Identification of novel pyrazoloquinazolinecarboxilate analogues to inhibit nerve growth factor in vitro

Nerve growth factor (NGF) is known to regulate the development and survival of select populations of neurons via its binding/activation of the TrkA and p75(NTR) receptors. However, in some physiological circumstances NGF dysregulation can result in debilitating pathologies, including diabetic neuropathies, interstitial cystitis and fibromyalgia. Thus, the identification of small molecules which inhibit NGF signalling have significant therapeutic potential. PD 90780, Ro 08-2750, and ALE 0540 are small molecules that have been reported to bind and inhibit NGF activity. Importantly, the docking site of these compounds is hypothesised to occur at the loop I/IV cleft of NGF-a region which is required for efficient and selective binding of this neurotrophin to its receptor(s). Molecular modelling predicts a number of previously reported NGF antagonists (PD 90780, ALE 0540, and Ro 08-2750) share conserved molecular features, and these drug-like small molecules have the ability to bind and modify the molecular topology of NGF. In order to understand the putative mechanism of binding, we synthesised a pyrazoloquinazolinecarboxilate analogue series and tested each compound in an NGF-dependent PC12 cell differentiation assay. In vitro data confirms that the pyrazoloquinazolinecarboxilate analogues functionally inhibit NGF's effects on PC12 cell differentiation. The results of this study provide evidence to refine the docking mode of pyrazoloquinazolinecarboxilate based compounds for the purposes of inhibiting NGF in vitro. In addition, we identified series analogue PQC 083 (IC50=7.0 µM; CI=5.4-10.1 µM) which displays markedly higher potency than previously described NGF antagonists.