A putative bioactive 3-dimensional conformation of the PAT series identified by CoMFA was used to guide synthesis of a set of PAT analogues which were designed to explore the central B-ring and its role in the alignment of the molecule and to probe the nature of the steric pocket adjacent to the pyridine A-ring. Previous exploration of the basic SAR around the aniline C-ring had shown that small, lipophilic substituents were optimal for activity hence we chose to use the 3-methylaniline ring.20
The ligand model gave a consistent curved orientation with the thiazole N
placed in the inner surface and the thiazole S
on the exposed rim (). Replacement of the thiazole ring with any other 5-membered-ring heterocycle led to either a large drop in potency and selectivity or complete loss of activity (). Replacement of the thiazole S with another heteroatom (NH and O) led to inactive analogues (3
). Incorporation of an additional N
within the central ring gave much less potent derivatives (e.g., thiadiazole 5
) while retaining a hint of selectivity, although lack of aqueous solubility prevent a full determination in the RCC4/VHL cell line (). Similarly, triazole analogues displayed low potency and selectivity. It was not possible to fully evaluate the influence of the thiazole N
on the biological activity as our efforts towards the synthesis of a thiophene analogue were unsuccessful with the instability of the desired thiophene product precluding purification. We compared the effect of the substitution of O
by overlaying minimised structures of thiazole 2
, oxazole 4
and thiadiazole 8
and noted that only a small conformational shift was seen in the case of the oxazole (Supplementary data, Figure S3
Evaluation of the 6-membered ring analogues showed that introduction of a phenyl ring totally abolished selectivity (e.g., 13). Retention of an N-atom in the 2-position of the central ring was necessary for activity against the VHL-deficient RCC4 line (e.g., 9, 11, 12), although it was not sufficient for selectivity with pyrimidine 9 being nonselective. There was no correlation between the pKa of either the pyridyl N, or the aniline NH, and cytotoxic potency across the series 1–13. Taken together, it seems likely that the thiazole N plays a key role in the binding to the target; isosteric replacement of the thiazole S with =CH is tolerated in six-membered rings, but other minor modifications of the central ring result in a loss in potency, thus these analogues were not pursued further.
The putative space around the pyridine ring was explored with analogues bearing fused rings to provide an extended aromatic region. We had previously demonstrated20
that a 4-pyridine was the only regioisomer that retained activity and so the heterocycles were designed to place an N
-atom in a similar spatial location. These rigid, bulky aromatic systems generally gave low potency and selectivity (), suggesting that optimising the steric interaction might require a more directed approach.
Hence, a set of aryl, alkynyl alkyloxy and alkylamino analogues were synthesized by elaboration of the pyridine with a variety of functional groups conveniently introduced by use of Suzuki and Sonogashira reactions, Click chemistry, as well as direct nucleophilic displacement of aryl halides. Functionalization of the pyridine at the 3-position was examined first with simple aryl 18 and pyridyl 19 substituents resulting in decreased potency and selectivity relative to 2. Addition of a range of lipophilic substituents at the 4-position of the phenyl group (20–27) gave a range of activities and suggested the potential for a lipophilic H-bond acceptor such as sulphone 25 or methyl ketone 27 to provide potency in the low micromolar range and modest selectivity. In contrast, more polar H-bond acceptor/donor carboxamides 22 and 24 were less potent and non-selective.
We had previously demonstrated the 3-propargyl analogues 28
displayed increased potency while maintaining selectivity,20
thus the higher homologues 30
were prepared. However, increasing alkyl chain length was not favourable and resulted in decreasing potency and selectivity and increasing the terminal bulk was also disfavoured, with only the morpholine 32
being comparable to 28
. Incorporation of the benzyltriazole unit 34
Pyridines bearing flexible solubilising basic amine side chains generally retained potency but displayed low selectivity (e.g., 35–38). Curiously, the oxygen-linked amines 37 and 38 were superior to the nitrogen-linked amines 35 and 36. The data on the 3-series show that while substituted phenyl groups at the 3-position of the pyridine gave some active compounds, the most potent analogues were obtained with the introduction of a small H-bond acceptor (e.g., CH2OH, CH2OMe) linked via a rigid acetylene.
We finally examined substitutions at the 2-position of the pyridine to see if these could also provide positive steric interactions. In contrast with the 3-series, use of a variety of 6-membered rings (40–44), gave compounds with low potency and selectivity. Interestingly, the introduction of a range of 5-membered ring heterocycles (45–50) gave analogues that generally were active and selective with the only anomaly being the 1-methyl pyrazole 48. The 1-benzylpyrazole derivative 49 had low micromolar activity and a high selectivity ratio of 30, which was similar to the benzyltriazole analogue 50, whereas the unsubstituted pyrazole analogue 47 showed a four-fold increase of potency while preserving a modest selectivity of 14-fold. Together the data suggests the presence of a second domain adjacent to the 2-position of the pyridine ring which can accommodate a five-membered ring. The exact definition of this domain and the associated H-bond contacts remain to be fully characterized. Introduction an alkyl chain at the 2-position with solubilising tertiary amines gave compounds (51–54) with low selectivity.
A series of active compounds (2, 32, 38, 47, and 49) were examined to confirm that the mechanism of cell death involved the induction of autophagy. Treatment of RCC4 cells with 5 μM of each drug for 24 h induced the formation of intracytoplasmic vesicles confirming VHL-selective induction of autophagy resulting in cell death ().
CoMFA was used with the data from this and an earlier study to better understand the effects of substitutions around the PAT core on activity. The 3D-QSAR model generated could only explain approximately 30% of the variance in the IC50 data, and this was reflected in its lack of predictivity of an external test set of compounds. A similar result was also found when a 2D-QSAR method (HQSAR) was used, indicating that the alignment strategy used to generate the CoMFA model was appropriate, and that the SAR data of the PAT series with respect to autophagy may be quite complex.
Examination of the CoMFA contour maps generated from Model 6 using the complete training set () shows that, within the training set, the contouring around the aniline C-ring is similar to that previously published. The new PAT CoMFA model exhibits a large region adjacent to the sulfur of the thiazole B-ring of 28 that suggests a positive electrostatic effect will favor activity. Interestingly, this region has three distinct, yet interconnected regions. Two of these are found around either side of the B-ring while the third is more accessible by substitution from the pyridyl ring 3-position substitution, as substitutions on this ring were all seen to reduce activity. Around the pyridyl group, increased steric bulk is positively associated with activity around the 2- and 3-positions, consistent with the previous model. From the compounds characterized in , this region is better accessed by substitutions at the 3-position as suggested by the higher potency across a number of homologues. Two distinct regions around the pyridyl 2-position and nitrogen atom indicate contrasting effects of steric bulk on activity. A region adjacent to the 2-position indicates that increased steric bulk is associated with decreased activity (yellow contour); although this is contrasted by the 2-pyrazole that has increased bulk at this position yet has the strongest activity recorded for a 2-position substitution. This region of disfavored steric bulk is also accessed by the fused ring compounds () e.g. 15. The pyrazole moiety may contribute to an additional favorable steric bulk zone adjacent to the N-atom (green contour).
The further development of the CoMFA alignment model has not produced gains in the ability of the model to explain the QSAR observed. Despite generating six different models we are unable to explain more than 31% of the variance observed and did not significantly improve on this using the alternative HQSAR approach. This low predictivity may be a consequence of the complexity of the phenotypic readout which reports the product of many combined parameters rather than just the interaction of the PAT with the molecular target. Although we have further explored the SAR around the pyridyl A-ring and B-ring, the relatively small gains in potency and selectivity seen with the 2- and 3-substituted pyridyl analogues, coupled with the relatively flat SAR profile observed in our previous study, suggest that the PAT series may occupy a local minimum and a large random leap may be necessary to improve potency and selectivity substantially. We will endeavour to use the alignment model generated in this study to conduct a virtual screen in an effort to identify new chemotypes with VHL-selective activity. In another approach, we plan to exploit the steric tolerance adjacent to the pyridyl ring to introduce linker moieties to assist in the preparation of molecular probes to isolate and identify the molecular target of this PAT chemotype.