Hypoxic tumour cells in solid tumours are oxygen deficient, this is due to their own proliferation as they grow too quickly and become too large and distanced from the blood-oxygen supply. This makes solid tumours impervious to most chemotherapy and radiotherapy treatments. Therefore, to selectively target the solid tumour prodrugs need to be utilised. Prodrugs are bioreductive compounds in which a portion is activated in oxygen deficient environments (usually by reductive activation) releasing an active substituent to the site of intended action. Quinones are the archetypical moieties used in bioreductive compounds, this is due to their reductive activation by endogenous reductases in the body.
The general template for prodrugs is the naturally occurring compound Mitomycin C (MMC). MMC is a naturally occurring prodrug which was first isolated from the broth of streptomyces caespitosus in 1958 by the Kyowa Hakko corporation. MMC contains an indolequinone which can be involved in reductive activation, an aziridine ring for DNA alkylation and a carbamate group which leads to cross linking of DNA. The mechanism for MMC's biological activity was devised in 1963 by Iyer and Szybalski, this mechanism shows its DNA alkylation and cross-linking activity. MMC is not reactive with DNA at pH 7-8 but with enzymatic, metabolic, electrochemical or chemical reduction alkylation is observed with DNA cross-linking commencing in less than one minute.
Reduction of MMC's quinine can happen in two ways, either one electron reduction or two electron reduction, It is suggested that two electron reduction is the prevalent reaction but it is not fully known which biological reducing agent is used with MMC as several enzymes have the potential to reductively activate it. Reduction of the quinine can happen due to one electron reductants such as NADPH-cytochrome c reductase producing a semiquinone or by a two electron reductant such as DT Diaphorase producing a hydroquinone. This converts the heterocyclic nitrogen from a vinologous amide nitrogen into an amine nitrogen which is nucleophillic and can eliminate the -methoxide ion. Tautomerisation of the immonium ion gives fig1.4, which lets the strained aziridine ring open. Once this activation step is completed the drug is active and the electrophillic site at C-1 alkylates DNA at the NH2 site of Guanine (one of the four nucleotide bases DNA is constructed by). The carbamate at C-10 is then displaced at a slower rate than C-1 and causes cross-linking of the DNA at the NH2 site of Guanine on the opposite strand to the C-1 alkylation. Both semiquinone and hydroquinone forms are capable of initiating the cascade of reactions that follow, it has been shown through reactions in water both one and two electron reducing agents produce similar alkylation products which suggests a common intermediate. There has been further proof shown by disproportionation of the semiquinone that hydroquinone is the common intermediate in anaerobic conditions. In aerobic conditions however, the semiquinone is reoxidised by o2 to the quinine preventing reductive activation and DNA alkylation, this shows that MMC is selective in targeting only hypoxic cells. Although MMC is very effective against hypoxic cells it is still one of the most toxic anti-cancer drugs in clinical use. This is due to the formation of hydrogen peroxide (H2O2), Superoxide (O?) and Hydroxide radicals (HO?) during the reoxidation of semiquinone under aerobic conditions.
It is the aim of this project to synthesize an antitumour agent that is hypersensitive and selective with as little toxicity as possible. With the same objectives in mind a number of bioreductive anti tumour agents have previously been synthesized. Skibo and co-workers made PBI compounds which had an alternative hydrolytic strand cleavage mechanism in which the aziridine ring opened upon nucleophillic attack by DNA phosphate. These PBI's possessed an aziridine ring attached directly to a quinone moiety, they showed considerable toxicity especially from the 7 methoxy PBI's. Aldabbagh and co. workers have reported a series of bioreductive anti tumour agents which show that five membered fused ring compounds are more toxic towards human fibroblast cell line (GM00637). The most important in terms of this project is the synthesis and testing of N-[(aziridin-2-yl)methyl]benzimidazolequinone 1 and its synthetic precursor 4,7-dimethoxy-N-[(aziridin-2-yl)methyl]benzimidazole 2. 1 was shown to be marginally more cytotoxic than MMC. In a later publication by the same group it was shown that 10 was significantly less toxic than 9. 10 is also more selective for FA than both 9 and MMC. This information is of extra importance as 10 is hypersensitive towards FA cells and can kill the cells more effectively than MMC even though it cannot cross link DNA as it only has one position for DNA alkylation (the opening of the aziridine ring). This research effectively shows a quinine moiety is not needed for this operation but an aziridine ring is essential, for these reasons the title compound was chosen as it contains the aziridine ring for DNA alkylation and doesn't contain a quinine, in theory this should make the target compound less toxic due to no quinine and still remain hypersensitive towards Fanconi anaemia.
Fanconi Anaemia was first discovered by a Swiss paediatrician called Guido Fanconi in 1927 it is a rare autosomal ressesive cancer susceptibility syndrome classified as a chromeosome break-up syndrome or DNA repair disorder. It is characterised by progressive bone marrow failure, cellular hypersensitivity to alkylating agents and congenital abnormalities such as skeletal malformations, hyperpigmentation, urogenital, renal and cardiac anomalies. People with FA can also develop acute myeloid leukemia (AML) and squamous cell carcinomas, frequently of the neck and head or of the gynecolegic system. F.A. does not have a predisposition to gender or ethnicity, it usually reveals itself in children before they are twelve years old in rare cases patients do not present any symptoms until adulthood. The total number of F.A. patients worldwide is not documented but experts estimate that the carrier frequency (people carrying the defect in a F.A. gene, whose matching F.A. gene is normal) is somewhere between 1 in 600 and 1 in 1,000. The international F.A. registry is currently managed by Dr. Arleen Aurerbach at the Rockerfeller University where case data on over 3,000 patients is stored. F.A. patients usually have a smaller stature than average, experience chronic fatigue and have frequent infections, nosebleeds or gentle bruising. These along with alow white cell, red cell or platelet count can be the first sign of the disease. The most common test for F.A. however is exposure of cells to MMC, treatment of FA patients with MMC causes increased chromosome breakage due to the cellular hypersensitivity of the syndrome. Due to this fact researchers have speculated that a link exists between FA and DNA repair. Recent cell fusion studies have shown at least twelve complementation groups (FANC-A,B,C,D1,D2,) of FA. Eight of these proteins have been defined by somatic cell fusion studies and six have been successfully cloned. These eight proteins have been shown to assemble and form a core protein complex in the nucleus upon replicative stress/DNA damage in the cell caused by cross linking agents, ROS or during the S phase of the cell cycle. The FANC protein complex then causes monoubiquitation of the FANC-D2 protein thereby targeting it to BRCA1 (the breast cancer susceptibility gene) containing nuclear foci, loss of FANC-D2 foci can happen if a mutation of an FA gene disrupts this monoubiquitation which causes hypersensitivity. The interaction of FANC-D2 and BRCA1 suggests that the complex does in fact have a role in DNA repair. FANC-B and FANC-D1 (the unidentified subtypes) have been shown to contain biallelic mutations in BRCA2 and express truncated BRCA2 protein. It now appears that FANC-D1 and possibly FANC-B are in fact BRCA2. All of this shows that FA proteins and BRCA1 operate in a common pathway the DNA repair pathway which when mutated can cause a variety of cancers most commonly breast cancer. These pathways have been proposed by other researchers as BRCA pathway and FA-BRCA network. Defects of the FA genes have been found in a variety of human cancers not only breast cancer to which the BRCA genes are most commonly associated this includes patients without FA. Research into the area of the FA pathway is hoping to yield possible mechanisms for genomic instability in cancer. This new connection means that if the target compound does in fact kill FA cells with hypersensitivity and minor normal cell toxicity then it could also be used as a possible anti-breast cancer drug to kill hypoxic cancer cells.