Synthesis, biological evaluation, and molecular modelling studies of potent human neutrophil elastase (HNE) inhibitors

Article abstract of DOI:10.1080/14756366.2018.1480615

We report the synthesis and biological evaluation of a new series of 3- or 4-(substituted)phenylisoxazolones as HNE inhibitors. Due to tautomerism of the isoxazolone nucleus, two isomers were obtained as final compounds (2-NCO and 5-OCO) and the 2-NCO derivatives were the most potent with IC₅₀ values in the nanomolar range (20–70 nM). Kinetic experiments indicated that 2-NCO 7d and 5-OCO 8d are both competitive HNE inhibitors. Molecular modelling on 7d and 8d suggests for the latter a more crowded region about the site of the nucleophilic attack, which could explain its lowered activity. In addition molecular dynamics (MD) simulations showed that the isomer 8d appears more prone to form H-bond interactions which, however, keep the reactive sites quite distant for the attack by Ser195. By contrast the amide 7d appears more mobile within the active pocket, since it makes single H-bond interactions affording a favourable orientation for the nucleophilic attack.

Full text of DOI:10.1080/14756366.2018.1480615

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis, biological evaluation, and molecular
modelling studies of potent human neutrophil
elastase (HNE) inhibitors
Maria Paola Giovannoni, Igor A. Schepetkin, Mark T. Quinn, Niccolò Cantini,
Letizia Crocetti, Gabriella Guerrini, Antonella Iacovone, Paola Paoli, Patrizia
Rossi, Gianluca Bartolucci, Marta Menicatti & Claudia Vergelli
To cite this article: Maria Paola Giovannoni, Igor A. Schepetkin, Mark T. Quinn, Niccolò
Cantini, Letizia Crocetti, Gabriella Guerrini, Antonella Iacovone, Paola Paoli, Patrizia
Rossi, Gianluca Bartolucci, Marta Menicatti & Claudia Vergelli (2018) Synthesis, biological
evaluation, and molecular modelling studies of potent human neutrophil elastase (HNE)
inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 1108-1124, DOI:
10.1080/14756366.2018.1480615
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 33, NO. 1, 1108–1124
https://doi.org/10.1080/14756366.2018.1480615
RESEARCH PAPER
Synthesis, biological evaluation, and molecular modelling studies of potent human
neutrophil elastase (HNE) inhibitors
a
a
Maria Paola Giovannonia , Igor A. Schepetkinb , Mark T. Quinnb , Niccolo Cantini , Letizia Crocetti ,
ꢀ
Gabriella Guerrinia , Antonella Iacovonea, Paola Paolic , Patrizia Rossic , Gianluca Bartoluccia
Marta Menicattia and Claudia Vergellia
,
b
aNEUROFARBA, Pharmaceutical and Nutraceutical Section, University of Florence, Sesto Fiorentino, Italy; Department of Microbiology and
c
Immunology, Montana State University, Bozeman, MT, USA; Department of Industrial Engineering, University of Florence, Florence, Italy
ABSTRACT
ARTICLE HISTORY
Received 6 April 2018
Revised 21 May 2018
Accepted 21 May 2018
We report the synthesis and biological evaluation of a new series of 3- or 4-(substituted)phenylisoxazo-
lones as HNE inhibitors. Due to tautomerism of the isoxazolone nucleus, two isomers were obtained as
final compounds (2-NCO and 5-OCO) and the 2-NCO derivatives were the most potent with IC50 values in
the nanomolar range (20–70 nM). Kinetic experiments indicated that 2-NCO 7d and 5-OCO 8d are both
competitive HNE inhibitors. Molecular modelling on 7d and 8d suggests for the latter a more crowded
region about the site of the nucleophilic attack, which could explain its lowered activity. In addition
molecular dynamics (MD) simulations showed that the isomer 8d appears more prone to form H-bond
interactions which, however, keep the reactive sites quite distant for the attack by Ser195. By contrast the
amide 7d appears more mobile within the active pocket, since it makes single H-bond interactions afford-
ing a favourable orientation for the nucleophilic attack.
KEYWORDS
Isoxazol-5(2H)-one; HNE;
molecular model-
ling; stability
Introduction
secretory leucocyte protease inhibitor (SLPI), and elafin13.
a1-Antitrypsin is a water-soluble glycoprotein classified as systemic
HNE inhibitor that is synthesised in the liver and is particularly
abundant in the lungs14. In contrast, SLPI and elafin are classified
as alarm inhibitors because they are produced and released dir-
ectly into the airway epithelium in response to the release of cyto-
kines, regulating the immune response and inflammatory
Proteases are enzymes implicated in cellular reactions involving
the cleavage of protein substrates1. Serine proteases are character-
ised by the presence of a serine residue at the active site2. They
are divided into four classes: chymotrypsin, subtilisin, carboxypep-
tidase Y, and caseinolytic protease3. Human neutrophil elastase
(HNE), proteinase 3 (PR3), cathepsin G, and the recently discovered
NSP44 are serine proteases belonging to the chymotrypsin family
and represent neutrophil serine proteases (NSP)5,6. NSP are syn-
thesised and expressed in neutrophil azurophilic granules.
Neutrophils play a pivotal role in host defence, inflammation and
tissue remodelling, and HNE is a key mediator of neutrophil-driven
inflammation7.
processes15–17
.
Under physiological conditions, the balance
between protease and anti-protease supports the maintenance of
tissue homeostasis. However, if this balance fails, excessive HNE
activity can cause tissue the damage associated with some serious
chronic diseases4,6
. Among the pathologies associated with
increased HNE activity are adult respiratory distress syndrome
(ARDS)18, chronic obstructive pulmonary disease (COPD)19,20, cystic
fibrosis (CF)21,22, and other disorders with an inflammatory compo-
nent, such as rheumatoid arthritis23, atherosclerosis24, psoriasis,
and dermatitis25. Recently, it was demonstrated that HNE is also
implicated in the progression of non-small cell lung cancer26.
Although a large number of molecules have been reported as
HNE inhibitors27,28, only two drugs are currently available for clin-
ical use: Prolastin (purified a1-AT), a peptide drug synthesised by
recombinant DNA techniques29 and used for the treatment of a1-
HNE is a small, basic, and soluble glycoprotein of about
30 kDa8 that performs many functions in our body. For example,
HNE is involved in the maintenance of tissue homeostasis; it
degrades a variety of structural proteins of the extracellular matrix,
such as elastin, fibronectin, collagen, proteoglycan, and laminin;
and it repairs damaged tissue9. Moreover, HNE plays an important
and dual role in inflammation by degrading pro-inflammatory
cytokines to reduce the intensity of the inflammation but also
increasing the secretion of pro-inflammatory factors10. In the case
of infection, HNE plays as an intracellular function in destroying
phagocytosed pathogens, as well as an extracellular function
through the formation of neutrophil extracellular traps, which can
trap and kill microorganisms11,12. The powerful HNE activity is
tightly controlled by the presence of extracellular neutralizing
R
antitripsin deficiency (AATD)30, and Sivelestat (ElaspolV100), a non-
peptide low molecular weight compound, belonging to the
second generation of HNE inhibitors31. Sivelestat has an
IC50 ¼ 44 nM and is currently marketed only in Japan and South
Korea32,33 (Figure 1). AZD9668 (Alvelestat, AstraZeneca)34 and BAY
85-8501 (Bayer HealthCare)35 are two potent HNE inhibitors,
endogenous serine protease inhibitors, such as a-1 antitrypsin, belonging to the third and fifth generations of HNE inhibitors31,
CONTACT Letizia Crocetti
letizia.crocetti@unifi.it
Dipartimento di NEUROFARBA, Via Ugo Schiff 6, Sesto Fiorentino 50019 Firenze, Italy
Supplemental data for this article can be accessed here.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1109
Figure 1. Potent HNE inhibitors.
respectively, that have recently reached Phase II of clinical trials and were calculated using TopSpin 1.3 software (Nicolet Instrument
for COPD, CF, and BE (Figure 1).
Corp., Madison, WI) and are rounded to the nearest 0.1vHz. Mass
In recent research focused on the development of new HNE spectra (m/z) were recorded on an ESI-TOF mass spectrometer
inhibitors, we investigated various bicyclic scaffolds, such as inda- (Brucker Micro TOF, Bruker Inc., Billerica, MA), and reported mass
zole36,37, indole38, and cinnolinone39, compounds. The most inter- values are within the error limits of
5 ppm mass units.
esting inhibitors were effective in the nanomolar range, with a Microanalyses indicated by the symbols of the elements or func-
tions were performed with a Perkin–Elmer 260 elemental analyser
(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.
potency comparable to Sivelestat. Subsequently, we focused our
research on the design and synthesis of monocyclic derivatives
with an isoxazol-5(2H)-one core, and the first series of 3/4-alkyl-
(di)substituted isoxazolones was recently published40. We report
here the synthesis of a new series of isoxazolone derivatives bear-
ing a (substituted)phenyl at positions 3 and 4 and biological evalu-
ation of their HNE inhibitory activity.
Chemistry
3-Methyl-2-(3-methylbenzyl)-4-phenylisoxazol-5(2H)-one (2)
A mixture of the appropriate intermediate (1a41) (0.57 mmol),
Material and methods
K2CO3
(1.14 mmol),
and
1-(chloromethyl)-3-methylbenzene
€
All melting points were determined on a Buchi apparatus (New
(0.86 mmol) in 2 ml of anhydrous acetonitrile was stirred at reflux
for 2 h. After cooling, the mixture was concentrated in vacuo,
diluted with ice-cold water (10 ml), and extracted with ethyl acet-
ate (3 ꢀ 15 ml). The organic phase was dried over sodium sulphate
and the solvent was evaporated in vacuo to afford the final com-
pound 2, which was purified by column chromatography using
cyclohexane/ethyl acetate 2:1 as eluent. Yield ¼57%; oil. 1H NMR
(CDCl3-d1) d 2.35 (s, 6H, 2 ꢀ CH3), 4.79 (s, 2H, CH2), 7.07–7.14 (m,
Castle, DE) and are uncorrected. Extracts were dried over Na2SO4,
and the solvents were removed under reduced pressure. Merck
F-254 commercial plates (Merck, Durham, NC) were used for analyt-
ical TLC to follow the course of reactions. Silica gel 60 (Merck
70–230 mesh, Merck, Durham, NC) was used for column chromatog-
raphy. 1H NMR, 13C NMR, HSQC, HMBC, and NOESY bidimensional
spectra were recorded on an Avance 400 instrument (Bruker Biospin
Version 002 with SGU, Bruker Inc., Billerica, MA). Chemical shifts (d) 3H, Ar), 7.22–7.28 (m, 2H, Ar), 7.38 (t, 2H, Ar, J ¼ 7.8 Hz), 7.45 (d,
are reported in ppm to the nearest 0.01 ppm using the solvent as 2H, Ar, J ¼ 7.2 Hz). 13C NMR (CDCl3-d1) d 12.44 (CH3), 21.63 (CH3),
an internal standard. Coupling constants (J values) are given in Hz 54.90 (CH2), 103.58 (C), 125.16 (CH), 127.17 (CH), 128.21 (CH),

1110
M. P. GIOVANNONI ET AL.
128.58 (CH), 128.82 (CH), 128.90 (CH), 129.40 (CH), 129.77 (C), (CH3), 108.39 (C), 127.08 (CH), 127.62 (C), 128.28 (CH), 128.50 (CH),
133.45 (C), 138.75 (C), 158.65 (C), 169.53 (C). ESI-MS calcd. for 128.81 (CH), 129.08 (CH), 130.28 (CH), 131.14 (C), 134.07 (CH), 138.32
C18H17NO2, 279.33; found: m/z 280.13 [M þ H]þ. Anal. C18H17NO2 (C), 154.62 (C), 163.79 (C), 165.90 (C). IR (t) ¼ 1690 cmꢂ1 (CO amide),
(C, H, N).
1750 cmꢂ1 (CO ester). ESI-MS calcd. for C18H15NO3, 293.32; found:
m/z 294.11 [M þ H]þ. Anal. C18H15NO3 (C, H, N).
General procedure for compounds (3a–c)
2-(Cyclopropanecarbonyl)-3-methyl-4-phenylisoxazol-5(2H)-one
(4b). Yield ¼ 63%; mp ¼ 83–86 ꢁC (EtOH). 1H NMR (DMSO-d6) d
1.02–1.07 (m, 2H, CH2 cC3H5), 1.09–1.15 (m, 2H, CH2 cC3H5),
2.36–2.41 (m, 1H, CH cC3H5), 2.58 (s, 3H, CH3), 7.35–7.41 (m, 1H,
Ar), 7.43–7.48 (m, 4H, Ar). 13C NMR (DMSO-d6) d 10.82 (CH2), 13.25
(CH3), 15.14 (CH), 106.28 (C), 128.25 (C), 128.58 (CH), 129.10 (CH),
129.35 (CH), 154.69 (C), 166.05 (C), 169.02 (C). IR (t) ¼ 1695 cmꢂ1
(CO amide), 1755 cmꢂ1 (CO ester). ESI-MS calcd. for C14H13NO3,
243.26; found: m/z 244.09 [M þ H]þ. Anal. C14H13NO3 (C, H, N).
To a suspension of the appropriate 4-substituted benzensulfonyl
chloride (0.16 mmol) in 3 ml of anhydrous pyridine, 0.79 mmol of
intermediate 1a41 was added. The mixture was stirred at room
temperature for 4 h. The solvent was concentrated in vacuo to
afford the final compounds 3a–c which were purified by column
chromatography using cyclohexane/ethyl acetate in different ratio
(2:1 for 3a, 4:1 for 3b) or toluene/ethyl acetate 9:1 for 3c
as eluents.
2-((4-Hydroxyphenyl)sulfonyl)-3-methyl-4-phenylisoxazol-5(2H)-one
3-Methyl-2-(4-methylbenzoyl)-4-phenylisoxazol-5(2H)-one (4c).
1
(3a). Yield ¼ 30%; mp ¼ 50–51 ꢁC (EtOH). H NMR (CDCl3-d1) d 2.57
1
Yield ¼ 35%; mp ¼ 134–136 ꢁC (EtOH). H NMR (CDCl3-d1) d 2.51 (s,
(s, 3H, CH3), 6.93 (d, 2H, Ar, J ¼ 8.8 Hz), 7.30–7.40 (m, 5H, Ar), 7.78
(d, 2H, Ar, J ¼ 8.8 Hz). 13C NMR (CDCl3-d1) d 14.59 (CH3), 113.94 (C),
116.39 (CH), 121.92 (C), 126.91 (C), 128.77 (CH), 128.87 (CH), 129.19
(CH), 131.88 (CH), 158.94 (C), 162.88 (C), 168.80 (C). ESI-MS calcd.
for C16H13NO5S, 331.34; found: m/z 332.05 [M þ H]þ. Anal.
C16H13NO5S (C, H, N).
3H, p-CH3-Ph), 2.87 (s, 3H, CH3), 7.38 (d, 2H, Ar, J ¼ 8.0 Hz),
7.43–7.48 (m, 1H, Ar), 7.51–7.58 (m, 4H, Ar), 7.92 (d, 2H, Ar,
J ¼ 8.4 Hz). 13C NMR (CDCl3-d1) d 15.37 (CH3), 22.02 (CH3), 108.19
(C), 127.85 (C), 128.46 (CH), 128.59 (CH), 128.95 (CH), 129.19 (CH),
128.19 (CH), 130.28 (CH), 144.55 (C), 154.60 (C), 158.00 (C), 163.62
(C), 166.13 (C). IR (t) ¼ 1672 cmꢂ1 (CO amide), 1755 cmꢂ1 (CO
ester). ESI-MS calcd. for C18H15NO3, 293.32; found: m/z 294.11
[M þ H]þ. Anal. C18H15NO3 (C, H, N).
4-((3-Methyl-5-oxo-4-phenylisoxazol-2(5H)-yl)sulfonyl)phenyl piva-
1
late (3b). Yield ¼ 15%; mp ¼ 115–116 ꢁC (EtOH). H NMR (CDCl3-d1)
d 1.35 (s, 9H, C(CH3)3), 2.58 (s, 3H, CH3), 7.26 (d, 2H, Ar, J ¼ 8.0 Hz),
7.32 (d, 2H, Ar, J ¼ 8.6 Hz), 7.35–7.40 (m, 3H, Ar), 7.95 (d, 2H, Ar,
J ¼ 8.6 Hz). 13C NMR (CDCl3-d1) d 14.50 (CH3), 26.97 (CH3), 29.37
(C), 39.36 (C), 114.27 (C), 122.81 (CH), 127.03 (C), 128.45 (CH),
128.81 (CH), 129.11 (CH), 130.39 (CH), 130.97 (CH), 156.83 (C),
157.77 (C), 167.45 (C), 175.75 (C). ESI-MS calcd. for C21H21NO6S,
415.46; found: m/z 416.11 [M þ H]þ. Anal. C21H21NO6S (C, H, N).
3-Methyl-2-(2-methylbenzoyl)-4-phenylisoxazol-5(2H)-one (4d).
Yield ¼ 42%; mp ¼ 99–100 ꢁC (EtOH). 1H NMR (CDCl3-d1) d 2.47 (s,
3H, o-CH3-Ph), 2.85 (s, 3H, CH3), 7.32 (d, 2H, Ar, J ¼ 6.8 Hz),
7.40–7.49 (m, 7H, Ar). 13C NMR (CDCl3-d1) d 15.37 (CH3), 20.22
(CH3), 108.95 (C), 126.27 (CH), 128.10 (C), 128.74 (CH), 129.14 (CH),
129.43 (CH), 129.60 (CH), 131.53 (CH), 132.10 (CH), 132.52 (C),
137.22 (C), 153.99 (C), 164.91 (C), 166.31 (C). IR (t) ¼ 1688 cmꢂ1 (CO
amide), 1767 cmꢂ1 (CO ester). ESI-MS calcd. for C18H15NO3, 293.32;
found: m/z 294.11 [M þ H]þ. Anal. C18H15NO3 (C, H, N).
N-(4-((3-methyl-5-oxo-4-phenylisoxazol-2(5H)-yl)sulfonyl)phenyl)pi-
valamide (3c). Yield ¼ 72%; mp ¼ 153–155 ꢁC (EtOH). 1H NMR
(CDCl3-d1) d 1.30 (s, 9H, C(CH3)3), 2.58 (s, 3H, CH3), 7.26 (d, 1H, Ar,
J ¼ 6.8 Hz), 7.36–7.42 (m, 4H, Ar), 7.70 (exch br s, 1H, NH), 7.79 (d,
2H, Ar, J ¼ 8.8 Hz), 7.84 (d, 2H, Ar, J ¼ 8.8 Hz). 13C NMR (CDCl3-d1) d
14.61 (CH3), 27.42 (CH3), 44.65 (C), 113.95 (C), 119.39 (CH), 125.36
(C), 125.85 (CH), 128.43 (CH), 128.81 (CH), 128.97 (CH), 129.88 (CH),
130.68 (CH), 130.91 (CH), 134.00 (C), 144.98 (C), 158.06 (C), 165.00
(C), 177.20 (C). ESI-MS calcd. for C21H22N2O5S, 414.47; found: m/z
415.13 [M þ H]þ. Anal. C21H22N2O5S (C, H, N).
3-Methyl-4-phenyl-2-(3-(trifluoromethyl)benzoyl)isoxazol-5(2H)-one
1
(4e). Yield ¼ 42%; mp ¼ 96–97 ꢁC (EtOH). H NMR (CDCl3-d1) d 2.85
(s, 3H, CH3), 7.40–7.45 (m, 1H, Ar), 7.48–7.54 (m, 4H, Ar), 7.69 (t,
1H, Ar, J ¼ 8.0 Hz), 7.90 (d, 1H, Ar, J ¼ 7.8 Hz), 8.15 (d, 1H, Ar,
J ¼ 8.0 Hz), 8.20 (s, 1H, Ar). 13C NMR (CDCl3-d1) d 15.61 (CH3),
109.53 (C), 125.39 (C), 127.34 (CH), 127.38 (C), 127.84 (CH), 129.33
(CH), 129.49 (CH), 129.63 (CH), 129.71 (CH), 130.28 (C), 131.69 (C),
132.75 (CH), 133.47 (CH), 154.88 (C), 162.74 (C), 166.07 (C). 19F
NMR (CDCl3-d1) d ꢂ 62.82. IR (t) ¼ 1689 cmꢂ1 (CO amide),
1738 cmꢂ1 (CO ester). ESI-MS calcd. for C18H12F3NO3, 347.29;
found: m/z 348.08 [M þ H]þ. Anal. C18H12F3NO3 (C, H, N).
General procedure for compounds (4a–h, 4n–t)
To a suspension of the appropriate substrates 1a–e (1a41, 1b42,
1c,d43, and 1e44) (0.86 mmol) in 10 ml of anhydrous THF,
1.72 mmol of sodium hydride (60% dispersion in mineral oil), and
1.03 mmol of the appropriate acyl/aroyl chloride were added. The
mixture was stirred at room temperature overnight. The solvent
was concentrated in vacuo to obtain the final compounds 4a–h
and 4n–t which were purified by column chromatography using
hexane/ethyl acetate (5:1 for 4a,c,d; 5:2 for 4e,g), cyclohexane/
ethyl acetate (1:1 for 4f; 3:1 for 4t; 4:1 for 4h; 5:1 for 4o–s; 6:1 for
4n), or toluene/ethyl acetate 9.5:0.5 (for 4b) as eluents.
3-Methyl-2-(3-(methylsulfonyl)benzoyl)-4-phenylisoxazol-5(2H)-one
(4f). Yield ¼ 15%; mp ¼ 185–186 ꢁC (EtOH). 1H NMR (CDCl3-d1) d
2.83 (s, 3H, CH3), 3.13 (s, 3H, CH3SO2), 7.40–7.45 (m, 1H, Ar),
7.48–7.55 (m, 4H, Ar), 7.75 (t, 1H, Ar, J ¼ 8.0 Hz), 8.19 (d, 2H, Ar,
J ¼ 7.6 Hz), 8.46 (s, 1H, Ar). 13C NMR (CDCl3-d1) d 14.98 (CH3), 44.52
(CH3), 109.12 (C), 127.11 (C), 128.84 (CH), 128.93 (CH), 129.02 (CH),
129.75 (CH), 131.47 (CH), 132.84 (C), 134.47 (CH), 141.35 (C), 154.22
(C), 161.55 (C), 165.36 (C). ESI-MS calcd. for C18H15NO5S, 357.38;
found: m/z 358.07 [M þ H]þ. Anal. C18H15NO5S (C, H, N).
3-Methyl-2-(3-methylbenzoyl)-4-phenylisoxazol-5(2H)-one (4a).
1
Yield¼ 52%; mp¼ 85–88 ꢁC (EtOH). H NMR (CDCl3-d1) d 2.43 (s, 3H,
3-(3-Methyl-5-oxo-4-phenyl-2,5-dihydroisoxazole-2-carbonyl)benzo-
m-CH3-Ph), 2.79 (s, 3H, CH3), 7.35–7.40 (m, 3H, Ar), 7.43–7.51 (m, 4H, nitrile (4g). Yield ¼ 7%; mp ¼ 118–119 ꢁC (EtOH). 1H NMR (CDCl3-
Ar), 7.70–7.75 (m, 2H, Ar). 13C NMR (CDCl3-d1) d 15.09 (CH3), 21.58 d1) d 2.83 (s, 3H, CH3), 7.45–7.55 (m, 5H, Ar), 7.67 (t, 1H, Ar,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1111
J ¼ 7.2 Hz), 7.89 (d, 1H, Ar, J ¼ 6.4 Hz), 8.16 (d, 1H, Ar, J ¼ 7.6 Hz), calcd. for C13H11NO3, 229.23; found: m/z 230.08 [M þ H]þ. Anal.
8.20 (s, 1H, Ar). 13C NMR (CDCl3-d1) d 14.94 (CH3), 107.22 (C),
C13H11NO3 (C, H, N).
112.76 (C), 118.63 (C), 127.98 (CH), 128.67 (CH), 128.91 (CH), 129.54
(CH), 130.71 (CH), 131.86 (CH), 134.54 (C), 134.95 (C), 135.62 (CH),
2-(3-Methylbenzoyl)-3-(4-nitrophenyl)isoxazol-5(2H)-one (4s).
Yield ¼ 40%; mp ¼ 177–180 ꢁC dec. (EtOH). 1H NMR (DMSO-d6) d
139.52 (C), 157.64 (C), 165.87 (C). ESI-MS calcd. for C18H12N2O3,
304.30; found: m/z 305.09 [M þ H]þ. Anal. C18H12N2O3 (C, H, N).
2.42 (s, 3H, m-CH3-Ph), 7.13 (s, 1H, CH), 7.54 (t, 1H, Ar, J ¼ 7.6 Hz),
7.64 (d, 1H, Ar, J ¼ 7.6 Hz), 7.95–8.00 (m, 2H, Ar), 8.20 (d, 2H, Ar,
J ¼ 8.8 Hz), 8.36 (d, 2H, Ar, J ¼ 8.8 Hz). 13C NMR (DMSO-d6) d 21.27
4-(3-Methyl-5-oxo-4-phenyl-2,5-dihydroisoxazole-2-carbonyl)benzo-
nitrile (4h). Yield ¼ 46%; mp ¼ 178–180 ꢁC dec. (EtOH). 1H NMR
(CH3), 124.60 (CH), 125.76 (C), 126.91 (CH), 127.99 (CH), 128.28
(CDCl3-d1) d 2.82 (s, 3H, CH3), 7.37–7.42 (m, 1H, Ar), 7.45–7.50 (m,
4H, Ar), 7.80 (d, 2H, Ar, J ¼ 8.4 Hz), 8.00 (d, 2H, Ar, J ¼ 8.4 Hz). 13C
NMR (CDCl3-d1) d 14.94 (CH3), 109.39 (C), 116.49 (C), 117.69 (C),
127.10 (C), 128.85 (CH), 128.93 (CH), 129.03 (CH), 130.41 (CH),
132.32 (CH), 135.19 (C), 154.20 (C), 161.73 (C), 165.45 (C). ESI-MS
calcd. for C18H12N2O3, 304.30; found: m/z 305.09 [M þ H]þ. Anal.
C18H12N2O3 (C, H, N).
(CH), 128.91 (CH), 130.18 (CH), 131.20 (C), 133.90 (CH), 136.50 (C),
138.35 (C), 148.54 (C), 160.45 (C), 167.84 (C). ESI-MS calcd. for
C17H12N2O5, 324.29; found: m/z 325.08 [M þ H]þ. Anal. C17H12N2O5
(C, H, N).
2-(Cyclopropanecarbonyl)-3-(4-nitrophenyl)isoxazol-5(2H)-one (4t).
Yield ¼ 23%; mp ¼ 160–163 ꢁC (EtOH). 1H NMR (CDCl3-d1)
d
1.16–1.21 (m, 2H, CH2 cC3H5), 1.27–1.32 (m, 2H, CH2 cC3H5),
1.87–1.94 (m, 1H, CH cC3H5), 6.43 (s, 1H, CH), 7.96 (d, 2H, Ar,
J ¼ 8.8 Hz), 8.31 (d, 2H, Ar, J ¼ 8.8 Hz). 13C NMR (CDCl3-d1) d 10.55
(CH2), 12.71 (CH), 86.27 (CH), 121.42 (C), 124.67 (CH), 127.37 (CH),
135.22 (C), 148.74 (C), 162.53 (C), 168.75 (C). ESI-MS calcd. for
C13H10N2O5, 274.23; found: m/z 275.06 [M þ H]þ. Anal. C13H10N2O5
(C, H, N).
2-(Cyclopropanecarbonyl)-3,4-diphenylisoxazol-5(2H)-one (4n).
Yield ¼ 21%; mp ¼ 100–103 ꢁC (EtOH). 1H NMR (CDCl3-d1)
d
1.06–1.11 (m, 2H, CH2 cC3H5), 1.16–1.21 (m, 2H, CH2 cC3H5),
1.80–1.86 (m, 1H, CH cC3H5), 7.17–7.22 (m, 2H, Ar), 7.31–7.40 (m,
5H, Ar), 7.39 (d, 1H, Ar, J ¼ 7.2 Hz), 7.46 (d, 2H, Ar, J ¼ 7.6 Hz). 13C
NMR (CDCl3-d1) d 10.39 (CH2), 12.51 (CH), 103.59 (C), 128.03 (CH),
128.31 (CH), 128.37 (CH), 128.72 (CH), 129.16 (CH), 129.84 (CH),
134.92 (C), 142.60 (C), 170.20 (C), 180.73 (C). ESI-MS calcd. for
C19H15NO3, 305.33; found: m/z 306.11 [M þ H]þ. Anal. C19H15NO3
(C, H, N).
General procedure for compounds (4i, 4l)
To a suspension of 4-(pivaloyoxy)benzoic acid45 or 4-(pivalamido)-
benzoic acid46 (0.32 mmol) in 1 ml of anhydrous toluene,
0.64 mmol of SOCl2, and a catalytic amount of DMF (0.05 mmol)
were added. The mixture was stirred at reflux for 2 h. The solvent
was concentrated in vacuo and the crude compound was used
without purification and added to a previously prepared solution
composed of 0.29 mmol of intermediate 1a41 and 0.64 mmol of
sodium hydride in 5 ml of anhydrous THF. The mixture was stirred
at room temperature overnight. After evaporation of the solvent,
the product was purified by column chromatography using tolu-
ene/ethyl acetate 9:1 for 4i and hexane/acetone 4:1 for 4l
as eluents.
4-Methyl-2-(3-methylbenzoyl)-3-phenylisoxazol-5(2H)-one (4o).
1
Yield ¼ 27%; oil. H NMR (CDCl3-d1) d 1.96 (s, 3H, CH3), 2.40 (s, 3H,
m-CH3-Ph), 7.32–7.40 (m, 2H, Ar), 7.44–7.50 (m, 5H, Ar), 7.70 (d, 2H,
Ar, J ¼ 6.8 Hz). 13C NMR (CDCl3-d1) d 7.57 (CH3), 21.63 (CH3), 105.74
(C), 127.37 (CH), 128.01 (CH), 128.33 (CH), 128.67 (CH), 128.82 (C),
130.51 (CH), 130.60 (CH), 131.07 (C), 134.31 (CH), 138.46 (C), 156.85
(C), 165.24 (C), 169.02 (C). ESI-MS calcd. for C18H15NO3, 293.32;
found: m/z 294.11 [M þ H]þ. Anal. C18H15NO3 (C, H, N).
2-(Cyclopropanecarbonyl)-4-methyl-3-phenylisoxazol-5(2H)-one
(4p). Yield ¼ 31%; oil. 1H NMR (CDCl3-d1) d 1.04–1.09 (m, 2H, CH2
cC3H5), 1.10–1.15 (m, 2H, CH2 cC3H5), 1.86 (s, 3H, CH3), 2.38–2.44
(m, 1H, CH cC3H5), 7.36–7.41 (m, 2H, Ar), 7.43–7.48 (m, 3H, Ar). 13C
NMR (CDCl3-d1) d 7.30 (CH3), 10.55 (CH2), 12.71 (CH), 104.82 (C),
127.78 (C), 128.21 (CH), 128.35 (CH), 128.51 (CH), 130.44 (CH),
154.89 (C), 168.03 (C), 168.99 (C). ESI-MS calcd. for C14H13NO3,
243.26; found: m/z 244.09 [M þ H]þ. Anal. C14H13NO3 (C, H, N).
N-(4-(3-methyl-5-oxo-4-phenyl-2,5-dihydroisoxazole-2-carbonyl)-
phenyl)pivalamide (4i). Yield ¼ 10%; mp ¼ 132–134 ꢁC (EtOH). 1H
NMR (CDCl3-d1) d 1.37 (s, 9H, C(CH3)3), 2.82 (s, 3H, CH3), 7.41–7.46
(m, 1H, Ar), 7.49–7.55 (m, 4H, Ar), 7.73 (d, 2H, Ar, J ¼ 8.4 Hz), 7.99
(d, 2H, Ar, J ¼ 8.8 Hz). 13C NMR (CDCl3-d1) d 15.11 (CH3), 27.56
(CH3), 39.95 (C), 118.86 (CH), 126.01 (C), 127.54 (C), 127.66 (CH),
128.47 (CH), 128.82 (CH), 129.05 (CH), 131.65 (CH), 142.83 (C),
154.74 (C), 162.65 (C), 166.04 (C), 176.92 (C). ESI-MS calcd. for
C22H22N2O4, 378.42; found: m/z 379.16 [M þ H]þ. Anal. C22H22N2O4
(C, H, N).
2-(3-Methylbenzoyl)-3-phenylisoxazol-5(2H)-one (4q). Yield ¼ 72%;
oil. 1H NMR (CDCl3-d1) d 2.46 (s, 3H, m-CH3-Ph), 6.54 (s, 1H, CH),
7.43–7.50 (m, 5H, Ar), 7.82–7.87 (m, 2H, Ar), 8.11–8.16 (m, 2H, Ar).
13C NMR (CDCl3-d1) d 21.24 (CH3), 85.88 (CH), 126.76 (CH), 126.98
(C), 127.94 (CH), 129.04 (CH), 129.18 (C), 130.38 (CH), 131.23 (CH),
135.69 (CH), 139.06 (C), 160.47 (C), 164.26 (C), 165.70 (C). IR
(t) ¼ 1600 cmꢂ1 (CO amide), 1757 cmꢂ1 (CO ester). ESI-MS calcd.
for C17H13NO3, 279.29; found: m/z 280.09 [M þ H]þ. Anal.
C17H13NO3 (C, H, N).
4-(3-Methyl-5-oxo-4-phenyl-2,5-dihydroisoxazole-2-carbonyl)phenyl
pivalate (4l). Yield ¼ 41%; mp ¼ 130–132 ꢁC (EtOH). 1H NMR
(CDCl3-d1) d 1.37 (s, 9H, C(CH3)3), 2.80 (s, 3H, CH3), 7.22 (d, 2H, Ar,
J ¼ 8.8 Hz), 7.35–7.40 (m, 1H, Ar), 7.45–7.50 (m, 4H, Ar), 7.99 (d, 2H,
Ar, J ¼ 8.8 Hz). 13C NMR (CDCl3-d1) d 15.07 (CH3), 27.07 (CH3), 39.26
(C), 108.25 (C), 121.70 (CH), 127.56 (C), 128.16 (CH), 128.53 (CH),
128.83 (CH), 129.04 (CH), 131.70 (CH), 154.55 (C), 155.05 (C), 162.54
(C), 165.80 (C), 176.34 (C). IR (t) ¼ 1678 cmꢂ1 (CO amide),
1753 cmꢂ1 (CO ester), 1768 cmꢂ1 (CO ester). ESI-MS calcd. for
C22H21NO5, 379.41; found: m/z 380.15 [M þ H]þ. Anal. C22H21NO5
2-(Cyclopropanecarbonyl)-3-phenylisoxazol-5(2H)-one (4r). Yield ¼ 57%;
oil. 1H NMR (CDCl3-d1) d 1.10–1.16 (m, 2H, CH2 cC3H5), 1.23–1.28
(m, 2H, CH2 cC3H5), 1.84–1.90 (m, 1H, CH cC3H5), 6.34 (s, 1H, CH),
7.42–7.47 (m, 3H, Ar), 7.74–7.79 (m, 2H, Ar). 13C NMR (CDCl3-d1) d
10.82 (CH2), 12.71 (CH), 85.75 (CH), 126.55 (CH), 128.87 (CH),
129.27 (C), 130.26 (CH), 164.15 (C), 165.51 (C), 168.48 (C). ESI-MS (C, H, N).

1112
M. P. GIOVANNONI ET AL.
General procedure for compounds (4m, 4u)
General procedure for compounds (6a–e)
To a solution of intermediate 1b42 or 1f47 (0.32 mmol) in 2 ml of t- A 3.00 mmol of appropriate intermediate 5a–c48–50 and 5e,f was
BuOH, 0.35 mmol of K2CO3 and 0.64 mmol of m-toluoyl chloride dissolved in 1.3 ml of water and heated at 80 ꢁC. To this solution,
3.3 mmol of hydroxylamine hydrochloride in 6.5 ml of methanol
was added. The mixture was stirred at reflux for 5 h. After evapor-
ation of the solvent, the residue was mixed with ice-cold water
(20 ml). Compounds 6a,c were recovered by extraction with ethyl
acetate (3 ꢀ 15 ml), while compounds 6b,d,e were recovered by
vacuum filtration. The final compounds 6b,d were purified by crys-
tallisation with ethanol, while the compounds 6a,c,e were purified
by column chromatography using dichoromethane/methanol 9:1
as eluent.
were added. The mixture was stirred at reflux for 3 h. The solvent
was concentrated in vacuo, diluted with ice-cold water (10 ml), and
extracted with DCM (3 ꢀ 15 ml). The organic phase was dried over
sodium sulphate and the solvent was evaporated in vacuo to
afford the final compounds 4m,u, which were purified by column
chromatography using cyclohexane/ethyl acetate in different ratio:
5:1 for 4m and 1:1 for 4u as eluent.
2-(3-Methylbenzoyl)-3,4-diphenylisoxazol-5(2H)-one (4m). Yield ¼ 14%;
mp ¼ 160–163 ꢁC (EtOH). 1H NMR (CDCl3-d1) d 2.42 (s, 3H, m-CH3-
Ph), 7.26–7.32 (m, 5H, Ar), 7.37–7.48 (m, 7H, Ar), 7.72–7.77 (m, 2H,
Ar). 13C NMR (CDCl3-d1) d 21.40 (CH3), 101.40 (C), 127.48 (CH),
128.31 (CH), 128.46 (CH), 128.53 (CH), 128.83 (CH), 130.74 (CH),
132.49 (C), 132.63 (C), 134.21 (C), 134.52 (CH), 138.50 (C), 142.65
(C), 157.66 (C), 165.80 (C). ESI-MS calcd. for C23H17NO3, 355.39;
found: m/z 356.12 [M þ H]þ. Anal. C23H17NO3 (C, H, N).
3-Methyl-4-(p-tolyl)isoxazol-5(2H)-one (6a). Yield ¼ 35%; oil. 1H
NMR (CDCl3-d1) d 2.17 (s, 3H, CH3Ph), 2.28 (s, 3H, CH3), 7.09 (d, 2H,
Ar, J ¼ 6.0 Hz), 7.28 (d, 2H, Ar, J ¼ 6.0 Hz), 9.03 (exch br s, 1H, NH).
ESI-MS calcd. for C11H11NO2, 189.21; found: m/z 190.08 [M þ H]þ.
Anal. C11H11NO2 (C, H, N).
4-(3-Methyl-5-oxo-2,5-dihydroisoxazol-4-yl)benzonitrile (6b). Yield ¼
72%; mp ¼ 114–116 ꢁC (EtOH). 1H NMR (CDCl3-d1) d 2.43 (s, 3H,
CH3), 7.70 (s, 4H, Ar). IR (t) ¼ 2270 cmꢂ1 (CN). ESI-MS calcd. for
C11H8N2O2, 200.19; found: m/z 201.06 [M þ H]þ. Anal. C11H8N2O2
(C, H, N).
N-(4-(2-(3-methylbenzoyl)-5-oxo-2,5-dihydroisoxazol-3-yl)phenyl)a-
cetamide (4u). Yield ¼ 21%; oil. 1H NMR (CDCl3-d1) d 2.20 (s, 3H,
CH3CO), 2.45 (s, 3H, m-CH3-Ph), 6.50 (s, 1H, CH), 7.42 (t, 1H, Ar,
J ¼ 7.8 Hz), 7.50 (d, 1H, Ar, J ¼ 7.6 Hz), 7.56 (exch br s, 1H, NH), 7.62
(d, 2H, Ar, J ¼ 8.0 Hz), 7.76 (d, 2H, Ar, J ¼ 8.4 Hz), 7.98–8.13 (m, 2H,
Ar). 13C NMR (CDCl3-d1) d 21.30 (CH3), 24.81 (CH3), 85.70 (C),
119.72 (CH), 127.45 (CH), 127.93 (CH), 128.88 (CH), 129.65 (C),
131.17 (CH), 134.11 (C), 135.75 (CH), 138.99 (C), 139.73 (C), 156.05
(C), 157.65 (C), 167.14 (C), 168.90 (C). ESI-MS calcd. for C19H16N2O4,
336.34; found: m/z 337.11 [M þ H]þ. Anal. C19H16N2O4 (C, H, N).
3-Methyl-4-(4-nitrophenyl)isoxazol-5(2H)-one (6c). Yield ¼ 27%;
mp ¼ 208–209 ꢁC (EtOH). 1H NMR (DMSO-d6) d 2.19 (s, 3H, CH3),
7.83 (d, 2H, Ar, J ¼ 9.2 Hz), 7.94 (d, 2H, Ar, J ¼ 8.4 Hz). ESI-MS calcd.
for C10H8N2O4, 220.18; found: m/z 221.05 [M þ H]þ. Anal.
C10H8N2O4 (C, H, N).
N-(4-(3-methyl-5-oxo-2,5-dihydroisoxazol-4-yl)phenyl)acetamide
(6d). Yield ¼ 75%; mp ¼ 210–212 ꢁC (EtOH). 1H NMR (DMSO-d6) d
2.01 (s, 3H, NHCOCH3), 2.27 (s, 3H, CH3), 7.43 (d, 2H, Ar, J ¼ 8.0 Hz);
7.56 (d, 2H, Ar, J ¼ 8.0 Hz), 9.92 (exch br s, 1H, NHCOCH3), 12.54
(exch br s, 1H, NH). ESI-MS calcd. for C12H12N2O3, 232.24; found:
m/z 233.09 [M þ H]þ. Anal. C12H12N2O3 (C, H, N).
General procedure for compounds (5e, 5f)
To a cooled (0 ꢁC) suspension of 5d48 (0.68 mmol) in anhydrous
CH2Cl2 (2 ml), Et3N (1.36 mmol) and 2.04 mmol of appropriate acyl
chloride were added. The mixture was stirred at 0 ꢁC for 2 h and
then at room temperature for an additional 2 h. The solvent was
evaporated, cold water was added and the mixture was neutral-
ised with 0.5 N NaOH. The reaction mixture was extracted with
CH2Cl2 (3 ꢀ 15 ml), the solvent was dried over sodium sulphate,
evaporated in vacuo, and compounds 5e and 5f were purified by
column chromatography using dichloromethane/methanol (9:1 for
5e; 99:1 for 5f) as eluents.
N-(4-(3-methyl-5-oxo-2,5-dihydroisoxazol-4-yl)phenyl)cyclopropane-
carboxamide (6e). Yield ¼ 58%; mp ¼ 208–210 ꢁC (EtOH). 1H NMR
(DMSO-d6) d 0.70–0.75 (m, 4H, 2 ꢀ CH2 cC3H5), 1.70–1.75 (m,1H,
CH), 2.12 (s, 3H, CH3), 7.40 (d, 2H, Ar, J ¼ 8.4 Hz), 7.47 (d, 2H, Ar,
J ¼ 8.4 Hz), 9.97 (exch br s, 1H, NHCO cC3H5), 12.50 (exch br s, 1H,
NH). ESI-MS calcd. for C14H14N2O3, 258.27; found: m/z 259.10
[M þ H]þ. Anal. C14H14N2O3 (C, H, N).
Ethyl 2-(4-acetamidophenyl)-3-oxobutanoate (5e). Yield ¼ 67%;
1
mp ¼ 112–113 ꢁC (EtOH). H NMR (CDCl3-d1) showed a 3:1 mixture
of aldo-enol tautomers: d 1.24 (t, 3H, CH2CH3, J ¼ 7.0 Hz), 1.61 (t,
1H, CH2CH3, J ¼ 7.0 Hz), 1.81 (s, 1H, COCH3), 2.12 (s, 3H, COCH3),
2.15 (s, 4H, NHCOCH3), 4.16 (q, 2.7H, CH2CH3, J ¼ 7.2 Hz), 4.65 (s,1H,
CH), 7.06 (d, 0.7H, Ar, J ¼ 8.0 Hz), 7.23 (d, 2H, Ar, J ¼ 8.4 Hz), 7.48 (d,
2.7H, Ar, J ¼ 8.0 Hz), 7.92 (exch br s, 1.3H, NH), 13.07 (exch br s,
0.3H, OH). ESI-MS calcd. for C14H17NO4, 263.29; found: m/z 264.12
[M þ H]þ. Anal. C14H17NO4 (C, H, N).
General procedure for compounds (7a–e, 8a,b,d,e)
Compounds 7a–e and 8a,b,d,e were obtained following the same
procedure performed for compounds 4a–h, 4n–t but starting from
precursors 6a–e. The solvent was concentrated in vacuo to obtain
the final compounds which were purified by column chromatog-
raphy using petroleum ether/ethyl acetate 10:1 for 7a/8a, hexane/
ethyl acetate (5:1 for 7b/8b; 5:2 for 7c), dichloromethane/metha-
nol (98:2 for 7d/8d; 99:1 for 7e/8e) as eluents.
Ethyl 2-(4-(ciclopropanecarboxamido)phenyl)-3-oxobutanoate (5f).
Yield ¼ 78%; oil. 1H NMR (CDCl3-d1) showed the only aldo tauto-
mer: d 0.72–0.77 (m, 2H, CH2 cC3H5), 0.80–0.85 (m, 2H, CH2 cC3H5),
1.20 (t, 3H, CH2CH3, J ¼ 7.2 Hz), 1.39–1.46 (m, 1H, CH cC3H5), 2.35
(s, 3H, COCH3), 4.17 (q, 2H, CH2CH3, J ¼ 7.2 Hz), 4.63 (s, 1H, CH),
7.19 (d, 2H, Ar, J ¼ 8.4 Hz), 7.46 (d, 2H, Ar, J ¼ 7.6 Hz), 7.52 (exch br
3-Methyl-2-(3-methylbenzoyl)-4-(p-tolyl)isoxazol-5(2H)-one (7a).
1
Yield ¼ 60%; oil. H NMR (CDCl3-d1) d 2.39 (s, 3H, p-CH3Ph), 2.43 (s,
3H, m-CH3Ph), 2.79 (s, 3H, CH3), 7.27 (d, 2H, Ar, J ¼ 8.4 Hz),
7.37–7.42 (m, 4H, Ar), 7.70 (d, 2H, Ar, J ¼ 6.8 Hz). 13 C NMR (CDCl3-
s, 1H, NH). ESI-MS calcd. for C16H19NO4, 289.33; found: m/z 290.13 d1) d 15.05 (CH3), 21.33 (CH3), 108.19 (C), 124.63 (C), 127.03 (CH),
[M þ H]þ. Anal. C16H19NO4 (C, H, N).
128.30 (CH), 128.89 (CH), 129.52 (CH), 130.24 (CH), 131.27 (C),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1113
134.00 (CH), 138.28 (C), 138.48 (C), 154.09 (C), 163.82 (C), 166.07 7.92 (s, 2H, Ar). 13 C NMR (CDCl3-d1) d 12.31 (CH3), 21.25 (CH3),
(C). ESI-MS calcd. for C19H17NO3, 307.34; found: m/z 308.12
29.70 (C), 111.68 (C), 118.37 (C), 126.33 (C), 128.02 (CH), 128.61
(CH), 128.95 (CH), 131.30 (CH), 132.75 (CH), 133.27 (C), 136.04 (CH),
139.11 (C), 160.61 (C), 161.90 (C). ESI-MS calcd. for C19H14N2O3,
318.33; found: m/z 319.10 [M þ H]þ. Anal. C19H14N2O3 (C, H, N).
[M þ H]þ. Anal. C19H17NO3 (C, H, N).
4-(3-Methyl-2-(3-methylbenzoyl)-5-oxo-2,5-dihydroisoxazol-4-
yl)benzonitrile (7b). Yield ¼ 26%; mp ¼ 108–110 ꢁC (EtOH). 1H NMR
(CDCl3-d1) d 2.44 (s, 3H, CH3Ph), 2.84 (s, 3H, CH3), 7.38–7.48 (m, 2H,
Ar), 7.65 (d, 2H, Ar, J ¼ 8.4 Hz), 7.71 (d, 2H, Ar, J ¼ 8.0 Hz), 7.76 (d,
2H, Ar, J ¼ 8.4 Hz). 13C NMR (CDCl3-d1) d 15.19 (CH3), 21.37 (CH3),
29.70 (C), 106.24 (C), 112.03 (C), 118.41 (C), 127.13 (CH), 128.43
(CH), 129.43 (CH), 130.34 (CH), 130.72 (C), 132.52 (CH), 132.74 (C),
134.45 (CH), 138.49 (C), 155.56 (C), 163.79 (C), 165.15 (C). ESI-MS
calcd. for C19H14N2O3, 318.33; found: m/z 319.10 [M þ H]þ. Anal.
C19H14N2O3 (C, H, N).
4-(4-Acetamidophenyl)-3-methylisoxazol-5-yl3-methylbenzoate (8d).
Yield ¼ 10%; mp ¼ 57–60 ꢁC (EtOH). 1H NMR (CDCl3-d1) d 2.15 (s,
3H, NHCOCH3), 2.36 (s, 3H, CH3), 2.41 (s, 3H, CH3Ph), 7.29 (d, 3H,
Ar, J ¼ 8.4 Hz), 7.38 (t, 1H, Ar, J ¼ 8.0 Hz), 7.46 (exch br s, 1H, NH),
7.51 (d, 2H, Ar, J ¼ 8.4 Hz), 7.91 (s, 2H, Ar). 13C NMR (CDCl3-d1) d
12.23 (CH3), 21.24 (CH3), 24.59 (CH3), 29.69 (C), 120.11 (CH), 123.90
(C), 126.71 (C), 127.97 (CH), 128.83 (CH), 129.67 (CH), 131.26 (CH),
135.70 (CH), 137.61 (C), 138.91 (C), 161.21 (C), 162.31 (C), 168.39
(C). ESI-MS calcd. for C20H18N2O4, 350.37; found: m/z 351.13
[M þ H]þ. Anal. C20H18N2O4 (C, H, N).
3-Methyl-2-(3-methylbenzoyl)-4-(4-nitrophenyl)isoxazol-5(2H)-one
(7c). Yield ¼ 6%; mp ¼ 152–154 ꢁC (EtOH). 1H NMR (CDCl3-d1) d
2.45 (s, 3H, CH3Ph), 2.86 (s, 3H, CH3), 7.39–7.46 (m, 2H, Ar), 7.72 (d,
4H, Ar, J ¼ 8.8 Hz), 8.32 (d, 2H, Ar, J ¼ 8.8 Hz). 13C NMR (CDCl3-d1) d
13.21 (CH3), 21.32 (CH3), 29.70 (C), 124.02 (CH), 127.16 (CH), 128.45
(CH), 129.56 (CH), 130.36 (CH), 130.65 (C), 134.51 (CH), 134.69 (C),
138.52 (C), 147.37 (C), 155.78 (C), 163.80 (C). ESI-MS calcd. for
C18H14N2O5, 338.31; found: m/z 339.09 [M þ H]þ. Anal. C18H14N2O5
(C, H, N).
4-(4-(Ciclopropanecarboxamido)phenyl)-3-methylisoxazol-5-yl3-me-
thylbenzoate (8e). Yield ¼ 8%; mp ¼ 80–82 ꢁC (EtOH). 1H NMR
(CDCl3-d1) d 0.80–0.90 (m, 4H, 2 ꢀ CH2 cC3H5), 1.35–1.40 (m, 1H,
CH), 2.37 (s, 3H, CH3), 2.41 (s, 3H, CH3Ph), 7.28 (d, 2H, Ar,
J ¼ 8.0 Hz), 7.38 (t, 1H, Ar, J ¼ 7.6 Hz), 7.40 (exch br s,1H, NH), 7.47
(d, 1H, Ar, J ¼ 7.6 Hz), 7.52 (d, 2H, Ar, J ¼ 7.2 Hz), 7.92 (s, 2H, Ar).
13C NMR (CDCl3-d1) d 8.15 (CH2), 12.24 (CH3), 14.90 (CH), 21.25
(CH3), 100.52 (C), 119.91 (CH), 127.97 (CH), 128.79 (CH), 128.85
(CH), 131.25 (CH), 132.05 (C), 135.68 (CH), 138.70 (C), 138.89 (C),
154.20 (C), 158.90 (C), 165.20 (C), 180.70 (C). ESI-MS calcd. for
C22H20N2O4, 376.41; found: m/z 377.15 [M þ H]þ. Anal. C22H20N2O4
(C, H, N).
N-(4-(3-methyl-2-(3-methylbenzoyl)-5-oxo-2,5-dihydroisoxazol-4-
1
yl)phenyl)acetamide (7d). Yield ¼ 37%; mp ¼ 159–160 ꢁC (EtOH). H
NMR (CDCl3-d1) d 2.14 (s, 3H, NHCOCH3), 2.41 (s, 3H, CH3Ph), 2.75
(s, 3H, CH3), 7.34–7.40 (m, 4H, Ar), 7.58 (d, 2H, Ar, J ¼ 8.4 Hz), 7.67
(s, 2H, Ar), 7.97 (exch br s, 1H, NH). 13C NMR (CDCl3-d1) d 15.06
(CH3), 21.35 (CH3), 24.53 (CH3), 120.11 (CH), 123.11 (C), 126.99 (CH),
128.33 (CH), 128.94 (CH), 129.61 (CH), 130.23 (CH), 131.11 (C),
131.27 (CH), 134.12 (CH), 138.29 (C), 154.40 (C), 163.79 (C), 166.26
(C), 168.87 (C). ESI-MS calcd. for C20H18N2O4, 350.37; found: m/z
351.13 [M þ H]þ. Anal. C20H18N2O4 (C, H, N).
General procedure for compounds (10a–c)
To suspension of the substrate 951 (0.37 mmol) in tert-Butanol
(3 ml), K2CO3 (0.41 mmol), and 0.74 mmol of the appropriate acyl
chloride were added. The mixture was stirred at reflux for 3 h.
After evaporation of the solvent, the residue was mixed with ice-
cold water (20 ml) and extracted with ethyl acetate (3 ꢀ 15 ml).
The organic phase was dried over sodium sulphate, and the solv-
ent was evaporated in vacuo to afford the final compounds 10a–c,
which were purified by column chromatography using cyclohex-
ane/ethyl acetate (5:1) as eluent.
N-(4-(3-methyl-2-(3-methylbenzoyl)-5-oxo-2,5-dihydroisoxazol-4-yl)
phenyl)ciclopropanecarboxamide (7e). Yield ¼ 6%; mp ¼ 120–123 ꢁC
(EtOH). 1H NMR (CDCl3-d1) d 0.84–0.89 (m, 2H, CH2 cC3H5),
1.07–1.12 (m, 2H, CH2 cC3H5), 1.24 (s, 1H, CH), 2.43 (s, 3H, CH3Ph),
2.79 (s, 3H, CH3), 7.36–7.41 (m, 2H, Ar), 7.46 (d, 2H, Ar, J ¼ 8.4 Hz),
7.61 (d, 2H, Ar, J ¼ 8.4 Hz), 7.70 (d, 2H, Ar, J ¼ 7.2 Hz). 13C NMR
(CDCl3-d1) d 8.24 (CH2), 15.13 (CH3), 15.88 (CH), 21.41 (CH3), 107.20
(C), 119.79 (CH), 127.03 (CH), 128.33 (CH), 129.68 (CH), 130.25 (CH),
130.30 (C), 134.09 (CH), 134.10 (C), 137.70 (C), 138.33 (C), 139.50
(C), 157.61 (C), 163.83 (C), 180.70 (C). ESI-MS calcd. for C22H20N2O4,
376.41; found: m/z 377.15 [M þ H]þ. Anal. C22H20N2O4 (C, H, N).
1-Propionylbenzo[c]isoxazol-3(1H)-one (10a). Yield ¼ 7%; oil. 1H
NMR (CDCl3-d1) d 1.28 (t, 3H, CH2CH3, J ¼ 7.4 Hz), 2.83 (q, 2H,
CH2CH3, J ¼ 7.2 Hz), 7.38 (t 1H, Ar, J ¼ 7.6 Hz), 7.78 (t 1H, Ar,
J ¼ 7.6 Hz), 7.89 (d, 1H, Ar, J ¼ 8.0 Hz), 8.11 (d, 1H, Ar, J ¼ 8.4 Hz).
13C NMR (CDCl3-d1) d 9.72 (CH3), 20.70 (CH2), 120.35 (CH), 122.50
(C), 124.09 (CH), 130.31 (CH), 133.90 (CH), 142.44 (C), 166.02 (C),
172.05 (C). ESI-MS calcd. for C10H9NO3, 191.18; found: m/z 192.06
[M þ H]þ. Anal. C10H9NO3 (C, H, N).
3-Methyl-4-(p-tolyl)isoxazol-5-yl 3-methylbenzoate (8a). Yield ¼ 12%;
oil. 1H NMR (CDCl3-d1) d 2.33 (s, 3H, p-CH3Ph), 2.38 (s, 3H, CH3),
2.41 (s, 3H, m-CH3Ph), 7.18 (d, 2H, Ar, J ¼ 8.0 Hz), 7.24 (d, 2H, Ar,
J ¼ 8.4 Hz), 7.38 (t, 1H, Ar, J ¼ 8.0 Hz), 7.47 (d, 1H, Ar, J ¼ 7.2 Hz),
7.93 (s, 2H, Ar). 13 C NMR (CDCl3-d1) d 12.21 (CH3), 21.23 (CH3),
100.50 (C), 127.97 (CH), 128.10 (CH), 128.75 (CH), 129.63 (CH),
130.10 (C), 131.26 (CH), 133.30 (C), 135.57 (CH), 138.84 (C), 154.20
1-Pentanoylbenzo[c]isoxazol-3(1H)-one (10b). Yield ¼ 12%; oil. 1H
NMR (CDCl3-d1)
d
0.87 (t, 3H, CH3CH2CH2CH2CO, J ¼ 6.8 Hz),
1.53–1.58 (m, 2H, CH3CH2CH2CH2CO), 1.98–2.03 (m, 2H,
(C), 158.90 (C), 165.20 (C). ESI-MS calcd. for C19H17NO3, 307.34; CH3CH2CH2CH2CO), 3.63 (t, 2H, CH3CH2CH2CH2CO, J ¼ 6.8 Hz), 7.22
found: m/z 308.12 [M þ H]þ. Anal. C19H17NO3 (C, H, N).
(d, 1H, Ar, J ¼ 8.4 Hz), 7.31 (t, 1H, Ar, J ¼ 7.4 Hz), 7.68 (t, 1H, Ar,
J ¼ 7.6 Hz), 7.86 (d, 1H, Ar, J ¼ 7.6 Hz). 13C NMR (CDCl3-d1) d 13.10
(CH3), 22.15 (CH2), 27.65 (CH2), 28.21 (CH2), 120.31 (CH), 122.49 (C),
124.00 (CH), 130.33 (CH), 133.90 (CH), 142.41 (C), 165.31 (C), 172.22
(C). ESI-MS calcd. for C12H13NO3, 219.24; found: m/z 220.09
4-(4-Cyanophenyl)-3-methylisoxazol-5-yl 3-methylbenzoate (8b).
Yield ¼ 20%; mp ¼ 98–100 ꢁC (EtOH). 1H NMR (CDCl3-d1) d 2.41 (s,
3H, CH3), 2.43 (s, 3H, CH3Ph), 7.43 (t, 1H, Ar, J ¼ 8.0 Hz), 7.47 (d, 2H,
Ar, J ¼ 8.4 Hz), 7.51 (d, 1H, Ar, J ¼ 7.6 Hz), 7.67 (d, 2H, Ar, J ¼ 8.4 Hz), [M þ H]þ. Anal. C12H13NO3 (C, H, N).

1114
M. P. GIOVANNONI ET AL.
1-(3-Methylbenzoyl)benzo[c]isoxazol-3(1H)-one (10c). Yield ¼ 53%; quantum chemical calculations (QC) and MD simulations with
mp ¼ 116–119 ꢁC (EtOH). 1H NMR (CDCl3-d1) d 2.44 (s, 3H, CH3), the receptor.
7.39–7.45 (m, 3H, Ar), 7.75–7.80 (m, 2H, Ar), 7.83 (t 1H, Ar,
GAUSSIAN09 (Rev. C01)55 was used for quantum chemical calcula-
J ¼ 8.4 Hz), 7.92 (d, 1H, Ar, J ¼ 8.0 Hz), 8.22 (d, 1H, Ar, J ¼ 8.4 Hz). tions (QC) on 7d and 8d by using the B3LYP56,57 and B97D58 func-
13C NMR (CDCl3-d1) d 21.40 (CH3), 115.83 (CH), 117.51 (C), 125.60 tionals. The basis set was 6-31 þ G(d,p)59, and the Berny algorithm
(CH), 126.03 (CH), 126.87 (CH), 128.34 (CH), 130.12 (CH), 133.40 (C), was used60. Reliability of the stationary points was assessed by
133.81 (CH), 136.54 (CH), 137.80 (C), 151.60 (C), 158.51 (C), 172.03 evaluation of the vibrational frequencies. For each inhibitor, differ-
(C). ESI-MS calcd. for C15H11NO3, 253.25; found: m/z 254.08 ent conformational isomers, chosen from amongst the low lying
[M þ H]þ. Anal. C15H11NO3 (C, H, N).
energy conformers (as found in MD simulations and subsequent
geometry optimisation) were considered.
Molecular plots were produced by the program Discovery
Studio Visualiser (v 4.5)61.
HNE inhibition assay
Compounds were dissolved in 100% DMSO at 5 mM stock concen-
trations. The final concentration of DMSO in the reactions was 1%,
and this level of DMSO had no effect on enzyme activity. The HNE
Results and discussion
inhibition assay was performed in black flat-bottom 96-well micro- Chemistry
titer plates. Briefly, a buffer solution containing 200 mM Tris–HCl,
It is well-known that the isoxazolone nucleus exhibits three tauto-
pH 7.5, 0.01% bovine serum albumin, and 0.05% Tween-20 and
20 mU/mL of HNE (Calbiochem) was added to wells containing dif-
ferent concentrations of each compound. The reaction was initi-
ated by addition of 25 mM elastase substrate (N-methylsuccinyl-
Ala-Ala-Pro-Val-7-amino-4-methylcoumarin, Calbiochem) in a final
reaction volume of 100 ml/well. Kinetic measurements were
obtained every 30 s for 10 min at 25 ꢁC using a Fluoroskan Ascent
FL fluorescence microplate reader (Thermo Electron, MA) with exci-
tation and emission wavelengths set at 355 and 460 nm, respect-
ively. For all compounds tested, the concentration of inhibitor that
caused 50% inhibition of the enzymatic reaction (IC50) was calcu-
lated by plotting % inhibition versus logarithm of inhibitor con-
centration (at least six points). The data are presented as the
mean values of at least three independent experiments with rela-
tive standard deviations of <15%.
mers43,62,63, as illustrated below. The NH form seems to be the
most representative, especially in polar solvents62,64–66. However, it
is possible to find examples of alkylation and acylation products in
the literature, as shown by the NH and the OH forms67. Taking
into account this information, we performed the synthesis of our
final compounds, as shown in Schemes 1–3, and the structures
were confirmed on the basis of analytical and spectral data.
Scheme 1 depicts the synthetic pathway followed to obtain the
final 2-N-substituted isoxazolones of type 2, 3, and 4, which have
different groups at positions 3 and 4. The previously described key
intermediate isoxazol-5-(2H)-ones of type 1 were treated under
various conditions to obtain the final compounds 2, 3, and 4. The
alkylation of 1a41 with 3-methylbenzyl chloride and K2CO3 in
anhydrous acetonitrile at reflux resulted in compound 2. On the
other hand, treatment of compounds 1a–f (1a41, 1b42, 1c,d43,
1e44, and 1f47) with the appropriate acyl/aroyl chloride and NaH
in anhydrous THF at room temperature (compounds 4a–l,n–t) or
with m-toluyl chloride, and K2CO3 in t-ButOH at 80 ꢁC (compounds
4m,u) resulted in the corresponding 2-NCO derivatives of type 4.
Likewise, we synthesised the sulfonamide derivatives 3a–c by
treatment of intermediate 1a41 with the appropriate commercially
available phenyl sulfonyl chloride in pyridine at room temperature.
All of these reactions led to a single derivative originating from
the NH form of the isoxazolone nucleus, in agreement with previ-
ous data reported in literature.
Analysis of compound stability
Spontaneous hydrolysis of selected derivatives was evaluated at
25 ꢁC in 0.05 M phosphate buffer, pH 7.3. Kinetics of hydrolysis
were monitored by measuring changes in the absorbance spectra
over time using a SpectraMax Plus microplate spectrophotometer
(Molecular Devices, Sunnyvale, CA). Absorbance (At) at the charac-
teristic absorption maxima of each compound was measured at
the indicated times until no further absorbance decreases
occurred (A )52. Using these measurements, we created semilogar-
1
Scheme 2 shows the synthetic procedures used to obtain the
final compounds of type 7 and 8, which have a 4-substituted phe-
nyl at position 4. b-Ketoesters 5a–d (5a49, 5b,d48, and 5c50) were
synthesised as described previously48–50, while compounds 5e,f
were obtained by acylation of 5d48 with the appropriate acyl
chloride and Et3N in dichloromethane. These compounds served
as the starting material for synthesis of the key intermediates of
ithmic plots of log(At–A ) versus time, and k0 values were deter-
1
mined from the slopes of these plots. Half-conversion times were
calculated using t1/2 ¼ 0.693/k0, as described previously36,37
.
Molecular modelling procedures
The programs used for the energy minimisation, MD, and docking type 6 with an isoxazolone nucleus. Cyclisation of 5a–c,e,f with
were the simulation protocols Minimisation, Standard Dynamics hydroxylamine hydrochloride in a mixture MeOH/H2O 1:1 at reflux
Cascade, Analyse Trajectory, and CDocker implemented in Accelrys resulted in the intermediates 6a–e which, in turn, were treated
Discovery Studio 2.153. The Force Field used for all simulations with m-toluoyl chloride under the same conditions reported in
was CHARMm54.
Scheme 1. Unexpectedly this last step resulted the pair of isomers
The following parameters were used for MD simulations, both of type 7 and 8 (NCO/OCO ratio 3:1), with the only exception
in vacuum and in implicit solvent (the latter was simulated by being the nitro derivative 7c, which was obtained only in the N-
using distance dependent dielectric constant set to 4r): time CO form.
step ¼ 1 fs, equilibration time ¼ 100 ps, production time ¼ 1000 ps
Assignment of the isomer structures was first performed using
(5000 ps for the inhibitor-HNE assembly), T ¼ 300 and 600 K. Ten the 1H NMR chemical shift value of the methyl at position 3.
snapshot conformations with evenly spaced intervals were Compounds 4a–l (Scheme 1) had shifts of 2.8 ppm, which is char-
extracted from each MD trajectory and subsequently minimised acteristic of NCO derivatives. Likewise, we attributed the NCO-
(using the steepest descent and conjugate gradient algorithms) in structure to compounds with a 2.8 ppm shift for 3-CH3 (7a–e) and
order to obtain the starting geometries for the subsequent the OCO-structure to compounds with a 2.4 ppm shift (8a,b,d,e)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1115
4
a
b
c
d
e
R2
m-CH3-Ph
cC3H5
p-CH3-Ph
o-CH3-Ph
m-CF3-Ph
m-SO2CH3-Ph
m-CN-Ph
p-CN-Ph
R3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Ph
Ph
Ph
Ph
Ph
Ph
R4
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
CH3
CH3
H
1
a
b
c
d
e
f
R3
CH3
Ph
Ph
Ph
R4
Ph
Ph
CH3
H
3
a
b
c
X
OH
OCOC(CH3)3
NHCOC(CH3)3
4-NO2-Ph
4-NHCOCH3-Ph
H
H
f
g
h
i
p-NHCOC(CH3)3-Ph
p-OCOC(CH3)3-Ph
m-CH3-Ph
cC3H5
l
m
n
o
p
q
r
m-CH3-Ph
cC3H5
m-CH3-Ph
cC3H5
H
s
t
u
m-CH3-Ph
cC3H5
m-CH3-Ph
4-NO2-Ph
4-NO2-Ph
4-NHCOCH3-Ph
H
H
H
Scheme 1. Reagents and conditions: (a) 3-methylbenzyl chloride, K2CO3, anhydrous CH3CN, 80 ꢁC, 2 h; (b) 4-X-Ph-SO2Cl, anhydrous pyridine, r.t., 4 h; (c) for 4a–l, n–t:
R2-COCl, NaH, anhydrous THF, r.t., 24 h; for 4m,u: m-toluoyl chloride, K2CO3, t-BuOH, 80 ꢁC, 3 h.
(see Supporting Information). In order to verify our findings, we possibility of a two point of attack for Ser195, 2-NCO, and 5-CO,
and docking studies confirmed that the endocyclic C ¼ O at pos-
performed additional techniques, such as IR spectroscopy and 2 D
1
1
NMR (1H–13C HSQC, H–13C HMBC, and H–1H NOESY).
ition 5 was involved in the catalysis process, whereas the amidic
C ¼ O group was important for anchoring to the sub-pocket of the
binding site. We could expect a similar trend in this new series of
isoxazolones.
In Scheme 3, we show the synthetic pathways used to obtain
the benzoisoxazolone derivatives 10a–c, which are an elaboration
of the previous isoxazolone scaffold. The acylation of benzoisoxa-
zolone intermediate 951 with the appropriate acyl chloride and
potassium carbonate in t-BuOH led to final compounds 10a–c.
Beginning analysis of the data with the 3-methyl-4-phenylisoxa-
zol-5(2H)-one derivatives (compounds 4a–l), the results reported in
Table 1 suggest that, similar to our previous series40, the best sub-
stituents at position N-2 are a m-methylbenzoyl or a cyclopropa-
necarbonyl fragment, which resulted in compounds 4a and 4b
that were active in the nanomolar range (IC50 ¼ 77 and 59 nM,
respectively). Displacement of the methyl group from the meta to
the para (4c) or ortho (4d) positions on the phenyl led to com-
pounds with activity one order of magnitude lower than 4a, which
Biological evaluation and structure–activity relationship
(SAR) analysis
All compounds were evaluated for their ability to inhibit HNE in
comparison with Sivelestat, a reference HNE inhibitor, and the
results presented in Tables 1–3. Previously40, we examined the is different than our previous series40, where moving the methyl

1116
M. P. GIOVANNONI ET AL.
5
a
b
c
d
e
f
R
CH3
CN
NO2
NH2
6-8
a
R
CH3
CN
b
c
NO2
d
NHCOCH3
NHCOcC3H5
NHCOCH3
NHCOcC3H5
e
Scheme 2. Reagents and conditions: (a) R-COCl, Et3N, anhydrous CH2Cl2, 0 ꢁC, 2 h, then r.t., 2 h; (b) NH2OH.HCl, H2O/MeOH 1:1, reflux, 5 h; (c) m-toluoyl chloride, NaH,
anhydrous THF, r.t., 24 h.
inhibitory activity and also probably involved in catalysis. The
insertion of the 3-methyl-4-phenylisoxazol-5(2H)-one scaffold of
the sulfonamide fragment of the drug Sivelestat at position 2 led
to compound 3b, which exhibited comparable activity to
Sivelestat (IC50 ¼ 59 nM, Table 1). In contrast, its pivalamide deriva-
tive (3c) was completely inactive, suggesting that the possible
point of attack of Ser195 is the CO of the pivalate function, which
is also present in Sivelestat. These data were also confirmed by
the inactivity of the hydrolysed derivative 3a. Moreover, we syn-
thesised compounds 4l and 4i by substituting the SO2 group at
position 2 (3b and 3c, respectively) with an amidic function. The
amide 4l had activity (IC50 ¼ 0.48 mM) that was one order of mag-
nitude higher than the corresponding sulfonamide 3b, while the
10
a
R1
C2H5
amide 4i had a higher potency (IC50 ¼ 8.5 mM) than its correspond-
ing sulfonamide 3c, which was completely inactive. These results
suggest that the SO2 group at position 2 of 3b could also be
important for anchoring the ligand to the sub-pocket of the bind-
ing site.
b
C4H9
c
m-CH3-Ph
Keeping m-methylbenzoyl and ciclopropanecarbonyl at pos-
ition N-2, we modified positions 3 and 4 of the isoxazolone
(Table 1). Generally, we observed a collapse of activity. In particu-
lar, the 3,4-diphenylisoxazol-5(2H)-one derivatives (compounds
4m and 4n), the 4-methyl-3-phenylisoxazol-5(2H)-one derivatives
(compounds 4o and 4p), and the 3-phenylisoxazol-5(2H)-one
derivatives (compounds 4q,r and 4u) all had decreased activity
(IC50 ¼ 10–50 mM). The insertion in the para position of the phe-
nyl ring at position 3 of compounds 4q,r with substituents that
in the previous series gave good results (such as a nitro group
or acetamide function) led to completely inactive (compounds
Scheme 3. Reagents and conditions: (a) R1-COCl, K2CO3, t-BuOH, 80 ꢁC, 3 h.
to the para position had no effect on HNE inhibitory activity.
Substitution of m-methyl with other groups, such as trifluoro-
methyl (4e), cyano (4g,h), or methylsulfonyl (4f), which are found
in other potent HNE inhibitors, was not favourable for activity, and
only compound 4e exhibited activity in the submicromolar
range (IC50 ¼ 200 nM).
To evaluate the importance of the 2-CO amidic group in this
series, we synthesised the alkyl derivative 2, which was completely
inactive, suggesting that the carbonyl group is important for HNE 4s,t). Based on the results in Table 1, we can conclude that

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1117
Table 1. HNE inhibitory activity of isoxazolone derivatives 2, 3a–c, and 4a–u.
Comp.
R2
R3
R4
IC50 (mM)a
2
–
–
–
–
–
–
–
–
–
NA
NA.
0.059 0.02
3a
3b
3c
4a
4b
4c
4d
4e
4f
4g
4h
4i
p-OH-Ph
p-OCOC(CH3)3-Ph
p-NHCOC(CH3)3-Ph
m-CH3-Ph
cC3H5
p-CH3-Ph
o-CH3-Ph
m-CF3-Ph
m-CH3SO2-Ph
m-CN-Ph
NA
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Ph
Ph
Ph
Ph
Ph
Ph
4-NO2-Ph
4-NO2-Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
CH3
CH3
H
0.077 0.027
0.059 0.018
0.23 0.034
0.35 0.036
0.20 0.027
29.6 7.2
6.3 1.4
p-CN-Ph
11.4 1.9
8.5 2.1
p-NHCOC(CH3)3-Ph
p-OCOC(CH3)3-Ph
m-CH3-Ph
cC3H5
m-CH3-Ph
cC3H5
m-CH3-Ph
cC3H5
m-CH3-Ph
cC3H5
4l
0.48 0.13
10.1 1.3
17.2 2.3
13.6 2.4
1.1 0.14
48.7 3.3
12.7 2.7
N.A.
4m
4n
4o
4p
4q
4r
H
H
H
4s
4t
N.A.
4u
Sivelestat
m-CH3-Ph
4-NHCOCH3-Ph
H
28.6 3.3
0.044 0.011
aIC50 values are presented as the mean SD of three independent experiments.
NA: no inhibitory activity was found at the highest concentration of compound tested (50 mM).
Table 2. HNE inhibitory activity of isoxazolone derivatives 7a–e and 8a,b,d,e.
Comp.
R
IC50 (mM)a
7a
8a
7b
8b
7c
7d
8d
7e
CH3
CH3
CN
CN
NO2
NHCOCH3
NHCOCH3
NHCOcC3H5
NHCOcC3H5
0.02 0.009
10.2 1.4
0.07 0.02
0.42 0.13
0.21 0.04
0.05 0.02
0.54 0.11
0.05 0.02
0.62 0.21
0.044 0.011
8e
Sivelestat
aIC50 values are presented as the mean SD of three independent experiments.
position 4 can bear bulky groups, such as an aromatic ring, In Table 2, we report the activity of the pair of isomers of
when there is a methyl group in position 3, probably to fit con- type 7 and 8, the amidic and ester derivatives, respectively,
straints of the lipophilic pocket. Moving the phenyl ring to pos- which were obtained by introducing a substituent to the para
ition 3 is not tolerated, either when there is a methyl or a position of the phenyl group at position 4. All N-benzoyl deriva-
hydrogen present in position 4.
tives (7a–e), with the exception of the nitro compound 7c,

1118
M. P. GIOVANNONI ET AL.
Table 3. HNE inhibitory activity of benzoisoxazolone
derivatives 10a–c.
Comp.
R1
C2H5
C4H9
IC50 (mM)a
10a
10b
25.1 3.6
NA
10c
Sivelestat
m-CH3-Ph
0.638 0.121
0.044 0.011
aIC50 values are presented as the mean SD of three
independent experiments.
NA: no inhibitory activity was found at the highest
concentration of compound tested (50 mM).
Figure 2. Panel (A): Superimposition of the minimised conformations of 7d
extracted from the MD trajectory (T ¼ 600 K, e ¼ 4r). Panel (B): Superimposition of
the minimised conformations of 8d extracted from the MD trajectory
(T ¼ 600K, e ¼ 4r).
Preliminary molecular dynamics (MD) simulations at 300 and
600 K were performed on 7d and 8d to evaluate their flexibility,
accessible conformational space, and preferred 3D arrangements
(low-energy conformations). As expected, the overall shape of
both ligands did not change significantly on changing the simula-
tion medium, as roughly determined by comparison of the dihe-
dral angles, which define the overall shape of the molecules (see
Figure S1, Supplementary Material). For 7d, MD trajectories (300
and 600 K) showed that s1 and s2 adopted a trans conformation
that is maintained throughout the simulations, while s3–s5 access
different conformations (Figures S2 and S3, Supplementary
Material). Overall, the molecule adopted an elongated cylindrical
shape (Figure S4, Supplementary Material). In particular, the con-
formational behaviour of dihedrals s1 and s4 seems to be quite
important (vide infra), given that the endocyclic C ¼ O at position
5 appears to be involved in the catalytic process, while the closest
C ¼ O group could play an important role in anchoring to the sub-
pocket of the binding site40. The superimposition (Figure 2, panel
A) of the minimised conformers of 7d extracted from the MD tra-
jectory (T ¼ 600 K, e ¼ 4r), which are comprised within about
2 kcal molꢂ1, exemplifies the conformational space accessible to
the molecule through rotations about the s3–s5 dihedrals.
exhibited good HNE inhibitory activity in the nanomolar range
(IC50 ¼ 20–70 nM). In addition, the activity seems to be independ-
ent of the electrophilic properties of the substituents (CH3, NO2,
CN, NHCOR). The best compound of this series was the p-methyl
derivative 7a, which had an IC50 of 20 nM. The O-benzoyl deriva-
tives 8b,d,e, which were derived from tautomerism of the isoxa-
zolone scaffold, surprisingly exhibited activity, although at one
order of magnitude lower than the corresponding N-benzoyl
type 7 derivatives (Table 2). Only the ester isomer of the potent
7a had a significant loss in activity (8a, IC50 ¼ 10 mM). The unex-
pected data related to esters 8a,b,d,e could indicate a different
interaction with the target, since the CO endocyclic implicated in
the catalysis is missing, and the only carbonyl group presented
in the molecule is an ester function at position 5. Further dock-
ing studies and kinetic experiments could help us to understand
the interaction of these compounds with the HNE catalytic site
and their mechanism of action.
Table 3 shows the HNE inhibitory activity of benzoisoxazolone
derivatives 10a–c, which are elaborations of the isoxazolone scaf-
fold. In this case, only the m-methylbenzoyl derivative 10c had
reasonable activity, with an IC50 of 638 nM, while the other com-
pounds were less active (10a) or inactive (10b).
Compound 8d appears to be a little bit more flexible compared
to 7d, as suggested by the larger variability of the distance sepa-
rating the centroids of the phenyl rings. While the s1, s2, and s3
dihedrals were almost frozen irrespective of the simulation tem-
perature and medium [they adopted cis, trans (with only few
exceptions), and cis (with only few exceptions) conformations,
respectively], increasing the temperature from 300 to 600 K made
the s5 and s6 dihedrals freely rotate (Figures S5 and S6,
Supplementary Material). Dihedral s4 showed a preference for a
gauche arrangement. Overall, 8d is V-shaped: in most cases both
of the phenyl rings were rotated with respect to the mean plane
defined by the heterocyclic ring (Figure S7, Supplementary
Material). The superimposition (Figure 2, panel B) of the minimised
conformers of 8d extracted from MD simulations (T ¼ 600 K, e ¼ 4r)
revealed two possible orientations for the carbonyl group of the
ester function at position 5, which is supposed to be involved in
Molecular modelling
Several attempts were made using different solvents in order to
obtain crystals of 7d and 8d. However, it was not possible to
obtain crystals suitable for X-ray diffraction (probably in part due
to their quite low melting points). As a consequence, the starting
3D geometry of these molecules was obtained by using as build-
ing blocks the solid state structures of 3,5-dicyano-4-(4-methoxy-
phenyl) isoxazole (QAQPON refcode)68 found in the Cambridge
Structural Database (CSD; v 5.37)69 and that of the isoxazolone
derivative 2j (3-ethyl-2–(3-methylbenzoyl)isoxazol-5(2H)-one)40. The
3D arrangement of 7d and 8d was then roughly improved by an
energy minimisation procedure, followed by molecular dynamics
simulations (details in the Supplementary Material).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1119
Figure 3. Panel (A): Accessibility of the carbonyl grouping involved in the catalytic process estimated by the dimension of a sphere (in red, r ¼ 2.5 Å) centred on the
carbon atom of 7d (left) and 8d (right). Panel (B): Accessibilities of the carbonyl groups in 7d involved in the catalytic process and in H-bond interactions with the
receptor points estimated by the dimension of a sphere centred on the carbon and oxygen atom (r ¼ 2.5Å, red and blue, respectively).
the catalytic process (energies are comprised within poses featured Gly193 as the anchor group, and the H-bond inter-
about 5 kcal molꢂ1).
action with this amino acid pushed the site of the nucleophilic
attack quite distant from Ser195. However, a few poses show
Gly193 involved as an H-bond donor to the ester function of 8d
and, in these cases, the distance HO(Ser195) … CO ranged from
3.3 to 4.8 Å (Figure 4, Panel A). The same amino acids are also
involved in the interaction with 7d. Anchoring of the terminal
NHCO group to Ser195 via H-bonds pushed the CO at position 5
distant from the nucleophilic –OH of Ser195, while H-bonds
In both inhibitors, accessibility of the carbonyl group involved
in the catalytic process was roughly estimated by the dimension
of a sphere centred on the C ¼ O carbon atom (r ¼ 2.5 Å, in red in
Figure 3, panel A). Similarly a sphere (r ¼ 2.5 Å, in blue in Figure 3,
panel B) centred on the oxygen atom, which could be involved in
H-bond interactions with the receptor points, was used to assess
the exposure to the environment of C ¼ O in 7d. Panel A of Figure
3 suggests a more crowded region about the site of the nucleo- between 5-CO and Gly193 brought the two partners of the
philic attack of 8d, which could account for its lower activity com-
nucleophilic attack a little bit closer (distances range from 4.3 to
pared to 7d (in 8d, the carbonyl group is directly bound to the 5.1 Å, Figure 4, Panel B). However, the mean distance between the
phenyl ring and faces the pending arm at position 4). In addition, inhibitor-HNE reacting sites, as determined from the saved poses,
the almost cylindrical shape of 7d caused, at least in principle, the was very long for both complexes (ca 7 Å).
In summary, docking results do not help to explain the differ-
ent activities of the two inhibitors towards HNE. The fact that the
CDocker protocol keeps the receptor rigid, while the inhibitor is
allowed to flex during the refinement, could however significantly
affect the results, and this is the reason why the ligand–receptor
binding affinity was also assessed by MD. On the other hand, MD
simulations confirmed the leading role of Gly193 as an anchoring
carbonyl carbon atom to be attacked from both the side of the
plane defined by the 5-membered ring (Figure S8, Supplementary
Material). In contrast, only one side of the ester group in 8d
appeared to be easily accessible to the hydroxyl group of Ser195
(Figure S9, Supplementary Material). Finally, in both cases the ter-
minal amide group appeared to be well exposed and thus prone
for anchoring to the sub-pocket of the binding site.
Two different approaches were used to gain an idea of the group for both inhibitors. However, in contrast with the docking
interaction of 7d and 8d with HNE. First, docking of the inhibitors results, MD shows the 5-CO grouping of 7d was significantly closer
(two different starting conformations were considered) into the to the HNE reactive site (distance less than 4.5 Å in a large fraction
active site of HNE using the CDocker protocol (in vacuum, target of the snapshot conformations) with respect to the corresponding
temperature 300 K, 20 poses retained) and secondly, MD simula- active site of 8d (d < 5 Å in few of the sampled conformations). In
tions on each inhibitor-HNE complex (in vacuum, simulation time contrast, a comparison of the total number of the intermolecular
5 ns, T ¼ 300K, with the atoms outside the binding sphere con- DH … A contacts (distances less than 2.5 Å) shows a greater pro-
strained to fixed points in space in order to save computational pensity for 8d to form H-bond interactions compared to 7d. In
addition, while multiple intermolecular H-bonds were present for
8d, most of the snapshot conformations of 7d exhibited only one
H-bond interaction. In other words, the net of intermolecular H-
bonds that involves 8d keeps the molecule quite distant from
Ser195. Finally, it is noteworthy that in the HNE complexes with
8d featuring closer distances between the active sites, the heter-
oatoms of the 5-membered ring acted as H-bond acceptors both
towards the Ser195 –OH group and Gly193. As a result of these
interactions, the relative 3 D arrangement of 8d in the HNE active
time). The structure of HNE complexed with a peptide chloro-
methyl ketone inhibitor was used for the docking study and MD
simulations (1HNE70 entry of the Protein Data Bank). The binding
site of HNE was defined as a sphere with a 12 Å radius centred at
the centroid of the five-membered ring of the peptide chloro-
methyl ketone inhibitor which covered all the active site amino
acids of the HNE enzyme. All water molecules and bound inhibitor
were removed from the macromolecule and hydrogen atoms
were added.
Interaction energies from docking protocols on 7d and 8d with site does not appear to be propitious for approach of the nucleo-
HNE did not significantly differ, and the two inhibitors showed phile of Ser195 (see for example Figure 5, Panel A). In contrast,
comparable binding modes. In particular for 8d, most of the saved the H-bond between 5-CO and Gly193, which in most cases held

1120
M. P. GIOVANNONI ET AL.
Figure 4. Panel (A): Pose from the CDocker protocol showing Gly193 involved as H-bond donor to the carbonyl function of 8d. Panel (B): Pose from the CDocker proto-
col showing Gly193 involved as H-bond donor to 5-CO in 7d.
together the 7d/HNE adduct, results in a favourable 3 D arrange- (Table 4). In general, the isoxazolones were more stable than our
previously described HNE inhibitors with cinnolinone39, N-benzoy-
ment of the reacting partners (see for example Figure 5, panel B)
for nucleophilic attack.
lindazole36,37, and N-benzoylpyrazole scaffolds71
.
The most potent isoxazolones were also selected for evaluation
of the reversibility of HNE inhibition over time. As shown in Figure
6, HNE inhibition was rapidly (ꢃ30 min) reversed for compounds
7d and 7b. The inhibition was maximal for up to 60 min with com-
pound 7e and >120 min for the other tested compounds (4a, 4b,
and 7a). However, inhibition by the compounds was eventually
reversed, and full recovery of HNE activity was observed by 4 h
after treatment with 8 mM of the compound (e.g. see Figure 6).
To better understand the mechanism of action of these isoxa-
Stability and kinetic features
The most potent isoxazolones with IC50 < 100 nM and their ester
analogs were further evaluated for chemical stability in aqueous
buffer using spectrophotometry to detect compound hydrolysis.
The compounds had t1/2 values from 2.9 to 9.6 h for spontaneous
hydrolysis, indicating that the amides were more stable than the
esters in the corresponding pairs of 7a/8a, 7d/8d, and 7e/8e zolone HNE inhibitors, we performed kinetic experiments. As

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1121
Figure 5. Panel (A): View of the 8d-HNE adduct from MD showing the H-bond interaction between the nitrogen atom of the 5-membered ring acting as H-bond
acceptor towards Gly193. Panel (B): View of the 7d-HNE adduct from MD showing the H-bond interaction between the oxygen of 5-CO acting as H-bond acceptor
towards Gly193.
shown in Figure 7, the representative double-reciprocal indicated this nucleus as an appropriate scaffold for this target.
Lineweaver–Burk plot of fluorogenic substrate hydrolysis by HNE
in the absence and presence of compounds 7d and 8d indicates
that these compounds are competitive HNE inhibitors.
The most potent compounds had a methyl group at position 3
and a (substituted)phenyl ring at position 4, and the higher HNE
inhibitory activity was found for compound 7a (IC50 ¼ 20 nM). In
addition to the 2-NCO derivatives, a number of 5-NCO compounds
were obtained, although with lower activity than the correspond-
ing amide. Studies of chemical stability in aqueous buffer indi-
cated that amides were generally more stable than esters, while
Conclusions
In the present study, we report a new series of isoxazolones as
potent HNE inhibitors, confirming our previous results40, which kinetic experiments confirmed that both amides and esters were

1122
M. P. GIOVANNONI ET AL.
Table 4. Half-life (t1/2) for the spontaneous hydrolysis of selected
competitive HNE inhibitors. Docking and molecular dynamics stud-
ies are the different approaches used to understand the interac-
tions of the two isomers 7d and 8d with HNE. Both studies
highlight the fundamental role of Gly193 as anchoring group for
both the inhibitors, but the MD simulations help us to explain the
difference in inhibitory activity between the inhibitors 7d and 8d.
The ester isomer 8d shows a greater propensity to form H-bond
interactions with respect to 7d and as a result the molecule is
quite distant from the Ser195, the amino acid responsible for the
nucleophilic attack. While the amide isomer 7b appears more
mobile within the active site of HNE, being held in place by single
H-bond interactions (vs. multiple in 8d), which leads the C ¼ O at
position 5 to obtain a favourable orientation for the nucleophilic
attack by Ser195.
derivatives.
Comp.
t1/2 (h)
Absorbance (nm)a
4a
4b
7a
8a
7d
8d
7e
8e
4.1
8.3
7.7
5.3
3.9
2.9
9.6
5.6
330
290
340
320
340
330
320
280
aAbsorption used for monitoring spontaneous hydrolysis.
vehicle
400
Disclosure statement
No potential conflict of interest was reported by the authors.
7a
300
200
100
0
4
a
7e
Funding
7b
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; the
Ministry of Education and Science of the Russian Federation (pro-
ject No. 4.8192.2017), Montana University System Research
Initiative: 51040-MUSRI2015–03; Montana State University
Agricultural Experiment Station.
7d
4b
Sivelestat
0
60
120
Time (min)
180
240
Figure 6. Evaluation of HNE inhibition by representative isoxazolones and
Sivelestat over extended periods of time. HNE was incubated with the indicated
compounds (8 mM), and kinetic curves monitoring substrate cleavage catalysed by
HNE over time are shown. Representative curves are from two independent
experiments.
ORCID
Maria Paola Giovannoni
http://orcid.org/0000-0003-0310-7850
Igor A. Schepetkin
Mark T. Quinn
Gabriella Guerrini
http://orcid.org/0000-0003-2139-8110
http://orcid.org/0000-0001-8114-5073
http://orcid.org/0000-0001-6711-7965
Paola Paoli
Patrizia Rossi
http://orcid.org/0000-0002-2408-4590
http://orcid.org/0000-0002-6316-338X
Gianluca Bartolucci
Claudia Vergelli
http://orcid.org/0000-0002-5631-8769
http://orcid.org/0000-0001-9774-8047
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