Mitomycin C Dna Mechanism

J HjO

CHj-CH* + Nj Vinyl Carbonium

C02+ 2 HNCHjC^a 2-Chlorethylamine

Figure 4-7. Proposed carmustine degradation (Montgomery et al., 1967).

carbonium ion—the alkylating agent. The chloroethyl isocyanate can act as a carbamoy-lating agent, or, after decomposing to 2-chloroethylamine, become a second alkylating species. A more general scheme for mono-alkyl functional nitrosoureas is outlined in Figure 4-8. Decomposition, possibly initiated by hydroxide ion, affords an isocyanate and hydroxy 2-chlorethyl diazine, which spontaneously decomposes to the 2-chloroethyl carbonium ion, the very active alkylating species that, in a two-step sequence, can actually cross-link DNA.

Because of their highly lipophilic nature, nitrosoureas are particularly useful against malignancies of the central nervous system such as brain tumors (gliomas), where response rates have exceeded 40%. They are also used (in combination therapy) against multiple myeloma, Hodgkin's disease, and non-Hodgkin's lymphoma.

CtCHjCHjfö^N—OH «- Q—CHj-CHj —N—C—NH—R -» 0=ON R Isocyanate

[OCHJCHJ*] 2-Chlorethyl carbonium Ion

J Protein-NHj ?

Protein.NH—C Nil R Protein carbamoyl derivative

[OCHJCHJ*] 2-Chlorethyl carbonium Ion

Na, Nj nprwant nudaophil* on Onnd A & B of DNA twlix

Monoalkylated DNA

Crocs-linked DNA

Figure 4-8. Possible nitrosourea degradation scheme with resultant alkylation and carbamoylation (adapted from Reed et al„ 1975, and Kohn, 1977).

Na, Nj nprwant nudaophil* on Onnd A & B of DNA twlix

Monoalkylated DNA

Crocs-linked DNA

Figure 4-8. Possible nitrosourea degradation scheme with resultant alkylation and carbamoylation (adapted from Reed et al„ 1975, and Kohn, 1977).

Streptozotocin is unique both in origin and pharmacology. It is an antibiotic obtained from Streptomyces achromogenes and contains a glucosamine component in its structure, which may contribute to its tendency to be taken up into the P-cells of the islets of Langerhans of the pancreas. Where it was initially used to induce diabetes in experimental animals, it is now used to treat pancreatic islet cell carcinoma, resulting in about a 50% response rate, but few survivors past 1 year.

Among alkylating agents cyclophosphamide should be considered as a true pro-drug, requiring extensive metabolic activation to obtain the actual cytotoxic alkylating agent (Fig. 4-9) (see also mitomycin, later). The drug is first oxidized by hepatic mixed-function oxidase (Chapter 3) to yield the 4-hydroxy metabolite. This compound is in tautomeric equilibrium with the acyclic form aldophosphoramide. The 4-hydroxy and acyclic aldehyde metabolites are further enzymatically oxidized to 4-ketocyclophos-phamide and carboxyphosphamide, respectively. Neither metabolite is cytotoxic. The ultimate cytotoxic alkylating agent is produced by a beta-elimination reaction of aldophosphoramide, yielding the phosphoramide mustard and the byproduct acrolein, a highly reactive volatile liquid with sharp irritating effects. Since the nonenzymic degradation of aldophosphoramide to the active alkylator most likely occurs extrahepatically (blood, tissues, and, one hopes, in tumor cells), as evidenced by lack of hepatotoxicity, this may in part explain cyclophosphamide's higher specificity of cytotoxicity in the S phase than other alkylators.

In addition to the clinical toxicities encountered with nitrogen mustard in general, cyclophosphamide exhibits considerable urinary tract toxicity in the form of cystitis with hematuria. This has been attributed to an accumulation of the corrosive acrolein in the bladder. The parenteral administration of fluids and acetylcysteine (believed to react with acrolein) prevents cystitis greatly. Another compound, the sodium salt of 2-mercap-toethanesulfonic acid, mesna (Mesnex) -HS-CH2-CH2-SO3H, also minimizes hemorrhagic cystitis due to acrolein by adding across the acrylic double bond of the aldehyde and detoxifying it. A newer analog of cyclophosamide is ifosfamide (Table 4-3). It too is metabolized to an active alkylating species, N,N'-bis(2-chloroethyl)phosphoric acid diamide (ifosphoramide) and acrolein. Another alkylating agent that must be activated to achieve its cytotoxicity is the antibiotic mitomycin C, which is obtained from Streptomyces caespito-sus. In this case NADPH-dependent enzyme reduction activates the antibiotic to a Afunctional alkylating species. Figure 4-10 outlines the reactions involved in the "bioreductive

Mitomycin Mechanism Action

4-Katocyctophosphamid* Carboxypbosphoramida Phosphoramida mustard

Figure 4-9. Cyclophosphamide metabolism and activation (proposed by Connors et al., 1974).

4-Katocyctophosphamid* Carboxypbosphoramida Phosphoramida mustard

Figure 4-9. Cyclophosphamide metabolism and activation (proposed by Connors et al., 1974).

Figure 4-10. Probable mechanism of action of mitomycin C (Iver and Szybalski, 1964).

alkylation" mechanism, by which mitomycin alkyates DNA. Mitomycin C5 has been shown to require benzoquinone to hydroquinone reduction preceding DNA cross-linkage alkylation. The reduction is effected by a cellular NADPH-dependent quinone reductase system, resulting in spontaneous expulsion of the methoxy group from its tertiary 9a position to form the 9-9a double bond. Protonation of the aziridine ring and the facile leaving of the carbamoyloxy group at position 10 results in two active alkylating carbonium ions at carbons 10 (CH2+) and one with which attacking purine and pyrimidine bases of DNA cross-link. Cross-linkage has been shown to be proportional to guanine and cytosine content of the DNA.

Probably the most "unorthodox" cross-linking alkylating agent is an inorganic platinum-coordinating compound ds-platinum (II) diamminedichloride, (c/'s-DDP, cisplatin, Platinol). It is a classic square-planar four-coordinate platinum (II) compound.

Cisplatin was discovered during electrolysis experiments of bacterial culture media utilizing platinum electrodes. An electrode product exhibited antibacterial activity at very low concentrations. The product, cisplatin, also revealed antitumor activity. Cisplatin crosslinks DNA via ring atoms of purines and possibly pyrimidines, with the elimination of CT analogously to the bifunctional alkylating agents discussed. Although ds-platinum can be

5 Mitomycin A and B differ in substituents at positions 7, 9a, and the aziridine nitrogen.

demonstrated to inhibit RNA and protein synthesis in mammalian cells in vitro, it is selective DNA inhibition in vivo that is the primary cause of cytocidal activity. The drug has shown clinical effectiveness against testicular, ovarian, head and neck, and bladder cancer. Combinations with other drugs such as bleomycin, vinblastine, and doxorubicin allow for decreased cisplatin dosages and resultant lessening of toxic effects.

Cisplatin exhibits intense emetic reactions, ototoxicity, and severe cumulative renal toxicity. Some success with "rescue" techniques, using compounds with high heavy metal affinity such as diethyldithiocarbamate, have been reported.

Figure 4-11 depicts a possible sequence whereby cisplatin, forming a reactive aquo intermediate, reacts with the N-7 nitrogen of two guanine bases on two different DNA chains, thus cross-linking them in a manner analogous to the nitrogen mustards. This "mechanism," of course, offers no explanation for the lesser bioactivity of the trans-diammin-dichloro-platinum stereoisomer since there seems no apparent reason for a similar reaction sequence not to occur.

The mystery may now be somewhat closer to a solution. Both cisplatin and the transisomer do share some biological properties, including binding covalently to chromosomal DNA. Both adducts inhibit DNA replication and are therefore cytotoxic. However, the cis- isomer seems more potent and appears preferentially more so to malignant cells. Lippard et al. (1985) utilized a circular chromosome from the DNA virus SV40, and discovered that cisplatin was 14 times more effective than the trans- isomer in inhibiting DNA synthesis to the same degree, yet the two isomers were bound in the same quantities. Cellular uptake was even at equal rates. However, kinetic measurements showed that after an initial six-hour period trans binding was decreased, while cis continued to accelerate. After 24 hours, when DNA replication is usually at maximal rates, little trans isomer could be found bound. The authors proposed that for geometric reasons the trans adduct damage is being more efficiently repaired. Additional X-ray structural determination of cisplatin bound to a dinucleotide of deoxyguanosine (linked via a phosphodiester) clearly shows the cross-linkage to require the two guanine rings to tilt away from the normally stacked relationship of the intact DNA double helical structure. Sherman et al. (1985) therefore proposed that this explains the interference with DNA replication and furthermore that such cross-linkage is, for stereochemical reasons, not possible with trans-DDP.

Figure 4-11. Possible cross-link of cisplatin with two guanine bases in DNA.

Since the success of cisplatin has been established, analogs have been synthesized and have reached human clinical trials. Effective compounds with decreased renal toxicity have been reported. Among the more promising leads are derivatives of 1,2-diamminocy-clohexano- and cycloheptanodichloroplatinum (II). A malonic derivative showed remission of leukemias and regression of solid tumors. A particularly intriguing new platinum complex is cis- 1,2-diamminocyclohexylplatinum (II) ascorbate. Unlike cisplatin, here both the cis and trans isomer exhibited antitumor activity, and were actually greater than cisplatin in some tumor screens. The activation mechanism by which cisplatin loses its chloride ligands during tissue transport is not possible here, considering the Pt-C bond to ascorbic acid. This raises the possibility of a new differing mechanism—or a rethinking of the currently accepted mechanism for platinum compounds.

Another organometallic platinum drug has been approved in the United States. It is a diammin platinum coordinate with 1,1-cyclobutanedicarboxylic acid named carboplatin (CBDA, Paraplatin). Its advantages over cisplatin are a lesser emetogenic effect as well as a possible decrease in nephro- and ototoxicity.

4.12. Antimetabolites

The rationale for utilizing organic compounds structurally similar to normal cellular metabolites as anticancer agents is simple: to interfere with the biosynthesis of these substances within the cell and thus inhibit cell proliferation. Among the precursors that cells require for normal and abnormal growth are the building blocks of nucleic acids, the pyrimidine and purine bases. The biosynthesis of the bases themselves requires, among other steps, the transfer of one-carbon units that are derived from folinic acid (Chapter 2). The synthesis of proteins requires various amino acids. Thus structural analogs of any of these bases and amino acids yield potential folic acid, purine, pyrimidine, and amino acid antagonists of chemical usefulness.6

6 Structurally unrelated compounds having the ability to interfere with the various enzymes that are involved in the biosynthesis of nucleosides, nucleotides, and amino acids, and their respective biopolymerization, can be another source of anticancer drugs.

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