(CANCER RESEARCH 58. 717-723. February 15. 1998) Novel Triterpenoids Suppress Inducible Nitric Oxide Synthase (iNOS) and Inducible Cyclooxygenäse(COX-2) in Mouse Macrophages1 Nanjoo Suh, Tadashi Honda, Heather J. Finlay, Aaron Barchowsky, Charlotte Williams, Nicole E. Benoit, Qiao-wen Xie, Carl Nathan, Gordon W. Gribble, and Michael B. Sporn2 Department of Pharmacology and Norris Cotton Cancer Center, Dartmouth Medical School [N. S.. A. B., C. W.. N. E. B., M. B. SJ and Department of Chemistry. Dartmouth College, Hanover, New Hampshire 03755 ¡T.H., H. J. F., G. Vi.G.], and Department of Médiane, Cornell University Medical College, New York, New York 10021 ¡Q-w.X., C.N.J ABSTRACT We have synthesized more than 80 novel triterpenoids, all derivatives of oleanolic and ursolic acid, as potential anti-inflammatory and chemopre- ventive agents. These triterpenoids have been tested for their ability to suppress the de novo formation of two enzymes, inducible nitric oxide synthase (¡NOS) and inducible cyclooxygenase (COX-2), using IFN-y- stimulated primary mouse macrophages or lipopolysaccharide (1,1'Si- activated RAW 264.7 macrophages as assay systems. Two synthetic oleananes, 3,12-dioxoolean-l-en-28-oic acid (TP-69) and 3,11-dioxoolean- l,12-dien-28-oic acid (TP-72), were highly active inhibitors of de novo formation of both ¡NOSand COX-2. Both TP-69 and TP-72 blocked the increase in iNOS or COX-2 mRNA induced by IFN-y or LPS. In addition, TP-72 suppressed NT-Kit activation in primary macrophages treated with the combination of II N-y and LPS or II N-y and tumor necrosis factor. The 3-«(axial)-epimer of ursolic acid suppressed de novo formation of COX-2, in contrast to naturally occurring 3-ß( equatorial )-ursolic acid. Inhibitory effects of TP-69 or TP-72 on ¡NOSformation were not blocked by the glucocorticoid receptor antagonist RU-486, indicating that these triterpenoids do not act through the glucocorticoid receptor, nor does TP-72 act as an ¡NOSor COX-2 enzyme inhibitor when added to RAW cells in which synthesis of these two enzymes in response to LPS has already been induced. It may be possible to develop triterpenoids as useful agents for chemoprevention of cancer or other chronic diseases with an inflammatory component. INTRODUCTION One of the major needs in cancer prevention is the development of effective and safe new agents for chemoprevention. In particular, there is a unique need for chemopreventive agents targeted at mech anisms known to be involved in the process of carcinogenesis ( 1). In recent years, there has been a resurgence of interest in the study of mechanisms of inflammation that relate to carcinogenesis and in the use of such mechanisms as the basis for development of new chemo preventive agents. The concept that inflammation and carcinogenesis are related phe nomena has been the subject of many previous studies that have attempted to link these two processes in a mechanistic fashion (2-4). The enzymes that mediate the constitutive synthesis of NO and prostaglandins from arginine and arachidonate, respectively, have relatively little significance for either inflammation or carcinogenesis. In contrast, iNOS3 (EC 1.14.13.39) and inducible cyclooxygenase (COX-2; EC 1.14.99.1) have critical roles in the response of tissues to Received 10/14/97: accepted 12/19/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This research was supported by Morris Cotton Cancer Center Grant CA-23108. NIH Grant ROI AI 34543. the Oliver S. and Jennie R. Donaldson Charitable Trust, and DOD/AMRD Award 17-96-1-6163. - To whom requests for reprints should be addressed, at Department of Pharmacology and Toxicology. Dartmouth Medical School. 7650 Remsen, Hanover, NH 03755. Phone: (603)650-6557: Fax: (603)650-1129; E-mail: Michael.Sporn@danmouth.edu. 1The abbreviations used are: iNOS, inducible nitric oxide synthase; TGF. transform ing growth factor; COX. cyclooxygenase; OA. oleanolic acid; UA. ursolic acid; PG. prostaglandin; LPS. lipopolysaccharide; TNF, tumor necrosis factor; NF, nuclear factor. injury or infectious agents. These inducible enzymes are essential components of the inflammatory response, the ultimate repair of injury, and carcinogenesis (5-10). Although physiological activity of iNOS and COX-2 may provide a definite benefit to the organism, aberrant or excessive expression of either iNOS or COX-2 has been implicated in the pathogenesis of many disease processes, as diverse as septic shock, cardiomyopathy, acute and chronic neurodegenerative disease, rheumatoid arthritis, and carcinogenesis (11-19). Immense effort has been devoted to developing new molecules that are direct inhibitors of the enzymatic activity of either iNOS or COX-2. However, an alternative approach is to find new agents that can prevent expression of the respective genes coding for these enzymes. Glucocorticoids and TGF-ßare such molecules; they both suppress transcription or translation of iNOS and COX-2 genes (20- 25). A rationale thus exists to develop more selective agents for suppression of genes that might be overexpressed during the inflam matory or carcinogenic process. In this report, we attempt to apply this strategy for the development and evaluation of new triterpenoids. Although triterpenoids are widely used for medicinal purposes in many Asian countries, this class of molecules, which resemble ste roids in their chemical structure, biogenesis, and pleiotropic actions, has not impacted on the practice of Western medicine. Triterpenoids, like the steroids, are formed in nature by the cyclization of squalene, with the retention of all 30 carbon atoms in molecules such as OA and UA (Fig. 1). Although OA and UA are known to have numerous pharmacological activities, the potency of these naturally occurring molecules is relatively weak. Chemical synthesis of new steroid analogues has provided many useful derivatives that are more potent and specific than natural parent structures. With this as a model and considering the known anti-inflammatory and anticarcinogenic activ ities of OA and UA (26-29), we have synthesized (30) and charac terized a new series of synthetic triterpenoid analogues as potential inhibitors of inflammation and carcinogenesis using suppression of the formation of nitric oxide and prostaglandins as assay systems. We report here the structures and activities of three of the most potent of these derivatives of OA and UA. These new agents are significantly more active than their parent molecules. MATERIALS AND METHODS Reagents Details of the synthesis of TP-69 and TP-72 (see Fig. I for structures) have been published (30). TP-52 (3-a-OH UA) was synthesized by Jones oxidation of UA. followed by Meenvein-Ponndorf reduction. Recombinant mouse IFN-y (LPS content. <10 pg/ml) was purchased from Genzyme (Cambridge, MA); NF-«B oligonucleotide was purchased from Promega Corp. (Madison, WI); goat polyclonal COX-1. COX-2 IgG. and anti-goat IgG peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); TGF-ß, was from R&D (Minneapolis. MN); and enzyme immu- noassay reagents for PGE2 assays were from Cayman Co. (Ann Arbor. MI). TNF-a was provided by Dr. Jan Vilcek (New York University Medical Center, New York, NY). LPS (from Escherichia coli 0111:B4 y-irradiated) and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). 717 Research. on February 9, 2016. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from