13 Alkylating agents

In this chapter we will discuss alkylating agents, in particular Cyclophosphamide and Melphalan.

13.1 A brief history of alkylating agents

Before their use in chemotherapy, alkylating agents such as sulphurmustard (mustard gas) were better known for their use as chemical weapons in World War I and II. Apparently, it is still used in some countries as a weapon today. The chemical structure of sulphur mustard, also called LOST, is shown in Figure 11.2. Sulphur mustards are blister agents or vesicants, causing severe skin, eye and mucosal pain and irritation. They also cause a variety of other side effects, which may lead to death.

The use and effects of alkylating agents during World War I. Even today mustard gas is still used in some countries as a chemical weapon.

Figure 13.1: The use and effects of alkylating agents during World War I. Even today mustard gas is still used in some countries as a chemical weapon.

Second generation mustards are the nitrogen mustards (Figure 13.2). Nitrogen mustards as chemical weapons were stockpiled but not used during World War II.

Chemical structures of sulphur mustard, known as Lost and nitrogen mustard. Please not the structural resemblance of both alkylating agents

Figure 13.2: Chemical structures of sulphur mustard, known as Lost and nitrogen mustard. Please not the structural resemblance of both alkylating agents

Two scientists, Goodman and Gilman started studying nitrogen mustards as potential anticancer agents in 1942 at Yale University. In a Landmark article (Goodman 1946), they described the treatment of 67 patients with nitrogen mustard. It is a very interesting article since it provides insights of how clinical trials were carried out in the 40th.

For example, after treatment with an alkylating agent, one of the patients showed complete remission (Figure 13.3), but the tumour relapsed after a month and the patient was given two further treatments. Eventually, the patient did not die from the tumour, actually the tumour was small after three rounds of treatment, but he died from side effects of the drug on the bone marrow, in particular thrombocytopenia.

Patient with terminal lymphosarcoma. a) Tumour masses are clearly visible in the axillas and thorax. b) Complete disappearance of tumour masses in axillas and the thorax after treatment with alkylating agents.  Pictures taken from (Goodman 1946).

Figure 13.3: Patient with terminal lymphosarcoma. a) Tumour masses are clearly visible in the axillas and thorax. b) Complete disappearance of tumour masses in axillas and the thorax after treatment with alkylating agents. Pictures taken from (Goodman 1946).

13.2 Common properties of alkylating agents

Alkylating agents are highly reactive compounds. They covalently bind to nucleophilic groups of cellular constituents such as DNA, RNA and proteins, in a process called alkylation. They are generally cytotoxic, mutagenic and carcinogenic. Alkylating agents are not only used to treat cancer, but they can also cause cancer. Structurally, the nitrogen mustards are very similar to the sulfur mustards.

Alkylating agents can be classified into different groups (Table 11.1). They can be monofunctional or bifunctional agents. We will concentration bifunctional agents, in particular Cyclophospamide and Melphalan. But as you can see from this list there are many different types of alkylating agents available.

Later we will also discuss cisplatin, which is classified as an alkylating-like agent. This is a very important difference, because in student exams we often read that cisplatin is an alkylating agent, which is wrong. Again, cisplatin is an alkylating-like agent.

Table 11.1 Different classes of alkylating agents. Please not that platinum containing compounds are alkylating-like compounds and do not belong to the class of alkylating agents.

Bifunctional agents Examples
Nitrogen mustards C yclophosphamide, Melphalan, Mustine
Aziridines Thiotepa
Alkyl sulfonates Busulphan
Nitrosoureas Carmusitne, Lomustine
Monofunctional agents
Add example Add example
Platinum compounds (alkylating-like) Cisplatin, Carboplatin

13.3 Mechanism of action of alkylating agents

What is the mechanism of action of alkylating agents? We want to understand it at a molecular level using nitrogen mustard as an example: (Figure 13.4).

Nitrogen mustard reacts via an initial cyclisation to the corresponding aziridinium ion, by intramolecular displacement of the chloride by the amine nitrogen. This is a SN1 reaction (1). The electron pair of the N-7 nucleophilic centre of the guanine base then attacks the aziridinium ion. This reaction leads to the monoalkylated adduct (2).

Please remember that nitrogen mustard is a bifunctional agent. The second chloride can then be displaced by the amine nitrogen to form a second aziridinium ion, as in step 1 (3).

A second attack of the lone electron pair of N-7 of a second guanine, after the displacement of the second chlorine, forms the second alkylation step that results in the formation of the cross-link between two guanines of for example double stranded DNA.

Detailed mechanism of action of bifunctional nitrogen mustard subsequently reacting with two guanine bases present in DNA leading to a guanine-guanine cross-link.

Figure 13.4: Detailed mechanism of action of bifunctional nitrogen mustard subsequently reacting with two guanine bases present in DNA leading to a guanine-guanine cross-link.

Excursion box: Aziridines are organic compounds containing the aziridine functional group, a three-membered heterocycle with one amine group and two methylene bridges. The formation of a carbocation occurs by separation of the leaving group (the chloride anion) from the carbon atom. This step is slow and reversible. The carbocation reacts with the nucleophile, the free electron pair of the amine nitrogen. This reaction step is fast.

A careful reader may have noticed that the chemical structure of guanine contains several reactive nitrogen atoms. In deed alkylation can also occur at other bases and positions. Although the reaction mainly involves the N-7 position of the guanine base, the reactivity of nucleophilic sites in bases is:

N-7 of guanine > N-3 of adenine > N-7 of adenine > N-3 of guanine

Hydrogen bond networks (dotted lines) of CG and AT base pairs found in double-stranded DNA. The reactive nitrogen atoms and their systematic numbering are shown in colour for both guanine and adenine. Please note that nitrogen atoms that are involved in forming hydrogen bond interactions are not reactive and therefore not available for cross-linking reactions.

Figure 13.5: Hydrogen bond networks (dotted lines) of CG and AT base pairs found in double-stranded DNA. The reactive nitrogen atoms and their systematic numbering are shown in colour for both guanine and adenine. Please note that nitrogen atoms that are involved in forming hydrogen bond interactions are not reactive and therefore not available for cross-linking reactions.

There are two different types of cross links that may occur. First, there can be intrastrand cross-linking, whereby two adjacent guanines of the same strand are cross-linked (Figure 13.6, left panel). Second, interstrand cross-linking can occur between two distinct DNA strands (Figure 13.6, right panel). However, only interstrand cross-linking has been correlated with antitumour activity.

Examples of a) intrastrand and b) interstrand cross-links in DNA. Please note that only interstrand cross-linking has been correlated with antitumour potency

Figure 13.6: Examples of a) intrastrand and b) interstrand cross-links in DNA. Please note that only interstrand cross-linking has been correlated with antitumour potency

So far we have looked at the level of individual bases in DNA and how cross-linking via bifunctional alkylating agents is achieved. But how does this excert anti cancer activity in cancer cells? In other words how can inter-strand cross-links lead to apoptotic cell death?

The replication machinery in eukaryotic DNA replication is a large multi-protein complex (Figure 13.7). Therefore, this model is a simplification, but sufficient to explain the mechanism. For DNA replication, the double stranded DNA is unwound by proteins called DNA helicases, which form a so-called replication fork containing two single-stranded templates. DNA replication is the task of DNA polymerases. The inter-strand cross-links prevent DNA helicases to separate or unwind the two DNA strands so that the DNA polymerases cannot replicate the DNA. Replication and transcription stops. DNA damage is sensed by the cell, the checkpoint is activated and if the DNA damage cannot be repaired, the cell is marked for programmed cell death.

Model of the inhibition of DNA replication by interstrand cross-linking by bifunctional alkylating agents. This simple model explains why the two strand of double-stranded DNA cannot be separated leading to inhibition of DNA replication.

Figure 13.7: Model of the inhibition of DNA replication by interstrand cross-linking by bifunctional alkylating agents. This simple model explains why the two strand of double-stranded DNA cannot be separated leading to inhibition of DNA replication.

13.4 Tweaking the reactivity of alkylating agents

Some of the problems associated in the early days with the clinical use of nitrogen mustards / Mechlorethamine were due to its high chemical reactivity. At physiological pH, this compound can rapidly cyclize to react with water or blood and tissue constituents. With time this type of alkylating agents could be further optimised into less reactive agents. How could reactivity be tweaked then? The placement of an aromatic ring system next to the nitrogen alters the rate of alkylation (Figure 13.8). A suitable substituent on the ring may further control reactivity of the nitrogen.

The chemical structure of the simple but reactive nitrogen mustard compared to a generic aniline mustard with reduced reactivity. The aromatic ring system (coloured in blue) can be further modified by adding distinct substituents (shaded in red).

Figure 13.8: The chemical structure of the simple but reactive nitrogen mustard compared to a generic aniline mustard with reduced reactivity. The aromatic ring system (coloured in blue) can be further modified by adding distinct substituents (shaded in red).

Because of the electron withdrawing effect of the ring moiety, and th eassociated delocalisation of the lone pair of electrons into the ringsystem, the rate of cyclization in these modified nitrogen mustards is slow, rendering them much less reactive than Mechlorethamine. This decreased reactivity allows time for absorption and wide distributionbefore extensive alkylation occurs.

Nevertheless, although the reaction is slower, these compounds alkylate DNA and other cellular constituents in a manner similar to that of nitrogen mustard and their biological effect is the same.

A variety of alkylating agents based on nitrogen mustards are currently in clinical use (Figure 13.9). Cyclophosphamide itself is not active. It is a prodrug that undergoes a microsomal cytochrome p450 dependent metabolic activation in the liver to the active metabolite4-hydroxycyclophospamide. Cyclophosphamide was developed in the hope it might preferentially be activated in neoplastic cells, but this is not the case. Melphalan is a phenylalanine derivate of Mechlorethamine. A third example is Chlorambucil.

Chemical structures of three distinct bifunctional alkylating agents cyclophosphamide, Melphalan and Chlorambucil in clinical use.

Figure 13.9: Chemical structures of three distinct bifunctional alkylating agents cyclophosphamide, Melphalan and Chlorambucil in clinical use.

13.5 Clinical uses, toxicities and route of administration

Alkylating agents can be used to treat a variety of cancers. Cyclophosphamide, which is also the most commonly used alkylating agent, can be used to treat lymphomas, breast, lung, ovarian and endometrium cancers. Melphalan is used to treat multiple myeloma as well as ovarian and breast cancers. Finally, Chlorambucil is used for the treatment of Chronic Lymphocytic Leukemia (CLL).

Common acute side effects of Cyclophosphamide and Melphalan are nausea and vomiting (level 3 to 4) as well as delayed bone marrow depression (Dose-limiting toxicity, DLT), in particular thrombocytopenia and leukopenia. Cyclophosphamide can also lead to alopecia (hair loss) and at high concentrations to Acute Myeloid Leukemia (AML) in about 1 to 2% of patients after three to nine years of treatment (add refs). Treatment with Cyclophosphamide can lead to hemorrhagic cystitis. MESNA, anorganosulfur compound, is used to reduce the incidence of haemorrhagiccystitis when patients receive Cyclophosphamide for cancer chemotherapy. In vivo, Cyclophosphamide may be converted to urotoxic metabolites, such as acrolein. Mesna assists to detoxify these metabolites.

The route of administration depends on different factors, such as the water solubility of the drug and the vesicant properties (blistering agent), so you do not want to apply this to the patients skin. It also depends on the rate of transformation to the active intermediate. Cyclophosphamide and Melphalan are given orally or intravenously. Mechlorethamine, which is very reactive, is given IV, and extravasation should be avoided, the leaking of the drug out of a blood vessel. Chlorambucil is given orally.

Table 11.2 Summary of clinical uses, common side effects and routes of administration for selected alkylating agents.

Alkylating agent Cancer Main side effects Administration
Cyclophosphamide Breast, lung, ovarian and endometrium cancer, lymphoid tumours Nausea and vomiting, bone marrow depression Orally or intravenously
Melphalan Multiple myeloma, ovarian and breast cancers Nausea and vomiting, bone marrow depression Orally or intravenously
Chlorambucil Chronic lymphocytic leukemia (CLL) Nausea and vomiting, bone marrow depression Orally

Bibliography

Louis S. Goodman, Maxwell M. Wintrobe,William Dameshek,Morton J. Goodman,Major Alfred Gilman, Margaret T. McLennan (1946). Nitrogen Mustard Therapy. Use of Methyl-Bis(Beta-Chloroethyl)amine Hydrochloride and Tris(Beta-Chloroethyl)amine Hydrochloride for Hodgkin’s Disease, Lymphosarcoma, Leukemia and Certain Allied and Miscellaneous Disorders. Jama 132, 126-132.

Add article on development of AML upon treatment with alkylating agents.

Add review or book chapter on alkylating agents, look at Nature reviews or good books.