ATP site-directed competitive and irreversible inhibitors of protein kinases

28

© 2000 John Wiley & Sons, Inc. CCC 0198-6325/00/010028-30

ATP Site-Directed Competitiveand Irreversible Inhibitors

of Protein Kinases

Carlos García-Echeverría, Peter Traxler, Dean B. Evans

Oncology Research, Novartis Pharma AG, CH-4002, Basel, Switzerland

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Abstract: Several tyrosine and serine/threonine protein kinases have emerged in the last few yearsas attractive targets in the search for new therapeutic agents being applicable in many differentdisease indications. Initially, inhibition of these protein kinases by ATP site-directed inhibitors wasconsidered less prone to success, but medicinal chemists from both academia and industry havebeen able to impart potency and selectivity to a limited number of scaffolds by modulating andfine-tuning the interactions of the modified template with the ATP binding site of the selected ki-nase. The chemical templates that have been used in the synthesis of ATP site-directed protein ki-nase inhibitors are reviewed with emphasis on the kinase inhibitors that have entered or are aboutto enter clinical trials. Examples have been selected to illustrate how structure-based design ap-proaches and new methods to increase compound diversity have had an impact on this area of re-search. © 2000 John Wiley & Sons, Inc. Med Res Rev, 20, No. 1, 28–57, 2000

Key words: kinase inhibiton; structure-based design; pharmacophore model; receptors; growthfactors

1. I N T R O D U C T I O N

The characterization of the processes involved in the regulation of individual signal transductionpathway components and the integration of these responses has been used to identify promising newroutes and targets for therapeutic intervention. From this basic research work, protein kinases haveemerged as attractive targets in the search for new therapeutic agents applicable in many differentdisease indications. Among the many different approaches reported in the medicinal chemistry lit-erature to inhibit protein kinases,* this review focuses on contributions in the inhibition of receptor

*Peptide-based active site-directed inhibitors of protein kinases have been described, but they exhibit, in general, low in-hibitory activity with Ki values in the 1–2 mM range.1 Recently, the use of bivalent peptide-based inhibitors to target the Srcfamily of protein tyrosine kinases has been reported.2 This new approach is based on the design of peptides capable of bind-ing simultaneously to the active site and to the SH2 domain of the Src kinase.

Correspondence to: Carlos García-Echeverría

ATP SITE-DIRECTED INHIBITORS OF PROTEIN KINASES • 29

and nonreceptor protein kinases by ATP site-directed compounds. Initially, this approach was con-sidered unlikely to result in selectivity due to the assumption that the ATP-binding site is highly con-served among protein kinases. The highly selective ATP site-directed inhibitors that are currently inearly phases of clinical trials3 – 8 and the available X-ray structures of protein kinases,** in most casescomplexed with inhibitors, have confirmed that the initial skeptism and concerns for this “adven-turous” medicinal chemistry approach were not well founded, at least for some specific kinases. Theuse of structural information obtained by X-ray crystallography or computer-assisted molecularmodeling based on kinase domain homology has been a key factor in the design of selective proteinkinase inhibitors. Pharmacophore models for ATP site-directed competitive inhibitors10 –13 havebeen obtained by combining three-dimensional structural information and structure-activity rela-tionship data providing directions for structure-based drug design approaches.11,14 –16 This workhighlights key medicinal chemistry achievements in the design and synthesis of ATP site-directedprotein kinase inhibitors and expands and partially overlaps recent reviews on this topic.3, 17–20 Itstarts with an overview of a few specific kinase targets in which the medicinal chemistry activitieshave advanced with some success.

2. P R O T E I N K I N A S E S A S T H E R A P E U T I CT A R G E T S — S C I E N T I F I C R A T I O N A L E

Over the years, many different tyrosine and serine/threonine protein kinases have been selected ascandidates for drug discovery activities based either on their over-expression and/or dysfunction inthe particular organ or tissue, or through their association in signal transduction/cell cycle pathwaysthat have been implicated in numerous disease processes. As a consequence of this altered function,inhibition of the particular kinase should lead to a modification of the functional cellular responseand, in turn, this should lead to the modification of the disease process in question.

The receptor protein tyrosine kinases (RPTKs) are typically activated following the binding oftheir ligand peptide growth factor to the receptor. The RPTKs have important roles in signal trans-duction pathways that regulate a number of cell functions such as cell differentiation and prolifera-tion occurring both under normal physiological conditions as well as in a variety of disease situa-tions. For example, many different tumor types have been shown to have dysfunctional RPTKs eitheras a consequence of excessive production of either the growth factor, the receptor, or both, or in somecases via mutations in the RPTK’s structure. Irrespective of the cause, this leads to the over-activi-ty of the particular RPTK system and, in turn, to the aberrant and inappropriate post-receptor cellu-lar signalling within the cell. RPTKs are attractive targets in the search for therapeutic agents notonly against cancers, where most attention has been focussed, but also against many other diseaseindications. The over-expression or activity of non-RPTKs can also lead to alterations in signal trans-duction pathways. For example, the src family of non-RPTKs has been implicated in pathology of anumber of tumor types, osteoclast-mediated bone resorption occurring in osteoporosis, and in dis-orders associated with the proliferation of T cells. In addition to the many different tyrosine kinasesbeing explored as molecular therapeutic targets, the serine/threonine kinases also represent a largeclass of kinases for further drug discovery opportunities. The first such approach with the serine/threonine kinases was typified by PKC with the rationale to block aberrant signal transductionprocesses leading to excessive cell proliferation. Subsequently, the MAPK family of signal trans-ducers and many cell cycle-associated serine/threonine kinases have gained attractiveness as thera-peutic targets.

**The reader is directed to this web site http://www.sdsc.edu/kinases, which covers a comprehensive list of protein kinasesincluding 3D structural information.9

30 • GARCÍA-ECHEVERRÍA, TRAXLER, AND EVANS

A. Epidermal Growth Factor Receptor (EGFR)

For a recent review on the EGFR refer to Voldborg et al.21 and the Activation of the EGF ReceptorKinase website at http://liba.ludwig.edu.au/modelling/pubegfr–f.html. About ten years ago, whenresearch in the signal transduction drug discovery field was initiated in many pharmaceutical com-panies, the epidermal growth factor receptor (EGFR) was chosen as a “prototype” drug discoveryRPTK target. Epidermal growth factor (EGF) was one of the earliest growth factors to be formallycharacterized, and this was later shown to bind to the EGFR. The EGFR is a growth factor tyrosinekinase receptor that induces cell proliferation and cell differentiation upon binding and activation byone of a number of its known ligands. The EGFR consists of a single polypeptide chain of 1186amino acids that is expressed on the cell membrane of numerous cell types. In addition to EGF, sev-eral other ligands have been shown to bind to the EGFR including transforming growth factor a(TGF-a), betacellulin (BTC), and Heparin-binding EGF-like growth factor (HB-EGF). Followingligand binding, the EGFR receptor dimerizes potentially either as a homodimer or as a heterodimerwith other members of the EGFR receptor family. Receptor dimerization results in alterations in theintracellular portion of the receptor leading to the activation of the intracellular tyrosine kinase ac-tivity and auto-phosphorylation of cytoplasmic located tyrosine residues on the EGFR, which serveas recognition sites for interactions of the SH2 (Src homology 2)-containing substrates required forfurther transduction of the signal.

Over recent years, much evidence has been gathered to implicate the EGFR and its family mem-bers in the development and progression of numerous human tumors. In a large proportion of tumors,aberrant activity of the EGFR can be changed as a result of over-expression or via mutations. In ad-dition, the increased expression of the EGFR ligands or co-expression of both receptor and ligand(s)within the tumor cells has the potential for autocrine regulation and EGFR activation leading to anover-active receptor pathway. Consequently, aberrant EGFR activity can lead to uncontrolled cellu-lar processes including cell proliferation, leading to the development of a malignant condition. Geneamplifications have also been detected in a number of major tumor types, and EGFR over-expres-sion has been documented in tumors such as breast, ovarian, bladder, lung, glioblastomas, and squa-mous carcinomas. The EGFR is often used as a tumor prognostic marker, because its over-expres-sion has been correlated with a poor prognosis in several tumor types. Based on the clinicalepidemiology and feasibility, the EGFR has been viewed as an ideal and viable target in drug dis-covery programs and represents one of the more advanced targets being explored clinically. The roleof EGFR in cell proliferation makes this an attractive target in other hyperproliferative disorders suchas psoriasis.

B. Platelet-Derived Growth Factor Receptor (PDGFR)

Refer to Heldin et al.22 for a recent review. Platelet-derived growth factor (PDGF) is a potent stim-ulator of cell growth and motility of several cell types. Structurally, PDGF is a dimeric protein com-posed of disulphide bond-linked A or B polypeptide chains that may combine to form homodimers(AA or BB) or heterodimers (AB). These peptide ligands bind to two structurally similar PDGF re-ceptors (PRGFR) termed a and b. One PDGF peptide is capable of binding to two PRGFRs at once.Due to the existence of two different ligands and receptors, a number of permutations exist for in-teractions: a/a PRGFR interacts with A/A, A/B, and B/B; a/b PRGFR interacts with A/B and B/B; while b/b PRGFR only interacts with B/B ligands. The PRGFRs initiate similar although not ful-ly identical cell effects. Activities regulated by the PGDFRs include stimulation of cell proliferation,actin filament rearrangements (stress fibers, cell margin veil-like structures, and circular ruffles),chemotaxis, Ca21 mobilization and protection against apoptosis. The PRGFRs both contain five Igdomains in their extracellular region, a single transmembrane spanning domain, and a cytoplasmicregion containing a split-insert kinase domain. The PDGF ligands bind to the three N-terminal Ig re-

gions of the receptor and induce receptor dimerization that is considered to occur via the fourth Igdomain. Alignment of the two receptors then allows for the autophosphorylation of receptor tyro-sine residues and regulation of the kinase activity. Tyrosine-857 represents the only autophospho-rylation site within the kinase domain of the b PRGFR and this residue is conserved in the aPRGFR(Y849) and is important for receptor kinase activity. The other eleven autophosphorylation sites inthe b PRGFR are found outside the kinase domain and constitute interaction sites for SH2-con-taining proteins, which mediate different cellular responses. Alterations in the PRGFR signalingcan, therefore, lead to the aberrant regulation of different signal transduction pathways implicatedin tumorigenesis.

C. Vascular Endothelial Growth Factor Receptor (VEGFR)

Refer to Neufeld et al.23 for a recent review. Angiogenesis represents an essential event in a varietyof physiological and pathological processes that involve the formation of new blood vessels from anexisting vascular network. Under normal physiological conditions, angiogenesis is restricted toprocesses associated with embryogenesis, ovulation, and wound healing. However, under patholog-ical conditions, angiogenesis is also associated with tumor growth and the formation of tumor metas-tases, inflammation, rheumatoid arthritis, ocular neovascularization, and in psoriasis. Many differ-ent cytokines and growth factors have been shown to exert angiogenic activities. One of the mainproteins involved is called vascular endothelial growth factor (VEGF), which appears to be a keyfactor in pathological situations involving neovascularization as well as with enhanced vascular per-meability. The family of VEGF receptors (VEGFR) include VEGFR1 (Flt-1, Fms-like tyrosine ki-nase) and VEGFR2 (KDR, kinase insert domain-containing receptor), which belong to a larger fam-ily including the related PRGFR, cKit, c-Fms, Flt-3, and Flt-4 receptors. Expression of VEGFRs isusually low in normal tissues and increases during the development of pathological states associat-ed with neovascularization. The VEGFRs are structurally characterized by the presence of seven im-munoglobulin-like domains in their extracellular domain, a single transmembrane-spanning region,and an intracellular split tyrosine kinase domain. The ligands that bind to VEGFR1 are VEGF-A,VEGF-B, and also the related placenta growth factors (PIGF); whereas VEGFR2 binds VEGF-A,VEGF-C, and VEGF-D. The VEGFRs form dimers that undergo autophosphorylation on cytoplas-mic tyrosine residues, which promotes the binding and phosphorylation of the adaptor proteins Shcand Nyc, binding of Grb2, and the protein tyrosine phosphatases SHP-1 and SHP-2 to the VEGFR2and to activation of the downstream MAP kinase activity. Subsequent effects associated with VEGFR’s function in the angiogenesis process include the production of proteases needed for thebreakdown of blood vessel basement membranes, expression of certain integrins associated with an-giogenesis, and stimulation of cell migration and proliferation.

D. Fibroblast Growth Factor Receptor (FGFR)

For a recent review on FGFR refer to Klint and Claesson-Welsh.24 The FGF family of proteins iscurrently composed of many different factors that show 30–70% identity in their primary amino-acid sequences. FGFs are potent mitogens for a wide variety of different cell types and bind to fourstructurally related FGF receptors (FGFRs) resulting in transmission of intracellular signal cascades.Dimerization of FGFR monomers occurs upon ligand binding, and this is required for the activationof the tyrosine kinase domain and receptor transphosphorylation. Receptor activation leads to au-tophosphorylation of cytoplasmic tyrosine residues that serve as high-affinity docking sites for a va-riety of SH2 domain-containing signal transduction molecules, which transduce the signal cascadesfrom the receptor and eventually result in the biological responses.

The four FGFRs have a similar overall structural organization. The extracellular domain is com-posed of two or three immunoglobulin (Ig)-like domains. The extracellular portion of the FGFRs is


connected via a single transmembrane spanning domain to the cytoplasmic region composed of ajuxtamembrane, kinase, and C-terminal domains. The juxtamembrane domain of the FGFRs is muchlonger than those found in other tyrosine kinase receptors. A single tyrosine phosphorylation site ex-ists within the juxtamembrane region of the FGFR1 and FGFR2. However, the FGFR3 and FGFR4lack this corresponding tyrosine residue. Like the VEGFRs, the tyrosine kinase domain of the FGFRsis divided into two parts separated via a short noncatalytic insert of approximately 15 amino acids.In this insert region of the FGFR1 and FGFR2, two phosphorylatable tyrosine residues are found. Inthe FGFR3, a single tyrosine phosphorylation site is found, while there are none in FGFR4. The C-terminal tail region of the FGFRs contains a number of tyrosine residues, of which some are locat-ed at identical positions in this receptor class.

Following the binding of FGF, this leads to either homo- or heterodimerization of FGFRs. Asfor many other RPTKs, dimerization followed by conformational alterations of the FGFR appearsto be an important requirement for activation of the tyrosine kinase activity. Excluding the kinasedomain, there are five cytoplasmic tyrosine residues located in the FGFR1. Whereas Y463, Y583,Y585, and Y766 are potential phosphorylation and SH2-docking sites, Y776 does not appear to bephosphorylated. Once phosphorylated, only Y766 has been shown to interact with SH2-containingproteins, in this case binding phospholipase Cg. Within the FGFR1 kinase domain, three tyrosinephosphorylation sites have been identified: namely, Y653, Y654, and Y730. Y653 and Y654 appearto be involved in the regulation of the kinase activity, since their mutation results in the loss of ki-nase function. FGFs are known to promote in vitro endothelial cell migration, proliferation, and dif-ferentiation. In a similar way to VEGF, FGFs appear to play a major role in vivo in the regulation ofangiogenesis.

E. p38 MAP Kinase

Refer to Garrington and Johnson25 and The Mammalian MAPK signaling pathway website at http://kinase.oci.utoronoto.ca/signallingmap.html for a general overview on MAPKs and to Refs. 26–28and individual references contained therein for specific details on p38 MAPK.

Mitogen-activated protein kinases have been shown to have an important role as intermediatesignal transducing molecules between the extracellular signals and cellular response. Several groupsof MAPKs have been identified in mammalian cells, and these are characterized by the canonicaldual phosphorylation site (TXY) or other conserved primary amino acid features. The p38 MAPKgroup is composed of a number of members that possess a TGY motif, share 60% amino acid iden-tity, and include p38, p38b, p38g, and p38d members. The p38 kinases are considered to play im-portant roles in stress and inflammatory responses and are activated by proinflammatory stimuli suchas lipopolysaccharides, interleukin-1, and tumor necrosis factor. Stimulation of certain gene prod-ucts such as inducible nitric oxide synthase and cyclo-oxygenase-2 (COX-2), which are implicatedin proinflammatory cytokine-mediated disease processes, appear to involve the induction of p38MAPK. For example, rheumatoid arthritis is a systemic disorder of the immuno-inflammatory sys-tem characterized by erosive, inflammatory joint disease, which develops in several or many di-arthrodial joints. A wide range of pro-inflammatory mediators have been identified in the rheuma-toid joint, the most important group being the cytokines. Although different cytokines can be detectedin synovial tissues, the most important include IL-1b, TNF-a. The p38 MAP kinase cascade in mono-cyte/macrophages is involved in TNF-a and IL-1 production. Moreover, TNF-a and IL-1 activatethe p38 MAP kinase cascade in fibroblasts and other cells relevant for the pathological process inrheumatoid arthritis. The inhibition of p38a MAP kinase in macrophages would inhibit the produc-tion of TNF-a and IL-1, thereby reducing the main cytokines, which are involved in the activationand proliferation of synoviocytes and fibroblasts. In synoviocytes and fibroblasts, the inhibition ofp38a would be expected to inhibit production of COX-2 reducing inflammation and destruction ofcartilage.



F. Cyclin-Dependent Kinases (CDKs)

For a recent review on CDK’s and cell cycle control refer to Pines29 and references contained there-in. Information on individual CDKs can also be found at http://geo.nihs.go.jp/csndb.html.

In multicellular organisms, a balance exists between cell proliferation and cell death, thus main-taining a homeostatic equilibrium; so regardless of whether a cell proliferates or dies, these eventsare tightly linked to the cell cycle activities. Hence, the fine-tuned regulation of cell cycle progres-sion is critical for normal development, while situations such as tumor formation may be consideredas direct consequences of the disturbance of these processes. Mitogen-induced progression throughthe first gap phase (G1) and initiation of DNA synthesis (S phase) during the mammalian cell cycleare regulated predominantly by a series of serine/threonine kinases, cyclin-dependent kinases(CDKs) whose activities are in turn regulated by CDK inhibitors (CKIs). A proliferating cell goesthrough four different phases of the cell cycle—G1 r S (DNA synthesis) r G2 r M (segregationof duplicated chromosomes in two daughter cells)—and phase transition can be controlled by dif-ferent CDKs. The initiation of the cell cycle in normal cells requires growth-stimulatory factors, andits progression depends on several CDKs whose driving forces are counter-balanced by growth-sup-pressive molecules (e.g., tumor suppressors such as p53 and pRb, CKIs). Critical cell cycle eventssuch as DNA replication and chromosomal segregation are rigorously controlled by a number of fac-tors including the concerted action of p53, pRb, as well as CDK1 and CDK2. Normal cells can ar-rest in G1, such as when DNA repair is needed, by the expression of the p53-mediated and intrinsicCDK2 inhibitor p21. The subsequent G1 block stresses the essential role of pRb and CDK2 in theG1/S control mechanism. The role that several other CDKs play in the cell cycle make them idealtargets for inhibition by kinase inhibitors for the treatment of tumors.

3. C H E M I C A L T E M P L A T E S U S E D I N T H E D E S I G NA N D S Y N T H E S I S O F A T P S I T E - D I R E C T E DC O M P E T I T I V E A N D I R R E V E R S I B L E P R O T E I NK I N A S E I N H I B I T O R S

A. Quinazolines

The quinazoline scaffold has undergone extensive structure–activity relationship and biologicalstudies, and at least three quinazolines derivatives (1–3) (see Fig. 1) are currently in clinical trials.PD 153035 (1) was originally published by Parke–Davis,30 but it is now in Phase I clinical trials bySugen (SU 5271) for the treatment of psoriasis.31 It is a potent (Ki 5 6 pM) and specific inhibitor ofthe EGFR tyrosine kinase that suppresses EGFR autophosphorylation in A431 cells at nanomolarconcentrations.32 Inhibiton of cell growth and induction of apoptosis in vitro was observed upontreatment of different tumor cells lines with this compound.33 – 34 The antitumor activity of PD153035 in cells was enhanced by co-treatment with C225, an anti-EGFR-blocking monoclonal an-tibody.35 To explore the in vivo characteristics of PD 153035 with positron emission tomography

Figure 1. Structures of the quinazoline derivatives in clinical trials.


(PET), [methoxy-11C]PD 153035 was prepared by O-alkylation of O-desmethyl PD 153035 with[11C]methyl iodide (due to the protocol used, the position of mono-demethylation is not known).36

The total synthesis time including derivatization, purification, formulation, and sterile filtration was45–50 min, and the radiochemical purity was .99% (radio-HPLC). The distribution of the labeledcompound has been examined in vivo in healthy and tumor-implanted rats with PET. The compoundshowed a promising initial biodistribution, but the problem of sustained delivery of PD153035 ob-served in A431 xenografts in immunodeficient nude mice may have discouraged the developmentof this compound as an anticancer agent.32

One of the most promising 4-phenylamino-quinazoline derivatives is ZD 1839 (2),37– 38 whichis under development by Astra–Zeneca for the treatment of cancers and is in Phase Ib/II clinical stud-ies (for Phase I data, see Ref. 5). It is a potent ATP site-directed inhibitor for the EGFR (Ki 5 2.1nM on purified receptor; IC50 5 23 2 79 nM), and it has shown excellent activity against a broadrange of human solid tumor xenografts in nude mice. When administerd orally at 10 mg/kg/day, a50% reduction in the growth of A431 tumor cells (epidermoid carcinoma) was observed, and com-plete regression of large tumors was obtained with 200 mg/kg/day for two weeks. Tumor growthwas suppressed for as long as four months, but regrowth occurred when treatment was suspended.Activity was also observed against A549 (lung adenocarcinoma), KB (epidermoid carcinoma), HT29(colon carcinoma), HX62 (ovarian carcinoma), MCF-7 (breast carcinoma), and Du 145 (prostate car-cinoma).

CP 358,774 (3) is a clinical candidate from Pfizer39 – 41 and is in early Phase II clinical studiesfor the treatment of cancers. It is also a potent ATP site-directed inhibitor of the EGFR tyrosine ki-nase (IC50 5 1–2 nM) and highly selective (ratio . 1000-fold) against other tyrosine kinases (e.g.,pp60v-src, pp145c-abl, IGF-IR, or InsR). In athymic nude mice bearing HN5 (head and neck tumor)xenografts, 50% inhibition was observed after oral administration at 10 mg/kg/day. The inhibitionof A431 derived tumors required a higher dose, 200 mg/kg/day. Uniform distribution into HN5 tu-mors as well as other target tissues was demonstrated using a radio-labeled compound.

Beside the above clinical candidates, a tremendous amount of work has been done in the syn-thesis and biological evaluation of a whole range of quinazoline derivatives. This includes 4-, 6-, or7-substituted quinazoline (e.g., 4–6) and tricyclic quinazoline analogs (e.g., 7–9),12,42– 52 (see Fig.2), which have been extensively described in previous reviews.18 –20

Recently, a new class of selective and irreversible inhibitors with subnanomolar potency for theEGF and erbB-2 receptors have been reported.53 – 55 The suicide inhibitors (e.g., 10–14) (see Fig. 3)

Figure 2. Examples of 4-, 6-, and 7-substituted quinazolines and tricyclic quinazoline analogues.


contain Michael acceptor-type substituents at the 6- or 7-positions of the quinazoline scaffold andexploit the presence of a cysteine residue at the “sugar pocket” of the ATP binding site to establishthe addition product when bound to the enzyme. Cysteine-773, which is located on the extended coilstretch of the EGFR, is unique for the EGFR family of kinases providing selectivity (ratio of nearly105-fold) against other receptor or intracellular kinases (e.g., InsR, PDGFR, bFGFR, and PKC). Thespecific alkylation of cysteine-773 by PD 168393 (12) was confirmed by a set of experimental data,including mass spectra analysis and site-directed mutagenesis (Cys773Ser).54 Molecular modelingstudies suggest that the g-carbon of the 6-acrylamido moiety is optimally placed for reaction withcysteine-733 (d 5 3.5 Å), whereas the 7-acrylamido side chain is not close enough to induce a rapidalkylation (d 5 8 Å). PD 183805 (13),56 – 57 a potent EGFR kinase inhibitor (IC50 5 1.5 nM, isolat-ed enzyme) that also inhibits heregulin induced cell proliferation (IC50 5 9 nM), is about to enterPhase I clinical trials.

Additional structure–activity studies with 6- and 7-substituted acrylamidoquinazolines andacrylamidopyrido[d]pyrimidines have shown that the pyrido[3,2-d]pyrimidine derivatives weresomewhat less potent (2- to 6-fold) in a cellular autophosphorylation assay for EGFR using A431cells53 (see Section 3.C). In addition, the quinazolines analogues were generally less potent againsterbB-2 than EGFR in the cellular assays, whereas the pyrido[d]pyrimidines (see Section 3.C) wereequipotent in both assays. Selected compounds showed good in vivo activity against A431 and H125xenografts, but they showed poor aqueous solubility requiring formulation as fine particulate emul-sions. More soluble derivatives (e.g., 14) were synthesised by Warner Lambert to improve thepharmokinetic properties of this class of inhibitors.58 For additional examples of irreversible quina-zoline derivatives, see Refs. 59–60.

Quinazoline derivatives have also been reported as inhibitors of RAF kinase (e.g., 15, IC50 5100 nM in a MEK phosphorylation assay),61 c-erbB2/c-erbB4/EGFR (e.g., 16, IC50

c-erbB2 5 16 nM,B4 5 nM, IC 5 1),42, 45, 62 CSF-1R (e.g., 17, IC50 5 0.5 mM),47, 51 and VEGFR (e.g., 18, IC50

KDR

5 30 nM and IC50Flt 5 0.7 mM; compound 18 is about to enter Phase I clinical studies).63 –72 The

above compounds (15–18) (see Fig. 4) are representative examples of how medicinal chemists havebeen able to impart potency and selectivity to the quinazoline scaffold by modulating and fine-tuning the interactions of the modified template with the selected protein kinase.

Figure 3. Examples of 4-phenylamino-quinazolines as irreversible inhibitors.


Beside classical synthetic approaches in solution, methods for the synthesis of 4-anilino substi-tuted quinazolines and oxindole quinazolines on solid phase have been reported.73 2-Carboxy-quinazolines are synthesized in two steps on a hydroxymethylpolystyrene resin and functionalizedat the C-4 position using an approach previously demonstrated in solution for quinazolinones.74 TheC-7 position of a resin bound quinazoline was modified by a Mitsunobu reaction, and the introduc-tion of the C-4 oxindole substituents was achieved by nucleophilic displacement of the thioether link-er. Purification of the final compounds was achieved by solid phase extraction using an acidic sul-fonic silica.73

B. Phenylamino-Pyrimidines

A representative example of this class of compounds is STI 571 (CGP 57148) (19), a dual inhibitorof the v-Abl (IC50 5 38 nM) and PDGFR (IC50 5 50 nM) tyrosine kinases75 that is currently inPhase I clinical trials by Novartis in patients with chronic myelogenous leukemia (CML). This com-pound exhibits a high selectivity against a panel of other protein–tyrosine kinases and serine/threo-nine kinases,75 –76 and inhibits v-Abl and PDGFR autophosphorylation in intact cells with similarIC50 values (0.3 mM). In addition, STI 571 showed potent antitumor activity in vivo against v-Abl-and v-Sis-transformed BALB/c 3T3 cells in nude mice.77

Phenylamino-pyrimidine derivatives78 –79 were originally identified as dual inhibitors acting onPDGFR80 and PKC-a kinases81 (e.g., 20). Selectivity for the PDGFR against serine/threonine ki-nases was achieved by introducing a methyl group at the six position of the phenyl ring. The loss ofactivity against PKCa was ascribed to a steric clash of the modified inhibitor with some of theresidues forming the ATP binding site of the protein kinase C family. Increased potency and selec-tivity for the v-Abl at the in vitro level was obtained after an extensive optimization work on thephenyl ring.75

4,6-Dianilinopyrimidine derivatives have been reported as potent inhibitors of the EGFR tyro-sine kinase (e.g., 21, IC505 1.0 nM).82 The kinase domains of Lck, Fyn, ZAP-70, Csk, EGFR, andPKC can be inhibited with substituted 2-anilinopyrimidines (e.g., 22, IC50

Lck 5 40 nM; 23, IC50Fyn

5 68 nM; 24, IC50PKC 5 22 nM) (see Fig. 5).83

Figure 4. Additional examples of quinazoline derivatives.


C. Pyrido[d]pyrimidines and Pyrimido[d]pyrimidines

Pyrido[d]pyrimidines were first patented by Warner Lambert,84 – 85 and since then pyrido[4,3-d]-,pyrido[3,4-d]-, pyrido[2,3-d], and pyrido[3,2-d]pyrimidine templates have been used to target sev-eral kinases (see Figs. 6 and 7). After an initial report on 4,7-diaminopyrido[4,3-d]pyrimidines aspotent inhibitors of EGFR (e.g., 25),86 an extensive SAR study of all four isomeric series of pyri-do[d]pyrimidines, which can be considered as aza analogues of quinazolines, showed significant dif-ferences between them as inhibitors of the EGFR.87 For the set of compounds evaluated, the [3,4-d]and [4,3-d] series were the most active EGFR inhibitors, followed by the [3,2-d] derivatives, whilethe [2,3-d] compounds were less potent. Furthermore, it was observed that the addition of steric bulk

Figure 5. Examples of phenylamino-pyrimidines.

Figure 6. Examples of pyrido[3,4-d ]pyrimidines and pyrido[4,3-d ]pyrimidines.


(e.g., methyl or dimethyl groups) to the 6-N of pyrido[3,4-d]pyrimidine (e.g, 26 and 27) or the 7-Nof pyrido[4,3-d]pyrimidines (e.g., 28 and 29) resulted in an impressive boost in inhibitory potencyin the isolated enzyme assay (IC50 5 8, 6, 130, and 91 nM for 26, 27, 28, and 29, respectively) andin the inhibition of autophosphorylation of EGFR in A431 cells in culture (IC50 5 15, 21, 16, and14 nM for 26, 27, 28, and 29, respectively). In spite of the above data, the in vitro antitumor activi-ty of PD 158780 (26) appeared disappointing.88 Delayed tumor growth was observed in oestrogen-dependent MCF-7 and A431 epidermoid tumors and weak activity was observed in other EGFR-transfected tumor models.

To improve the aqueous solubility of the above class of compounds, three types of solubilizinggroups were investigated: neutral (alcohols and polyols), cationic (amines), and anionic (carboxy-lates).89 The substituents were introduced at the 7 position, which, in accordance with previously re-ported modeling studies for the EGFR,12 lies in the entrance of the adenine binding cleft of theeynzme providing some steric freedom for the accommodation of large groups. The most effectivesubstituents in terms of aqueous solubility (. 40 mM) and high potency in both enzyme and cellu-lar assays were weakly basic amine groups (e.g., 30; IC50 5 1.9 nM in the EGFR isolated enzymeassay). Substantial delays in tumor growth were observed in A431 xenografts treated with compound30 (25 mg/kg/injection on days 7–21 after tumor implantation).

Pyrido[3,4-d]- and pyrido[4,3-d ]pyrimidines have also been described as potent inhibitors of c-erbB2 and c-erbB4 kinases (IC50 5 11–100 nM)90 – 92 and EGFR tyrosine kinase combined withanti-angiogenic properties (e.g., 31).93 – 94

Pyrido[2,3-d]pyrimidine derivatives (32 and 33)95 active against PDGFR, FGFR, and pp60c-src

kinases were identified through compound library screening (see Fig. 7). Extensive SAR data withthis template was obtained by the Parke–Davis/Warner Lambert team by introducing variable sub-

Figure 7. Examples of pyrido[2,3-d ]pyrimidines.

stituents at the C-2 , C-6, C-7, and N-8 positions.10, 96 – 98 The structure–activity relationship datatogether with X-ray structural information obtained from kinases ligand-bound to ATP or competi-tive inhibitors were used to develop a model for the binding mode of this class of compounds.10 Inthis model, the pyrido[2,3-d]pyrimidine inhibitors are proposed to have a hydrogen-bonding patternsimilar to that of olomoucine (see Section 3.G) with the N-3 and the exocyclic 2-amino hydrogenforming a bidentate hydrogen bond system with two amino residues of the hinge region. Moreover,the 6-phenyl substituent protudes away from the ATP-envelop and interacts with a deep pocket ad-jacent to the adenine binding pocket made up of strands 4, 5, and 8, and the aC helix. With this struc-tural information available, structure-based design of highly potent and selective inhibitors for anumber of different protein kinases has been accomplished. Thus, selectivity with respect to FGFRwas obtained by replacing the 2,6-dichlorophenyl group at the 6-position of the pyrido[2,3-d]pyrim-idine heterocycle template by the 3,5-dimethoxyphenyl moiety. Thus, compound PD 166866 (34)had an IC50 value of 60 nM against FGFR and did not inhibit PDGFR, pp60c-src, MAP, CDK4, EGFR,and InsR kinases at 40 mM.97– 98 Further optimization of PD 166866 (34) led to PD 173074 (35),which is a potent and selective inhibitor of FGFR1 and VEGFR2 kinase activity in in vitro assaysand in cultured cells.99 The efficacy of this compound as an inhibitor of bFGF or VEGF-stimulatedangiogenesis in vivo has been demonstrated using a mouse corneal model.100 A substantial inhibi-tion of bFGF- or VEGF-induced neovascularitation was observed at 1mg/kg/day (i.p.) for bFGF and2mg/kg/day (i.p.) for VEGF with no apparent toxicity.

The X-ray structure of the tyrosine kinase domain of the FGFR1 complexed with PD 173074(35) together with the previously reported SAR data97 provide a foundation for understanding themolecular basis for its high selectivity. The high complementarity observed between the inhibitorand the ATP-binding cleft of the protein and, in particular, the interactions established by the 3,5-dimethoxy phenyl group attached to the 6-position of the pyrido[2,3-d]pyrimidine template under-line the potency and selectivity of PD 173074 (35).

Modification of the amino group of the 2-amino-6-(2,6-dichlorophenyl)-8-methyl-8H-pyri-do[2,3-d]pyrimidin-7-one template resulted in the identification of potent and broadly active tyro-sine kinase inhibitors.96 For example, PD 166285 (36), which had IC50 values of 79, 43, 9, and 44nM against PDGFR, bFGFR, c-src, and EGFR, respectively, tyrosine kinase activity, inhibitedPDGF- and EGF-stimulated receptor autophosphorylation in a number of cells lines with IC50 val-ues of 1.6–0.0065 mM.101 In in vivo testing, PD 166285 (36) was effective only against a limitednumber of tumor models. In spite of this and because of the excellent potency of this compoundagainst selected protein kinases, PD 166285 (36) was described as a potential candidate for ad-vancement to clinical trials with therapeutic potential in cancer, atherosclerosis, and restenosis.101

Recently, the synthesis and biological evaluation of a series of 2-amino-6-(2,6-dichlorophenyl)-8-methyl-8H-pyrido[2,3-d]pyrimidin-7-one derivatives with different substituents at the C-6 and N-8 positions has been reported.102 Compound 37 is a somewhat more potent inhibitor of PDGFR thanPD-166285 (IC50 5 31 nM vs. 79 nM) and it also inhibits the tyrosine kinase activity of bFGFR(IC50 5 88 nM) and c-src (IC50 5 31 nM). It was active in several PDGF-dependent cellular assaysand blocked the in vivo growth of PDGF-dependent tumor lines at oral doses between 20–40 mg/kg.

In addition to the irreversible quinazoline-type inhibitors discussed in Section 3.A, irreversibleinhibitors of the ATP binding site of the epidermal growth factor receptor containing the 4-(phenyl-amino)pyrido[d]pyrimidine acrylamide template have been reported in the same publication.53

Quinazoline, pyrido[3,4-d]pyrimidine, and pyrido[3,2-d]pyrimidine 6-acrylamides all showed, ingeneral, similar potencies in the isolated enzyme assay; although the pyrido[3,2-d]pyrimidine de-rivatives were somewhat less potent than the former analogues in the EGFR autophosphorylation assay.

The comparative study of the four isomeric series of pyrido[d]pyrimidines discussed above87

was complemented by the same group by preparing a series of 6-substituted 4-anilinopyrimido[5,4-



d]pyrimidines as inhibitors of the EGFR.103 Compound 38 (see Fig. 8) was the most potent inhibitorin this series in the A431 human epidermoid carcinoma cellular EGFR autophosphorylation assay(IC50 5 3.1 nM). The SAR data of pyrimido[5,4-d ]pyrimidine derivatives more closely resemblethose of the [3,2-d ] rather than the pyrido[3,4-d ]pyrimidine isomers. The conformational effect ofN-5 in pyrido[3,2-d ]pyrimidines and pyrimido[5,4-d ]pyrimidines, and C-5 in pyrido[3,4-d ]pyrim-idines was illustrated using X-ray crystal structures. A carbon rather than a nitrogen atom leads tosignificant conformational changes in the molecule: (i) a longer C5a}C4 bond; (ii) and a 308 out-of-plane rotation of the phenyl group. These structural changes can relieve the nonbonding interac-tions between the C-5 and N-9 protons and can account for the observed similarities and differencesin SAR data.

Other reports have also described the synthesis of pyrimido[5,4-d ]pyrimidines and their evalu-ation as inhibitors of EGFR (e.g., 39, IC50 5 1 nM; 40, IC50 5 21 nM).104 –105

D. Pyrrolo[d]pyrimidines and a Pyrrolo[2,3-b]pyridine

The pyrrolo[d ]pyrimidine template has been used by several pharmaceutical companies to inhibitthe tyrosine kinase domain of EGFR and c-Src.

Optimization of 7H-pyrrolo-[2,3-d ]pyrimidines derivatives106 against EGFR was pursued atNovartis by utilizing an in-house pharmacophore model for the ATP binding site of the above re-ceptor.13,16,19,107 In accordance to this pharmacophore model, the NH of the pyrrole ring and the N-1 of the pyrimidine ring form a bidentate hydrogen bond donor-acceptor system with the Gln767 andMet769 residues, and the substituted anilino moiety at the C-4 position interacts with the sugar pock-et and, in particular, with the Cys773 residue, which is unique to the EGFR family of kinases. In ad-dition, the substituents at the 5 and 6 positions can establish van der Waals interactions with the hy-drophobic pocket not used by the natural ATP ligand. A representative member of this structural classis CGP 59326 (41) (see Fig. 9), which was selected as a development candidate. The compoundshowed a good potency and selectivity in the in vitro kinase assay (IC50 5 27 nM) and in the inhi-bition of EGF-stimulated tyrosine phosphorylation in cells (IC50 5 0.3 mM). It also exhibited goodantiproliferative activity (IC50 5 0.5 2 1.9 mM) against a panel of EGFR-positive epithelial celllines (e.g., NCI-H596, MDA-MB468, A431) and only weak activity against EGFR-negative celllines (e.g., NCI-H520, NCI-H69). Improved biological and physicochemical properties in this serieswas obtained by modifying the 4- and 6-positions of the pyrrolo[2,3-d ]pyrimide template. Thus, avariety of substituents in the 6-position [e.g., ester (42), amide (43), heterocylic- (44), and p- and m-substituted aromatic rings (45–49)] were introduced in order to increase the number of van der Waalscontacts with the hydrophobic region formed mainly by the Thr766 and Thr860 residues. These mod-ifications translated in most of the cases to improved activity in the EGFR kinase assay (IC50 5 125 nM). Furthermore, replacement of the m-chloroanilino moiety at the 4-position by a (R)-phenethylamino group substantially improved the pharmokinetic properties of the compounds (e.g.,50–53). The best derivatives in this new series (in vitro IC50 5 1 2 3 nM) were orally bioavailable

Figure 8. Examples of pyrimido[5,4-d ]pyrimidines.


(cmax . 10 mM, 50–100 mg/Kg/mice) and blocked the EGF-stimulated tyrosine phosphorylationin EGFR-positive A431 cells (IC50 5 10 2 50 nM). Antiproliferative activities were observed inEGFR-positive epithelial cell lines (IC50 5 0.1 2 0.4 mM), whereas the compounds were inactivein a panel of EGFR-negative cell lines. A derivative of this series is about to enter Phase I clinicaltrials.

Pyrrolo[3,2-d ]- and [2,3-d ]pyrimidine derivatives have also been described by Novartis as ty-rosine kinase inhibitors of pp60c-src.111–117 It is important to mention that for the 5,7-modified pyrro-lo[2,3-d ]pyrimidine chemical template an alternative binding mode in the ATP-binding site of pp60c-

src has been postulated. The bidentate hydrogen bond donor-acceptor with the protein should involvethe amino group at position 4 and N-3. The substituent at the pyrrole nitrogen interacts with the sug-ar pocket, whereas the substituent at position 5 can establish van der Waals interactions with the hy-drophobic pocket not used by the natural ATP ligand. CGP 77675 (54) inhibits the phosphorylationof substrates by c-Src kinase with an IC50 value of 20 nM and showed specificity (7.5- to 500-fold)against other tyrosine and serine/threonine kinases (e.g., EGFR, KDR, v-Abl, Cdc2, and Fak) or oth-er members of the Src kinase family (Lck and Yes). In addition, it blocks tyrosine phosphorylationof the Src substrates Fak and paxillin in a Src-overexpressing cell line IC8.1 with IC50 values of 0.3and 0.5 mM, respectively, but it had much less effect on the phosphorylation of Src (IC50 5 5.7 mM).When tested in the rat fetal long-bone cultures, inhibition of parathyroid hormone-induced bone re-sorption was observed (IC50 5 0.8 mM). The compound partially prevents bone loss and rescuesbone microarchitectural features in young ovariectomized rats.111

Pyrrolo[2,3-b]pyridine derivatives were screened in a cell assay (inhibition of TNF-a produc-tion) to identify inhibitors of the p38 kinase (see also Section 3.H).118 RWJ 68354 (55; R. W. John-son) is a potent inhibitor of cellular p38 kinase activity (IC50 5 9 nM) and LPS-induced TNF-a pro-duction in mice (ED50 , 10 mg/kg) and in rats (ED50 , 25 mg/kg). This compound was describedas a promising candidate for further preclinical evaluation.

Figure 9. Examples of pyrrolo[d ]pyrimidines and a pyrrolo[2,3-b ]pyridine.


E. Pyrazolo[d]pyrimidines

Pyrazolo[3,4-d ]pyrimidine derivatives have been reported to be moderate inhibitors of CSF-1R47

and potent inhibitors of Lck, FynT, and EGFR tyrosine kinases.Compound 56 (see Fig. 10) was synthesized as a bio-isosteric replacement of the quinazoline

moiety in the CSF-1R program by Rhône–Poulenc Rorer. The replacement of the template provid-ed in vitro selectivity against the EGFR (IC50 5 0.18 mM CSF-1R compared to IC50 . 50 mMEGFR).47

PP1 (57) was designed based on a parent compound identified in the course of a tyrosine kinaserandom screening. The compound showed high potency and selectivity in vitro against Lck (IC50 55 nM) and FynT (IC50 5 6 nM), and a low micromolar activity in intact cells was observed.119 How-ever, it shows complex kinetics for inhibition of Lck, and so PP1 appears to be an ATP site-directedcompetitive inhibitor only at certain concentrations of ATP. PP2 (58), a close analog of PP1 (57), isalso a potent inhibitor of Lck and FynT with IC50 values of 4 and 5 nM, respectively. This compoundwas co-crystallized with the kinase domain of Lck, and the X-ray structure revealed that PP2 (58)binds in the ATP-binding site of the enzyme.120 The specificity profile of PP2 (58) against other pro-tein kinases was interpreted120 on the basis of the interactions established by the 3-(4-chlorophenyl)substituent with a hydrophobic pocket that has a unique amino acid composition for the Src familyof kinases. The structure of the Lck-PP2 (58) complex was also used to propose a binding mode for1,3-diphenyl-pyrrolo[3,4-d ]pyrimidine inhibitors of c-Src, previously disclosed in a review paper.19

Recently, a panel of C3-derivatized PP1 (57) analogues was designed, synthesized, and screenedagainst the catalytic domain of a rationally engineered v-Src protein (Ile338Gly; this point mutationwas introduced to create a unique pocket in the ATP binding site of v-Src). From this series of de-rivatives, compound 59 was identifed as a highly potent and selective inhibitor for the mutated v-Srckinase domain (IC50 5 1.5 nM vs. IC50 5 1 mM for wild-type v-Src; article was originally publishedon the Web, see Ref. 220). This structure-based design approach for both the protein kinase and theinhibitor can have applications in target validation at the cellular level and in the elucidation of thebiological events associated with phosphorylation-dependent signal transduction pathways.

Figure 10. Examples of pyrazolo[3,4-d ]pyrimidines.


Starting from a lead compound identified by random screening (60, IC50 5 0.2 mM),15 struc-ture optimization of the 4-phenylamino-pyrazolo[3,4-d ]pyrimidine template in an interactiveprocess led to highly potent (61–66, IC50 , 10 nM in vitro; 61 and 62, IC50 , 50 nM in cells) andselective (data reported on c-Src, v-Abl, PKCa, and Cdk1) EGFR tyrosine kinase inhibitors.121–122

In agreement with the pharmacophore model, which was previously used for the pyrrolo[2,3-d ]pyrimidine class,16 the SAR data show a preference for bulky substituents in the 3 position of thepyrazole ring and for a m-chloro substituent in the 4-phenylamino moiety. Furthermore, a hydro-gen bond interaction between the backbone amide of Phe832 and the hydroxy or amino group of themoiety at the 3 position was proposed to explain the highest binding affinity observed for the com-pounds containing these substituents.

F. Indolin-2-Ones

Chemical series of 3-substituted indolin-2-ones have been designed, synthesized, and characterizedby Sugen as a novel class of receptor tyrosine kinase inhibitors including VEGFR, FGFR, EGFR,Her-2, and PDGFR. The compounds can be easily obtained by condensing substituted indolin-2-onesand aldehydes or ketones in the presence of bases.123 –124 In a recent study, a series of derivativescontaining the indolin-2-one template and different arylidenyl substituents at the C-3 position wereevaluated for their relative inhibitory properties against a panel of tyrosine kinase receptors in cell-based kinase assays.125 The SAR data showed that it is possible to obtain selective chemical leadsfor specific tyrosine kinases with this template.

A good example of this class of compounds is SU 5416 (67) (see Fig. 11), which is presentlyunder evaluation in Phase II/III clinical studies (i.v. administration) for the treatment of human can-cers. This compound is a potent and selective inhibitor of KDR/flk-1 that showed an IC50 of 1 mMin a cellular tyrosine kinase assay (flk-1-overexpressing NIH 3T3 cells). The compound showedaround 20-fold less inhibitory potency on PDGF-dependent autophosphorylation and complete lackof activity with EGFR, InsR, and FGFR. Furthermore, it inhibited VEGF-driven mitogenesis of HUVECs in a dose-dependent manner with an IC50 of 40 nM. Extensive data on the effect of SU5416 on subcutaneous growth of a panel of tumor cell lines in athymic mice have been reported

Figure 11. Examples of indolin-2-ones.


in a recent publication.126 SU6668,127 another member of this class of compounds, is in Phase I clin-ical trials (p.o. administration) for the same indications.

SU5402 (68) and SU4984, two compounds related to SU5416 (67), have been co-crystallizedwithin the catalytic core of the tyrosine kinase domain of FGFR.128 In the X-ray crystal structure,the oxindole moiety occupies the same site as the adenine moiety of ATP,129 forming with N-1 andO-2 a bidentate hydrogen bond donor-acceptor interaction with two residues of the hinge region(Glu562 and Ala564). The substituent at the C-3 position of SU5402 (68), which is a more specific in-hibitor of FGFR than SU4984, extends into the hinge region between the two kinase loops and formsa hydrogen bond interaction between the carboxyethyl group and the side chain of asparagine-568.In addition, a conformational change in the nucleotide-binding loop is induced upon binding of thisinhibitor. These two features can account for the specificity profile observed for SU5402. The avail-able structural information on the FGFR together with homology models of the catalytic domains ofthe VEGF and PDGF receptors have been exploited in structure-based drug design approaches toidentify new indolin-2-ones with broader tyrosine kinase inhibitory profiles against the above threereceptors. The rationale of this approach is to try to combine the cytostatic and anti-survival proper-ties of the inhibition of the PDGFR with the anti-angiogenic effect of inhibiting the other two re-ceptors. To accomplish this, 5- or 6-substituted indolin-2-one derivatives bearing different groups inthe pyrrole ring were synthesized. Several compounds (e.g., 69 and 70) showed a broad inhibitoryactivity against Flk-1/KDR, FGFR1, and PDGFR both in biochemical and cellular assays.130

3-Substituted indolin-2-ones have also been described as weak in vitro inhibitors of the v-Abltyrosine kinase (71, IC50 5 15 mM),131–132 and potent c-raf-1 (72, IC50 5 17 nM)133 –134 and Cdk1/Cdk2 inhibitors (73, IC50 values of 12 nM and 0.6 for Cdk1 and Cdk2, respectively).135 –136 In thelast two cases, parallel synthesis and classical medicinal chemistry lead optimization approacheswere used and, for the Cdk1/Cdk2 inhibitors, the X-ray structure of the ligand-bound Cdk2 was usedin the selection of the building blocks to be incorporated in the template.

G. Purines

Purine analogs have been screened for inhibition of a variety of protein kinases, particularly serine/threonine kinases. Olomoucine (74) (see Fig. 12), which was first described as an inhibitor of cy-tokinin 7-glucosyltransferase,137 is a moderately active ATP-competitive inhibitor of Cdk1, Cdc2/

Figure 12. Examples of purine analogues.


cyclin B, Cdk2/cyclin A, Cdk2/cyclin E, and Cdk5/p35 (IC50 53–7 mM).138 –139 The inhibitory ac-tivity profile of olomoucine for the above kinases was slightly improved with roscovitine (75, IC505 0.2–0.7 mM), another purine derivative.138, 140 Attempts have been undertaken to increase the po-tency and selectivity profile of the purine template for cyclin-dependent kinases by synthesizinganalogs with varying substituents at positions 2, 6, and 9141–146 and exploiting structural informa-tion obtained by X-ray crystallography.147–154

The solid and solution phase synthesis and biological screening of combinatorial libraries basedon the purine scaffold found in olomucine has been reported by P. Schultz and coworkers,145–146,155,157

and other laboratories.156, 158–159 The iteration of 2,6,9-trisubstituted library synthesis with structuralanalysis of the optimized hits allowed the identification of purvalanol B (76), a potent and highly se-lective*** inhibitor of Cdk2-cyclin A (IC50 5 6 nM). In addition, the structure of Cdk2 with purvalanolB (76) was determined by X-ray crystallography at 2.05 Å resolution.145 Several structural featurescould explain the increased inhibitor potency of the above compound relative to previously reportedinhibitors of Cdk2 (e.g., olomucine and flavopiridol): (i) additional interactions established by the iso-propyl side chain of the C2 substituent; (ii) improve packing and polar interactions of the 3-chloroanili-no group at N6; and (iii) reduced number of conformations due to an increase in conformational con-straints. The above synthetic and in vitro biological work was complemented by analyzing the cellulareffects of purvalanol A(77), a close analog of purvalanol B (76) with better membrane-permeable prop-erties in mammalian cells (60 human tumor cell lines from the NCI) and yeast.

Inhibitory activities in the low nanomolar range for Cdk1/cyclin B (IC50 5 28–90 nM) havebeen obtained by combining trans-4-amino-cyclohexanol or trans- and cis-cyclohexane-1,4-diaminesubstituents at position 2 of the purine template with a series of m- and p-substituted aniline moietiesat the 6 position (e.g., 78 and 79).141 Some of the most potent compounds were selective againstPKCa, PKA, and EGFR by a factor of 10–100.

H. Pyridinylimidazoles, Pyrimidinylimidazoles, and Related Compounds

The pyridinylimidazole derivative SKF-86002 (80) (see Fig. 13) was the original representative of anovel class of anti-inflammatory agents that inhibit the p38 MAP kinase.160 The X-ray structures of theligand-bound p38 MAPkinase26,161–162 have shown that for this class of compounds the pyridine grouplies in the adenine binding pocket of ATP. In addition, the pyridine nitrogen is engaged in a hydrogenbond interaction with the peptide backbone of Met109.† This moiety has been described in a recentpublication as responsible for the inhibition of the human liver P450 isozymes observed for severalpyridinylimidazole derivatives.164 The inhibition was ascribed to the interaction of the 4-pyridinylgroup with the heme iron of cytochrome P450, and it caused increased liver weight and significant el-evations of hepatic P450 enzymes in 10-day rat dose-ranging toxicological studies with SKF-86002(80) and SKF-203580 (81).†† This side effect was reduced by replacing pyridine by substituted pyrim-idines (e.g., 82, IC50 5 0.48 mM), which are known to be weak P450 binders relative to pyridine.164,†††

The in vitro potency in the pyrimidinylimidazole class has been improved by replacement of the mor-pholinylpropyl group with a piperidine moiety, SB-220025 (83, IC50 5 19 nM).169

During the biochemical and enzymological characterization of recombinant human p38 pro-duced in Drosophila S2 cells, Frantz et al.170 showed that pyridinylimidazoles are able to bind both

***Among the 22 human purified kinases tested, only cdc2-cyclin B, CDK2-cyclin A, CDK2-cyclin E, and CDK5-p35 weresignificantly inhibited (IC50 5 6 2 9 nM).145

†For additional information on the pharmaphore model of p38 MAP see Ref. 163.††This compound is a selective p38a and b MAP kinase inhibitor165 –166 that has been used to validate the p38 MAP kinaseconcept in cellular systems.167

†††Examples of pyrimidinylimidazoles have also appeared in a recent Merck patent.168


the active and inactive forms of the enzyme. The latter effect, which is noncompetitive with ATP,may block the biological activity of the enzyme by reducing its rate of activation and, if this mech-anism is correct, ATP would not effectively compete with this class of inhibitors in vivo.

Several patents and publications have reported the replacement of the central imidazole inpyridinylimidazole p38 MAP kinase inhibitors by oxazoles,171 pyrroles,172–176 pyrazoles,177–179

pyrimidines,180 –182 and indoles.183 Potent in vitro and in vivo compounds containing the above mo-tifs were identified proving the nonessential character of the imidazole ring for inhibiting the p38MAP kinase protein.

In a recent publication from Merck,184 an extensive structure–activity relationship study of aseries of imidazoles allowed them to identify 84, which is an extremely potent (IC50 5 0.19 nM) andselective inhibitor of p38 MAP kinase (data reported on c-Raf, JNK2a1, JNK2a2, Lck, EGFR, MEK,PKA, and PKC). The compound inhibits the lipopolysaccharide-induced release of TNF-a from hu-man whole blood with an IC50 value of 2.8 nM, shows oral bioavailability (85% in rat, 86% in fast-ed male rhesus monkey), and produces a significant inhibition of secondary paw swelling in a func-tional model of arthritis.

Further modifications of the imidazole template have led to the identification of L-779,450 (85;Merck), which proved to be a potent (IC50 5 2.0 nM) and selective inhibitor (10 other kinases test-ed) of the Raf kinase.185 L-779,450 (85) also inhibited the Ras-mediated activation of the MAP ki-nase cascade in cancer cells with an IC50 value of 0.7 mM. The anchorage-independent growth ofdifferent cells lines was inhibited at concentrations ranging from 0.3–2 mM.

I. Various Structural Classes

1. Balanol

Balanol (86) (see Fig. 14) is a natural product isolated from the fungus verticullium balanoides thatshows high inhibitory activity for specific serine and threonine kinases (e.g., most PKC isoforms),186

Figure 13. Examples of pyridinylimidazoles, pyrimidinylimidazoles, and related compounds.


while being ineffective against tyrosine kinases. The compound has been subjected to extensivechemical modifications, including the removal of functional groups187 and the synthesis ofacyclic,188 perhydroazepine,189 –190 conformational constrained,191 and indane192 analogs of bal-anol. Prodrug approaches have been performed to increase the activity of this class of compounds incellular assays.193 –194 Recently, the X-ray structure of balanol in complex with the catalytic subunitof cAMP-dependent protein kinase has been reported.195

Figure 14. Various structural classes of protein kinase inhibitors.


2. Flavopiridol

Flavopiridol (87, HMR 1275, NSC 649890 or L 86-8275)196 is a flavonoid structurally related to analkaloid isolated from Dysoxylum binectariferum. The compound inhibits most cyclin-dependent ki-nases (e.g., cdk1, cdk2, cdk4, and cdk7), suppresses the growth of a panel of tumor cell lines,196 –

201 and has antitumor activity on human tumor xenografts198 similar to standard cytostatic drugs andsuperior to them at least in prostate carcinoma.196 Based in part on its behavior in the anticancer drugscreening system of the National Cancer Institue, flavopiridol was selected for further studies. In aPhase I clinical trial,6 flavopiridol, which was administered as 72-h infusion every two weeks,showed antitumor effects in certain patients with renal, prostate, and colon cancer, and non-Hodgkin’s lymphoma. Phase II clinical trials are currently ongoing. The X-ray structure of the de-schloro analogue of flavopiridol (88) in complex with Cdk2 has been recently determined.202

3. Staurosporine, Indolocarbazole, and Related Analogues

Staurosporine (89) is a microbial alkaloid that is a potent, but nonspecific protein kinase inhibitor.203

Several staurosporine derivatives with improved specificity profiles have emerged in recent years aspotential anticancer drugs.204 PKC 412 (CGP41251) (90) is a potent PKC inhibitor (IC50 5 50 nM)with an improved specificity profile compared to staurosporine.205 The compound has been shownto exhibit antitumor activity in vitro and in vivo and to reverse multidrug resistance.206 PKC 412 (90)is in Phase II clinical studies by Novartis for the treatment of low grade non-Hodgkin’s lymphomaand chronic lymphocytic leukemia.7 UCN-01 (91; Kyowa Hakko) is another staurosporine deriva-tive. It is a potent PKC inhibitor (IC50 5 4.1 nM)203, 207 that was isolated from a Streptomyces strain(N-126).208 –209 The compound is in Phase I clinical trials for the treatment of advanced cancer inpatients who have not responded to previous treatment.

A series of indolocarbozole derivatives were licensed by Cephalon Inc. from Kyowa Hakko sev-eral years ago for development against several diseases, including head and spinal injury, neurode-generative disorders, cancer, and cardiovascular diseases. CEP-751 (92, KT-6587) is a potent in-hibitor (IC50 5 5 nM) of the tyrosine kinase domain of the NGF receptor TrkA, as well as the relatedneurotrophin receptors TrkB and C.210 It caused impressive tumor regression in Dunning H prostatecancer in Copenhagen rats.211 The corresponding water-soluble prodrug ester CEP-2563 (93)212 isin Phase I clinical studies against prostate cancer. Indolocarbozole compounds have also been de-scribed as potent and selective inhibitors of PDGR. 3744W (94) inhibited PDGFR autophosphory-lation in the intact A10 cell assay with an IC50 value of 14.5 nM.213 In human smooth muscle cells,this compound blocked (c 5 1 mM) the autophosphorylation of the a and b isoforms of the PDGRby 85% and 80%, respectively.

LY-333531 (95)214 is a potent and selective inhibitor of PKCbI (IC50 5 4.7 nM, Ki 5 2 nM)and PKCbII (IC50 5 5.9 nM). The compound is in Phase II clinical trials by Ely Lilly for the treat-ment of diabetic complications.215 –216 It has been shown that PKCbII is preferentially activated inthe retina, heart, and aorta of diabetic rats. In addition, the activated PKC enzyme may play an im-portant role in diabetic retinopathy leading together with other disease states to blindness.

Recently, the synthesis of some cyclopentane-bridged indolocarbazole derivatives using a con-vergent route has been described. This approach may open a new avenue in the synthesis of potentand selective PKC inhibitors (e.g., 96, IC50 5 53 nM).217

4. Phthalazines

Phthalazine derivatives (e.g., 97, 98) have been described as inhibitors of the VEGF and PDGF re-ceptor tyrosine kinases in a recent patent by Novartis.218 The compounds showed submicromolar in-hibitory potencies in the isolated enzyme assay and in cellular based assays. A series of compounds

from this class inhibited in vivo the growth of a broad panel of human carcinomas and the formationof microvessels.219 A lead compound from this series (PTK 787/ZK 222584; 98) has already passedpreclinical development (a joint effort of Novartis with Schering-Berlin and Tumor Klinik Freiburg).

4. C O N C L U S I O N

Inhibition of protein kinases by ATP site-directed competitive and irreversible inhibitors is a rapid-ly evolving area of research in academia and industry. Rather than attempt an exhaustive review ofall the published chemical templates, this work has put more emphasis on the kinase inhibitors thathave entered or are about to enter clinical trials. After overcoming research and development chal-lenges, clinical trials are the ultimate test for these “highly promising” therapeutic agents. It is ob-vious that the results of these studies will have a tremendous impact on the future of this medicinalchemistry approach. Positive results will greatly stimulate further research in the protein kinase in-hibition field. In the meantime, new protein kinases are emerging as targets for therapeutic inter-vention. Structure-based design approaches and new methods to increase compound diversity willplay an important role in the successful derivatization of the “classical kinase templates” and in theidentification of new lead structures.

A B B R E V I A T I O N S

Abl, v-abl, Abelson leukemia virus tyrosine kinaseATP, adenosine triphosphatebFGFR, basic FGFRBTC, betacellulinCdk, CDK, cyclin-dependent kinaseCKIs, cyclin-dependent kinase inhibitorsCML, chronic myelogenous leukemiaCOX-2, cyclo-oxygenase-2CSBP, cytokine-suppressive anti-inflammatory drug binding proteinCSF-1R, colony stimulating factor-1 receptorCsk, C-terminal src kinaseEGF, epidermal growth factorEGFR, epidermal growth factor receptorFak, focal adhesion kinaseFGF, fibroblast growth factorFGFR, fibroblast growth factor receptorFlk-1, fetal liver kinase-1Flt, fms-like tyrosine kinaseHB-EGF, heparin-binding EGF-like growth factorHer-2, p185neu, erbB-2, p185erbB-2HPLC, high-pressure liquid chromatographyHUVEC, human umbilical vein endothelial cellIGF-IR, insulin-like growth factor-I receptorInsR, insulin receptorJNK, c-Jun N-terminal kinaseFyn, fyn, p59fyn

KDR, kinase insert domain-containing receptorLck, lck, p56lck


MAP, mitogen-activated proteinMAPK, mitogen-activated protein kinaseNGF, nerve growth factorPET, positron emission tomographyPDGF, platelet-derived growth factorPDGFR, platelet-derived growth factor receptorPKA, cAMP-dependent protein kinasePKC, protein kinase CPKCa, protein kinase C-aRPTK, receptor protein tyrosine kinaseSAR, structure–activity relationshipSCCH, squamous cell carcinoma of the head and neckSrc, c-src, pp60c-src

TGF-a, transforming growth factor aTNF, tumor necrosis factorVEGF, vascular endothelial growth factorVEGFR, vascular endothelial growth factor receptorZAP-70, j chain associated protein 70 KDa

We thank our colleagues Drs. P. Imbach, G. Bold, P. Manley, and M. Lang for helpful suggestions and for kindly reviewingthe manuscript.

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Carlos García-Echeverría received his B.S. degree and Ph.D. in organic chemistry at the School of Chem-istry—University of Barcelona (Spain) working with Prof. F. Albericio and M. Pons. After a three-year post-doctoral stay with Prof. D. H. Rich at the University of Madison–Wisconsin, he joined the Exploratory Researchgroup of Ciba–Geigy AG (now Novartis Pharma AG) in 1993. Since his incorporation at the Oncology Re-search Group in 1995, he has been the chemistry sponsor of different programs linked to disruption of cellgrowth signaling.

Peter Traxler received his Ph. D. degree in organic chemistry (natural products) at the University of Basel(Switzerland). Following a postdoctoral stay with Prof. G.R. Pettit at Arizona State University (Tempe), hejoined Ciba–Geigy AG (now Novartis Pharma AG) in 1972 to work as medicinal chemist. Dr. Traxler has beenactively involved in pharmaceutical drug discovery in the fields of antibiotics and protein tyrosine kinase in-hibitors (over 40 publications and 30 patents).

Dean B. Evans graduated from the University of Salford (UK) with a B. Sc. (Hons) degree in Applied Biology.


His Ph.D. thesis was conducted at the University of Sheffield Medical School (UK) investigating the regulationof human osteoblast-like cell metabolism by bone active peptides and pharmacological agents. During subse-quent postdoctoral positions, he continued his research aimed at understanding the mechanisms of how boneactive factors regulate osteoblast-like cell function in regard to the disease osteoporosis. He joined the Oncol-ogy Research Group within Ciba–Geigy AG (now Novartis Pharma AG), Basel, Switzerland as a laboratoryhead in April 1994, where he is currently. In addition, he is the deputy program team head of a program aimedat discovering and developing new inhibitors of tyrosine kinase receptors, and he is also the International Pro-ject Team Representative of Research for Femarat, a marketed breast cancer product.

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ATP site-directed competitive and irreversible inhibitors of protein kinases