key: cord-0055143-l9x8bswr
authors: Xu, Yanzhao; Qi, Na; Wen, Hui; Zhang, Gang; Wang, Yuchen; Cui, Huaqing
title: Synthesis and evaluation of benzenesulfonic acid derivatives as human neutrophil elastase (hNE) inhibitors
date: 2021-01-12
journal: Med Chem Res
DOI: 10.1007/s00044-020-02684-4
sha: 213c2b8d194a451c09885c3d31c9c76576a789b0
doc_id: 55143
cord_uid: l9x8bswr

Herein we report our investigation concerning the development of Human neutrophil elastase (hNE) inhibitors for the treatment of Acute Respiratory Distress Syndrome (ARDS). Various benzenesulfonic acid derived compounds were synthesized and evaluated as competitive inhibitors of hNE. Biological screening revealed that compound 4f shows moderate inhibitory activity (IC(50) = 35.2 μM) against hNE. Compound 4f was also superimposed onto the active center of hNE to understand the binding mode.

Coronavirus Disease 2019 (COVID-19) is a pneumonia disease, which is caused by a virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1] . Clinically, around 41.8% of severe COVID-19 patients will develop symptoms associated with acute respiratory distress syndrome (ARDS), such as, severe hypoxia and eventual respiratory failure [2] . Therefore, new treatments that treat the roots of severe hypoxia and respiratory failure should be addressed. Currently, mechanical ventilation is the most commonly used treatment for ARDS, while suppression of the strong immune response is a potential strategy to alleviate ARDS [3] [4] [5] .

Human neutrophil elastase (hNE, EC 3.4.21.37) is a globular glycoprotein with a molecular weight of about 30 kDa [6, 7] . hNE is a serine proteinase and the proteolysis occurs through the Ser195-Asp102-His57 catalytic triad [6, 8, 9] . Elegant studies have shown that the proteolysis is triggered via His57, which in turn activates the hydroxyl group of Ser195. This nucleophilic hydroxyl group subsequently attached the carbonyl functionality of the target peptide bond and forms the classical tetrahedral intermediate. Subsequent proton transfers, mediated by His57, onto the amine of this tetrahedral intermediate results in a covalent acyl-enzyme complex. Finally, a water molecule attacks this complex to generate a new tetrahedral intermediate, which collapses and leads to the regeneration of activated hNE [8, 9] . hNE is an active proteinase and has been shown to degrade a range of proteins including all extracellular matrix proteins and many important plasma proteins [6, 10] . Under normal physiological conditions, various immune cells use hNE as important protective species [6] . When the body undergoes inflammation or infection, neutrophils are the first called immune cells [11] . hNE and other proteases are secreted by neutrophils to destroy the pathogens and to regulate the inflammation. It was reported that every minute there was around 30 billion neutrophils distributed in lung capillaries, and each neutrophil can release up to 3 pg hNE [6, 12] . Usually the activity of hNE was strictly controlled by endogenous inhibitors [13] . However, excessive secretion of hNE breaks the balance and further causes the overdegradation of the extracellular matrix in the inflammation site [6, 14] . Furthermore, the increased damage of the lung and the continuous release of pro-inflammatory cytokines will result in an acute lung injury (ALI) [4] , the early stage of ARDS [15] . hNE is known to contribute the production of mucin in airway epithelial cells [16] . Therefore, inhibition of hNE is considered to be one of the potential strategies to alleviate ARDS [17, 18] .

Efforts have been made to discover potent and selective hNE inhibitors. Many naturally derived products have been reported exhibiting pronounced hNE inhibitory activity [19] [20] [21] . Laboratory designed hNE inhibitors fall into two key categories: peptides and heterocyclic compounds. Prolastin is a known peptide type hNE inhibitor, and has been shown to be successful in the treatment of α1-antitrypsin deficiency [22] , however challenges remain bringing the treatment to market. Compared to peptides, heterocyclic compounds have many advantages, including metabolic stability, oral bioavailability, modularity in their synthesis, and more controlled ADME properties. Sivelestat (Fig. 1) is a selective and potent hNE inhibitor, which has been marketed in Japan and Korea for the treatment of ALI/ARDS related to systemic inflammatory response syndrome [23, 24] . However, the performance of Sivelestat for the treatment of ALI/ARDS in the clinic remains controversial [25, 26] . Therefore, it is necessary to develop additional safe and potent hNE small molecule inhibitors. At present, two potent and selective small molecule hNE inhibitors ( Fig. 1 ) are in clinical trials: Alvelestat (AZD9668, AstraZeneca, Cambridge, Germany) [27, 28] and Bay 85-8501 (Bayer HealthCare, Leverkusen, Germany) [29] .

A series of benzenesulfonyl fluoride (SuEFx-1, Fig. 2 ) was previously reported as covalent inhibitors of hNE (IC 50 = 3.3 μM) [30] . However the chemical instability and less than ideal hNE inhibitory profile of these benzenesulfonyl fluorides mean that further optimization is required (Fig. 2) . Interestingly, due to the small size of SuEFx-1, the analogs can be used as the initial fragment for fragment based drug design. Further installment of additional effective fragments can lead to potent hNE inhibitors. Thus, taking advantage of the binding interaction between benzenesulfonyl fluoride and the target hNE enzyme, we utilized benzenesulfonic acids as a direct replacement to the benzenesulfonyl fluoride moiety with the aim to develop a competitive hNE inhibitor.

Design and synthesis of amide series Based on the structure of ortho-subtituted benzenesulfonyl fluoride (SuEFx-1), we designed a series of amide substituted benzenesulfonic acids (4a-h) including both orthoand metasubstituents. The synthesis of compounds 4a-h is described in Scheme 1. Acids chlorides, derived from the The mode of action of hNE covalent inhibitor, SuEFx-1 [30] . hNE competitive inhibitors were also designed to mimic the transition state of covalent inhibitor, taking advantage of the binding mode of SuEFx-1 reaction various carboxylic acids (1a-h), with thionyl chloride were reacted with a range of sulfonic anilines 3 to give functionalized amide (4a-h) in synthetically useful yields.

Initially, we attempted to perform the condensation between compounds (1a-h) and sulfonic aniline to directly obtain the desired amides, however this resulted in uniformly low yields. Then we decided to firstly transform the carboxylic acid into acyl chloride, the reaction between acyl chloride and the sulfonic acid substituted aniline will give the desired product. In order to prepare the acyl chloride, oxalyl chloride was firstly reacted with various carboxylic acid (1a-h). However, this protocol did not give a decent amount of acyl chloride. Then, we directly dissolved various carboxylic acid (1a-h) into thionyl chloride as the solvent and this protocol gave excellent transformation of acyl chloride. Due to DIPEA was used in the reaction as the organic base, the benzenesulfonic acid will form salt with DIPEA. The solvent of DCM was removed from the reaction mixture and washed with the ether to quickly obtain the final products 4c-f and 4h, which are DIPEA salts.

Next we designed a series of urea substituted benzenesulfonic acids (6a-j), which also includes both orthoand metasubstituents. The compounds (6a-j) were obtained by reacting various isocyanates 5a-j with sulfonic aniline 3 (Scheme 2). All of the urea substituted benzenesulfonic acid (6a-j) were purified as white solids with the yields above 80%.

The urea was prepared with the reactants of the isocyanates and the amine. Various commercially available isocyanates reacted directly with sulfonic anilines to form the desired urea products. The reaction processed finely with decent yields. In addition, the final products were also existed as DIPEA salt. After the DCM was evaporated, the 

We also designed and synthesized two benzenesulfonyl fluorides (9a, 9b) (Schemes 3 and 4). These two compounds contain the reactive group of sulfonyl fluoride, which can form covalent bond with the hydroxyl group of serine residue. 9a was synthesized via the Suzuki coupling of 7a and 7b, while 9b was synthesized in two steps starting from the sulfonic acid 10.

In Scheme 3, Suzuki-Miyaura reaction was initially performed under the condition of Pd(PPh 3 ) 4 , sodium carbonate at 100°C under argon. However, we did not find the desired product under this condition. Then, we adopted relatively mild reaction conditions, we used palladium acetate as the catalyst, triethylamine as the base, and the reaction was carried out at room temperature under argon. Next, because the direct transformation of benzenesulfonic acid to benzenesulfonic fluorine is difficult, we first prepared the benzenesulfonyl chloride, which was then mixed with potassium fluoride and 18-crown-6 in anhydrous acetonitrile. The reaction needed to be refluxed. Moreover, in the process water was strictly eliminated to avoid hydrolysis of the final product benzenesulfonyl fluoride.

Once we obtained our desired compounds, they were evaluated for hNE inhibitory activity (Table 1 ). In general, most of the compounds exhibited low hNE inhibitory activity, with the majority displaying inhibitory activity at very high concentrations (100 μM). For the amide series (4a-h), the substitution pattern of the amides (ortho-and meta-) showed no discernible difference in their activity against hNE.

However, we observed that the substituents attached to the amide had a clear impact on the hNE inhibitory activity. Most notable of these was the meta-amide-(4-biphenyl) substituted compound (4f), which exhibited decent potency against the target, with an IC 50 of 35.2 μM. For the urea series (6a-j), both orthoand metaureas did not show significant difference in inhibitory activity against hNE. Interestingly, hydrophobic groups substituted at the paraor metapositions of the phenyl ring were favorable, with clear inhibitory activity observed. The two benzenesulfonyl fluorides (9a, 9b), which we initially used as a direct comparison between covalent inhibitors and competitive inhibitors, showed no discernible inhibition profile against hNE.

It is worth noting that compounds 9b and 4a have similar structures with the only difference at sulfonyl acid or sulfonyl fluoride and both exhibited similar hNE activity. We therefore reason that the amide moiety plays an important role in hNE binding. Moreover, compound 4f containing the meta-amide moiety was discovered as the most potent hNE inhibitor. This emphasized the importance of the fragment of meta-amide-(4-biphenyl).

In addition, we also measured cell cytotoxicity of these compounds against five mammalian cancer cell lines, MCF-7, BGC823, A549, HepG2, and HCT116, and no obvious growth inhibition towards all five cell lines were observed for all the tested compounds at the concentration of 50 μM. This indicates that there is no inherent cell cytotoxicity for the core structure of benzenesulfonyl acids, and they can be further developed as the potential therapeutic agents of ARDS.

In summary, we evaluated 18 benzenesulfonyl acids and 2 benzenesulfonyl fluorides for their hNE inhibitory activities. Most of these compounds show low inhibition activities against hNE. We observed that our amide series exhibited slightly better inhibitory activities than the urea series. Compound 4f was the most active hNE inhibitor with the IC 50 of 35.2 μM, and suggests that the amide-(4-biphenyl) 

Due to the performance of compound 4f, and in order to ascertain key design principles, we decided to probe the key binding interaction with hNE with the hit compound. The experimental results showed that compound 4f occupied the same site as the original ligand SuEFx-1, benzene-1, 2disulfonyl fluoride, in the crystal structure 6e69 (Fig. 2A) .

Compound 4f formed key hydrogen bond interactions with His57, Ser214, and Val216. The phenyl ring of Phe192 had ππ stacking interaction with the middle phenyl ring of compound 4f. The hydrophobic tail of compound 4f interacted with pocket formed by Leu35, Phe41, and Val62 (Fig. 2B, C) . Interestingly, the benzenesulfonic acid moiety of 4f exhibited similar binding mode as SuEFx-1. The residues of His57, Ser214, and Val216 interacted with the sulfonyl group in both compounds, while the covalent bond formed between Ser195 and the sulfonyl group of SuEFx-1 was not observed in the docking study of 4f. Moreover, benzenesulfonic acid moiety is a relative small fragment, which cannot maintain a strong binding with hNE. Thus, the place of 4-diphenyl moiety in the right pocket of hNE further increases the overall inhibitory activity of the molecule.

In this work, starting from hNE covalent inhibitor SuEFx-1, we designed and synthesized two series of orthoand metasubstituted benzenesulfonic acids and the inhibitory activities of these compounds were ascertained through in vitro experiments. The majority of these benzenesulfonic acids showed low inhibition against hNE, however 4f exhibited moderate hNE activity with an IC 50 of 35.2 μM. We found that the substituents, particularly the amide-(4-diphenyl) moiety, of these compounds is important for the hNE inhibition. Computational docking revealed a similar binding position of compound 4f and SuEFx-1 in the active center of hNE, and also revealed the importance of the amide-4-diphenyl moiety for the binding. Taken together, we discovered a promising hit compound which can be further optimized to increase hNE inhibitory activity.

Chemistry All solvents and chemicals were purchased from commercial sources: Sigma-Aldrich Chemical Co., Shanghai Bepharm Science&Technology Co., Shanghai Macklin Biotechnology Co., Tianjin Heowns Biotechnology Co., and Shanghai Jingke Chemistry Technology Co. with the purity of more than 95%. Flash column chromatography was performed on a Biotage Isolera one. 1 H NMR, 13 C NMR, and 19 F NMR were recorded on Mercury400, Bruker AVANCEΙΙΙ 400 spectrometer. Chemical shifts are referenced to the residual solvent peak and reported in ppm (δ scale) and all coupling constant (J) values are given in Hertz (Hz) . The following multiplicity abbreviations are used: (s) singlet, (d) doublet, (t) triplet, (q) quartet, (m) multiplet. ESI-HRMS data were measured using a Thermo Exactive Orbitrap plus spectrometer. Purity was determined using LCMS. All of the synthesized compounds have a purity of >95%.

Carboxylic acid (1a-h) (679.04 μmol) was dissolved in SOCl 2 (2 mL). The mixture was stirred at room temperature for 2 h. After evaporation of the solvent, the intermediate (2a-h) was dissolved in anhydrous DCM (2 mL) and then added dropwise to another anhydrous DCM (10 mL) in which sulfonic aniline (3) (565.87 μmol) and DIPEA (188.94 μL) were dissolved. The mixture was stirred at room temperature for 4 h and the precipitate was recovered by vacuum filtration. The final compound was purified by column chromatography using methanol/dichloromethane 1:19 as eluents (4a, 4b, and 4g) or purified by stirring with the ether (4c-f and 4h). 13 13 13 135.41, 134.76, 132.63, 131.00, 129.76, 129.47, 128.81,  128.65, 128.14, 127.29, 125.64, 124.99, 121.41, 120. General procedure for compounds 6a-j To a suspension of the sulfonic aniline (3) (565.87 μmol) and DIPEA (188.94 μL) in anhydrous DCM (10 mL), another anhydrous DCM (2 mL) with the appropriate commercially available isocyanate (5a-j) (679.04 μmol) were added dropwise. The mixture was stirred at room temperature for 8 h and the precipitate was recovered by vacuum filtration. The final compound 6a-j was purified by stirring with ether. 13 

Benzenesulfonic acid (4a) (171.63 μmol) was dissolved in SOCl 2 (2 mL). The mixture was stirred at room temperature for 2 h. After evaporation of the solvent, the intermediate (8) was dissolved in anhydrous CH 3 CN (10 mL) in which KF (3.23 mmoL) and 18-crown-6-ether (8.07 μmoL) were dissolved. The mixture was stirred at reflux for 12 h in an argon-filled environment. After the solvent was evaporated in vacuo, ethyl acetate was added, and washed with saturated brine (3 × 15 mL). The organic layer was dried with anhydrous Na 2 SO 4 . The final compound 9a was purified by column chromatography using ethyl acetate/petroleum ether 1:9 as the eluent.

2-(4-methylbenzamido)benzenesulfonyl fluoride (9b) Yield = 26%; yellow flaky solid. 1 

The X-ray crystallographic structure of the adduct of the neutrophil elastase and benzene-1,2-disulfonyl fluoride (PDB Code: 6e69) was downloaded from the Protein Data Bank. The PDB protein and small molecule were prepared by adding hydrogen and missing residues in Accelrys Discovery Studio 2018 (Accelrys Software Inc., San Diego, CA) and converted to pdbqt format by Openbabel [31] . In vitro HNE inhibition studies [29] Assays were performed at 37°C in buffer (200 mM NaCl, 200 mM Tris-HCl, pH = 7.5, 0.01% BSA, 0.05% Tween-20). The peptide substrate MeOsuc-AAPV-AFC (ab142178) and hNE enzyme (ab91099) were purchased from Abcam. Reactions were started by the addition of various inhibitors in 200 μL of enzyme assay containing 1 mU of hNE and 25 μM peptide substrates. The fluorescence measurement was immediately performed with E x = 380 nm and E m = 460 nm for 10 min to calculate the kinetic changes of the fluorescence. The inhibition was calculated using Graphpad Prism. Sivelestat was used as the reference compound.

Human colon cancer cell HCT-116, human liver cancer cell HepG2, human gastric cancer cell BGC-823, human lung cancer cell A549, and human breast adenocarcinoma cell MCF-7 were obtained from cell center of Chinese Academy of Medical Sciences & Peking Union Medical College. They were cultured in DMEM medium (Invitrogen) with 10% fetal bovine serum (Gibco) at 37°C with 5% CO 2 . All compounds were assessed the cyto-toxicity in five human cancer cell lines, namely HCT-116, HepG2, BGC-823, MCF-7, and A549 following the protocol of MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay [33, 34] . 

-chlorophenyl) ureido)benzenesulfonate (6b) Yield = 89%

76 (s, 1H)

Hz, 1H), 7.57 (d, J = 8.9 Hz, 2H), 7.30 (d, J = 8.9 Hz, 2H), 7.28-7.24 (m, 1H), 6.95 (t, J = 7.5 Hz, 1H)

-(trifluoromethyl) phenyl)ureido)benzenesulfonate (6c) Yield = 85%; white solid. 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.06 (s, 1H)

Hz, 2H), 7.67 (dd, J = 7.7, 1.5 Hz, 1H)

Hz, 2H), 7.31-7.25 (m, 1H), 7.02-6.92 (m, 1H), 3.60 (dp, J = 10.5, 3.9 Hz, 2H), 3.13 (dq, J = 7.3, 3.0 Hz, 2H), 1.25 (t, J = 6.2 Hz, 15H). 13 C NMR (101 MHz

121.60 (d, J = 30

-(trifluoromethoxy) phenyl)ureido)benzenesulfonate (6d) Yield = 91%; white solid. 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.83 (s, 1H)

3.60 (td, J = 6.6, 3.9 Hz, 2H), 3.14-3.08 (m, 2H), 1.26-1.23 (m, 15H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 152.88, 142.90 (d, J = 1.6 Hz)

5-dichlorophenyl)ureido)benzenesulfonate (6e) Yield = 85%; white solid. 1 H NMR (400 MHz, DMSO-d 6 ) δ 9

Hz, 1H), 7.48 (q, J = 8.7 Hz, 2H), 7.29 (t, J = 7.4 Hz, 1H), 6.98 (t, J = 7.3 Hz, 1H), 3.67-3.50 (m, 2H), 3.17-3.02 (m, 2H), 1.23 (t, J = 6.1 Hz, 15H). 13 C NMR (101 MHz

5-bis(trifluoromethyl)phenyl)ureido)benzenesulfonate (6f) Yield = 90%

62 (s, 1H), 7.57 (dt, J = 6.5, 2.4 Hz, 1H), 7.30-7.24 (m, 2H), 3.61 (pd

dichlorophenyl)ureido)benzenesulfonate (6g) Yield = 82%; white solid. 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.04 (s, 1H), 9.01 (s, 1H), 7.93-7.84 (m, 1H), 7.75-7.68 (m, 1H)

07 (s, 2H), 6.97-6.91 (m, 1H), 6.91-6.87 (m, 1H), 6.79-6.72 (m, 1H), 6.59 (s, 1H), 6.51-6.44 (m, 1H), 3.61 (dq, J = 13.1, 6.5 Hz, 2H), 3.12 (dt

2-yl)ureido)benzenesulfonate (6i) Yield = 84%; white solid. 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.09 (s, 1H), 8.21 (s, 1H), 7.94 (d, J = 8.2 Hz, 1H), 7.60 (s, 1H), 7.57 (s, 1H), 7.52 (t, J = 7.4 Hz, 2H), 7.42 (t, J = 8.2 Hz, 4H), 7.37-7.30 (m, 1H), 7.23-7.19 (m, 1H), 7.18 (s, 1H)

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Acknowledgements This work was financially supported by The CAMS Innovation Fund for Medical Sciences (2017-I2M-1-010), The Drug Innovation Major Project (2018ZX09711001). This paper is dedicated to Prof. Robert Vince's 80th birthday.

Conflict of interest The authors declare that they have no conflict of interest.Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.