Boc-D-Pra-OH

D-alpha-Hydroxyacyl-prolyl-arginals, a new type of analogues of D-Phe-Pro-Arg-H (R1), have been prepared and evaluated. Unlike R1, whose terminal group is NH2, the new analogues with a terminal OH group are stable, as are the N-substituted derivatives of R1, that is, D-MePhe-Pro-Arg-H (R2), the highly potent and selective thrombin inhibitor, and Boc-D-Phe-Pro-Arg-H (R3), the much less favorable analogue. The most notable of the new analogues corresponds to the general formula D-Xaa-Pro-Arg-H, wherein Xaa means the acyl residue of mandelic acid (Man, 1), diphenyllactic acid (Dpl, 2), hexahydrophenyllactic acid (Hpl, 3), or hexahydromandelic acid (Hma, 4). In plasma clotting assays, 1 to 4 appeared to inhibit thrombin as well as some other clotting enzymes involved in thrombin generation, whereas R1 and R2 seemed to produce anticoagulation through inhibition of thrombin only. In the fibrin plate assay, 1 to 4 possessed even more moderate antifibrinolytic activities than R2. In in vivo evaluation in rats and rabbits, 2 to 4 proved to be potent anticoagulants/antithrombotics even on oral administration in a dose of 5 mg/kg. In view of these findings with the alpha-hydroxyacyl-prolyl-arginals, it is very likely that the less favorable biologic properties of Boc-D-Phe-Pro-Arg-H are due to the hydrophobicity and bulkiness of the terminal Boc-NH rather than its neutrality.

Relatively few cyclic peptides have reached the pharmaceutical marketplace during the past decade, most produced through fermentation rather than made synthetically. Generally, this class of compounds is synthesized for research purposes on milligram scales by solid-phase methods, but if the potential of macrocyclic peptidomimetics is to be realized, low-cost larger scale solution-phase syntheses need to be devised and optimized to provide sufficient quantities for preclinical, clinical, and commercial uses. Here, we describe a cheap, medium-scale, solution-phase synthesis of the first reported highly potent, selective, and orally active antagonist of the human C5a receptor. This compound, Ac-Phe[Orn-Pro-d-Cha-Trp-Arg], known as 3D53, is a macrocyclic peptidomimetic of the human plasma protein C5a and displays excellent antiinflammatory activity in numerous animal models of human disease. In a convergent approach, two tripeptide fragments Ac-Phe-Orn(Boc)-Pro-OH and H-d-Cha-Trp(For)-Arg-OEt were first prepared by high-yielding solution-phase couplings using a mixed anhydride method before coupling them to give a linear hexapeptide which, after deprotection, was obtained in 38% overall yield from the commercially available amino acids. Cyclization in solution using BOP reagent gave the antagonist in 33% yield (13% overall) after HPLC purification. Significant features of the synthesis were that the Arg side chain was left unprotected throughout, the component Boc-d-Cha-OH was obtained very efficiently via hydrogenation of d-Phe with PtO(2) in TFA/water, the tripeptides were coupled at the Pro-Cha junction to minimize racemization via the oxazolone pathway, and the entire synthesis was carried out without purification of any intermediates. The target cyclic product was purified (>97%) by reversed-phase HPLC. This convergent synthesis with minimal use of protecting groups allowed batches of 50-100 g to be prepared efficiently in high yield using standard laboratory equipment. This type of procedure should be useful for making even larger quantities of this and other macrocyclic peptidomimetic drugs.

N-formyl peptide receptors (FPRs) are G protein-coupled receptors involved in the recruitment and activation of immune cells in response to pathogen-associated molecular patterns. Three FPRs have been identified in humans (FPR1-FPR3), characterized by different ligand properties, biological function and cellular distribution. Recent findings from our laboratory have shown that the peptide BOC-FLFLF (L-BOC2), related to the FPR antagonist BOC2, acts as an angiogenesis inhibitor by binding to various angiogenic growth factors, including vascular endothelial growth factor-A165 (VEGF). Here we show that the all-D-enantiomer of L-BOC2 (D-BOC2) is devoid of any VEGF antagonist activity. At variance, D-BOC2, as well as the D-FLFLF and succinimidyl (Succ)-D-FLFLF (D-Succ-F3) D-peptide variants, is endowed with a pro-angiogenic potential. In particular, the D-peptide D-Succ-F3 exerts a pro-angiogenic activity in a variety of in vitro assays on human umbilical vein endothelial cells (HUVECs) and in ex vivo and in vivo assays in chick and zebrafish embryos and adult mice. This activity is related to the capacity of D-Succ-F3 to bind FRP3 expressed by HUVECs. Indeed, the effects exerted by D-Succ-F3 on HUVECs are fully suppressed by the G protein-coupled receptor inhibitor pertussis toxin, the FPR2/FPR3 antagonist WRW4 and by an anti-FPR3 antibody. A similar inhibition was observed following WRW4-induced FPR3 desensitization in HUVECs. Finally, D-Succ-F3 prevented the binding of the anti-FPR3 antibody to the cell surface of HUVECs. In conclusion, our data demonstrate that the angiogenic activity of D-Succ-F3 is due to the engagement and activation of FPR3 expressed by endothelial cells, thus shedding a new light on the biological function of this chemoattractant receptor.