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Clinical perspectives for irreversible tyrosine kinase inhibitors in cancer

Biochemical Pharmacology, 11, 84, pages 1388 - 1399

Graphical abstract

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Abstract

Irreversible inhibitors provide potent and selective inhibition of tyrosine kinase enzymes. Use of such inhibitors has proved promising in overcoming the tumor resistance encountered with reversible tyrosine kinase inhibitors. Irreversible inhibitors inactivate their protein target through covalent interaction with a nucleophilic cysteine residue within the nucleotide binding pocket of the kinase domain.

Different irreversible tyrosin kinase inhibitors directed against epidermal growth factor receptor (EGFR), Bruton's tyrosine kinase (BTK), vascular endothelial growth factor receptor (VEGFR) and fibroblast growth factor receptor tyrosine kinase (FGFR) have been developed and some of them have been employed clinically as anticancer agents. This review focuses on recent preclinical and clinical progress with currently available irreversible tyrosine kinase inhibitors. The chemical structures of the candidates, structure-activity relationships, biological activities and results of current clinical investigations are described.

Abbreviations: AE - adverse event, BTK - Bruton's tyrosine kinase, CI - confidence interval, CLL - chronic lymphocytic leukemia, CR - complete response, DTL - dose limiting toxicity, EGFR - epidermal growth factor receptor, FGFR - fibroblast growth factor receptor, HR - hazard ratio, MTD - maximum tolerated dose, NSCLC - non small cell lung cancer, NHL - Non-Hodgkin's lymphoma, ORR - overall response rate, OS - overall survival, PD - progressive disease, PDGFR - platelet derived growth factor receptor, PFS - progression free survival, PR - partial response, SCCHN - squamous cell carcinoma of the head and neck, SD - stable disease, TK - tyrosine kinase, TKI - tyrosine kinase inhibitor, VEGFR - vascular endothelial growth factor receptor.

Keywords: Tyrosine kinase, Irreversible inhibitors, EGFR, BTK, Covalent interaction.

1. Introduction

The human genome encodes for 518 protein kinases [1] , of which approximately 100 are tyrosine kinases (TKs). These kinases regulate several physiological mechanisms, including cell proliferation, differentiation, migration and metabolism, by transferring the ATP terminal phosphate to tyrosine residues of protein substrates. TKs have been divided into two major groups: transmembrane receptor TKs [2] , characterized by membrane localization and by the presence of an extracellular domain and non-receptor TKs [3] , mainly located in the cell cytoplasm as integral components of the signaling cascades triggered by receptor TKs and other cell-surface receptors.

Dysfunction in kinase activity disrupts the normal control of cellular phosphorylation signaling pathways and leads to numerous pathologies ranging from inflammation to cancer [4] . This observation has stimulated the development of numerous small molecule kinase inhibitors targeting kinases such as breakpoint cluster region-Abelson proteine kinase (Bcr-ABL), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and platelet derived growth factor receptor (PDGFR) [5] . To date, a number of tyrosine kinase inhibitors (TKIs) have been employed in the treatment of cancer and several are in various stages of clinical development, demonstrating the importance of tyrosine kinases as prime targets for novel antitumor agents. The majority of TKIs developed thus far target the ATP binding site, which is highly conserved across the human protein kinases. ATP-competitive inhibitors typically consist of a heterocyclic ring system that occupies the purine binding site, where it serves as a scaffold for side chains that occupy adjacent hydrophobic regions. These ATP-competitive compounds inhibit protein catalytic activity in a reversible manner, binding to the kinase domain of the target through weak interactions (hydrogen-bonds, van der Waals and hydrophobic interactions).

In the past decade, much progress has been made in the development of a new class of potent and selective TKIs, that irreversibly inhibit their target protein via the formation of covalent bonds [6], [7], [8], and [9]. Covalent irreversible TKIs are characterized by a heterocyclic core structure (driving portion), generally resembling that of reversible inhibitors, carrying at a proper position an electrophilic functionality (warhead) that covalently interacts with a nucleophilic cysteine residue located near the ATP binding pocket [4] and [10]. There are several potential advantages for irreversible kinase inhibitors over conventional reversible ATP-competitive ones. An irreversible inhibitor would be expected to have prolonged pharmacologic effects relative to systemic exposure. In fact, when the target enzyme is deactivated by covalent bond, the biological effect should persist even after the inhibitor has left the circulation. Furthermore, covalent bond formation can circumvent competition with high ATP concentrations in the cell and is less affected by changes in the ATP binding affinity, which can occur with mutant forms of the enzyme. As a consequence, time-dependent inactivation gives irreversible inhibitors the potential to overcome resistance. A further advantage is improved selectivity, since only those kinases that form the covalent bond with the inhibitors should be inhibited.

On the other hand, the presence of chemical groups affording irreversible binding to proteins raises the issue of selectivity, both within the kinome and in competition with other cell constituents. Promiscuity is recognized as a major issue in the development of new irreversible TKI drugs. The catalytic domains of all kinases present similar three-dimensional structures, making selectivity within the kinome a difficult goal. However, the large number of available inhibitor-kinase co-crystal structures [11] and [12] has enabled development of selective driving portions endowed with high target affinity, by tailoring the structural elements required for interaction with the ATP-binding site of the protein. The introduction of a reactive warhead at the proper position on a specific recognition portion should increase target selectivity, at least on a time-resolved scale. In fact, irreversible TKIs target specific cysteine residues, and only a limited group of kinases has a cysteine at corresponding positions [7] and [10]. Thus, among the kinases showing affinity for the recognition (driver) portion, only those having a thiol group that can react with the warhead would be inhibited irreversibly, while the activity of others will be affected in a competitive manner. A drawback of covalent binding kinase inhibitors is that the intrinsic reactivity of cysteine-reactive groups leads to non-selective reactions with off-target proteins, giving rise to increased toxicity and lack of target specificity [13] and [14]. On the other hand, higher selectivity against off-targets can be achieved by combining a low intrinsic reactivity of the electrophilic warhead with a suitable arrangement of the driving portion, so that the reaction with the thiol can only occur when preceded by specific non-covalent binding of the inhibitor, presenting the reactive counterparts at a favorable distance and orientation.

The number of irreversible TKIs entering clinical trials studies is steadily increasing, although the kinases targeted by irreversible inhibitors only represent a small fraction of all kinases targeted by therapeutic agents. This review focuses on clinical and preclinical progress in irreversible TKIs. Structures, biological properties and results of clinical trials are described for the irreversible ErbB and Bruton's tyrosine kinase (BTK) inhibitors currently under clinical investigation. In addition, we present an overview of VEGFR and FGFR irreversible inhibitors as preclinical candidates to discuss the potential of these compounds in a broader application of this approach.

2. Medicinal chemistry of irreversible TKIs

2.1. Introduction

The recognition of the potential reactivity of cysteine residues within the catalytic site of protein kinases has opened the way for a rational design of inhibitors that terminally inhibit such kinases [15] . Alignment of all the kinases in the human genome [1] , followed by bioinformatic analysis of the kinome [6] , revealed that approximately 200 different kinases have a cysteine located in the vicinity of the ATP binding pocket. Cysteines that are accessible for covalent modification have been recently classified according to their locations in the ATP active site [4] and [16]. Within the tyrosine kinase subfamily of protein kinases, three groups of cysteines have been addressed by irreversible inhibitors ( Fig. 1 ): (i) a cysteine located close to the hinge region and present in 11 kinases is targeted by covalent irreversible EGFR [17] and BTK [18] inhibitors; (ii) a cysteine located in the glycine-rich loop, at the top of the ATP binding site of FGFR TK, has recently been targeted by a covalent irreversible inhibitor [19] ; (iii) a cysteine near the bottom of the ATP pocket, present in 48 kinases, has been explored as a target for VEGFR [20] and [21] covalent inhibitors. The ability to exploit unconserved cysteine in the kinase family [22] affords the ability to refine their selectivity profile and provides a strategy for overcoming the major drawbacks of reversible TKIs in clinical practice or under clinical investigation (i.e. lack of efficacy and resistance). Covalent irreversible TKIs require careful optimization of both the non-covalent binding affinity and the reactivity of the electrophilic warhead. To date, the majority of irreversible TKIs has been designed by appending to a highly specific recognition portion, common to that of reversible inhibitors, an electrophilic functionality at that position with a geometry that is compatible with the formation of the critical bond.

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Fig. 1 Schematic representation of cysteines targeted by irreversible TKIs in the ATP-binding site of tyrosine kinases (based on the crystal structure of EGFR in complex with the reversible inhibitor Tarceva (green carbons) (PDB ID: 1M17). The protein backbone at the ATP-binding pocket is displayed in white ribbons. The spheres indicated the locations of the three groups of cysteines that have been addressed by irreversible inhibitors: (i) a cysteine located close to the hinge region (Cys797 in EGFR); (ii) a cysteine located in the glycine-rich loop (Cys486 in FGFR); (iii) a cysteine near the bottom of the ATP pocket (Cys1045 in VEGFR). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

2.2. ErbB inhibitors

2.2.1. ErbB inhibitors in clinical development

Irreversible inhibitors of EGFR (ErbB1) and human epidermal growth factor receptor 2 (HER-2 or ErbB2) tyrosine kinases are the most advanced drug candidates under clinical evaluation. EGFR and ErbB2 are transmembrane-receptor TKs belonging to the ErbB family, which also includes related members ErbB3 and ErbB4. The overexpression of EGFR and ErbB2 has been observed in many human cancers, such as lung, head and neck, colorectal, ovarian, breast and bladder cancers [23] , and a strong correlation has been found between solid tumors with high levels of EGFR/ErbB2 and poor prognosis [24] . Therefore, compounds targeting the ErbB family TKs have been extensively investigated as antitumor agents. The first generation of EGFR-targeting therapeutic agents includes the reversible EGFR inhibitors gefitinib (Iressa) and erlotinib (Tarceva), approved for the treatment of non-small-cell lung cancer (NSCLC), and the dual EGFR/ErbB2 reversible inhibitor lapatinib (Tykerb), approved for the treatment of ErbB2-positive breast cancer. Although these drugs have been extremely effective in patient populations with tumor containing mutated oncogenic forms of TKs (e.g. L858R and del 746–750 of EGFR), their usefulness has been diminuished by the significant number of non-responding patients and by the emergence of resistance during treatment [25] . Secondary mutation of a single amino acid in the catalytic domain of EGFR, i.e. conversion of gatekeeper threonine 790 to methionine (T790M), is the most common mechanism of acquired resistance to reversible tyrosine kinase inhibitors [26] . The increased ATP affinity showed by T790M mutants with respect to the L858R mutant of EGFR, which prevents reversible inhibitors from binding at cellular high ATP concentrations, can be overcome by second generation irreversible EGFR inhibitors able to covalently alkylate a specific cysteine residue (Cys797 in EGFR, Cys805 in ErbB2, and Cys803 in ErbB4) close to the ATP-binding site of the receptor [11] and [15]. Despite the high sequence homology of the ATP-binding sites, a cysteine residue at the position corresponding to that of Cys797 in EGFR (or Cys805 in ErbB2, or Cys803 in ErbB4) is rare among other protein kinases, so that it can represent a selectivity filter to achieve irreversible inhibition of ErbB TKs only.

Acrylamides are Michael-acceptor compounds that can readily react with nucleophiles, such as thiols of cysteines, giving the conjugate-addition products ( Fig. 2 ). The introduction of an acrylamide fragment on the heterocyclic scaffold of known reversible EGFR/ErbB2 binders, mainly 4-anilinoquinazoline or 4-anilino-3-cyanoquinoline, has led to potent and selective irreversible ErbB inhibitors. This strategy has prompted the development of a number of effective covalent inhibitors, which have progressed to late stage clinical development [10] . All irreversible TKIs currently in clinical development (ErbB and BTK inhibitors in Table 1 ), whose structures have been disclosed (compounds 19 in Fig. 2 ), share a common electrophilic acrylamide warhead that undergoes Michael addition reaction with cysteine residue within the ATP-binding pocket of the enzyme ( Fig. 2 ). On the other hand, alternative electophilic functionalities with different reaction mechanisms toward cysteine have been exploited in the past decade. Non-acrylamide irreversible TKIs (such as compounds 11, 12, 19, and 20 in Fig. 3 ) are currently under preclinical evaluation, their structures and activities are discussed hereinafter (Sections ErbB inhibitors in preclinical development and Other TK inhibitors).

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Fig. 2 Michael reaction mechanism of acrylamide toward cysteine and chemical structures of acrylamide-based irreversible TKIs in clinical development. In the Michael addition the electron-rich sulphur atom of cysteine reacts at the electophilic β-carbon of the acrylamide warhead. The intermediate enolate evolves to the final 1,4-addition product.

Table 1 Irreversible tyrosine kinase inhibitors in clinical development.

Drug Target kinase(s) Company Cancer Development Phase Ref
Canertinib (CI-1033) EGFR, HER2-4 Pfizer NSCLC Phase II (no ongoing trials) [51]
Breast cancer Phase II (no ongoing trials) [52]
Pelitinib (EKB-569) EGFR, HER2 Wyeth/Pfizer NSCLC Phase II (no ongoing trials) NCT00067548 *
Colorectal cancer Phase II (no ongoing trials) NCT00072748 *
Neratinib (HKI-272) EGFR, HER2 Wyeth/Pfizer NSCLC Phase II (no ongoing trials) [56]
Breast cancer Phase III NCT00878709 *
Solid tumours Phase II NCT00706030 *
Dacomitinib (PF00299804) EGFR, HER2-4 Pfizer NSCLC Phase III [64]
Gastric cancer Phase I/II NCT01152853 *
Head and neck cancer Phase I/II NCT01449201 *
Glioblastoma Phase I/II NCT01112527 *
Afatinib (BIBW2992) EGFR, HER2 Boerhringer Ingelheim NSCLC Phase III [68]
  Phase III NCT01085136 *
  Phase III NCT01523587 *
Breast cancer Phase III NCT01125566 *
Head and neck cancer Phase III NCT01345682 *
  Phase III NCT01345669 *
Prostate cancer Phase II NCT00706628 *
Esophagogastric cancer Phase II NCT01522768 *
Colorectal cancer Phase II NCT00801294 *
Glioma Phase II NCT00727506 *
Glioblastoma Phase I NCT00977431 *
HM781-36B EGFR, HER2-4 Hanmi Pharmaceutical Co.,Ltd Solid tumours Phase I [74]
AV-412/MP-412 EGFR AVEO Pharmaceuticals, Inc. Solid tumours Phase I [75]
CO-1686 Mutant EGFR Clovis Oncology, Inc. NSCLC Phase I/II NCT01526928 *
Ibrutinib (PCI-32765) BTK Pharmacyclics, Inc and Janssen Research & Development, LLC CLL Phase III NCT01578707 *
B cell Lymphoma Phase II NCT01236391 *
  Phase II NCT01325701 *
Multiple Myeloma Phase II NCT01478581 *
AVL-292 BTK Avila Therapeutics CLL, NHL Phase I [48]
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Fig. 3 Other irreversible TKIs under preclinical investigation.

A first group of covalent irreversible EGFR inhibitors under clinical evaluation is represented by the quinazoline-based inhibitors canertinib [27] (1, CI-1033, Fig. 2 ), AV-412/MP-412 [28] (2, Fig. 2 ), afatinib [29] (3, BIBW2992, Fig. 2 ), dacomitinib [30] (4, PF00299804, Fig. 2 ), and HM781-36B [31] (5, Fig. 2 ). The acrylamide warhead is at the 6-position on the heterocyclic structure, designed to enable formation of a covalent bond with the thiol group of cysteine in the ATP binding pocket of the kinase domain. Co-crystallization of a 6-acrylamidoquinazoline irreversible inhibitor, namely 6-acryalmide-4-(3-bromoanilino)quinazoline (PD168393), within the kinase domain of human EGFR [11] showed the crucial interactions of this class of inhibitors within the catalytic site of EGFR. The 4-anilinoquinazoline driving portion adopts a conformation similar to that observed for several reversible quinazoline inhibitors in complexes with kinases, in particular: (i) the N1 and N3 atoms form two crucial hydrogen bonds with the backbone nitrogen of Met793 and with the side chain of Thr790 (via a water bridge), respectively; (ii) the 4-anilino substituent points toward an hydrophobic pocket located at the top of the adenosine binding site; and (iii) substituents at the 6- and 7-positions are directed toward the solvent. When the 4-anilinoquinazoline portion is accommodated within the ATP pocket of ErbB receptors, the acrylamide side-chain is brought into close proximity with Cys797 of EGFR (or the analogous Cys805 and Cys803 in ErbB-2 and -4, respectively).

Canertinib (1) is a selective irreversible pan-ErbB inhibitor, active on EGFR, ErbB-2, and ErbB-4 [27] . ErbB-3 has no intrinsic kinase activity and it signals only when dimerized with another ErbB receptor; therefore, ErbB-3 signaling is also precluded by blocking TK activity of EGFR, ErbB-2, and ErbB-4. The acrylamide warhead is at the 6-position on a 4-(3-chloro-4-fluoroanilino)quinazoline driving nucleus bearing a water-solubilizing group at the 7-position. Canertinib shows inhibition of EGFR and ErbB2-4 at nanomolar concentrations and it has been shown to be effective against a variety of human lung and breast carcinomas [32] . The 4-anilinoquinazoline AV-412 (2) is a potent dual inhibitor of EGFR and ErbB2 TKs, including mutant EGFR (L858R/T790M), with selectivity against other kinases (more than 100-fold selective for ErbB TKs). Moreover, AV-412 abrogates EGFR signaling in cell lines harboring the T790M mutation of EGFR, responsible for resistance to gefitinib [28] . Within the 4-anilinoquinazoline class, afatinib (3) is the most advanced clinical candidate, currently under phase III evaluation for the treatment of NSCLC, breast, and head and neck cancers (Table 2). This derivative is a selective dual EGFR/ErbB2 inhibitor active on wild-type and mutant EGFR, including gefitinib-resistant isoforms [29] . The improved pharmacokinetic properties of afatinib are provided by hydrophilic side chain, pendant off the Michael acceptor group through a methylene spacer ( Fig. 2 ). The dimethylamino substituent has been proposed to act both as a solubilizing group and an intramolecular catalyst, by de-protonation of the sulfydryl group of the nucleophilic cysteine residue. Afatinib inhibits EGFR and ErbB2 at nanomolar concentration and it has proved to be more effective than erlotinib or gefitinib in inhibiting survival of lung cancer cell lines resistant to first-generation inhibitors. Dacomitinib (4) is a quinazoline-based irreversible pan-ErbB inhibitor active against EGFR, ErbB2 and ErbB4. In common with other members of the class, this 6-acrylamidoquinazoline exhibits nanomolar inhibition of both wild-type and mutated EGFR and ErbB2, and it has antiproliferative activity in preclinical cell culture models harboring gefitinib-resistant mutated EGFR as well as anti-tumor activity in xenograft models of T790M-mediated gefitinib resistance [30] . Finally, HM781-36B (5) is a quinazoline-based irreversible pan-ErbB inhibitor, belonging to the general formula shown in Fig. 2 [33] . The Michael acceptor group, linked at the 6-position of the recognition scaffold through a conformationally constrained spacer ( Fig. 2 ), allows covalent modifications of the EGFR kinase domain active site in a manner similar to that of other irreversible EGFR inhibitors. HM781-36B inhibits the ErbB family TKs at nanomolar concentrations and shows excellent inhibitory activities on mutated EGFRs [31] . Moreover, with the exception of Tec family kinases (such as BLK, BMX and BTK), HM781-36B is highly selective for EGFR family members: its IC50s for other TKs 100- to 1000-fold greater than ErbB ones. HM781-36B strongly suppresses proliferation of a variety of EGFR-dependent cancer cell lines, including gefitinib-resistant NSCLC cell lines, and it also shows selectivity toward cancer cells over normal ones.

The insertion of the acrylamide warhead on a 4-anilino-3-cyanoquinoline core scaffold led to the clinical candidates pelitinib [34] (6, EKB-569, Fig. 2 ) and neratinib [35] (7, HKI-272, Fig. 2 ) and The driving portion undergoes the same interactions of the quinazoline ring within the active site of the kinase, with the exception of the 3-cyano group, that interacts with the side chain of Thr830 with the displacement of the water-molecule bridge. Both pelitinib and neratinib carry a basic dimethylamino substituent on the acrylamide side chain, enhancing solubility and improving reactivity toward cysteine. Pelitinib (6) is a dual EGFR/ErbB2 inhibitor with antitumor activity in EGFR and ErbB2-dependent cell lines. Neratinib (7) is an irreversible dual EGFR/ErbB2 inhibitor carrying a 3-chloro-4-(3-fluorobenzyloxy)aniline side chain at position 4 similar to that of the EGFR/ErbB2 inhibitor lapatinib ( Figure 2 ). It has proved effective in inhibiting cell proliferation, EGFR activity and downstream signaling in gefitinib-resistant cell lines (human NSCLC cells H1975), which possess the L858M and T790M mutations in EGFR. This agent has also demonstrated activity in cells harboring various EGFR/ErbB2 mutants. However, a study in NSCLC cell cultures showed the emergence of T790M-mediated resistance to neratinib at a concentration similar to the maximum concentration observed in a phase I clinical trial [36] , indicating that this agent could overcome T790M-resistance only at concentrations above those employed in clinical trials.

Another clinically advanced EGFR irreversible inhibitor is CO-1686 [37] (8), a representative of third generation EGFR inhibitors, characterized by selectivity toward mutant T790M EGFR over wild-type protein. CO1686, the structure of which has not been disclosed, inhibits cell proliferation and EGFR signaling in NSCLC cell lines harboring activating mutation, as well as the double mutation L858R/T790M of EGFR. Interestingly, no effect on EGFR signaling is observed in normal lung or skin tissues with CO1686 treatment, confirming that it does not inhibit wild-type EGFR. This compound significantly suppresses tumor growth of H1975 cells (L858R/T790M) in a dose-dependent manner and shows tumor regression in lung cancer xenograft models.

2.2.2. ErbB inhibitors in preclinical development

The described acrylamide-based 4-anilinoquinazoline or 4-anilino-3-cyanoquinoline inhibitors are currently undergoing clinical trials to evaluate safety and efficacy on several solid tumors and studies are summarized in Table 1 and reviewed in Section 3 . However, in the past decade several warhead-driver portion combinations have been explored to identify new drug-like leads for the development of ErbB TKIs as candidates for anticancer therapy. One strategy focused on the replacement of the acrylamide function by different and less reactive electrophiles, in order to improve selectivity and reduce non-specific toxicity [17] . A variety of alternative electrophilic warheads, different from acrylamide, have been investigated, ranging from those potentially reactive toward generic nucleophiles, such as butynamides, vinylsulfonamides, and epoxides [17] and [38], to less reactive functionalities that only react upon target binding. This second group includes the recently reported 3-aminopropanamides [39] and [40] (11, Fig. 3 ). These compounds possess a particular reactivity profile, being nonreactive directly, but able to covalently bind to their target after bioconversion to the corresponding acrylamide compound. A less reactive warhead is also the acetylene moiety on a thienopyrimidine scaffold as in compound 12 [17] ( Fig. 3 ). The pan-ErbB irreversible inhibitor 12 is able to covalently bind EGFR, as demonstrated by X-ray crystallography and mass spectroscopy.

Parallel to the exploration of the warhead, several driving portion and linkers between the heterocyclic scaffold and the warhead have been explored, in order to optimize fitting of the molecule in the ATP-binding pocket and warhead orientation with respect to the position of the nucleophilic residues. Various warhead-driving portion linkers with limited conformational flexibility have been explored [31] (general structure 5, Fig. 2 ), leading to compounds endowed with potent and selective dual EGFR/ErbB2 inhibitory activity, favorable pharmacokinetic profile, and significant antitumor effect in xenograft models. These compounds are included within the general formula indicated for the clinical candidates HM781-36B (5). Additionally, completely new scaffolds have recently been reported, as furanopyrimidines [41] and tetrahydro-benzothienopyrimidines [42] , where the presence of the acrylamide assures covalent irreversible inhibition of wild-type and mutated EGFR and antiproliferative activity on human lung cancer cell lines. In principle, major improvements in safety and efficacy could be achieved by third-generation irreversible EGFR inhibitors based on a substituted pyrimidine scaffold which displays good selectivity for mutated EGFR over wild-type kinase. One of the most active and selective inhibitors, the acrylamide-substituted 2,4-dianilino-5-chloropyrimidine WZ4002 [43] (13, Fig. 3 ), has around 100-fold more affinity for L858R/T790M EGFR than for the wild-type kinase. The crystal structure of WZ4002 in complex with EGFR T790M shows that the compound binds within the ATP-binding pocket of the enzyme, forming a covalent bond with Cys797. WZ4002 potently inhibits proliferation of cancer cells harboring different EGFR mutations, while it is less potent against the growth of wild-type EGFR cells. Moreover, WZ4002 exhibits promising in vivo efficacy in gefitinib-resistant NSCLC models [43] . Many efforts have been directed toward the development of this new class of mutant-selective EGFR inhibitors and a number of pyrimidine-based compounds with promising efficacy, selectivity and pharmacokinetic profiles have recently been reported (14 [44] and 15 [45] , Fig. 3 ).

Covalent irreversible ErbB inhibitors have recently been developed as novel biomarkers and diagnostic imaging tools for positron emission tomography (PET). The ability of PET to quantitatively and non-invasively image the distribution of radiolabeled drugs within the body makes this technique a valuable tool at several stages of drug development and application. A number of 11C, 18F, and 124I labeled reversible and irreversible EGFR inhibitors have been developed in order to further understanding of the in vivo behavior and efficacy of these drugs in animal models and individual patients. Interestingly, an irreversible PEG-ylated anilinoquinazoline derivative labeled with 18F (16, [18F]F-PEG6-IPQA, Fig. 3 ) has shown increased selectivity and irreversible binding to the L858R active mutant EGFR kinase compared to wild-type and T790M mutant EGFR kinases [46] . The ability to discriminate tumor-containing gefitinib-sensitive L858R EGFR could open the possibility to select patients for individualized therapy with small molecule inhibitors of ErbB kinase currently in clinical use.

2.3. BTK inhibitors

BTK is a nonreceptor cytoplasmic tyrosine kinase, belonging to the Tec family, predominantly expressed in hematopoietic cells. BTK plays a key role in the B-cell receptor signal pathway (BCR) and is a mediator of proinflammatory signals. Inhibition of BTK is a promising strategy for the treatment of B-cell malignancies and autoimmune diseases. BTK has been recognized as a member of the group of 11 tyrosine kinases (the Tec-family kinases, EGFR, ErbB2, ErbB4, Jack3, and BLK) that contain a conserved cysteine residue adjacent to the ATP-binding site [6] and [16]. This cysteine (Cys481 in BTK) has been exploited as nucleophilic site to form a covalent adduct with electrophilic inhibitors [47] . From the exploration of various types of Michael acceptors, acrylamide, vinylsuofonamide, and propiolamide have emerged as effective Cys481 covalent binders when inserted on a suitable BTK-recognition scaffold. Ibrutinib [48] (9, PCI-32765, Fig. 2 ) is a potent and selective BTK inhibitor characterized by a 4-aminopyrazolo[3,4-d]pyrimidine recognition portion carrying an acrylamide group through a pyrazole spacer. Ibrutinib shows potent inhibition of enzyme activity and of BCR signaling downstream of BTK. It selectively blocks B-cell activation, and has shown efficacy in animal models of arthritis, lupus, and B-cell lymphoma [48] . This compound is under clinical development in patients with B-cell lymphoma and chronic lymphocytic leukemia (CLL). A second clinically advanced BTK irreversible inhibitor is AVL-292 [49] (10, structure not disclosed), which also targets Cys481. AVL-292 is probably based on a diaminopyrimidine scaffold decorated with acrylamide warheads, corresponding to compounds which have been described in patent [50] . AVL-292 shows potent inhibition of BTK kinase activity and reduction of B-cell proliferation, and is currently undergoing clinical development for the treatment of non-Hodgkin's lymphoma (NHL) and CLL patients.

Other classes of recently reported irreversible BTK inhibitors are: (i) pyrrolotriazines, such as 17 ( Fig. 3 ), that shows potent inhibition of BTK kinase and murine B-cell proliferation; (ii) imidazo[1,5–1]quinoxalines, such as 18 ( Fig. 3 ) with good biochemical BTK potency, good selectivity over other TKs (except EGFR), favorable pharmacokinetic proprieties and nanomolar inhibition of B-cell proliferation [18] .

2.4. Other TK inhibitors

VEGFR is a receptor tyrosine kinase expressed on endothelial cells, including blood vessels, involved in angiogenesis and tumor neovascularization. VEGFR represent a promising target for antitumor therapy as demonstrated by the approval of reversible TKIs sorafenib, sunitinib, pazopanib, vandetanib, and axitinib for the treatment of advanced renal cell carcinoma [51] . In addition to VEGFR tyrosine kinases, these agents potently inhibit a wide range of tyrosine kinases and other targets (such as PDGFR, c-kit, Flt3, RET, CSF1R, c-Raf, and B-Raf), and this lack of specificity results in a series of adverse effects unrelated to efficient VEGF blockade. In order to increase target selectivity, new quinazoline-based covalent VEGFR-2 inhibitors have been reported [20] . These irreversible inhibitors, including compound 19 ( Fig. 3 ), contain a reactive quinone group at the 4-position on the heterocyclic ring, able to covalently interact with Cys1045 in the hydrophobic pocket at the top of the ATP-binding site of the kinase ( Fig. 1 ). In compound 19, the presence of the electron-withdrawing fluorinated substituent on the quinone moiety increases reactivity toward nucleophiles, while the water-solubilizing basic side chain at the 7-position enhances pharmacokinetic properties. Compound 19 inhibits VEGFR-2 at nanomolar concentration and shows antitumor activity in in vivo models. Also hypotemicine (20, Fig. 3 ) and resorcylic acid lactones (RAL), that contain a cis-enone Michael acceptor, have been shown to irreversibly inhibit VEGFR, as well as the majority of kinases with a cysteine at a position corresponding to that of Cys1045 in VEGFR-2 (e.g. PDGFR) [21] . Hypotemicine analogues show good inhibition of VEGFR in vitro, and inhibition of tumor growth in xenograft models with comparable efficacy to the reversible drug sunitinib [21] .

The FGFR family of receptor tyrosine kinases, which consists of four members (FGFR 1–4), plays a critical role in tumor formation and progression and appears to be a plausible target for anticancer therapeutics. Besides FGFR reversible inhibitors directed to the ATP binding site of the kinase, generally affected by poor pharmacokinetic properties and limited in vivo activity, acrylamide-based irreversible FGFR inhibitors have been described recently [19] . The pyrimidopyridine 21 (FIIN-1, Fig. 3 ) irreversibly blocks FGFR and its downstream signals with reasonable selectivity over other kinases bearing cysteine at position corresponding to that of Cys486 in FGFR ( Fig. 1 ). Compound 21 inhibits proliferation and survival of FGFR-expressing cancer cell lines, included cells expressing mutant isoforms of the enzyme [19] .

3. Clinical efficacy of irreversible tyrosine kinase inhibitors

3.1. ErbB family irreversible tyrosine kinase inhibitors

Currently, eight irreversible tyrosin kinase inhibitors directed against ErbB family receptors have entered the clinic as anticancer agents ( Table 1 ). Except canertinib and pelitinib, discontinued from further clinical development, neratinib, dacomitinib, afatinib have reached phase III development and CO-1686, a third generation mutant–selective EGFR inhibitor, is under investigation in a phase I/II clinical trial.

3.1.1. Canertinib

Canertinib (CI-1033, 1 in Fig. 2 ) is an oral irreversible inhibitor of all four members of the ErbB receptor family. In a phase I dose-escalation trial of 32 patients with solid tumors refractory to standard therapy, the maximum tolerated dose (MTD) was determined to be 450 mg per day. None of the patients achieved objective responses and six of them had SD (stable disease) [32] . A phase II randomized open-label trial evaluated canertinib as single agent in patients with advanced NSCLC who had failed prior platinum-based combination chemotherapy [52] . A total of 166 patients were allocated to receive three different dose levels (50, 150, 450 mg per day). The incidence of 3/4 adverse events (AEs), diarrhea and rash, was dose-related and the 450 mg arm was soon discontinued. The clinical activity, however, did not differ among the three dose arms, only 4 patients (2.5%) achieved PR (partial response) and 30 patients (19%) had SD.

Canertinib was also evaluated in a randomized phase II study in pretreated metastatic breast cancer [53] . Overall, 194 patients were treated with the same dose levels tested in the above-mentioned clinical trial. Canertinib did not exhibit clinically meaningful activity, the toxicity was strongly dose-dependent and was considered unacceptable at the highest dose level. So far, clinical results have not been very encouraging and there are no ongoing clinical trials with this agent.

3.1.2. Pelitinib

Pelitinib (EKB-569, 6 in Fig. 2 ) is an irreversible selective inhibitor of EGFR which also binds to the ErbB2 receptor at higher concentrations. A phase I dose-escalation study was performed in patients with advanced solid tumors overexpressing EGFR [34] . Dose limiting toxicity (DTL) was grade 3 diarrhea and the MTD was 75 mg per day. Two other phase I studies examined combination therapy in advanced metastatic colorectal cancer with capecitabine alone or FOLFIRI regimen, respectively. A dose of 50 mg pelitinib could be safely combined with conventional doses of capecitabine with tolerable gastrointestinal and cutaneous side effects. Although no patient had a complete response (CR) or PR, 48% had SD [54] . In combination with FOLFIRI the MTD was reduced to 25 mg per day due to overlapping toxicities of the two treatments. Grade 3 to 4 toxicities were diarrhea, neutropenia and asthenia [55] . In conclusion, the addition of pelitinib to FOLFIRI did not improve clinical efficacy.

Results of a phase I trial using pelitinib and tensirolimus in 48 patients with advanced solid tumors have been reported recently [56] . The combination resulted in partial responses at the MTD (35 mg daily) with 4 patients having a PR and 15 SD. The most common toxicities were nausea, diarrhea, fatigue, anorexia, stomatitis, rash, anemia, neutropenia, thrombocytopenia, and hypertriglyceridemia.

Two phase II trials have been completed with pelitinib as single agent in advanced colorectal cancer ( NCT00072748 ) and in advanced NSCLC ( NCT00067548 ) and results are awaited. At present no further clinical trials are planned.

3.1.3. Neratinib

Neratinib (HKI-272, 7 in Fig. 2 ) is an orally administered irreversible pan-ErbB TKI [35] . A phase I study in patients with advanced–stage solid tumors expressing EGFR or ErbB-2 (predominantly breast cancer, 40%, and NSCLC, 21%) reported 320 mg once daily as MTD with diarrhea being the dose limiting toxicity (DTL). Efficacy results were only promising in breast cancer patients with a partial response observed in 32% of them [36] . Results of a phase II trial in 165 advanced NSCLC patients [57] , divided into three arms based on prior reversible TKI treatment and EGFR mutation status, showed that neratinib had low activity in all patients tested, 2% ORR (overall response rate), and, significantly, no patients with T790M mutation (7%) had responses. The initial dose, 320 mg per day, caused excessive diarrhea (grade 3 in 50% of patients) and was reduced to 240 mg per day with a decrease grade 3 diarrhea to 25% (in any case an high rate compared to erlotinib toxicity). Currently, there are no ongoing studies of neratinib in patients with NSCLC.

The reason underlying the inefficacy of neratinib in NSCLC are unknown, however preclinical in vitro and in vivo experimental findings suggested that neratinib can only overcome T790M resistance at suprapharmacologic concentrations, while treatment at maximally tolerated doses may lead to the emergence of T790M-mediated resistance [58] . By contrast, significant responses to neratinib have been observed in breast cancer. In an open-label, multicenter, phase II study, neratinib (240 mg per day) was evaluated in two cohorts of patients with ErbB2 positive advanced breast cancer pretreated (66 patients) or trastuzumab-naïve (70 patients) [59] . The 16-week progression free survival (PFS) rates were 59% in patients pretreated with trastuzumab and 78% in patients with no prior trastuzumab treatment. The median PFS were 22.3 and 39.6 weeks, with objective response rates of 24% and 56% among patients with prior trastuzumab treatment and trastuzumab-naïve cohort, respectively. The most common AE was diarrhea, occurring in 30% of patients with prior trastuzumab treatment and in 13% of patients trastuzumab-naïve, but was manageable and only one patient discontinued therapy.

At present (June 2012), several active, not recruiting and recruiting clinical trials are under evaluation essentially in subjects with advanced breast cancer testing neratinib alone ( NCT00300781 ; NCT01494662 ; NCT00878709 ), or neratinib in combination with trastuzumab ( NCT00398567 ), paclitaxel ( NCT00445458 ; NCT00915018 ), paclitaxel and trastuzumab NCT01423123 ), capecitabine ( NCT00741260 ), vinorelbine ( NCT00706030 ), and tensirolimus ( NCT01111825 ; NCT00838539 )

3.1.4. Dacomitinib

Dacomitinib (PF00299804, 4 in Fig. 2 ) is a potent, highly selective, irreversible inhibitor of EGFR, ErbB2 and ErbB4 [60] . Two phase I dose-escalation studies on dacomitinib, conducted in Western ( NCT00225121 ) and Japanese (NCT007833328) patients with advanced solid tumors, have been completed and results recently published [61] and [62]. In the Western trial, the first-in-human study, 121 patients were enrolled including a large cohort of NSCLC patients (47%), the majority of whom had previously received gefitinib or erlotinib. This study demonstrated that dacomitinib is generally safe and well tolerated. DLT included stomatitis, rash, palmar-plantar erythrodysesthesia syndrome, dehydration and diarrhea. The MTD was established as 45 mg per die and most AEs (skin toxicity and gastrointestinal disorders) were mild and consistent with side effects observed with erlotinib. Pharmacokinetic analyses revealed a very large apparent volume distribution and a much longer half-life than those obtained with neratinib, canertinib and pelitinib. Forty four patients had SD, 55 had PD (progressive disease) and 8 patients had clinical benefit defined as CR, PR or SD for at least 24 weeks. Interestingly, four patients (3.6%), all with NSCLC previously treated with erlotinib or gefitinib, had a PR, however patients with documented T790M mutation (4 patients) did not have PR probably because, as the authors discussed, the concentration clinically achieved with the reported regimen may be insufficient to obtain a full inhibition of T790M based on preclinical studies [30] . In the Japanese study, dacomitinib appeared to be generally well tolerated with a safety profile consistent with that observed in the Western study. None of the 13 treated patients at the three dose levels tested (15, 30 or 45 mg once daily) had a DTL, and AEs were generally of grade 1/2 severity and manageable. Antitumor activity was also reported, particularly in NSCLC. It is noteworthy that one patient with L858R and T790M mutations had sustained SD.

Phase II clinical trials active not recruiting in advanced NSCLC patients who failed chemotherapy and erlotinib (US: NCT00548093 ; Korea: NCT00553254 ) may clarify the efficacy of dacomitinib in patients pretreated with erlotinib or gefitinib. Results from 62 evaluable patients [63] indicated a clinical benefit despite prior erlotinib failure: 3 patients achieved PR and 35 had SD for ≥6 weeks. Common manageable AEs included diarrhea (86%), fatigue (40%), rash (45%) and stomatitis (23%).

A parallel phase II trial ( NCT00769067 ) comparing dacomitinib (45 mg daily) vs erlotinib (150 mg daily) after failure of at least one prior chemotherapy regimen, involving 188 patients, showed that dacomitinib induced significantly prolonged PFS vs erlotinib in the overall population (12.4 vs 8.3 weeks; hazard ratio, HR = 0.66, p = 0.012) and also ORR favored the irreversible inhibitor (17% vs 4%, p = 0.008). However, common EGFR TKI AEs were more frequent with dacomitinib (diarrhea 71% vs 48% with erlotinib; acne 53% vs 34%) and 6 patients treated with the irreversible inhibitor discontinued therapy vs 2 patients treated with erlotinib [64] . Based on this study, a randomized double-blinded phase III clinical trial ( NCT01360554 ) has been designed to compare the efficacy of dacomitinib with erlotinib in advanced NSCLC patients after failure of at least one chemotherapy. As of January 2012, 117 of a planned 800 patients have been enrolled. The primary endpoint is PFS [65] .

A phase II trial ( NCT00818441 ), testing the safety and efficacy of the inhibitor in the first-line treatment of NSCLC selected patients either non-smokers or former light smokers or with known EGFR activating mutation, is recruiting participants. Among 92 enrolled patients, 47 had EGFR mutations in exons 19 or 21. 34/46 evaluable patients with EGFR mutations had a PR (74%) and preliminary median PFS was 17 months (95% CI: 13–24). For all 92 patients, common AEs were diarrhea (grade 3; 14%) and dermatitis acneiform (grade 3; 17%). 3/46 patients with EGFR mutations discontinued treatment due to drug-related toxicity. No data are available on T790M patients [66] .

Finally, dacomitinib is under investigation in several Phase I/II clinical trials in other solid tumors (i.e. gastric cancer NCT01152853 , head and neck squamous cell carcinoma NCT01449201 , glioblastoma NCT01112527 ) and in NSCLC in combination with the c-MET inhibitor crizotinib (PF02341066) ( NCT01121575 ).

3.1.5. Afatinib

Afatinib (BIBW2992, 3 in Fig. 2 ) is the most advanced second generation TKIs in clinical development. The LUX trials (LUX-Lung, LUX-Breast and LUX-Head and Neck) are a program of clinical trials investigating afatinib in a range of solid tumor types. The LUX-Lung program is evaluating afatinib as a second- or third-line treatment in enriched EGFR mutation positive/TKI pretreated NSCLC patients (LUX-Lung 1, 4 and 5), as a first–line treatment in patients with activating mutations (LUX-Lung 2, 3, 6 and 7), and in EGFR wild-type squamous cell carcinoma/TKI naïve patients (LUX-Lung 8). The results of LUX-Lung 1 and LUX-Lung 2 have been published recently [67] and [68].

The phase IIb/III LUX-Lung 1 trial ( NCT00656136 , [67] ), enrolled patients with stage IIIB or IV adenocarcinoma whose disease progressed after previous chemotherapy and at least 12 weeks of erlotinib or gefitinib treatment. The trial comparing afatinib (50 mg per die) plus best supportive care with placebo plus best supportive care, was done in 86 centers in 15 countries from Asia, Europe and North America. More that 50% of patients were of Asian origin and two/third were never smokers. 585 patients were enrolled (390 allocated to afatinib and 195 to placebo). Despite OS (overall survival), the primary endpoint, was 10.8 months in the afatinib group and 12 months in the placebo (HR = 1.08, 95% confidence interval, CI: 0.86–1.35; p = 0.74), median PFS was significantly prolonged in afatinib vs placebo (3.3 months vs 1.1 months; HR = 0.38, 95% CI 0.31–0.48; p < 0.0001). The PR was 7% (29 patients) in the afatinib group. The response in patients with T790 M (8 patients: 4 for each group) was not reported probably because of the small number. Diarrhea (87%; 17% grade 3) and rash or acne (78%; 14% grade 3) were the most common AEs in the afatinib group and 38% of patients needed a dose reduction.

The LUX-Lung 2 ( NCT00525148 , [68] ) was a single arm phase II study of afatinib in patients with advanced or metastatic lung adenocarcinoma harboring EGFR mutations who progressed after one line of chemotherapy or were chemotherapy naïve [68] . 129 patients, enrolled from 30 centers in Taiwan and in the USA, were treated with afatinib, 99 with a starting dose of 50 mg and 30 with a starting dose of 40 mg daily. 61 patients received afatinib as first-line treatment, and 68 after one line of chemotherapy.

Afatinib was more active in patients with two common EGFR mutations (exon deletion 19 or exon 21 L858R) than in patients with less common mutations. 70 (66%) of 106 patients with common EGFR mutations had an OR (CR (2%) or PR (64%)); 9 (39%) of 23 patients with other mutations had PR, and the single patient with T790M had PD. Median PFS was longer for patients with common EGFR mutations (13.7 months 95% CI: 8.31–19.35 for Del 19, 95% CI: 6.37–15.57 for L858R) than for patients with uncommon mutations (3.7 months 95%, CI: 1.74–6.37). 61% of patients had an objective response irrespective of starting dose. However, grade 3 AEs (diarrhea and rash or acne) were more common in patients receiving a starting dose of 50 mg daily (22% and 28% respectively) than in patients treated with 40 mg of afatinib as initial dose (7% for both types of AEs).

Based on these results, two first-line phase III trials are ongoing, comparing afatinib at a starting dose of 40 mg daily with cisplatin/pemetrexed (LUX-Lung 3, NCT00949650 ) or cisplatin/gemcitabine (LUX-Lung 6, NCT01121393 ). Data obtained in the LUX-Lung 3 trial on 345 patients randomized 2:1 in favor of afatinib (230 vs 115), recently presented at the 48th ASCO meeting [69] , indicated that treatment with the drug significantly prolonged PFS vs pemetrexed/cisplatin treatment (median 11.1 vs 6.9 months; HR 0.58 (0.43–0.78), p = 0.0004) suggesting that afatinib could represent a valuable first-line treatment option. Interestingly, in 308 patients with common mutations (Del119/L858R), median PFS was 13.6 vs 6.9 (HR 0.47 (0.34–0.65), p < 0.0001). ORR was higher with afatinib (56% vs 23%, p < 0.001) and a significant delay in time to deterioration in lung cancer-related symptoms (cough, dyspnea and pain) was observed. AEs with afatinib (diarrhea, rash and paronychia) were manageable and with a lower discontinuation rate than pemetrexed/cisplatin treatment (8% vs 12%).

Afatinib has been tested in Japanese patients in advanced NSCLC in a phase I/II trial equivalent to LUX-Lung 1 (LUX-Lung 4, NCT00711594 ) [70] and [71]. 62 patients, (73% EGFR mutation-positive) received afatinib 50 mg daily. 8% had PR and 69% had disease control for more than 8 weeks. The median PFS was 4.6 months. 82% of patients met the criteria for acquired resistance to erlotinib/gefitinib and they were similarly responders, indicating a possible efficacy of afatinib in increasingly resistant disease.

LUX-Lung 5 ( NCT01085136 ) is a phase III trial currently evaluating afatinib 40 mg daily in combination with paclitaxel vs chemotherapy alone (Investigator's choice) following afatinib monotherapy in patients with stage IIIB/IV progressed after erlotinib or gefitinib treatment.

Two newly initiated trials are currently recruiting patients. LUX-Lung 7 ( NCT01466660 ) is a phase IIb study evaluating afatinib vs gefitinib as a first-line treatment in EGFR mutated NSCLC patients and LUX-Lung 8 ( NCT01523587 ) is a phase III evaluating afatinib vs erlotinib in second line treatment of squamous cell carcinoma of the lung.

Finally, an interesting combination of afatinib and cetuximab, significantly effective in a mouse xenograft model of a T790M NSCLC tumor [72] , is under evaluation in a phase Ib clinical trial of NSCLC patients with progression following prior erlotinib ( NCT01090011 ). 22 Patients received afatinib 40 mg daily and cetuximab at 500 mg/m2 biweekly. Disease control was observed in all patients enrolled with tumor size reduction up to 76% and treatment duration up to 5+ months when reported [73] . Common AEs were grade 1/2 rash and diarrhea and 3 patients had grade 3 rash. PR was noted in 36% of patients including 4 patients with T790M mutation.

The LUX-Breast program is recruiting patients to evaluate afatinib in ErbB2 positive metastatic patients previously treated with trastuzumab in a phase III trial of afatinib plus vinorelbine vs trastuzumab plus vinorelbine (LUX-Breast 1, NCT01125566 ); in ErbB2 positive metastatic patients failing ErbB2-targeted treatment in the neoadjuvant and/or adjuvant treatment (phase II LUX-Breast 2, NCT01271725 ) and in ErbB2-positive patients with brain metastases alone or in combination with vinorelbine (phase II LUX-Breast 3 NCT, NCT01441596 ).

Results of a phase II study ( NCT00431067 ) which evaluated afatinib activity in ErbB2-positive breast cancer patients progressing after trastuzumab treatment has been reported recently [74] . 41 patients received 50 mg of afatinib, 73% discontinued due to disease progression, and 22% due to AEs. 20 patients required dose reduction to 40 mg and 6 patients had a further reduction to 30 mg. 19 patients achieved clinical benefit (4 patients (11%) had a PR, 15 (43%) had SD). In the total population, the median PFS was 15.1 weeks (95% CI: 8.1–16.7). Grade 3 AEs were diarrhea (24.4%) and rash (9.8%).

LUX-Head and Neck 1 ( NCT01345682 ) and LUX-Head and Neck 2 ( NCT01345669 ) are two phase III ongoing trials evaluating afatinib vs methotrexate in patients with platinum-refractory metastatic/recurrent SCCHN (squamous cell carcinoma of the head and neck) and afatinib vs placebo as adjuvant therapy after chemoradiotherapy in patients with unresected locoregional SCCHN, respectively.

Finally, afatinib is under evaluation in other advanced solid tumors including esophageal and gastric cancer (Phase II study: NCT01522768 ), colorectal cancer (Phase II study: NCT00801294 ), glioblastoma (Phase I study: NCT00977431 ), glioma (Phase II study: NCT00727506 ) and prostate cancer (Phase II study: NCT00706628 ).

3.1.6. HM781-36B

HM781-36B (general structure 5 in Fig. 2 ) is a novel irreversible pan-ErbB inhibitor. Preclinical results indicate that it is the most potent pan HER inhibitor with an excellent antitumor activity in in vivo models at much lower doses than those with afatinib [31] . Currently, phase I clinical trials of HM781-36B to determine the MTD and to assess the safety and pharmacokinetic profile are recruiting patients with advanced solid tumors in South Korea ( NCT01455584 ; NCT01455571 ). Preliminary evidence of anticancer activity has been reported recently: among 41 patients with malignancies refractory to standard therapies four (2 ErbB2-positive breast cancer patients) achieved PR and 19 had SD. The MTD was determined as 24 mg with diarrhea, stomatitis, rash, pruritus and anorexia as most common AEs [75] .

3.1.7. AV-412/MP-412

AV-412 (MP-412, 2 in Fig. 2 ) was evaluated in phase I dose escalation studies daily or intermittently ( NCT00381654 , terminated since it did not meet pre-specified objectives; NCT00551850 , completed) in patients with advanced malignancies. Preliminary data from the last clinical study indicated that among 36 patients, two experienced DLT at 200 mg three times a week and two at 500 mg once weekly. Common AEs were nausea (69%), diarrhea (55.6%) and vomiting (41.7%). Eight patients had SD [76] .

3.1.8. CO-1686

CO-1686 (8) is a mutant-selective, wild-type sparing irreversible EGFR inhibitor whose structure has not been disclosed yet [37] . It is the first third-generation EGFR inhibitor in clinical development. A Phase I/II, open-label, safety, pharmacokinetic and preliminary efficacy study of oral CO-1686 in patients with previously treated mutant-EGFR NSCLC is recruiting patients ( NCT01526928 ).

3.2. BTK irreversible inhibitors

Irreversible inhibitors in tumor clinical development are not limited to target the EGFR/ErbB2–4 receptor family. A nonreceptor tyrosine kinase of the Tec kinase family, a central player in BCR signaling, the BTK, is irreversibly inhibited by ibrutinib [47] and by AVL-292.

3.2.1. Ibrutinib and AVL-292

Ibrutinib (PCI-32765, 9 in Fig. 2 ) has shown encouraging clinical activity in patients with B-cell malignancies. There have been two active non-recruiting phase I clinical trials evaluating ibrutinib in CLL ( NCT01105247 ) and in recurrent B-cell lymphoma ( NCT00849654 ). The first study evaluated two cohorts of patients (previously untreated patients over 65 years of age and with relapsed/refractory disease following at least two prior therapeutic regimens), treated with 420 or 840 mg daily for 28-day cycles until PD. Interim results on 31 treatment-naïve patients [77] indicated that treatment was well tolerated with grade 3–4 AEs in 19% of patients. At a median follow up of 10.7 months on 420 mg cohort, 65% had PR and 8% CR with marrow clearance. In the second trial, 56 patients with multiple histological subtypes of B cell NHL were enrolled [78] . Possible related AEs grade ≥ 3 occurred in 16% of patients. 30/50 patients (60%) achieved objective response (CR 33%; PR 76%) seen at all dose levels and across all histologies.

Phase II studies of ibrutinib as single agent in relapsed or refractory mantle cell lymphoma for patients who had or had not been treated with bortezomib ( NCT01236391 ), in subjects with relapsed or refractory or de novo diffuse large B-cell lymphoma ( NCT01325701 ), and in relapsed and refractory patients with CLL/Small Lymphocytic Lymphoma and B-cell Prolymphocytic Leukemia ( NCT01589302 ) are ongoing or planned. A phase III study of ibrutinib vs ofatumumab in CLL ( NCT01578707 ) is planned, but is not yet recruiting participants.

Although down-regulated in normal plasma cells, BTK is highly expressed in the malignant cells from many myeloma patients and some cell lines [79] . Based on these data, a phase II clinical trial which will evaluate the safety and preliminary efficacy of ibrutinib in relapsed or relapsed and refractory Multiple Myeloma is currently recruiting patients ( NCT01478581 ).

Finally, ibrutinib is under evaluation, mainly in CLL, in combination with other agents i.e. ofatumumab ( NCT01217749 ), rituximab ( NCT01520519 ), FCR (fludarabine/cyclophosphamide/rituximab) and BR (bendamustine/rituxumab) ( NCT01292135 ).

Another BTK-targeting irreversible inhibitor, AVL-292 (10, structure not disclosed), is in phase I clinical trial for B-malignancies (CLL, B-NHL and Waldenstrom's macroglobulinemia) ( NCT01351935 ).

Clinical data from 12 patients (8 CLL and 4 B-NHL) indicated that AVL-292 was well tolerated until 400 mg daily (full BTK occupancy achieved with dose ≥ 250 mg); the most AEs included diarrhea, rash/skin infection, upper respiratory infection, nausea and fatigue. To date all CLL patients and 2 NHL patients have SD [49] .

4. Conclusions

Irreversible TKIs have been designed to achieve potent and selective inhibition of tyrosine kinase enzymes. A number of irreversible inhibitors of ErbB family (such as afatinib, dacomitinib), and of BTK (ibrutinib, AVL-292) are rapidly progressing to late stage clinical development, demonstrating encouraging clinical activity and safety margins. Clinical data confirm that irreversible inhibition provides additional weapons for fighting drug-resistant tumors, even if the potential advantages (efficacy, ability to overcome competition, within-class selectivity) of new drugs with this mechanism are counterbalanced by intrinsic liabilities, including target- and mutation-dependent responses and toxicity. The positive outcome of clinical trials for afatinib (LUX-Lung 3) demonstrates that covalent inhibition of TKIs can be effective toward lung cancer, making afatinib a suitable candidate for first line anticancer therapy. However, specific advantages of irreversible drugs over reversible ones will be only clarified by results of currently ongoing clinical trial LUX-Lung 7, where afatinib is compared to gefitinib as a first-line treatment in EGFR mutated NSCLC patients. Finally, the usefulness of irreversible TKIs as second line agents for the treatment of tumor became resistant to first generation inhibitors has been essentially demonstrated at preclinical level, with results that are very encouraging.

In our opinion, irreversible inhibition by covalent binding provides potential advantages in terms of selectivity over competitive reversible inhibition, since only a limited group of kinases has a cysteine at proper position. Moreover, the turnover rate required to restore target function, knocked down by irreversible inhibition, gives further advantages by making TKIs able to overcome competition by ATP and suitable for less frequent dosing regimens. On the other hand, a possible drawback of covalent binding kinase inhibitors is that the intrinsic reactivity of cysteine-reactive groups can in principle give rise to increased toxicity due to off-target related effects. In this context, clinical data on EGFR-TKIs have not shown non-specific additional side effects for irreversible compounds, compared to reversible marketed drugs (e.g. gefitinib). Usually, diarrhea and rash are the most frequent reported adverse events, and when higher toxicity (in terms of frequency and grade) has been reported, this might be attributed to more prolonged inhibition of the primary targets. Correspondingly, at the chemical level, the presence of reactive groups can be exploited to design new drugs with favorable efficacy and safety, but it also requires careful study during all the development phases, to balance reactivity and chemical recognition in drug structure, efficacy toward targets and selectivity vs off-targets. Chemotherapy agents include many compounds that take covalent bonds with tissue constituents, and this feature is generally related to their non-specific toxicity. On the other hand, irreversible TKIs are the first clinically relevant example of drugs where a reactive group has been directly linked to a recognition portion, with proper geometry and reactivity, finely tuned to react preferentially with the primary target that with other tissue constituents. At the moment, while we wait for further results from ongoing clinical studies, irreversible TKIs should be considered a promising class of anti-tumor drugs, with the first-in-class agents showing encouraging clinical outcomes. Moreover, the development of new compounds characterized by alternative warheads, less reactive than acrylamides (e.g. 3-aminopropanamides), could address the improvement of therapeutical index, combining increased efficacy with lower toxicity.

Acknowledgements

This work was partially supported by: Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan grant IG 8856. The authors thank Dr. Marcello Tiseo and Dr. Andrea Ardizzoni for their medical support and critiques. The authors thank Dr. Alessio Lodola for helpful comments on the manuscript. None of the authors have any financial conflict of interest.

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Footnotes

a Pharmaceutical Department, University of Parma, V.le G. P. Usberti 27/A, 43124 Parma, Italy

b Experimental Oncology Section, Clinical and Experimental Medicine Department, University of Parma, Via Volturno 39, 43126 Parma, Italy

lowast Corresponding author. Tel.: +39 521 903768; fax: +39 521 903742.