Modified Salicylanilide and 3-Phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione Derivatives as Novel Inhibitors of Osteoclast Differentiation and Bone Resorption

Abstract

Inhibition of osteoclast formation is a potential strategy to prevent inflammatory bone resorption and to treat bone diseases. In the present work, the purpose was to discover modified salicylanilides and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione derivatives as potential antiosteoclastogenic agents. Their inhibitory effects on RANKL-induced osteoclastogenesis from RAW264.7 cells were evaluated by TRAP stain assay. The most potent compounds, 1d and 5d, suppressed RANKL-induced osteoclast formation and TRAP activity dose-dependently. The cytotoxicity assay on RAW264.7 cells suggested that the inhibition of osteoclastic bone resorption by these compounds did not result from their cytotoxicity. Moreover, both compounds downregulated RANKL-induced NF-κB and NFATc1 in the nucleus, suppressed the expression of osteoclastogenesis-related marker genes during osteoclastogenesis, and prevented osteoclastic bone resorption but did not impair osteoblast differentiation in MC3T3-E1. Therefore, these modified salicylanilides and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-diones could be potential lead compounds for the development of a new class of antiresorptive agents.

The Simultaneous Inhibitory Effect of Niclosamide on RANKL-Induced Osteoclast Formation and Osteoblast Differentiation

“…To assess the effect of niclosamide on RANKL-induced bone resorption, we performed a pit formation assay as described previously 31 . The BMMs or RAW264.7 were seeded into a 24-well Corning Osteo assay plate.…”

Discovery of 5-(2′,4′-difluorophenyl)-salicylanilides as new inhibitors of receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis

“…Absorbance was measured at 540 nm by using an ELISA reader. [40,41] RAW264.7 cells were seeded into 96-well culture plate at a density of 1 × 10 3 Absorbance was measured at 405 nm by using an ELISA reader. The intracellular protein level was measured at 562 nm by using a BCA protein assay kit (Pierce, USA).…”

“…The intracellular protein level was measured at 562 nm by using a BCA protein assay kit (Pierce, USA). [40,41] RAW264.7 cells were seeded into 6-cm dish at a density of 1 × 10 5 …”

“…These cells were cultured with Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL, NY, USA) and supplemented with 10% heat-inactivated fetal bovine serum (FBS) in 95% air, 5% CO 2 humid atmosphere at 37 °C.5.2.2. Cell viability assay[40,41] RAW264.7 cells were seeded into a 96-well culture plate at a density of 1 × 10 4 per well with 200 µL medium (DMEM) supplemented with 10% FBS and treated with a series of synthesized compounds for 24 h. After 24 h, 96-well plate were washed three times with phosphate-buffered saline (3 × PBS) and then treated with medium containing 500 µg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent for 1 h at 37 °C.…”

Rapid, Simple and Efficient Microwave‐Assisted Alkylation of 6‐Acetyl‐2H‐Benzo[e][1, 3] Oxazine‐2, 4(3H)‐Dione

“…Among them, benzoxazine and benzoxazinone (I) are an important class of benz fused heterocyclic compounds which has been found a wide range of biological and pharmacological activities. The substituent pattern of these class of compounds are exhibit significant activity against so many therapeutic agents like anti‐bacterial, anti‐fungal(I),, anti‐inflammatory(II), antitumor, anticancer, antidepressants, ALK2 Inhibitor, RANKL inhibitors(III), anti‐tuberculosis, anti‐mycobacterial, antioxidant activity, anti‐nociceptive(IV) ,. The 1,3‐benzoxazinone moieties is also found a same structural pattern in a drug like efavirenz(V) .…”