Development of an efficient strategy for the synthesis of the ET_B receptor antagonist BQ-788 and some related analogues

Abstract

BQ-788 [N-cis-2,6-dimethylpiperidine-1-carbonyl-l-γ-methylleucyl-d-1-methoxycarbonyltryptophanyl-d-norleucine sodium salt] is a very potent and selective ET_B receptor antagonist. The formation of the highly hindered trisubstituted urea functionality in the peptide chain and the carbamination on the indole nitrogen of the tryptophan side chain are major challenges in the synthesis of this particular antagonist. Furthermore, the high cost of the unnatural amino acids in the sequence of BQ-788 and its reported synthesis render this pseudopeptide very expensive to produce. In order to improve the yield and to reduce the number of steps compared to previous reported syntheses, we developed an efficient strategy involving a novel one-pot procedure for the synthesis of a highly hindered trisubstituted urea. Under very mild conditions, the urea was obtained by using triphosgene and sodium iodide. This strategy allowed us to synthesize BQ-788 in seven steps with an overall yield of 53%. We also generalized the use of this powerful methodology by creating some new structural analogues of the cis-2,6-dimethylpiperidine moiety by replacing it with other bulky secondary amines. We evaluated the antagonist properties of those three new analogues of BQ-788 in two bioassays in vitro. These new antagonists were less potent than BQ-788 in an ET_B rich preparation and inactive in an ET_A rich preparation.

Introduction

Endothelin-1 (ET-1), initially isolated from endothelial cells, is a very potent vasoconstrictive peptide [32]. Subsequently, two other isoforms of ET-1 (ET-2 and ET-3) were reported [14]. ET-1 shows a very strong affinity for two types of G-protein-coupled receptors (GPCRs): ET_A and ET_B. ET_A receptors [1] mediate vasoconstriction and are predominantly located in vascular smooth muscles. ET_B receptors [27] are functionally linked to vasodilation on endothelial cells; however, in certain low resistance vessels they can also mediate vasoconstriction. In the last 10 years, ET_B receptors antagonists such as BQ-788 [16], [26] (Fig. 1) have been used towards the identification of the role of the ET_B receptor subtype in physiological and/or pathophysiological conditions. As reviewed by D’Orléans-Juste et al. [9], ET_B receptors modulate vascular tone and clearance of circulating ET-1 in several species including humans. Thus, powerful antagonists such as BQ-788 have contributed to the pharmacological characterization of ET receptor subtypes in several assays in vitro and in vivo.

On the other hand, the high cost of the unnatural amino acids present in the sequence of BQ-788 as well as its reported syntheses [13], [15] (see Table 2) render this commercially available compound very expensive to produce. The highly hindered urea in the peptide chain and the substitution on the indole nitrogen of the tryptophan side chain are the key elements of this very demanding synthesis.

As pointed out by He et al. [13], Ishikawa et al. encountered several synthetic problems regarding BQ-788 as described in their patent [15]. The main difficulties were related to the formation of the sterically hindered urea functionality, the racemization of the tryptophan during carbamination and the facile reduction of the indole ring of the tryptophan during catalytic hydrogenation. Nevertheless, the general strategy of limiting the number of steps to a minimum, by performing the carbamination directly on the dipeptide moiety in a C → N-terminal approach, was a positive point. In doing so, they achieved the synthesis of BQ-788 in eight steps starting from the corresponding amino acid esters with the exception of Boc-d-Trp-OH. However, the overall yield was not reported.

On the other hand, He et al. [13] published a synthesis of BQ-788 in 15 steps from the corresponding Boc amino acids, with an overall yield of 23%. The downsides of their synthesis included the uncommon N → C-elongation, the repetitive protection/deprotection procedures that could have been avoided and the formation of the urea bond in two steps. Nonetheless, in contrast to Ishikawa et al. [15], He et al. [13] succeeded in developing very mild and racemization-free conditions for the carbamination on the indole nitrogen of the tryptophan as well as reliable conditions for the hydrogenolysis of the benzyl ester groups.

In order to improve the yield and to reduce the number of steps, we have developed what we believe to be a more efficient and straightforward synthesis of BQ-788. Our approach involves a novel one-pot procedure for the synthesis of a highly hindered trisubstituted urea based on the work of Streith et al. [29]. Moreover, we adopted this new synthetic route towards novel analogues of BQ-788 by using different bulky secondary amines. Finally, the pharmacodynamic properties of the synthesized BQ-788 and of the new analogues made were compared to the same commercially available ET_B antagonist in ET_A and ET_B selective bioassays in vitro [7].