Drug Selectivity DOI: 10.1002/anie.201102971 Increasing avb3 Selectivity of the Anti-Angiogenic Drug Cilengitide by N-Methylation** Carlos Mas-Moruno, Johannes G. Beck, Lucas Doedens, Andreas O. Frank, Luciana Marinelli, Sandro Cosconati, Ettore Novellino, and Horst Kessler* The drug Cilengitide, c(RGDf(NMe)V), is a cyclic RGD pentapeptide (R = arginine, D = aspartic acid, G = glycine) currently in clinical phase III for the treatment of brain tumors and in phase II for other cancer types. [1] The anti- tumoral properties of this peptide are based on its antago- nistic activity for pro-angiogenic integrins, such as avb3, avb5, or a5b1. However, the specific roles of these integrin subtypes in angiogenesis and cancer are not yet clear and fully understood. In this work, we present di-N-methylated ana- logues of the stem peptide c(RGDfV) which retain an avb3- binding activity in the nanomolar range but have lost most of the activity for integrins avb5 and/or a5b1. Highly active and selective peptides for avb3 are important tools to study the specific role of this integrin in angiogenesis and cancer. Integrins are heterodimeric receptors that govern cell–cell and cell–extracellular matrix (ECM) interactions, and play crucial roles in a plethora of cellular functions. [2] The fact that many integrins are involved in pathological processes, such as tumor angiogenesis, has stimulated their study as therapeutic targets. [3] A number of integrin receptors recognize and bind the tripeptide sequence RGD, which is a prominent cell- adhesion motif present in ECM proteins. [4] Mimicking this tripeptide sequence with RGD-peptides or peptidomimetics is hence a promising approach to target integrins involved in angiogenesis and to develop anti-cancer agents. [1, 3b, 5] It is known that avb3 and avb5 are involved in two different angiogenic pathways. [6] Whereas angiogenesis induced by basic fibroblast growth factor (bFGF) or tumor necrosis factor-a depends on avb3, angiogenesis triggered by vascular endothelial growth factor (VEGF) or transforming growth factor-a is avb5-dependent. These two integrins are also described to be important mediators in the regulation of hypoxia in glioblastomas. [7] However, mice lacking either av or b3 and b5 integrins showed extensive angiogenesis. [8] These intriguing results were a matter of debate and challenged our understanding about the role of these two integrins in angiogenesis. [9] The integrin a5b1 is also highly expressed in angiogenic vasculature by several angiogenic stimuli, such as bFGF but not by VEGF. [10] Since avb3, avb5 and a5b1 have partially overlapping ligand affinities, [4b] it is plausible that a5b1 might substitute the pro-angiogenic activity of the other integrins. Paradoxically, another recent study showed that low concentrations of Cilengitide stimulates VEGF-mediated angiogenesis. [11] Although the doses used in this study are far lower than therapeutic concentrations [12] and hence such a “pro-angiogenic” effect is not likely to be observed in the clinical studies, it becomes evident that a better understand- ing of anti-angiogenic agents is necessary. [13] It has been shown by us and others that N-methylation can increase the selectivity towards specific receptor subtypes. [14] These biological effects are often caused by the induction of conformational constraints in the peptide backbone, which lead to preferred single conformers essential for biological activity. [14a,d,h, 15] Thus, we envisioned that further N-methyl- ation of Cilengitide could result in enhanced selectivity profiles. For this reason we designed a library containing all the di-N-methylated analogues of c(RGDfV) (Figure 1). Note that the synthesis of NMe peptides (especially if they are cyclic) is not without challenges that need to be carefully considered. [14a, 16] In the first place, although many N-methyl amino acids are commercially available, most of them are still expensive. Therefore, we synthesized, in solution, the NMe residues of Gly, Val, and d-Phe by reduction of the corresponding oxazolidinone using Freidinger conditions. [17] Alternatively, Arg and Asp were methylated on resin using the Miller and Scanlan method, [18] later optimized by Biron et al. , [19] which is compatible with acid-sensitive side-chain Figure 1. Schematic representation of our library of di-N-methylated analogues of c(RGDfV). [*] Dr. C. Mas-Moruno, J. G. Beck, Dr. L. Doedens, Dr. A. O. Frank, Prof. Dr. H. Kessler Institute for Advanced Study and Center of Integrated Protein Science, Department Chemie, Technische Universität München 85747 Garching (Germany) E-mail: kessler@tum.de Prof. Dr. H. Kessler Chemistry Department, Faculty of Science, King Abdulaziz University, 21589 Jeddah (Saudi Arabia) Prof. Dr. L. Marinelli, Dr. S. Cosconati, Prof. Dr. E. Novellino Dipartimento di Chimica Farmaceutica e Tossicologica, Universitµ di Napoli “Federico II”, 80131 Napoli (Italy) [**] This work was partially supported by the International Graduate School of Science and Engineering. We thank B. Cordes for technical assistance with mass spectroscopy. C.M.M. thanks the Generalitat de Catalunya for a Beatriu de Pinós postdoctoral fellowship. J.G.B thanks the TUM Graduate School for support. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201102971. Communications 9496  2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2011, 50, 9496 –9500