Valor absoluto promedio

To begin, a literature search on pre-existing crystal data for FGFRs was carried out. A recent crystal structure (PDB code: 3WJ6 – 2.15 Å) of human FGFR1 co-crystallised with CH5183284 (1) was identified.80 _{The PDB file was loaded into SPROUT} (Figure 2.1).

Compound 1 is observed to occupy the ATP binding pocket within FGFR1 and several interactions between the inhibitor and the protein are apparent (Figure 2.2). Two H-bonds form with the benzimidazole moiety; one with the backbone nitrogen of Asp641 and the other with a side chain carboxy oxygen of Glu531. Another H-bond forms between the pyrazole NH2 and the backbone carbonyl of Glu562. An H-bond is

a)

Figure 2.1: a) SPROUT image of co-crystal structure of 1 within ATP binding site of FGFR1. b) Co-crystal structure of 1 within FGFR1 showing predicted H-bonding interactions. Acceptor and donor sites present within the FGFR1 active site are shown in blue and red respectively. c) 2D representation of binding pose of 1 within FGFR1 showing intermolecular interactions. Amino acids, H-bonds and hydrophobic pockets are shown in green, red, and blue respectively.

c)

b)

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also present between the ketone moiety and the backbone NH of Ala564. The benzimidazole methyl group occupies a small hydrophobic pocket, known as H1. Occupation of this pocket by the methyl group has been shown to increase the selectivity of compound 1 for FGFR kinases over structurally similar kinases such as VEGFRs. This raises significant implications for designing selective inhibitors of FGFR kinases.80

De novo design of novel FGFR inhibitor scaffolds applied to this crystal structure was

carried out using SPROUT, with compound 1 acting as a template to guide the design process. Three of the four interaction sites (Glu531, Glu562 and Asp641) were selected in HIPPO (Section 1.6.5). Appropriate target and spacer templates were then chosen to generate 6-phenylindole (14) as a fragment predicted to bind to FGFR1.

Figure 2.2: ‘End-on’ view of compound 1 occupying the ATP binding site within FGFR1. H-bonding interactions are indicated using cyan dashes and hydrophobic pockets are indicated by H1 and H2.

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Binding Pose of Compound 14

Compound 14 is predicted to bind in a similar way to that of compound 1 with the indole NH forming an H-bond with the backbone carbonyl of Glu562 (Figure 2.3). In order to strengthen the predicted SPROUT pose, compound 14 was subjected to consensus docking whereby multiple docking programs (Glide and eHiTS) were used in conjunction with each other to validate the predicted binding pose. Both docking solutions were visualised using PyMOL and showed good overlap with each other and the binding pose of compound 1 (Figure 2.3).

Consensus docking was carried out for all future compounds with Glide being the chosen docking software; with results visualised in PyMOL. Upon inspection of the docking pose of compound 14 (Figure 2.4), it was determined that several modifications could be made in order to increase the number of intermolecular bonding interactions between FGFR1 and compound 14. Substitution from an indole to an indazole would open up the opportunity for an H-bond to form between the indazole 2-position nitrogen and the backbone NH of Ala564. Substitution of the 6-

Figure 2.3: Overlay of compound 1 and docking poses of compound 14 using eHiTS and Glide. Compound 1 is shown in green and the eHiTS and Glide poses for compound 14 are shown in purple and yellow respectively.

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phenyl ring to a 6-pyridyl derivative could also induce an H-bond with the pyridine nitrogen and the backbone NH of Asp641.

Furthermore, placement of a small hydrophobic group in the 3-position of the 6-phenyl ring could also allow H2 to be occupied. Manual manipulation of the docked pose of compound 14 was carried out to test what substituents could be tolerated in the H2 pocket. Halogenated compounds such as the iodo, bromo, and chloro derivatives were chosen due to their small size. The iodo and bromo derivatives were found to be too large and overlap with the boundary surface and therefore the chloro derivative was chosen, leading to target compound 15 (Figure 2.5).

Figure 2.5: 2D representation of proposed binding mode of compound 15 bound within FGFR1. Figure 2.4: De novo designed ligand 14 docked within the ATP binding site of FGFR1 using Glide. An H-bond is predicted to form between the indole NH and the backbone carbonyl of Glu562. Modifications that could be made to increase potency of compound 14 against FGFR1 are outlined.

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Compound 15 was then subjected to docking to see whether the proposed binding mode matched the docked binding mode; the docked solution is outlined (Figure 2.6).

As predicted by the proposed binding mode of compound 15 (Figure 2.5), a new H-bond is predicted to be possible between the indazole 2-position nitrogen and the backbone NH of Ala564. In contrast to the proposed binding mode of compound 15, docking has resulted in the 6-position ring becoming ‘inverted’ relative to that proposed originally. This places the Cl atom orientated towards the H1 pocket instead of the H2 pocket. The Cl atom may be too large to occupy the H2 pocket and therefore is predicted to bind more favourably in the H1 pocket; this places the pyridine nitrogen away from the Asp641 residue and instead is orientated towards a hydrophobic wall, and is therefore unfavourable. To validate these hypotheses, a small library of compounds was targeted for synthesis and is outlined below.

Figure 2.6: Glide docking model of compound 15 bound within FGFR1. H-bonds are indicated using cyan dashes.

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As well as compounds 14 and 15, compounds 16-19 were also included to give a thorough SAR study for this small library.

2.2 ‘First Generation’ Library Synthesis

Retrosynthetic Analysis

Retrosynthetic analysis of structures 14-19 indicated that the desired compounds could be made very simply from Pd-catalysed Suzuki couplings (Scheme 2.1).

Scheme 2.1: Retrosynthetic analysis of indole-based structures.

Suzuki Mechanism

Suzuki chemistry has become a very useful approach for forming carbon-carbon bonds in the medicinal chemist’s toolbox. A general mechanism for the process is outlined below (Scheme 2.2).108

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In the initial step Pd(0) undergoes oxidative addition with a halogenated aryl species. The Pd inserts into the carbon-halogen bond which results in oxidation of the palladium to give a Pd(II) species. Substitution of the halogen with base gives an intermediate that can then undergo transmetallation with a base-activated boronic acid/ester species, forming the penultimate intermediate. This intermediate can then undergo reductive elimination giving the desired product and the Pd(0) species which can then take part in the catalytic cycle again.

Pd-Catalysed Suzuki Couplings

Attempts at synthesising compounds 14 and 18 via Suzuki couplings using an adaptation of a method outlined by Liu et al are summarised below (Scheme 2.3).109

Scheme 2.3: Attempted Suzuki coupling using thermal conditions.

The syntheses of compounds 14 and 18 were unsuccessful; there are several possible reasons why this was the case. Some nitrogen-containing heterocycles have been known to interfere with Pd-catalysed Suzuki chemistry through their inherent ability to donate lone pairs to the metal centre, rendering the catalyst inefficient, which could be the case for compound 22.110 The unprotected free NH in compounds 21 and 22 may have the capability to participate in unwanted Buchwald coupling, leading to the failure of the reaction. Another reason could be due to the electron rich nature of the halogenated heterocyclic ring; oxidative insertion will be hindered allowing other competing pathways to take place.

To avoid the unwanted Buchwald side products, compound 22 was protected as the benzenesulfonyl derivative using an adaptation of a method outlined by Baldwin et al as summarised below (Scheme 2.4).111

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Scheme 2.4: Indazole protection using a benzenesulfonyl protecting group.

Deprotonation of the indazole 22 using NaH initiates nucleophilic displacement of the chlorine atom in 23 resulting in sulfonamide 24 in a yield of 43%. Prior to subjecting

24 to Suzuki coupling, a literature search outlined the use of microwave energy to

facilitate the reaction without the need of protecting groups. Therefore, compounds

14-19 were synthesised using an adaptation of a method outlined by Baldwin et al as

summarised below (Scheme 2.5).112

Scheme 2.5: Pd-catalysed microwave Suzuki couplings.

One common issue with this synthetic procedure is the appearance of an impurity that was assumed to be polymeric material. Removal of this material was difficult which may account for the low yields. Sonication of the purified solid in pentane was found to be the best way to significantly reduce the impurity to acceptable levels of <5% (1H NMR) for biological evaluation.

Biological Evaluation of ‘First Generation’ Fragments

The biological evaluation of compounds 14-19 was carried out by Life Technologies, Paisley, Scotland. Compounds 14-19 were screened against FGFR1 at an initial concentration of 500 µM using a fluorescence resonance energy transfer (FRET)-based assay (Section 8.1). The results are outlined below (Table 2.1).

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Table 2.1: Biological results for ‘first generation’ fragments when screened against FGFR1.

a _{% Inhibition and IC}

50 values are given as the mean ± standard deviation (SD) of all data points, n = 2. b _{No difference in measured data points.} c _{NT = not tested.}

The initial de novo designed fragment 14 was found to be inactive at 500 µM. The other two indole-based compounds 16 and 17 were marginally more active but still low considering the high screening concentration. An IC50 value of >500 µM for compound 17 confirmed that the indole compounds were indeed inactive against FGFR1. Interestingly, all the indazole derivatives showed >50% increase in activity than their corresponding indole counterparts. IC50 measurements confirmed that compounds 15, 18 and 19 have modest double-digit µM activity against FGFR1. This outlines that the 2-position nitrogen present in the indazole compounds is crucial for inhibition of FGFR1. The ligand efficiency (LE) is a measurement of the binding energy per atom of a ligand to its binding partner and can be calculated using the equation outlined below (Equation 2.1).113

LE = 1.4(-logIC50)/N Compound No. Structure % Inhibitiona (500 µM) IC50a (µM) LE 14 1 ± 0.0b _NTc _N/A 16 16 ± 0.5 NT N/A 17 21 ± 3.5 >500 N/A 18 53 ± 0.0 b _{77 ± 0.9 0.38} 19 66 ± 1.5 90 ± 0.9 0.38 15 73 ± 1.0 36 ± 0.9 0.39

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The LE for compounds 15, 18 and 19 has been calculated. A reasonable LE starting point is considered to be >0.3114 and therefore compounds 15, 18 and 19 satisfy this. Compound 15 is the most active fragment against FGFR1 at 36 µM. This could be due to the Cl atom occupying the H1 pocket as predicted from the Glide docking of compound 15 (Figure 2.6). Compound 18 is more active than compound 19. This outlines the detrimental effect that the pyridine nitrogen has upon the binding of compound 19 to FGFR1, an aspect that can be rationalised from the docking of compound 15 (Figure 2.6).