Total Synthesis of the Potent Marine-Derived Elastase Inhibitor Lyngbyastatin 7 and in Vitro Biological Evaluation in Model Systems for Pulmonary Diseases

Abstract

Lyngbyastatin 7 (1) is a marine cyanobacteria-derived lariat-type cyclic depsipeptide of which the macrocyclic core possesses modified amino acids, including a featured 3-amino-6-hydroxy-2-piperidone (Ahp) moiety and a (Z)-2-amino-2-butenoic acid (Abu) moiety. The first total synthesis of 1 was successfully established via 31 steps, and the conditions of several crucial steps were optimized to ensure smooth operations. The previously reported structural assignment and elastase inhibitory activity of the isolated natural product were confirmed. According to the extensive in vitro biological evaluation, compound 1 displayed low nanomolar IC50 in blocking elastase activity and strong ability in protecting bronchial epithelial cells against elastase-induced antiproliferation and abrogating the elastase-triggered induction of pro-inflammatory cytokine expression. Its overall performance was superior over sivelestat, the only approved small molecule drug targeting elastase, which indicated its potential in developing as a pharmacotherapeutic against elastase-mediated pathologies. The success in total synthesis, designed with a novel convergent strategy, not only overcame the supply issue for thorough preclinical studies but also paved the way for convenient synthesis of analogues with improved potency and druglike properties.

Figure 1

Chemical structure of lyngbyastatin 7 (1), somamide A, symplostatin 5 and sivelestat.

Figure 2

Comparison of the reported synthetic strategy of somamide A with our novel designed synthetic strategy for lyngbyastatin 7 (1).

Figure 3

In vitro biological evaluation of compound 1, symplostatin 5 and sivelestat at the enzyme, cellular and transcriptional level. (A) In PPE enzyme assay, PPE was first incubated with compound 1, symplostatin 5 and sivelestat respectively for 15 min in Tris-HCl (pH 8.0) and then N-succinyl-Ala-Ala-Ala-p-nitroanilide was used as substrate to monitor the enzyme activity. The IC₅₀ value for each compound is 88, 73 and 3080 nM respectively. (B) In HNE enzyme assay, HNE was first incubated with compound 1, symplostatin 5 and sivelestat respectively for 15 min in 0.1 M Tris-0.5 M NaCl (pH 7.5) and then N-(OMe-succinyl)-Ala-Ala-Pro-Val-p-nitroanilide was used as substrate to monitor the enzyme activity. The IC₅₀ value for each compound is 100, 161 and 123 nM respectively. (C) In the cell viability assay, BEAS-2B cells were cotreated with compound 1, symplostatin 5, sivelestat or solvent control and 100 nM HNE or vehicle for 24 h. The cell viability was monitored by using MTT reagent. The IC₅₀ value for each compound is 70, 106 and 861 nM respectively. (D) In order to monitor the changes in the transcript levels of IL1B, IL1A and IL8, total RNA was extracted after BEAS-2B cells were cotreated with 10 μM compound 1 or solvent control and 100 nM HNE or vehicle for 3 h. After cDNA synthesis, qPCR was carried out while using GAPDH as endogenous control. (E) In order to monitor the changes in transcript level of IL1B in the presence of different compounds in various concentrations, total RNA was extracted after BEAS-2B cells were cotreated with compound 1, symplostatin 5 and sivelestat or solvent control and 100 nM HNE or vehicle for 3 h. After cDNA synthesis, qPCR was carried out while using GAPDH as endogenous control. Data are presented as mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared to HNE-treated cells using ANOVA, Dunnett's t test (n=3).

Scheme 1

Retrosynthetic Analysis of 1

Scheme 2

Synthesis of Building Block 6

Scheme 3

Synthesis of Building Block 7

Scheme 4

Synthesis of Building Block 8

Scheme 5

Synthesis of Building Block 4

Scheme 6

Total synthesis of 1