DOI: 10.1002/cmdc.201402075 Development of Novel Selective Peptidomimetics Containing a Boronic Acid Moiety, Targeting the 20S Proteasome as Anticancer Agents Kety Scarbaci, [b] Valeria Troiano, [b] Roberta Ettari,* [a] Andrea Pinto, [a] Nicola Micale, [b] Carmen Di Giovanni, [c] Carmen Cerchia, [c] Tanja Schirmeister, [d] Ettore Novellino, [c] Antonio Lavecchia,* [c] Maria Zappalà, [b] and Silvana Grasso [e] Introduction The 20S proteasome is an essential component of the adeno- sine triphosphate-dependent proteolytic pathway in eukaryotic cells, and it plays a key role in the degradation of most cellular proteins. It has a barrel-like structure and is composed of four stacked rings, each of which contains seven subunits. Whereas the two inner rings contain seven b subunits (b1–b7), the two outer rings are characterized by a subunits (a1–a7). [1] The cata- lytic activities of the proteasome are classified into three major categories: chymotrypsin-like (ChT-L), trypsin-like (T-L), and post glutamyl peptide hydrolase or caspase-like (PGPH or C-L), associated with the b5, b2, and b1 subunits that cleave after hydrophobic, basic, and acidic residues, respectively. [2] Defects in proteasome activity can induce tumor development owing to anarchic cell proliferation, and thus a promising strategy for cancer treatment is the inhibition ChT-L activity. [3] Recent stud- ies clearly point out that an efficient reduction in abnormal proteasome activity can be achieved only by the simultaneous inhibition of ChT-L activity, together with a second active site, [4] whereas inhibition of all three catalytic subunits is strongly cy- totoxic. Over the last years our research group has been involved in the development of peptidomimetic 20S proteasome inhibi- tors, [5, 6] structurally related to bortezomib (Velcade, Figure 1), the first proteasome inhibitor approved by the US Food and Drug Administration (FDA) for the treatment of multiple mye- loma [7] and mantle cell lymphoma. [8] Although bortezomib has been shown to provide significant benefits to patients with multiple myeloma in clinical trials, its efficacy and its adminis- tration have been limited by toxic side effects, [7] the most severe ones include hematological toxicity and peripheral neu- ropathy, characterized by decreased sensation, paresthesia, and a high rate of shingles. As a matter of fact, bortezomib is able to activate the mitochondrial-based apoptotic pathway, in which mitochondrial and endoplasmic reticulum damage may play a key role in bortezomib-induced side effects. Taking into account all of these problems, the development of peptidomimetics could present many pharmacokinetic and pharmacodynamic advantages, as also described in the case of selective peptidomimetic 20S proteasome inhibitors. [9] This strategy can be accomplished, for example, by simple replace- This paper describes the design, synthesis, and biological eval- uation of peptidomimetic boronates as inhibitors of the 20S proteasome, a validated target in the treatment of multiple myeloma. The synthesized compounds showed a good inhibi- tory profile against the ChT-L activity of 20S proteasome. Com- pounds bearing a b-alanine residue at the P2 position were the most active, that is, 3-ethylphenylamino and 4-methoxy- phenylamino (R)-1-{3-[4-(substituted)-2-oxopyridin-1(2H)-yl]pro- panamido}-3-methylbutylboronic acids (3c and 3d, respective- ly), and these derivatives showed inhibition constants (K i ) of 17 and 20 nm, respectively. In addition, they co-inhibited post glu- tamyl peptide hydrolase activity (3c, K i = 2.57 mm ; 3d, K i = 3.81 mm). No inhibition was recorded against the bovine pan- creatic a-chymotrypsin, which thus confirms the selectivity to- wards the target enzyme. Docking studies of 3c and related in- hibitors into the yeast proteasome revealed the structural basis for specificity. The evaluation of growth inhibitory effects against 60 human tumor cell lines was performed at the US National Cancer Institute. Among the selected compounds, 3c showed 50 % growth inhibition (GI 50 ) values at the sub-micro- molar level on all cell lines. [a] Dr. R. Ettari, Dr. A. Pinto Dipartimento di Scienze del Farmaco e Prodotti per la Salute Università degli Studi di Messina, Viale Annunziata, 98168 Messina (Italy) E-mail : roberta.ettari@unimi.it [b] Dr. K. Scarbaci, Dr. V. Troiano, Dr. N. Micale, Prof. M. Zappalà Dipartimento di Scienze Farmaceutiche Università degli Studi di Milano, Via Mangiagalli, 25, 20133 Milano (Italy) [c] Dr. C. Di Giovanni, Dr. C. Cerchia, Prof. E. Novellino, Prof. A. Lavecchia Drug Discovery Laboratory, Dipartimento di Farmacia Università degli Studi di Napoli, Via Montesano 49, 80131 Napoli (Italy) E-mail : antonio.lavecchia@unina.it [d] Prof. Dr. T. Schirmeister Institut für Pharmazie & Biochemie, Johannes Gutenberg Universität Mainz Staudinger Weg 5, 55099 Mainz (Germany) [e] Prof. S. Grasso Dipartimento di Scienze Chimiche, Università degli Studi di Messina Via F. Stagno D’Alcontres 31, S. Agata, 98166 Messina (Italy) # 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 2014, 9, 1801 – 1816 1801 CHEMMEDCHEM FULL PAPERS