《药物化学》课程文献资料（Medicinal Chemistry）GLIVEC（STI571, IMATINIB）,A RATIONALLY DEVELOPED,TARGETED ANTICANCER DRUG，Nat Rev Drug Disc 2002.pdf

© 2002 Nature Publishing Group REVIEWS Until the early 1980s, drug discovery programmes for cancer were focused almost exclusively on DNA synthesis and cell division, and resulted in agents such as antimetabolites, alkylating agents and microtubule destabilizers. These drugs showed efficacy, but at the price of high toxicity due to lack of selectivity. Also, resistance was frequently observed after initial stabilization or regression of the disease. The discovery of cancer-causing genes, later called oncogenes, represented a radical departure — all of a sudden, genes were identified that were uniquely associated with cancerous cells. The molecular epidemiology of these genes was established over many years of studying clinical tumour samples, but as described below, it was clear at the outset that chronic myelogenous LEUKAEMIA (CML) — a haematological stem-cell disorder that is characterized by excessive proliferation of cells of the myeloid lineage — represented a particularly interesting case. Target selection: BCR–ABL CML is characterized by a reciprocal translocation between chromosomes 9 and 22 (REF. 1). The shortened version of chromosome 22, which is known as the Philadelphia chromosome, was discovered by Nowell and Hungerford2 , and provided the first evidence of a specific genetic change associated with human cancer. The molecular consequence of this inter-chromosomal exchange is the creation of the BCR–ABL gene, which encodes a protein with elevated tyrosine-kinase activity. The demonstration that Bcr–Abl as the sole oncogenic event could induce leukaemias in mice3–5 has established BCR–ABL as the molecular pathogenic event in CML. As the tyrosine-kinase activity of BCR–ABL is crucial for its transforming activity 6 , the enzymatic activity of this deregulated gene could plausibly be defined as an attractive drug target for addressing BCR–ABL-positive leukaemias. For the first time, a drug target was identified that clearly differed in its activity between normal and leukaemic cells. It was conceivable that this enzyme could be approached with classical tools of pharmacology, as its activity — the transfer of phosphate from ATP to tyrosine residues of protein substrates — could clearly be described and measured in biochemical as well as cellular assays. Furthermore, cell lines that were derived from human leukaemic cells with the same chromosomal abnormality were available. Such cell lines were instrumental for in vitro and animal studies, which laid the groundwork for the clinical trials. So, the essential tools were assembled to go forward with the aim of identifying potent and selective inhibitors of the ABL tyrosine kinase. GLIVEC (STI571, IMATINIB), A RATIONALLY DEVELOPED, TARGETED ANTICANCER DRUG Renaud Capdeville, Elisabeth Buchdunger, Juerg Zimmermann and Alex Matter In the early 1980s, it became apparent that the work of pioneers such as Robert Weinberg, Mariano Barbacid and many others in identifying cancer-causing genes in humans was opening the door to a new era in anticancer research. Motivated by this, and by dissatisfaction with the limited efficacy and tolerability of available anticancer modalities, a drug discovery programme was initiated with the aim of rationally developing targeted anticancer therapies. Here, we describe how this programme led to the discovery and continuing development of Glivec (Gleevec in the United States), the first selective tyrosine-kinase inhibitor to be approved for the treatment of a cancer. NATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | JULY 2002 | 493 Novartis Oncology, Novartis Pharma AG, S-27 2.033, CH-4002 Basel, Switzerland. Correspondence to R.C. e-mail: renaud.capdeville@ pharma.novartis.com doi:10.1038/nrd839 LEUKAEMIA Leukaemia is an uncontrolled proliferation of one type of white blood cell (leukocyte)

© 2002 Nature Publishing Group APOPTOSIS Programmed cell death. SYNGENEIC MODEL An animal model in which the injected tumour cells are derived from the same animal species as the host animal. 494 | JULY 2002 | VOLUME 1 www.nature.com/reviews/drugdisc REVIEWS and low solubility in water. The attachment of a highly polar side chain (an N-methylpiperazine) was found to improve markedly both solubility and oral bioavailability. To avoid the mutagenic potential of aniline moieties, a spacer was introduced between the phenyl ring and the nitrogen atom. The best compound from this series was a methylpiperazine derivative that was originally named STI571 (imatinib, now known as Glivec or Gleevec), which was selected as the most promising candidate for clinical development9,10 (FIG. 1d). Docking studies11 and X-ray crystallography12 showed that binding of Glivec occurs at the ATP-binding site. Analysis of the crystal structure12 showed that Glivec inhibits the ABL kinase by binding with high specificity to an inactive form of the kinase. The need for the kinase to adopt this unusual conformation, which favours binding, might contribute to the high selectivity of the compound. Unexpectedly, these analyses indicated that the N-methylpiperazine group (added to increase drug solubility) also interacted strongly with ABL by means of hydrogen bonds to the backbone carbonyl group of isoleucine (Ile)360 and histidine (His)361. In an in vitro screen against a panel of protein kinases, the compound was found to inhibit the autophosphorylation of essentially three kinases: BCR–ABL, c-KIT and the platelet-derived growth factor (PDGF) receptor (TABLE 1). More recently, activity against ARG kinase has also been reported13. Pharmacological profile In collaboration with Brian Druker, the selective inhibitory activity of Glivec was shown at the cellular level on the constitutively active p210BCR–ABL tyrosine kinase14. Subsequently, a similar inhibitory activity was also shown on other ABL fusion proteins, such as p185BCR–ABL (REFS 15,16) and TEL (ETV6)–ABL15. The inhibition of autophosphorylation of BCR–ABL was closely related to the antiproliferative activity of Glivec. Incubation with submicromolar concentrations of Glivec selectively induced APOPTOSIS in BCR–ABL-positive cell lines, and induced cell killing in primary leukaemia cells from patients with Philadelphia-chromosome-positive (Ph+) CML and acute lymphoblastic leukaemia14,16–20. In in vivo experiments, once daily intraperitoneal treatment with 2.5–50 mg kg–1 of Glivec, started one week after injecting BCR–ABL-transformed 32D cells into SYNGENEIC mice, caused dose-dependent inhibition of tumour growth14. In nude mice implanted with KU812 cells, oral treatment with 160 mg kg–1 daily in three divided doses for 11 consecutive days was associated with continuous blockage of p210BCR–ABL tyrosine phosphorylation, and resulted in tumour-free survival of the animals20. The antitumour effect of Glivec was specific for BCR–ABL-expressing cells, as no growth inhibition occurred in mice that were given injections of U937, a BCR–ABL-negative myeloid cell line. Recently, Glivec was shown to be orally active in a mouse model of CML, based on retroviral p210BCR–ABL Medicinal chemistry The starting point for every medicinal-chemistry project is a lead compound with a given pharmacological activity. However, the biological activity of a molecule must be complemented by other properties that make the molecule a good drug — it is estimated that a large proportion of molecules fails in late stages of drug development due to drug–drug interactions or poor ADME (absorption, distribution, metabolism and excretion) features. Not detecting these liabilities early in the drug discovery process can be extremely costly and time consuming. On the basis of physical and calculated properties for known drugs, criteria for ‘drug-likeness’ have been established7 . In the case of Glivec, a lead compound was identified in a screen for inhibitors of protein kinase C (PKC). This compound — a phenylaminopyrimidine derivative — had promising ‘lead-like’ properties8 and a high potential for diversity, allowing simple chemistry to be applied to produce compounds with more potent activity or selectivity. Strong PKC inhibition in cells was obtained with derivatives bearing a 3′-pyridyl group at the 3′-position of the pyrimidine (FIG. 1a). During the optimization of this structural class, it was observed that the presence of an amide group on the phenyl ring provided inhibitory activity against tyrosine kinases, such as the BCR–ABL kinase (FIG. 1b). At this point, a key observation from analysis of structure–activity relationships was that a substitution at position 6 of the diaminophenyl ring abolished PKC inhibitory activity completely. Indeed, although the introduction of a simple ‘flag-methyl’ led to loss of activity against PKC, the activity against protein tyrosine kinases was retained or even enhanced (FIG. 1c). However, the first series of selective inhibitors that was prepared originally showed poor oral bioavailability N N N N H N N N N N R1 H H 6 O N N N N N R1 H H O H3C N N N N H H N O N N a b c d Figure 1 | Summary of the chemical optimization. The core structure of the lead compound, a phenylamino derivative, is indicated in black. a | The addition of a 3′-pyridyl group (blue) at the 3′-position of the pyrimidine enhanced the cellular activity. b | An amide group (red) attached to the phenyl ring provided activity against tyrosine kinases. c | A ‘flag methyl’ (purple) attached to the diaminophenyl ring abolished the undesirable protein-kinase-C inhibitory activity. d | The final attachment of an N-methyl piperazine moiety (green) markedly increased the solubility and oral bioavailability

© 2002 Nature Publishing Group NATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | JULY 2002 | 495 REVIEWS Fundamental phenotypic features in BCR–ABLpositive cells involve resistance to apoptosis, enhanced proliferation and altered adhesion properties. The impact of Glivec on some known downstream signalling molecules of BCR–ABL has been examined. A link between constitutive activation of STAT5 (signal transducer and activator of transcription 5) and enhanced viability of BCR–ABL-transformed cells has been shown22,23. Glivec had a profound inhibitory effect on STAT5 activation in vitro and in vivo21–23. Furthermore, inhibition of the BCR–ABL kinase activity by Glivec in BCR–ABL-expressing cell lines and fresh leukaemic cells from CML patients induced apoptosis by suppressing the capacity of STAT5 to activate the expression of the anti-apoptotic protein BCL-XL 23. The adaptor molecule CRKL is a prominent target of BCR–ABL, and its tyrosine phosphorylation has been a useful marker of BCR–ABL kinase activity24. As expected, a decrease in tyrosine phosphorylation of CRKL has been observed in Glivec-treated cell lines, and has also served as an indicator of BCR–ABL kinase activity in patients (see below). There is increasing evidence that cell-cycle regulation is disturbed in BCR–ABL-positive cells; however, the underlying molecular mechanisms are poorly understood. Recently, BCR–ABL has been shown to promote cell-cycle progression and activate cyclindependent kinases by interfering with the regulation of the cell-cycle inhibitory protein p27 (REF. 25). Glivec prevented downregulation of p27 levels in BCR–ABLexpressing cells25,26. The effects of Glivec on cytoskeletal changes and adhesion have been investigated using BCR–ABLtransfected fibroblasts27. Glivec was shown to restore normal architecture and to increase adhesion in this model of BCR–ABL expression. Clinical development in CML Because of the three known targets of Glivec, many potential cancers can be speculated to be good candidates for clinical testing of this new drug. However, in most cancers, tumorigenesis is complex and involves the disruption of multiple genes and signalling pathways. By contrast, CML can be considered as one of the few examples of a malignancy in which a single signallingpathway defect is thought to cause the disease. In addition, in contrast to most of the solid tumours, for which the measurement of tumour response is complex, pharmacodynamic response in CML can be measured easily using blood leukocyte count as the end point. For these reasons, CML was selected as the first indication for Phase I clinical testing. Clinically, CML is a chronic disease that evolves through three successive stages, from the chronic phase to the end stage of blast crisis that resembles acute leukaemia (FIG. 2). Overall, the median survival time of patients with newly diagnosed CML is approximately 5–6 years with an interferon-based treatment regimen. The first trial with Glivec was a Phase I study in patients with chronic-phase, and subsequently also with blastphase, CML. In this trial, patients were treated with doses transduction of transplanted bone marrow. Survival of animals was significantly prolonged, together with a marked improvement in peripheral-white-blood-cell counts and splenomegaly21. Table 1 | Cellular profile of Glivec Assay IC50 (µM) Inhibition of autophosphorylation v-ABL 0.1–0.3 p210BCR–ABL 0.25 p185BCR–ABL 0.25 TEL–ABL 0.35 TEL–ARG 0.5 PDGF receptor 0.1 TEL–PDGF receptor 0.15 c-KIT 0.1 FLT3 > 10 c-FMS and v-fms > 10 EGF receptor > 100 c-ERBB2 > 100 Insulin receptor > 100 IGF-1 receptor > 100 v-SRC > 10 JAK2 > 100 Inhibition of MAPK activation PDGF dependent 0.1–1 SCF dependent 0.1–1 Inhibition of AKT activation SCF dependent 0.1–1 Inhibition of IP release PDGF induced 0.25 Inhibition of c-FOS mRNA expression PDGF induced 0.3 –1 EGF, FGF or PMA induced > 100 Antiproliferative activity* 32D, MO-7e, BaF3 cells > 10 BCR–ABL-transfected 32D, MO-7e, BaF3 cells < 1 BCR–ABL-positive human leukaemia lines|| 0.1–1 BaF3 TEL-ARG 0.5 BALB/c 3T3 v-SIS (PDGF autocrine) 0.3 BaF3 TEL–PDGF receptor < 1 U-87 human glioma‡ ~1.5 U-343 human glioma‡ ~1.5 MO-7e, SCF stimulated ~0.1 H526 human SCLC, SCF stimulated§ 0.8 Human GIST882 line¶ < 1 Human mast-cell leukaemia line HMC-1# 0.01–0.1 Glivec concentrations that cause 50% inhibition (IC50) are given13–20,47,48,53,54,61,63,66. EGF, epidermal growth factor; FGF, fibroblast growth factor; FLT3, fms-related tyrosine kinase 3; IGF-1, insulin-like growth factor-1; IP, inositol phosphate; MAPK, mitogen-activated protein kinase; PDGF, plateletderived growth factor; PMA, phorbol 12-myristate 13-acetate; SCF, stem-cell factor; SCLC, small-cell lung cancer. *Antiproliferative experiments were carried out in 10% fetal calf serum, except for those that were carried out in ‡ 5% human-platelet poor plasma or under § serum-free conditions. ||K562, KU812, MC-3, MBA-1, KBM-5, Z-33, Z-119, Z-181. ¶Expresses the activating KIT mutation K642E (lysine 642 to glutamic acid). # Expresses the activating KIT mutation V560G (valine 560 to glycine)

© 2002 Nature Publishing Group 496 | JULY 2002 | VOLUME 1 www.nature.com/reviews/drugdisc REVIEWS Subsequently, three large multinational studies have been carried out in 532 patients with late chronic-phase CML in whom previous interferon therapy had failed31, in 235 patients with accelerated-phase CML32, and in 260 patients with myeloid blast crisis33. Treatment was given at a dose of 400 mg in the chronic-phase trial and 600 mg in the two other studies. The results of these three studies indicated that the rate of both haematological and cytogenetic response increased as the treatment was started earlier in the course of the disease (FIG. 4). Importantly, the achievement of a haematological and/or cytogenetic response was associated with improved survival and progression-free survival31–33. In the chronic-phase study, in which patients started treatment within a median of 32 months after their initial diagnosis, the estimated probability of being free of progression at 18 months was 89.2%31. The most frequently reported adverse events were mild nausea, vomiting, oedema and muscle cramps. However, rare but serious adverse events, such as liver ranging from 25 to 1,000 mg per day, and no maximal tolerated dose was identified, despite a trend for a higher frequency of GRADE III–IV ADVERSE EVENTS at doses of 750 mg or higher. On the other hand, a clear dose–response relationship with respect to efficacy was described in patients with chronic-phase CML.At doses of 300 mg or higher, 98% of the patients achieved a complete haematological response, and trough serum levels were above the concentrations required for in vitro activity28,29. Subsequently, a mathematical modelling of the relationship between dose and response, as measured by leukocyte counts after four weeks of therapy, confirmed that doses of 400 mg and higher were optimal in inducing a haematological response30 (FIG. 3). In addition, effective inhibition of the BCR–ABL kinase was documented in patient samples by inhibition of the phosphorylation status of the downstream target CRKL27. From this study, doses ranging from 400 mg (for chronic-phase patients) to 600 mg (for advanced-phase CML) were recommended for subsequent studies. GRADE III–IV ADVERSE EVENTS For each adverse event that is associated with a specific treatment, grades are assigned and defined using a scale from 0 to V. Grade III, severe and undesirable adverse event; grade IV, life-threatening or disabling adverse event. 1,000 0 200 400 600 Dose (mg) Model-fitted line WBC counts (× 109 –l 1) 800 1,000 100 10 1 Chronic patients Upper limit of normal range of WBCs Figure 3 | Dose–response relationship of Glivec in CML (Phase I study). Using the leukocyte (white blood cell; WBC) count after 28 days of treatment as a pharmacodynamic marker, the relationship between dose and response was modelled using an Emax model , which makes the assumption that once the maximal effect is achieved (Emax), increasing the dose further does not translate into additional benefit. The data indicate that at doses of 400 mg per day or higher, all the patients are predicted to achieve a reduction of their leukocyte counts within normal range below 10 x 109 l –1 . Adapted with permission from REF. 30 © (2001) American Society of Clinical Oncology. CML, chronic myelogenous leukaemia. Chronic phase Median 4–6 years stabilization Accelerated phase Advanced phases Median duration up to 1 year Blastic phase (blast crisis) Median survival 3–6 months Figure 2 | Clinical course of chronic myelogenous leukaemia

© 2002 Nature Publishing Group NATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | JULY 2002 | 497 REVIEWS observed by Kuriyan and co-workers12 in a complex between mouse c-Abl and a Glivec analogue, and cannot bind ATP. The knowledge of the crystal structure allows a better understanding of the decreased sensitivity of mutated BCR–ABL to Glivec, and can be a powerful tool in the design of new BCR–ABL inhibitors that maintain inhibitory activity against these mutated kinases. Resistance to Glivec might also be related to pharmacokinetic factors. Glivec is a substrate of the multidrug-resistance-associated P-glycoprotein (PgP). toxicity or fluid-retention syndromes, were also reported. Neuropaenias and thrombopaenias were more common in patients with advanced disease, which indicates that haematological toxicity might be related more to an underlying compromised bonemarrow reserve than to toxicity of the drug itself through inhibition of c-KIT-driven haematopoiesis. Taken together, these findings have established Glivec as a safe and effective therapy in all stages of CML, and were the basis for marketing approval by the FDA on 10 May 2001 — less than three years after the start of the first Phase I study (FIG. 5). Resistance. In CML blast crisis, even though the rate of haematological responses with Glivec is high, these responses are usually short lived, and most patients will ultimately develop resistance and undergo disease progression. A prerequisite to optimally develop strategies to prevent or overcome this resistance is to get a good understanding of the potential mechanisms of resistance in these patients. On the basis of preclinical and clinical data that are available at present, several potential mechanisms of resistance have been described, which are summarized in BOX 1. They can be categorized into two main groups: mechanisms whereby BCR–ABL is reactivated and cell proliferation remains dependent on BCR–ABL signalling, and mechanisms whereby the BCR–ABL protein remains inhibited by Glivec, but alternative signalling pathways become activated. BCR–ABL overexpression and BCR–ABL gene amplification has been shown in p210BCR–ABL-transformed mouse haematopoietic Ba/F3 cells that are resistant to Glivec34,35, as well as in human BCR–ABL-positive leukaemia lines LAMA84 and AR230 (REFS 35,36). In treated patients, there is now increasing evidence that amplification of the BCR–ABL gene and mutations in the BCR–ABL kinase domain are two common mechanisms of resistance to Glivec. The occurrence of these mechanisms was first reported by Sawyers’ group37. In a study of 11 patients with blast crisis and overt clinical resistance when treated with Glivec, 3 had amplification of the BCR–ABL gene and 6 had a point mutation in the ABL kinase domain, which resulted in a T315I (threonine 315 to isoleucine) amino-acid substitution. Following this initial report, the T315I mutation as well as further mutations in the ABL kinase domain have been reported by various investigators38–41. Even though these mutations vary in their type and frequency, it is speculated that they might all lead to a reactivation of BCR–ABL-driven signal transduction. To understand the molecular mechanism by which such mutations might cause resistance to Glivec, current studies are using X-ray crystallography to analyse the three-dimensional structure of a complex between the drug and the human c-ABL kinase domain. Glivec binds to an unusual, inactive conformation of ABL with the amino terminus of the activation loop, which contains the highly conserved DFG (asparagine-phenylalanine-glycine) motif, folded into the ATP-binding site42. This conformation has been Haematological response Major cytogenic response % of patients 100 80 60 40 20 0 Late chronic phase, IFN failure (n = 524) Accelerated phase (n = 181) Blast crisis (n = 229) 95 69 30.6 60 24 16.2 Figure 4 | Haematological and cytogenetic response in CML: Phase II data. In all studies, results are expressed as the percentage of responding patients among the patients for whom the diagnosis of the correct phase of chronic myelogenous leukaemia (CML) was confirmed after a central review of the data. A major cytogenetic response combines both complete (0% Ph+ metaphases) and partial (1–35% Ph+) responses. Haematological response was defined as complete haematological response (CHR) in the chronicphase study, and as either a CHR, a marrow response or a return to chronic phase (RTC) in the advanced-phase studies, all to be confirmed after at least four weeks. In the chronic-phase study, CHR was defined as white blood cells <10 x 109 l –1, platelets <450 x 109 l –1, myelocytes and metamyelocytes <5% in blood, no blasts and promyelocytes in blood, basophils <20% and no extramedullary involvement. In advanced-phase studies, CHR was defined as neutrophils = 1.5 x 109 l –1, platelets = 100 x 109 l –1, no blood blasts, marrow blasts <5% and no extramedullary disease. A marrow response was defined by the same criteria as for CHR, but with neutrophils = 1 x 109 l –1 and platelets = 20 x 109 l –1 . An RTC was defined as <15% blasts in marrow and blood, <30% blasts and promyelocytes in marrow and blood, <20% basophils in blood and no extramedullary disease. IFN, interferon; Ph+, Philadelphia chromosome positive