New 3-unsubstituted isoxazolones as potent human neutrophil elastase inhibitors: Synthesis and molecular dynamic simulation

Article abstract of DOI:10.1002/ddr.21625

Human neutrophil elastase (HNE) is a proteolytic enzyme belonging to the serine protease family and is involved in a variety of pathologies. Thus, compounds able to inhibit HNE represent promising therapeutics for the treatment of inflammatory diseases. H

Full text of DOI:10.1002/ddr.21625

Received: 26 September 2019
DOI: 10.1002/ddr.21625
Revised: 28 October 2019
Accepted: 9 November 2019
R E S E A R C H A R T I C L E
New 3-unsubstituted isoxazolones as potent human neutrophil
elastase inhibitors: Synthesis and molecular dynamic simulation
Maria Paola Giovannoni1
| Letizia Crocetti1
| Niccolò Cantini1
|
Gabriella Guerrini1 | Claudia Vergelli1 | Antonella Iacovone1
|
Elisabetta Teodori1 | Igor A. Schepetkin2 | Mark T. Quinn2 | Samuele Ciattini3
Patrizia Rossi4 | Paola Paoli4
|
1Neurofarba, Pharmaceutical and
Abstract
Nutraceutical Section, University of Florence,
Sesto Fiorentino, Italy
Human neutrophil elastase (HNE) is a proteolytic enzyme belonging to the serine pro-
tease family and is involved in a variety of pathologies. Thus, compounds able to
inhibit HNE represent promising therapeutics for the treatment of inflammatory dis-
eases. Here, we report the further elaboration of our previously reported
3-methylisoxazolone derivatives, synthesizing a new series of 3-nor-derivatives bear-
ing different substituents at the 4-phenyl ring. The most potent compounds 3a, 3g,
and 3h, had IC50 values of 16, 11, and 18 nM, respectively. Molecular modeling stud-
ies and molecular dynamic (MD) simulations demonstrated no substantial differences
between the 3-methylisoxazole derivatives previously tested and the corresponding
3-unsubstituted derivatives in the snapshot conformations sampled during the MD
simulations, which is consistent with their similar levels of HNE inhibitory activity.
Thus, we conclude that the isoxazolone scaffold is a good scaffold for developing
HNE inhibitors, as it tolerates several modifications when adhering to basic scaffold
requirements, and the resulting derivatives are quite potent HNE inhibitors.
2Department of Microbiology and
Immunology, Montana State University,
Bozeman, Montana
3Center of Structural Crystallography,
University of Florence, Sesto Fiorentino, Italy
4Department of Industrial Engineering,
University of Florence, Florence, Italy
Correspondence
Letizia Crocetti, Dipartimento di Neurofarba,
University of Florence, Via Ugo Schiff 6, Sesto
Fiorentino, Firenze 50019, Italy.
Email: letizia.crocetti@unifi.it
Funding information
Montana State University Agricultural
Experiment Station; National Institutes of
Health IDeA Program COBRE, Grant/Award
Number: GM110732; USDA National Institute
of Food and Agriculture Hatch project, Grant/
Award Number: 1009546
K E Y W O R D S
human neutrophil elastase inhibitors, molecular and crystal structures, molecular modeling
1
|
INTRODUCTION
plays a central role in several inflammatory diseases, such as chronic
obstructive pulmonary disease (Lucas, Costa, Guedes, & Moreira,
Neutrophils are one of primary immune cell populations in humans
and produce inflammatory mediators that target invading microorgan-
isms and foreign materials (Korkmaz, Horwitz, Jenne, & Gauthier,
2010; Stapels, Gijsbrecht, & Rooijakkers, 2015). Neutrophils adhere to
the endothelium, migrate to sites of infection/inflammation, and
release granule components, such as human neutrophil elastase
(HNE). HNE is a small, basic, and soluble glycoprotein of about 30 kDa
(Sinha et al., 1987) and is stored in neutrophil azurophilic granules
with other serine proteases. HNE and other serine proteases are key
enzymes involved in inflammation, coagulation, and host defense
(Massberg et al., 2010; Pham, 2006). On the other hand, HNE also
2013; Pandey, De, & Mishra, 2017), cystic fibrosis (Twigg et al., 2015),
rheumatoid arthritis (Hilbert, Schiller, Arnhold, & Arnold, 2002), cancer
(Akizuki et al., 2007; Lerman et al., 2017; Lerman & Hammes, 2017;
Vaguliene, Zemaitis, Lavinskiene, Miliauskas, & Sakalauskas, 2013),
and other disorders with an inflammatory component (Bronze-da-
Rocha & Santos-Silva, 2018; Marto et al., 2018). Because of their
potential for proteolytic tissue damage, neutrophil protease activity is
regulated by specific endogenous (α-1 antitrypsin, secretory leucocyte
protease inhibitor and elafin) and exogenous inhibitors, thereby
preventing several inflammatory diseases with severe impact on organ
tissue integrity (von Nussbaum et al., 2016; von Nussbaum & Li,
Drug Dev Res. 2019;1–12.
wileyonlinelibrary.com/journal/ddr
© 2019 Wiley Periodicals, Inc.
1

2
GIOVANNONI ET AL.
2015). Therefore, HNE is an attractive therapeutic target, and the
design of new small molecule inhibitors of this enzyme is an area of
extensive investigation. Nevertheless, only two HNE inhibitors are
commercially available: Sivelestat (Elaspol® 100), which is in use for
the treatment of ALI and ARDS only marketed in Korea and Japan
(Iwata et al., 2010), and Prolastin (purified α1-AT), which is use for the
treatment of α1-antitrypsin deficiency (Bayer Corporation, 2002). In
order to develop new compounds that modulate the HNE proteolytic
activity, our research group has investigated several molecular scaf-
folds, such as indazoles (Crocetti et al., 2011, 2013), indoles (Crocetti
et al., 2016), azaindoles (Crocetti et al., 2018; Giovannoni et al., 2019),
cinnolinones (Giovannoni et al., 2016), and thiazolones (Crocetti et al.,
2017). Most recently, we focused on the synthesis isoxazol-5(2H)-one
derivatives (Giovannoni et al., 2018; Vergelli et al., 2017). These new
isoxazolone-based derivatives exhibited HNE inhibitory activity in the
nanomolar range (IC50 = 20–96 nM), and molecular modeling studies
allowed us to determine that the endocyclic carbonyl at Position 5 of
the isoxazolone was the group attacked by Ser195 of the catalytic
triad. The most potent isoxazolones were further evaluated for chemi-
cal stability, and the results showed that these derivatives were more
stable than our previously described HNE inhibitors with a bicyclic
scaffold. Finally, kinetic experiments demonstrated that these com-
pounds acted as competitive HNE inhibitors, and structure–activity
relationship (SAR) analyses established that the amide function at
Position 2 was important for the activity, and the best substituents at
this position were m-tolylcarbonyl and cyclopropylcarbonyl groups
(Giovannoni et al., 2018). In order to complete SAR analysis for this
class of HNE inhibitors, we designed and synthesized a new series of
4-phenyl-3-unsubstituted derivatives (Figure 1) bearing different sub-
stituents on the 4-phenyl ring while maintaining the best fragments at
commercial plates (Merck, Durham, NC) were used for analytical TLC
to follow the course of the reactions. Silica gel 60 (Merck 70–230
mesh, Merck, Durham, NC) was used for column chromatography. 1H-
NMR, 13C-NMR, 1H-13C HSQC, and 1H-13C HMBC bidimensional
spectra were recorded on an Avance 400 instrument (Bruker Biospin
Version 002 with SGU, Bruker Inc., Billerica, MA). Chemical shifts (δ)
are reported in ppm to the nearest 0.01 ppm using the solvent as an
internal standard. Coupling constants (J values) are given in Hz and
were calculated using the TopSpin 1.3 software (Nicolet Instrument
Corp., Madison, WI) and are rounded to the nearest 0.1 vHz. Mass
spectra (m/z) were recorded on an ESI-TOF mass spectrometer
(Brucker Micro TOF, Bruker Inc., Billerica, MA), and reported mass
values are within the error limits of 5 ppm mass units. Microanalyses
indicated by the symbols of the elements or functions were per-
formed with a Perkin–Elmer 260 elemental analyzer (PerkinElmer,
Inc., Waltham, MA) for C, H, and N, and the results were within 0.4%
of the theoretical values, unless otherwise stated. Reagents and
starting material were commercially available.
2.2
|
Chemistry
2.2.1
|
Synthesis of 4-(5-Oxo-2,5-dihydroisoxazol-
4-yl)benzonitrile (2h)
A suspension of intermediate 1h (Alves, Dazinger, Veiros, & Kirchner,
2010; 1.38 mmol) in about 6 ml of MeOH/H2O (10:1) was heated at
100ꢀC, and 2.76 mmol of hydroxylamine hydrochloride was added.
The mixture was stirred at reflux for 4 hr. After cooling, the solvent
was evaporated under vacuum to obtain the crude compound, which
was purified by column chromatography using dichloromethane/
methanol 8:2 as the eluent. Yield = 82%; mp = 141–143ꢀC (EtOH).
1H-NMR (400 MHz, DMSO-d6) δ 8.72 (s, 1H, CH), 7.79 (d, 2H, Ar,
J = 8.0 Hz), 7.58 (d, 2H, Ar, J = 8.0 Hz). 13C-NMR (100 MHz, CDCl3) δ
169.2 (C), 147.1 (CH), 136.8 (C), 132.1 (2 × CH), 127.1 (2 × CH),
111.8 (C), 111.6 (C), 96.4 (C). ESI-MS calcd. For C10H6N2O2, 186.17;
found: m/z 187.05 [M + H]+. Anal. C10H6N2O2 (C, H, N).
Position
2 of the isoxazolone scaffold (i.e., m-tolylcarbonyl or
cyclopropylcarbonyl groups).
2
|
EXPERIMENTAL SECTION
| Material and methods
2.1
All melting points were determined on a Büchi apparatus (New Castle,
DE) and are uncorrected. Extracts were dried over Na2SO4, and the
solvents were removed under reduced pressure. Merck F-254
2.2.2
4a-i
|
General procedure for compounds 3a-i and
To a suspension of the appropriate substrate 2a-i (Beccalli, La Rosa, &
Marchesini, 1984; Snyder et al., 2004; 0.86 mmol) in 10 ml of anhy-
drous THF, 1.72 mmol of sodium hydride (60% dispersion in mineral
oil) and 1.03 mmol of cyclopropanecarbonyl chloride or m-toluoyl
chloride were added. The mixture was stirred at room temperature
overnight. The solvent was concentrated in vacuo and the resulting
mixture was quenched by adding cold water (10 ml). The suspension
was extracted with ethyl acetate (3 × 15 mL), dried on anhydrous
sodium sulfate, and then filtered to obtain the final compounds 3a-i
and 4a-i, which were purified by crystallization from ethanol (3a, 3c,
3e-3i, 4d-f, and 4h) or by column chromatography using cyclohexane/
FIGURE 1 Previously synthesized isoxazolones (Giovannoni et al.,
2018; Vergelli et al., 2017) and new derivatives

GIOVANNONI ET AL.
3
ethyl acetate 6:1 (3b, 3d, 4b, 4c, 4g, and 4i) or hexane/acetone 4:1
(4a) as eluents.
4-(4-Chlorophenyl)-2-(cyclopropanecarbonyl)isoxazol-5(2H)-one (3f).
Yield = 64%; mp = 144–146ꢀC (EtOH). 1H-NMR (400 MHz, CDCl3) δ
8.71 (s, 1H, CH), 7.73 (d, 2H, Ar, J = 8.4 Hz), 7.38 (d, 2H, Ar, J = 8.4 Hz),
2.40–2.35 (m, 1H, CH cC3H5), 1.34–1.29 (m, 2H, CH2 cC3H5),
1.21–1.16 (m, 2H, CH2 cC3H5). 13C-NMR (100 MHz, CDCl3) δ 166.7
(C), 165.8 (C), 136.0 (CH), 134.5 (C), 129.2 (CH), 127.0 (CH), 125.9 (C),
107.0 (C), 11.8 (CH), 11.3 (CH2). IR ѵ (cm−1): 1,606 (C C isox), 1,693
(C O amide), 1,759 (C O lactone), 3,099 (CH isox). ESI-MS calcd. for
2-(Cyclopropanecarbonyl)-4-phenylisoxazol-5(2H)-one
(3a).
Yield = 28%; mp = 171–172ꢀC (EtOH). 1H-NMR (400 MHz, CDCl3) δ
8.72 (s, 1H, CH), 7.78 (d, 2H, Ar, J = 7.2 Hz), 7.42 (t, 1H, Ar, J = 7.2 Hz),
7.36 (d, 2H, Ar, J = 6.0 Hz), 2.45–2.35 (m, 1H, CH cC3H5), 1.31–1.26
(m, 2H, CH2 cC3H5), 1.20–1.15 (m, 2H, CH2 cC3H5). 13C-NMR
(100 MHz, CDCl3) δ 166.7 (C), 165.9 (C), 136.0 (CH), 128.9 (CH),
128.6 (CH), 127.4 (C), 125.8 (CH), 108.1 (C), 11.8 (CH), 11.2 (CH2). IR
ѵ (cm−1): 1,591 (C C isox), 1,693 (C O amide), 1,759 (C O lactone),
3,103 (CH isox). ESI-MS calcd. for C13H11NO3, 229.24; found: m/z
230.08 [M + H]+. Anal. C13H11NO3 (C, H, N).
C
C
13H10ClNO3, 263.68; found: m/z 264.04 [M + H]+. Anal.
13H10ClNO3 (C, H, N).
2-(Cyclopropanecarbonyl)-4-(4-methoxyphenyl)isoxazol-5(2H)-one
(3g). Yield = 25%; mp = 198–200ꢀC (EtOH). 1H-NMR (400 MHz,
CDCl3) δ 8.62 (s, 1H, CH), 7.72 (d, 2H, Ar, J = 8.2 Hz), 6.94 (d, 2H, Ar,
J = 8.2 Hz), 3.83 (s, 3H, OCH3), 2.40–2.35 (m, 1H, CH cC3H5),
1.30–1.25 (m, 2H, CH2 cC3H5), 1.20–1.14 (m, 2H, CH2 cC3H5). 13C-
NMR (100 MHz, CDCl3) δ 166.7 (C), 166.2 (C), 159.9 (C), 134.8 (CH),
127.2 (CH), 119.7 (C), 114.4 (CH), 108.2 (C), 55.3 (CH3), 11.7 (CH),
11.0 (CH2). IR ѵ (cm−1): 1,597 (C C isox), 1,676 (C O amide), 1,762
2-(Cyclopropanecarbonyl)-4-(p-tolyl)isoxazol-5(2H)-one
(3b).
Yield = 41%; mp = 130–133ꢀC (EtOH). 1H-NMR (400 MHz, CDCl3) δ
8.67 (s, 1H, CH), 7.67 (d, 2H, Ar, J = 8.2 Hz), 7.22 (d, 2H, Ar, J = 8.2 Hz),
2.45–2.35 (m, 4H, 1H CH cC3H5 + 3H CH3), 1.31–1.26 (m, 2H, CH2
cC3H5), 1.19–1.14 (m, 2H, CH2 cC3H5). 13C-NMR (100 MHz, CDCl3) δ
166.7 (C), 166.0 (C), 138.7 (C), 135.5 (CH), 129.6 (CH), 125.7 (CH),
124.4 (C), 108.3 (C), 21.3 (CH3), 11.7 (CH), 11.0 (CH2). IR ѵ (cm−1):
1,600 (C C isox), 1,678 (C O amide), 1,770 (C O lactone), 3,095
(CH isox). ESI-MS calcd. for C14H13NO3, 243.26; found: m/z 244.09
[M + H]+. Anal. C14H13NO3 (C, H, N).
(C
O lactone), 3,095 (CH isox). ESI-MS calcd. for C14H13NO4,
259.26; found: m/z 260.09 [M + H]+. Anal. C14H13NO4 (C, H, N).
4-(2-(Cyclopropanecarbonyl)-5-oxo-2,5-dihydroisoxazol-4-yl)benzonitrile
(3h). Yield = 31%; mp = 208–210ꢀC (EtOH). 1H-NMR (400 MHz, CDCl3)
δ 8.82 (s, 1H, CH), 7.92 (d, 2H, Ar, J = 8.2 Hz), 7.70 (d, 2H, Ar, J = 8.2 Hz),
2.43–2.38 (m, 1H, CH cC3H5), 1.35–1.30 (m, 2H, CH2 cC3H5), 1.23–1.18
(m, 2H, CH2 cC3H5). 13C-NMR (100 MHz, CDCl3) δ 166.7 (C), 165.8 (C),
136.8 (C), 132.1 (CH), 127.1 (CH), 126.0 (CH), 118.6 (C), 111.8 (C), 109.6
2-(Cyclopropanecarbonyl)-4-(m-tolyl)isoxazol-5(2H)-one
(3c).
Yield = 22%; mp = 155–157ꢀC (EtOH). 1H-NMR (400 MHz, CDCl3) δ
8.69 (s, 1H, CH), 7.61 (s, 1H, Ar), 7.57 (d, 1H, Ar, J = 8.0 Hz), 7.30 (t,
1H, Ar, J = 7.6 Hz), 7.16 (d, 1H, Ar, J = 7.2 Hz), 2.44–2.38 (m, 4H, 1H
CH cC3H5 + 3H CH3), 1.28–1.23 (m, 2H, CH2 cC3H5), 1.21–1.16 (m,
2H, CH2 cC3H5). 13C-NMR (100 MHz, CDCl3) δ 166.7 (C), 166.0 (C),
138.7 (C), 135.9 (CH), 129.4 (CH), 128.8 (CH), 127.3 (C), 126.4 (CH),
122.9 (CH), 108.2 (C), 21.4 (CH3), 11.8 (CH), 11.1 (CH2). IR ѵ (cm−1):
1,589 (C C isox), 1,647 (C O amide), 1,768 (C O lactone), 3,103
(CH isox). ESI-MS calcd. for C14H13NO3, 243.26; found: m/z 244.09
[M + H]+. Anal. C14H13NO3 (C, H, N).
(C), 11.9 (CH), 11.6 (CH2). IR ѵ (cm−1): 1,587 (C C isox), 1,712 (C
amide), 1,753 (C O lactone), 2,223 (CN), 3,082 (CH isox). ESI-MS calcd.
for
14H10N2O3, 254.24; found: m/z 255.07 [M + H]+. Anal.
O
C
C14H10N2O3 (C, H, N).
2-(Cyclopropanecarbonyl)-4-(4-(trifluoromethyl)phenyl)isoxazol-5(2H)-
one (3i). Yield = 31%; mp = 210–212ꢀC (EtOH). 1H-NMR (400 MHz,
CDCl3) δ 8.80 (s, 1H, CH), 7.92 (d, 2H, Ar, J = 8.0 Hz), 7.67 (d, 2H, Ar,
J = 8.0 Hz), 2.44–2.39 (m, 1H CH cC3H5), 1.35–1.30 (m, 2H, CH2
cC3H5), 1.24–1.19 (m, 2H, CH2 cC3H5). 13C-NMR (100 MHz, CDCl3) δ
166.8 (C), 165.7 (C), 136.8 (CH), 134.9 (C), 131.1 (C), 125.9 (CH), 124.1
(C), 106.5 (C), 11.8 (CH), 11.5 (CH2). IR ѵ (cm-1): 1,598 (C C isox),
1,693 (C O amide), 1,759 (C O lactone), 3,089 (CH isox). ESI-MS
calcd. for C14H10F3NO3, 297.23; found: m/z 298.06 [M + H]+. Anal.
2-(Cyclopropanecarbonyl)-4-(o-tolyl)isoxazol-5(2H)-one
(3d).
Yield = 60%; mp = 90–92ꢀC (EtOH). 1H-NMR (400 MHz, CDCl3) δ
8.49 (s, 1H, CH), 7.48 (d, 1H, Ar, J = 7.2 Hz), 7.28–7.22 (m, 3H, Ar),
2.45–2.35 (m, 4H, 1H CH cC3H5 + 3H CH3), 1.30–1.25 (m, 2H, CH2
cC3H5), 1.20–1.15 (m, 2H, CH2 cC3H5). 13C-NMR (100 MHz, CDCl3) δ
166.8 (C), 165.2 (C), 138.5 (CH), 136.7 (C), 130.8 (CH), 129.6 (CH),
129.0 (CH), 126.4 (C), 126.1 (CH), 108.5 (C), 20.6 (CH3), 11.8 (CH),
11.0 (CH2). IR ѵ (cm−1): 1,602 (C C isox), 1,697 (C O amide), 1,761
(C O lactone), 3,097 (CH isox). ESI-MS calcd. for C14H13NO3,
243.26; found: m/z 244.09 [M + H]+. Anal. C14H13NO3 (C, H, N).
4-(4-Bromophenyl)-2-(cyclopropanecarbonyl)isoxazol-5(2H)-one (3e).
Yield = 30%; mp = 143–145ꢀC (EtOH). 1H-NMR (400 MHz, CDCl3) δ
8.72 (s, 1H, CH), 7.66 (d, 2H, Ar, J = 8.0 Hz), 7.55 (d, 2H, Ar, J = 8.0 Hz),
2.40–2.35 (m, 1H, CH cC3H5), 1.34–1.28 (m, 2H, CH2 cC3H5),
1.22–1.17 (m, 2H, CH2 cC3H5). 13C-NMR (100 MHz, CDCl3) δ 166.1
(C), 165.8 (C), 136.0 (CH), 132.1 (CH), 127.2 (CH), 126.4 (C), 122.6 (C),
109.5 (C), 11.8 (CH), 11.3 (CH2). IR ѵ (cm−1): 1,606 (C C isox), 1,685
(C O amide), 1,759 (C O lactone), 3,086 (CH isox). ESI-MS calcd. for
C14H10F3NO3 (C, H, N).
2-(3-Methylbenzoyl)-4-phenylisoxazol-5(2H)-one (4a). Yield = 27%;
mp = 120–121ꢀC (EtOH). 1H-NMR (400 MHz, CDCl3) δ 8.89 (s, 1H,
CH), 7.85–7.80 (m, 4H, Ar), 7.45–7.37 (m, 5H, Ar), 2.45 (s, 3H, m-CH3-
Ph). 13C-NMR (100 MHz, CDCl3) δ 166.1 (C), 160.7 (C), 138.7 (C),
138.0 (CH), 134.6 (CH), 130.5 (CH), 129.6 (C), 129.2 (CH), 129.0 (CH),
128.8 (CH), 128.6 (CH), 127.3 (CH), 126.9 (CH), 125.9 (CH), 124.2 (C),
108.7 (C), 21.3 (CH3). IR ѵ (cm−1): 1,589 (C C isox), 1,672 (C
amide), 1,768 (C O lactone), 3,099 (CH isox). ESI-MS calcd. for
17H13NO3, 279.29; found: m/z 280.09 [M + H]+. Anal. C17H13NO3
(C, H, N).
2-(3-Methylbenzoyl)-4-(p-tolyl)isoxazol-5(2H)-one (4b). Yield = 47%;
O
C
C
C
13H10BrNO3, 308.13; found: m/z 308.98 [M + H]+. Anal.
mp = 90–92ꢀC (EtOH). 1H-NMR (400 MHz, CDCl3) δ 8.83 (s, 1H, CH),
13H10BrNO3 (C, H, N).
7.80 (d, 2H, Ar, J = 8.2 Hz), 7.71 (d, 2H, Ar, J = 8.2 Hz), 7.46–7.39 (m,

4
GIOVANNONI ET AL.
2H, Ar), 7.23 (d, 2H, Ar, J = 7.6 Hz), 2.44 (s, 3H, m-CH3-Ph), 2.37 (s,
3H, p-CH3-Ph). 13C-NMR (100 MHz, CDCl3) δ 166.2 (C), 160.6 (C),
138.9 (C), 138.6 (C), 137.5 (CH), 134.5 (CH), 130.5 (CH), 129.6 (CH),
129.3 (C), 128.5 (CH), 127.2 (CH), 125.8 (CH), 124.3 (C), 108.8 (C),
2H, Ar, J = 8.8 Hz), 3.87 (s, 3H, OCH3), 2.48 (s, 3H, m-CH3-Ph). 13C-
NMR (100 MHz, CDCl3) δ 165.8 (C), 157.0 (C), 159.8 (C), 138.5 (C),
134.4 (CH), 130.4 (CH), 129.3 (CH), 129.0 (CH), 128.6 (C), 128.5 (CH),
127.4 (CH), 127.2 (CH), 114.4 (C), 114.1 (CH), 109.8 (C), 55.3 (CH3),
21.4 (CH3). IR ѵ (cm−1): 1,604 (C C isox), 1,668 (C O amide), 1,763
21.4 (CH3), 21.3 (CH3). IR ѵ (cm−1): 1,597 (C C isox), 1,660 (C
amide), 1,759 (C O lactone), 3,099 (CH isox). ESI-MS calcd. for
18H15NO3, 293.32; found: m/z 294.11 [M + H]+. Anal. C18H15NO3
(C, H, N).
2-(3-Methylbenzoyl)-4-(m-tolyl)isoxazol-5(2H)-one (4c). Yield = 42%;
O
(C
O lactone), 3,093 (CH isox). ESI-MS calcd. for C18H15NO4,
C
309.32; found: m/z 310.10 [M + H]+. Anal. C18H15NO4 (C, H, N).
4-(2-(3-Methylbenzoyl)-5-oxo-2,5-dihydroisoxazol-4-yl)benzonitrile
(4h). Yield = 28%; mp = 165–167ꢀC (EtOH). 1H-NMR (400 MHz,
CDCl3) δ 9.02 (s, 1H, CH), 7.96 (d, 2H, Ar, J = 8.0 Hz), 7.83 (d, 2H, Ar,
J = 8.4 Hz), 7.72 (d, 2H, Ar, J = 8.4 Hz), 7.49 (d, 1H, Ar, J = 7.2 Hz),
7.44 (t, 1H, Ar, J = 7.0 Hz), 2.46 (s, 3H, CH3). 13C-NMR (100 MHz,
CDCl3) δ 165.4 (C), 160.6 (C), 139.6 (CH), 138.8 (C), 135.0 (CH), 132.7
(CH), 132.1 (C), 130.6 (CH), 128.7 (CH), 127.4 (CH), 126.2 (CH), 118.4
(C), 112.0 (C), 106.4 (C), 21.3 (CH3). IR ѵ (cm−1): 1,678 (C C isox),
1,722 (C O amide), 1,737 (C O lactone), 2,222 (CN), 3,080 (CH isox).
ESI-MS calcd. for C18H12N2O3, 304.30; found: m/z 305.09 [M + H]+.
Anal. C18H12N2O3 (C, H, N).
mp = 135–137ꢀC (EtOH). 1H-NMR (400 MHz, CDCl3) δ 8.86 (s, 1H,
CH), 7.81 (d, 2H, Ar, J = 8.0 Hz), 7.64 (s, 1H, Ar), 7.61 (d, 1H, Ar,
J = 8.0 Hz), 7.46–7.39 (m, 2H, Ar), 7.31 (t, 1H, Ar, J = 7.6 Hz), 7.17 (d,
1H, Ar, J = 7.2 Hz), 2.44 (s, 3H, m-CH3-Ph-CO), 2.39 (s, 3H, m-CH3-
Ph). 13C-NMR (100 MHz, CDCl3) δ 165.8 (C), 157.6 (C), 138.6 (C),
138.0 (CH), 134.5 (CH), 132.4 (C), 130.5 (CH), 129.6 (CH), 129.3 (C),
128.8 (CH), 128.5 (CH), 127.2 (CH), 126.5 (CH), 125.0 (C), 123.0 (CH),
109.6 (C), 21.4 (CH3), 21.3 (CH3). IR ѵ (cm−1): 1,610 (C C isox), 1,672
(C O amide), 1,759 (C O lactone), 3,105 (CH isox). ESI-MS calcd. for
C
18H15NO3, 293.32; found: m/z 294.11 [M + H]+. Anal. C18H15NO3
(C, H, N).
2-(3-Methylbenzoyl)-4-(o-tolyl)isoxazol-5(2H)-one (4d). Yield = 60%;
2-(3-Methylbenzoyl)-4-(4-[trifluoromethyl]phenyl)isoxazol-5(2H)-one
(4i). Yield = 72%; mp = 164–166ꢀC (EtOH). 1H-NMR (400 MHz,
CDCl3) δ 9.00 (s, 1H, CH), 7.96 (d, 2H, Ar, J = 8.0 Hz), 7.83 (d, 2H, Ar,
J = 8.8 Hz), 7.69 (d, 2H, Ar, J = 8.0 Hz), 7.49–7.41 (m, 2H, Ar), 2.45 (s,
3H, CH3). 13C-NMR (CDCl3) δ 165.83 (C), 157.0 (C), 139.0 (CH),
138.5 (C), 135.8 (C), 134.4 (CH), 134.1 (C), 131.0 (CH), 130.6 (CH),
130.2 (C), 129.8 (CH), 128.6 (CH), 127.4 (CH), 126.0 (CH), 125.9 (CH),
124.1 (C), 109.6 (C), 21.3 (CH3). IR ѵ (cm−1): 1,598 (C C isox), 1,693
(C O amide), 1,759 (C O lactone), 3,089 (CH isox). ESI-MS calcd. for
mp = 95–96ꢀC (EtOH). 1H-NMR (400 mHz, CDCl3) δ 8.68 (s, 1H, CH),
7.83 (d, 2H, Ar, J = 8.4 Hz), 7.51 (d, 1H, Ar, J = 7.2 Hz), 7.45–7.40 (m,
2H, Ar), 7.34–7.29 (m, 3H, Ar), 2.45 (s, 3H, m-CH3-Ph), 2.42 (s, 3H, o-
CH3-Ph). 13C-NMR (100 MHz, CDCl3) δ 166.8 (C), 160.8 (C), 140.6
(CH), 138.6 (C), 136.3 (C), 134.6 (CH), 130.9 (CH), 130.5 (CH), 129.6
(C), 129.1 (CH), 128.5 (CH), 127.3 (CH), 126.3 (C), 126.2 (CH), 102.2
(C), 21.3 (CH3), 20.7 (CH3). IR ѵ (cm−1): 1,605 (C C isox), 1,681 (C
amide), 1,766 (C O lactone), 3,097 (CH isox). ESI-MS calcd. for
18H15NO3, 293.32; found: m/z 294.11 [M + H]+. Anal. C18H15NO3
(C, H, N).
4-(4-Bromophenyl)-2-(3-methylbenzoyl)isoxazol-5(2H)-one
O
C
C
18H12F3NO3, 347.29; found: m/z 348.08 [M + H]+. Anal.
18H12F3NO3 (C, H, N).
C
(4e).
2.3
|
Pharmacology
Yield = 62%; mp = 159–162ꢀC (EtOH). 1H-NMR (400 MHz, CDCl3) δ
8.90 (s, 1H, CH), 7.81 (d, 2H, Ar, J = 8.0 Hz), 7.71 (d, 2H, Ar, J = 8.4 Hz),
7.56 (d, 2H, Ar, J = 8.4 Hz), 7.46–7.40 (m, 2H, Ar), 2.45 (s, 3H, CH3).
13C-NMR (100 MHz, CDCl3) δ 165.8 (C), 160.6 (C), 138.7 (C), 138.1
(CH), 134.7 (CH), 132.2 (CH), 130.5 (CH), 129.0 (C), 128.6 (CH), 127.4
(CH), 127.3 (CH), 126.3 (C), 122.8 (C), 109.6 (C), 21.3 (CH3). IR ѵ
(cm−1): 1,604 (C C isox), 1,670 (C O amide), 1,757 (C O lactone),
3,105 (CH isox). ESI-MS calcd. for C17H12BrNO3, 358.19; found: m/z
359.00 [M + H]+. Anal. C17H12BrNO3 (C, H, N).
Compounds were dissolved in 100% DMSO at 5 mM stock con-
centrations. The final concentration of DMSO in the reactions was
1%, and this level of DMSO had no effect on enzyme activity. The
HNE inhibition assay was performed in black flat-bottom 96-well
microtiter plates. Briefly, a solution containing 200 mM Tris–HCl,
pH 7.5, 0.01% bovine serum albumin, 0.05% Tween-20, and
20 mU/ml of HNE (Calbiochem) was added to wells containing
different concentrations of each compound. The reaction was initi-
ated by addition of 25 μM elastase substrate (N-methylsuccinyl-
Ala-Ala-Pro-Val-7-amino-4-methylcoumarin, Calbiochem) in a final
reaction volume of 100 μl/well. Kinetic measurements were
obtained every 30 s for 10 min at 25ꢀC using a Fluoroskan Ascent
FL fluorescence microplate reader (Thermo Electron, MA) with exci-
tation and emission wavelengths at 355 and 460 nm, respectively.
For all compounds tested, the concentration of inhibitor that cau-
sed 50% inhibition of the enzymatic reaction (IC50) was calculated
by plotting % inhibition versus logarithm of inhibitor concentration
(at least six points). The data are presented as the mean values of
at least three independent experiments with relative standard devi-
ations of <15%.
4-(4-Chlorophenyl)-2-(3-methylbenzoyl)isoxazol-5(2H)-one
(4f).
Yield = 32%; mp = 135–137ꢀC (EtOH). 1H-NMR (400 MHz, CDCl3) δ
8.90 (s, 1H, CH), 7.81–7.76 (m, 4H, Ar), 7.46–7.39 (m, 4H, Ar), 2.45 (s,
3H, CH3). 13C-NMR (100 MHz, CDCl3) δ 165.9 (C), 160.6 (C), 138.7
(C), 138.1 (CH), 134.7 (CH), 132.1 (CH), 130.5 (CH), 129.2 (CH), 129.1
(C), 128.6 (CH), 127.3 (CH), 127.1 (CH), 125.8 (C), 107.5 (C), 21.3
(CH3). IR ѵ (cm−1): 1,605 (C C isox), 1,660 (C O amide), 1,770 (C
O
lactone), 3,096 (CH isox). ESI-MS calcd. for C17H12ClNO3, 313.74;
found: m/z 314.05 [M + H]+. Anal. C17H12ClNO3 (C, H, N).
4-(4-Methoxyphenyl)-2-(3-methylbenzoyl)isoxazol-5(2H)-one
(4g).
Yield = 28%; mp = 168–170ꢀC (EtOH). 1H-NMR (400 MHz, CDCl3) δ
8.82 (s, 1H, CH), 7.84–7.77 (m, 4H, Ar), 7.49–7.44 (m, 2H, Ar), 6.98 (d,

GIOVANNONI ET AL.
5
2.4
|
Single crystal X-ray diffraction
Minimization, Standard Dynamics Cascade, and Analyze Trajectory,
implemented in Accelrys Discovery Studio 2.1 (Accelrys Discovery,
2019). The Force Field used for all simulations was CHARMm (Brooks
et al., 1983). Molecular plots were produced by the program Discov-
ery Studio Visualizer (v 4.5; Dassault Systemes BIOVIA, 2015).
GAUSSIAN09 (Rev. C01; Frisch et al., 2016) was used for QC calcula-
tions by using the B97D (Grimme, 2006) functional. The basis set was
6–31 + G(d,p) (Hehre, Ditchfield, & Pople, 1972). The Berny algorithm
was used (Peng, Ayala, Schlegel, & Frisch, 1996). The reliability of the
stationary points was assessed by evaluation of the vibrational fre-
quencies. For each inhibitor, different conformational isomers chosen
among the low-energy lying conformers (as found in MD simulations
and subsequent geometry optimization) were considered in QC calcu-
lations. Finally, CrystalExplorer17 (Turner et al., 2017) was used to
compute the Hirshfeld surface (HS).
Data for Compound A were collected at 100 K on an Xcalibur PX
Ultra CCD four-circle diffractometer using a graphite monochromator
and Cu Ka radiation.
A reference frame was monitored every
50 frames to control for stability of the crystal, and the system rev-
ealed no intensity decay. The data set was corrected for Lorentz,
polarization effects, and absorption corrections were performed by
the ABSPACK multiscan procedure of the CrysAlis (CrysAlis RED, n.d.,
version 171.37.35) data reduction package. The structure was solved
using the direct method with the SHELXS-2011 (Sheldrick, 1990) soft-
ware, and refinement was performed using the SHELXL-2013
(Sheldrick, 2008) software package. All nonhydrogen atoms were
located from the initial solution or from subsequent electron density
difference maps during the initial course of the refinement. After
locating the nonhydrogen atoms, the models were refined against F2,
first using isotropic and finally anisotropic thermal displacement
parameters. Hydrogen atoms have been introduced from subsequent
electron density difference maps at the end of the refinement. Pro-
grams used in the crystallographic calculations included WinGX
(Farrugia, 2012) and ORTEP (Farrugia, 1997) for graphics. Crystal and
refinement data are listed in Table S1. Crystal structural data are avail-
able from the Cambridge Crystallographic CCDC 1937512.
3
|
RESULTS AND DISCUSSION
| Chemistry
3.1
All new compounds were synthesized as reported in Figure 2, and the
structures were confirmed on the basis of analytical and spectral data.
Starting from 2-aryl-2-formylethylacetate derivatives 1a-i (Alves et al.,
2010; Beccalli et al., 1984; Liu, Chen, Wu, & Tang, 2016; Rajput et al.,
2014), we performed cyclization with hydroxylamine hydrochloride
and piperidine in ethanol at reflux temperature (for compounds 2a-g;
Beccalli et al., 1984; Snyder et al., 2004) or with hydroxylamine hydro-
chloride in a mixture MeOH/H2O 10:1 at reflux (for compounds 2h
and 2i [Snyder et al., 2004]) to obtain the key intermediates with an
isoxazolone core of Type 2. Despite the possibility that this nucleus
can exist in three different tautomers (Cencioni, Franchini, & Orienti,
1968; Franchini, 1968; Frolund et al., 2005), we confirmed in our pre-
vious work (Giovannoni et al., 2018) and here that acylation of inter-
mediates 2a-i (Beccalli et al., 1984; Snyder et al., 2004) resulted
exclusively in the N CO derivatives 3a-i and 4a-i when used the
appropriate acyl/aroyl chloride and sodium hydride in anhydrous tet-
rahydrofuran at room temperature. This result indicates that the NH
form is the only product formed under these reaction conditions. In
order to confirm our findings, assignment of the structure of final
compounds of Types 3 and 4 was performed using 1H-NMR, 13C-
NMR, IR spectroscopy, and 2D NMR (1H-13C HSQC, 1H-13C HMBC).
2.5
|
Molecular modeling procedures
Compounds 3a, 4a, A, and B were considered in the modeling studies.
The molecular structure of A, as obtained from X-ray diffraction
(details in the related sections), was used as the starting geometry in
the computational study for Compound A and as a building block for
3a. The starting geometry for 4a and B was derived by the structure
of
the
closely
related
isoxazolone
derivative
3-ethyl-
2-(3-methylbenzoyl)isoxazol-5(2H)-one (Vergelli et al., 2017). The 3D
arrangement of all molecules was improved by geometry optimization
procedures, followed by molecular dynamic (MD) simulations at
300 and 600 K (time step = 1 fs, equilibration time = 100 ps, produc-
tion time = 1,000 ps). MD simulations were performed both in vac-
uum and in implicit solvent, the latter was simulated using distance-
dependent dielectric constants set to 4r. Ten snapshot conformations
with evenly spaced intervals were extracted from MD trajectories
(performed at 600 K with ε = 4r) and subsequently minimized (by using
the steepest descent and conjugate gradient algorithms) in order to
obtain starting geometries for the subsequent quantum chemical cal-
culations (QC) and MD simulations with HNE. The structure of HNE
complexed with a peptide chloromethyl ketone inhibitor was used for
the MD simulations (1HNE [Navia et al., 1989] entry of the Protein
Data Bank). The bound inhibitor was removed, as well as all of the
water molecules, and hydrogen atoms were added prior to starting
simulations. MD simulations on each ligand-HNE assembly were per-
formed at 300 K under vacuum (time step = 1 fs, equilibration
time = 100 ps, production time 5,000 ps). The programs used for the
energy minimization and MD were the simulation protocols
3.2
|
Biological evaluation and SAR analysis
All compounds were evaluated for their ability to inhibit HNE in com-
parison with Sivelestat. All the biological results are reported in
Table 1, as compounds of Type 3 (N-cyclopropylcarbonyl derivatives)
and of Type 4 (N-m-tolylcarbonyl derivatives) containing the same
substituents R in the phenyl ring at Position 4 (R is shown in the cen-
tral column). For comparison, we also include our previous reported
compounds
A and B, which belong to the series of the

6
GIOVANNONI ET AL.
FIGURE 2 Reagents and
conditions. (a) for 2a-g: EtOH,
piperidine, NH2OHÁHCl, 80ꢀC, 2 hr;
for 2h,i: MeOH/H2O (10:1),
NH2OHÁHCl, 100ꢀC, 4 h; (b) m-CH3-
Ph-COCl or cC3H5-COCl, dry THF,
NaH (60% dispersion in mineral oil),
r.t., 24 hr
3-methylisoxazolones (Giovannoni et al., 2018). Our biological results
verify that all compounds of Type 3 and 4 are potent HNE inhibitors,
as their HNE inhibitory activity was in the low nanomolar range
(IC50 = 11–81 nM), with most compounds being more active than
Sivelestat (IC50 = 44 nM). Starting from compounds 3a and 4a, which
are derivatives of the reference compounds A and B, respectively,
elimination of the methyl group at Position 3 increased activity of
both compounds ~fourfold for 3a and twofold for 4a (3a vs. A:
IC50 = 16 nM vs. 59 nM; 4a vs. B: IC50 = 46 nM vs. 77 nM). On the
other hand, the introduction of different substituents on the phenyl
ring at Position 4 led to compounds which, regardless of the group at
Position 2 of the amide function (i.e., m-tolyl or cyclopropyl) had the
same potency, such as when the substituent was a methyl group (3b-
d and 4b-d), but also when Cl, Br, or methoxy groups were present
(3e-g and 4e-g). Slightly greater differences were observed for 3h and
4h, where the N-cyclopropylcarbonyl derivative 3h was about ~four-
fold more potent than the N-m-tolylcarbonyl derivative 4h (IC50 = 18
vs. 78 nM, respectively). The same trend was observed for the p-CF3-
phenyl derivatives 3i and 4i (IC50 = 33 and 81 nM, respectively).
Overall, we found that the nature of the substituent (CH3, Br, Cl,
OCH3, CN, CF3) and/or their position (para, meta, or ortho) in the phe-
nyl ring do not seem to significantly influence HNE inhibitory potency,
indicating that the pocket of the enzyme hosting the aromatic ring is
wide and able to hold bulky groups with different chemical and elec-
tronic properties.
3.3
|
Molecular structure from single crystal X-ray
diffraction and molecular modeling
The solid-state molecular structure of compound A (Giovannoni et al.,
2018) was obtained from single crystal X-ray diffraction. A crystallizes
in the P212121 with one independent molecule in the asymmetric unit.
Figure
3 shows an ORTEP-3 representation of the compound
together with atom labeling. Bond distances and angles are within the
expected ranges. The conformation of the compound, as defined by
the dihedral angles τ1 (−179.1[2]), τ2 (−38.5[5]), and τ3 (−175[2]) (see
Figure 4a), can be described as anti-gauche-anti (Klyne & Prelog,
1960). A search in the Cambridge Structural Database 5.40 (CSD;
Groom, Bruno, Lightfoot, & Ward, 2016) investigating the usual range
of values for τ1, τ2, and τ3 found that 53 hits with the fragment
sketched in Figure 4b are present in the CSD; of these, only two
(AMIXPO and GUJJIF refcodes) feature a C O bound to 2-N. The
related torsion angle τ1 adopts an eclipsed and anti-conformation,

GIOVANNONI ET AL.
7
TABLE 1 HNE inhibitory activity of compounds 3a-i and 4a-i
Comp.
3a
IC50 (nM)a
R
IC50 (nM)a
Comp.
4a
16
26
22
5
4
5
H
46 12
3b
3c
p-CH3
m-CH3
o-CH3
p-Br
p-Cl
22
31
28
47
25
23
78
7
3
3
7
2
7
2
4b
4c
3d
3e
39 11
4d
4e
31
42
11
18
33
3
8
3
5
1
3f
4f
3g
p-OCH3
p-CN
p-CF3
–
4g
3h
3i
4h
4i
81 28
77 27
59 18
Note: Sivelestat IC50 = 44 20 nM.
Abbreviation: HNE, human neutrophil elastase.
aThe experiments were performed in triplicate using N-methylsuccinyl-Ala-Ala-Pro-Val-7-amino-4-cumarin as substrate for HNE. *Giovannoni et al., 2018.
respectively; finally, an anti-conformation for τ1 was also observed in
the isoxazolone derivative 3-ethyl-2-(3-methylbenzoyl)isoxazol-5(2H)-
one (Vergelli et al., 2017). No comparisons were made for τ2 due to
the lack of suitable reference structures. The τ3 dihedral adopted the
usual conformation.
temperature (300 and 600 K), as shown by the values adopted by the
dihedral angles τ1, τ2, and τ3 (Figure 4a,c) throughout the MD simula-
tions. In particular, snapshot conformations collected from MD trajec-
tories at 300 and 600 K all had τ1 dihedrals in an anti-conformation,
which was maintained throughout the simulations. As expected, a
larger variety of conformations was observed for τ2 and τ3 (-
Figures S1-S4). In addition, all the molecules had an almost identical
overall shape that can be roughly described as an elongated cylinder,
as shown in Figure 5 where superimposition of the minimized con-
formers obtained from a sampling of the corresponding MD trajecto-
ries (T = 600 K, ε = 4r) is shown for each molecule. As expected,
results for compounds 4a and B correlated well with those of the
closely related isoxazolone derivatives, as we reported previously
(Giovannoni et al., 2018).
Molecular dynamic simulations at 300 and 600 K were performed
in order to explore the conformational space accessible to compound
A and, eventually, to find other low-energy conformers. For compari-
son, the same approach (MD at 300 and 600 K) was used to sample
the conformational space and identify low-energy conformers of
closely related compounds 3a, 4a, and B (Giovannoni et al., 2018;
sketched in Table 1).
The overall shape of each molecule was not affected by either
the different simulation medium (vacuum and
ε = 4r) or the

8
GIOVANNONI ET AL.
FIGURE 3 ORTEP-3 view of the asymmetric unit of A (ellipsoid
probability 20%)
FIGURE 5 Superimposition of the minimized conformers of
A (top left), 3a (top right), B (bottom left), and 4a (bottom right)
all the cases close to 180ꢀ and τ2 was gauche). Regardless of the orien-
tation of the m-toluoyl group, differences in the energy content were
negligible. Finally, as estimated by the Mullikan and APT charge-
derivation schemes, there were not significant differences in atomic
charge of the 5-C O carbon atom, which is involved in the attack of
Ser195, on moving from the 3-methylisoxazoles (A and B) to the
corresponding nor-derivatives (3a and 4a).
In summary, conformation of the τ1 dihedral in the cyclopropyl
(3a and A) and m-tolyl (4a and B) derivatives appears frozen, regard-
less of the environment (MD simulation in vacuum and ε = 4r, crystal)
or the temperature (MD simulations at 300 and 600 K). In contrast, τ2
and τ3 accessed almost all conformations during MD simulations
(300 and 600 K). For 4a and B, small differences in orientation of the
m-tolouyl group did not affect the relative energy content of the
corresponding conformational isomers. All of the compounds had a
cylindrical overall shape.
3.4
|
Intermolecular binding propensity from single
crystal X-ray diffraction and molecular modeling
Given that N
C O plays the role of anchoring the molecule to the
subpocket of the binding site through H-bond interactions with the
receptor, we analyzed the intermolecular contacts involving this group
in the crystal lattice of compound A to assess its propensity to act an
H-bond acceptor. In the crystalline solid (Figure 6), the oxygen atom
FIGURE 4 (a) Schematic of the cyclopropyl derivatives A and 3a
and the related dihedral angles defining the molecule's conformation;
(b) fragment searched in the Cambridge Structural Database;
(c) schematic of the m-tolylcarbonyl derivatives B and 4a and the
related dihedral angles defining the molecule's conformation
belonging to the N
C O group acts as an acceptor for the hydrogen
atom provided by a symmetry-related molecule: O(3)Á Á ÁH(4)C(4)iv
(iv = 1.5 − x,1 − y, −0.5 + z). This interaction is quite weak (Desiraju &
Steiner, 1999) as determined by the OÁ Á ÁH distance (2.41(3)Å) and the
related angle (143(3)ꢀ).
The different conformational isomers optimized by the QC
method converge to the same conformer for compounds 3a and
A (anti-eclipsed-anti and anti-gauche-anti, respectively), which, in the
case of compound A, was identical to that found in the solid state
(Figure S5a). For the m-toluoyl derivatives, the optimization procedure
led to different conformers (Figure S5b) that in both cases (i.e., 4a and
B) essentially differed in the conformations adopted by τ3 (τ1 was in
The oxygen atom of the 5-C=O group (whose carbon atom
appears to be involved in the attack of Ser195) also contributes to the
net intermolecular contacts acting as a tri-furcated acceptor for the
hydrogen atoms provided by three distinct symmetry-related mole-
cules: O(1)Á Á ÁH(13A)C(13)i (i = 2 − x, 0.5 + y, 0.5 − z), O(1)Á Á ÁH(10C)C
(10)ii (ii = −0.5 + x, 1.5 − y, 1 − z) and O(1)Á Á ÁH(3)C(3)iii (iii = 1.5 − x,
2 − y, −0.5 + z). These interactions also are quite weak [39], as

GIOVANNONI ET AL.
9
FIGURE 8 (a) Accessibility of the 5-C O and N
C O groups
estimated by the dimension of a sphere (r = 2.5 Å) centered on the
carbon atom (red) and on the oxygen atom (blue). Left: (a) right: 3a;
(b) accessibility of the 5-C O and N
C O groups estimated by the
FIGURE 6 Crystal packing of compound A highlighting the
carbonyl oxygen atoms (space filling representation) involved in
intermolecular contacts with the hydrogen atoms of neighboring
molecules (space filling representation)
dimension of a sphere (r = 2.5 Å) centered on the carbon atom (red)
and on the oxygen atom (blue). Left: (b) right: 4a
Following the same approach previously adopted (Giovannoni
et al., 2018), the exposure to the environment of both carbonyl
groups in molecule A and 3a was further assessed by the dimension
of two spheres centered on the 5-C=O carbon atom and on the
N
C
O oxygen atom. In both cases, a sphere having radius 2.5 Å
appears suitable to map the accessible region about the two carbonyl
groups (Figure 8A). Thus, as with the N O oxygen atom, the dif-
C
ferent substituents at the 3-position ( CH3 vs H) did not seem to
affect its accessibility to the enzyme. Identical results were found for
the m-tolyl derivatives 4a and B (Figure 8B). Finally, we can also con-
clude that substitution with cyclopropyl or m-tolyl did not appear to
have consequences on exposure of the anchoring carbonyl group. In
summary, no significant differences were detected both within each
class of inhibitors (3a vs A; 4a vs B) and also between the two families
(cyclopropyl vs m-toluoyl derivatives) in terms of the accessibility of
the two functional groups more specifically involved in the interaction
with HNE.
According to our previous modeling (Giovannoni et al., 2018), the
ligand–receptor binding modes were assessed by MD simulations
(in vacuum, simulation time 5 ns, T = 300 K), with the atoms outside
of the binding sphere constrained to fixed points in space in order to
save computational time.
Regarding the ligand-enzyme interactions, no substantial differ-
ences were observed between the 3-methylisoxazole derivatives
(A and B) and the corresponding nor-derivatives (3a and 4a) in the
snapshot conformations sampled during the MD simulations; the
same applied for the cyclopropyl/m-toluoyl families (A vs B and 3a vs
4a). In all cases, the ligand-enzyme interaction involved anchoring of
FIGURE 7 Hirshfeld surface of A and neighboring molecules
associated with close contacts
determined by the OÁ Á ÁH distances (2.55(4), 2.65(4), and 2.67(3)Å,
respectively, and the related angles (160(3), 124(3), and 136(3)ꢀ). The
relative strength of these interactions is clearly evident in the HS of
A shown in Figure 7 (the brightest red spots are associated with the
closest intermolecular contacts), which also shows the closest neigh-
boring molecules involved in the H-bonds.
the N
C O group to Gly193 via H-bonding. Consequently, the 5-CO
group is quite close to the HNE active site (distance <5 Å in a large
fraction of the snapshot conformations), which is consistent with our
previously studies on the closely related isoxazolone derivatives
(Giovannoni et al., 2018).

10
GIOVANNONI ET AL.
4
|
CONCLUSION
ORCID
Maria Paola Giovannoni
https://orcid.org/0000-0003-0310-7850
With the aim to understand the importance of substituents at Position
3 of the potent isoxazolones previously published by us (Giovannoni
et al., 2018; Vergelli et al., 2017), we synthesized a new series of
3-unsubstituted isoxazolones containing a phenyl group at Position
4 with various substituents. Elimination of the substituent at Position
3, as well as the introduction of various groups (CH3, Br, Cl, OCH3,
CN, CF3) at different positions (para, meta, and ortho) of the 4-phenyl
ring did not influence HNE inhibitory activity. In fact, all new
isoxazolones were highly active, with IC50 values comparable to those
of the three-substituted analogues. The most potent compounds were
3a, 3g, and 3h, which had IC50 values of 16, 11, and 18 nM, respec-
tively. The crystal structure of compound A shows that the carbonyl
oxygen atoms, which are both involved in the inhibitory activity, act
as H-bond acceptors toward symmetry-related molecules. Molecular
modeling on the closely related 3a, A, 4a, and B suggests that the
Letizia Crocetti
https://orcid.org/0000-0003-3473-2683
https://orcid.org/0000-0001-6711-7965
Gabriella Guerrini
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Alves, L. G., Dazinger, G., Veiros, L. F., & Kirchner, K. (2010). Unusual anion
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ACKNOWLEDGMENTS
This research was supported in part by National Institutes of Health
IDeA Program COBRE Grant GM110732; USDA National Institute of
Food and Agriculture Hatch project 1009546; and the Montana State
University Agricultural Experiment Station.
Crocetti, L., Giovannoni, M. P., Schepetkin, I. A., Quinn, M. T.,
Khlebnikov, A. I., Cantini, N., … Vergelli, C. (2018). 1H-pyrrolo[2,3-b]
pyridine: A new scaffold for human neutrophil elastase (HNE) inhibitor.
Bioorganic & Medicinal Chemistry, 26, 5583–5595. https://doi.org/10.
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CONFLICT OF INTEREST
The authors declare no conflict of interest.
Crocetti, L., Schepetkin, I. A., Ciciani, G., Giovannoni, M. P., Guerrini, G.,
Iacovone, A., … Vergelli, C. (2016). Synthesis and pharmacological eval-
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trophil elastase inhibitors. Drug Development Research, 77(6), 285–299.
https://doi.org/10.1002/ddr.21323
AUTHOR CONTRIBUTIONS
C.L. and G.M.P. designed the compounds and wrote the manuscript;
C.N., G.G., I.A., V.C., and T.E. synthesized the compounds, wrote the
experimental section, and checked the final version of the manuscript.
in vitro studies (inhibition essay) were performed by S.I.A. and
Q.M.T. and they wrote the pharmacological section. The crystal struc-
ture was made by C.S.; molecular dynamic (MD) simulations and the
corresponding analysis of data were performed by R.P. and P.P. All of
the authors have given approval to the final version of the manuscript.
Crocetti, L., Schepetkin, I. A., Cilibrizzi, A., Graziano, A., Vergelli, C.,
Giomi, D.,
… Giovannoni, M. P. (2013). Optimization of N-
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
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How to cite this article: Giovannoni MP, Crocetti L, Cantini N,
et al. New 3-unsubstituted isoxazolones as potent human
neutrophil elastase inhibitors: Synthesis and molecular
dynamic simulation. Drug Dev Res. 2019;1–12. https://doi.org/
10.1002/ddr.21625
von Nussbaum, F., Li, V. M. J., Meibom, D., Anlauf, S., Bechem, M.,
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neutrophil elastase inhibitors with novel equatorial ring topology: