Alkylating Agents PDF

Alkylating Agents Yam Mun Fei School of Pharmaceutical Sciences Universiti Sains Malaysia Lecture outcomes: Define the classification of alkylating agents Identify the generic names of alkylating agents Explain the mechanism of action of alkylating agents Describe drug r...

Alkylating Agents Yam Mun Fei School of Pharmaceutical Sciences Universiti Sains Malaysia Lecture outcomes: Define the classification of alkylating agents Identify the generic names of alkylating agents Explain the mechanism of action of alkylating agents Describe drug resistance mechanisms How a chemical weapon can lead to the discovery of anti-cancer agents? World war 1 Sulfur mustard, or mustard gas has the dubious distinction of being one of the original chemical weapons Bis-(2-chloroethyl) sulfide was first synthesized by London Institute of Physics co-founder Frederic Guthrie in 1860 The malevolent use of mustard gas may be responsible for nearly 100000 deaths and approximately five times as many wartime injuries. How powerful is mustard gas? Mustard Gas Powerful vesicant and blistering effects Mutagenic and carcinogenic Skin irritation and intense itching Conjunctivitis (pink eye), swell and temporary blindness Bleeding and blistering of the respiratory system, damaging mucous membranes and pulmonary edema Historical of development s as less toxic alkylating agents first ALIPHATIC - chlormethine or mustine The figure shows the historic development hydrochloride — is still in clinical use today of the mustard family of agents, from mustard gas through to molecules that have progressively decreasing toxicity to normal cells, to a molecule designed to target the oestrogen receptor in tumour cells. It was realized that if the electrophilicity of mustard gas could be reduced, then less- toxic drugs might be obtained that could be administered orally. This led to the development of the subsequent compounds chlormethine, chlorambucil, melphalan, cyclophosphamide and estramustine, which selective uptake by tumour cells are all still in use today. Melphalan is in which rapid protein synthesis composed of a phenylalanine attached to the mustard, enhancing the selective uptake by tumour cells. Estramustine is the reducing testosterone levels- combination of a mustard and an oestrogen. metastatic prostate cancer cytochrome enzymes (CYP)- release of the bis(2-chloroethyl) group, which is responsible for generating the active cation  Mechanisms of action of alkylating agents transfer of an alkyl group from one molecule to another Nucleophilic molecule or group are rich in electrons and, as thus, donate electron pairs. The principle behind alkylation is that highly electrophilic species, looking for nucleophilic sites to attack, and forming covalent bonds to bases in DNA. Chemical structures of DNA bases, nucleosides and nucleotides, (b) Sites of alkylation on DNA bases, with the major sites shown in red and blue, and with the minor sites shown in green. What is the principle behind alkylation? Alkylating agents are electrophilic agents (electron deficient, unstable). It looks seeks nucleophilic sites on DNA bases to form covalent bonds. Most common site is N7 Guanine. Alkylating agents can form various interactions with DNA bases. They can: bind to a single base on a single DNA strand fit into the DNA groove via intercalation - occurs when ligands of an appropriate size and chemical nature fit themselves in between base pairs of DNA bind within the same strand (intrastrand crosslink) or across strands (interstrand crosslink) block enzymatic functions of DNA-dependent proteins bind to nucleic acid bases results in miscoding and distortion Alkylating agents are considered non-cell cycle specific because its activity is not restricted to a specific cell-cycle phase, although some cells are more susceptible to alkylation in post-mitotic (G1) and synthetic (S) phases. This unspecific activity increases their cytotoxic efficiency Different targets of DNA Alkylation major groove is approximately 50% wider than the minor. Alkylating agents are either monofunctional (react with only one DNA The sugar-phosphate backbones spiral strand) or bifunctional (react with an atom on each of the two DNA strands The different kinds of alkylation products induced by mono-functional (a–c) or bi- functional (d–h) alkylating agents. The major groove of the DNA is the main target of alkylation but N2G and N3A alkylation is also observed for minor groove binding (MGB alkylating agents) Different sites of base alkylation In general base alkylation predominantly occurs on position: guanine N7 and O6, adenine N1 and N3, and cytosine N3 adenine N6, N7, thymine O2, N3, O4, or cytosine O2 alkylation on O6G, N1G, N2G, or O4T results in stable DNA adducts, alkylation on other positions lead to chemically unstable adducts that are converted to DNA damage by opening of the base ring Different sites of base alkylation. The size of arrows is proportional to the frequency of alkylation, regardless of the stability of the alkylated base. (*) The N2G position is the target of minor groove alkylators. Mechanism of action of alkylating agent N-7-guanine residues are located in the same DNA strand, it leads to limpet attachment of the drug molecule to the DNA Formation of cross-bridges, bonds Alkylated G bases may erroneously DNA fragmentation might occur as between atoms in the DNA results in pair with Ts. If this altered pairing is a result of attempt to replace inhibiton of replication or transcription. not corrected it may lead to a alkylate bases by DNA repair permanent mutation enzymes. Chemical structures of main classes of alkylating agents Reactive cationic active electrophilic molecule that will seek out neutrophils from DNA bases or proteins. Different classes produces different species of cationic molecules (+ charged ions)  Nitrogen mustards leukemias and lymphomas The mono-alkylation product can lead to Alkyl-N7G:T base mispairing or can be the target of a second alkylation leading to N7G–N7G inter-strand cross-links Chemical structures of nitrogen mustards. (B) Generation of the aziridinium ion responsible for the alkylation of guanine N7 by nitrogen mustards.  Oxazaphosphorines or oxazaphorines enhance the stability and to reduce the toxicity of nitrogen mustards. interconversion phosphorus nitrogen bond which prevents the direct ionization of the bis(2-chloroethyl) moiety, preventing the formation of aziridinium ion (which is highly toxic) palliative care 4-hydroxy-cyclophosphamide pancreatic cancers A) Chemical structures of oxazaphosphorines. (B) The mechanism of DNA alkylation by cyclophosphamide. Cyclophosphamide is first oxidized by cytochrome P450 leading to 4-hydroxycyclophosphamide, acrolein and a dichloro intermediate, hydrolysis of which generates chloroethylaziridine that is responsible for DNA alkylation and the formation of N7G:N7G cross-links.  Polyaziridines and mitomycin C one or more aziridine cycles mono-alkylation in N7 & N2 position guanine bis-alkylation in N2 position (in the DNA minor groove) leading to intra- or inter-strand cross-links. same mechanism as nitrogen mustards except that the covalent links between most aziridine cycle base nucleophiles and can also form inter-strand N7G:N7G cross-links Streptomyces bacteria (A) Chemical structures of the two electrophilic centers: C1 and polyaziridines used in the clinic, C10 thiotepa and altretamine. The red rectangle indicates the aziridine cycle. (B) Chemical structure of mitomycin C and the enzymatic Thiotepa is rarely used for the treatment of reduction leading to its active ovarian and breast cancers, and as metabolite. intravesical instillation for bladder cancers, (C) The two types of alkylation as well as altretamine for the treatment of ovarian cancers and small cell lung on guanines: mono-alkylation in cancers N7 position and bis-alkylation in N2 Mitomycin C is still used for the treatment of various adenocarcinomas (stomach, position (in the DNA minor pancreas, colon, rectum, breast and groove) leading to intra- or inter- bladder) strand cross-links.  lipid soluble, non-ionized, cell-cycle-nonspecific agents that readily cross the blood– Nitrosoureas brain barrier active intermediates, a chloroethyl diazohydroxide and an isocyanate group chloroethylation of guanines in position O6 that is followed by a cyclization with guanine N1 unstable and rearranges in a product where guanine N1 is cross-linked to nitrogen N3 of the complementary cytosine Isocyanate is responsible for the carbamoylation of the arginine or lysine residues of proteins (A) Chemical structures of the main nitrosoureas. (B) Generation of isocyanate and diazohydroxyde, the two active metabolites of nitrosoureas responsible for protein and DNA alkylation, respectively. Diazohydroxyde leads to chloroethylation of guanines in position O6 that is followed by a cyclization with guanine N1. This cyclic intermediate is unstable and rearranges in a product where guanine N1 is cross-linked to nitrogen N3 of the complementary cytosine. Isocyanate is responsible for the carbamoylation of the arginine or lysine residues of proteins, leading to the inactivation of their biological functions. Alkyl alkane sulfates only compound chronic myeloid leukemias and prior to hematopoietic stem cell transplant in grafts Structure of busulfan (alkyl alcane sulfonates) and of its mechanism of DNA or protein alkylation.  Triazines and hydrazine initial step of enzymatic activation 5-(3-methyl-1- triazenyl)imidazole-4- carboxamide (MITC) Structures of the main triazenes and hydrazines, (A) dacarbazine and temozolomide and (B) procarbazine. They share a common mechanism of alkylation with an initial step of enzymatic Alkylation: O6 position of guanines activation by cytochrome P450 leading to the formation of methyldiazonium ion (red rectangle). Alkylation mainly takes adenine N3 and place on O6 position of guanines. guanine N7 and O6 Platinum derivatives A) Chemical structures of the main platinum derivatives used in the clinic. DACH: diaminocyclohexyl. (B) Intra-strand and inter-strand crosslinks induced by platinum derivatives and their associated frequency. (*)  How cancer cells evade the toxicity effects of these drugs? Cells have a variety of mechanisms to prevent mutations, or permanent changes in DNA sequence. If DNA gets damaged, it can be repaired by various mechanisms, including chemical reversal, excision repair, and double-stranded break repair. In some cancer cells (which are resistant to the effects of alkylating agents), a DNA repair enzyme (O6-MGMT) are highly expressed which exerts a protective mechanism Other mechanisms drug efflux pump P-glycoprotein Reduced DDP and melphalan accumulation Mitomycin C Bleomycin hydrolase cyclophosphamide bind cytotoxic agents such as platinum compounds glutathione S-transferases-conjugate  O-6-methylguanine-DNA methyltransferase (06-MGMT)- mediated direct DNA repair MGMT restores DNA integrity by removing the alkyl groups from the bases Protective mechanism Highly expressed in some tumors Cyclophosphamide CP undergoes complicated metabolism involving several pathways in vivo. The main pathway involves hydroxylation of CP by hepatic microsomal oxidases to form 4-hydroxy-cyclophosphamide (4OH-CP) which is in equilibrium with its acyclic tautomeric form, aldophosphamide (ALDO). These metabolites are transport forms of CP. Oxidation of ALDO by aldehyde dehydrogenase results in the noncytotoxic metabolite carboxyphosphamide Dehydrogenation of 4OH-CP gives another inactive metabolite, 4-keto cyclophosphamide. The final stage of activation, which takes place in cells that are susceptible, involves cleavage of 4OH-CP/ALDO by a b-elimination reaction to yield phosphoramide mustard and acrolein , both of which are highly cytotoxic and represent active forms of the drug. One of the major dose-limiting side-effects of CP is hemorrhagic cystitis which has been attributed to the urinary excretion of its metabolites, particularly acrolein Ifosfamide