The Onyx Review: The Interdisciplinary Research Journal © 2017 Center for Writing and Speaking 2017, Vol. 3, No. 1, pp. 53–63 Agnes Scott College

Exploiting Nucleophilic Attack in Pooja Venkatesh Agnes Scott College

Alkylating agents - a large class of clinically used chemotherapy drugs - target tumors via various DNA-drug inter/intrastrand linking mechanisms. These drugs form electrophilic compounds under physiological conditions, allowing for nucleophilic attack by intracellular macromolecules, including DNA. This review will focus on electrophilic addition by the nucleotide , whose nitrogen and oxygen atoms facilitate covalent linkage between the reactive pharmaceutical and the DNA strand. Three examples of alkylating agents, mechlorethamine, , and , will be discussed. The first chemotherapeutic drug, mechlorethamine, forms reactive aziridinium rings, following loss of a chloride ion in aqueous solution. The resultant cation is then free to alkylate N-6 of guanine. The second compound, mitomycin C, undergoes bioreduction and ring-opening steps, ultimately forming DNA adducts after bond formation to N-2 of guanine. Finally, lomustine spontaneously decomposes in cells forming an alkylating and carbamoylating agent. While the carbamoylating compound can react with lysine residues to inactivate DNA repair enzymes, the alkylating agent is free to attach to O-6 of guanine. While this class of drugs has proven to be highly effective in chemotherapy, alkylating agents are notorious for their cytotoxicity and poor molecular selectivity because of their reactive electrophilic nature. Significant research into drug development must be conducted to improve clinical outcomes and reduce the deleterious side effects of these agents.

ntroduction to alkylating agents: tissue.2, 3 The early 1900s marked a time Nonhormonal alkylating agents period of significant were the first class of effective research and the first large scale uses of I 4 anticancer drugs developed. Pharmaceuticals toxic mustard gases. Nitrogen mustards within this class form covalent linkages to were developed in the 1930s and found to DNA, thereby halting DNA synthesis and inhibit cell division of lymphoid tissue, bone cancerous cell proliferation.1 Of note, the marrow, and gastrointestinal cells in affected term “alkylating agent” inaccurately soldiers.4 This led scientists to investigate describes the chemistry of these drugs, as the potential of nitrogen mustards in they do not add organic alkyl groups to inhibiting proliferation. A group of target nucleotides. Instead, the entirety of Yale scientists conducted the first the electrophilic, active form of the drug, chemotherapy clinical trials in 1942 using attaches to DNA in a nucleophilic addition nitrogen mustards on a sample of terminal reaction.1 Often, the portion of the molecule lymphosarcoma patients.4 Success of these directly involved in the reaction is composed first clinical trials began common use of of inorganic elements. The mechanisms of chemotherapy agents in the treatment of three subclasses of alkylating agents will be tumors.5 Today, mustards are frequently detailed here. used as anticancer agents with common Research into alkylating, anticancer drug drugs including , development began after physicians , and - all of which observed that World War I soldiers exposed share a bischloroethyl substituent (see to sulfur gases displayed lymphoid aplasia, Figure 1).1 An additional example, or disrupted development of lymphoid mechlorethamine (also referred to as

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) will be reviewed here. specialized forms of cancer.6 Drugs of this Chlormethine is in clinical use for treatment class react in the body to form highly of chronic lymphocytic and myelogenous electrophilic aziridinium ions that are then , bronchogenic carcinoma, and capable of DNA crosslinking, thereby advanced Hodgkin among other terminating mitosis in dividing cells.

Figure 1: Examples of Currently Used Nitrogen Mustards 7 Naturally occurring -containing crosslinking DNA at specific adenine and compounds are a second subclass of widely guanine nucleotide motifs during the late G1 used chemotherapeutic agents that are or S phases of cell division, as well as closely related to the nitrogen mustards. One preventing gene expression by binding of the most toxic antitumor drugs in current inducible promoter sequences.9 MMC is clinical use is the aziridine mitomycin C considered a prodrug as it is converted into (MMC) (see Figure 2).8 MMC was the active electrophile after intracellular discovered and purified from the gram- reduction and ring-opening steps.8 The negative bacterium Streptomyces pharmaceutical is currently being used for caespitosus in 1958 and has been studied for treatment of advanced or metastasized its significant antibacterial and anticancer gastric and pancreatic properties.8, 9, 10 The compound is capable of adenocarcinoma.11

Figure 2: Structure of Mitomycin C 12 As a third subclass, examples of consists of prodrugs that decompose after will also be analyzed as administration to form an electrophilic commonly used chemotherapy drugs. This alkylating agent and a carbamoylating agent. class of chloroethyl-containing anticancer While the alkylating compound forms agents was first developed in the 1960s and interstrand DNA linkages, the second

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compound carbamoylates lysine residues on lomustine (structure shown in Figure 3) will intracellular proteins and has been be outlined in this paper. This drug is demonstrated to inactivate DNA repair currently used in the treatment of multiple enzymes.8 Examples of frequently used myeloma, primary brain tumors, and nitrosoureas include lomustine, , .1, 13 and . The mechanism of

Figure 3: Structures of lomustine 8 While there are multiple nucleophilic preferred, allowing for correct base pairing sites on DNA where attachment to with cytosine. However, addition of certain alkylating agents can occur, this review will alkylating agents onto guanine shifts the focus on addition to nitrogen-7, nitrogen-2, molecule’s equilibrium such that the enol and oxygen-6 of guanine (see Figure 4 for tautomer is preferred. While this may seem numbering convention). Covalent bonding inconsequential, the enol isomer is likely to to DNA not only results in steric blocking, base pair with thymine, rather than cytosine, preventing transcriptional and replication resulting in miscoded messenger RNA enzymes from accessing the strands, but can molecules and dysfunctional protein also result in incorrect base pairing. This is products.8 This paper will discuss the possible because guanine exists as two mechanisms by which the chemotherapeutic tautomers (constitutional isomers), one keto drugs mechlorethamine, mitomycin C, and and one enol form. Under normal lomustine add covalent linkages to guanine physiological conditions, the keto isomer is residues in actively replicating cells.

Figure 4: Structure of the Guanine Nucleotide 8 Discussion: N-7 readily occur, they are not considered As mentioned prior, the DNA pro-mutagenic as linkages at this site are nucleophilic sites of focus in this paper are unstable and often do not disrupt normal N-7, N-2, and O-6 of guanine (see Figure 4). base pairing. Half lives of N-7 guanine Of these sites, N-7 is of particular interest in adducts typically range from 2 hours to 150 cancer research because of its high reactivity hours, a relatively short period compared to relative to other sites of alkylation. This is other DNA-chemical bonds.14 so because the lone pair on the nitrogen can Unlike N-7 of guanine, covalent linkages readily be donated to electrophilic at O-6, methylation in particular, is highly substances, like the nitrogen mustards to be mutagenic. This is so because addition of discussed. While guanine adducts at location electron donating groups at oxygen shifts

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equilibrium of the nucleotide such that the intramolecular rearrangement.19 Under enol tautomer is preferred. Consequently, physiological conditions, chlormethine polymerases wrongly insert a thymine, spontaneously cyclizes to form an instead of cytosine, opposite the guanine aziridinium ion.20 As depicted in Figure 6, resulting in miscoded DNA, mRNA, and the lone pair on the central nitrogen of the protein.15 Alkylation by drugs and drug forms a bond to a distal carbon, environmental at this site is displacing the previously attached .8 uncommon, however when adducts are Of note, this type of intramolecular formed to the oxygen they are most potent. displacement is an example of anchimeric Unfortunately, extended clinical use of assistance, a key characteristic of alkylating agents that target O-6 often chlormethine that allows the drug to be such results in chemoresistant tumors that an effective alkylating agent.21 Having upregulate expression of the DNA repair formed this highly electrophilic ion, enzyme, methylguanine-DNA- chlormethine can now induce DNA-protein methyltransferase (MGMT), capable of or DNA-DNA crosslinks. Covalent linkages transferring attached methyl groups, on to DNA can form after N-7 of guanine guanine, to cytosine residues.16 attacks the slightly positive carbon, breaking Lastly, N-2 adducts also promote the C-N bond in the molecule and incorrect guanine base pairing, but not to the neutralizing the prior +1 charge. Because extent of O-6 adducts. Studies have chlormethine is a symmetrical molecule, the demonstrated that the “Y” family of lone pair on the central nitrogen can repeat translesion polymerases in mammals is the above process by attacking the capable of synthesizing error-free, remaining chlorine-bonded carbon, causing complementary DNA strands despite the removal of the chloride ion. Once the presence of covalent linkages at N-2.17 This aziridinium ion is formed, N-7 of a guanine means while N-2 adducts often occur due to residue on the other DNA strand can now environmental exposure or are attack the slightly positive carbon forming a specifically targeted in chemotherapy, second covalent drug-DNA linkage. This tumors can upregulate expression of interstrand bond formation results in severe translesion polymerases to bypass the steric hindrance of the region of DNA, mutagenic effects of alkylation at N-2.18 preventing binding of replication and A prime example of anticancer nitrogen transcription enzymes to the site of mustards is 2-chloro-N-(2-chloroethyl)-N- alkylation.8, 20 This mechanism of action is methylethanamine (mechlorethamine), depicted visually in Figure 5 below.8 shown in Figure 5 below. With a molecular Structurally symmetrical drugs within the weight of 156.05 g/mol, this molecule acts family follow a similar as an alkylating agent following cross linking mechanism.

Figure 5: Structure of mechlorethamine 22

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Figure 6: Chlormethine Mechanism of Action 8 The aziridine-containing drug, Encoded by the NQO1 gene, this mitomycin C, is converted into an active enzyme forms an active homodimer with alkylating agent under physiological two FAD molecules bound per subunit. Two conditions, making the compound a reduced NADH or NADPH cofactors are prodrug.8 Compared to nitrogen mustards, then utilized to catalyze a reductive two MMC has a far more complicated, electron transfer from NAD(P)H to cytosolic asymmetrical molecular structure composed quinones.23 In vitro this reaction was of a quinone ring system, pyrrolo[1,2- determined to be pH-sensitive, with reaction a]indole, , and aziridine (see rates being highest under slightly acidic Figure 7).10 Activation of MMC begins with conditions.24 Xenograft mouse models found intracellular reduction of the quinone ring to correlations between NQO1 genetic a hydroquinone. Under normoxic conditions, overexpression and MMC sensitivity, as reduction is catalyzed by the flavoenzyme well as correlations with cytochrome P450 NAD(P)H:quinone oxidoreductase I. reductases, suggesting multiple enzymes can catalyze MMC quinone reduction in vivo.23

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Under acidic conditions, the methanol and DNA (see Figure 7 for MMC carbon substituent on MMC is protonated and numbering). Release of NH3 and CO2 from cleaved from the ring.10 Following loss of the ring carbamate group occurs as N-2 of a the methanol, the reduced compound guanine residue on the other strand of DNA undergoes a spontaneous aziridine ring attacks C-10 of MMC. Completion of this opening to form the active alkylating agent. step results in MMC being covalently The nucleophilic NH2 substituent of guanine attached to both strands of DNA at guanine N-2 can then attack C-1 of MMC, forming motifs.8 This mechanism of action is the first covalent linkage between the drug illustrated in Figure 8 below.

Figure 7: Structure of Mitomycin C 10

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Figure 8: Mitomycin C Mechanism of Action 8 As a last mechanism for review, the group undergoes further decomposition. nitrosourea alkylating agent lomustine has a Release of a hydroxide ion and N2 from 2- third unique method of DNA covalent chloroethyl diazene hydroxide leaves a linkage. Following spontaneous highly electrophilic carbonium ion. Unique decomposition in the body, lomustine forms to the nitrosoureas, O-6 of guanine is the an alkylating and carbamoylating agent. In preferred alkylation site. Acting as a Lewis aqueous media, the process begins when the base, oxygen donates a pair of electrons to oxygen of a hydroxide ion attacks the amide an empty p orbital on the carbonium ion hydrogen causing spontaneous release of an resulting in a DNA-drug crosslink. isocyanate group and 2-chloroethyl diazene Dissociation of a chloride ion from the hydroxide.25 While the isocyanate group can attached drug allows for a second DNA now carbamoylate lysine residues on nearby strand linkage.8, proteins, inactivating them, the alkylating 25

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Figure 9: Lomustine Mechanism of Action 8 Overall, while the chemotherapeutic components of cells. Furthermore, antitumor agents reviewed have significant organic drugs are poorly selective and tend to have components, the majority of their alkylating drastic effects on all proliferating cells in the activity originates from their inorganic body. substituents. Covalent addition to DNA While the drugs reviewed have well residues is further facilitated by inorganic established mechanisms of action, new nitrogen and oxygen nucleophiles present on drugs must regularly be developed to guanine nucleotides. While the three combat the increasing chemoresistance described drugs all alkylate DNA, their displayed by aggressive cancers. Resistance mechanisms are quite different from one to mechlorethamine often occurs due to another. Nitrogen mustards have cellular adaptations decreasing active symmetrical bischloroethyl substituents that transport of the molecule into the cell. An initiate aziridinium ion formation, alternative mechanism for resistance is mitomycin C undergoes bioreduction and inactivation of the agent, as performed by aziridine ring-opening, and lomustine aldehyde dehydrogenase enzymes on spontaneously decomposes in solution to nitrogen mustards. This enzyme detoxifies form an alkylating and carbamoylating the primary metabolites of the mustard agent. Alkylating agents have proven to be compounds rendering them non-reactive. highly effective in treating cancers, however Strong associations between resistance and further drug development is necessary to glutathione (GSH) levels have also been reduce cytotoxicity and increase molecular made, suggesting that GSH may protect selectivity of these drugs. cancer cells from exposure to highly Conclusion: electrophilic substances.1 Lu et al. Ultimately, research into the demonstrated overexpression of gamma- development and anticancer properties of glutamylcysteine synthetase, a GSH the nitrogen mustards, aziridine compounds, synthesizing enzyme, in -resistant and nitrosoureas has allowed for great liver cancer cells.26 Furthermore, three strides in chemotherapy. However, the drugs isozymes of glutathione S-transferase (GST) reviewed, mitomycin C in particular, are have been shown to be capable of known for their serious side effects and high conjugating GSH to aziridinium ions, toxicities. Chemotherapy, as a whole, must preventing alkylation of nitrogen mustards continue to be heavily researched. While to DNA. Tumor resistance to nitrosourea electrophiles are effective alkylating agents, alkylation is possible through upregulation they are also highly reactive molecules that of the DNA repair enzyme O6-alkylguanine- can alkylate proteins, lipids, and other vital DNA-alkyltransferase (O6-AT) that

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catalyzes the removal of covalently attached receiving patients including hematopoietic, groups at O-6 of guanine residues, undoing gastrointestinal, gonadal, and pulmonary the interstrand DNA crosslinks of toxicities.1 As mentioned, extended research nitrosoureas drugs.1 must be conducted to alleviate the severe side effects of chemotherapy drug use and Severe toxicity due to alkylating agents improve drug efficacy. affects multiple physiological systems in

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