A Novel Dicyanotriterpenoid, 2-Cyano-3,12-dioxooleana- 1,9(11)-dien-28-onitrile, Active at Picomolar Concentrations for Inhibition of Nitric Oxide Production Tadashi Honda, a Yukiko Honda, a Frank G. Favaloro, Jr., a Gordon W. Gribble, a, * Nanjoo Suh, b Andrew E. Place, b Mara H. Rendi b and Michael B. Sporn b, * a Department of Chemistry, Dartmouth College, Hanover, NH 03755, USA b Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755, USA Received 14 November 2001; accepted 31 January 2002 Abstract—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-g in mouse macrophages. This potency is about 100 times and 30 times more potent than CDDO and dexamethasone, respectively. # 2002 Elsevier Science Ltd. All rights reserved. In previous papers, we reported that 2-cyano- 3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) (1), its methyl ester 2 and methyl 2-carboxy-3,12-dioxo- oleana-1,9(11)-dien-28-oate (3) show high inhibitory activity against production of nitric oxide (NO) induced by interferon-g (IFN-g) in mouse macrophages (IC 50 =0.1 nM level). 1 4 We also reported that CDDO is a potent, multifunctional agent in various in vitro assays. 5 For example, CDDO induces monocytic differ- entiation of human myeloid leukemia cells and adipo- genic differentiation of mouse 3T3-L1 fibroblasts. CDDO also inhibits proliferation of many human tumor cell lines, and blocks de novo synthesis of indu- cible nitric oxide synthase (iNOS) and inducible cyclo- oxygenase (COX-2) in mouse macrophages. The above potencies have been found at concentrations ranging from 10 6 to 10 9 M in cell culture. Mechanism studies revealed that CDDO is a ligand for peroxisome pro- liferator-activated receptor g (PPARg) 6 and induces apoptosis in human myeloid leukemia cells. 7 Modifications of rings A and C of oleanolic acid (30), a commercially available naturally occurring triterpene, led to the synthesis of CDDO. However, we had not modified the carboxyl group at C-17 of CDDO, which is very important from the perspective of structure– activity relationships (SARs). Because the synthesis of CDDO involves 11 steps from oleanolic acid, this has limited the preparation of sufficient quantities of CDDO to allow such modifications. However, we have recently produced a sufficient amount to be able to synthesize various CDDO derivatives with modified carboxyl groups (i.e., nitrile, esters, glycosides, and amides) at C-17 (see Table 1). As a result, we found that 2-cyano- 3,12-dioxooleana-1,9(11)-dien-28-onitrile (4) shows extremely high inhibitory activity (IC 50 =1 pM level) against production of NO in mouse macrophages. This potency is about 100 times and 30 times more potent than that of CDDO and dexamethasone, respectively. In this communication, we report the synthesis, inhibi- tory activity and SARs of these new analogues. Dinitrile 4 was synthesized from CDDO by the method as shown in Scheme 1. Addition of oxalyl chloride to CDDO gave acyl chloride 31 in quantitative yield. Amide 15 was prepared in 91% yield from 31 with ammonia gas in benzene. Dehydration of 15 with thio- nyl chloride gave 4 in 89% yield. 8 Because the C-17 carboxyl group of CDDO is hindered, esterifications of CDDO with alcohols under acidic conditions were not successful. We found that a nucleophilic substitution 0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0960-894X(02)00105-1 Bioorganic & Medicinal Chemistry Letters 12 (2002) 1027–1030 *Corresponding authors. G.W. Gribble Tel.: +1-603-646-3118; fax: +1-603-646-3946; M.B. Sporn Tel.: +1-603-650-6557; fax: +1-603- 650-1129; E-mail: grib@dartmouth.edu (G.W. Gribble); michael. sporn@dartmouth.edu (M.B. Sporn).