A cell-permeable benzotropolone compound that selectively antagonizes agonist-dependent TLR1/TLR2 heterodimer activation (IC50 = 580 nM against Pam3CSK4- (Cat. No. 506350) stimulated NO production in RAW264.7) by competing for TLR1/TLR2 binding (Ki = 410 nM against Pam3CSK4 in competition binding assays), displaying no cytotoxicity in RAW264.7 cultures (100 µM for 24 h) and exhibiting little inhibitory activity against a panel of 10 kinases (by <11% at 5 µM) or agonist-dependent activation of other TLRs (0% inhibition of TLR2/TLR6, TLR3, TLR4, TLR7 activation at 0.5 µM). Computer-aided in silico structural analysis reveals that the highly TLR1/TLR2-over-TLR2/TLR6 selective nature of CU-CPT22 is due to the exclusive location of CU-CPT22 R6 hexyl ester and Pam3CSK4 amide-linked N-pamitoyl fatty acid side chain competition site in TLR1, but not in TLR6.