Design, synthesis, and biological activity of second-generation synthetic oleanane triterpenoids

Liangfeng Fu a, Qi-xian Lin a, Evans O. Onyango a, Karen T. Liby *b, Michael B. Sporn *b and Gordon W. Gribble *a
aDepartment of Chemistry, Dartmouth College, Hanover, New Hampshire 03755, USA. E-mail:
bDepartment of Pharmacology and Toxicology, Geisel School of Medicine, Hanover, New Hampshire 03755, USA. E-mail:;

Received 12th June 2017 , Accepted 28th June 2017

First published on 28th June 2017

We report the synthesis and biological activity of C-24 demethyl CDDO-Me 2 and the C-28 amide derivatives 3 and 4, which are analogues of the anti-inflammatory synthetic triterpenoid bardoxolone methyl (CDDO-Me) 1. Demethylation of the C-24 methyl group was accomplished via “abnormal Beckmann” rearrangement and subsequent ring A reformation. Amides 3 and 4 were found to be potent inhibitors of the production of the inflammatory mediator NO in vitro.

Bardoxolone methyl (2-cyano-3,12-dioxooleane-1,9(11)-dien-28-oic acid methyl ester; CDDO-Me) (1, Fig. 1) is the most advanced of a series of highly potent anti-inflammatory synthetic oleanane triterpenoids that our group synthesized several years ago.1 Subsequent work in our laboratories revealed the extraordinary biological activity of 1 in numerous assays evaluating inflammation, cytotoxicity, apoptosis, and oxidative stress.2 Recent studies of 1 and related analogues demonstrate their profound activity in chemoprevention of pancreatic,3 lung,4 and breast5 cancer as well as inflammation of the colon.6
image file: c7ob01420a-f1.tif
Fig. 1 Derivatives of CDDO-Me.

Originally tested as a cancer drug,7 bardoxolone methyl exhibited improved kidney function in type 2 diabetes patients with advanced chronic kidney disease in Phase 1 and 2 clinical trials.8 Unfortunately, a Phase 3 trial in stage 4 diabetes patients was terminated in 2012 due to increased cardiac toxicity in the group treated with this drug.9 Despite this disappointing result, clinical trials with bardoxolone methyl or related derivatives are ongoing for pulmonary hypertension, mitochondrial myopathies, and cancer.

We now describe the synthesis of the novel C-24 demethylated analogue 2 of bardoxolone methyl, and the synthesis and biological activity of the corresponding C-24 demethyl CDDO-ethyl amide 3 and C-24 demethyl CDDO-trifluoroethyl amide 4, which display improved biological activity and pharmacokinetic properties compared to the bardoxolone amides. These original amides were designed to improve drug delivery into the brain, and they are active in a variety of preclinical models of neurodegenerative diseases.10 While exploring chemical space and designing second generation triterpenoids, we now report that the removal of the axial C-24 methyl group (at the C-4 position of ring A) improves the biological activity and exposure levels in vivo of a subset of these triterpenoids.

Our synthesis of C-24 demethyl CDDO-Me 2 and the target amides 3 and 4 is summarized in Schemes 1 and 2. As we previously reported, diketo ester 5 was prepared in six steps from oleanolic acid (69% overall yield).1a Oximation of 5 afforded oxime 6 (91% yield).1a After considerable experimentation, we effected an “abnormal Beckmann” rearrangement11 of 6 with p-toluenesulfonyl chloride in DMF at 60 °C to give the desired nitrile 7 (75% yield). Epoxidation (m-CPBA/CH2Cl2) of the alkene double bond in 7 gave oxirane 8 in 79% yield. Although initial attempts to transform cyano epoxide 8 to the desired C-24 demethylated 9 were mediocre using TiCl4, SnCl4, ZnCl2, or MgBr2, we eventually found that boron trifluoride etherate in refluxing toluene gave 9 in 88% yield.11a Following our standard isoxazole methodology for the synthesis of CDDO-Me, we converted ketone 9via ketoaldehyde 10 to isoxazole 11 (76% yield), and thence, via cyano enol 12 and a sequence of bromination (1,3-bromo-5,5-dimethylhydantoin) [DBDMH]-dehydrobromination to the desired 24-demethyl CDDO-Me 2 in 88% yield from 11 (Scheme 1).12

image file: c7ob01420a-s1.tif
Scheme 1 Synthesis of C-24 demethyl CDDO-Me 2.

image file: c7ob01420a-s2.tif
Scheme 2 Synthesis of amides 3 and 4.

The synthesis of amides 3 and 4 is shown in Scheme 2. Demethylation of 2 with LiI/pyridine afforded 24-demethyl CDDO 13 in 77% yield. Subsequent acid chloride formation with oxalyl chloride and in situ reaction with appropriate amines gave the desired ethyl amide 3 and trifluoroethyl amide 4 in good yield.

The biological activity of 24-demethyl CDDO amides 3 and 4, relative to their CDDO counterparts (CDDO-ethyl amide 14 and CDDO-trifluoroethyl amide 15), is summarized below. As shown in Fig. 2, the demethyl derivatives are more potent at inhibiting nitric oxide (NO) production in RAW264.7 macrophage-like cells stimulated with either interferon gamma (IFN-γ) or lipopolysaccharide (LPS) than CDDO-EA 14 or CDDO-TFEA 15, respectively. Under basal conditions, these cells do not express inducible nitric oxide synthase (iNOS), the enzyme that catalyzes the production of NO from L-arginine. Both IFN-γ and LPS cause a robust induction of iNOS, and low nanomolar concentrations of the triterpenoids inhibit production of this inflammatory molecule. We have used the NO assay as primary screening assay for all newly synthesized triterpenoids. In contrast to the CDDO amides (14 and 15) and their demethyl derivatives (3 and 4), there was no difference in the activity of 24-demethyl CDDO-Me 2vs. CDDO-Me 1 in this assay (data not shown).

image file: c7ob01420a-f2.tif
Fig. 2 Triterpenoids inhibit NO production in RAW264.7 macrophage-like cells. Cells were treated with drug and either IFNγ (10 ng ml−1) or LPS (5 ng ml−1) for 24 hours, and supernatants were assayed by the Griess reaction. Independent NO experiments were repeated at least three times.

An increase in activity in the in vitro NO assay is not always observed in vivo, so we next measured drug levels in various tissues. When mice were gavaged with these triterpenoids in a protocol we have used previously to monitor drug levels of the amides,10e higher tissue concentrations in the liver, kidney, lung, pancreas, whole blood and plasma levels were found with both 24-demethyl CDDO-EA 3 and 24-demethyl CDDO-TFEA 4 (Table 1). In the lung, pancreas, plasma and whole blood, drug levels were at least two-fold higher with these new derivatives. In contrast to the other tissues, there were no significant differences in drug levels in the brain with the new derivatives. Notably, drug levels were no different in adipose tissue and were slightly lower in the mammary gland with the new demethyl derivatives 3 and 4. As observed in the NO assay, these differences in tissue concentrations were not found with CDDO-Me 1vs. 24-demethyl CDDO-Me 2 (data not shown). Given that the presumptive mode of action for the cyano enone triterpenoids is a thia-Michael (reversible) addition of sulfur amino acids to the C-1 position,13 which mediates the activation of the transcription factor Nrf2,14 we measured enzyme activity of NQO1 in select tissues. NQO1 is a prototypical target gene of the Nrf2 cytoprotective pathway, which is important for many of the biological activities of the triterpenoids.2d As shown in Fig. 3, NQO1 enzyme activity was again higher in mice gavaged with 24-demethyl CDDO-EA 3 and 24-demethyl CDDO-TFEA 4 in the liver, kidney, and lung.

image file: c7ob01420a-f3.tif
Fig. 3 Triterpenoids induce NQO1 enzyme activity in vivo. Female CD-1 mice were gavaged once daily for 3 days with 1 μmol of triterpenoid. Six hours after the final gavage, tissues were harvested and analyzed for NQO1 activity. Values are mean ± SD; n = 5 per group; V = DMSO vehicle; dm = demethyl.
Table 1 Blood and tissue levels
Female CD-1 mice were gavaged once daily for 3 days with 1 μmol of triterpenoid. Six hours after the final gavage, tissues were harvested, extracted in acetonitrile, and analyzed by LC-MS. Values are mean ± SEM; n = 10 per group.
Liver 6.0 ± 0.6 8.9 ± 1.1 2.8 ± 0.3 5.6 ± 0.6
Kidney 7.7 ± 0.4 11.5 ± 1.2 5.3 ± 0.5 10.3 ± 1.0
Lung 1.1 ± 0.1 2.3 ± 0.4 0.84 ± 0.12 2.0 ± 0.2
Pancreas 1.0 ± 0.1 2.2 ± 0.4 0.73 ± 0.08 1.4 ± 0.2
Brain 0.19 ± 0.03 0.21 ± 0.04 0.57 ± 0.07 0.71 ± 0.08
Adipose 2.3 ± 0.3 2.9 ± 0.6 7.1 ± 1.2 7.1 ± 0.8
Mammary gland 2.6 ± 0.4 2.2 ± 0.3 5.3 ± 0.8 3.8 ± 0.5
Whole blood, μmol L−1 1.8 ± 0.5 5.0 ± 0.8 1.0 ± 0.08 2.3 ± 0.3
Plasma, μmol L−1 0.30 ± 0.04 0.77 ± 0.13 0.23 ± 0.05 0.66 ± 0.16

In summary, employing a novel “abnormal Beckmann” rearrangement, we have synthesized a new synthetic triterpenoid scaffold related to bardoxolone methyl and its analogues. Our rationale in the design of 24-demethyl CDDO was that removal of the C-24 (axial) methyl group should facilitate the thia-Michael nucleophilic addition to the C-1 position, an event that is believed to be responsible for the profound biological activity of these triterpenoids. The reason for the differing biological activity between the 24-demethyl CDDO amides 3 and 4 and the 24-demethyl CDDO-Me is unclear. Nevertheless, the increased drug levels in liver, lung, pancreas, and whole blood/plasma are intriguing as they suggest that lower doses could be used, and we already have promising data in models of lung and pancreatic cancer with CDDO-EA. The differences observed in brain with 24-demethyl CDDO-TFEA 3 also suggest this compound might be useful for neurodegenerative diseases. It remains to be seen if our new 24-demethyl CDDO amides are better tolerated and show less toxicity than CDDO-Me as observed in the Phase 3 trial for chronic kidney disease.


We gratefully acknowledge support from Reata Pharmaceuticals. We are grateful to Renee Risingsong, Darlene Royce and Charlotte Williams of the Department of Pharmacology and Toxicology, Geisel School of Medicine for assistance with the biological assays.


  1. (a) T. Honda, T. Janosik, Y. Honda, J. Han, K. T. Liby, R. C. Williams, D. R. Couch, A. C. Anderson, M. B. Sporn and G. W. Gribble, J. Med. Chem., 2004, 47, 4923 CrossRef CAS PubMed; (b) For a more efficient recent synthesis, see: L. Fu and G. W. Gribble, Org. Lett., 2013, 15, 1622 CrossRef CAS PubMed.
  2. For reviews, see: (a) K. T. Liby, M. M. Yore and M. B. Sporn, Nat. Rev. Cancer, 2007, 7, 357 CrossRef CAS PubMed; (b) M. B. Sporn, K. Liby, M. M. Yore, N. Suh, A. Albini, T. Honda, C. Sundararajan and G. W. Gribble, Drug Dev. Res., 2007, 68, 174 CrossRef CAS; (c) M. B. Sporn, K. T. Liby, M. M. Yore, L. Fu, J. M. Lopchuk and G. W. Gribble, J. Nat. Prod., 2011, 74, 537 CrossRef CAS PubMed; (d) K. T. Liby and M. B. Sporn, Pharmacol. Rev., 2012, 64, 972 CrossRef CAS PubMed; (e) Y.-Y. Wang, Y.-X. Yang, H. Zhe, Z.-X. He and S.-F. Zhou, Drug Des., Dev. Ther., 2014, 8, 2075 CAS; (f) R. D. Couch, N. J. Ganem, M. Zhou, V. M. Popov, T. Honda, T. D. Veenstra, M. B. Sporn and A. C. Anderson, Mol. Pharmacol., 2006, 69, 1158 CrossRef CAS PubMed.
  3. K. T. Liby, D. B. Royce, R. Risingsong, C. R. Williams, A. Maitra, R. H. Hruban and M. B. Sporn, Cancer Prev. Res., 2010, 3, 1427 CrossRef CAS PubMed.
  4. K. Liby, D. B. Royce, C. R. Williams, R. Risingsong, M. M. Yore, T. Honda, G. W. Gribble, E. Dmitrovsky, T. A. Sporn and M. B. Sporn, Cancer Res., 2007, 67, 2414 CrossRef CAS PubMed.
  5. (a) C. To, C. S. Ringelberg, D. B. Royce, C. R. Williams, R. Risingsong, M. B. Sporn and K. T. Liby, Carcinogenesis, 2015, 36, 769 CrossRef CAS PubMed; (b) M. S. Ball, E. P. Shipman, H. Kim, K. T. Liby and P. A. Pioli, PLoS One, 2016, 11, e0149600 Search PubMed; (c) A. Refaat, C. Pararasa, M. Arif, J. E. P. Brown, A. Carmichael, S. S. Ali, H. Sakurai and H. R. Griffiths, Free Radical Res., 2017, 51, 211 CrossRef CAS PubMed.
  6. (a) C. H. L. Dinh, Y. Yu, A. Szabo, Q. Zhang, P. Zhang and X.-F. Huang, J. Histochem. Cytochem., 2016, 64, 237 CrossRef CAS PubMed; (b) L. R. Fitzpatrick, E. Stonesifer, J. S. Small and K. T. Liby, Inflammopharmacology, 2014, 22, 341 CrossRef CAS PubMed.
  7. D. S. Hong, R. Kurzrock, J. G. Supko, X. He, A. Naing, J. Wheler, D. Lawrence, J. P. Eder, C. J. Meyer, D. A. Ferguson, J. Mier, M. Konopleva, S. Konoplev, M. Andreeff, D. Kufe, H. Lazarus, G. I. Shapiro and B. J. Dezube, Clin. Cancer Res., 2012, 18, 3396 CrossRef CAS PubMed.
  8. (a) P. E. Pergola, P. Raskin, R. D. Toto, C. J. Meyer, J. W. Huff, E. B. Grossman, M. Krauth, S. Ruiz, P. Audhya, H. Christ-Schmidt, J. Wittes and D. G. Warnock, N. Engl. J. Med., 2011, 365, 327 CrossRef CAS PubMed; (b) D. S. Hong, R. Kurzrock, J. G. Supko, X. He, A. Naing, J. Wheler, D. Lawrence, J. P. Eder, C. J. Meyer, D. A. Ferguson, J. Mier, M. Konopleva, S. Konoplev, M. Andreeff, D. Kufe, H. Lazarus, G. I. Shapiro and B. J. Dezube, Clin. Cancer Res., 2012, 18, 3396 CrossRef CAS PubMed.
  9. (a) H. J. L. Heerspink, G. M. Chertow, T. Akizawa, P. Audhya, G. L. Bakris, A. Goldsberry, M. Krauth, P. Linde, J. J. McMurray, C. J. Meyer, H.-H. Parving, G. Remuzzi, H. Christ-Schmidt, R. D. Toto, N. D. Vaziri, C. Wanner, J. Wittes, D. Wrolstad and D. de Zeeuw, Nephrol., Dial., Transplant., 2013, 28, 2841 CrossRef PubMed; (b) D. Camer and X.-F. Huang, Am. J. Nephrol., 2014, 40, 288 CrossRef CAS PubMed; (c) D. de Zeeuw, T. Akizawa, P. Audhya, G. L. Bakris, M. Chin, H. Christ-Schmidt, A. Goldsberry, M. Houser, M. Krauth, H. J. L. Heerspink, J. J. McMurray, C. J. Meyer, H.-H. Parving, G. Remuzzi, R. D. Toto, N. D. Vaziri, C. Wanner, J. Wittes, D. Wrolstad and G. M. Chertow, N. Engl. J. Med., 2013, 369, 2492 CrossRef CAS PubMed; (d) C. Camer and X.-F. Huang, J. Card. Failure, 2015, 21, 258 CrossRef PubMed.
  10. (a) D. J. Graber, P. J. Park, W. F. Hickey and B. T. Harris, J. Neuroimmune Pharmacol., 2011, 6, 107 CrossRef PubMed; (b) C. Stack, D. Ho, E. Wille, N. Y. Calingasan, C. Williams, K. Liby, M. Sporn, M. Dumont and M. F. Beal, Free Radicals Biol. Med., 2010, 49, 147 CrossRef CAS PubMed; (c) A. Neymotin, N. Y. Calingasan, E. Wille, N. Naseri, S. Petri, M. Damiano, K. T. Liby, R. Risingsong, M. Sporn, M. F. Beal and M. Kiaei, Free Radicals Biol. Med., 2011, 51, 88 CrossRef CAS PubMed; (d) T. K. Pareek, A. Belkadi, S. Kesavapany, A. Zaremba, S. L. Loh, L. Bai, M. L. Cohen, C. Meyer, K. T. Liby, R. H. Miller, M. B. Sporn and J. J. Letterio, Sci. Rep., 2011, 1, 201 CrossRef PubMed; (e) N. A. Kaidery, R. Banerjee, L. Yang, N. A. Smirnova, D. M. Hushpulian, K. T. Liby, C. R. Williams, M. Yamamoto, T. W. Kensler, R. R. Ratan, M. B. Sporn, M. F. Beal, I. G. Gazaryan and B. Thomas, Antioxid. Redox Signaling, 2013, 18, 139 CrossRef CAS PubMed.
  11. (a) A. Wahhab, M. Ottosen and F. W. Bachelor, Can. J. Chem., 1991, 69, 570 CrossRef CAS; (b) L. Cao, J. Sun, X. Wang, R. Zhu, H. Shi and Y. Hu, Tetrahedron, 2007, 63, 5036 CrossRef CAS; (c) A. Roy, F. G. Roberts, P. R. Wilderman, K. Zhou, R. J. Peters and R. M. Coates, J. Am. Chem. Soc., 2007, 129, 12453 CrossRef CAS PubMed.
  12. For an early use of DBDMH to effect bromination/dehydrobromination, see: (a) J. Redel, C. Rebut-Bonneton and F. Delbarre, J. Steroid Biochem., 1978, 9, 1179 CrossRef CAS PubMed; (b) For a review, see: S. C. Virgil, e-EROS Encyclopedia of Reagents for Organic Synthesis, 2001, p. 1 Search PubMed.
  13. R. D. Couch, R. G. Browning, T. Honda, G. W. Gribble, D. L. Wright, M. B. Sporn and A. C. Anderson, Bioorg. Med. Chem. Lett., 2005, 15, 2215 CrossRef CAS PubMed.
  14. M. B. Sporn and K. T. Liby, Nat. Rev. Cancer, 2012, 12, 564 CrossRef CAS PubMed.


Electronic supplementary information (ESI) available. See DOI: 10.1039/c7ob01420a
Present address: Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824, United States.

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