Synthesis and evaluation of benzenesulfonic acid derivatives as human neutrophil elastase (hNE) inhibitors

Article abstract of DOI:10.1007/s00044-020-02684-4

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 (IC₅₀ = 35.2 μM) against hNE. Compound 4f was also superimposed onto the active center of hNE to understand the binding mode.

Full text of DOI:10.1007/s00044-020-02684-4

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:387–398
https://doi.org/10.1007/s00044-020-02684-4
ORIGINAL RESEARCH
Synthesis and evaluation of benzenesulfonic acid derivatives
as human neutrophil elastase (hNE) inhibitors
Yanzhao Xu1 Na Qi Hui Wen Gang Zhang Yuchen Wang Huaqing Cui
1 ●
1 ●
1 ●
1 ●
1
●
Received: 25 October 2020 / Accepted: 2 December 2020 / Published online: 12 January 2021
©
The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
Abstract
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 = 35.2 μM) against hNE. Compound 4f was also superimposed onto the active center of
50
hNE to understand the binding mode.
●
●
●
●
Keywords Human neutrophil elastase Inhibitor Benzenesulfonic acid ARDS Drug design
Introduction
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 subse-
quently attached the carbonyl functionality of the target
peptide bond and forms the classical tetrahedral inter-
mediate. 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 inter-
mediate, 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
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 alle-
viate ARDS [3–5].
Human neutrophil elastase (hNE, EC 3.4.21.37) is a
globular glycoprotein with a molecular weight of about
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02684-4) contains supplementary
material, which is available to authorized users.
*
*
Yuchen Wang
wangyuchen@imm.ac.cn
Huaqing Cui
[
6, 12]. Usually the activity of hNE was strictly controlled
hcui@imm.ac.cn
by endogenous inhibitors [13]. However, excessive secre-
tion of hNE breaks the balance and further causes the over-
degradation 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
1
State Key Laboratory of Bioactive Substances and Function of
Natural Medicine, Institute of Materia Medica, Peking Union
Medical College and Chinese Academy of Medical Sciences, 1
Xiannongtan Street, Beijing 100050, China

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Medicinal Chemistry Research (2021) 30:387–398
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, inhibi-
tion of hNE is considered to be one of the potential stra-
tegies 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
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.
[
19–21]. Laboratory designed hNE inhibitors fall into two
key categories: peptides and heterocyclic compounds. Pro-
lastin 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 mar-
keted in Japan and Korea for the treatment of ALI/ARDS
related to systemic inflammatory response syndrome
Results and discussion
Design and synthesis of amide series
[
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
Based on the structure of ortho-subtituted benzenesulfonyl
fluoride (SuEFx-1), we designed a series of amide sub-
stituted benzenesulfonic acids (4a–h) including both ortho-
and meta- substituents. The synthesis of compounds 4a–h is
described in Scheme 1. Acids chlorides, derived from the
[
(
Fig. 1) are in clinical trials: Alvelestat (AZD9668,
Fig. 1 Current known hNE inhibitors
Fig. 2 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

Medicinal Chemistry Research (2021) 30:387–398
389
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.
DIPEA. The solvent of DCM was removed from the reac-
tion mixture and washed with the ether to quickly obtain the
final products 4c–f and 4h, which are DIPEA salts.
Initially, we attempted to perform the condensation
between compounds (1a–h) and sulfonic aniline to directly
obtain the desired amides, however this resulted in uni-
formly 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 var-
ious 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
Design and synthesis of urea series
Next we designed a series of urea substituted benzenesulfonic
acids (6a–j), which also includes both ortho- and meta- sub-
stituents. 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 iso-
cyanates 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
Scheme 1 Reaction conditions: a, SOCl2, 2 h; b, anhydrous DCM, DIPEA, 4 h
Scheme 2 Reaction conditions:
a, anhydrous DCM, DIPEA, 8h

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Medicinal Chemistry Research (2021) 30:387–398
Scheme 3 Reaction conditions:
a, 1, 4-dioxane:H2O = 4:1,
EtN3, Pd(OAc)2, 2 h
Scheme 4 Reaction conditions:
a, SOCl2, 2 h; b, KF, 18-crown-
6
-ether, anhydrous CH3CN,
reflux, Ar, 12 h
reaction mixture was washed with the ether to obtain the
final product 6a–j.
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 IC50 of 35.2 μM. For the urea
series (6a–j), both ortho- and meta- ureas did not show
significant difference in inhibitory activity against hNE.
Interestingly, hydrophobic groups substituted at the para- or
meta- positions of the phenyl ring were favorable, with clear
inhibitory activity observed. The two benzenesulfonyl
fluorides (9a, 9b), which we initially used as a direct com-
parison between covalent inhibitors and competitive inhibi-
tors, 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 sul-
fonyl 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
IC50 of 35.2 μM, and suggests that the amide-(4-biphenyl)
Synthesis of benzenesulfonyl fluoride series
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 ) , sodium car-
3
4
bonate 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 pre-
pared 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.
In vitro hNE inhibitory activity
Once we obtained our desired compounds, they were eval-
uated 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.

Medicinal Chemistry Research (2021) 30:387–398
391
Table 1 hNE inhibitory activity of compounds 4, 6, and 9
Compound
Structure
Inhibition@100 μM
4
a
10.2 ± 1.0%
4
4
b
c
21.8 ± 5.2%
0
4
4
d
e
0
10.3 ± 4.9%
4
4
f
35.2 μM (IC50)
g
21.1 ± 2.3%
4
h
29.9 ± 2.1%
6
6
a
19.4 ± 2.2%
35.3 ± 2.8%
b

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Medicinal Chemistry Research (2021) 30:387–398
Table 1 (continued)
Compound
Structure
Inhibition@100 μM
6
c
41.2 ± 3.8%
6
6
d
e
18.6 ± 0.1%
0
6
f
33.5 ± 1.5%
6
6
6
g
h
i
17.8 ± 1.4%
41.3 ± 1.2%
25.2 ± 6.8%
6
9
9
j
32.5 ± 2.6%
30.1 ± 0.2%
30.2 ± 2.5%
33 nM (IC )
a
b
Sivelestat
50

Medicinal Chemistry Research (2021) 30:387–398
393
moiety in this scaffold plays a key role in providing hNE
inhibitory activity.
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
Molecular modeling
1
13
was performed on a Biotage Isolera one. H NMR,
C
1
9
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, 2-
disulfonyl 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 π–
π stacking interaction with the middle phenyl ring of com-
pound 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, benzene-
sulfonic 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.
NMR, and F NMR were recorded on Mercury400, Bruker
AVANCEΙΙΙ 400 spectrometer. Chemical shifts are refer-
enced 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%.
General procedure for compounds 4a–h
Carboxylic acid (1a–h) (679.04 μmol) was dissolved in
SOCl (2 mL). The mixture was stirred at room temperature
2
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).
Conclusion
In this work, starting from hNE covalent inhibitor SuEFx-1,
we designed and synthesized two series of ortho- and meta-
substituted benzenesulfonic acids and the inhibitory activ-
ities 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 IC50 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 bind-
ing 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.
2-(4-methylbenzamido)benzenesulfonic acid (4a) Yield =
1
84%; white solid. H NMR (400 MHz, CD OD) δ 8.51 (dd,
3
J = 8.3, 2.0 Hz, 1H), 7.94–7.88 (m, 3H), 7.52–7.43 (m,
1H), 7.34 (d, J = 8.0 Hz, 2H), 7.22–7.14 (m, 1H), 2.42 (s,
1
3
3H). C NMR (101 MHz, DMSO-d ) δ 164.48, 142.49,
6
135.83, 135.76, 132.39, 130.30, 129.87, 127.53, 127.44,
122.99, 120.13, 21.52. HRMS (ESI): m/z calcd for
+
C H O NS [M + H] , 292.06381; found, 292.06351.
1
4
14
4
2-(2-(4-(trifluoromethyl)phenyl)acetamido)benzenesulfonic
1
acid (4b) Yield = 79%; white solid. H NMR (400 MHz,
DMSO-d ) δ 10.71 (s, 1H), 8.23 (d, J = 8.0 Hz, 1H), 7.70
6
(s, 1H), 7.67 (d, J = 7.6 Hz, 2H), 7.57 (d, J = 8.0 Hz, 2H),
7.29 (t, J = 7.9 Hz, 1H), 7.08–6.99 (m, 1H), 3.77 (s, 2H).
1
3
C NMR (101 MHz, DMSO-d ) δ 168.24, 140.63, 135.89,
6
135.42, 130.76, 130.11, 127.44, 125.64 (q, J = 3.8 Hz),
Experimental section
123.03, 120.19, 44.57. HRMS (ESI): m/z calcd for
+
C H O NF S [M + H] , 360.05119; found, 360.05170.
1
5
13
4
3
Chemistry
N-ethyl-N-isopropylpropan-2-aminium 2-(2-(naphthalen-2-yl)
All solvents and chemicals were purchased from commer-
cial sources: Sigma-Aldrich Chemical Co., Shanghai
acetamido)benzenesulfonate (4c) Yield = 85%; white
1
solid. H NMR (400 MHz, DMSO-d ) δ 10.77 (s, 1H), 8.29
6

394
Medicinal Chemistry Research (2021) 30:387–398
(
(
d, J = 8.0 Hz, 1H), 8.15 (s, 1H), 7.94–7.89 (m, 2H), 7.88
d, J = 4.7 Hz, 2H), 7.71 (dd, J = 7.8, 1.5 Hz, 1H),
HRMS (ESI): m/z calcd for C H O NS [M + H]+,
410.14206; found, 410.14246.
2
3
24
4
7
1
.55–7.45 (m, 3H), 7.34–7.27 (m, 1H), 7.04 (td, J = 7.7,
.0 Hz, 1H), 3.82 (s, 2H), 3.54 (p, J = 6.5 Hz, 2H), 3.06 (q,
3-(2-naphthamido)benzenesulfonic acid (4g) Yield = 90%;
13
1
J = 7.2 Hz, 2H), 1.19 (t, J = 7.3 Hz, 15H). C NMR
101 MHz, DMSO-d ) δ 168.93, 135.82, 135.61, 133.54,
white solid. H NMR (400 MHz, DMSO-d ) δ 13.08 (s,
6
(
1H), 8.61 (s, 1H), 8.11 (d, J = 7.6 Hz, 2H), 8.08–8.04 (m,
1H), 8.03–7.97 (m, 4H), 7.67–7.58 (m, 3H), 7.41–7.30 (m,
6
1
1
4
33.47, 132.43, 130.13, 128.32, 128.21, 128.19, 128.02,
27.98, 127.44, 126.61, 126.18, 122.93, 120.17, 54.08,
1
3
1H). C NMR (101 MHz, DMSO-d ) δ 167.93, 139.13,
6
5.34, 42.33, 18.50, 17.13, 12.94.HRMS (ESI): m/z calcd
135.41, 134.76, 132.63, 131.00, 129.76, 129.47, 128.81,
+
for C H O NS [M + H] , 342.07946; found, 342.07913.
128.65, 128.14, 127.29, 125.64, 124.99, 121.41, 120.76,
1
8
16
4
+
1
18.35. HRMS (ESI): m/z calcd for C H O NS [M + H] ,
1
7
14
4
N-ethyl-N-isopropylpropan-2-aminium 2-(2-([1,1′-biphenyl]-4-
328.06381; found, 328.06241.
yl)acetamido)benzenesulfonate (4d) Yield = 75%; white
1
solid. H NMR (400 MHz, DMSO-d ) δ 10.74 (s, 1H),
N-ethyl-N-isopropylpropan-2-aminium 3-(2-(6-methoxy-
naphthalen-2-yl)propanamido)benzenesulfonate (4h)
6
8
.29–8.26 (m, 1H), 7.68 (dd, J = 8.4, 1.3 Hz, 2H), 7.66–7.63
1
(
m, 2H), 7.62 (d, J = 1.8 Hz, 1H), 7.47 (dd, J = 7.1, 1.5 Hz,
Yield = 86%; white solid. H NMR (400 MHz, DMSO-
d6) δ 10.18 (s, 1H), 7.86 (s, 1H), 7.80 (d, J = 9.0 Hz, 2H),
7.77 (d, J = 8.7 Hz, 1H), 7.68–7.62 (m, 1H), 7.52 (dd, J
= 8.5, 1.6 Hz, 1H), 7.29–7.25 (m, 2H), 7.22 (t, J =
7.7 Hz, 1H), 7.14 (dd, J = 8.9, 2.5 Hz, 1H), 3.97 (q, J =
6.9 Hz, 1H), 3.85 (s, 3H), 3.55 (dt, J = 12.5, 5.9 Hz, 2H),
3.07 (q, J = 7.2, 6.6 Hz, 2H), 1.49 (d, J = 7.0 Hz, 3H),
2
8
3
H), 7.45–7.43 (m, 2H), 7.38–7.33 (m, 1H), 7.30 (td, J = 8.5,
.1, 1.6 Hz, 1H), 7.03 (td, J = 7.6, 1.2 Hz, 1H), 3.67 (s, 2H),
.59 (p, J = 6.5 Hz, 2H), 3.11 (q, J = 7.3 Hz, 2H), 1.22 (t,
1
3
J = 7.5 Hz, 15H). C NMR (101 MHz, DMSO-d ) δ 168.86,
6
1
1
5
40.41, 139.09, 135.80, 135.58, 135.08, 130.32, 130.07,
29.39, 127.80, 127.42, 127.16, 127.05, 122.85, 120.00,
1
3
4.01, 44.81, 42.28, 18.55, 17.20, 12.97. HRMS (ESI): m/z
1.21 (t, J = 8.1 Hz, 15H). C NMR (101 MHz, DMSO-
d6) δ 172.79, 157.52, 149.15, 139.14, 137.48, 133.70,
129.63, 128.87, 128.43, 127.27, 126.81, 125.86, 120.82,
+
calcd for C H O NS [M + H] , 368.09511; found,
2
0 18 4
3
68.09616.
1
19.60, 119.13, 117.16, 106.17, 55.63, 53.70, 46.35,
N-ethyl-N-isopropylpropan-2-aminium 3-(4′-methyl-[1,1′-
42.06, 21.63, 19.24, 13.06. HRMS (ESI): m/z calcd for
+
biphenyl]-2-carboxamido)benzenesulfonate (4e) Yield =
C H O NS [M + H] , 386.10567; found, 386.10602.
2
0
20
5
1
8
9%; white solid. H NMR (400 MHz, DMSO-d ) δ 10.27
6
(
s, 1H), 8.99 (s, 1H), 7.85 (s, 1H), 7.52 (t, J = 9.3 Hz, 3H),
General procedure for compounds 6a–j
7
7
.48–7.39 (m, 2H), 7.34 (d, J = 7.7 Hz, 2H), 7.28 (d, J =
.5 Hz, 1H), 7.24–7.12 (m, 3H), 3.68–3.52 (m, 2H), 3.12
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.
(
dd, J = 6.9, 4.6 Hz, 2H), 2.29 (s, 3H), 1.27 (dd, J = 11.8,
1
3
6
1
1
1
.4 Hz, 15H). C NMR (101 MHz, DMSO-d ) δ 168.42,
6
49.15, 139.55, 139.03, 137.63, 137.50, 136.99, 130.35,
30.14, 129.41, 128.61, 128.26, 127.41, 121.17, 120.06,
17.49, 53.75, 42.03, 21.16, 18.44, 17.17, 12.66. HRMS
+
(
ESI): m/z calcd for C H O NS [M + H] , 368.09511;
2
0
18
4
found, 368.09378.
N-ethyl-N-isopropylpropan-2-aminium
biphenyl]-4-carboxamido)benzenesulfonate (4f) Yield =
3-(4′-butyl-[1,1′-
N-ethyl-N-isopropylpropan-2-aminium 2-(3-(4-methoxyphenyl)
ureido)benzenesulfonate (6a) Yield = 84%; white solid. H
1
7
1
7
8
1
1
7%; white solid. 1H NMR (400 MHz, DMSO-d ) δ
NMR (400 MHz, DMSO-d ) δ 9.40 (s, 1H), 9.26 (s, 1H),
6
6
0.33 (s, 1H), 8.09 (s, 1H), 8.07 (d, J = 2.3 Hz, 2H),
.89–7.84 (m, 1H), 7.81 (d, J = 8.4 Hz, 2H), 7.68 (d, J =
.1 Hz, 2H), 7.37–7.29 (m, 4H), 3.57 (s, 1H), 3.08 (s,
H), 2.64 (t, J = 7.6 Hz, 2H), 1.60 (p, J = 7.5 Hz, 2H),
.35 (dt, J = 14.8, 7.3 Hz, 2H), 1.28–1.15 (m, 12H), 0.92
7.99 (d, J = 8.1 Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.43 (d,
J = 8.9 Hz, 2H), 7.24 (t, J = 7.6 Hz, 1H), 6.91 (t, J = 7.5 Hz,
1H), 6.84 (d, J = 8.9 Hz, 2H), 3.71 (s, 3H), 3.65–3.56 (m,
1
3
2H), 3.18–3.07 (m, 2H), 1.25 (t, J = 6.1 Hz, 15H). C NMR
(101 MHz, DMSO-d ) δ 154.72, 153.08, 136.78, 135.59,
6
1
3
(
t, J = 7.3 Hz, 3H). C NMR (101 MHz, DMSO-d ) δ
133.78, 129.59, 127.20, 121.10, 121.00, 120.72, 114.23,
6
1
1
1
65.52, 149.19, 143.53, 142.94, 139.11, 136.95, 133.70,
29.48, 128.86, 128.33, 127.25, 126.76, 121.33, 120.80,
18.33, 53.55, 41.97, 34.94, 33.55, 22.25, 21.60, 14.29.
55.59, 54.07, 42.32, 18.54, 17.19, 12.96. HRMS (ESI): m/z
+
calcd for C H O N S [M + H] , 323.06962; found,
1
4
15
5
2
323.06921.

Medicinal Chemistry Research (2021) 30:387–398
395
N-ethyl-N-isopropylpropan-2-aminium 2-(3-(4-chlorophenyl)
N-ethyl-N-isopropylpropan-2-aminium 3-(3-(3,5-bis(trifluoro-
ureido)benzenesulfonate (6b) Yield = 89%; white solid.
methyl)phenyl)ureido)benzenesulfonate (6f) Yield = 90%;
1
1
H NMR (400 MHz, DMSO-d ) δ 9.76 (s, 1H), 9.37 (s, 1H),
white solid. H NMR (400 MHz, DMSO-d ) δ 9.49 (s, 1H),
6
6
8
1
2
.18 (s, 1H), 7.97 (d, J = 8.1 Hz, 1H), 7.65 (dd, J = 7.7,
.2 Hz, 1H), 7.57 (d, J = 8.9 Hz, 2H), 7.30 (d, J = 8.9 Hz,
H), 7.28–7.24 (m, 1H), 6.95 (t, J = 7.5 Hz, 1H), 3.61 (ddp,
9.24 (s, 1H), 8.16 (s, 2H), 7.81–7.76 (m, 1H), 7.62 (s, 1H),
7.57 (dt, J = 6.5, 2.4 Hz, 1H), 7.30–7.24 (m, 2H), 3.61 (pd,
J = 6.6, 3.9 Hz, 2H), 3.13 (qd, J = 7.3, 4.3 Hz, 2H), 1.25 (t,
1
3
J = 10.3, 6.4, 3.8 Hz, 2H), 3.13 (qd, J = 7.3, 4.5 Hz, 2H),
J = 6.3 Hz, 15H). C NMR (101 MHz, DMSO-d ) δ
6
13
1
.25 (t, J = 6.1 Hz, 15H). C NMR (101 MHz, DMSO-d6)
152.84, 148.74, 142.45, 139.16, 131.50 (d, J = 32.5 Hz),
130.85 (d, J = 32.5 Hz), 128.80, 125.17, 122.45, 120.11,
119.30, 118.29, 116.45, 54.02, 42.28, 18.54, 17.20, 12.94.
δ 152.83, 139.78, 136.34, 135.88, 129.65, 128.87, 127.22,
1
1
3
25.55, 121.44, 121.27, 120.43, 54.07, 42.32, 18.55, 17.19,
+
+
2.96. HRMS (ESI): m/z calcd for C H O N ClS [M + H] ,
HRMS (ESI): m/z calcd for C H O N F S [M + H] ,
13
12
4
2
15 11
4
2 6
27.02008; found, 327.01978.
429.03382; found, 429.03354.
N-ethyl-N-isopropylpropan-2-aminium 2-(3-(4-(trifluoromethyl)
N-ethyl-N-isopropylpropan-2-aminium 3-(3-(3,5-dichlorophe-
phenyl)ureido)benzenesulfonate (6c) Yield = 85%; white
nyl)ureido)benzenesulfonate (6g) Yield = 82%; white
1
1
solid. H NMR (400 MHz, DMSO-d ) δ 10.06 (s, 1H), 9.46
solid. H NMR (400 MHz, DMSO-d ) δ 9.04 (s, 1H), 9.01
6
6
(
8
8
s, 1H), 8.18 (s, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.75 (d, J =
.6 Hz, 2H), 7.67 (dd, J = 7.7, 1.5 Hz, 1H), 7.61 (d, J =
.7 Hz, 2H), 7.31–7.25 (m, 1H), 7.02–6.92 (m, 1H), 3.60 (dp,
(s, 1H), 7.93–7.84 (m, 1H), 7.75–7.68 (m, 1H), 7.52 (dd,
J = 9.0, 5.6 Hz, 2H), 7.36 (dd, J = 8.8, 2.5 Hz, 1H), 7.25 (d,
J = 5.0 Hz, 2H), 3.61 (pd, J = 6.6, 4.0 Hz, 2H), 3.13 (qd,
1
3
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). C NMR (101 MHz, DMSO-d ) δ 152.76,
J = 7.4, 4.3 Hz, 2H), 1.24 (q, J = 6.7 Hz, 15H). C NMR
1
3
(101 MHz, DMSO-d ) δ 152.78, 148.71, 140.60, 139.42,
6
6
1
45.57, 144.56, 136.12, 129.86 (d, J = 27.6 Hz), 127.47 (d,
131.47, 131.04, 128.76, 123.44, 119.83, 119.61, 118.95,
118.69, 116.14, 53.95, 42.20, 18.59, 17.25, 13.00. HRMS
J = 44.7 Hz), 126.35 (d, J = 3.8 Hz), 123.77, 121.98 (d, J =
3
+
1.7 Hz), 121.60 (d, J = 30.2 Hz), 118.55, 115.83, 114.77,
(ESI): m/z calcd for C H O N Cl S [M + H] ,
1
3
11
4
2
2
5
4.08, 42.34, 18.53, 17.17, 12.94. HRMS (ESI): m/z calcd
360.98111; found, 360.98083.
+
for C H O N F S [M + H] , 361.04644; found,
1
4
12
4
2 3
3
61.04568.
N-ethyl-N-isopropylpropan-2-aminium 3-(3-(3,5-dimethylphe-
nyl)ureido)benzenesulfonate (6h) Yield = 80%; white
1
N-ethyl-N-isopropylpropan-2-aminium 2-(3-(4-(trifluoromethoxy)
solid. H NMR (400 MHz, DMSO-d ) δ 8.57 (s, 1H), 7.07
6
phenyl)ureido)benzenesulfonate (6d) Yield = 91%; white
(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, J = 11.0, 5.4 Hz, 2H), 2.22 (s,
1
solid. H NMR (400 MHz, DMSO-d ) δ 9.83 (s, 1H), 9.40 (s,
6
1
1
H), 8.55 (s, 1H), 7.99 (d, J = 8.2 Hz, 1H), 7.67 (d, J =
.5 Hz, 1H), 7.66–7.61 (m, 2H), 7.29 (d, J = 1.5 Hz, 1H), 7.25
1
3
6H), 1.25 (t, J = 6.3 Hz, 15H). C NMR (101 MHz,
(
3
s, 2H), 6.95 (t, J = 7.5 Hz, 1H), 3.60 (td, J = 6.6, 3.9 Hz, 2H),
.14–3.08 (m, 2H), 1.26–1.23 (m, 15H). C NMR (101 MHz,
DMSO-d ) δ 152.95, 149.90, 140.14, 138.12, 137.05,
129.45, 123.77, 121.86, 120.71, 118.08, 116.29, 54.06,
6
13
DMSO-d ) δ 152.88, 142.90 (d, J = 1.6 Hz), 140.07, 136.34,
42.31, 21.62, 18.55, 17.21, 12.94. HRMS (ESI): m/z calcd
6
+
135.86, 129.67, 128.82 (d, J = 224.2 Hz), 127.24, 121.96,
for C H O N S [M + H] , 321.09035; found,
1
5
17
4
2
1
21.47, 121.23, 120.08, 53.98, 42.24, 18.51, 17.18, 12.87.
321.09039.
+
HRMS (ESI): m/z calcd for C H O N F S [M + H] ,
14 12 5 2 3
3
77.04135; found, 377.04102.
N-ethyl-N-isopropylpropan-2-aminium 3-(3-([1,1′-biphenyl]-
-yl)ureido)benzenesulfonate (6i) Yield = 84%; white
2
1
N-ethyl-N-isopropylpropan-2-aminium 2-(3-(3,5-dichlorophe-
solid. H NMR (400 MHz, DMSO-d ) δ 9.09 (s, 1H), 8.21
6
nyl)ureido)benzenesulfonate (6e) Yield = 85%; white
(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), 7.16–7.09 (m,
1H), 3.61 (dq, J = 6.7, 4.2 Hz, 2H), 3.14 (dd, J = 7.3,
1
solid. H NMR (400 MHz, DMSO-d ) δ 9.98 (s, 1H), 9.47
6
(
s, 1H), 8.20 (s, 1H), 8.04–7.90 (m, 2H), 7.68 (d, J =
7
1
.5 Hz, 1H), 7.48 (q, J = 8.7 Hz, 2H), 7.29 (t, J = 7.4 Hz,
H), 6.98 (t, J = 7.3 Hz, 1H), 3.67–3.50 (m, 2H), 3.17–3.02
1
3
4.2 Hz, 2H), 1.25 (d, J = 6.2 Hz, 15H).
C NMR
13
(
m, 2H), 1.23 (t, J = 6.1 Hz, 15H). C NMR (101 MHz,
(101 MHz, DMSO-d ) δ 153.14, 149.30, 139.68, 138.92,
6
DMSO-d ) δ 152.69, 141.04, 136.07, 135.93, 131.30,
136.17, 133.15, 130.76, 129.63, 129.34, 128.55, 127.99,
123.74, 123.10, 119.42, 118.27, 115.84, 114.31, 113.64,
111.93, 54.07, 42.33, 18.54, 17.19, 12.94. HRMS (ESI): m/
6
1
1
30.91, 129.76, 127.25, 123.31, 121.75, 121.30, 119.89,
18.91, 54.07, 42.33, 18.52, 17.16, 12.93. HRMS (ESI): m/
+
+
z calcd for C H O N Cl S [M + H] , 360.98111; found,
z calcd for C H O N S [M + H] , 369.09035; found,
1
3
11
4
2
2
19 17
4
2
3
60.98172.
369.08954.

396
Medicinal Chemistry Research (2021) 30:387–398
N-ethyl-N-isopropylpropan-2-aminium 3-(3-benzylureido)ben-
zenesulfonate (6j) Yield = 90%; white solid. H NMR
(s, 1H), 8.92–8.82 (m, 1H), 8.08–8.00 (m, 1H), 7.85 (d, J =
8.2 Hz, 2H), 7.81 (t, J = 7.4 Hz, 1H), 7.34 (d, J = 8.0 Hz,
2H), 7.31 (d, J = 8.0 Hz, 1H), 2.45 (s, 3H). C NMR
1
1
3
(
400 MHz, DMSO-d ) δ 8.62 (s, 1H), 8.20 (s, 1H), 7.60 (s,
6
1H), 7.44 (d, J = 7.3 Hz, 1H), 7.32 (d, J = 6.5 Hz, 4H), 7.23
(101 MHz, DMSO-d ) δ 166.48, 142.81, 137.72, 137.56,
6
(
5
2
t, J = 6.6 Hz, 1H), 7.15 (q, J = 7.2 Hz, 2H), 6.58 (t, J =
131.16, 130.85, 130.80, 129.62, 128.37 (d, J = 21.7 Hz),
1
9
.7 Hz, 1H), 4.34–4.25 (m, 2H), 3.61 (dd, J = 10.1, 6.2 Hz,
128.20, 128.00, 21.54. F NMR (376 MHz, CDCl ) δ 66.47.
3
13
+
H), 3.16–3.10 (m, 2H), 1.24 (d, J = 6.2 Hz, 15H). C NMR
HRMS (ESI): m/z calcd for C H O NFS [M + H] ,
1
4 13 3
(
101 MHz, DMSO-d ) δ 155.64, 149.15, 140.85, 140.35,
294.05947; found, 294.05960.
6
128.76, 128.35, 127.59, 127.15, 118.09, 115.57, 54.07, 43.19,
4
2.33, 18.54, 17.19, 12.95. HRMS (ESI): m/z calcd for
Docking study
+
C H O N S [M + H] , 307.07470; found, 307.07449.
1
4 15 4 2
The X-ray crystallographic structure of the adduct of the
General procedure for compound 9a
neutrophil elastase and benzene-1,2-disulfonyl fluoride
(PDB Code: 6e69) was downloaded from the Protein Data
The starting materials 7a (418.31 μmol), 7b (627.46 μmol),
EtN (175.48 μL) and Pd(OAc) (4.18 μmol) were sequen-
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]. A
grid box with dimensions of 40 × 40 × 40 Å (-24.348, 1.759,
8.542) with a spacing of 0.375 Å was constructed around
the docking area using Autogrid 4.2 software. The docking
was performed using Vina with exhaustiveness grade 8,
with up to nine poses saved per molecule [32]. Accelrys
Discovery Studio Client 2018 (Accelrys, San Diego, CA,
USA) was used for interaction visualization, and the lowest
energy conformation was selected. The ligand interactions
with the neutrophil elastase was determined by PyMOL
0.99 and Accelrys Discovery Studio Client 2018 (Accelrys,
San Diego, CA, USA).
3
2
tially added to the solvent (1,4-dioxane:H O = 4:1)
2
(
2
10 mL). The mixture was stirred at room temperature for
h. After the solvent was evaporated in vacuo, ethyl acetate
was added, and washed with 1 N dilute hydrochloric acid
15 mL) and saturated brine (3 × 15 mL) successively. The
organic layer was dried with anhydrous Na SO . The final
(
2
4
compound 9a was purified by column chromatography
using ethyl acetate/petroleum ether 1:19 as the eluent.
4
′-fluoro-[1,1′-biphenyl]-3-sulfonyl fluoride (9a) Yield =
1
3
1
1
8
2
1
2
4%; white solid. H NMR (400 MHz, CDCl ) δ 8.22 (t, J =
3
.8 Hz, 1H), 8.06 (d, J = 7.9 Hz, 1H), 7.99 (d, J = 7.9 Hz,
H), 7.78 (d, J = 1.8 Hz, 1H), 7.76 (s, 2H), 7.72 (d, J =
1
3
.4 Hz, 2H). C NMR (101 MHz, CDCl ) δ 163.24 (d, J =
3
49.0 Hz), 142.22, 134.66–133.52 (m), 133.90, 130.21,
In vitro HNE inhibition studies [29]
28.97 (d, J = 8.1 Hz), 126.87, 126.66, 116.28 (d, J =
19
1.5 Hz). F NMR (376 MHz, CDCl ) δ 66.07, −112.89.
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
3
+
HRMS (ESI): m/z calcd for C H O F S [M + H] ,
12 8 2 2
255.02858; found, 255.02861.
General procedure for compound 9b
200 μL of enzyme assay containing 1 mU of hNE and 25 μM
Benzenesulfonic acid (4a) (171.63 μmol) was dissolved in
peptide substrates. The fluorescence measurement was
immediately performed with E = 380 nm and E = 460 nm
SOCl (2 mL). The mixture was stirred at room temperature
2
x
m
for 2 h. After evaporation of the solvent, the intermediate
for 10 min to calculate the kinetic changes of the fluorescence.
The inhibition was calculated using Graphpad Prism. Sive-
lestat was used as the reference compound.
(
8) was dissolved in anhydrous CH CN (10 mL) in which
3
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 satu-
rated brine (3 × 15 mL). The organic layer was dried with
anhydrous Na SO . The final compound 9a was purified by
Cell viability assay
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
2
4
column chromatography using ethyl acetate/petroleum ether
:9 as the eluent.
1
2
2
-(4-methylbenzamido)benzenesulfonyl fluoride (9b) Yield =
1
6%; yellow flaky solid. H NMR (400 MHz, CDCl ) δ 9.75
10% fetal bovine serum (Gibco) at 37 °C with 5% CO . All
3
2

Medicinal Chemistry Research (2021) 30:387–398
397
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-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell
proliferation assay [33, 34].
14. Rubio F, Cooley J, Accurso FJ, Remold-O’Donnell E. Linkage of
neutrophil serine proteases and decreased surfactant protein-A (SP-
A) levels in inflammatory lung disease. Thorax. 2004;59:318–23.
1
5. Fan E, Brodie D, Slutsky AS. Acute respiratory distress syn-
drome: advances in diagnosis and treatment. JAMA. 2018;319:
698–710.
1
6. Fischer BM, Voynow JA. Neutrophil elastase induces MUC5AC
gene expression in airway epithelium via a pathway involving
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.
reactive oxygen species. Am
002;26:447–52.
J Respir Cell Mol Biol.
2
1
1
7. Mohamed MMA, El-Shimy IA, Hadi MA. Neutrophil elastase
inhibitors: a potential prophylactic treatment option for SARS-
CoV-2-induced respiratory complications? Crit Care. 2020;24:311.
8. Lee WL, Downey GP. Leukocyte elastase: physiological functions
and role in acute lung injury. Am J Respir Crit Care Med.
2001;164:896–904.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
19. Siedle B, Hrenn A, Merfort I. Natural compounds as inhibitors of
human neutrophil elastase. Planta Med. 2007;73:401–20.
2
0. Martins FT, Assis DM, Dos Santos MH, Camps I, Veloso MP,
Juliano MA, Alves LC, Doriguetto AC. Natural polyprenylated
benzophenones inhibiting cysteine and serine proteases. Eur J
Med Chem. 2009;44:1230–9.
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Competitive neutrophil elastase inhibitory isoflavones from the
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Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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