chemical approaches to the discovery and

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CHEMICAL APPROACHES TO THE DISCOVERY AND DEVELOPMENT OF CANCER THERAPIES Stephen Neidle* and David E. Thurston‡ Abstract | The chemical sciences are essential for the process of anticancer-drug discovery, and a range of chemical research techniques is needed to develop clinically effective drugs. Improved understanding of the cellular, molecular and genetic basis of cancer has increased the number of drug targets available. What chemical approaches are used to develop agents that target specific features of cancer cells and make these therapeutics more effective? We outline the roles that chemical synthesis and understanding of drug uptake have had in drug discovery over the past 100 years, as well as the chemical insights derived from knowledge of the threedimensional structure of targets. SYNTHETIC CHEMISTRY

The creation of new molecules by means of a series of defined chemical reactions. CREATIVE CHEMISTRY

The intellectually driven application of chemical principles.

*Cancer Research UK Biomolecular Structure Group and ‡ Gene Targeted Drug Design Research Group, The School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX, UK. Correspondence to S.N. e-mail: stephen.neidle@ ulsop.ac.uk doi:10.1038/nrc1587

Chemistry is a very broad subject, and can justly claim to encompass many aspects of the study of biological molecules. To most researchers in the cancer field, the term ‘chemistry’ is often used in a much narrower way and is synonymous with SYNTHETIC CHEMISTRY as a tool for the discovery of anticancer drugs. However, the role of the chemical sciences in cancer therapeutics covers far more than synthesis per se; the active involvement of many aspects of chemistry is needed for our knowledge of the cellular, molecular and genetic basis of cancer to be translated into effective therapies. Drug discovery requires knowledge of the structure and reactivity of small molecules and macromolecules, and of the ways in which the molecules interact by means of both covalent and noncovalent recognition during signal transfer. Several effective clinical agents have been discovered by chemists, who possess a deep understanding of these topics and, in particular, knowledge of the relationships between structure and reactivity/activity properties for particular classes of molecules, leading to insights into potential anticancer agents. More recently, CREATIVE CHEMISTRY has started to overlap with biology, with considerable success and promise for the future. Looking back at the history of cancer therapies, we find that chemistry has had several different roles in the discovery and development of most new anticancer

NATURE REVIEWS | C ANCER

drugs. We outline these roles at the outset because, in our view, some of these are still relevant to drug discovery today. The dawning of cancer chemotherapy is generally accepted to have been the serendipitous discovery of the mustard family of agents in the first half of the twentieth century. The application of medicinal chemistry to sulphur mustard gas led to agents that are still clinically useful today1. Although ‘serendipity’ is not a reliable source of new anticancer-drug leads, more molecules with interesting anticancer properties might still appear through chance in the future, especially from natural products. Second, synthetic chemistry has been used to modify drug leads discovered in plant material — the so-called ‘semi-synthetic’ approach. For example, in the 1960s the natural product paclitaxel was identified by Monroe Wall as a potent and clinically useful anticancer agent2,3, yet synthetic chemistry was needed to make it (and subsequent paclitaxel-related compounds) available in sufficient quantities before they could become clinically available drugs. A third way in which chemistry has generated anticancer-drug leads is through screening programmes that use cancer cell lines in vitro. Organic chemists often produce large numbers of new compounds for the purpose of developing novel synthetic pathways, rather than for the intrinsic value of the compounds per se. In

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PRODRUG

Summary

An inactive compound that is activated to a reactive drug species, preferably within target tumour cells, possibly by metabolism, selective action of a cancer-cell-specific enzyme, or by differences in pH/oxygenation between tumour and non-tumour tissue.

• The diverse roles of chemistry in the discovery of anticancer drugs include not only chemical synthesis, but an understanding of drug–target interactions and of the features of drug molecules that govern uptake and metabolism. • The role of chemistry in the development of anticancer drugs began with the mechanism-driven modification and synthesis of the nitrogen mustards. • Since the 1950s, several important drugs have been discovered by screening novel organic compounds and natural products using in vitro cell lines. • Structural biology and chemistry represent new approaches for discovering anticancer drugs, and are being used to determine the molecular aspects of kinase and protein–protein inhibition. • In silico screening can be used to screen large virtual libraries of compounds against the known structure of a target. • Molecules are being developed that selectively target a unique DNA sequence to inhibit transcription. • Synthetic medicinal chemistry has made important contributions to the development of targeted therapies and prodrugs — otherwise inactive compounds that are converted in tumour cells to active species. • Chemistry is essential for transforming ‘lead molecules’ into drugs. This requires optimizing the distribution, metabolism and excretion properties of a molecule as early as possible in the drug-discovery cycle.

the 1950s, the National Cancer Institute (NCI) in the United States recognized this as a valuable resource and set up a series of screening programmes that invited chemists from around the world to submit their novel compounds for screening against a range of in vitro tumour cell lines4–6. The fourth, and most recent, application of chemistry has been to generate drug leads following the discovery of a new target; for example, the specific identification of a cancer-related gene from the sequence of the human genome. Such targets can be proteins, enzymes or nucleic acids. Knowledge of the three-dimensional Mustard gas (sulphur mustard)

Chlormethine (mustine)

Chlorambucil

Cl

Cl

Cl

H3C N

S

HOOC

N

Cl

Cl

Too toxic to be used in humans

Cl

Aliphatic mustards have sufficient therapeutic index to be used in humans

Estramustine

Aromatic mustards are less electrophilic and react with DNA more slowly. Can be administered orally.

Cyclophosphamide

Melphalan

OH

Phenylalanine

Cl

Oestrogen O Cl

An attempt to target oestrogen-dependant tumour cells

Cl N

H2N Cl

O Cl

NH P N O O

Represents an attempt to release mustard agent through enzymatic degradation

COOH

Cl

Represents an attempt to enhance cellular uptake through the phenylalaninetransport mechanism

Figure 1 | Structures of mustards. The figure shows the historic development 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 are all still in use today. Melphalan is composed of a phenylalanine attached to the mustard, enhancing the selective uptake by tumour cells. Estramustine is the combination of a mustard and an oestrogen.

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structure of a target, obtained using X-ray crystallography, can lead to the rational design of specific inhibitor molecules that target functionally important parts of the structure. A fruitful extension of this is the generation of virtual libraries of potential drug molecules (for which the three-dimensional structure has been defined) to be used for in silico screening against a threedimensional target structure. Structure-based design is currently considered to be among the most productive approaches for generating drug leads, whereas drug discovery through screening is now often difficult to justify and fund in either the academic or industrial sectors. Most drugs that have been discovered through screening are cytotoxic agents, but now the goal of most efforts to discover anticancer drugs is to target molecular aberrations that are specific to tumour cells. Whether this goal will be attained for common human solid cancers that have become established is still unclear, in view of the widespread misregulation of signalling pathways. Finally, there have been many advances in PRODRUG development and in other so-called ‘targeted approaches’ to treating cancer. We will outline the crucial role that chemistry continues to have in, for example, the design of prodrugs and photoactive agents and the production of novel linkers to couple drugs to antibodies. Finally, we will illustrate how knowledge of the chemical properties of drug molecules is important for transforming lead anticancer molecules into real drugs, by considering features such as hydrophobicity (relevant to cellular uptake) and reactivity (relevant to metabolism). Serendipity and chemistry

The nitrogen mustards were developed as derivatives of sulphur mustard gas (FIG. 1), which was first synthesized in 1886 but made its debut as a war gas in 1917. Astute clinical observation of military personnel exposed to sulphur mustard showed that it lowered the whiteblood-cell count, and so it was cautiously tested in humans as a treatment for leukaemia and found to be antimitotic. Although it proved too toxic for this purpose, Gilman1 hypothesized that the toxicity was related to the high electrophilicity of the agent, which made it www.nature.com/reviews/cancer

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REVIEWS very chemically reactive towards electron-rich groups such as the phosphates in nucleic acids. He made some less electrophilic analogues by exchanging the sulphur atom for a substituted nitrogen. This led to the nitrogen mustards, which have an acceptable THERAPEUTIC INDEX in humans, and were introduced into the clinic in 1946 (REF. 7). The first ALIPHATIC example of a mustard — mechlorethamine hydrochloride (now called chlormethine or mustine hydrochloride) — is still in clinical use today. The aromatic nitrogen mustards (for example, chlorambucil) were developed through the application of medicinal chemistry and were introduced in the 1950s as less toxic alkylating agents than the nitrogen mustards. The aromatic ring acts as an electron sink, withdrawing electrons from the nitrogen atom and discouraging aziridinium ion formation (FIG. 1). Unlike aliphatic mustards, the central nitrogen atom of an aromatic mustard is not sufficiently basic to form a cyclic aziridinium ion because the nitrogen electron pair is delocalized by interaction with the π electrons of the aromatic ring. Therefore, alkylation most likely proceeds through an S 1 MECHANISM, with normal carbocation formation (resulting from chloride-ion ejection) providing the rate-determining step. Therefore, aromatic analogues such as chlorambucil are sufficiently deactivated that they can reach their target DNA sites before being degraded by reacting with collateral nucleophiles. This means that they can be taken orally, a significant advantage. Further chemistry-based design work on the mustard family has improved tumour-cell selectivity and reduced bone-marrow toxicity. For example, melphalan (also called phenylalanine mustard; Alkeran) was designed to be more selective based on the hypothesis that the attached phenylalanine might allow selective uptake by tumour cells in which rapid protein synthesis occurs, through an L-phenylalanine active transport mechanism. In view of the significant clinical activity of the mustards, considerable effort has been devoted to attaching mustards to other therapeutic moieties, either to achieve combination therapy, or to target the mustard to a particular organ or cell type. The most commercially successful of these approaches has been estramustine — a combination of a mustard and an oestrogen, which seems to provide both an antimitotic and (by reducing testosterone levels) a hormonal effect. This drug, which is given orally, is used as primary and secondary therapy in patients with metastatic prostate cancer. The discovery of mustards is not the only example in which serendipity and chemistry have together led to the discovery of clinically effective anticancer agents. Cisplatin was discovered in 1963 through studies by Rosenberg and co-workers on the passage of an electric current (using platinum electrodes) through suspensions of Escherichia coli bacteria 8 using ammonium chloride as an electrolyte. Analytical chemical expertise was then used to establish that the platinum electrodes used in the experiment had reacted with constituents of the culture N

THERAPEUTIC INDEX

Ratio of the drug dosage that is required for toxic effect to the dosage required for therapeutic effect. ALIPHATIC

An organic molecule that contains fully saturated carbon atoms. That is, a molecule without any aromaticity. SN1 REACTION

Displacement of an atom or group by a nucleophilic atom or group; the process occurs as a unimolecular or bimolecular reaction, respectively. STEREOCHEMISTRY

The three-dimensional relationship of atoms to each other in a molecule. TOTAL CHEMICAL SYNTHESIS

The multistep synthesis of complex molecules, usually natural products, from simple precursors.


medium to form diamminetetrachloroplatinum(IV) (PtIV(NH3)2Cl4) (REF. 9), which inhibits division of bacterial cells. Rosenberg et al. then hypothesized that the precursor compound, cisplatin, would also affect cell division in mammalian systems, and found that it showed selective toxicity both in vitro and in vivo against experimental tumours. Semi-synthetic agents from natural products

Small molecules derived from plant and microbial sources have long had a significant role in cancer therapy. As a class, they have the advantage of having greater chemical diversity than typical synthetic chemical libraries (reviewed in REF. 10). They also tend to have greater chemical complexity, often with complex STEREO11 CHEMISTRY . It is these features that make natural products very specific for particular targets, and it is these features that combinatorial libraries of relatively simple synthetic compounds try to mimic. However, complex natural products are normally difficult, if not impossible, to produce on a commercial scale by TOTAL CHEMICAL SYNTHESIS, except by using fermentation technologies, which have been used for many antibiotics and the promising anticancer agents called the epotholones. One way around this problem is to use understanding of the chemistry of the complex natural product to complete its synthesis from a more easily obtained naturally occurring precursor; this ‘semi-synthesis’ is the approach used to manufacture the anticancer agent paclitaxel, starting with 10-deacetyl baccatin, which is readily obtained from needles of the English Yew. This is a renewable source, in contrast to natural paclitaxel, which is found in the bark of the rarer Pacific Yew tree. The screening of extracts of plants, bacteria and fungi for cytotoxicity in cancer cell lines is still an active area of research throughout the world, and it is entirely possible that novel anticancer agents, including those amenable to a semi-synthetic approach, will be discovered in the future. Plants are used in traditional Chinese medicine to treat diseases including cancer, indicating that there might still be value in the so-called ‘ethnopharmacology’ approach to drug discovery — the study of natural products described in folklore. Organic synthesis and screening

Ever since Wöhler reported the first synthesis of an organic compound from inorganic constituents in 1828, organic chemists have synthesized novel compounds to explore structural diversity and new synthetic reactions and pathways. When biologists learnt to grow cancer cells in culture, the practice of screening novel compounds for cytotoxic action began. Although in vitro screening is practiced in numerous laboratories throughout the world, one of the best-known screening facilities has been developed by the NCI, who routinely screen large numbers of novel compounds against 60 cancer cell lines (the so-called ‘60-cell-line screen’)4,5. This in vitro screen is funded by the United States government and accepts compounds from both academic and industrial chemists worldwide. (For further information on this screen, see the In Vitro Cell Line Screening Project link in the Online


REVIEWS

Temozolomide

Monomethyl triazine

O

H2N

N N

N

H2O

O

H2N

N

N N

N CH3

O

H 2N

H N

N

NH HN

N CH3

O

NH

+ CO2

N N

CH3

H+

Tetrazinone ring Methyldiazonium ions (DNA-methylating species) N2 + CH3 DNA

DNA

O

H2N

N N

5-aminoimidazole-4carboxamide

Molecular targeting of enzymes and proteins NH2

CH3

N

NH

Methylated DNA

Repair?

Figure 2 | Temozolomide and its chemical reaction with DNA. Temozolomide acts as a prodrug, transporting a methylating agent (the methyldiazonium ion) to guanine bases within the major groove of DNA. The mechanism of activation involves chemical (as opposed to enzymatic) hydrolytic cleavage of the tetrazinone ring at physiological pH to give the unstable monomethyl triazene, which then undergoes further cleavage to liberate the stable 5-aminoimidazole-4carboxamide and the highly reactive methyldiazonium methylating species. After methylating DNA (or otherwise decomposing by reacting with water to give methanol), the latter forms N2. The generation of the small stable molecules 5-aminoimidazole-4-carboxamide, CO2 and N2 provides the driving force for the mechanism of action of temozolomide. The antitumour activity of temozolomide correlates with its accumulation in tumours, where it methylates guanine O6 and N7 positions in DNA. Cellular selectivity might be attributable to the slightly different pH environments of normal versus malignant tissues in the brain, coupled with differential capacities to repair the methylated lesions by O6-alkyl-DNA alkyltransferase or other repair processes. A fragment of duplex DNA is shown with a methylated guanine base.

HIGH-THROUGHPUT

Automated processes of biological, biochemical or biophysical assay that examine very large numbers of compounds (or compound mixtures) on a short time scale, and enable active compounds to be rapidly identified. SYNCHROTRON

A particle accelerator that can produce extremely intense X-rays, used for studying very small and/or poorly diffracting macromolecular crystals.

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Although it is likely that organic chemists will continue to produce novel compounds and have them screened for cytotoxicity and other biological activities for many years to come, the current predominant view is that this approach is no longer the high-yielding source of anticancer-drug leads that it was considered to be in the 1970s and 1980s. Nevertheless, there is a good chance that chemists with biological intuition will still identify important cancer-selective agents in the future.

links box.) Compounds with interesting activity profiles across the 60 cells lines then progress to in vivo studies and preclinical development if appropriate. One of the best recent examples of a new chemical entity discovered through a combination of organic chemistry and screening is temozolomide (Temodal; FIG. 2). In the 1980s, a wide range of analogues of the heterocyclic imidazotetrazine family were prepared and studied by Stevens and co-workers6, with the intention of developing anticancer drugs. Novel compounds were routinely evaluated in cancer cell lines and two analogues (temozolomide and mitozolomide) were discovered that had interesting biological activity. Temozolomide took 15 years to reach the market in the late 1990s but it is now a well-established agent for the treatment of brain tumours (gliomas), with very good oral bioavailability, uptake and distribution properties, and is also used experimentally to treat melanoma. The considerations of chemistry during its design led to several important features, including unusually low molecular weight (194 Da), hydrophobicity, and its ability to undergo conversion to the active ring-opened DNA-methylating species at physiological pH without requiring metabolism.

Today, the combination of modern structural biology and chemistry represents the most sophisticated and productive means for discovering new anticancer-drug lead compounds (FIG. 3). The three-dimensional arrangement of atoms in a molecule defines its stereochemistry and reactivity. Therefore, knowledge of the geometry of an enzyme’s active site and the environment of a reactive base in a nucleic acid provides direct information about the distribution and availability of hydrogen-bond donors and acceptors, about hydrophobic regions, and about the distribution of net charge and chemical reactivity. All of these can be exploited through the design of small-molecule inhibitors. The determination of the structures of putative macromolecular targets, principally by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, is increasingly automated, owing to advances in key technologies such as HIGH-THROUGHPUT protein expression and crystallization screening12, which are coming from structural genomics. The ease and speed of ab initio crystal-structure analysis has been markedly improved over the past few years by several technological advances, not least owing to the widespread application of powerful structure-determining methods. The sole caveat is that a suitable source of high-intensity X-rays is required, normally at a SYNCHROTRON facility. However, technological advances in laboratory high-intensity X-ray sources promise to further increase the number of macromolecular structures being reported. At present, the Research Collaboratory Structural Bioinformatics Protein Data Bank — a worldwide repository for the processing and distribution of three-dimensional structure data for proteins and nucleic acids — receives about 2,000 structures per annum, although as of February 2005 only a small fraction of the total 29,733 X-ray and NMR entries in the public domain represent structures of human proteins and enzymes. There is no doubt that the combination of structural biology and chemistry will continue to grow as a major source of novel anticancer drugs as more macromolecular structures become available. However, at present, the gulf between the number of experimentally determined macromolecular structures and the number of proteins encoded by the human genome remains wide. Even the much smaller goal of working with those 1% of proteins implicated in human cancers is still distant, not least because of the large size of many of the proteins and their functional complexes; for example, BRCA1 and BRCA2 — the breast cancer susceptibility proteins — are 190 kDa and 384 kDa, respectively. www.nature.com/reviews/cancer


REVIEWS

• Define the target macromolecule

• Isolate target molecule directly from cells, by cloning the gene, or by protein expression and purification (if a protein or enzyme) • Crystallize molecule

• Determine three-dimensional structure of the native target

• Find a plausible inhibitor from a known starting point or from screening • Model its interactions with target

• Co-crystallize inhibitor and target • Determine its structure

• Model used to suggest improved inhibitor • Synthesize improved inhibitor

• Optimal compound obtained • Undertake cell-based and other assays

Figure 3 | The principal steps in structure-based drug design using crystallography. Structure-based drug design normally starts from an experimental structure, with a lead inhibitor either being derived from the natural substrate, or, increasingly, from in silico analysis. A purely screening approach to a known target using large libraries of compounds will not succeed except by chance or when a focused library is used following the discovery of a chemical lead. This is because there are estimated to be 1062–1065 distinct possible chemical entities with drug-like molecular weights. Only 108 of these exist at present, either because they have been synthesized or because they occur naturally. Therefore, combinatorial approaches can only ever sample a vanishingly small percentage of total chemical space.

PHARMACOPHORE

The group of atoms in a drug molecule that are responsible for the pharmacological effects of the drug.

Targeting kinases. There are several ways in which small molecules can be used to inhibit the function of an enzyme, preferably of known three-dimensional structure. The traditional and well-tested approach is to design small molecules to interact with an active site or another operationally important location such as an ATP-binding site. Recent examples of the successful application of this approach are the development of kinase inhibitors such as imatinib (Glivec) and erlotinib (Tarceva). Such design must always take into account the feasibility of synthesizing the inhibitor, using as few steps as possible, and preferably in a way that enables scaling up for manufacture to be achieved. Large libraries of compounds can also be tested using simple in vitro or cell-based high-throughput assays against the presumed target. The kinase superfamily comprises an important set of targets for specialized


libraries; there are well over 500 kinases encoded in the human genome, and many have significant roles in particular human cancers. Considerable effort has therefore been put into investigating several cancer-relevant kinase subcategories, such as growth-factor receptor kinases13 and cell-cycle inhibitors (cyclindependent kinases)14. Specificity for a particular kinase has been the goal of most drug-discovery programmes in the kinase field, especially those based on structural biology and chemistry15. Although it is inherently difficult to differentiate between the well-conserved ATPbinding sites of the various members in a particular kinase family, this problem has not hindered many research groups from attempting to discover agents that have high specificity for single kinases16. However, relative non-selectivity is turning out to be therapeutically advantageous, as is now apparent in the case of imatinib. This compound was originally designed to be a specific inhibitor of the ATPbinding site of the ABL kinase component of the BCR–ABL translocation gene product in chronic myeloid leukaemia, binding to an inactive conformation of the enzyme 17. However, it also inhibits the platelet-derived growth-factor receptor (PDGFR) tyrosine kinase, and has more recently been shown to be an effective (and clinically useful) inhibitor of the KIT receptor tyrosine kinase that is overexpressed in stromal tumours of the gastrointestinal tract18,19 and this contributes to its clinical efficacy. The determination of the crystal structure of imatinib bound to the KIT tyrosine kinase ATP site has revealed that the drug maintains this kinase in a conformation that disrupts its membrane association 20. So, relatively non-selective kinase inhibitors might be of greater use in the clinic than highly selective ones, in part because of the redundancy inherent in many signalling pathways and therefore the potential involvement of several kinases in a particular cancer type. Screens for multiple kinases are now widely available (see, for example, the Kinasource link in the Online links box). There is considerable effort 21 being invested in developing second-generation ABL (and KIT) ATPsite inhibitors that overcome the problem of clinical resistance to imatinib. Resistance is a serious barrier to the continuing effectiveness of imatinib in many patients and is due to mutations in the ATP-binding site that effectively block imatinib from binding. A search for potential drugs that can show anticancer activity in the resistant phenotype has resulted in the synthesis of compounds such as BMS-354825 (REF. 22). This compound is a dual SRC–ABL inhibitor that was predicted on the basis of structural considerations to bind ABL in both active and inactive conformations, as was subsequently shown by the crystal structure of its complex with the ABL kinase. Therefore, BMS-354825 does not contact the same mutated residues in the active site as imatinib. It is 100-fold more active than imatinib, and shows activity against a panel of imatinib-resistant BCR–ABL mutants23. It is now in clinical trial.


REVIEWS a Br

OH O

N

b

N

N O N

CH3 O

Br

Figure 4 | Nutlin-2. a | The chemical structure of the p53–MDM2 inhibitor Nutlin-2. b | A view of the crystal structure31 of the Nutlin-2–p53–MDM2 complex, showing a bound Nutlin-2 molecule in the p53 pocket, with its two bromophenyl groups buried in the hydrophobic clefts.

Homology modelling to obtain a three-dimensional structure in the absence of an experimental structure, using known kinase structures as starting points, can be moderately successful when homology is high, in the case of individual subfamilies of kinases, because there are now well over 150 kinase crystal structures in the public domain. A recent study used the kinase-domain structures of PDGFR, vascular endothelial growth factor receptor, epidermal growth factor receptor, fibroblast growth factor receptor, p38 and SRC, together with libraries of known inhibitors and random compounds to select some of the known inhibitors from a random library16. Inevitably, their accuracy is low compared with experimentally determined complexes. Targeting protein–protein complexes. The functional roles of most proteins and enzymes involve macromolecular interactions. Intervention in crucial protein–protein interactions in signalling pathways is therefore a major goal for cancer therapy. The design of small molecules that can block the interaction of two proteins has been problematic for a long time, but there has been recent success in the discovery of inhibitors of the important p53–MDM2 interaction. The tumour-suppressor protein p53, which is mutated in over 50% of human cancers, has a pivotal role in regulating the growth of many tumours, and in the response to cytotoxic agents. Those tumours with mutated p53 generally respond poorly to cytotoxic agents, and there is a loss of apoptosis in affected cells. p53 is itself regulated by the oncogene MDM2, which binds to p53, causing its inactivation and the targeting of the complex for destruction by ubiquitylation. Inhibition of the p53–MDM2 association leads to normal p53 function and the reactivation of apoptotic pathways. Inhibition of the p53–MDM2 complex was demonstrated several years ago using potent synthetic peptides24, and the crystal structure of a p53–MDM2 complex has identified the key interactions involved in its inhibition, notably through a hydrophobic cleft in the MDM2-recognition surface25. However, transforming these into drug-like small molecules has proved challenging.

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The fungal metabolite chlorofusin has been identified as a moderately potent p53–MDM2 inhibitor26, which binds directly to the amino-terminal domain of MDM2 (REF. 27). The high molecular weight (1,363 Da) and chemical complexity of chlorofusin have, until recently, precluded its development as a drug. Synthesis of the chlorofusin cyclic peptide moiety has recently been described28,29, although it is biologically inactive in the absence of the more complex part of the molecule, the absolute stereochemistry of which has not yet been reported. Several small-molecule leads have been reported to also produce low-µM inhibition, including a sulphonamide (NSC 279287) from the NCI database, found through in silico screening of virtual libraries30 (discussed below) and which also shows p53-dependent transcriptional activity in a MDM2-overexpressing cell line. Inhibitors that are two orders of magnitude more potent and yet have lower molecular weights (300 analogues synthesized

N

• Metabolically stable

Imatinib H N

N N

N

H N

N

CH3

H N

N N

O

H N O

N

N

• The piperazine group confers aqueous solubility, and its salts are water soluble • The mesylate salt is used in clinical formulations, because it is non-corrosive during manufacture

• The methyl group at the 9-position confers selectivity for the ABL kinase • This is an insoluble compound • On hold since 1996

Figure 7 | The principal steps in the medicinal chemistry of the discovery of imatinib (Glivec). The figure shows the development of the final active molecule from the initial discovery of a lead weak-binding inhibitor. The successive steps illustrate how medicinal chemistry was used to modify this initial molecule to enhance affinity and selectivity to its target ABL kinase, as well as metabolic stability, aqueous solubility and manufacturing characteristics.

‘Drugability’

The transformation of a lead molecule into a drug is a key step in the process of drug discovery, requiring the application of knowledge of the compound’s absorption, distribution, metabolism and excretion (ADME) profile67 to optimize its ‘drugability’. Most compounds that fail to reach the clinic, even though they might be high-affinity in vitro inhibitors of the desired target, often do so because of insufficient attention to these issues. Therefore, there is now widespread agreement68 that drug-like and ADME properties should be incorporated as early as possible in the discovery cycle, rather than ‘retro-fitted’, after the optimization of molecular recognition features. It is striking that few of the anticancer drugs currently in the clinic have optimized ADME features. For example, all too often the cancer pharmacologist is presented with an aqueous-insoluble compound that has to be taken up in a non-aqueous solvent such as dimethylsulphoxide (DMSO), and evaluated in cells as a DMSO solution. Evidence of in vivo activity in a xenograft model based on a DMSO suspension of an agent is encouraging5,69, but DMSO itself is toxic so in this case there might have to be a significant effort in formulating anticancer drugs before clinical trials can commence. Alternatively, molecular re-engineering has to be used to produce an active analogue with sufficient aqueous solubility. Several software packages are now available to provide guidance on solubility and ADME properties, but an experienced chemist can, often on inspection, provide insights into solubility properties as well as likely patterns of metabolism and potential features that might result in adverse toxic effects. It is not always feasible to screen large numbers of compounds for absorption and metabolism properties, so retro-fitting or, better still,

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incorporation of desirable ADME features at the outset, is also made easier when structure-based design is possible. The importance of selecting lead molecules with drug-like properties at the start of a synthetic-chemistry programme has been highlighted by various surveys of chemical databases, often used as library starting points, compared with actual drug databases. Only 36% of the compounds in the widely used Available Chemical Database are drug-like70, as defined by Lepinski-like features71 (BOX 1), whereas in a typical drug database, more than 60% of compounds will pass such a test. A molecule that is effectively taken up into cells and has oral bioavailability (a highly desirable feature for an anticancer drug) needs to be soluble in aqueous media, with a balance of hydrophobic features that facilitate uptake and transport through cellular membranes. For example, the possession of formal cationic charges confers good aqueous solubility, but can only be useful in conjunction with a significantly sized hydrophobic group such as the benzimidazole or acridine-ring system. In the general class of marketed orally active drugs across all disease types, physico-chemical properties of drug molecules have been found to correlate with variables such as the number of oxygen and nitrogen atoms72, and these show a preference for particular motifs and substituent groups. Where these medicinal chemistry principles have been applied to the design process, such as in the development of imatinib (FIG. 7), there are clear practical advantages in terms of ease of formulation, administration and bioavailability, and especially of oral availability. This is an important goal for anticancer agents in the future, as the oral administration of lowtoxicity drugs will be accompanied by much greater patient tolerability and reduced associated cost. Concluding remarks

The traditional role of chemistry as the driving force behind the discovery of anticancer drugs has, in recent years, been eclipsed by major advances in our understanding of cancer biology and genetics, which have led to an almost ‘factory-like’ approach to finding new agents — an over-reliance on the screening of libraries of compounds. This is now changing, in part driven by the lack of new clinically efficacious agents for treating common human cancers. There is not only the beginning of a renaissance in creative chemistry, for example with the synthesis of complex potent natural products with anticancer activity73, but also with the re-emergence of medicinal chemists who can understand the biology of drug action as well as the chemistry of the drugs themselves. A new breed of chemists are pioneering chemistry-driven studies that they themselves are using to probe biological systems74,75, and in time new drug-candidate molecules will undoubtedly emerge from this one-shop approach. The emergence of relatively small yet highly diversified chemical libraries76 is an important step towards enabling multiple pathways to be effectively probed. Furthermore, the success of a novel chemicalgenetics approach using screens for multiple disease combinations77 indicates that this might be useful in www.nature.com/reviews/cancer


REVIEWS the selection of anticancer-drug combination therapies, especially in the development of combinations of cytotoxic and oncogene-targeted agents. The goal of research into cancer therapeutics is to produce new drugs that significantly affect the common solid tumour types, as well as overcome the two major clinical problems of metastatic disease and the emergence of resistance. Whether this goal can be met with single agents targeted against individual proteins or genes is doubtful, except for those rare cancers where there is a single dominant molecular abnormality, (for example, in chronic myelogenous leukaemia) or perhaps in the very early stages of solid tumours. However, even this is unlikely given the large number of mutated genes involved in human cancers78. The 2004 survey of the 291 known cancer genes found that some protein families are over-represented; for example, the number of kinases is four times greater than expected78. Protein motifs for transcriptional regulation are also over-represented. This has potential therapeutic significance and indicates that relatively non-selective kinase inhibitors might be of greater use in the clinic than highly selective ones. The same could be true for inhibitors of heat-shock protein 90 or even new types of DNA-binding agents79 that have only modest sequence selectivity combined with the ability to produce adducts that are poorly repaired. Modulation of transcription at the protein level by the use of small molecules that mediate between activation domains can be achieved, for example, by mimicking the stereochemical features of endogenous functional groups in activation domains, such as been achieved with isoxazolidines using an in vitro transcription

1.

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Acknowledgements We are grateful to Cancer Research UK and its predecessor, Cancer Research Campaign, for its support of cancer-related chemistry over many years, and, in particular, to their support of work in our laboratories.

Competing interests statement The authors declare competing financial interests: see web version for details.

Online links DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene ABL | BCR | BRCA1 | BRCA2 | CYP3A4 | FBP | KIT | MDM2 | p53 | PDGFR | SRC National Cancer Institute: http://cancer.gov/ brain tumours | breast cancer | chronic myeloid leukaemia | melanoma | prostate cancer FURTHER INFORMATION In Vitro Cell Line Screening Project: http://dtp.nci.nih.gov/branches/btb/ivclsp.html Kinasource: http://www.kinasource.com/ PDBbind database: www.pdbbind.org Research Collaboratory Structural Bioinformatics Protein Data Bank: http://www. rcsb. org/pdb Zinc database: http://blaster.docking.org/zinc/ Access to this interactive links box is free online.

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