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1.5.2.1 Type I

Currently, several type I FGFR inhibitors are in clinical use, with some acting as selective FGFR inhibitors and some as pan-kinase inhibitors (Figure 1.12).

Figure 1.12: Current FGFR inhibitors that exhibit DFG-in binding conformations.

Compound 1 is a potent inhibitor of the FGFR kinases (Section 1.5.1.1) exhibiting IC50 values of 9.3, 7.6 and 22 nM respectively. Compound 1 was found to display anti-tumour activity against a panel of 327 cell lines that harbour FGFR genetic alterations with xenografts models also reflecting this. Compound 1 was discovered

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using high-throughput screening (HTS) followed by structure-based drug design (SBDD) taking advantage of X-ray crystal data.⁸⁰

Nintedanib (2) is a triple angiokinase inhibitor targeting proangiogenic pathways that are mediated by TKs such as FGFR, VEGFR, and PDGFR. In vitro studies outline potent IC50 values ranging from 13-108 nM for the targeted TKs, however, 2 lacks selectivity amongst the different sub-types of each of these TKs. Compound 2 is currently in phase III clinical trials and has shown significant efficacy in the treatment of non-small-cell lung carcinomas (NSCLCs) and ovarian cancer.⁸⁴

Dovitinib (3) is a multi-targeted TK inhibitor that is currently being investigated in phase II clinical trials for a wide range of FGFR related cancers.⁸⁵ It has been found to have activity against FGFR, VEGFR, PDGFR, Fms-like tyrosine kinase 3 (FLT3) and tyrosine-protein kinase Kit (KIT) with IC50 values in the range of 1-50 nM.⁸⁶ Recently this molecule has been shown to have anti-tumour activity in FGFR-amplified breast cancer cell lines but not in FGFR-normal cell lines indicating a selective preference for cancerous cells.⁸⁷

Lucitanib (4) is a dual inhibitor of VEGFR1-3 and FGFR1-2 exhibiting IC50 values in the range of 7-83 nM. In vitro studies looking at ligand-dependant signal transduction showed that compound 4 inhibited this process. In vivo studies using mice showed that compound 4 completely inhibited FGF-induced angiogenesis as well as a reduction in tumour vessel density and increased tumour necrosis. Compound 4 is currently in phase II clinical trials for the treatment of VEGFR and FGFR related diseases.⁸⁸ AZD4547 (5) is a potent and selective inhibitor of FGFR1-3 exhibiting in vitro IC50

values of 0.2, 2.5 and 1.8 for FGFR1-3 respectively. A selectivity screen indicated that compound 5 is selective for FGFR1-3 against other kinases such as CDK2, PI3K and PKB.⁸⁹ In vivo studies on mice treated with compound 5 show 99% tumour growth inhibition. AZD4547 is now in phase II clinical trials for the treatment of gastroesophageal cancer.⁹⁰

PD173074 (6) is a potent and selective inhibitor of FGFR1/3 with in vitro efficacies of 21.5 and 5 nM respectively.^91,92 In vitro studies on related kinases such as VEGFR, PDGFR and insulin growth factor receptor (IGFR) show a 1000-fold decrease in potency.⁹¹ A study has shown that compound 6 suppresses cell proliferation in cell lines that harbour FGFR3 mutations when compared to WT FGFR3.⁹²

BGJ398 (7) is a potent pan-FGFR inhibitor that is currently in phase I clinical trials for FGFR1-2 amplified cancers and FGFR3 mutated cancers.⁸⁵ In biochemical assays,

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compound 7 was found to give IC50 values of 0.9, 1.4 and 1.0 nM for FGFR1-3 respectively. Over fifteen other RTKs were tested but were found to be unaffected by compound 7 revealing this molecule to be a selective FGFR inhibitor.⁹³

SU4984 (8) was the first inhibitor to inhibit TKs that possessed an oxindole core. It inhibits FGFR1 with a moderate activity between 10-20 µM and was also shown to inhibit autophosphorylation of FGFR1. Compound 8 also inhibited related kinases such as PDGFR and IGFR with similar potencies to that observed agaist the FGFRs, which demonstrates the use of an oxindole core as a general TK pharmacophore.⁹⁴ Compound 9 is a potent FGFR1 inhibitor exhibiting an IC50 of 2.9 nM and comparable single concentration inhibition against FGFR2. Cellular assays also outline this compound to have an activity of 40.5 nM against SNU-16 cell lines. The construction of this compound was based on both compounds 5 and 7 using a scaffold hop and molecular hybridisation strategy respectively.⁹⁵

1.5.2.2 Type II

There are two examples of type II inhibitors in the literature at present; ARQ069 (10) and ponatinib (11) (Figure 1.13). Compound 10 is a moderately potent inhibitor of FGFR1/2 with a potency of 0.84 and 1.23 µM activity against the inactive (unphosphorylated) form. In vitro assays looking at inhibition of the phosphorylated forms of both FGFR1/2 show a complete loss of activity and a drop to 24.8 µM respectively. This outlines that compound 10 prefers to target the DFG-out binding mode. These results have also been reflected in a cellular environment.⁹⁶

Figure 1.13: Current FGFR inhibitors that exhibit DFG-out binding conformations.

Currently, compound 11 is the only food and drug administration (FDA) approved type II inhibitor and is used for the treatment of chronic myeloid leukaemia (CML) and Philadelphia chromosome positive (Ph*) acute lymphoblastic leukemia (ALL).⁹⁷ Compound 11 is a pan-kinase inhibitor that targets Abelson murine leukaemia viral oncogene homolog 1 (ABL), Lyn, VEGFR2 and FGFR1 with potencies of 0.37, 0.24,

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1.5 and 2.2 nM respectively. Compound 11 is used as a first-line treatment for patients who suffer from cancers that have the breakpoint cluster region protein (BCR)-ABL^T315I mutation as other drugs such as imatinib fail to be effective against this particular mutant.⁹⁷

1.5.2.3 Type III

Irreversible inhibitors are often seen as a last resort due to their propensity for receptor promiscuity, however, a couple of examples do exist (Figure 1.14).

Figure 1.14: Current FGFR inhibitors that exhibit covalent binding interactions.

FIIN-2 (12) is an irreversible inhibitor of FGFR1-4 with EC50 activities of 1, 4, 93 and 32 nM respectively.⁹⁸ Compound 12 is a derivative of compound 6 where an acryl-amido-benzyl substituent has been substituted on the N of the cyclic urea. It was tested against a panel of 456 kinases and showed good overall selectivity. X-ray crystal data of compound 12 bound with FGFR4 (PDB-4QQC) shows that the covalent bond is formed between Cys-477, a residue conserved between FGFR1-4, deep within the ATP-binding pocket.⁹⁸

BLU9931 (13) is an irreversible, potent, and selective inhibitor of FGFR4 with an IC50

value of 3 nM and IC50 values of 591, 493 and 150 nM for FGFR1-3 respectively.

Compound 13 differs to that of compound 12 in that it reacts with Cys552.⁹⁹ This explains the difference in selectivity as the residue at this position in FGFR1-3 exists as a tyrosine (Figure 1.10). Further validation into the importance of this covalent bond in selectivity targeting FGFR4 is demonstrated with a non-covalent analogue of compound 13 which shows a much lower potency of 938 nM against FGFR4.⁴⁴

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