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Kappa opioid receptor (KOR) antagonists have recently shown potential for treating drug addiction and mood disorders. The linear acetylated dynorphin A analog arodyn (Ac[Phe1,2,3,Arg4,d-Ala8]dynorphin A-(1-11)NH2), synthesized in our laboratory, demonstrated potent and selective KOR antagonism. Cyclization of arodyn could potentially stabilize the bioactive conformation and enhance its metabolic stability. The cyclization strategy employed involved ring closing metathesis between adjacent meta- or para-substituted Tyr(allyl) residues in the "message" sequence that were predicted in a docking study to yield analogs that would bind to the KOR with binding poses similar to arodyn. Consistent with the modeling, the resulting analogs retained KOR affinity similar to arodyn; the peptides involving cyclization between para O-allyl groups also retained high KOR selectivity, with one analog exhibiting KOR antagonist potency (KB = 15 nM) similar to arodyn. These promising cyclized analogs with constrained aromatic residues represent novel leads for further exploration of KOR pharmacology.

We are exploring constraining aromatic residues in the kappa opioid receptor selective antagonist arodyn (Ac[Phe(1,2,3),Arg(4),D-Ala(8)]dynorphin A(1–11)-NH(2)) by ring closing metathesis (RCM) involving tyrosine(O-allyl) (Tyr(All)), but desallyl products limited the yields of the desired cyclic peptide. The model dipeptide Fmoc-Tyr(All)-Tyr(All) was used to explore different reaction conditions, including the use of isomerization suppressants, to minimize formation of the desallyl products and enhance formation of the desired RCM product. Reaction conditions were identified that enhanced the RCM product yield while suppressing desallyl products using both second-generation Grubbs and second-generation Hoveyda Grubbs catalysts. These optimized reaction conditions were then applied to the cyclization of a tripeptide and an arodyn analog resulting in ≥70% conversion to the desired cyclic peptides. These strategies should be applicable to RCM involving Tyr(All) and similar residues in peptide and peptidomimetic cyclizations performed on solid phase.

The oncoprotein c-Myc is often overexpressed in cancer cells, and the stability of this protein has major significance in deciding the fate of a cell. Thus, targeting c-Myc levels is an attractive approach for developing therapeutic agents for cancer treatment. In this study, we report the anti-cancer activity of the macrocyclic peptides [D-Trp]CJ-15,208 (cyclo[Phe-D-Pro-Phe-D-Trp]) and the natural product CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]). [D-Trp]CJ-15,208 reduced c-Myc protein levels in prostate cancer cells and decreased cell proliferation with IC50 values ranging from 2.0 to 16 µM in multiple PC cell lines. [D-Trp]CJ-15,208 induced early and late apoptosis in PC-3 cells following 48 hours treatment, and growth arrest in the G2 cell cycle phase following both 24 and 48 hours treatment. Down regulation of c-Myc in PC-3 cells resulted in loss of sensitivity to [D-Trp]CJ-15,208 treatment, while overexpression of c-Myc in HEK-293 cells imparted sensitivity of these cells to [D-Trp]CJ-15,208 treatment. This macrocyclic tetrapeptide also regulated PP2A by reducing the levels of its phosphorylated form which regulates the stability of cellular c-Myc protein. Thus [D-Trp]CJ-15,208 represents a new lead compound for the potential development of an effective treatment of prostate cancer.

Kappa (κ) opioid receptor (KOR) ligands, particularly antagonists, have received increasing attention due to their therapeutic potential in stress related conditions [1]. The acetylated dynorphin A (Dyn A) analog arodyn (Ac[Phe,Arg,D-Ala]Dyn A(1-11)-NH2, Figure 1), shows potent KOR antagonism in vitro. Arodyn has high affinity (Ki = 10 nM) and selectivity (Ki ratio (κ/μ/δ) = 1/174/583) for KOR [2]. Clinical development of KOR antagonists has been hampered partly because prototypical small molecule KOR antagonists such as JDTic exhibit long-lasting antagonism, lasting for weeks after a single dose [1]. Conversely, linear peptide KOR antagonists such as arodyn are rapidly metabolized and inactivated. Conformational constraint of the flexible linear peptide by ring closing metathesis (RCM) cyclization was expected to enhance its metabolic stability and potentially stabilize the bioactive conformation. Aromatic residues in arodyn contribute to affinity at KOR [4]; we are exploring RCM cyclization employing Tyr(All) residues which retain the aromatic nature of the N-terminal region. In contrast to high yields observed in RCM using allylglycine in Dyn A analogs [5], a mixture of side products (Figure 2) compromised the RCM product yield of arodyn analogs containing Tyr(All) [4]. Olefin isomerization due to degradation products of the Grubbs catalyst could lead to the observed side products [6]. A model dipeptide, Fmoc-Tyr(All)Tyr(All), was employed to explore reaction conditions to enhance yields of the cyclic RCM product, conditions which were subsequently applied to arodyn analogs. Here, we present the results of different strategies to minimize the side reaction, including examination of reagents reported to suppress isomerization [6,7] as well as microwave heating.