Synthesis of Nitro Esters of Prednisolone, New Compounds Combining Pharmacological Properties of Both Glucocorticoids and Nitric Oxide Pier Giovanni Baraldi,* ,² Romeo Romagnoli, ² Maria del Carmen Nun˜ ez, ² Mauro Perretti, § Mark J. Paul-Clark, § Massimiliano Ferrario, ‡ Mirco Govoni, ‡ Francesca Benedini, ‡ and Ennio Ongini ‡ Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara, 44100 Ferrara, Italy, Nicox Research Institute, Via Ariosto 21, 20091 Bresso, Milan, Italy, and The William Harvey Research Institute, Pharmacology Division of St. Bartholomew’s & The Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, United Kingdom Received July 21, 2003 Glucocorticoids (GC) are widely used in therapy for their many pharmacological properties including antiinflammatory and immunosuppressive actions. However, their use over long periods is hampered by a number of severe side effects. Given the biological properties of nitric oxide (NO) and previous experience with nonsteroidal antiinflammatory agents, we synthesized new chemical entities combining both NO and GC properties. Here we report the synthesis of nitro esters of prednisolone obtained through the esterification, with different linkers, on the hydroxy group at C-21 position of the corticosteroid structure. The alkyl chain, as of the nitrooxy derivative (2), or aromatic linkers, as of o-, m-, and p-nitrooxymethylbenzoate derivatives (3- 5), respectively, furnish stable compounds that release NO and inhibit the GC receptors in biological assays. To improve solubility we introduced a more water-soluble linker such as the nitrooxyalkylpiperidine or -piperazine group (6-9). Also these compounds retained properties of both NO and prednisolone. Compound 5 (NCX 1015) was selected for its better profile: enhanced antiinflammatory properties and reduced side effects compared with prednisolone. NCX 1015 is currently under preclinical development. Introduction Glucocorticoids (GC) have been used successfully for more than 50 years for their potent antiinflammatory and immunosuppressive action. Use of GC in therapy is limited by their side effects, which depend on the dose used and duration of treatment. The continued use of GC leads to an array of unwanted effects ranging from alteration of the endocrine homeostasis to a number of complications such as hypertension, hyperglycemia, fluid and electrolyte abnormalities, osteoporosis, gastro- intestinal damage, increased susceptibility to infection, etc. 1,2 Based on the knowledge of the mechanism of action of the GC, a medicinal chemistry effort was done in order to identify new compounds possessing potent antiinflammatory properties and reduced complications. Specifically, GC bind to a specific member of the nuclear receptor superfamily, the glucocorticoid receptor (GR), which has been cloned and more recently the crystal structure has been determined. 3 Upon ligand binding, the GR translocates to the nucleus where it interacts with specific DNA sequences. The net result is a regulation of gene transcription, through either activa- tion or repression mechanisms. Currently, it is believed a better drug profile can be achieved through a degree of dissociation between transactivation and trans- repression, for example, strong transrepression/low transactivation. Recent reviews illustrate the ongoing effort toward the realization of more selective gluco- corticoid-related therapeutic agents. 2,4 Prompted by the successful results with nitric oxide- (NO-) releasing nonsteroidal antiinflammatory agents (NSAIDs), 5-7 we identified a strong rationale to apply the same synthetic approach to glucocorticoids. More than a decade of intense research has demonstrated that NO exerts antiinflammatory effects, has cytoprotective actions on gastric mucosa, and is relevant to bone metabolism, to name a few of its biological actions. 8,9 Thus, we introduced a NO-releasing moiety into the GC structure so as to have new chemical entities that could release NO slowly over time, the first step being the ester bond cleavage. Structural changes were introduced in position 21 of the GC so as to avoid interference with molecular recognition of the GC. Here, we describe a group of NO-releasing derivatives of prednisolone (1) that appear of interest as a new class of improved GC-related agents. On the basis of previous experience with NO-releasing NSAIDs, we started with the introduction of the nitro- oxybutyryl (2) and the isomers of nitrooxymethylbenzoyl groups (3-5) into the 21 position of prednisolone. The compounds displayed inhibitory activity on GR, there- fore showing that they retain GC-related effects. As a further step, with the aim to improve water solubility, we synthesized the compounds 6-9 by linking the nitrooxy (-ONO 2 ) moiety to C-21 of prednisolone through heterocycles such as piperidine (6 and 7) and piperazine (8 and 9). To determine the effect of the nitrooxymethyl * To whom correspondence should be addressed at the Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy. Tel +39-0532-291293; fax +39- 0532-291296; e-mail pgb@ifeuniv.unife.it. ² Universita` di Ferrara. ‡ Nicox Research Institute. § The William Harvey Research Institute, Pharmacology Division of St. Bartholomew’s & The Royal London School of Medicine and Dentistry. 711 J. Med. Chem. 2004, 47, 711-719 10.1021/jm030964b CCC: $27.50 © 2004 American Chemical Society Published on Web 12/20/2003