Synthesis and pharmacological characterization of 2-aminobenzaldehyde oxime analogs as dual inhibitors of neutrophil elastase and proteinase 3

doi:10.1016/j.bmc.2014.12.056

Bioorganic & Medicinal Chemistry

Volume 23, Issue 5, 1 March 2015, Pages 1123-1134

https://doi.org/10.1016/j.bmc.2014.12.056 Get rights and content

Abstract

Proteinase 3 (Pr3), and human neutrophil elastase (HNE) are two major neutrophilic serine proteases (NsPs) expressed in neutrophil azurophil granules. Emerging data suggest that excessive release of proteases mediates tissue damage, and therefore prolonged neutrophil accumulation has an important role in the pathogenesis of many diseases. Thus, HNE and Pr3 inhibitors may prove to be targets for the generation of agents in the treatment of neutrophilic inflammatory disease. Sivelestat is the only commercially available selective HNE inhibitor. Therefore, sivelestat was chosen as the model structure in an attempt to obtain more potent anti-NsPs agents. In the present study, a series 2-aminobenzaldehyde oxime and 2-aminobenzoate analogs were synthesized and their inhibitory effects on NsPs (CatG, Pr3, and HNE) were determined, respectively. The results of structure–activity relationships studies concluded that a hydroxyl oxime moiety plays an important role in ligand–enzyme affinity through hydrogen bonding. As compound 6 had more potency and showed dual inhibitory effects on NE and Pr3, both in vitro and in vivo experiments were carried out to evaluate its selectivity, effects in cell-based assays, and efficacy in models of inflammation and damage. Compound 6 had the potential to reduce paw edema induced by LPS and HNE, as well as acute lung injury, and may be approved as a candidate for the development of new agents in the treatment of neutrophilic inflammatory diseases.

Graphical abstract

Eleven 2-aminobenzaldehyde oxime analogs were synthesized. Of these, 6 had the potential to inhibit the activities of neutrophil elastase and proteinase 3. In addition, it also reduce paw edema induced by LPS and HNE, as well as acute lung injury. Therefore, 6 may be approved as a candidate for the development of new agents in the treatment of neutrophilic inflammatory diseases.

Introduction

Neutrophils are a major type of blood leukocyte, and are essential for host defense against invading microorganisms.¹ In addition, neutrophils are involved in the activation, regulation and effector function of innate and adaptive immune cells.² In response to microbial attack, neutrophils are activated and recruited to the infection site, thereby releasing reactive oxygen species (ROS) and serine proteases into the extracellular space.1, 3 However, emerging data suggest that oxidant stress and excessive release of proteases mediate tissue damage. Thus, prolonged neutrophil accumulation has an important role in the pathogenesis of chronic inflammation, autoimmunity, and cancer.1, 2, 3, 4

Serine proteases are the largest known family of protein-cleaving enzymes, and modulate blood coagulation, apoptosis and inflammation.⁵ Of these, cathepsin G (CatG), proteinase 3 (Pr3), and human neutrophil elastase (HNE) are three major neutrophilic serine proteases (NsPs) expressed in neutrophil azurophil granules.6, 7 In particular, HNE and Pr3 are two major NsPs which play crucial roles in antimicrobial defense with overlapping and potentially redundant substrate specificity.⁶ However, recent reports suggest that excess proteolysis which results from an influx of neutrophils into the airways is pivotal in the progressive destruction of lung parenchyma, for example, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and chronic obstructive pulmonary disease (COPD).7, 8, 9, 10 Therefore, HNE and Pr3 may prove to be targets for the generation of agents in the treatment of neutrophilic inflammatory disease.6, 11

In 2000, an acetylating inhibitor, sivelestat, was developed for the treatment of ALI in Japan, and is the only nonpeptidic inhibitor marketed.12, 13, 14 However, the use of sivelestat was limited by its poor pharmacokinetics and serious risks of organ toxicity because it irreversibly inhibits HNE.¹⁵ In addition, the clinical trials of sivelestat in the western world were terminated due to a lack in proof of therapeutic effect.¹² Therefore, sivelestat was chosen as the model structure in the present study in an attempt to obtain more potent anti-neutrophil serine protease agents. As sivelestat has hydrophobic and hydrogen-bonding interactions with HNE through pivalate ester, sulfonamide, and glycine moieties,¹⁶ structural modification was carried out by changing the glycine moiety to hydrogen, aliphatic chains, or esters, and replacing the anthranilate moiety with 2-aminobenzaldehyde oximes, as well as exchanging pivalate ester with isostere. In the present study, we report the synthesis of target components (Scheme 1, Scheme 2, Scheme 3, Scheme 4, Scheme 5, Scheme 6, Scheme 7), as well as their structure–activity relationships (SARs) and both in vitro and in vivo pharmacologic data.

Section snippets

Effects of synthetics on neutrophil serine proteases

In attempt to investigate the SARs of the synthetics, the effects of all the synthetics on neutrophil serine proteases (HNE, Pr3 and CatG) were determined.9, 12, 17 Most of these compounds had a potent and dual inhibitory effect on HNE and Pr3. Of these synthetics, a new 2-aminobenzaldehyde oxime analog, compound 6, exhibited the most potent inhibitory effects on the activity of HNE and Pr3 with IC₅₀ values of 0.05 and 0.22 μM, respectively, and was slightly potent than sivelestat (Table 1,

Conclusion

In conclusion, we synthesized twenty-three 2-aminobenzaldehyde oxime analogs and compared their inhibitory effects on NsPs (CatG, Pr3, and HNE), respectively. The results of SARs studies concluded that the hydroxyl oxime moiety was favorable for HNE and Pr3 activity, and indicated that hydrogen bonding interactions play an important role in ligand–enzyme affinity. As compound 6 had significant potency and showed dual inhibitory effects on HNE and Pr3, both in vitro and in vivo experiments were

Chemicals and reagents

α-Chymotrypsin from bovine pancreas, trypsin from bovine pancreas, thrombin from bovine plasma, elastase from human leukocytes, N-succinyl-Ala-Ala-Pro-Phe-7-amido-4-methylcoumarin, N_α-benzoyl-L-arginine-7-amido-4-methylcoumarin, porcine pancreas elastase, lipopolysaccharides (Escherichia coli 055:B5), sodium azide, o-dianisidine dihydrochloride, phenol, and 2-aminoacetophenone were purchased from Sigma–Aldrich Chemicals (St. Louis, MO, USA). Human neutrophil elastase and Cathepsin G, and N

Acknowledgements

The investigation was supported by research grants to P. W. Hsieh from the National Science Council of the Republic of China (NSC101-2320-B-182-008) and Chang Gung Medical Research Foundation (CMRPD1B0301–CMRPD1B0303) in Taiwan.

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^†: These authors contributed equally to this work.

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Synthesis and pharmacological characterization of 2-aminobenzaldehyde oxime analogs as dual inhibitors of neutrophil elastase and proteinase 3

Abstract

Graphical abstract

Introduction

Section snippets

Effects of synthetics on neutrophil serine proteases

Conclusion

Chemicals and reagents

Acknowledgements

Int. J. Biochem. Cell Biol.

Lancet

Biochem. Pharmacol.

Phytochemistry

Int. J. Biol. Macromol.

Eur. J. Med. Chem.

Am. J. Pathol.

Tetrahedron

Inflamm. Res.

Nat. Rev. Immunol.

Mol. Immunol.

J. Clin. Invest.

Pharmacol. Rev.

Am. J. Respir. Cell Mol. Biol.

J. Med. Chem.