Diagnósticos sobre la adaptabilidad de la educación a las necesidades

Molecular docking was conducted using the method described in Section 4.3, using the Peakdale Molecular99 database as the ligand files and the NDM-1 crystal structure PDBID: 3Q6X.38 A set of ten possible inhibitors of NDM-1 was identified from the Peakdale Molecular database for purchase. Details of compound structures and predicted binding affinities are shown in Appendix A2.2.

4.4.1 Preliminary ‘hit’ molecule

Compound 4.1 (Figure 40) was identified from a preliminary biological evaluation, which was conducted at the University of Bristol by Dr. James Spencer. The preliminary biological evaluation was to give an indication of an inhibitory effect but not to quantify it. Of the top ten scoring compounds from the vHTS runs only compound 4.1 showed inhibition at 100 μM. Compound 4.1 showed limited solubility in 10% DMSO solution.

Figure 40: Structure of inhibitor 4.1 identified from Peakdale screen

Figure 41 shows that the main interactions between the ‘hit’ molecule and

NDM-1 are predicted to come from the coumarin portion of the molecule interacting through the carbonyl to Gln-123 and the sulfonyl to Lys-211, Asn- 220 and Zn2 respectively. The indole portion of the scaffold is seen to extend into a hydrophobic region of the binding site and is therefore a good place to investigate the effects of substituents in this region and attempt to make the molecule more soluble and more potent.

Figure 41: Binding interactions of 4.1 in a) 2D skeletal representation and b) 3D representation

In order to confirm these results, a re-synthesis of the hit molecule 4.1 was conducted as shown below (Scheme 4).

Coumarin 4.2 was reacted with chlorosulfonic acid to form sulfonyl chloride

4.3. The reaction proceeded with moderate yields due to the formation of the

undesired isomer with the sulfonyl chloride group on the 8 position of the coumarin (41%). In order to form nitro indole 4.5, a Fisher indole synthesis124 was conducted from hydrazine 4.4 and butan-2-one in moderate yield (52%). Nitro indole 4.4 was reduced under an atmosphere of hydrogen to produce amine 4.6 in high yield (83%). Finally sulfonyl chloride 4.3 and amine 4.6 were coupled in the presence of base to produce 4.1 in good yield (70%)(Scheme 4).

MC = Main chain SC = Side chain

Scheme 4: Synthetic route to 4.1

4.4.2 SAR investigation

The ‘Hit’ molecule 4.1 was re-examined in SPROUT125

to look at the predicted interactions with NDM-1 and sites of potential interactions where a SAR investigation could be conducted to increase potency and solubility. SPROUT identified three potential areas where a structure activity relationship exploration could be conducted around 4.1 with the aim of identifying a more potent inhibitor (Figure 42)

Figure 42: Areas for SAR investigation of 4.1

All synthesised compounds (4.13-4.18, 4.20, 4.22, 4.25, 4.27) were biologically evaluated against NDM-1 by Dr Jürgen Brem at the University of Oxford, and the results are reported in Section 4.4.2. IC50 values were

determined on compounds which showed less than 30% residual enzyme activity at 100 μM.

4.4.2.1 Structure activity relationship of the N-aryl ring substituent.

The first SAR series containing compounds (4.13-4.18) was designed to examine the effects of changing the electron density of the aromatic ring attached directly to the amine side of the sulfonamide (previously the indole in 4.1).

Compound 4.13 was synthesised as an analogue of 4.1 with the methyl groups on the indole portion of the molecule removed in an attempt to reduce the hydrophobicity of the molecule and make it more soluble in the assay without reducing the potency. Compounds 4.14-4.18 are simpler analogues of 4.1 looking at the effects of changing the electron density of the ring.

Scheme 5: Synthetic route to compounds 4.13-4.18

The synthesis of compounds (4.13-4.18) investigating the N-aryl substituent was conducted as shown in Scheme 5. Each amine (4.7-4.12) was coupled to sulfonyl chloride 4.3 in the presence of base to produce compounds 4.13-

4.18 respectivly in good yields (90-68%)(Scheme 5)

Biological evaluation of compounds 4.13-4.18 against NDM-1 was conducted by Dr Jürgen Brem at the University of Oxford using the nitrocefin based assay described in Appendix A1.1.

From the residual activity it appears that the addition of electron withdrawing groups (rendering the aromatic ring electron deficient) helps to slightly increase the potency (Table 3). The substitution to a pyridine ring helps to both increase the potency and solubility (as in compound 4.15). Although there is a significant reduction in the residual activity it is not below the desired level of <30% and therefore the IC50 of the compound was not

Table 3: SAR Study of N-aryl ring substituent of 4.1 against NDM-1

Compound Number Structure RA[%], 100 μM

4.1 83 4.13 86 4.14 89 4.15 40 4.16 89 4.17 81 4.18 92

4.4.2.2 Structure activity relationship of the dihedral angle of the linker.

The second series of compounds was designed to investigate the role of the dihedral angle of the N-aryl ring from the coumarin in order to explore if there are specific π stacking interactions occurring within the binding site due to the position of these groups.

The nitrogen from the sulfonamide was substituted for a carbon (4.20) and an oxygen (4.22). The substitutions were conducted in order to change the dihedral angle due to the change in bond angles around the individual atoms.

The synthesis of compound 4.20 investigating the role of the dihedral angle of the N-aryl ring from the coumarin where the nitrogen was substituted for a carbon was conducted as shown in Scheme 6. Benzyl bromide (4.19) was coupled to sulfonyl chloride 4.3 in the presence of base to produce compound 4.20 in moderate yield (29%)(Scheme 6).

Scheme 6: Synthetic route to compound 4.20

The synthesis of compound 4.22 investigating the role of the dihedral angle of the N-aryl ring from the coumarin where the nitrogen was substituted for a oxygen was conducted as shown in Scheme 7. Phenol (4.21) was coupled to sulfonyl chloride 4.3 in the presence of base to produce compound 4.22 in good yield (73%)(Scheme 7).

Scheme 7: Synthetic route to compound 4.22

Biological evaluation of compounds 4.20 and 4.22 against NDM-1 was conducted by Dr Jürgen Brem at the University of Oxford using the nitrocefin based assay described in Appendix A1.1.

Table 4: SAR investigation of linker against NDM-1

Compound Number Structure RA[%], 100 μM

4.14 83

4.20 95

4.22 98

It can be seen that substitution of the sulfonamide nitrogen for an oxygen or a carbon has a negative effect on the potency of the compound (Table 4). The dihedral angle created by the sulfonamide must be required for good binding.

4.4.2.3 Structure activity relationship of the aryl sulfonyl group.

The third series was designed to investigate the importance of the predicted interaction between the coumarin carbonyl to the main chain amine of the Gln-123 residue.

Compound 4.27 was therefore designed to remove this interaction completely and compound 4.25 to change the nature of the bonding due to the tautomerisation to the hydroxyquinoline. The synthesis of compound

4.25 investigating the importance of the predicted interaction between the

coumarin carbonyl to the main chain amine of the Gln-123 residue by changing the nature of the bonding was conducted as shown in Scheme 8. 1,2-dihydroquinolin-2-one 4.23 was reacted with chlorosulfonic acid to form sulfonyl chloride 4.24. The reaction proceeded with moderate yields due to the formation of the undesired isomer with the sulfonyl chloride group on the 8 position of the quinoline (39%). Sulfonyl chloride 4.24 and amine 4.6 were coupled in the presence of base to produce 4.25 in moderate yield (31%)(Scheme 8).

Scheme 8: Synthetic route to compound 4.25

The synthesis of compound 4.27 investigating the importance of the predicted interaction between the coumarin carbonyl to the main chain amine of the Gln-123 residue by completely removing the interaction was conducted as shown in Scheme 9. Benzene sulfonyl chloride (4.26) was coupled to amine 4.6 in the presence of base to produce compound 4.27 in good yield (57%)(Scheme 9).

Scheme 9: Synthetic route to compounds 4.27

Biological evaluation of compounds 4.25 and 4.27 against NDM-1 was conducted by Dr Jürgen Brem at the University of Oxford using the nitrocefin based assay described in Appendix A1.1.

Table 5: SAR investigation of coumarin portion of 4.1 against NDM-1

Compound Number Structure RA[%], 100 μM

4.1 83 4.25 82 4.27 93

The substitution of the coumarin to 2-hydroxyquinoline and a simple phenyl ring appeared to have no effect upon binding to the enzyme (Table 5). The residual activity of the enzyme remained almost constant for all of the compounds.

4.4.3 Biological evaluation of the hits identified from the Peakdale Molecular screening collection

Biological evaluation of the identified potential inhibitors against NDM-1 was conducted by Dr Jürgen Brem at the University of Oxford using the nitrocefin based assay described in Appendix A1.1.

The top four results from the biological evaluation of the selected inhibitors from the Peakdale molecular library against NDM-1 are shown in Table 6. As the residual activity was greater than 30% at 100 μM in all cases, no IC50

values were determined. Although these compounds showed some activity against the enzyme, they were not pursued due to the activity levels being well above the desired levels of less than 30% residual activity at 100 μM.

Table 6: Peakdale molecular potential inhibitors and residual activities against NDM-1

Inhibitor Structure RA[%], 100 μM

4.28 75

4.29 78

4.30 80

4.5 Molecular docking of the Chembridge screening