Quinone-related hexacyclic by-products in the production process of exemestane Giovanni Battista Giovenzana a,⇑ , Norberto Masciocchi b , Roberto Negri a , Giovanni Palmisano b , Andrea Penoni b , Lucio Toma c a Università del Piemonte Orientale ‘‘A. Avogadro”, Dipartimento di Scienze del Farmaco, Largo Donegani 2, I-28100 Novara, Italy b Università degli Studi dell’Insubria, Dipartimento di Scienza e Alta Tecnologia, Via Valleggio 11, I-22100 Como, Italy c Università degli Studi di Pavia, Dipartimento di Chimica, Via Taramelli 12, I-27100 Pavia, Italy article info Article history: Received 8 September 2016 Received in revised form 5 January 2017 Accepted 18 January 2017 Available online xxxx Keywords: Exemestane p-Chloranil DDQ Cycloaddition abstract Exemestane, a 3rd-generation aromatase inhibitor, is clinically used in the treatment of breast cancer in postmenopausal women. The key step of the industrial synthetic process, i.e., a dehydrogenation to intro- duce the D 1 -unsaturation, is normally performed with quinones such as p-chloranil or DDQ. We observed the formation of two different hexacyclic by-products, depending on the quinone used in the oxidation step. These compounds arise from an initial [4+2] cycloaddition between the precursor 6-methylenan- drost-4-ene-3,17-dione and the quinone reagent, followed by a twofold dehydrohalogenation (with p- chloranil) or dehydrogenation (with DDQ). The structures of these unprecedented hexacyclic adducts were determined by a combination of mass spectrometry, NMR techniques and crystallographic analysis. Ó 2017 Elsevier Inc. All rights reserved. 1. Introduction Estradiol, the most potent endogenous estrogen, is biosynthe- sised from androgens by the P450 complex called ‘‘aromatase” [1]. The inhibition of this enzyme, blocking the estrogenic biosyn- thesis, has been demonstrated to reduce postmenopausal breast cancer. 6-Methylenandrosta-1,4-diene-3,17-dione (1) (INNS exemestane; Aromasin Ò )(Scheme 1) is a suicide inhibitor of aro- matase, acting as a structurally analogue of the natural androgenic substrate, i.e.: androstenedione [2]. Exemestane binds irreversibly to the enzyme leading to its inactivation, and it is currently in clin- ical use for the treatment of postmenopausal women with early or advanced breast cancer [3]. The interest of pharmaceutical industries in 1 prompted an intensive research activity devoted to identify efficient synthetic processes, most of them retrievable in the patent literature. Several steroidal precursors containing the skeleton of interest were identified as suitable starting materials, among them: 17a-acetoxy-6-methyleneprogesterone [4], dihydrotestosterone [5], androsta-1,4-diene-3,17-dione [6,7], or androst-4-ene-3,17- dione [8–20]. These syntheses must cope with the introduction of the exo-methylene moiety in position 6 [21], key functional group for the biological activity, and with the optional adjustment of the unsaturation degree of ring A. In particular, the introduction of the D 1 -unsaturation has been widely investigated, resulting in a massive patenting of experimental conditions for this specific transformation. The dehydrogenation reaction is usually performed by powerfully oxidising quinones (i.e., DDQ [8–12], p-chloranil [13–15]), but processes using SeO 2 [16] as well as IBX [17] as oxidants or bromination/dehydrobromination sequence [18] have been developed, too. Alternative processes recruit microorganisms, as Nocardioides simplex VKM As-2033D [19] or Arthrobacter simplex [20], endowing specific enzymes for this oxidative transformation. One of the most convenient synthetic processes for the preparation of 1, starting from androst-4-ene-3,17-dione, is reported in Scheme 1. Androst-4-ene-3,17-dione 2 is first treated with triethyl orthoformate in acid conditions, giving the corre- sponding D 3,5 dienolether 3. Treatment with paraformaldehyde and N-methylaniline allows to introduce the exo-methylene group through a combined Mannich/elimination sequence, leading to 6-methylenandrost-4-ene-3,17-dione 4 [21]. The final step of the synthesis is the selective introduction of the D 1 -unsaturation and, as previously highlighted, preferably per- formed in industrial processes with the oxidising quinones DDQ or p-chloranil. During a screening of experimental conditions for this oxidation, we routinely observed the formation of two different by-products, strictly related to the nature of the quinone employed. The amount of these by-products ranged from negligi- ble to significant potentially affecting either the yield or the quality http://dx.doi.org/10.1016/j.steroids.2017.01.007 0039-128X/Ó 2017 Elsevier Inc. All rights reserved. ⇑ Corresponding author. E-mail address: giovannibattista.giovenzana@uniupo.it (G.B. Giovenzana). Steroids xxx (2017) xxx–xxx Contents lists available at ScienceDirect Steroids journal homepage: www.elsevier.com/locate/steroids Please cite this article in press as: G.B. Giovenzana et al., Quinone-related hexacyclic by-products in the production process of exemestane, Steroids (2017), http://dx.doi.org/10.1016/j.steroids.2017.01.007