Yukiko Honda

The Synthetic Triterpenoids, CDDO and CDDO-Imidazolide, Are Potent Inducers of Heme Oxygenase-1 and Nrf2/ARE Signaling

The synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its derivative 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) are multifunctional molecules with potent antiproliferative, differentiating, and anti-inflammatory activities. At nanomolar concentrations, these agents rapidly increase the expression of the cytoprotective heme oxygenase-1 (HO-1) enzyme in vitro and in vivo. Transfection studies using a series of reporter constructs show that activation of the human HO-1 promoter by the triterpenoids requires an antioxidant response element (ARE), a cyclic AMP response element, and an E Box sequence. Inactivation of one of these response elements alone partially reduces HO-1 induction, but mutations in all three sequences entirely eliminate promoter activity in response to the triterpenoids. Treatment with CDDO-Im also elevates protein levels of Nrf2, a transcription factor previously shown to bind ARE sequences, and increases expression of a number of antioxidant and detoxification genes regulated by Nrf2. The triterpenoids also reduce the formation of reactive oxygen species in cells challenged with tert-butyl hydroperoxide, but this cytoprotective activity is absent in Nrf2 deficient cells. These studies are the first to investigate the induction of the HO-1 and Nrf2/ARE pathways by CDDO and CDDO-Im, and our results suggest that further in vivo studies are needed to explore the chemopreventive and chemotherapeutic potential of the triterpenoids.

A novel dicyanotriterpenoid, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-onitrile, active at picomolar concentrations for inhibition of nitric oxide production

New oleanane triterpenoids with various substituents at the C-17 position of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and methyl 2-carboxy-3,12-dioxooleana-1,9(11)-dien-28-oate were synthesized. Among them, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-onitrile shows extremely high inhibitory activity (IC(50)=1 pM level) against production of nitric oxide induced by interferon-gamma in mouse macrophages. This potency is about 100 times and 30 times more potent than CDDO and dexamethasone, respectively.

Tricyclic compounds containing nonenolizable cyano enones. A novel class of highly potent anti-inflammatory and cytoprotective agents.

Forty-four novel tricycles containing nonenolizable cyano enones (TCEs) were designed and synthesized on the basis of a semisynthetic pentacyclic triterpenoid, bardoxolone methyl, which is currently being developed in phase II clinical trials for the treatment of severe chronic kidney disease in diabetic patients. Most of the TCEs having two different kinds of nonenolizable cyano enones in rings A and C are highly potent suppressors of induction of inducible nitric oxide synthase stimulated with interferon-γ and are highly potent inducers of the cytoprotective enzymes heme oxygenase-1 and NAD(P)H:quinone oxidoreductase-1. Among these compounds, (±)-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile ((±)-31) is the most potent in these bioassays in our pool of drug candidates including semisynthetic triterpenoids and synthetic tricycles. These facts strongly suggest that an essential factor for potency is not a triterpenoid skeleton but the cyano enone functionality. Notably, TCE 31 reduces hepatic tumorigenesis induced with aflatoxin in rats. Further preclinical studies and detailed mechanism studies on 31 are in progress.

Efficient synthesis of (-)- and (+)-tricyclic compounds with enone functionalities in rings A and C. A novel class of orally active anti-inflammatory and cancer chemopreventive agents.

Novel tricyclic compounds with enone functionalities in rings A and C [tricyclic-bis-enone (TBE) compounds] were designed on the basis of the structure of a synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO)(1), which is a promising drug candidate for prevention and/or treatment of cancer and inflammatory diseases whose pathogenesis may involve excessive production of nitric oxide (NO) and/or prostaglandins. A series of TBE compounds in racemic form shows high inhibitory activity against production of NO induced by interferon-[gamma](IFN-[gamma]) in mouse macrophages. One of these compounds, (+/-)-(4a[small beta],8a[small beta],10a[small alpha])-1,2,4a,6,8a,9,10,10a-octahydro-1,1,4a,8a-tetramethyl-2,6-dioxophenanthrene-3,7-dicarbonitrile ((+/-)-3), is orally active at 15 mg kg(-1)(single administration) in a preliminary study using mouse peritoneal inflammation induced by thioglycollate and IFN-[gamma]. Therefore, we desired to synthesize optically active TBE compounds for a comparison of the biological potency of both enantiomers. We now describe the synthesis of both enantiomers of (4a[small beta],8a[small beta],10a[small alpha])-1,2,4a,6,8a,9,10,10a-octahydro-1,1,4a,8a-tetramethyl-2,6-dioxophenanthrene-3-carbonitrile (2) and 3 from commercially available simple compounds. Interestingly, (+)-3 having the same configuration as the CDDO antipode shows about 10 times higher inhibitory activity than (-)-3 on NO production in mouse macrophages. In contrast, (-)-3 inhibits proliferation of MCF-7 breast cancer cells, whereas (+)-3 does not.

AN EFFICIENT SYNTHESIS OF TRICYCLIC COMPOUNDS, (+/-)-(4abeta8abeta10aalpha)-1,2,3,4,4a,6,7,8,8a,9,10,10a-DODECAHYDRO-1,1,4a-TRIMETHYL-2-OXOPHENANTHRENE-8a-CARBOXYLIC ACID, ITS METHYL ESTER, AND (+/-)-(4abeta,8abeta10aalpha)-3,4,4a,6,7,8,8a,9,10,10a-DECAHYDRO-8a-HYDROXYMETHYL-1,1,4a-TRIMETHYLPHENANTHREN-2(1H)-ONE.

Our ongoing efforts for the improvement of anti-inflammatory and antiproliferative activity of oleanolic acid analogues led us to discover 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO, 1) and related compounds.1 In connection with these investigations, we have found that tricyclic compounds with similar enone functionalities in rings A and C are also a novel class of highly active inhibitors of nitric oxide (NO) production in mouse macrophages.2 In particular, bis-cyano enone (±)-2 is orally active in a preliminary in vivo inflammation model.2 In addition, we have found that (+)-2 having the opposite configuration to that of CDDO shows 10 times higher inhibitory activity than (-)-2 on NO production in mouse macrophages.3 These results encouraged us to design and synthesize analogues of 2. Thus, we focused our attention on the modifications of the C-8a position, because some biologically active natural products have functionalities at the same position (e.g., anti-tumor quassinoids4). For our projected synthesis of C-8a functionalized TBE compounds, the simple tricycles 3-5 are potentially very desirable intermediates. We envisioned preparing 3-5 from the known acid 65,6 by standard reductive methylation.7 However, attempts to reductively methylate acid 6 with 5-7 equivalents of lithium in liquid ammonia containing no proton donor, followed by esterification with diazomethane gave 4 in 30% yield (average of 7 experiments, the yield fluctuates) along with many by-products. These by-products caused serious difficulty for the purification of 4. An attempt with one equivalent of tert-butanol gave similar results as without a proton donor. Attempts to reductively methylate methyl ester 7,6 which is prepared from 6 with diazomethane, using 10 equivalents of lithium in liquid ammonia containing no proton donor gave the desired compounds 3-5 in low yield along with several by-products including enones 6 and 8. After much experimentation, we have found that the addition of one equivalent of water dramatically improves this reductive methylation reaction. Thus, the reductive methylation of 7 using 7.2 equivalents of lithium and one equivalent of water followed by quenching the excess lithium with isoprene, and then methyl iodide at -78 °C cleanly produced 3-5 in 38%, 15%, and 36% yields (total 89%), respectively. The yields are reproducible and we have prepared 3-5 several times by this procedure. These compounds can be easily separated by extracting the acid with aqueous base, followed by column chromatography (see Experimental Section). Also, they were easily converted to a single compound. For example, oxidation (e.g., Jones reagent and RuO2-NaIO4 etc.) of alcohol 5 gave acid 3, and both acid 3 and methyl ester 4 were converted to alcohol 5 in three steps (ketalization, reduction with LiAlH4, and deketalization). Acid 3 may be an important intermediate for the synthesis of abietane and totarane diterpenoids.