Irreversible inhibition of BTK kinase by a novel highly selective inhibitor CHMFL-BTK-11 suppresses inflammatory response in rheumatoid arthritis model

BTK plays a critical role in the B cell receptor mediated inflammatory signaling in the rheumatoid arthritis (RA). Through a rational design approach we discovered a highly selective and potent BTK kinase inhibitor (CHMFL-BTK-11) which exerted its inhibitory efficacy through a covalent bond with BTK Cys481. CHMFL-BTK-11 potently blocked the anti-IgM stimulated BCR signaling in the Ramos cell lines and isolated human primary B cells. It significantly inhibited the LPS stimulated TNF-α production in the human PBMC cells but only weakly affecting the normal PBMC cell proliferation. In the adjuvant-induced arthritis rat model, CHMFL-BTK-11 ameliorated the inflammatory response through blockage of proliferation of activated B cells, inhibition of the secretion of the inflammatory factors such as IgG1, IgG2, IgM, IL-6 and PMΦ phagocytosis, stimulation of secretion of IL-10. The high specificity of CHMFL-BTK-11 makes it a useful pharmacological tool to further detect BTK mediated signaling in the pathology of RA.

Excess POCl 3 was removed under reduced pressure and the crude residue was neutralized with saturated sodium bicarbonate solution (100 mL) and extracted with ethyl acetate.
The organic layer was dried over Na 2 SO 4 , filtered and evaporated under reduced pressure.

Supplemental Materials
Supplemental Table 3 The evaluation standards of arthritis index* of AA rats points Symptoms 0 normal 1 Erythema and slight swelling of ankle 2 Erythema and slight swelling of ankle and metatarsophalangeal joints (or metacarpal joints)

3
Erythema and moderate swelling of ankle and metatarsophalangeal joints (or metacarpal joints)

4
Erythema and severe swelling of ankle and metatarsophalangeal joints *Each rat has 4 paw, and is awarded 16 points at most. Table 4 The evaluation standards of quantification of H&E staining points Symptoms 0 normal ankle joint 1 occasional synovial hyperplasia 2 definite arthritis with a few layers of flat to rounded synovial lining 3 clear hyperplasia of the synovium with three or more layers of loosely arranged lining cells