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48 | Chemistry World | July 2009 www.chemistryworld.org Temozolomide – birth of a blockbuster The history of anticancer drug temozolomide can be traced back over 30 years – and it all started with some novel nitrogen chemistry, says Clare Sansom

The pharmaceutical industry ‘chemistry led’ drug discovery. When the method with his own molecules. reserves the term ‘blockbuster’ for a new research student, Robert In short This synthesis produced the first its biggest-selling drugs – those Stone, joined the laboratory in 1978,  New blockbuster imidazotetrazinones – which, like that achieve sales of more than on a PhD studentship part-funded anticancer agent the triazene drugs, contains three $1 billion during a calendar year. It by pharmaceutical company May temozolomide can trace consecutive nitrogen atoms, although is an accolade that very few drugs & Baker, Stevens’ instructions to its history back to 1970s this time in cyclic form. ‘We formed a achieve. Of the many thousands him were: ‘Make some interesting heterocycle research at brand new ring system that had never of prescription drugs on the molecules’. Aston University, UK been seen before, and made a series of market, only about a hundred are Interesting molecules, to Stevens,  Malcolm Stevens’ variants with different substituents’, blockbusters. They account, however, were reactive heterocyclic ring research initially remembers Stevens. ‘We had a lot of for about a third of all revenue earned systems rich in nitrogen atoms. produced anticancer chemistry to do, to determine their from prescription drugs. Two strands of nitrogen chemistry agent azolastone – but structure and physical properties, One of the latest drugs to gain led to the eventual discovery of the molecule proved and, particularly, if they had any blockbuster status is also the temozolomide’s nucleus, the highly toxic to humans interesting pharmaceutical activity.’ treatment of choice for some of the imidazotetrazine ring system. Firstly,  Temozolomide was The first structure Stone most malignant and intractable by the late 1970s, it was recognised subsequently developed, synthesised contained a chloroethyl of brain tumours. Temozolomide, that some triazenes – molecules and eventually licensed group, and was also found to have marketed in the US as Temodar containing a linear chain of three by Schering-Plough extremely interesting anti-tumour and Europe as Temodal, made nitrogen atoms – had anti-tumour  The drug is now the properties. Another PhD student in $1.02 billion (£650 million) activity. One triazene, dacarbazine treatment of choice for Stevens’ group, Neil Gibson – along for Schering-Plough in 2008. (DTIC), was on the market against certain brain tumours, with Aston academics John Hickman Temozolomide is a DNA alkylating malignant melanoma, (and is still and shows wider promise and Andreas Gescher – showed that agent, an unfashionable molecule in clinical use); it is a pro-drug, as part of an anticancer it was effective with a single dose in this era of targeted anticancer forming the alkylating agent MTIC combination therapy against almost all contemporary treatment. The story of its (5-[3-monomethyl-1-triazeno]​ mouse models of cancer. Not development is a remarkable mixture imidazole-4-carboxamide) in vivo. surprisingly, the Aston group thought of scientific insight, serendipity, The second set of nitrogen-rich they had found the elusive ‘magic persistence and luck. molecules inspiring Stevens were the bullet’ against cancer. The compound Temozolomide’s beginnings can tetrazine heterocycles. By the time was given a lab name, azolastone, and be traced back over 30 years, to Stone arrived at Aston, the group was preparations were made for a Phase I when medicinal chemist Malcolm exploring bicyclic ring systems with clinical trial through the Cancer Stevens set up a multi-disciplinary ‘bridgehead’ nitrogen atoms between Research Campaign (CRC; now drug discovery laboratory in the the rings. Stevens read a paper by Cancer Research UK). ‘It was a great pharmacy department at Aston Günter Ege and Karlheinz Gilbert name, incorporating the azo group, University in central Birmingham, at the University of Heidelberg, Bioactive heterocycles: the name of the lab (Aston) and of UK. In the 1970s, little was known Germany, describing a new synthetic (left to right) DTIC, the student who synthesised it’, says about the nature and mechanisms route to fused tetrazine ring systems MTIC, azolastone Stevens. of most proteins involved in cancer via the interaction of diazoazoles (mitozolomide), and development. This was the era of and isocyanates, and decided to try temozolomide Reality check The bad news started almost immediately, with a controversy over the compound’s name. Carl Schwalbe, a crystallographer colleague, remembers what happened next. ‘May & Baker thought the name too similar to the common antihistamine, azelastine, and made www.chemistryworld.org Chemistry World | July 2009 | 49 Drug discovery

Malcolm Stevens is synthesis is still used to manufacture still looking for novel temozolomide today. heterocycles with antitumour activity Dose dependent MALCOLM STEVENS MALCOLM The first Phase I trial, testing single doses of temozolomide in patients with advanced cancer in different sites, yielded no positive responses. One of the clinical investigators, Ed Newlands of Charing Cross Hospital, London, then suggested a change of dose schedule. Giving temozolomide on five consecutive days changed the picture completely. An unusual number of positive responses for a Phase I trial were recorded, mainly in patients with brain tumours. ‘One or two Phase I patients saw almost biblical cures [with multiple doses of temozolomide], although this is not indicative of reliable clinical efficacy,’ says Sally Burtles, director of drug development at CRC and now Cancer Research UK. ‘These were glioma patients who had been bed- bound, but became able to get up, go about their normal business, and even go on holiday.’ After the initial study, CRC took the compound us change it. It was understandable – agent” – Malcolm [Stevens] was the into Phase II studies in glioma and the idea of a patient being mistakenly only one of us who was prepared to melanoma to demonstrate its activity prescribed a toxic anticancer drug champion it into clinical trials.’ more clearly. for hayfever gave us nightmares – but Mitozolomide’s severe bone But if this very promising molecule we were sorry to lose azolastone. marrow toxicity had been associated was to progress beyond early And I remember Malcolm being with its ability to cross-link DNA. Phase II, Cancer Research furious that the alternative name, The replacement of its chloroethyl Technology needed a mitozolomide, seemed to have no group with the methyl group of partner from the big chemical rationale behind it,’ he says. temozolomide produced a compound pharma sector. More serious disappointment that was less active but gave no ‘We set off on a was to come. The published Phase I possibility of cross-linking DNA; it “roadshow” round clinical trial of mitozolomide was, therefore, expected at least to the US, presenting revealed disappointing results. be free of that toxicity. It might, the the early clinical The molecule that had proved group proposed, ring-open in vivo data to potential so successful in mice was found to form an alkylating agent like partners. The drug to be extremely toxic in humans, DTIC, but under chemical rather was eventually licensed producing profound bone marrow than metabolic control. It was on to Schering-Plough, who still toxicity (thrombocytopenia). An that basis that temozolomide was markets it,’ says Stevens. unkind competitor even named the submitted to the CRC’s newly While temozolomide was molecule ‘Azo-last-one’. launched Phase I / II committee for progressing through the phases Those poor results could consideration as a clinical candidate. of clinical trials to registration, have been the end of the The entire project might have academics were continuing imidazotetrazinone series. May & been stalled before then, however, to elucidate the details of its Baker eventually dropped out of the by events thousands of miles away. mechanism of action. Phil Lowe, programme, wanting nothing more Stone’s original synthesis involved a PhD student working with to do with such cytotoxic drugs, so methyl isocyanate, the compound Schwalbe, had already determined Stevens appointed Cancer Research that caused the Bhopal disaster the crystal structures of both Technology (CRT). Even at Aston in India in December 1984. For mitozolomide and temozolomide, there was no consensus as to how to some time after that it was almost confirming the molecular proceed. Another colleague, John impossible to source this ‘pariah configuration that Stevens Slack, remembers meetings to decide molecule’, and the project could only had proposed. ‘Computational which candidate drugs to put forward go forward at all with bulk supplies chemistry, along with an analysis for clinical trials. ‘According to our of both methyl isocyanate and of hydrogen bonding patterns,

NATURE colleague Simon Langdon, there was temozolomide. Fortunately, May & yielded some unexpected only one other imidazotetrazine that Baker had manufactured enough information,’ says Schwalbe. demonstrated anti-tumour activity, temozolomide for pre-clinical and ‘We found that the carbonyl and that was the methyl derivative: Phase I studies, and the formulation carbon atom in the ring had an temozolomide. There wasn’t much and development work was At physiological pH, unusually high positive charge, support for it, as it was likely to be undertaken at Aston by an academic temozolomide generates supporting a mechanism in which “just another toxic DNA alkylating group funded by the CRC. The Stone the DNA-alkylating MTIC the ring system is broken down 50 | Chemistry World | July 2009 www.chemistryworld.org temozolomide with an MGMT inhibitor could improve the response of these patients, if one could be developed. However, one of the advantages of temozolomide that contributes to its premier position in

CNRI / SCIENCE PHOTO LIBRARY PHOTO SCIENCE / CNRI the market is that it can be delivered orally. Many drugs for advanced cancer cannot be formulated for oral delivery and must be given in hospital: co-prescribing temozolomide with any non-oral drug would be less convenient for the patient and so would detract from the drug’s significant advantage. Temozolomide has proved a very significant advance for many brain tumour patients, but it is not universally successful, and it can only lengthen life, not cure the cancer. Rees still describes brain tumours as ‘a core area of unmet medical need… we still need new treatments.’ So, where will ‘the next temozolomide’ – or even the next blockbuster anticancer agent – come from? It may be from the same stable. Stevens, now an emeritus professor at the University of Nottingham, is still developing temozolomide analogues at this position.’ Experiments by Although temozolomide was Temozolomide is now a and molecules with similar, nitrogen- biochemist Mike Tisdale established developed before the target-driven first line treatment for rich, heterocyclic structures that that temozolomide is a pro-drug, approach to drug design became glioblastoma multiforme might have even better antitumour a ‘molecular delivery device’ that fashionable, it does have a very activity. He is now also chief is stable under acid conditions but distinct molecular target – the O6 scientific officer of an innovative ring-opens at physiological pH to oxygen atom of guanine residues drug discovery company, Pharminox, methylate, specifically, oxygen O6 of within DNA, particularly those which has further molecules with guanine residues, particularly those within poly-guanine sequences. novel mechanisms in late stage pre- within runs of guanines. It can therefore be expected to clinical development and Phase I Temozolomide was first licensed interact with proteins that repair clinical trials. in 1999, initially as a second- damaged DNA. This can be used Above all, the temozolomide line treatment for glioblastoma both to predict the response of story shows the importance of multiforme, the most malignant individual patients to temozolomide, collaboration in drug development. grade of glioma. The indications for and to develop drugs that might act It was a textbook example of a which it can be prescribed have been synergistically with it. development strategy, led by increasing since, along with its sales. chemistry, that also involved It is now given with radiotherapy Doubling up disciplines as diverse as as the first line treatment for The enzyme 6-O-methylguanine- crystallography, pharmacology glioblastoma multiforme, and a DNA methyltransferase (MGMT) is and pharmaceutics, and, once the second line treatment for other a DNA repair protein that removes importance of the DNA methylating malignant gliomas after relapse. methyl groups from the O6 position agents was discovered, molecular Jeremy Rees, consultant neurologist of guanine and can thus ‘undo’ the biology and pharmacogenetics. at the UCL Institute of Neurology, action of temozolomide. Like many And it shows the importance of National Hospital for Neurology and proteins, MGMT can be ‘switched leadership. ‘Every drug discovery Neurosurgery, London, explains how off’ or silenced by methylation of group needs someone who believes it has altered the outlook for these DNA residues in its promoter region, in their molecules and will champion very sick patients. ‘When given with upstream of the coding sequence. them through difficult times. radiotherapy, temozolomide has Recent clinical trials have Malcolm Stevens was just such a improved the two-year survival rates suggested that those glioma patients champion for temozolomide,’ says for these glioma patients from 10 per with tumours in which the MGMT ‘Stevens was Burtles. cent to 25 per cent and the four- to gene has been silenced by promoter five-year survival rate has improved methylation are more sensitive the only one Clare Sansom is a freelance science from almost negligible to 10 per to temozolomide given with prepared to writer based in London and cent.’ Rees is currently involved in a radiotherapy. One study showed Cambridge, UK. Phase III Cancer Research UK trial clinical response to temozolomide champion comparing temozolomide against in 68 per cent of glioma patients temozolomide Further reading radiotherapy as a first-line treatment with MGMT silencing compared to M F G Stevens, Temozolomide: From cytotoxic to into clinical molecularly targeted agent. In: S Neidle, Cancer for lower-grade, less malignant 25 per cent of patients with active Drug Design and Discovery, p157-172. New York: gliomas. MGMT. It is possible that giving trials’ Academic Press, 2008 www.chemistryworld.org Chemistry World | July 2009 | 51