Resultados en el caso de seguimiento de trayectoria

In order to increase the potency of the FGFR inhibitor fragments, SPROUT was used to extend the structures of the fragments giving rise to larger compounds predicted to inhibit FGFR1. Lead fragment 31 was chosen as the representative compound to extend. The modelled fragment/FGFR complex was loaded into SPROUT.

Previous literature reports suggest that substitution at the 3-position of the indazole ring would lead to more favourable binding.84,95,125 Inspection of the residues extending out of the active site was carried out. The adjacent residue to Ala564, Ser565, was chosen as the next amino acid in which additional H-bonding contacts could be made via suitable extension of the indazole fragment structure. Appropriate target and spacer templates were chosen and several solutions found. These were analysed for ease of synthesis; the docking pose of the top-ranked solution (85) is outlined below (Figure 3.1).

In order to independently check the validity of the SPROUT-designed inhibitor-protein complex, molecular scaffold 85 was docked using Glide. The

a) b)

Figure 3.1: a) Glide docking model of compound 85 bound within FGFR1; b) 2D representation of predicted binding pose of compound 85.

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resulting pose of compound 85 corresponded very well to that generated using SPROUT. The indazole nitrogens of compound 85 are predicted to bind in the same way as compound 31 (Figure 2.9). The benzylamine moiety located at the indazole 3-position is predicted to be located at the entrance of the active site. The protonated amine moiety is predicted to make an H-bond with the backbone carbonyl of Ser565.

Target Library

Based on the new predicted H-bond between compound 85 and Ser565, an extended library looking to exploit this interaction was developed and is outlined below (Figure 3.2).

Figure 3.2: Focus library showing all the chosen extended variants of the ethoxy series.

In addition to inclusion of the benzylamine moiety within the extended inhibitor structure, the hydroxymethyl and ethyl analogues were also identified as targets for synthesis. It was reasoned that comparison of the binding of these derivatives to the FGFRs with that shown by the aminomethyl-based molecule 85; would prove the existence of the predicted H-bond between the benzylamine amino group and the Ser565 backbone carbonyl (Figure 3.1). The extended versions of compound 66 were also targeted for synthesis as this fragment showed promise in achieving selectivity for FGFR2 over FGFR1/3. It was also planned to prepare the extended versions of compound 67 in order for these systems to act as a control series; as fragment 67 was inactive it is hypothesised that the larger systems should also be inactive. The extended versions of compound 69 were also targeted for synthesis as this was the second most potent fragment.

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Retrosynthetic Analysis of Structure 85

Structure 85 was subjected to retrosynthetic analysis (Scheme 3.1).

Scheme 3.1: Retrosynthetic analysis of structure 85.

It was reasoned that the extended versions of compounds 31, 66, 67 and 69 could be made very simply by two consecutive Suzuki coupling reactions starting from compound 87.

Synthesis of the Extended Ethoxy Series

In order to gain access to the extended variants, iodide 87 was identified as a key intermediate. Compound 87 was synthesised using a procedure developed in-house and is summarised below (Scheme 3.2).

Scheme 3.2: Selective bromination of compound 46 to make compound 87.

Compound 46 can be brominated selectively at the 3-position of the indazole ring using NBS. The reaction is very high yielding and no over-bromination or undesired regiomers are observed. Compound 87 was then subjected to a selective Suzuki coupling using the conditions outlined previously (Scheme 2.4) as summarised below (Scheme 3.3). The presence of two different halogens in compound 87 allowed selective substitution of each halogen, yielding compounds 86 and 88-90 in the initial step. In all cases, formation of the bis-arylated compounds (91-94) was observed as the minor product with the exception of compound 93 which forms as the major product. Compounds 86 and 88-90 were then further reacted under Suzuki conditions using the desired boronic acids. The yields for the second Suzuki steps were found to be somewhat variable but in general, the yields of the methyl amino-based compounds (85, 97, 100 and 103) were low as purification of these proved troublesome. A total of sixteen final compounds were synthesised.

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Scheme 3.3: Synthesis of extended ethoxy series using selective Suzuki couplings.4

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Biological Evaluation of the Extended Ethoxy Series

Compounds 85 and 91-105 were screened against FGFR1-3 at a concentration of 10 µM using the FRET-based assay. The results are outlined (Table 3.1a/b).

Table 3.1a: Biological results for compounds 85 and 91-105 when screened against FGFR1-3.

a _{% Inhibition values are given as the mean ± SD of all data points, n = 2.} b _{No difference in measured}

data points.

Entry No. Structure % Inhibition

a _{(10 µM)} R R’ 1 2 3 1 (91) 4.0 ± 2.5 5.0 ± 11 6.0 ± 5.0 2 (85) 52 ± 2.0 71 ± 4.0 48 ± 0.5 3 (95) 30 ± 0.5 59 ± 1.5 13 ± 4.0 4 (96) -9.0 ± 1.0 7.0 ± 5.5 -8.0 ± 4.5 5 (92) -4.0 ± 2.0 8.0 ± 1.5 19 ± 3.0 6 (97) 59 ± 1.5 47 ± 4.0 56 ± 4.0 7 (98) 27 ± 1.0 31 ± 3.0 22 ± 2.5 8 (99) 1.0 ± 3.0 -10 ± 0.0b _{2.0 ± 2.5}

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Table 3.1b: Biological results for compounds 85 and 91-105 when screened against FGFR1-3.

a _{% Inhibition values are given as the mean ± SD of all data points, n = 2.} b _{No difference in measured}

data points.

Analysis of the above results show that addition of the substituted aromatic ring in the 3-position of the indazole ring is generally unfavourable for binding to the FGFR enzymes, with compounds 91-94 (entries 1, 5, 9 and 13) being inactive, possibly because the R’ groups are too large. Compounds 96, 99, 102 and 105 (entries 4, 8, 12 and 16) all contain the ethyl moiety and are also inactive. Interestingly, the hydroxymethyl and the aminomethyl derivatives for all compounds show better inhibition against FGFR1-3 than the corresponding ethyl derivatives. This is consistent with the predicted H-bond that forms between the amine in compound 85

Entry No. Structure % Inhibition

a _{(10 µM)} R R’ 1 2 3 9 (93) -4.0 ± 1.5 2.0 ± 1.0 -12 ± 6.5 10 (100) 23 ± 4.0 28 ± 1.5 -3.0 ± 1.5 11 (101) 7.0 ± 0.5 13 ± 1.0 4.0 ± 12 12 (102) -15 ± 0.5 2.0 ± 1.5 -4.0 ± 1.5 13 (94) -9.0 ± 0.5 2.0 ± 0.5 4.0 ± 4.5 14 (103) 73 ± 0.0 82 ± 2.0 70 ± 4.5 15 (104) 37 ± 0.0 61 ± 2.0 21 ± 6.5 16 (105) -3.0 ± 0.0 4.0 ± 0.5 6.0 ± 5.0

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and Ser565 (Figure 3.1). IC50 measurements were conducted and are outlined below (Table 3.2).

Table 3.2: Biological results for compounds 31, 85, 91, 95, 96, 97 and 103 when screened against FGFR1-3.

a _IC

50 values are given as the mean ± SD of all data points, n = 2. b NT = not tested.

IC50 measurements confirmed that compounds 91, 95, and 96 are inactive against FGFR1 suggesting the other bis-arylated, ethyl, and hydroxymethyl derivatives are also inactive. Compounds 85, 97, and 103 are active against most of the FGFRs but do not show an improvement in potency when compared to lead fragment 31. Therefore, it is very unlikely that the benzylamine amino group in compounds 85, 97 and 103 is forming an H-bond with Ser565, a predicted H-bond from the docking of compound 85 within FGFR1 (Figure 3.1). A potential reason for the retained activity of compounds 85, 97 and 103 could be due to better solvation within the active site of

Compound No. Structure IC50a (10 µM) R R’ 1 2 3 31 H 2.0 ± 0.4 0.8 ± 0.4 4.5 ± 1.6 91 >10 NTb _NT 95 >10 >10 >10 96 >10 NT NT 85 >10 2.1 ± 0.9 2.6 ± 1.1 97 8.4 ± 1.4 3.7 ± 1.0 5.8 ± 0.7 103 3.7 ± 0.6 2.4 ± 0.9 5.2 ± 0.9

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the compound. The 3-position substituent, in particular the aminomethyl moiety, is predicted to protrude out of the active site towards solvent (Figure 3.1). The aminomethyl group (compounds 85, 97 and 103) will be charged at physiological pH and will therefore help solvate the compound in a water-filled environment, which won’t be reflected for the hydroxymethyl or ethyl variants (compounds 95 and 96).