Dartmouth chemistry professor Gordon Gribble recently published an article in collaboration with Michael Sporn in the pharmacology and medicine department at Dartmouth Medical School. Sporn and Gribble focused on a set of compounds called triterpenoids, which are found in virtually all plants. Their findings were published in the Journal of Natural Products.
Oxidative stress in a cell is caused by reactive nitrogen and oxygen species, which are capable of damaging DNA, proteins, and lipids within a cell. Mechanisms evolved over time to prevent such damage from invading and keeping DNA regulation under control, specifically through the action of enzymes. Synthetic oleanane triterpenoids (SO’s) serve a similar function of helping to relieve oxidative stress by repressing expression and formation of these reactive nitrogen and oxygen species, which are often produced by metabolic action in the cell itself.
Gribble and Sporn have worked with SO’s since 1995, a class of molecules characterized by a five-ring skeletal structure. Sporn came to Gribble looking for a chemist, knowing that natural triterpenoids had modest anti-inflammatory activity, but hoped to modify them to make them even more effective. Gribble started with the naturally occurring triterpenoids, oleanolic acid (found in olive oil) and ursolic acid (found in cranberries), and “did every possible chemical reaction to them [he] could think of” alongside Tadashi Honda, he said, some of which are shown in Figure 1.
Figure 1: Proposed structural modifications of oleanolic acid. Starting with oleanolic acid, Gribble and Honda performed a battery of chemical reactions until Sporn confirmed a compound was biologically successful.
One small change of the hydroxyl to a ketone on ring A in the 39th compound they synthesized made it 1000x more reactive in inhibiting the synthesis of the enzyme “inducible nitric oxide synthase” (iNOS), which makes nitric oxide. When overexpressed, “nitric oxide destroys cartilage, causes inflammation, Parkinson’s, Crohn’s…,” explained Gribble, “and our compounds prevent that.” Even further, an addition of an electron withdrawing group on the 139th compound (CN–) increased its reactivity by a factor of 10.
Gribble’s lab synthesized Bardoxolone-methyl, or CDDO-Me (2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid) in an 11-step process (see Fig. 2). The steps involved include esterification, epoxidation with rearrangement to form a ketone, alpha-bromination, hydrolysis, Jones oxidation, addition of an OHC group, formation of a cyclic oxime, breaking of the oxime to give a nitrile and hydroxyl group, and oxidation with DDQ to give a conjugated enone on ring A. Again, this electron withdrawing nitrile on the enone increases its reactivity, particularly because it can act as a Michael acceptor.
Figure 2: CDDO-Me. The double bonds conjugated to the ketone give it great biological significance.
This Michael reactivity is very significant, in that it may undergo reversible addition with all the possible nucleophiles in a cell, but it can “soak up” the reactive, toxic species. The enone in ring A combined with the enone in ring C (middle ring) make it “400,000x more reactive than the oleanolic acid.”
On the biological side, CDDO-Me, with its anti-inflammatory properties, is able to relieve symptoms of chronic kidney disease caused by diabetes and pancreatic cancer near the end of life, essentially reversing kidney damage. CDDO-Me is now in Phase III of clinical trials; Gribble and Sporn hope to see effective results as this could potentially be “the first drug ever put on the market by Dartmouth.” He anticipates that the Food and Drug Administration will make the final decision sometime late 2012.
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