A novel synthetic tricyclic compound with an acetylene group at C-8a and cyano enones in rings A and C is a potent multifunctional agent in vitro and suppresses the formation of aflatoxin-DNA adducts in vivo (2007)

B66 TBE-31 is the lead molecule in a series of tricyclic compounds containing cyano enone functionalities in the A and C rings. Originally screened for its anti-inflammatory activity, TBE-31 inhibits the production of nitric oxide (IC50 = 1 nM) and the inducible nitric oxide synthase protein in RAW264.7 macrophage-like cells stimulated with either interferon-gamma or lipopolysaccharide. Low nanomolar concentrations of TBE-31 also induce the phase 2 cytoprotective proteins heme oxygenase-1 and NAD(P)H quinone oxidoreductase and reduce the formation of reactive oxygen species in cells treated with the tert-butyl hydroperoxide. In U937 human leukemia cells, TBE-31 increases expression of CD11b, a marker of monocytic differentiation; inhibits proliferation; and induces apoptosis, as shown by increased PARP cleavage and annexin V staining. These results are seen with higher concentrations (100-1000 nM) than needed for the anti-inflammatory activity and phase 2 enzyme induction. The multifunctional activities of TBE-31 are similar to those of the synthetic oleanane triterpenoids, including CDDO-methyl ester and CDDO-imidazolide; CDDO-methyl ester is currently in phase II clinical trials for the treatment of melanoma and pancreatic cancer. The alpha, beta-unsaturated carbonyl groups in TBE-31 suggest it can form Michael adducts with protein targets containing reactive cysteine residues. Indeed, TBE-31, like the triterpenoids, directly interacts with dithiothreitol, as determined by a spectral shift when dithiothreitol is added to TBE-31. Identifying the direct molecular targets of TBE-31 is an area of active investigation. To determine whether biologically meaningful tissue levels could be obtained in vivo, CD-1 mice were gavaged with 1 micromole TBE-31. Six hours later, blood and tissues were harvested, extracted in acetonitrile and analyzed by liquid chromatography-mass spectrometry. The levels of TBE-31 in blood were 1 micromolar. Of the organs surveyed, the highest concentrations were found in the liver and pancreas, with levels of 0.7 and 0.5 micromole/kg, respectively. Because of the high drug levels in the liver, rats were gavaged with TBE-31 and then, 48 hours later, challenged with aflatoxin. At both 10 and 100 micromole/kg, TBE-31 significantly reduced the formation of aflatoxin-DNA adducts. In these studies, TBE-31 was more potent than CDDO-imidazolide, one of the most active compounds ever tested in this assay. Studies evaluating the ability of TBE-31 to block hepatic pre-neoplastic lesions induced by aflatoxin are ongoing. The exquisite potency of TBE-31 in vitro and in vivo suggest it should be tested for both chemoprevention and chemotherapy in relevant models of cancer and other chronic, degenerative diseases where inflammation and oxidative stress are known factors in the pathogenesis.  Supported by NIH grant R01 CA78814, R03 CA105294, R01 CA39416, the National Foundation for Cancer Research and Reata Pharmaceuticals, Inc.