.1.1. Mechanism of Action
L 1210 leukemia are also sensitive to two new
purine antagonists: 5-carbamoyl-1H-imidazol- Nitrogen mustard (NSC-762) [51-75-2],
4-yl piperonylate and 4-carbamoylimidazolium- mechloroethamine, HN2, CH3 N(CH2 CH2 Cl)2 ,
5-olate [213]. These two new purine analogs C5 H11 Cl2 N, M r 156.07, is activated through
kill 6-MP-resistant cells by suppressive de novo loss of one of its chlorines. The α carbon then
purine synthesis. The activation of those new reacts with the nucleophilic nitrogen to form the
purine analogs is mediated by adenine phospho- positively charged, highly reactive, cyclic aziri-
ribosyltransferase. dinium compound, which is attacked by nucle-
ophiles to give the initial alkylated product. The
second chlorine can also leave, initiating a sec-
3. Alkylating Agents ond alkylation, which produces a cross-linked
alkylation between two nucleophiles.
Historically, alkylating agents were important Because HN2 bonds covalently to many bio-
in the early development of cancer chemothera- logic molecules, such as DNA, RNA, and pro-
pies. Victims of sulfur mustard gas exposure in teins, the alkylated sites responsible for its cy-
World War I were found to have severe lymphoid totoxicity are difficult to determine. However,
aplasia as well as pulmonary irritation [214]. studies have shown that cytotoxicity is likely to
This led to clinical trials of the related, but less result from inhibition of DNA synthesis by dam-
toxic, nitrogen mustard derivative, which pro- aging the DNA template [217–219]. The DNA
duced tumor regressions in lymphoma patients molecule is rich in potential sites for alkylation,
[215]. including the phosphate groups in the sugar
Clinical use of nitrogen mustard today is phosphate backbone structure, and the oxygen
mostly limited to the treatment of lymphomas, and nitrogen sites in the purine and pyrimidine
especially Hodgkin’s disease, where it is used bases. However, the tendency for alkylation to
in a multidrug regimen called MOPP (nitrogen occur in the N-7 position of guanine is enhanced.
mustard, vincristine, procarbazine, and pred- This may be mediated by the increased nucle-
nisone). Three widely used derivatives of nitro- ophilic characteristics of the N-7 deoxyguano-
gen mustard used in patients with malignancies sine due to base stacking and charge transfer
today include melphalan, cyclophosphamide, [220]. Other preferred sites of alkylation in de-
and chlorambucil (Sections 3.2, 3.3, 3.4). creasing order are the N-1 of adenosine, the N-3
The mechanism by which alkylating agents of cytidine, and the N-3 of thymidine [221].
act can be classified as either SN 1 or SN 2. In Bifunctional alkylating agents, such as HN2,
produce intra- and interstrand cross-linking bet-
ween DNA in the double-helix structure pref- 305.20, was rationally designed and synthe-
erentially at the N-7 guanosine site. Thus, these sized as a phenylalanine derivative of nitrogen
bifunctional compounds are more effective anti- mustard with the aim of obtaining increased
tumor agents than their monofunctional analogs; specificity against melanoma tumor cells that
however, increasing the number of alkylating utilize phenylalanine or tyrosine to produce
sites on the agent beyond two does not appear melanin.
to increase antitumor activity [222]. This evi-
dence suggests that DNA cross-linking is crit-
ical for alkylator activity. Further evidence for
the importance of cross – linking to cytotoxic-
ity comes from alkaline elution studies that can
detect low levels of cross-linking in cells ex- Melphalan
posed to minimal doses of bifunctional alkylat-
ing agents [223]. Although this compound does not exhibit spe-
In contrast, monofunctional agents, such as cific antimelanoma activity, it has a broad-
procarbazine and dacarbazine, do not produce spectrum cytotoxicity in multiple myeloma,
DNA interstrand cross-links and appear to ex- breast cancer, and lymphomas.
ert toxicity by producing single-strand DNA
breaks. The increased carcinogenicity seen with
some monofunctional alkylating agents may be 3.2.1. Mechanism of Action
due to incorrect base pair substitution by DNA Melphalan is a bifunctional alkylating agent but
repair enzymes that could result in malignant differs from HN2 mainly by the presence of
transformation. an aromatic ring. This ring reduces the nucle-
ophilicity of the nitrogen atom by withdrawing
electrons, making the drug less reactive. Thus, it
3.1.2. Mechanisms of Drug Resistance can be taken orally. It retains its alkylating activ-
ity but is more selective than nitrogen mustard
Several mechanisms have been elucidated for ni- because it is less likely to form the unstable and
trogen mustard and other bifunctional alkylating highly reactive aziridine intermediate indiscrim-
agents. First, resistant cells with defective drug inately. Like nitrogen mustard, it forms DNA
transport have been described. Nitrogen mustard cross-links that are critical for cytotoxic effect.
enters cells by an active transport system that Its cellular uptake is also mediated by an active,
is physiologically utilized for choline transport. energy-dependent transport mechanism shared
Lymphoma cells resistant to nitrogen mustard with leucine and glutamine uptake [218]. Thus,
demonstrate decreased drug uptake by this spe- high concentrations of leucine or glutamine can
cific active transport site, which also decreases reduce the cytotoxicity of melphalan in a mar-
its uptake of choline [224]. Second, cytosolic in- row colony-forming unit assay [229]. Another
crease in nonprotein sulfhydryl levels [225] and transport mechanism, although less active, can
higher nonprotein-bound thiol compounds that be used by melphalan, and this mechanism is
could inactivate the drugs before they reach the shared by the neutral amino acids alanine, cys-
nucleus have been found in nitrogen mustard- teine, and serine [218].
resistant cells [226]. Third, enhanced repair of
DNA cross-linking by repair enzymes has been
demonstrated in vitro [227] and in vivo [228]. 3.2.2. Mechanism of Resistance
Decreased transport of melphalan into drug-
3.2. Melphalan resistant leukemia cells has been demonstrated
and correlated to the melphalan-resistant pheno-
Melphalan (NSC-8806) [148-82-3], 4- type. Specifically, a mutation in the higher ve-
[bis(2-chloroethyl)amino]-l-phenylalanine, l- locity transport system has been suggested that
phenylalanine mustard, C13 H18 Cl2 N2 O2 , M r results in a decreased affinity of the carrier pro-
tein for melphalan and leucine [230]. Evidence
for the other two mechanisms of resistance seen 3.3.2. Mechanism of Resistance
with nitrogen mustard, i.e., increased intracellu-
lar thiol compounds and increased DNA cross- Similar mechanisms of resistance occur for cy-
linking repair also, exists. clophosphamide as for nitrogen mustard. Also,
defective metabolic conversion by the hepatic
microsomal system could serve to decrease the
3.3. Cyclophosphamide bioavailability of aldophosphamide to tumor tis-
sue, but it is not known whether this is signifi-
Cyclophosphamide (NSC-26271) [50-18-0], cant.
N,N-bis(2-chloroethyl)tetrahydro-2H-1,3,2-
oxazaphosphorin-2-amine 2-oxide,
C7 H15 Cl2 N2 O2 P, M r 261.10. 3.4. Chlorambucil
Chlorambucil (NSC-3088) [305-03-3], 4-

[bis(2-chloroethyl)amino]benzenebutanoic
acid, C14 H19 Cl2 NO2 , M r 304.23. This drug is a
close congener of melphalan and exhibits simi-
Cyclophosphamide
lar stability because of the electron-withdrawing
properties of the aromatic ring. It is given orally
This widely used bifunctional, cyclic alkylat- and has proven efficacy in treating chronic lym-
ing agent has important clinical use in lymphocytic leukemias, multiple myeloma, and
phomas, leukemias, sarcomas, carcinomas of lymphomas.
breast and ovary, as well as childhood malig- Chlorambucil is thought to have activation
nancies. The compound was rationally designed properties and mechanisms of resistance similar
based on data that tumor cells possess high to melphalan.
concentrations of enzymes capable of cleaving
the P−N bond. This reaction would activate
drug by release of the potent antitumor agent
phosphoramide mustard. Cyclophosphamide
requires hepatic activation by oxidase enzymes.
First, it is metabolized by liver microsomes Chlorambucil
to hydroxycyclophosphamide, which is spon-
taneously tautomerized to aldophosphamide.
Aldophosphamide reaches peripheral tissues
and tumors where it is hydrolyzed to yield the 3.5. Thio-TEPA
active antitumor agent phosphoramide mustard
and acrolein. Acrolein has very weak antitumor Triethylenethiophosphoramide (NSC-6396)
activity and, when concentrated within the blad- [52-24-4], 1,1 ,1 -phosphinothioylidyne-
der by excretion, can cause hemorrhagic cystitis trisaziridine, thio-TEPA, C6 H12 N3 PS, M r
[216]. 189.23. This agent is representative of alky-
lating agents that have two or more aziridine
rings. It has clinical activity against the same
3.3.1. Mechanism of Action tumors as nitrogen mustard and has been used
clinically in carcinomas of the breast and ovary.
Phosphoramide mustard can undergo similar bi- Thio-TEPA is also indicated for intrathecal ther-
functional alkylation as nitrogen mustard. Also, apy of meningeal carcinomas. The reactivity of
because of its need for metabolic conversion the aziridine groups is increased by protonation;
for biologic activity, cyclophosphamide can be thus thio-TEPA is most active at low pH and
given orally or intravenously. has been used to cause sclerosis of malignant
pleural effusions that often have an acidic pH.
mustine [489-15-0] was successfully launched

for treatment of prostate cancer [234].
Trade name: Estracyt (Pharmacia/Upjohn)
Thio-TEPA
3.8. Nitrosoureas
The mechanisms of action and resistance are
similar to those of nitrogen mustard. The clinically useful nitrosoureas in-
clude carmustine (NSC-409962) [154-93-8],
N,N -bis(2-chloroethyl)-N-nitrosourea,
3.6. Ifosfamide BCNU,C5 H9 Cl2 N3 O2 , M r 214.04; lo-
mustine (NSC-79037) [13010-47-4], N-
Ifosfamide, or isofosphamide (NSC-109724) (2-chloroethyl)-N -cyclohexyl-N-nitroso-
[3778-73-2], N,3-bis(2-chloroethyl)-tetra- urea, CCNU, C9 H16 ClN3 O2 , M r 233.69;
hydro-2H-1,3,2-oxazaphosphorin-2-amine 2- methylcyclohexylchloroethylnitrosourea (NSC-
oxide, C7 H15 Cl2 N2 O2 P, M r 261.07, is an ana- 94941) [52662-76-7], 1-(2-chloroethyl)-3-
log of cyclophosphamide [231]. It has approx- (4-methylcyclohexyl)-1-nitrosourea, methyl-
imately one-third the alkylating activity of cy- CCNU, C10 H18 ClN3 O2 , M r 248; strep-
clophosphamide and requires hepatic microso- tozotocin (NSC-85998) [18883-66-4], 2-
mal conversion to its active form. Also, the rates deoxy-2{[(methylnitrosoamino)carbonyl]ami-
of conversion by metabolism are similar in both no}-d-glucopyranose, C8 H15 N3 O7 , Mr
drugs, but less biologically active alkylating 265.22; and chlorozotocin (NSC-178248)
moieties are formed in ifosfamide [232]. It has [54749-90-5], 2-({[(2-chloroethyl)nitrosoami-
shown promising results in refractory pediatric no]carbonyl}amino)-2-deoxy-d-glucose,
bone and soft tissue sarcomas, refractory tes- DCNU, C9 H16 ClN3 O7 , M r 313.69. Streptozo-
ticular tumors, and Wilms’ tumor in children tocin is a naturally occurring nitrosourea derived
[233], [235]. from Streptomyces acromogenes.
Trade names: Ifex, Holoxan (ASTA Medica)
Ifosfamide
3.7. Estramustine This group of agents was developed by careful

structure – function studies based on the antitu-
mor activity of methyl-CCNU [235–237]. The
chloroethyl derivatives were found to possess
increased activity and a capacity to cross the
blood – brain barrier because of their lipophilic
nature. Each of these agents is capable of un-
dergoing alkylating reactions with biologic
molecules in a manner similar to the classic
mustards through the formation of highly re-
Estramustine active chloroethyl carbocations. With BCNU,
each molecule of drug may undergo two such
From the numerous analogs few found a way reactions to produce nucleic acid strand breaks,
into the clinic or to the market. However, estra- and DNA – DNA and DNA – protein cross-links
[238]. Monofunctional CCNU and methyl- to inactivation of the DNA template, may well be
CCNU cross-link by initial carbocation for- the key mechanism of cytotoxicity [252–254].
mation and alkylation followed by loss of the Monofunctional DNA alkylations also occur fol-
chloride substituent and formation of a second lowing nitrosourea exposure and these must also
reactive carbocation. These alkylation reactions be considered cytotoxic as monofunctional alky-
appear to be the major mode of cytotoxicity for lating agents, incapable of cross-linking, retain
these agents [239], [240]. With the exception of cytotoxic activity. Monofunctional alkylations
chlorozotocin, the nitrosoureas may also form may produce single-strand DNA breaks by en-
an isocyanate compound that may play a role donuclease cleavage at apurinic sites produced
in the toxic side effects of these agents [241], by the alkylation and repair process [255].
[242], but has little importance in the antitumor
effect. In support of this view is the fact that
chlorozotocin retains its potent cytotoxic capac- 3.8.2. Mechanisms of Resistance
ity while producing little or no isocyanate com-
pound and reportedly has less marrow toxicity. The mechanisms of cellular resistance to nitro-
Experimental evidence raises additional ques- soureas remain unclear, although defective drug
tions concerning the role of isocyanates, which transport, as has been demonstrated for the clas-
may enhance the antitumor activity of these sic alkylating agents, would be unlikely given
compounds. Methylnitrosourea, which cannot the lack of need for an active membrane trans-
alkylate DNA to produce cross-linking, pro- port system. In human glioblastoma cells, in-
duces alterations of nuclear protein in a manner creased activity of a specific excision enzyme,
similar to BCNU, and isocyanates are consid- guanine-O6 -alkyltransferase, is correlated with
ered a possible explanation for these effects from in vitro resistance to nitrosoureas [256], and re-
both agents [243]. Also, sensitivity of a Walker pair mechanisms would seem a likely mecha-
tumor line made resistant to bifunctional alky- nism since mammalian cells are capable gener-
lating agents can be restored by simultaneous ally of such repair [257].
treatment with an isocyanate-producing agent
[244].
3.8.3. Analogs
3.8.1. Mechanism of Action New agents have been synthesized that con-
tain a sugar moiety similar to streptozotocin.
Because of their lipophilicity, nitrosoureas enter Analogs that contain mannose, glucose, ribose,
cells by passive diffusion as opposed to an ac- maltose, and galactose have all been produced,
tive transport mechanism common to the clas- with the maltosyl derivative being exceptionally
sic alkylating agents [245]. Once inside the cell, active in a variety of tumors tested [258], [259].
alkylating agent exposure results in pancellu- Sugar alcohols, such as mannitol, linked to nitro-
lar covalent binding of drug to proteins, nu- soureas retain their cytotoxicity but appear to
cleic acids, and to a variety of smaller intra- protect against the marrow toxicity induced by
cellular molecules. Which of these reactions is the parent nitrosourea compounds. A number of
critical for cytotoxicity remains uncertain, but derivatives with di- and tripeptides containing
the majority of evidence points to interaction di- alkyl nitrosoureas have also been introduced but
rectly with DNA as the focal point of cytotoxic- have met with only moderate preclinical success
ity [246–248]. The 7 position of guanine is par- [260].
ticularly susceptible to alkylation, and accounts There is continuous effort to obtain new nitro-
for the majority of the total alkylation of DNA soureas with higher efficiency and/or lower tox-
[249], [250]. Since the chloroethyl nitrosoureas icity and several new compounds are undergo-
are each capable of two independent alkylations ing clinical trials. In 1987 ranimustine (NSC-
[238], DNA can be cross-linked by either inter- 270561, MCNU) [058994-96-0], was marketed
strand or intrastrand processes [251]. Multiple as Cymerin (Tokyo Tanabe) [261].
studies using a variety of alkylating agents have The phosphonoalanine derivative
confirmed that DNA cross-linking, which leads fotemustine (S-10036) [092118-27-9],
(±)-diethyl[1-[3-(2-chloroethyl)-3-nitroso- that, through a variety of mechanisms, are capa-

ureido]ethyl]phosphonate, C9 H19 ClN3 O5 P, M r ble of cytotoxicity [271].
315.69, mp 85 ◦ C was approved as Muphoran A crucial step in the activation of procar-
(Servier) for treatment of disseminated malig- bazine appears to be the production of the
nant melanoma [262], [263]. Side effects of azo analog N-isopropyl-α-(2-methyldiazeno)-
the related compound BCNU (carmustine) have p-toluamide, which is catalyzed by hepatic mi-
been linked to the inhibitory effect on a major crosomal cytochrome P450 [272], [273]. Fur-
enzyme of the glutathione pathway, the cytoso- ther metabolism by hepatic microsomes leads
lic glutathione reductase. Fotemustine has no to the production of methyl- and benzyl-azoxy
inhibitory effect on cytosolic glutathione reduc- metabolites [272], [273]. Cytotoxic alkylating
tase, indicating that fotemustine has a lower compounds may be formed from these metabo-
toxicity than carmustine [264–266]. In addition lites via hydroxylation reactions, and these may
it has been shown to have lower mutagenicity in play the major role in cytotoxicity. This se-
the Ames and micronucleus tests compared to quence of metabolism and formation of the alky-
BCNU [267]. In the clinical studies the major lating intermediates is consistent with the time
toxic effects of fotemustine were thrombocy- course of appearance of the active species iso-
topenia and leukopenia, which were delayed lated in the serum and then excreted [274],
and reversible, nausea and vomiting being mild [275]. In addition to the formation of these alky-
[268]. lating intermediates, methyl- and benzyl-azoxy
metabolites may result in free radicals through
the formation of diazenes [276], which in the
presence of oxygen form free radicals and ni-
trogen. However, free-radical formation is not
likely a major cause of cytotoxicity, because
drug activity is preserved when cells are exposed
to procarbazine under conditions that do not sup-
port the formation of free radicals [277]. The
degree of toxicity induced by alkylation and by
Ranimustine free-radical formation is unknown.
3.9.1. Mechanism of Action
The exact mechanism by which procarbazine

produces cytotoxicity is unknown; however, the
Fotemustine effects of its action have been well studied
at the cellular level. Chromosome breaks and
translocations have been demonstrated in vivo in
Ehrlich ascites and L 1210 leukemia cells [278].
3.9. Procarbazine
Inhibition of nucleic acid and protein synthe-
sis and of a variety of enzymes has also been
Procarbazine (NSC-77213) [671-16-9], N-(1-
documented following procarbazine treatment.
methylethyl)-4-[(2-methylhydrazino)methyl]-
Inhibition of transfer and nuclear RNA synthe-
benzamide, C12 H19 N3 O, M r 221.30, is one
sis occurs 2 h after procarbazine exposure and
of a number of substituted hydrazine com-
lasts for up to 24 h [279]. Thymidine incorpora-
pounds originally synthesized as monoamine
tion into DNA is inhibited concomitantly with
oxidase inhibitors in the early 1960s [269], and
protein synthesis, reaching maximum inhibition
found to possess antineoplastic activity, par-
in 12 – 16 h [271], [280]. Although many poten-
ticularly in the treatment of Hodgkin’s disease
tially cytotoxic events have been associated with
[270]. Procarbazine is nontoxic as the parent
procarbazine administration, it is not clear which
compound but undergoes rapid chemical and
of these effects causes cell death.
metabolic degradation to intermediate products
3.9.2. Mechanisms of Resistance 3.11. Hexamethylmelamine

Resistance mechanisms for procarbazine are Hexamethylmelamine (NSC-13875) [645-05-6],
poorly understood, and no detailed studies have N,N,N ,N ,N ,N -hexamethyl-1,3,5-triazine-
been reported that illustrate the typical cellular 2,4,6-triamine, altretamine, C9 H18 N6 , M r
resistance encountered with alkylating agents. 210.27, was synthesized in 1951 [287]. It has
Since the drug enters cells by simple diffusion, consistent antitumor activity in a variety of solid
resistance is unlikely to involve altered transport tumors, including ovarian, lung, and breast can-
mechanisms [281]. New pieces of chromosomal cer.
material were found in Ehrlich ascites cells made The compound is almost insoluble in wa-
resistant to procarbazine [271], and these may ter, and thus, must be administered orally. Nei-
represent gene amplification, perhaps encoding ther the mechanism of action nor the products
for a target enzyme or detoxifying enzyme. of metabolic breakdown have been firmly es-
tablished. Following administration of hexam-
ethylmelamine, a spectrum of N-demethylation
3.10. Dacarbazine
species has been isolated in the urine. The tri-
Dacarbazine (NSC-45388) [4342-03-4], 5- azine ring appears unaffected by metabolism, as
(3,3-dimethyl-1-triazenyl)-1H-imidazole-4- is evident by almost complete recovery of the
carboxamide, DTIC, C6 H10 N6 O, M r 182.18, intact ring in the urine using ring-labeled com-
was synthesized in the late 1950s as an analog pound [288]. Existing evidence suggests possi-
of 5-aminoimidazole-4-carboxamide, an inter- ble formation of an alkylating species through
mediate in de novo purine synthesis. The drug N-demethylation [289] or the formation of N-
is a product of rational synthesis, designed as methylol intermediates by hydroxylation of the
a false intermediate capable of inhibiting de parent compound [290] to account for the cyto-
novo purine synthesis. Despite this theoreti- toxic effects of the drug. N-Methylol derivatives
cal basis for antitumor action, DTIC does not have cytotoxic effects in vitro, but whether or not
function as a purine analog; instead, it is ex- these derivatives are formed in vivo is unclear.
tensively metabolized to a methylating agent
[282]. Similar to procarbazine, DTIC must un- Analogs. Pentamethylmelamine (NSC-
dergo activation by a microsomal oxidase to 118742) [35832-09-8], N,N,N ,N ,N -
form a compound that can spontaneously pro- pentamethyl-1,3,5-triazine-2,4,6-triamine,
duce a methyl diazonium ion intermediate that is C8 H17 N6 , M r 196, is the most commonly used
probably the active metabolite. However, more analog of hexamethylmelamine and differs by
recent evidence suggests a further metabolism the absence of a single methyl group. Its major
to N-hydroxymethyl diazonium ion may be re- advantage is aqueous solubility, allowing an in-
sponsible for the selective antitumor effect of travenous formulation. Its metabolism, toxicity,
the drug [283]. Evidence for methylation of and antitumor activity parallel those of hexam-
nucleic acids has been demonstrated in tissue ethylmelamine [291], [292]. Other metabolites
culture [284]. An additional metabolic pathway of hexamethylmelamine with varying numbers
involves the light sensitivity of the drug. Ex- of methyl groups also possess antitumor activity
posure to ultraviolet energy converts the parent that, in general, is directly proportional to the
compound to metabolites with moderate cyto- number of methyl groups on the triazene ring
toxicity in vitro [285], but this probably does not [292], [293].
represent an important pathway for cytotoxicity
in vivo.
3.12. Mitomycin-C
Mechanism of Action. The mechanism of
action of DTIC has not been systematically in- The mitomycins are a family of antibiotics iso-
vestigated, but it appears that the drug may act lated from Streptomyces caespitosus.
during any phase of the cell cycle [286] and can
produce inhibition of RNA, DNA, and protein
synthesis.
Mitomycin-C (NSC-26980, MIT-C) Meanwhile epirubicin (4’-epiadriamycin,

[50-07-7] [1aR-(1aα,8β,8aα,8bα)]-6-amino- pidorubicin, IMI-28) [56420-45-2], (8S-
8-[(aminocarbonyl)oxymethyl]-1,1a,2,8,8a,8b- cis)-10-[(3-amino-2,3,6-trideoxy-ß-l-arabino-
hexahydro-8a-methoxy-5-methylazirino- hexopyranosyl)oxy]-7,8,9,10-tetrahydro-
[2’,3’:3,4]pyrrolo[1,2-a]indole-4,7-dione, 6,8,11-trihydroxy-8-(hydroxyacetyl)-1-
C15 H18 N4 O5 , has DNA-alkylating properties methoxy-5,12-naphthacenedione, is the most
[294]. Mitomycin is applied for treatment of commonly used antineoplastic antibiotic for
stomach, breast, and gynecological cancers breast cancer treatment. The total turnover ex-
[295], [296]. Dose-limiting are leucopenia and ceeded $ 200 × 106 in 1996. The compound can
thrombopenia. be synthesized by classical chemical synthesis
[297–299].
Trade names: Farmorubicin and Pharmoru-
bicin (Farmitalia).
Mitomycin
4. Anthracyclines (→ Antibiotics,
Chap. 3.2.3.) Epirubicin
Doxorubicin (NSC-123127) [23214-92-8], In addition idarubicin (NSC-256439, IMI-

10-[(3-amino-2,3,6-trideoxy-α-l-lyxo-hexo- 30, DMDR) [058957-92-99] and piraru-
pyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-tri- bicin (THP-ADM) [072496-41-4], (8S-cis)-
hydroxy-8-(hydroxyacetyl)-1-methoxy-5,12- 10-[[3-amino-2,3,6-trideoxy-4-O-(tetrahydro-
naphthacenedione, adriamycin, C27 H29 NO11 , 2H-pyran-2-yl]-α-L-lyxo-hexopyranosyl]oxy]-
M r 543.54; and daunorubicin (NSC-82151 7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hy-
for HCl salt) [20830-81-3], 8-acetyl- droxyacetyl)-1-methoxy-5,12-naphthacene-
10-[(3-amino-2,3,6-trideoxy-α-l-lyxo-hexo- dione, C32 H37 NO12 , M r 627,64 are available
pyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-tri- as idamycin (Adria) and pinorubicin (Nippon
hydroxy-1-methoxy-5,12-naphthacenedione, Kayaku), respectively.
daunomycin, C27 H29 NO10 , M r 527.51. Dox-
orubicin is used in the treatment of breast can-
cer, sarcoma, lymphoma, and small-cell lung
cancer. Daunorubicin is used more commonly
in acute myelocytic and lymphocytic leukemias.
Idarubicin
Pirarubicin is the 4’-O-tetrahydropyranyl

analog of adriamycin and can be synthesized
R = COCH2 OH, Doxorubicin from adriamycin [300], [301]. The acute cardiac
R = COCH3 , Daunorubicin
toxicity was significantly less than that of adri-
amycin and general toxicity lower than that of has been established that the anthracyclines can
other analogs [302, 303, 303]. The main indica- affect every DNA function, including initiation,
tions for pirarubicin are cancer of the bladder, chain elongation, DNA synthesis, DNA repair,
head and neck, and cervix. and RNA synthesis [308], [309]. Experimentally
these compounds can cause sister chromatid ex-
changes, single- and double-strand breaks, and
alkylation of DNA [310], [311]. Interestingly,
some results strongly suggest that inhibition of
DNA synthesis is not essential for cell kill. New
anthracycline analogs, such as aclacinomycin
A, which selectively inhibits preribosomal RNA
synthesis and not DNA synthesis, retain cytotox-
icity. This suggests other non-DNA-mediated
mechanisms of cytotoxicity, such as preriboso-
mal RNA synthesis [309].
Pirarubicin
Free-Radical Generation. Free-radical for-
The anthracyclines are derived from Strep- mation (highly reactive compounds with an un-
tomyces species and are structurally tetracyclic paired electron) occurs during the metabolism
chromophore antibiotics [305]. They are classi- of anthracyclines. When the microsomal en-
fied by their chromophore, otherwise known as zyme P 450 reductase or xanthine oxidase in-
aglycone, structure. The sugar most commonly teracts with and reduces the ketone oxygen in
attached to the aglycones is daunosamine, but ring B to O− , a semiquinone radical interme-
other sugars may be involved, and these are men- diate is formed. This interacts with oxygen to
tioned in Section 4.3. The basic tetracyclic agly- produce the superoxide radical with regenera-
cone structure of the anthracyclines shares many tion of the original anthracycline structure [312].
characteristics with the hydroxyanthraquinones, The superoxide radical can serve as a substrate
which are ubiquitous in nature. for superoxide dismutase to form hydrogen per-
oxide, which can interact with the superoxide
molecules to form hydroxyl radicals [313]. The
4.1. Mechanism of Action superoxide and hydroxyl radicals can interact
with and damage cells, especially the hydroxyl
DNA−RNA Binding. The exact mecha- radical, which is one of the most reactive sub-
nism of cytotoxicity by anthracyclines is un- stances known. Hydroxyl radicals can also re-
known, but they do have multiple and distinct act with purine or pyrimidine bases, amines, and
toxic effects that may kill a tumor cell in one or thiols. This free-radical formation is responsible
more ways [306]. for cardiac damage seen with chronic use of the
Initially these drugs were found to bind DNA anthracyclines in the treatment of human malig-
by intercalation between base pairs perpendicu- nancies. Evidence exists in many animal mod-
lar to the long axis of the double helix, with the els, as well as in humans, that superoxide and hy-
major binding occurring between the B and C droxyl radical formation occurs in cardiac tissue,
rings of the drugs with the bases above and be- leading to lipid peroxidation of mitochondria
low [307]. The daunosamine sugar is thought to and sarcosomes [314], [315]. Since mitochon-
bind ionically with the sugar-phosphate back- dria account for more than 40 % of cardiac mus-
bone of DNA. The binding association con- cle mass, as well as being the major source for
stant is between 105 and 106 M−1 . Intercalation ATP needed for contraction, and are coupled to
of the DNA causes a partial unwinding of the calcium release during the action potential, one
helix and thus disrupts DNA polymerases and can easily visualize how these agents mediate
transcription. However, these experiments were cardiac toxicity. However, it now seems apparent
done with DNA in vitro; DNA in vivo is orga- that cardiac tissue lacks catalase and that dox-
nized into chromatin, which is DNA wrapped orubicin destroys glutathione peroxidase [315],
around a series of histone core particles. Also, it [316]. Agents that can scavenge free-radicals are
under active investigation. Interestingly, there is 4.2. Mechanism of Resistance

no evidence to date to link anthracycline free-
radical formation to its antitumor activity so that Probably the most common and important mech-
analogs incapable of free-radical formation may anism by which tumor cells become resistant to
demonstrate improved therapeutic index. the anthracyclines is decreased net intracellu-
lar accumulation. Many studies have shown that
Membrane Interactions. Anthracycline doxorubicin-resistant tumor cells are capable of
binding to cell membranes appears to be an im- effluxing the drug more efficiently than their
portant mechanism for cytotoxicity. Changes in parent sensitive cells [355]. In fact, this mecha-
membrane glycoproteins, transmembrane flux nism of drug resistance may be responsible for
of ions, and membrane morphology have been resistance to a variety of structurally unrelated
demonstrated in a variety of cells. Doxorubicin compounds with different modes of antitumor
binds most tightly to cardiolipin, a phospholipid action, such as the vinca alkaloids and actino-
found in high concentration in mitochondrial mycin D. This has been termed pleiotropic drug
and tumor cell membranes but in low concen- resistance, and several laboratories have shown
tration in normal cell membranes [317]. Also, the reversal of this resistant phenotype by co-
membrane redox potential changes occur with incubation with various calcium channel block-
drug binding, and may promote free-radical ers with doxorubicin [356]. These compounds
generation. Perhaps the most interesting and increase the net intracellular drug accumulation
important finding to date on this subject is that in resistant cells, but the precise mechanism of
doxorubicin, covalently linked to beads to pre- this action is uncertain.
vent cell entry of the drug, retains cytotoxic
effects [318]. This suggests that doxorubicin
does not need to enter cells or interact with 4.3. Analogs
DNA to mediate cell kill.
Another potential mechanism of action may As mentioned in Section 4.1, anthracycline tu-
be the dissociation of the cytochrome oxidase mor toxicity is probably related to intercala-
electron transport chain for ATP generation tion with DNA, preribosomal RNA inhibition,
[319]. Cytochrome c oxidase requires cardi- and/or membrane binding effects, while car-
olipin for activity, and doxorubicin can remove diac toxicity may be due to free-radical forma-
cardiolipin from the enzyme complex, thus in- tion by drug and drug – metal complex. Thus,
activating the enzyme. analogs have been developed with less poten-
tial for free-radical formation. One such drug in
Metal Ion Chelation. The anthracyclines early clinical development is aclacinomycin A
are capable of chelating ions, including cop- (NSC-208734), which has an aklavinone agly-
per, calcium, magnesium, zinc, and iron. Of the cone structure and is derived as a fermenta-
metal ions, the tightest complex seems to be tion product of Streptomyces [321]. This drug
with iron(III). Doxorubicin – iron chelates can lacks the 11-hydroxyl, and therefore has about
act as a redox catalyst for electron transfer from 10 % the potential to generate free-radicals in
glutathione to oxygen, which leads to formation the P 450 reductase system. Although it does not
of cytotoxic oxygen radicals. This reaction can bind DNA as well as doxorubicin, it retains sig-
also utilize hydrogen peroxide and superoxide, nificant antitumor activity.
leading to hydroxyl radical formation. Thus, ev- The National Cancer Institute (USA) has
idence exists that the anthracycline−iron com- screened hundreds of anthracycline analogs for
plex can mediate free-radical formation capable antitumor activity. From these studies it can be
of lipid peroxidation and cell damage [320]. concluded that (1) the amino sugar is not re-
Whether this is a mechanism of tumor cell kill quired for activity, (2) disaccharide analogs are
is unknown, but the phenomenon may be im- generally more active than the parent saccharide
portant to cardiac toxicity. in inhibiting RNA synthesis, and (3) in the agly-
cone, substituents in the 7 and 9 positions are
important for activity. Studies have also shown
that if the 4 -hydroxyl group is removed, car- compared to doxorubicin on an equimolar ba-
diac toxicity is lessened significantly [322–324]. sis mitoxantrone proved to be six to seven times
Thus, it seems that the amino sugar and/or the 4 - more potent in inhibiting the incorporation of
hydroxyl group are major determinants of car- uridine and thymidine into DNA [330]. Mitox-
diac toxicity in the anthracyclines. antrone is a cell phase nonspecific agent and
has wide spectrum of activity against several
experimental animal tumors. Cross-resistance
5. Intercalating Anthracenes and to adriamycin was not always seen [331–333].
Adriamycin-like cardiac toxicity was not found
Analogs in comparative studies using rats, dogs, rabbits,
and monkeys maybe partially due to inhibition
5.1. Mitoxantrone of free radical formation and due to the lack of
the amino sugar moiety [334], [335]. Although
Mitoxantrone hydrochloride (NSC-301739, mitoxantrone is not entirely free of cardiac tox-
DAD, CL-232315) [070476-82-3], 1,4- icity in humans, minimal nausea and vomiting
dihydroxy-5,8-bis[(2-(2-hydroxyethyl)ami- was observed [336–341].
no)ethyl]amino-9,10-anthracenedione, is a new
type of antineoplastic agent. Mitoxantrone is
active against breast cancer, acute leukemia, 5.2. Analogs
lymphoma, cervix carcinoma, and liver cell
cancer. Unlike the anthracyclines that have a Bisantrene hydrochloride (NSC-337766,
red color, the anthracenediones are deep blue. ADD, CL-216942) [071439-68-4], 9,10-
Mitoxantrone is structurally similar to adri- anthracenedicarboxaldehyde bis[(4,5-di-
amycin but without the aminosugar at C9 and hydro-1H-imidazol-2-yl] dihydrochloride,
can be synthesized starting from 1,8-dihydroxy- C22 H22 N8 ’2 HCl, M r 471.39, is an intercalating
anthrachinone [325–329]. anthracenebishydrazine cytostatic [342–344].
Trade names: Novanthrone (Lederle / Amer- The product was launched for treatment of acute
ican Cyanamide); Onkotrone (ASTA Medica non-lymphocytic leukemia [345].
AWD). Trade names: Zantrene and Cyabin (Led-
erle).
As solubility is a problem, bisantrene pro-
drugs with enhanced water solubility (e.g.,
199344, see below) were developed [346–348].
Mitoxantrone
Mechanism of Action. The quinone struc-

ture of mitoxantrone was recognized as being
similar to that of adriamycin, having a lower car-
diotoxic potential. However, its discovery was Bisantrene
both a result of serendipity and of rational drug
development [325]. The exact mechanism of ac-
tion by which mitoxantrone exerts its cytotoxic
effects has not been fully defined. The cytotoxi-
city is most likely associated with the action on
chromosomal elements, resulting in DNA dam-
age and leading to inhibition of nucleic acid syn-
thesis and the eventual death of the cell. When
199344
Further intercalating agents such as am- 1940s [357]. Structurally, compounds in this
sacrine (NSC-156303/NSC-249992, m-AMSA, class share a common phenoxone ring and two
SN-11841) [051264-14-3] [349], [350], or ni- cyclic pentapeptides. The natural products dif-
tracin [4533-39-5] [351] have the acridine struc- fer among themselves in the amino acid com-
ture in common. position of the peptide chains, but only actino-
Trade names: Amsacrine is marketed as Am- mycin D is used in the clinical treatment of can-
sidine, Amecrin, and Ansidyl (Parke-Davis) cer, where it demonstrates reproducible activity
[352–354]. against gestational choriocarcinoma and Wilms’
tumor.
Actinomycin D (NSC-3053) [50-76-0],
dactinomycin, C62 H86 N12 O16 , M r 1255.47.
Mechanism of Action. Actinomycin inter-

acts with DNA through “pseudo-intercalation”
at deoxyguanylyl-3 ,5 -deoxycytidine se-
quences [358], [359], resulting in inhibition
Amsacrine of DNA-directed RNA synthesis and inhibition
of protein synthesis [360]. Resistance to acti-
nomycin D is linked to cross-resistance against
6. Antitumor Antibiotics Other than other structurally unrelated amphiphilic drugs
with dissimilar mechanisms of action as part
Anthracyclines
of the phenomenon of pleiotropic drug resis-
The antitumor antibiotics are a group of mi- tance (see Section 4.2). Certainly it had been
crobial products capable of inhibiting tumor previously demonstrated that the accumulation
growth. This class of antitumor agents has been of drug is greater in sensitive cells [361], [362],
extensively studied as to mechanism of action suggesting that alterations in transport at the
and has a rather broad spectrum of activity. In membrane level might be important to resist-
contrast to antibacterial antibiotics, the thera- ance.
peutic index of these drugs tends to be narrow
and toxicity to normal host tissues is consider-
able. 6.2. Bleomycin (→ Antibiotics, Chap. 3.7.1.)
Bleomycins (NSC-125066) are a group of anti-

6.1. Actinomycin D (→ Antibiotics, tumor antibiotics initially isolated from broths
Chap. 3.7.6.) of Streptomyces verticillus [363].
The actinomycins are a family of antibiotics

derived from Actinomyces broths during the
Actinomycin D
[365]. In addition, total synthesis of bleomycin

described in the 1980s will likely lead to other
analogs and a further understanding of the drug’s
mechanism of action [367]. A new drug delivery
system which comprises peplomycin absorbed
on to small activated carbon particles was intro-
duced as slow release formulation suggesting to
decrease the systemic toxicity [366].
6.2.2. Mechanism of Action

Bleomycin enters cells slowly. Labeling stud-
ies demonstrate that the drug is first detected on
the cell membrane and reaches the nucleus only
after several hours [368]. Bleomycin kills cells
by producing single- and double-strand DNA
breaks. Bleomycin first binds guanine bases in
DNA through the amino terminal peptide of
the drug [369]. Free-radical formation occurs
through oxidation of a bleomycin – Fe(II) com-
plex to Fe(III), which catalyzes the reduction of
molecular oxygen to superoxides and free hy-
droxyl radicals [370]. In vitro resistance to the
drug appears to be mediated either by defective
accumulation [371] or by increased intracellular
The fermentation product consists of approxi-
drug degradation by a specific bleomycin hydro-
mately 12 different components clinically mar-
lase [372–374].
keted as bleomycin. Each is a peptide with
The clinical toxicity of bleomycin is unusual.
a low relative molecular mass (ca. 1500), all
The drug has little hematopoietic toxicity; its
containing bleomycinic acid but differing in
major dose-limiting toxicities are to the lungs
terminal alkylamine groups. The clinical prod-
and skin. Acute and chronic pneumonitis fol-
uct is approximately 60 – 70 % bleomycin A2
lowed by progressive pulmonary fibrosis ap-
(NSC-146842), N 1 -[3-(dimethylsulfonio)pro-
pears to be dose dependent, with risk increas-
pyl]bleomycinamide, C55 H84 N17 O21 S3 , M r
ing significantly in patients receiving a cumula-
1416; and 20 – 30 % bleomycin B2 . Other
tive dose of more than 450 mg. This toxic end-
analogs comprise about 5 % of the total. The
point may be due to the terminal amines of the
drug is highly active against germ cell neoplasm
parent compound. This hypothesis is supported
of the testis.
by preclinical studies [375] as well as by early
clinical studies of peplomycin. The maximum-
tolerated cumulative dose of this terminal ami-
6.2.1. Analogs
no acid-substituted bleomycin analog has yet to
be determined, although during phase I analy-
Peplomycin [68247-85-8], N 1 -{3-[(1-
sis, pulmonary toxicity was not observed until
phenylethyl)amino]propyl}bleomycinamide,
weekly doses exceeded 10 mg/m2 [376].
C61 H88 N18 O21 S2 , M r 1473.62, a biosynthetic
bleomycin analog, demonstrates significantly
reduced pulmonary toxicity in rodents [364]. 6.3. DNA Interactive Natural Products
The toxicology studies are supported by clinical
trials in Japan (initiated in 1981) and France Analogs related to the natural product CC-1065
(1983), and suggest that peplomycin has a like adozelesin, bizelesin, and carzelesin bind
greater therapeutic index than the parent com- in the minor grove of the DNA and form a co-
pound and may replace bleomycin in the clinic valent adduct with adenine [377]. Adozelesin
Adozelesin
CC-1065
Bizelesin
Carzelesin
Pyrindamycin A
Duocarmycin C1
[110314-48-2] [378] is the clinically farthest A series of new DNA cleaving molecules
advanced agent, while the prodrug carzelesin based on the reactive enediyne moiety
[119813-10-4] [379] and the dimer bizelesin are the anticancer antibiotics calicheam-
[129655-21-6] [380] demonstrated impressive icin [113440-58-7] [385], esperamicin
preclinical activity. [114797-28-3] [386], and dynemicin.
Further modified cyclopropylpyrroloindoles Simplified enediyne-type compounds dam-
(CPI) forming covalent adducts with DNA are age DNA upon activation by chemical or biolog-
duocarmycin A [118292-35-6] [381], [382], ical means and are extremely potent cytotoxic
pyrindamycin [118292-36-7] and FCE 24517 agents in vitro [387], [388].
[383], [384].
Esparamicin A1
7.1. Vinca Alkaloids (→ Alkaloids,

Chap. 11.3.4.)
Vinca alkaloids are dimeric indole derivatives

isolated from the periwinkle plant, Catharantus
roseus. Of the whole family of more than 70
naturally occurring alkaloids, only a few have
cytotoxic activity.
Dynemicin A 7.1.1. Vincristine and Vinblastine

The molecular structures of the dimeric Catha-
rantus roseus alkaloids vincristine (NSC-
67574) [57-22-7], 22-oxovincaleukoblastine,
C46 H56 N4 O10 , M r 824.94, Oncovin (sulfate);
7. Antitubulin Agents and vinblastine (NSC-49842) [865-21-4], vinca-
leukoblastine, C46 H58 N4 O9 , M r 811.00, Vel-
Tubulin-containing structures are important for ban (sulfate hydrate), are very similar. Both
diverse cellular functions, including chromo- are formed of multiringed units, vindoline and
some segregation during cell division, intracel- catharantine, linked by a carbon – carbon bridge.
lular transport, development and maintenance They differ only in the nature of the substituent
of cell shape, cell motility, and possibly dis- on the vindoline nitrogen atom.
tribution of molecules on cell membranes. The
drugs that interact with tubulin are heteroge-
neous in structure. A common characteristic of
these agents is binding to tubulin, causing its
precipitation and sequestration to interrupt many
important biologic functions that depend on the
microtubular class of subcellular organelles. The
tubulin – drug aggregates can be visualized by
the indirect immunofluorescence technique as
brightly stained cytoplasmic paracrystals. Of the
tubulin binders, those that are important in can-
cer medicine include vinca alkaloids, podophyl- R1 = CHO, R2 = CO(OCH3 ), R3 = CO(CH3 ), Vinristine
lotoxins, and taxoids. R1 = CH3 , R2 = CO(OCH3 ), R3 = CO(CH3 ), Vinblastine
R1 = CH3 , R2 = CO(NH2 ), R3 = OH, Vindesine
Cellular Pharmacology. As yet it is not nism of this pleiotropic drug resistance remains
clear how vinca alkaloids cross cell membranes. to be clarified. However, tubulin is not respon-
Some data suggest an energy-dependent trans- sible for that phenomenon because there is very
port system [389], while other data suggest sim- little difference in affinity binding of colchicine
ple diffusion across membranes [390]. However, to tubulin isolated from drug-sensitive and drug-
passive diffusion is important only at drug con- resistant cells [397]. The appearance of a novel
centrations exceeding 100 µM. Transport of vin- glycoprotein on the membrane of resistant cells
cristine is completely inhibited by vinblastine, and the accelerated drug efflux – leading to im-
suggesting a common carrier. paired drug accumulation – seem, at present,
Vincristine and vinblastine exert their bio- to be the possible mechanisms responsible for
logic effect through binding to tubulin. This oc- resistance [398].
curs in interphase (late S and G2 ), producing
a transient G2 block [391]. They do not affect
DNA synthesis directly. The metabolic conse- 7.1.2. Vindesine
quences of tubulin binding include polyploidy,
nuclear fragmentation, and inhibition of cytoki- Vindesine (NSC-245467) [53643-48-4], 3-
nesis, which occurs after prolonged drug expo- (aminocarbonyl)-O4 -deacetyl-3-de(methoxy-
sure. Present studies suggest that sensitivity to carbonyl)vincaleukoblastine, deacetylvin-
these drugs increases progressively as cells ap- blastineamide, C43 H55 N5 O7 , M r 753.95 (struc-
proach mitosis and that cells at the end of the ture, Section 7.1.1), is a vinblastine metabolite.
cycle at the time of exposure are likely to ex- It possesses antitumor activity that is similar
hibit the greatest degree of mitotic disorganiza- to vincristine’s rather than that of its parent
tion [391]. compound, vinblastine. Vindesine demonstrates
Vincristine and vinblastine share a common better activity than vinblastine in some tumor
binding site on each tubulin monomer, with the models (Gardner lymphosarcoma, Ridgway os-
binding affinity (K d ) of about 1.6 × 10−6 M. teogenic sarcoma, and mammary carcinoma).
The drug concentrations necessary to produce In the past several years vindesine has attracted
50 % cell kill in vitro are ca. 4 × 10−8 M [392]. considerable attention among clinical investi-
Malignant lymphocytes appear more susceptible gators and has been intensively investigated.
than normal lymphocytes, presumably because Response rates vary, with the highest responses
of high tubulin content expressed on the surfaces observed in the highest lymphatic malignancies
of leukemic cells [393]. Other metabolic effects [399]. The drug is less neurotoxic than vin-
include inhibition of DNA and RNA and pro- cristine and appears to be active in vincristine-
tein synthesis. However, these effects are exerted resistant tumors [400], [401].
only at very high drug concentrations (1000
times greater than those achieved in vivo) and
probably are secondary phenomena. 7.1.3. Vinorelbine
Drug Interactions. Both vincristine and Vinorelbine (KW-2307, NVB) [071486-22-1]

vinblastine potentiate the effect of methotrex- 3’,4’-didehydro-4’-deoxy-C’-norvincaleuko-
ate through their blockade of methotrexate exit blastine, C45 H54 N4 O8 , M r 778.94, is a semisyn-
from cells [394], [395]. Some amino acids (glu- thetic vinca alkaloid. Starting from anhydrov-
tamine, aspartic acid, ornithine, citrulline, and inblastine the compound is obtained as 5’-
arginine) completely reverse the cytotoxic effect noranhydrovinblastine in three steps [402],
of vinblastine in tissue culture [396]. [403]. The product was launched for the treat-
ment of non-small cell lung cancer.
Mechanisms of Resistance. Vinca resis- Trade names: Navelbine (Pierre-
tance may arise through mutations in tubu- Fabre/Glaxo), Ennades (Farmitalia).
lin, leading to decreased drug binding. Vinca-
resistant cells may also share cross-resistance Mechanism of Action. Vinorelbine was se-
with antitumor antibiotics through a separate lected for drug development due to its high affin-
mechanism of resistance. The precise mecha- ity for tubulin and its ability to prevent tubulin
polymerization [403–405]. The compound in-

duced total depolymerization of microtubules
in P 388 murine leukemia cells, possibly via
stimulation of microtubular protein synthesis
[403]. Vinorelbine and vincristine were equally
active against L 1210 leukemia in mice, while
vinblastine had no significant effect. Vinorel-
bine exerted significant antitumor activity in Teniposide (VM-26)
the vincristine-resistant cell line P 377/VCR and

low cross-resistance to other vinca alkaloids was Despite structural similarities between the
observed [406], [407]. semisynthetic derivatives and parent natural
In clinical studies leukopenia was dose- product, neither VP-16 nor VM-26 binds tubu-
limiting, no thrombocytopenia was observed lin. Evidence from a number of laboratories
[408]. Efficacy in non-small cell lung cancer was suggests that VP-16 exerts cytotoxic effects by
demonstrated [409–411]. causing DNA breakage [414], [415]. Selective
double-strand DNA breaks, which are particu-
larly lethal, appear to be caused by these agents
[416].
The precise mechanism for VP-16 and VM-
26 DNA damage is unknown, but the fact that
the presence of a 4 -hydroxyl group is critical for
cytotoxicity suggests formation of free-radical
intermediates. Indeed, free-radical scavengers
appear to be able to protect from podophyllin
derivative cytotoxic effects in vitro [417]. For-
Vinorelbine
mation of DNA breaks may also be due to spe-
cific inhibition of the DNA repair enzyme topoi-
somerase II by both VP-16 and VM-26 [418]. It
7.2. Podophyllotoxin and Its Derivatives is likely that further characterization of topoi-
somerase II and the effects of its inhibition will
Podophyllin is a complex mixture of crystalline result in the development of new leads in antitu-
compounds derived from the mayapple plant. mor drug development.
The active agent derived from this plant prod-
uct, podophyllotoxin [518-28-5], shares a com-
mon binding to tubulin with colchicine, and 7.3. Camptothecin and Analogs
the morphological effects of podophyllotoxin
and colchicine exposure are indistinguishable Camptothecin (→ Alkaloids, Chap. 11.3.5.)
[412]. The semisynthetic glycoside derivatives (CPT) [7689-03-4], an alkaloid isolated from the
of podophyllotoxin, VP-16-213 (NSC-141540) Chinese plant Camptotheca accuminata in 1966
[33419-42-0], etoposide, and VM-26 (NSC- [419] was proved to have antineoplastic activ-
122819) [29767-20-2], teniposide, have repro- ity in various tumor systems [420]. The clinical
ducible clinical activity against testicular cancer, use of CPT was, however, limited because of its
small-cell lung cancer, and lymphomas [413]. high toxicity and low solubility in water. New
derivatives have been synthesized carrying sub-
stitutents in 7,9,10- or 11-position of the ring
A.
R = CH3 Etoposide (VB-16-213)
Mechanism of Action. Topoisomerase

(topo I) is an ubiquitous nuclear enzyme in-
volved in the regulation of essential cellular
functions by relieving torsional DNA strain. Re-
laxation of supercoiled DNA is achieved through
a series of topo I-mediated reactions. The main
physiological role of topo-I is in sensing and
Campothecin subsequently releasing the positive supercoil-
ing generated ahead of the moving transcription
Irinotecan (NSC-616348, CPT-11, DQ- apparatus. A number of malignancies including
2805) [097682-44-5] [421], (+)-7-ethyl-10-[4- acute leukemia, blasts, colon, esophageal, and
(1-piperidyl)-1-piperidyl]carbonyloxycampto- ovarian cancers contain increased topo-I lev-
thecin, C33 H38 N4 O6 , M r 586.69 and topotecan els as compared to normal tissues [424], [425].
(NSC-609699, SKF-S-104864-A, E-89/001) Topotecan binding to topo-I-DNA adducts re-
[119413-54-4] [422], (S)-10-dimethylamino- sults in markedly decreased rates of nick reseal-
methyl-9-hydroxycamptothecin, C23 H23 N3 O5 , ing and in delayed enzyme release, leading to
M r 457.91 are camptothecin derivatives with increased numbers of strand breaks [426].
topoisomerase I-inhibitory activity. As anti- In animal tumor models including xenografts
neoplastic alkaloids, they can be synthesized of human cancer lines topotecan and irinotecan
semisynthetically starting from CPT [423]. In exhibit a wide spectrum of antineoplastic ac-
addition both agents have intercalating prop- tivity [427–429]. Cell lines overexpressing P-
erties and are water soluble. Irinotecan hydro- glycoprotein display low levels of resistance
chloride was launched for the treatment of small to topotecan through decreased drug retention,
cell and non-small cell lung cancer and cancers more pronounced resistance is found in cell lines
of the uterine, cervix, and ovaries. containing low levels of topo I [430], [431].
Trade names: Campto (Rhône-Poulenc Dose-limiting toxicity in patients were stom-
Rorer/Yakult Honsha) Topotecin (Daiichi atitis and esophagitis as well as neutropenia
Seiyaku). [432].
Unlike irinotecan, topotecan is not a prodrug
and does not require metabolic activation.
7.4. Taxoids
Paclitaxel (NSC-125973, BMS-181339)
[33069-62-4], C47 H51 NO14 , M r 853.9, is a
natural antineoplastic taxane derivative origi-
nally isolated from the plant Taxus brevifolia
[433] and subsequently synthesized due to lim-
ited supply from the bark of endangered yew
trees. Starting from 10-deacetyl baccatin III (iso-
lated from the renewable twigs and needles of
Irinotecan
Taxus baccata) the compound can be prepared
in three steps utilizing protected N-benzoyl-
(2R,3S)-3-phenylisoserine as key building block
[434–436]. Various synthetic approaches are
described and patented, including three total
synthesis of paclitaxel [437–440].
Paclitaxel has a novel mechanism of action.
Unlike vincristine or vinblastine, which bind
tubulin to inhibit tubulin polymerization, pacli-
taxel stabilizes microtubular structures [441].
Indicated for the treatment of primary ovarian
Topotecan cancer in combination with cisplatin and for
metastatic ovarian cancer where standard ther- Mechanism of Action. More than two
apy has failed, paclitaxel has been marketed decades after its isolation and the elucidation
since 1993 as Taxol (Bristol-Myers Squibb) of its complex structure and cytotoxic activity
and was the best-selling anticancer agent in [433] interest in paclitaxel raised again when
1996. Taxol does not share a common binding S. Horwitz et al. [446] reported on the novel
site with other antitubulin agents and may in- mechanism of action. As spindle poison pacli-
stead bind to and stabilize polymerized tubulin taxel promotes the polymerization of tubulin
[442]. to microtubules and stabilizes them against de-
A semisynthetic analog of pacli- polymerization, whereas vinca alkaloids induce
taxel, docetaxel (NSC-628503, RP- microtubule disassembly (see Fig. 2). Thus, with
56976) [114977-28-5], (2R,3S)-N-carboxy- paclitaxel the dynamic equilibrium of assem-
3-phenylisoserine, N-tert-butyl ester, 13- bly and disassembly of microtubules is shifted
ester with 5β, 20-epoxy-1,2α-4,7β,10β,13α- in favor of the polymer, preventing cell divi-
hexahydroxytax-11-en-9-one 4-acetate 2- sion [447]. Paclitaxel binds preferentially to the
benzoate, C43 H53 NO14 , M r 807.9, is a β-tubulin subunit. This binding is reversible
derivative with an N-tert-butyloxycarbonyl- and the site is different from the binding sites
(2R,3S)phenylisoserine C-13 side chain. Do- of vinca alkaloids, colchicine and podophyllo-
cetaxel is more potent than paclitaxel in vitro toxin [448–450]. If paclitaxel is present, tubu-
[443], [444]. Impressive clinical results have lin polymerizes without exogenous GTP and
been reported for the treatment of ovarian, these stabilized, rigid microtubules cannot be
breast, and bronchial cancers with docetaxel disassembled. As a result, the dynamic orga-
[445]. The compound was launched in 1996 for nization of the cell is interrupted which leads
the treatment of locally advanced breast can- to irreversible damage in rapidly dividing cells.
cer or relapse during anthracycline therapy of Further evidence has been reported that the an-
NSCLC and breast cancer. tiproliferative activity of paclitaxel is caused by
Trade name: Taxotere (Rhône-Poulenc additional effects [451–453].
Rorer). A major impediment in the development of
taxol as a drug was its poor water solubil-
ity [454]. With the solubility enhancer Cre-
mophor EL hypersensitivity reactions, including
hypotension, urticaria, and dyspnea, occurred in
patients during rapid infusion. To cope these al-
lergic reactions, 24-hour infusions and pretreat-
ment with dexamethasone, diphenhydramine, or
cimetidine was recommended [455].
The dose-limiting toxicity of paclitaxel is
neutropenia. Several other toxic effects such as
diarrhea, nausea, and emesis are less common.
Paclitaxel
Docetaxel shares many toxic effects with pacli-
taxel such as dose-limiting neutropenia, alope-
nia, myalgias, and mucositis. In addition fluid
retention and cutanous toxicities are observed
[445], [460], [461]. Because of the low water sol-
ubility of paclitaxel and docetaxel, the synthe-
sis of more soluble taxoid prodrugs and smaller
analogs has become an interesting area of re-
search (see Section 7.5).
Structure – Activity Relationship. A large

Docetaxel number of taxoid analogs has been synthesized
with emphasis to enhance biological activity
and to improve the water solubility [436]. C-13
Figure 2. Mechanism of action of paclitaxel and vinca alkaloids
side chain depleted analogs such as baccatin III Their unique capability to inhibit taxol resistant
and its derivatives [456] as well as N-benzoyl- tumor cell lines [470] and their good solubility
(2R,3S)-phenylisoserine are inactive. Simplified in water are the biggest advantages as compared
side chains at C-13 (like acetic, crotonic, or to paclitaxel. Their in vivo activity is similar to
phenylacetic acid) possess reduced activity. that of paclitaxel [469].
3’-Cyclohexyl-3’-dephenylpaclitaxel has a
similar cytotoxicity to paclitaxel. Further mod-
ifications in the aromatic 3’-phenyl group gave
compounds that were equipotent with paclitaxel.
The compound with a 3’-(p-methoxyphenyl) has
a slightly increased activity. Compounds with
different substituents in the N-benzoyl part were
similar to paclitaxel if these substituents were R = H, Epothilone A
aromatic, aliphatic substituents reduced cyto- R = CH3 , Epothilone B
toxicity.
As the 2’-hydroxyl group is essential for max-
imal biological activity [457] esterification leads
to a total loss of activity in the microtubule assay, 8. Heavy-Metal Complexes
whereas cytotoxicity remains unchanged. Thus
various amino acid esters were produced as pro- 8.1. cis-Platinum
drugs.
Notable loss of cytotoxicity was observed cis-Platinum (NSC-119875) [15663-27-1],
with A-ring modified analogs and oxidation at cis-diamminedichloroplatinum, cisplatin,
C-10 or C-7. 7-Acetylpaclitaxel and the C-7 PtCl2 (NH3 )2 , M r 300.05, was the first heavy-
epimer were similar in their ability to inhibit cell metal compound to be introduced into clinical
proliferation. All derivatives without the intact cancer chemotherapy. The discovery of this
oxetane moiety are inactive [458]. Further, all unique agent was predicated on the serendipi-
C-4 modified analogs were devoid of activity tous observation that bacterial growth was in-
underscoring the vital importance of these func- hibited when culture medium was subjected to
tional groups [459]. an alternating current using platinum electrodes
[471]. Moreover, the spent medium itself devel-
oped bacteriocidal characteristics, even in the
7.5. Epothilone A and B absence of electrical current. Detailed analysis
confirmed that, of the several platinum species
The high cytotoxicity and good stabiliza- produced by electrolysis, it was the cis isomer of
tion of microtubule raised interest in the PtCl2 (NH3 )2 that had antibacterial activity. In
natural products epothilone A [152044-53-6] 1969, Rosenberg and co-workers reported that
and B [152044-54-7] originally isolated from cis-platinum also had potent antitumor activ-
myxobacteria Sorangium cellulosum [462]. ity in murine tumors [472], and phase I clinical
Since Bollag et al. [463] reported on the mech- studies with the drug were initiated two years
anism of action, which resembles that of pacli- later [473], [474]. Cis-platinum, in combination
taxel numerous reports were published on total with other agents, has led to highly active and
synthesis [464–468] and biology of epothilones. often curative regimens in patients with testic-
ular, ovarian, and head and neck cancer [475], hemolytic anemia, hypomagnesemic tetany, al-
[476]. lergic reactions, and hepatotoxicity. Nephrotox-
icity was dose limiting in early clinical trials
and appeared to be due to tubular reabsorption
8.1.1. Mechanism of Action of active platinum species causing proximal and
distal tubule necrosis [483–485]. Tubular dam-
As with the alkylating agents, one of the ma- age has been reported to cause defective reab-
jor factors mediating the cytotoxicity of cis- sorption of magnesium, resulting in hypomagne-
platinum is probably the formation of cross-links semia (which may result in tetany) [486], [487],
between opposing strands of DNA (interstrand as well as inappropriate renal loss of calcium,
cross-links), linkage within a single strand of potassium, and phosphorus [488]. The acute re-
DNA (intrastrand cross-links), or the formation nal toxicities of cis-platinum appear to be sec-
of linkages dependent on the hydrolysis of cis- ondary to activation of the renin – angiotensin
platinum in solution. While the covalent stabil- system, resulting in reduced renal blood flow and
ity of Pt−NH3 bonds is high, both chlorides glomerular filtration [484], [489]. Thus, this tox-
are good leaving groups and can be displaced icity can be mitigated by high-volume diuresis
by water or hydroxyl ions to form positively [490]. In 1984 it has been demonstrated that even
charged, aquated platinum species that avidly re- high doses of cis-platinum are well tolerated
act with nucleophilic sites on macromolecules, when administered with high-volume chloresis,
especially the N 7 position of guanine and the which not only dilutes urinary platinum levels
N 3 position of cytosine [477]. Formation of the but also prevents leaving of the chloride groups
active intermediate is inhibited in the presence to form the toxic aquated molecule [491]. When
of Cl− ; in plasma the Cl− concentration is suffi- administered by this regimen, the limiting side
cient to inhibit aquation of the drug, which has an effects of cis-platinum become neurotoxicity
in vitro plasma half-life of several hours [478]. and myelosuppression, while renal function is
However, this process occurs rapidly in the in- remarkably spared.
tracellular milieu, where Cl− concentrations are
low.
The cis configuration is central to cytotox- 8.1.2. Mechanisms of Resistance
icity; the trans isomer is devoid of antitumor
effects. This fact suggests that of the reactions Although bacterial resistance to cis-platinum ap-
caused by the aquated platinum species, forma- pears to be due to increased efficiency of DNA
tion of intrastrand cross-links is most important repair [492], the mechanisms of tumor cell re-
(intrastrand cross-links cannot be formed by the sistance are less clear. Alkaline elution stud-
inactive trans compound). ies have demonstrated a direct relation between
As a result of nucleophilic attack on macro- DNA cross-linking and tumor cell resistance
molecules, cis-platinum causes changes in the [493–495], and a cis-platinum-resistant murine
structural conformation of DNA [479] as well as leukemia line has been described in which cross-
intra- and interstrand cross-links. These changes links are formed at a reduced rate [496]. How-
inhibit RNA transcription from the DNA tem- ever, it remains uncertain whether this resis-
plate [480] and, probably more important, di- tance is secondary to accumulation, impaired
rectly inhibit DNA synthesis itself [481]. In ad- activation, or altered DNA repair processes. As
dition, cis-platinum can react with tumor cell with the alkylating agents in general, high lev-
membrane to cause presentation of new anti- els of metallothionein have been reported in cis-
genic determinants [482]. Whether this mech- platinum-resistant cells [497]. This sulfhydryl-
anism is related to tumor cell recognition and rich protein is known to protect from metal tox-
immune response remains to be determined. icity by specifically binding to platinum, cad-
As a heavy-metal-based compound, cis- mium, and other heavy metals [498].
platinum has unique clinical toxicities, including Because the cellular mechanisms of resis-
renal tubular damage, severe nausea and vomit- tance to cis-platinum are so poorly character-
ing, high-tone hearing loss, and peripheral neu- ized, new platinum analogs in clinical trial have
ropathy. Less common are myelosuppression,
been selected in an attempt to reduce host toxic- 418 [514], nedaplatin (NSC-375101D, 254-
ity with the aim of improving therapeutic index S), [095734-82-0], cis-diamine(glycolato-
[499]. O1 ,O2 )platinum, C2 H8 N2 O3 Pt, M r 303.19
[515], and oxaliplatin (OHP) [61825-94-3],
[Sp-4-2-(1R-trans)](1,2-cyclohexanediamine-
8.2. Carboplatin N,N’)[ethanedioxato(2− )-O,O’]-platinum,
C8 H14 N2 O4 Pt [516] were clinically investi-
Carboplatin (NSC-241240, CBDCA) gated in depth. Nedaplatin entered the market
[839805-03-3], cis-diammine[1,1-cyclo- in 1997 for the treatment of head and neck,
butanedicarboxylato-(2)-O,O’]platinum(II), small-cell lung, non-small cell lung, esophageal,
C6 H12 N2 O4 Pt, M r 371.3, is a second genera- bladder, testicular, ovarian, and uterine cervical
tion cisplatin analog without significant nephro- cancers [517–520].
toxicity or neurotoxicity and with less emetic Trade name: Agupla (Shionogi).
potential than the parent compound [500–503]. Further studies of additional “third-
Clinical trials show activity against several tu- generation” compounds are centering
mor types. Carboplatin is especially effective on the elimination of toxicity, en-
in treatment of ovarian and small-cell lung can- hanced therapeutic activity, non-cross-
cer [504], [505]. The minimal emetogenic dose resistance and selective drug delivery
of cis-platinum in dogs is 9 mg/m2 , whereas [521]. Ormaplatin (NSC-363812, U-77233)
for carboplatin the dose is 624 mg/m2 . In addi- [62816-98-2] [±(trans)]-tetrachloro(1,2-cyclo-
tion, this compound retains antitumor activity in hexanediamine-N,N’]platinum, C6 H14 Cl4 N2 Pt
cis-platinum-restistant murine leukemia [506], and Lobaplatin (D-19466) [135558-11-1] are
demonstrating consistent antitumor activity in representatives of a series of platinum com-
patients with ovarian cancer in the absence of plexes in clinical development [522–524].
either ototoxicity, nephrotoxicity, or neuropathy
[507], [508]. Carboplatin entered the market in
1989 and is the leading platinum complex can-
cer drug with sales of $ 373 × 106 worldwide
in 1996. Carboplatin is indicated for the treat- Nedaplatin Lobaplatin
ment of ovarian cancer and sales have benefited
from the drug’s use in combination with Taxol
[509–512].
Trade names: Paraplatin (Bristol-Myers
Squibb), Carboplat (BMS).
Iproplatin
Carboplatin Oxaliplatin Ormaplatin
Orally active cisplatin-analogs with higher

therapeutic ratio and specifically high anticancer
8.3. Analogs activity are the challenge in the search for new
“fourth-generation” derivatives. Platinum(IV)-
The synthesis and development of “second- complexes like JM 216, drug targeting ap-
generation” platinum compounds such as proaches, e.g., the use of ligands with hormone
carboplatin has modified the problem of receptor-binding affinity [525] or intercalating
nephrotoxicity. Iproplatin (NSC-256927, structures [526], and special formulation tech-
CHIP) [34348-60-2], dichlorodihydroxy-bis- niques are the major focus.
(2-propanamine)platinum C6 H20 Cl2 N2 Pt, M r
9. Hormonally Active Anticancer However, primary and secondary resistance

Drugs/Antihormones to tamoxifen treatment requires the introduc-
tion of new analogs with improved therapeu-
Hormones and in particular, the sex hormones tic activity for hormon-dependent neoplasia.
were the first growth factors discovered to be Toremifene (NK-622, Fc-1157a) [089778-26-7]
involuntary helpers of cancer. Female breast [530], [531], 2-[p-[(Z)-4-chloro-1,2-diphenyl-
cancer and male prostate cancer are the best 1-butenyl]phenoxy]-N,N-dimethylethylamine,
known examples of tumors acknowledged to C26 H28 ClNO, M r 405.97, raloxifene [532], and
be hormone-dependent. Shutting down the main droloxifene [533] were investigated clinically.
production site of the sex hormones estrogen Toremifene is a novel triphenylethylene
and testosterone either by removing the ovaries derivative structurally related to tamoxifen
or by castration is a well-known and often ef- [527–529]. Almost all compounds related to
fective therapy; however, these procedures can tamoxifen contain an alkylaminoethoxy side
be problematic due to the concomitant psycho- chain, which seems to be essential for their bind-
logical stress. Modern hormone therapy for ad- ing to the estrogen-receptor (ER) and antiestro-
vanced breast cancer and prostate cancer at- genic activity [536].
tempts to spare the patient such irreversible op- Trade names: Toremifene has been launched
erative procedures for as long as possible by as Fareston (Farmos, ASTA Medica) and Es-
using hormone antagonists. Examples are the trimex (Adria) for treatment of postmenopausal
antiestrogens or LHRH antagonists, which hin- breast cancer.
der deployment of the hormone itself and thus
its growth-promoting activity.
9.1. Antiestrogens (→ Hormones,

Chap. 3.5.4.)
9.1.1. Antagonists Tamoxifen
Estrogens can induce hormone-dependent hu-

man breast carcinoma and stimulate tumor
growth. Reduced estrogen production is corre-
lated with a lower risk of breast cancer and in
particular with tumor regression. Hormonally
active drugs are considered to be the treatment
of first choice for advanced breast cancer, unless
metastatic complications require immediate ag- Toremifene
gressive chemotherapy.
Mechanism of Action. Tamoxifen and

9.1.2. Tamoxifen, Toremifene toremifene bind to estrogen receptors in the cy-
tosol, are translocated to the nucleus, and block
The antiestrogen tamoxifen (ICI 46474) estrogen-induced cell proliferation. However,
[10540-29-1], (Z)-2-[p-(1,2-diphenyl-1- specific differences in the drug profiles exist,
butenyl)phenoxy]-N,N-dimethylethylamine, indicating improved therapeutic properties of
C26 H29 NO, has become the standard first- toremifene compared with tamoxifen. These
line agent in postmenopausal patients [527– differences include lower intrinsic estrogenic
529]. The product is indicated for the treat- activity, longer nuclear retention, no retinal
ment of breast cancer with worldwide sales of damage or neoplastic liver changes. Toremifene
$ 561 × 106 in 1996. is more active against dimethylbenz[a]anthra-
Trade name: Nolvadex (Zeneca). cene (DMBA)-induced rat mammary cancer
and unlike tamoxifen, it inhibited the growth

of an ER-negative transplantable mouse uterus
sarcoma, although the antitumor effect of this
compound was preferentially directed against
estrogen-dependent tumors of the mammary
gland and endometrium [534], [535]. In clinical
trials no concrete side effects or pathological
clinical chemistry values were observed in most
patients [537], [538]. Some patients complained
of light hot waves, sweating, nausea, and tran-
Raloxifene
sient vertigo.
Cytotoxicity in vitro, dose related activ-
ity in ER-positive (i.e., cancer cells contain-
ing estrogen receptors) and ER-negative tumor
models suggest that additional mechanism like
growth-factor production may be triggered by
toremifene [539].
Panomifene
9.1.3. Analogs
Raloxifene (LY-139481) [82640-04-8] is a

benzo(b)thien-3yl-antiestrogen formerly under
development for treatment of breast can-
cer. It mimics the effects of estrogen on
the skeleton and is therefore effective in
the prevention of postmenopausal osteoporo-
sis [540]. Faslodex (ICI-182780, ZD-182780) Miproxifene phosphate
[129453-61-8] is a pure, steroidal estrogen an-
tagonist with oral anticancer activity [541],
[542]. The in vivo antitumor activity of faslodex
in xenografts of MCF-7 and Br 10 human
breast cancers in mice was equivalent to that
of tamoxifen. Miproxifene phosphate (Tat-
59) [115767-74-3] [543], panomifene (Gyki-
13504) [77599-17-8], idoxifene [544] (SB-
223030, CB-7432) [116057-75-1] and drolox-
ifene [83647-29-4] are more potent than tamox- Droloxifene
ifen against estrogen-dependent tumors in mice.
Faslodex
Idoxifene
9.2. Aromatase Inhibitors (→ Hormones,

Chap. 3.5.4.)
All endocrine therapies inhibit endogenous es-

trogen production or the interaction between es-
trogens and cellular estrogen receptors. Both ab-
lative and additive hormone therapies are equal
in efficiency. Exemestane
Aromatase inhibitors block cellular estrogen
synthesis and thus induce, particularly during
postmenopause, a marked decrease in estrogen
production. The inhibition of estrogen synthesis
is caused by a suppression of the enzyme aro-
matase which converts androstenedione to es-
trone [545].
Aminoglutethimide [125-84-8], (±)-2-(4-
aminophenyl)-2-ethylglutarimide, an unspecific NSK-01
aromatase inhibitor which also suppresses
In addition to steroidal also nonsteroidal, in
adrenal desmolase and 11-β-hydrolase, was the
particular, imidazole/triazole derivatives were
first aromatase inhibitor in the clinic and on the
investigated as aromatase inhibitors. Anas-
market.
trazole (ICI-D1033, ZD-1033) [120511-73-1]
Trade name: Orimeten (Ciba-Geigy).
[554], 2,2’-[5-(1H-1,2,4-triazol-1-ylmethyl)-
Aminoglutethimide induces a decrease in
1,3-phenylene]-bis(2-methylpropionitrile),
cortisol, followed by an increase in ACTH [546].
C17 H19 N5 , M r 293.37, fadrozole (CGS-
Therefore new powerful specific aromatase in-
16949A) [102676-96-0] [555], (±)-4-(5,6,7,8-
hibitors without influence on adrenal steroid
tetrahydroimidazo[1,5-a]pyridin-5-yl)benzo-
synthesis were developed.
nitrile, C14 H13 N3 , M r 259.74, letrozole
Formestane (4-OHA; CGP-32349)
(CGS-20267) [112809-51-5] [556], 4,4’-
[000566-48-3], 4-hydroxyandrost-4-ene-3,17-
(1H-1,2,4-triazol-1-ylmethylene)bis[benzoni-
dione, C19 H26 O3 , M r 302.41, is an androstane
trile], C17 H11 N5 , and vorozole (R83842),
derivative with highly specific aromatase in-
[129731-10-8] [557], (+)-6-[(4-chlorophenyl)-
hibition [547]. The substance is used for the
1H-1,2,4-triazol-1-ylmethyl]-1-methyl-1H-
treatment of advanced breast cancer in post-
benzotriazole, C16 H13 ClN6 , are highly selec-
menopausal women [548–553].
tive nonsteroidal aromatase inhibitors without
Trade name: Lentaron (Ciba-Geigy).
intrinsic androgenic or estrogenic properties.
Atamestane, exemestane, and NKS-01 are
Trade names: Anastrazole and Fadrazole are
orally active steroids.
marketed as Arimedex (Zeneca) and Arensin
(Ciba-Geigy), respectively, for treatment of
postmenopausal breast cancer.
Another aminoglutethimide analog in de-
velopment is rogletimide [558] (pyridog-
lutethimide) [121840-95-7], (±)-3-ethyl-3(4-
pyridinyl)-2,6-piperidinedione, C12 H14 N2 O2 .
Formestane
Fadrazole Letrozole
Atamestane
the commonly used initial treatment for prostate

cancer [568], [569].
The major circulating androgen in man is
testosterone, 90 % of which is produced in the
testis. In addition, a small amount of androgen is
produced by the adrenal gland under the control
Vorozole of ACTH. (see Fig. 3).
Four ways for androgen deprivation exist:
1) Removal of organs by surgery
2) Interference with control mechanism
3) Inhibition of biosynthesis
4) Competitive inhibition of androgens at the
Rogletimide Anastrazole receptor site
The group 2 approach with, e.g., LHRH
agonists and antagonists will be discussed in
Section 9.4. Examples of group 3 include com-
pounds (e.g., ketoconazole), which inhibit the
synthesis of adrenal androgens [570] as well as
YM-511 Aminoglutethimide inhibitors of the enzymes 5α-reductase [571]
and aromatase [572]. “True” antihormones are
compounds of group 4, based on the definition
Mechanism of Action. Fadrazole [559] and that an antiandrogen is a substance which binds
anastrazole [560–567] for example, were found to the target tissue androgen receptor and pre-
to be potent and specific aromatase inhibitors vents the stimulatory effects of androgens. Al-
with neither androgenic nor estrogenic activity. though the exact mechanism of action of an-
Fadrazole was 180 times more potent than ami- tiandrogens is not totally understood, an impor-
noglutethimide as an aromatase inhibitor. Anas- tant feature is the competitive inhibition of the
trazole inhibited human placental aromatase binding to the cytosol receptor. The first an-
with an IC50 = 15 nM (IC50 = inhibitory concen- tiandrogen to be used clinically was the syn-
tration). In animal studies fadrazole was found thetic steroidal antiandrogen cyproterone ac-
to lower serum estrogen, raise luteinizing hor- etate [427-51-0] [573]. However, cyproterone
mone (LH) level, and reduce uterine weight as acetate also exhibits other steroidal activities, it
a result of aromatase inhibition. In addition, ad- is, e.g., a potent progestin, exhibits weak anti-
ministration of 2 mg per kilogram body weight gonadotrophic activity and has glucocorticoid
caused regression of DMBA-induced mammary like properties. Beyond the success in treat-
tumors in female rats. In humans half-life of, ing prostate cancer the steroidal properties are
e.g., anastrazole was more than 30 h. No seri- largely responsible for the fluid retention and
ous side effects were reported and no significant thrombosis seen in patients [574]. Therefore,
effects on cortisol or aldosterone secretion was nonsteroidal antiandrogens are expected to have
observed. advantages by avoiding steroid-related side ef-
fects [575].
9.3. Antiandrogens
9.3.1. Flutamide
Prostate cancer is primarily a disease of the el-
derly and the second most common cancer in Flutamide (Sch-13521, NK-601, FTA)
men in the United States. Ever since Huggins [013311-84-7], 2-methyl-N-[4-nitro-3(trifluor-
and Hodges demonstrated the partial androgen methyl)phenyl]-propanamide, C11 H11 F3 N2 O3
dependence of most prostatic tumors more than was discovered in the early 1970s. Unlike the
50 years ago, androgen deprivation has become steroids it can easily be synthesized [576], [577]
and is devoid hormonal activities. Total sales of Trade name: Anandron (Roussel-Uclaf).
the product were $ 271 × 106 in 1996.
Trade names: Eulexin and Drogenil Mechanism of Action. Nilutamide compet-
(Schering-Plough). itively inhibits binding of androgens to the cy-
tosolic androgen receptor. Administration over
Mechanisms of Action. In a comparative 7 days to immature, castrated male rats, nilu-
study in castrated rats flutamide was shown to tamide inhibited the increase of prostate weights
be equipotent to cyproterone acetate as an an- induced by testosterone in a dose-dependent
tiandrogen [578]. Several groups had observed manner [588]. In rat pituitary cells nilutamide
that flutamide was a more potent antiandrogen in reverses the inhibition of LHRH-induced LH re-
vivo than in vitro and suggested the involvement lease elicited by dihydrotestosterone. It is prob-
of an active metabolite [579], [580]. The major ably because of these effects that nilutamide
metabolite was identified as 2-hydroxyflutamide is recommended for use only in surgically or
analog (Sch-16423) [52806-53-8] and high lev- medically castrated males [589]. Nilutamide has
els of 2-hydroxyflutamide in the plasma led demonstrated an antiandrogenic action in sev-
to the conclusion that this was the active eral animal tumor models [590].
form of flutamide [581]. Although the plasma Single dose kinetics of nilutamide in volun-
levels of testosterone increased on flutamide teers (100 mg) indicate a half-life of 43 ± 3 h,
treatment, the levels of testosterone and di- compared to 5.2 h for flutamide (200 mg) [591].
hydrotestosterone in androgen target tissue were Clinical trials with nilutamide have concen-
reduced [582]. Flutamide exerts its antiandro- trated on combination therapy with surgical or
genic action by blocking the binding of andro- medical castration. The side effects observed in
gens to the cytosolic androgen receptor and / or patients include hot flushes, nausea, vomiting,
inhibiting the nuclear binding of androgens in and visual problems [592].
the target tissue [581].
In patients improvements were seen in pain
relief, prostatic enlargement and induration, re-
duction of metastases, and increase in body
weight and phosphatase. Most common side ef-
fects of flutamide therapy are gynecomastia and
breast tenderness [584]. More recent trials use a Nilutamide
combination of flutamide with LHRH agonists
(see Section 9.4). This is the concept of maxi-
mal androgen withdrawal in which the LHRH 9.3.3. Bicalutamide
agonist wipes out androgens of testicular origin
and the antiandrogen blocks the action of andro- Bicalutamide (ICI-176334) [090357-06-5],
gens of adrenal origin at the androgen receptor (±)-4-[3-(4-fluorophenylsulfonyl)-2-hydroxy-
[585–587]. The therapeutic benefits seem to be 2-methylpropionamido]-2-(trifluoromethyl)benzo-
greatest in patients with minimal disease at the nitrile, C18 H14 F4 N2 O4 S, M r 430.37 was dis-
start of treatment. covered at ICI / Zeneca and selected from more
than 1000 compounds as having the desired
properties of a pure, nonsteroidal, peripher-
ally selective antiandrogen [593], [594]. The
product was launched for the treatment of ad-
vanced prostate cancer in combination with
Flutamide LHRH analogs or surgical castration.
Trade name: Casodex (Zeneca).
9.3.2. Nilutamide Mechanism of Action. Bicalutamide in-

hibits the binding of the synthetic androgen
Nilutamide has been discovered bei Roussel- [3 H]-R-1881 to both rat prostate and pitu-
Uclaf. itary cytosol androgen receptors. The substance
binds some 50 times less effectively than di- LHRH agonists (so-called superagonists) are
hydrotestosterone and about 100 times less ef- used in a depot form, which bring about desen-
fectively than R-1881 to the prostate androgen sitization of the pituitary receptors and thus in-
receptor, Its affinity for the prostate receptor terrupt the signal cascade. This results in a bio-
is about four-fold higher, that for the pituitary chemical “castration”, which opens up new ther-
receptor ten times higher than that of hydrox- apeutic possibilities for hormone-dependent dis-
yflutamide [595]. In vivo studies revealed that eases such as prostate cancer, breast cancer, and
bicalutamide is about five times as potent as endometriosis. Although superagonists are gen-
flutamide after oral application. Bicalutamide erally well tolerated, they have the disadvantage
did not cause a significant elevation in LH or that hormone secretion (estrogen, testosterone)
testosterone at any of the doses tested, whereas is initially stimulated before the depletion of re-
flutamide elicited increases. Half-life of bicalu- ceptors or down regulation can take place, and
tamide in prostate cancer patients who received thus the illness temporarily worsens [605]. This
10, 30 or 50 mg/d bicalutamide was around 6 d. has led to the development of the third concept:
The compound was well tolerated in all doses use of LHRH antagonists [606] (see Fig. 3). In
[596–598]. the late 1980s about 5000 LHRH analogs had
Nilutamide and bicalutamide offer advan- been synthesized worldwide and tested in vitro
tages over flutamide because of their long half- or in vivo [607–609]. Whilst LHRH agonists
lifes and sustained serum levels on once-daily have been on the market for about ten years,
dosing. The latter is essential to prevent andro- the LHRH antagonists that have been developed
gen stimulation. farthest are still in clinical testing [610–614].
9.4.1. LHRH Agonists
The first years after the discovery of the go-

Bicalutamide nadorelins were marked by the search for more
active agonists, since the therapeutic potential of
gonadorelins as, for example, antitumor agents
or in gynecology, was apparent [606], [615].
9.4. LHRH Analogs (→ Peptide and Such superagonists bring about a very effective
Protein Hormones, Chap. 2.1.) reversible inhibition of the release of steroidal
sex hormones. The exchange of glycine at pos-
The releasing hormone gonadorelin (GnRH, tion 6 (glycine6 ) of the native LHRH for other,
synonymous with LHRH, luteinizing hormone- always d-configured, amino acids, is common
releasing hormone or gonadoliberin), together to all modern superagonists; some have a C-
with its specific receptor, plays a central role terminal ethylamide (buserelin, leuprorelin) or
in neuroendocrinology [599]. The decapeptide azaglycinamide (goserelin) residue instead of
LHRH is formed in the cell bodies of hypotha- glycinamide. Eight to ten amino acids of the
lamic neurons and is secreted in pulses into the LHRH sequence are thus conserved in all clin-
blood stream [600–603]. Ultimately it stimulates ically relevant superagonists; by exchange at a
secretion of the sex-specific hormones in the maximum of two positions, the biological activ-
testes and ovaries. Specific receptors for LHRH ity or hormone suppression in tumor patients,
and synthetic analogs are also present in the pitu- can be increased by a factor of up to 100 on
itary gland and other tissues (for example, tumor subcutaneous application [606], [615]. Table 1
cells) [604] and organs. summarizes the most important derivatives.
Three concepts for therapeutic application Buserelin (Profact, Suprecur) [57982-77-1],
have emerged. The first is the restoration of nor- leuprorelin (Lupron, Carcinil, Enatone)
mal physiology by administration of LHRH by [53714-56-0], triptorelin (Decapeptyl)
infusion pump to promote fertility in men and [57773-63-4], and goserelin (Zoladex)
women who are infertile due to defective en- [65807-02-5] (trade names in Germany in paran-
dogenous LHRH secretion. Second, long-lasting theses) are the products on the market with the
Figure 3. Antitumor activity of LHRH agonists and antagonists in femaleFSH = follicle stimulating hormone;
ACTH = adrenocorticotropic hormone
Table 1. Structure of LHRH agonists on the market (given are only those amino acid residues that are different in LHRH).
Name (Co.) Structure
1 2 3 4 5 6 7 8 9 10
LHRH Glp[a] His Trp Ser Tyr Gly Leu Arg Pro Gly-NH2
Buserelin (Hoechst) d-Ser(tBu) Gly-NHEt
Nafarelin d-(2)Nal
Leuprorelin (Abbott, d-Leu Gly-NHEt
Takeda)
Goserelin (Zeneca) d-Ser(tBu) Azagly-NH2
Histrelin (Ortho) d-His(Bzl) Gly-NHEt
Triptorelin (Ferring) d-Trp
[a] Glp = pyroglutamic acid.
highest turnover. The Lupron line of products is responding C-terminal heptapeptide, and then
the leading anticancer hormone drug with world- the N-terminal dipeptide is condensed with this
wide sales in 1996 of $ 810 × 106 followed by to form the complete sequence. Control of the
Zoladex with sales of $ 563 × 106 . physicochemical process parameters, such as
Annual production is less than 100 kg for concentrations, precipitation, separations, reac-
buserelin and significantly over 100 kg for tion temperature profiles, and purification tech-
the market leader, leuprorelin. At this or- niques, is important for successful scale-up of
der of magnitude, substances are only pro- peptide synthesis to a technical scale [617].
duced by classical organic preparative syn-
thesis (fragment condensation in solution).
Hoechst, for example, synthesizes the non- 9.4.1.1. Leuprorelin Acetate
apeptide buserelin from the units pyrog- Because leuprorelin inhibits the synthesis of
lutamic acid-histidine (Glp-His), tryptophan- androgen and estrogen, the drug blocks the
serine-tyrosine (Trp-Ser-Tyr), and d-Serine- growth of hormone-dependent tumors by shut-
tert-butyl ether-leucine-arginine-proline-NHEt ting down testosterone production. Leuprore-
(D-Ser(tBu)-Leu-Arg-Pro-NHEt); the tri- and lin is indicated for the treatment of advanced
tetrapeptide units are coupled to form the cor- prostate cancer, as an alternative to castration
for the treatment of endometriosis, and for the extremities in animal experiments, due in part
presurgical management of patients with anemia to massive histamine release by mast cell de-
caused by benign fibroid tumors. granulation. Cyanosis and respiratory impair-
Trade name: Lupron (Abbott, Takeda). ment were also observed [620]. The cause of
these intolerable side effects is thought to be the
combination of d-arginine at position 6 with the
9.4.1.2. Goserelin three aromatic amino acids at the N-terminus of
the sequence [621]. For the desired biological
As certain prostate tumors grow in response to antagonist potency, a d-configured basic amino
testosterone, goserelin prevents the production acid is necessary at position 6. A. V. Schally
of testosterone in testes and is therefore indi- et al. achieved the breakthrough to highly ac-
cated for the treatment of prostate cancer, ad- tive antagonists free of side effects with the
vanced breast cancer, and endometriosis. derivatives SB-75 (cetrorelix) [120287-85-6]
Trade name: Zoladex (Zeneca). and SB-88 [120287-93-6], by incorporating hy-
drophilic, nonbasic amino acids with side-chain
carbamoyl functions at position 6 (d-Citrulline6 ,
9.4.2. LHRH Antagonists d-homocitrueline) [621], [622].
Today, e.g., cetrorelix is manufactured ex-
Common to the intrinsic activity of all superag- clusively by classical fragment condensation on
onists is the initial temporary stimulation of go- a kilogram scale. Two synthesis strategies pro-
nadotropin release. Soon after the use of highly ceed via either the N-terminal tripeptide (d-
active agonists became an established therapy, Nal1 -d-Cpa2 -d-Pal3 ) or the C-terminal tripep-
a search began for corresponding antagonists, tide (Arg8 -Pro9 -d-Ala10 ) and the complemen-
which do not bring about an initial hormone tary heptapeptide (Ser4 -d-Ala10 ) or (Nal1 -Leu7 )
release, to avoid this therapeutically counter- to the protected decapeptide with tert-butyl side
productive effect. A final big hurdle for clini- chain protection. The deprotection with hy-
cal use of highly active antagonists was the in- drochloric acid is followed by final purification
herent anaphylactic potential of these peptides. by preparative HPLC. The C- and N-terminal
Starting from the sequence of native LHRH, functionalization of the acetylated decapeptide
the individual positions of the peptide chain amide, necessary for biological activity and to
were examined in rapid succession for their avoid rapid enzymatic degradation, is introduced
contribution to biological activity. Particular at- at the level of the terminal tripeptide. Function-
tention was paid to side effects. The most ef- alization is achieved by acetylation of the free
fective early improvements in antagonistic ac- α-amino group of naphthylalanine1 with acetyl-
tivity were achieved by using d-phenylalanine hydroxysuccinimide, or amminolysis of the re-
(d-Phe) instead of histidine at position 2, by sulting alanine10 methyl ester in alcoholic am-
d-amino acids at position 6 instead of Gly, monia [623].
and the exchange of C-terminal glycine for d- Folkers et al. were able to improve active
alanine (d-Ala10 ). Further stepwise optimiza- antagonists successively by consistent and sys-
tion led to the sequence scheme now usual tematic modifications both at the relevant se-
for all modern antagonists of d-Nal1 -d-Cpa2 -d- quence positions and the side chain substituents;
Pal3 (Nal = 2-naphthylalanine, Cpa = Phe(4-Cl), the most successful were complex substitutions
Pal = 3-pyridylalanine) as hydrophobic cluster, at the positions 5, 6 and, in part, 8 [624–628].
a d-configured aromatic or aliphatic, yet hy- An important contribution was made by Rivier
drophilic, aminocarboxylic acid at position 6, et al., who synthesized the decapeptide “aza-
and the C-terminal hydrophilic sequence Xxx8 - line” with novel modifications at positions 5
Pro9 -d-Ala10 where Xxx is either arginine or and 6, where aminotriazole-substituted p-ami-
isopropyllysine. Excellent documentation of the nophenylalanine or lysine are positioned [629–
stepwise optimization can be found in [618], 631]. Azaline B [134457-28-6] is probably one
[619]. of the most active antagonists presently available
Antagonists of the second generation caused worldwide.
formation of temporary edemas of the face and
Deghenghi published a highly active de- bioavailability), yet have a high binding affin-
capeptide sequence with minimal histamine re- ity. Ideally, such substances should be able to be
lease and good water solubility. This structure, administered orally, be sufficiently stable in the
known as antarelix [151277-78-5], differs from organism, and possess favorable pharmacolog-
SB-75 (cetrorelix) in that it has homocitrulline6 ical parameters comparable to peptide antago-
instead of citrulline6 and isopropylysine8 in- nists. Clinical investigations of the influence of
stead of arginine8 [633]. By using Lys(iPr)8 , the antimycotic ketoconazole on prostate can-
residual potential for histamine release can be cer and testosterone suppression indicate that it
further reduced. Organon is developing the an- may have a LHRH-mimetic effect [648], [649].
tagonist ganirelix (RS26306) [124904-93-4] un- Further investigations by Abbott showed weak
der license from Syntex; this is a decapep- antagonistic activity both in vitro and in vivo for
tide with novel alkyl-modified d- and l- ketoconazole and its modified analogs; however,
homoarginine units at positions 6 and 8 [634]. the biological activity of such derivatives is most
With ramorelix (HOE 013) [127932-90-5], probably not mediated mainly by LHRH an-
Hoechst (HMR) has a peptide antagonist with tagonism [650], [651]. Complex, highly substi-
a sugar – amino acid unit (O-α-l-rhamnosyl- tuted nitrogen heterocycles seem to have LHRH
d-serine6 ), that has improved water solubility antagonistic potential [652]. In patent litera-
[632]. Schering has also synthesized peptide an- ture benzodiazepines, benzodiazepinones, het-
tagonists, using nonproteinogenic amino acids, erocyclic benzo-substituted alkylamines, and
such as ε-dialkylated lysine or benzodiazepine thienopyridine carboxylic acid derivatives are
aminocarboxylic acids [635], [636]. Abbott’s A- described as LHRH receptor antagonists with
76154 [136989-30-5] is an octapeptide antago- receptor-binding inhibition at submicromolar
nist with LHRH receptor affinity of a similar or- concentrations, suitable amongst other uses as
der of magnitude to that of active decapeptides antitumor agents for hormone-dependent tumors
such as A-75998 [135215-95-1] or “Nal-Glu” [647], [653]. A lead structure from the latter sub-
[103733-02-4] [644]. stance class is in extensive pharmacological tri-
als [654–656].
9.4.2.1. Receptor Assays
At present, selected peptides are being tested in

vivo by measuring the testosterone concentra-
tion in male rat serum. Here, a single subcuta-
neous application of a potent LHRH antagonist
leads to persistent testosterone suppression. The
activity in animals generally correlates well with
the binding affinity determined in vitro on hu-
man receptors. In cases where no in vivo activity McNeil, US 4 678 784
was observed despite high binding affinity, the
peptide probably had pharmacokinetic charac-
teristics that played a decisive role. In the future,
transgenic animals will also be very important
for in vivo testing [645–647].
9.4.2.2. Peptidomimetics
For some years, increased efforts have also been

made to find substances with affinity to the
LHRH receptor that do not have the character-
Takeda, WO 95/28405
istic substance-specific properties and also dis-
advantages of peptides (short half-life, lack of
Specific natural and synthetic kinase in-

hibitors are in clinical development as anticancer
agents [668–670].
Takeda, EP 679 642 A1
10. Signal Transduction Inhibitors

Anticancer drug discovery has been directed
away from agents that affect cells by produc-
ing DNA damage towards modulators of signal
transduction pathways that have become unreg-
ulated or aberrant in malignant transformation
[657]. Bryostatin 1
10.1. Enzyme Inhibitors

10.2. Phospholipid – Based
Binding of growth factors [658], such as epi-
dermal growth factor (EGF) [659] to a mem- Antineoplastics
brane bound receptor tyrosine kinase results
in dimerization and autophosphorylation of ty- Ether phospholipids and lysophospholipids are
rosine residues on the protein surface. As re- naturally occurring derivatives of phospho-
sult the GTP-bound form of ras (→ Toxicology, lipids of the cell membrane with many in-
Chap. 3.7.3.1.) [660] undergoes a conforma- teresting properties. For example, the ether
tional change on its surface, enabling it to bind phospholipid 1-O-alkyl-2-acetyl-sn-glycero-3-
to several effector molecules leading to the acti- phosphocholine (platelet activating factor, PAF)
vation of transcription factors involved in DNA [671] causes platelet aggregation and dilation of
synthesis [661]. An overexpression of the EGF- blood vessels and lysophosphatidic acid (LPA),
receptor is observed in various types of human the simplest natural phospholipid, is a potent mi-
cancers. togen. The observation that alkyllysophospho-
Mechanism-based screens have identified the lipids exerted experimental antitumor activity in
natural products bryostatin [662] [83314-01-6] vitro and in vivo [672], [673] led to system-
as partial agonist of protein kinase C and fu- atic modification of the structures and subse-
magillin [663] [23110-15-8] as interfering with quent clinical investigation of, e.g., ilmofosine
tumor-induced neovascularization thus inhibit- [83519-04-4] [675]. In the course of modifica-
ing angiogenesis. FR-111142 as analog of fu- tion it was found that an analog without glycerol
magillin is reported to be less toxic and more backbone was equally effective.
active than the parent compound [664]. A po-
tent inhibitor of ras protein farnesylation [665] Miltefosine (D-18506, hexadecylphos-
is pepticinnamin E [666], and peptides related to phocholine), [058066-85-6] [676], 2-
the CAAX-tetrapeptide (where C is cysteine, A [[(hexadecyloxy)-hydroxyphosphinyl]oxy]-
is valine, isoleucine, or leucine, and X is me- N,N,N-trimethylethanaminium hydroxide (in-
thionine or serine). In order to prepare com- ner salt); C21 H46 NO4 P, M r 407.57 is a new
pletely nonpeptide and potentially more stable phospholipid-based antineoplastic agent which
inhibitors a series of potent peptidomimetic in-
hibitors were designed [667].
exerts substantial antitumor activity in appro- treated with oral capsules were terminated early
priate models. Because of its special pharma- due to gastrointestinal intolerance [682].
cology – high activity against mammary carci-
noma and low toxicity to normal tissue – it is
an ideal candidate for topical treatment of cu- 11. Economic Aspects
tanous breast cancer metastases. The substance
was launched in 1993 as a topical formulation The world cancer drug market is expected to
for palliative treatment of refractory skin metas- reach $ 14 × 109 by 2000 [10], [14]. Total sales
tases of breast cancer [676]. of the top 100 anticancer drugs generated a
Trade name: Miltex (ASTA Medica). turnover of $ 4.66 × 109 in 1996. Of the worlds
A variety of derivatives were synthesized and eight top-selling anticancer drugs, four – the
characterized, some of which appeared to be prostate cancer-drugs flutamide (Eulexin), bica-
considerably less toxic to the gastrointestinal lutamide (Casodex), leuprorelin (Lupron) and
tract [677]. One of them, perifosine (D-21266) goserelin (Zoladex) – are merely palliative,
[157716-52-4] is currently in clinical trials. yet have combined annual sales of $ 1.7 × 109 ,
Mechanism of Action. Miltefosine shows whereas sales of the breast cancer drugs tamox-
highly selective antineoplastic activity in ifen (Nolvadex) and paclitaxel (Taxol) are ap-
DMBA- and N-methyl-N-nitrosourea (MNU)- proaching $ 500 × 106 and $ 850 × 106 , respec-
mammary carcinoma of the rat. The activity tively.
is not due to an antiestrogenic effect in these Taxol, BMS’s drug for treating ovarian and
estrogen-dependent tumor models [678]. The breast cancers, was the biggest selling anticancer
treatment was tolerated and no overt toxic symp- agent in 1996, with sales of $ 813 × 106 .
toms were observed. The mechanism of action
of miltefosine is most probably different from
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Cancer Chemotherapy 1

1. Introduction . . . . . . . . . . . . . . 2 3.8. Nitrosoureas . . . . . . . . . . . . . . 17

c 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

8.1.2. Mechanisms of Resistance . . . . . . 34 9.4. LHRH Analogs . . . . . . . . . . . . 41

1. Introduction $ 100 × 109 per year in treatment expenses and

ease that has spread beyond the primary site at

trexate, an important dose-limiting toxicity of ther lipophilic DHFR-inhibitors are trimetrex-

folates is a competitive process. The reasons

2.1.2. Analogs Trimetrexate

Many new analogs to methotrexate have been

Figure 1. Pathways for ﬂuoropyrimidine activation

2.3. 5-Azacytidine [87]; methylation appears to inhibit gene expres-

Mechanism of Action. Fludarabine and its Pentostatin

Mechanism of Action. The highest activity of 6-Mercaptopurine is used for maintenance

2.7.3. New Analogs the SN 1 reaction, a highly reactive intermedi-

Chlorambucil (NSC-3088) [305-03-3], 4-

mustine [489-15-0] was successfully launched

3.7. Estramustine This group of agents was developed by careful

(±)-diethyl[1-[3-(2-chloroethyl)-3-nitroso- that, through a variety of mechanisms, are capa-

3.9.1. Mechanism of Action

The exact mechanism by which procarbazine

3.9.2. Mechanisms of Resistance 3.11. Hexamethylmelamine

Mitomycin-C (NSC-26980, MIT-C) Meanwhile epirubicin (4’-epiadriamycin,

Doxorubicin (NSC-123127) [23214-92-8], In addition idarubicin (NSC-256439, IMI-

Pirarubicin is the 4’-O-tetrahydropyranyl

under active investigation. Interestingly, there is 4.2. Mechanism of Resistance

Mechanism of Action. The quinone struc-

Mechanism of Action. Actinomycin inter-

Bleomycins (NSC-125066) are a group of anti-

The actinomycins are a family of antibiotics

[365]. In addition, total synthesis of bleomycin

6.2.2. Mechanism of Action

7.1. Vinca Alkaloids (→ Alkaloids,

Vinca alkaloids are dimeric indole derivatives

Dynemicin A 7.1.1. Vincristine and Vinblastine

Drug Interactions. Both vincristine and Vinorelbine (KW-2307, NVB) [071486-22-1]

polymerization [403–405]. The compound in-

the vincristine-resistant cell line P 377/VCR and

Mechanism of Action. Topoisomerase

Structure – Activity Relationship. A large

Figure 2. Mechanism of action of paclitaxel and vinca alkaloids

Carboplatin Oxaliplatin Ormaplatin

Orally active cisplatin-analogs with higher

9. Hormonally Active Anticancer However, primary and secondary resistance

9.1. Antiestrogens (→ Hormones,

9.1.1. Antagonists Tamoxifen

Estrogens can induce hormone-dependent hu-

Mechanism of Action. Tamoxifen and

and unlike tamoxifen, it inhibited the growth

Raloxifene (LY-139481) [82640-04-8] is a

9.2. Aromatase Inhibitors (→ Hormones,

All endocrine therapies inhibit endogenous es-

the commonly used initial treatment for prostate

9.3.2. Nilutamide Mechanism of Action. Bicalutamide in-

9.4.1. LHRH Agonists

The ﬁrst years after the discovery of the go-

Name (Co.) Structure

[a] Glp = pyroglutamic acid.