Somatostatin-14 (somatostatin) and its clinically available analogues octreotide, lanreotide, and vapreotide are potent inhibitors of growth hormone, insulin, and glucagon release. Recently, a novel backbone cyclic somatostatin analogue c(GABA-Phe-Trp-(D)Trp-Lys-Thr-Phe-GlyC3-NH(2)) (analogue 1, PTR 3173) that possesses in vivo endocrine selectivity was described. This long-acting octapeptide exhibits high affinity to human recombinant somatostatin receptors (hsst) hsst2, hsst4, and hsst5. Its novel binding profile resulted in potent in vivo inhibition of growth hormone but not of insulin release. We report the synthesis, bioactivity, and structure-activity relationship studies of compounds related to 1. In these analogues, the lactam bridge of 1 was replaced by a backbone disulfide bridge. We present a novel approach for conformational constraint of peptides by utilizing sulfur-containing building units for on-resin backbone cyclization. These disulfide backbone cyclic analogues of 1 showed significant metabolic stability as tested in various enzyme mixtures. Receptor binding assays revealed different receptor selectivity profiles for these analogues in comparison to their prototype. It was found that analogues of 1, bearing a disulfide bridge, had increased selectivity to hsst2 and hsst5; however, they exhibited weaker affinity to hsst4 as compared to 1. These studies imply that ring chemistry, ring size, and ring position of the peptide template may affect the receptor binding selectivity.
General methods for the preparation of protected Nalpha(omega-thioalkyl) amino acids building units for backbone cyclization using reductive alkylation and on-resin preparation are described. The synthesis of non-Gly Fmoc-protected S-functionalized N-alkylated amino acids is based on the reaction of readily prepared protected omega-thio aldehyde with the appropriate amino acid. Preparation of Fmoc-protected S-functionalized N-alkylated Gly building units was carried out using two methods: reaction of glyoxylic acid with Acm-thioalkylamine and an on-resin reaction of bromoacetyl resin with Trt-thioalkylamines. Three model peptides were prepared using these building units. The GlyS2 building unit was incorporated into a backbone cyclic analog of somatostatin that contains a disulfide bridge. Formation of the disulfide bridge was performed by on-resin oxidation using 12 or Tl(CF3COO-)3. Both methods resulted in the desired product in a high degree of purity in the crude. The AspS3 building unit was also successfully incorporated into a model peptide. In addition, the in situ generation of sulfur containing Gly building units was demonstrated on a Substance P backbone cyclic analog containing a thioether bridge.
We present a new approach for the conversion of active sequences of proteins and peptides into small molecules. A library of macrocyclic disulfide molecules was made, in which the active pharmacophores of the parent peptide are preserved while the size of the macromolecular scaffold on which the pharmacophores are arranged is varied. This enables a systematic search for macromolecules in which the pharmacophores are in an appropriate conformation for biological activity. We developed two procedures for the synthesis of such libraries from building blocks that include commercial amino acids and functionalized aldehydes. Chemical synthesis using the "tea-bag" method gave a library with higher diversity, but low yields, compared to the manual synthesis of the library, in which the compounds were synthesized in individual vessels and the yield and purity improved dramatically. As a proof of concept, we synthesized a 34-member library derived from the sequence of the activation loop of insulin-like growth factor-1 receptor. Selected compounds were screened, and one was found to be biologically active in the low micromolar range. The concept presented here may prove particularly useful in cases where the pharmacophores are known but need to be systematically screened for a spatial arrangement that will enable biological activity.
A cyclic somatostatin analog [structure: see text] (1) has been synthesized. Biological assays show that this compound has strong binding affinities to somatostatin hsst2 and hsst5 receptor subtypes (5.2 and 1.2 nM, respectively, and modest affinity to hsst4 (41.1 nM)). Our conformational analysis carried out in DMSO-d6 indicates that this compound exists as two structures arising from the trans and cis configurations of the peptide bond between Phe7 and N-alkylated Gly8. However, neither conformer exhibits a type II' beta-turn. This is the first report of a potent bioactive somatostatin analog that does not exhibit a type II' beta-turn in solution. Molecular dynamics simulations (500 ps) carried out at 300 K indicate that the backbone of compound 1 is more flexible than other cyclic somatostatin analogs formed by disulfide bonds.
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