Alkylating Agents

Kenneth D. Tew

PERSPECTIVES

Alkylating agents were the first anticancer molecules developed, and they are still used today. After more than 50 years of use, the basic chemistry and pharmacology of this drug family is well understood and has not changed substantially. The family contains six major classes: nitrogen mustards, aziridines, alkyl sulfonates, epoxides, nitrosoureas, and triazene compounds, although a few nonstandard agents have recently been developed. Most epoxides tend to be quite nonspecific with respect to their reactivity and, as such, few have useful clinical characteristics. This chapter provides perspective on how the limited varieties of alkylating agents continue to be useful in the therapeutic management of cancer patients.

The alkylating agents are a diverse group of anticancer agents with the commonality that they react in a manner such that an electrophilic alkyl group or a substituted alkyl group can covalently bind to cellular nucleophilic sites. Electrophilicity is achieved through the formation of carbonium ion intermediates and can result in transition complexes with target molecules. Ultimately, reactions result in the formation of covalent linkages by alkylation with a broad range of nucleophilic groups, including bases in DNA, and these are believed responsible for ultimate cytotoxicity and therapeutic effect. Although the alkylating agents react with cells in all phases of the cell cycle, their efficacy and toxicity result from interference with rapidly proliferating tissues. From a historical perspective, the vesicant properties of mustard gas used during World War I were shown to be accompanied by the suppression of lymphoid and hematologic functions in experimental animals¹ and led to the development of mechlorethamine as the first alkylating agent used in the management of human cancer.² Subsequently, a number of related drugs have been developed, and these have roles in the treatment of a range of leukemias, lymphomas, and solid tumors. Most of the alkylating agents cause dose-limiting toxicities to the bone marrow and, to a lesser degree, the intestinal mucosa, with other organ systems also affected contingent on the individual drug, dosage, and duration of therapy. Despite the present trend toward targeted therapies, this class of “nonspecific” drugs maintains an essential role in cancer chemotherapy.

Because of the classic nature of the drug family, there have been relatively few advances in either their use or utility since publication of the previous edition of this book.

CHEMISTRY

Alkylating reactions are generally classified through their kinetic properties as S_N1 (nucleophilic substitution, first order) or S_N2 (nucleophilic substitution, second order) (Fig. 17.1). The first-order kinetics of the S_N1 reactions depend on the concentration of the original alkylating agent. The rate-limiting step is the initial formation of the reactive intermediate, and the rate is essentially independent of the concentration of the substrate. The S_N2 alkylation reaction is a bimolecular nucleophilic displacement with second-order kinetics, where the rate depends on the concentration of both alkylating agent and target nucleophile. Reactivity of electrophiles³ suggests that the rates of alkylation of cellular nucleophiles (including thiols, phosphates, amino and imidazole groups of amino acids, and various reactive sites in nucleic acid bases) are most dependent on their potential energy states, which can be defined as “hard” or “soft,” based on the polarizability of their reactive centers.⁴ Although the metabolism and metabolites of nitrogen mustards and nitrosoureas differ, the active alkylating species of each is the alkyl carbonium ion (see Fig. 17.1), a highly polarized hard electrophile as a consequence of its highly positive charge density at the electrophilic center. Alkyl carbonium ions will react most readily with hard nucleophiles (possessing a highly polarized negative charge density), where the high-energy transition state (a potential energy barrier to the reaction) is most favorable. In specific terms, an active alkylating species from a nitrogen mustard will demonstrate selectivity for cellular nucleophiles in the following order: (1) oxygen in phosphate groups of RNA and DNA, (2) oxygens of purines and pyrimidines, (3) amino groups of purine bases, (4) primary and secondary amino groups of proteins, (5) sulfur atoms of methionine, and (6) thiol groups of cysteinyl residues of protein and glutathione.³ The least favored reactions will still occur, but at much slower rates unless they are catalyzed.

Alkylation through highly reactive intermediates (e.g., mechlorethamine) would be expected to be less selective in their targets than the less reactive S_N2 reagents (e.g., busulfan). However, the therapeutic and toxic effects of alkylating agents do not correlate directly with their chemical reactivity. Clinically useful agents include drugs with S_N1 or S_N2 characteristics, and some with both.⁵ These differ in their toxicity profiles and antitumor activity, but more as a consequence of differences in pharmacokinetics, lipid solubility, penetration of the central nervous system (CNS), membrane transport, metabolism and detoxification, and specific enzymatic reactions capable of repairing alkylation sites on DNA.

CLASSIFICATION

The major classes of clinically useful alkylating agents are illustrated in Table 17.1 and summarized in the following sections. Doses and schedules of the various agents are shown in Table 17.2.

Alkyl Sulfonates

Busulfan is used for the treatment of chronic myelogenous leukemia. It exhibits S_N2 alkylation kinetics and shows nucleophilic selectivity for thiol groups, suggesting that it may exert cytotoxicity through protein alkylation rather than through DNA. In contrast to the nitrogen mustards and nitrosoureas, busulfan has a greater effect on myeloid cells than lymphoid cells, thus the reason for its use against chronic myelogenous leukemia.⁶

Aziridines

Aziridines are analogs of ring-closed intermediates of nitrogen mustards and are less chemically reactive, but they have
equivalent therapeutic properties. Thiotepa has been used in the treatment of carcinoma of the breast, ovary, for a variety of CNS diseases, and with increasing frequency as a component of high-dose chemotherapy regimens.⁷ Thiotepa and its primary desulfurated metabolite triethylenethiophosphoramide (TEPA) alkylate through aziridine ring openings, a mechanism similar to the nitrogen mustards.

Figure 17.1 Comparative decomposition and metabolism of a typical nitrogen mustard compared to a nitrosourea. Although intermediate metabolites are distinct, the active alkylating species is a carbonium ion in each case. This electrophilic moiety reacts with target cellular nucleophiles.

Triazines

Perhaps the newest clinical development in the alkylating agent field is the emergence of temozolomide (TMZ). This agent acts as a prodrug and is an imidazotetrazine analog that undergoes spontaneous activation in solution to produce 5-(3-methyltriazen-1-yl) imidazole-4-carboxamide (MTIC), a triazine derivative. It crosses the blood-brain barrier with concentrations in the CNS approximating 30% of plasma concentrations.⁸ Resistance to the methylating agent occurs quite frequently and has adversely affected the rate and durability of the clinical responses of patients. However, because of its favorable toxicity and pharmacokinetics, TMZ is being combined with numerous other classes of anticancer drugs in an effort to improve response rates in diseases such as malignant melanomas, gliomas, brain metastasis from solid tumors, and refractory leukemias. Many of these trials are currently underway.⁹

Nitrogen Mustards

Bischloroethylamines or nitrogen mustards are extensively administered in the clinic. As an initial step in alkylation, chlorine acts as a leaving group and the β-carbon reacts with the nucleophilic nitrogen atom to form the cyclic, positively charged, reactive aziridinium moiety. Reaction of the aziridinium ring with an electron-rich nucleophile creates an initial alkylation product. The remaining chloroethyl group achieves bifunctionality through the formation of a second aziridinium. Melphalan (L-phenylalanine mustard), chlorambucil, cyclophosphamide, and ifosfamide (see Table 17.1) replaced mechlorethamine as primary therapeutic agents. These derivatives have electron-withdrawing groups substituted on the nitrogen atom, reducing the nucleophilicity of the nitrogen and rendering them less reactive, but enhancing their antitumor efficacy.

TABLE 17.1 Major Classes of Clinically Useful Alkylating Agents

Drug	Main Therapeutic Uses	Clinical Pharmacology	Major Toxicities	Notes
ALKYL SULFONATES
Busulfan	Bone marrow transplantation, especially in chronic myelogenous leukemia	Bioavailability, 80%; protein bound, 33%; t_1/2, 2.5 h	Pulmonary fibrosis, hyperpigmentation thrombocytopenia, lowered blood platelet count and activity	Oral or parenteral; high dose causes hepatic venoocclusive disease
ETHYLENEIMINES/METHYLMELAMINES
Altretamine		Protein bound, 94%; t_1/2, 5-10 h	Nausea, vomiting, diarrhea, and neurotoxicity	Not widely used
Thio TEPA	Breast, ovarian, and bladder cancer; also bone marrow transplant	t_1/2, 2.5 h; urinary excretion at 24 h, 25%; substrate for CYP2B6 and CYP2C11	Myelosuppression	Nadirs of leukopenia, occur 2 wk; thrombocytopenia, 3 wk (correlates with AUC of parent drug)
NITROGEN MUSTARDS
Mechlorethamine	Hodgkin lymphoma		Nausea, vomiting, myelosuppression	Precursor for other clinical mustards
Melphalan (L-phenylalanine mustard)	Multiple myeloma and ovarian cancer, and occasionally malignant melanoma	Bioavailability 25%-90%; t_1/2, 1.5 h; urinary excretion at 24 h, 13%; clearance, 9 mL/min/kg	Nausea, vomiting, myelosuppression	Causes less mucosal damage than others in class
Chlorambucil	Chronic lymphocytic leukemia	t_1/2, 1.5 h; urinary excretion at 24 h, 50%	Myelosuppression, gastrointestinal distress, CNS, skin reactions, hepatotoxicity	Oral
Cyclophosphamide	Variety of lymphomas, leukemias, and solid tumors	Bioavailability, >75%; protein bound, >60%; t_1/2, 3-12 h; urinary excretion at 24 h, <15%	Nausea and vomiting, bone marrow suppression, diarrhea, darkening of the skin/nails, alopecia (hair loss), lethargy, hemorrhagic cystitis	IV; primary excretion route is urine
Ifosfamide	Testicular, breast cancer; lymphoma (non-Hodgkin); soft tissue sarcoma; osteogenic sarcoma; lung, cervical, ovarian, bone cancer	t_1/2, 15 h; urinary excretion at 24 h, 15%	As for cyclophosphamide	Ifosfamide is often used in conjunction with mesna to avoid cystinuria
NITROSOUREAS
Carmustine	Glioma, glioblastoma multiforme, medulloblastoma and astrocytoma, multiple myeloma and lymphoma (Hodgkin and non-Hodgkin)	Bioavailability, 25%; protein bound, 80%; t_1/2, 30 min	Bone marrow and pulmonary toxicities are a function of lifetime cumulative dose	Clinically, nitrosoureas do not share cross-resistance with nitrogen mustards in lymphoma treatment
Streptozotocin	Cancers of the islets of Langerhans	t_1/2, 35 min; excreted in the urine (15%), feces (<1%), and in the expired air	Nausea and vomiting; nephrotoxicity can range from transient protein urea and azotemia to permanent tubular damage; can also cause aberrations of glucose metabolism	A natural product from Streptomyces achromogenes
TRIAZENES
Dacarbazine	Malignant melanoma and Hodgkin lymphoma	t_1/2, 5 h; protein bound, 5% hepatic metabolism	Nausea, vomiting, myelosuppression	IV or IM
Temozolomide	Glioblastoma; astrocytoma; metastatic melanoma	Protein bound, 15%; t_1/2, 1.8 h; clearance, 5.5 l/h/m²	Nausea, vomiting, myelosuppression	Oral; derivative of imidazotetrazine, prodrug of dacarbazine; rapidly absorbed
t_1/2, half-life; TEPA, triethylenethiophosphoramide; AUC, area under curve; CNS, central nervous system; IV, intravenous; IM, intramuscular.

TABLE 17.2 Dose and Schedules of Clinically Useful Alkylating Agents

Alkylating Agent	Disease Sites and Dose Ranges Used Clinically	Notes
BCNU (Carmustine)	General antineoplastic 150-200 mg/m² (IV, every 6 wks) Cutaneous T-cell lymphoma 200-600 mg (topical solution) Adjunct to surgical resection of brain tumor 61.6 mg (implant)	Infusion 1-2 h; in combination, dose usually reduced by 25%-50% Side effects include irritant dermatitis, telangiectasia, erythema, and bone marrow suppression Up to 8 wafers (7.7 mg of carmustine) implanted
Busulfan	Chronic myelogenous leukemia and myeloproliferative disorders 4-8 mg (daily PO) 1.8 mg/m² (daily PO) Bone marrow transplant 640 mg/m² (daily PO)	Dispensed over 3-4 d, with cyclophosphamide
Carboplatin	Advanced ovarian cancer—monotherapy 360 mg/m² (IV, every 4 wks) Ovarian cancer—combination 300 mg/m² (IV, every 4 wks for 6 cycles) Ovarian cancer—IP 200-500 mg/m² (IP, 2 L dialysis fluid) Ovarian and other sites phase 1/2 setting— high-dose therapy 800-1,600 mg/m² (IV)	With cyclophosphamide Patients usually receive marrow transplantation or peripheral stem cell support
Cisplatin	Metastatic testicular cancer: 20 mg/m²/d for 5 d of each cycle (IV) Metastatic ovarian cancer: 75-100 mg/m² (IV, once every 4 wks) Head and neck cancer: 100 mg/m² (IV) Bladder cancer: (combination prior to cystectomy) 50-70; initiate dosing at 50 mg/m² (IV, once every 3-4 wks) Metastatic breast cancer: 20 mg/m² (IV, days 1-5 every 3 wks) Cervical cancer: 70 mg/m² (IV, dosing cycled every 4 wks) Non-small-cell lung cancer: 75 mg/m² (IV, every 3 wks) Esophageal cancer: 75 mg/m² on day 1 of wks 1, 5, 8, and 11 (IV)	With other antineoplastic agents With cyclophosphamide (600 mg/m² once every 4 wks) With vincristine, bleomycin, and fluorouracil With methotrexate and fluorouracil MVAC regimen (methotrexate, vinblastine, doxorubicin, and cisplatin) used for cervical cancer Administration preceded by paclitaxel 135 mg/m² every 3 wks With radiation therapy
Cyclophosphamide	General antineoplastic 1-5 mg/kg (daily PO) 40-50 mg/kg (IV, in divided doses over 2-5 d) 40-50 mg/kg (IV, in divided doses over 2-5 d) 10-15 mg/kg (IV, every 7-10 d) 10-15 mg/kg (IV, every 7-10 d) 3-5 mg/kg (IV twice per wk) High-dose regimen in bone marrow transplantation and for other autoimmune disorders 200 mg/kg (IV) 1-2.5 mg/kg (daily PO 7-14 d/mo)	Dose used as monotherapy for patients with no hematologic toxicity
Dacarbazine	General antineoplastic 2-4.5 mg/kg/d (IV) 150 mg/m²/d (IV)	Administered for 10 d, may be repeated at 4-week intervals With other anticancer agents; treatment lasts 5 d, may be repeated every 4 wks
Etoposide	Testicular cancer 50-100 mg/m²/day (IV, slow infusion over 30-60+ min for 5 d) Small cell lung cancer 35-50 mg/m²/day (IV, slow infusion over 30-60+ min for 4-5 d)	Alternatively, 100 mg/m²/d on days 1, 3, and 5 may be used; doses for combination therapy and are repeated at 3- to 4-wk intervals after recovery from hematologic toxicity Doses are for combination therapy and repeated at 3- to 4-wk intervals after recovery from hematologic toxicity; oral dose is twice the IV, rounded to the nearest 50 mg
Ifosfamide	General antineoplastic 1.2 g/m²/d (IV, for 5 consecutive days)	Repeat every 3 wks
Melphalan	Multiple myeloma: 16 mg/m² (IV, infusion over 15-20 min) 6 mg (daily PO) Epithelial ovarian cancer: 0.2 mg/kg (daily PO)	2-week intervals for 4 doses, 4-wk intervals thereafter After 2-3 wks treatment, should be discontinued for up to 4 wks, then reinstituted at 2-4 mg/d Daily dose for a 5-d course, repeated every 4-5 wks
Streptozotocin	Pancreatic tumors 500 mg/m²/d; 1,000 mg/m²/d (IV; IV)	500 mg for 5 consecutive days every 6 wks, 1,000 mg is for 2 wks, followed by an increase in weekly dose not to exceed 1,500 mg/m²/wk
Temozolomide	Brain tumors 150 mg/m² (daily PO)	Dose adjusted on the basis of blood counts
Thiotepa	General antineoplastic: 0.3-0.4 mg/kg (IV) Papillary carcinoma of the bladder: 60 mg/wk for 4 wks (bladder catheter) Control of serous effusions: 0.6-0.8 mg/kg (intracavitary)	Rapid administration given at 1- to 4-wk intervals 30 or 60 mL should be retained for 2 h, so the patient is usually dehydrated prior to administration of the drug
IV, intravenously; PO, by mouth; IP, intraperitoneal.

One distinguishing feature of melphalan is that an amino acid transporter responsible for uptake influences its efficacy across cell membranes.¹⁰ Although a number of glutathione (GSH) conjugates of alkylating agents are effluxed through adenosine triphosphate-dependent membrane transporters,¹¹ specific uptake mechanisms are generally rare for cancer drugs. Cyclophosphamide and ifosfamide are prodrugs that require cytochrome P-450 metabolism to release active alkylating species. Cyclophosphamide continues to be the most widely used alkylating agent and has activity against a variety of tumors.¹² A cost saving with equivalent therapeutic activity was recently shown in a modified regimen of high-dose cyclophosphamide plus cyclosporine in patients with severe or very severe aplastic anemia.¹³

Nitrosoureas

The nitrosoureas form a diverse class of alkylating agents that have a distinct metabolism and pharmacology that separates them from others.¹⁴ Under physiologic conditions, proton abstraction by a hydroxyl ion initiates spontaneous decomposition of the molecule to yield a diazonium hydroxide and an isocyanate (see Fig. 17.1). The chloroethyl carbonium ion generated is the active alkylating species. Through a subsequent dehalogenation step, a second electrophilic site imparts bifunctionality.¹⁵ Thus, while cross-linking may occur similar to those lesions caused by nitrogen mustards, the chemistry leading to the endpoint is distinct. The isocyanate species generated are also electrophilic, showing nucleophilic selectivity toward sulfhydryl and amino groups that can inhibit a number of enzymes involved in nucleic acid synthesis and thiol balance.¹⁶ Because carbamoylation is considered of minor importance to the therapeutic efficacy of clinically used nitrosoureas, chlorozotocin and streptozotocin were designed to undergo internal carbamoylation at the 1- or 3-OH group of the glucose ring, with the consequence that no carbamoylating species are produced.¹⁷^,¹⁸ Streptozotocin is also unusual in that most methylnitrosoureas have only modest therapeutic value. However, its lack of bone marrow toxicity and strong diabetogenic effect in animals led to its use in cancer of the pancreas (see Table 17.1).¹⁹ The dose-limiting toxicities in humans are gastrointestinal and renal, but the drug has considerably less hematopoietic toxicity than the other nitrosoureas. Because of their lipophilicity and capacity to cross the blood-brain barrier, the chloroethylnitrosoureas were found to be effective against intracranially inoculated murine tumors. Indeed, early preclinical studies showed that many mouse tumors were quite responsive to nitrosoureas. The same extent of efficacy was not found in humans. Subsequent analyses demonstrated that an enzyme responsible for repair of O-6-alkyl guanine (O⁶-methylguanine-DNA methyltransferase [MGMT], or the Mer/Mex phenotype)²⁰ was expressed at low levels in mice, but at high levels in humans, a contributory factor in the reduced clinical efficacy of nitrosoureas in humans. In the 1980s, in particular, a number of new nitrosoureas were tested in patients in Europe and Japan, but none established a regular role in standard cancer treatment regimens.

MGMT promoter methylation is crucial in MGMT gene silencing and can predict a favorable outcome in glioblastoma patients receiving alkylating agents.²¹ This biomarker is on the verge of entering clinical decision making and is currently used to stratify or even select glioblastoma patients for clinical trials. In other subtypes of glioma, such as anaplastic gliomas, the relevance of MGMT promoter methylation might extend beyond the prediction of chemosensitivity, and could reflect a distinct molecular profile. At this time, the standardization of MGMT assays will be critical in establishing prospective prognostic or predictive effects. In addition, eventual clinical trials will need to determine, for each subtype of glioma, the extent to which methylation patterns are predictive or prognostic and whether such assays could be incorporated into an individualized approach to clinical practice.²¹

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