BENZYLIDENE DERIVATIVES OF 12,13-DIHYDROANDROGRAPHOLIDE: SYNTHESIS, AND EVALUATION OF IN VITRO PHARMACOKINETICS AND NEUROPROTECTIVE PROPERTIES

Abstract

Andrographolide (AGP) is the major ent-labdane diterpenoid isolated from Andrographis paniculata (known locally in Malaysia as Hempedu Bumi). AGP was found to exhibit neuroprotective effects by inhibiting inflammation and oxidative stress. As such, AGP could act as a novel alternative for the treatment of neurological disorder. However, its oral bioavailability is reduced due to inactivation by liver glutathione S-transferase (GST). To tackle this shortcoming, a series of 12,13-dihydroandrographolide (HAGP) analogues were semisynthesised in the previous project where the C12-C13 double bond was reduced to avoid glutathione (GSH) conjugation. Besides that, substituted benzylidene moiety was introduced at C-3 and C-19 hydroxyl groups to increase the lipophilicity of the compounds for better blood-brain-barrier penetration. Among the analogues, 3,19-(3-ethoxy,4-hydroxylbenzylidene)-12,13-dihydroandrographolide (HAGP34) and 3,19-(3-flurobenzylidene)-12,13-dihydroandrographolide (HAGP38) showed better activity in the initial bioassay screening against pheochromocytoma (PC12) cells. In view of this, both compounds were selected to evaluate their in vitro pharmacokinetics and neuroprotective properties in this study. HAGP34 and HAGP38 were shown to avoid GSH conjugation (phase II metabolism). However, in the rat microsomes metabolism experiment to mimic Phase I metabolism in the liver, AGP (intrinsic clearance, CLint = 30 μL/min/mg) was found to be more stable than HAGP34 (CLint = 67 μL/min/mg), but less stable than HAGP38 (CLint = 16 μL/min/mg). These two in vitro pharmacokinetics studies evealed that HAGP38 was more stable than AGP in phase I and phase II metabolism. HAGP38 and AGP showed comparable reactive oxygen species (ROS) inhibition in rat pheochromocytoma cells (PC12), where the percentages of inhibition were 18% and 23% respectively at 10 μM. In mouse microglial cells (BV2), AGP exhibited strong nitric oxide (NO) inhibition where its IC50 value was 5 μM. When compared with HAGP38, the NO inhibition was only 17% at 5 μM. The Trans-AM assay revealed that HAGP38 inhibited NO production via disruption of NF-κB signalling pathway, similar to AGP as reported in the literature. On the other hand, HAGP34 showed weak ROS and NO inhibition in both cell lines. These accumulating findings revealed that the C12-C13 double bond or C-3 and C-19 hydroxyl groups of AGP may play strong roles as the pharmacophore of anti-inflammatory and antioxidant activities. Although the biological activities of HAGP38 were not as potent as than AGP, its improved in vitro pharmacokinetics may give better bioavailability compared to AGP. Therefore, HAGP38 is may be a promising molecule that should be further tested in animal models.