Cinnoline derivatives as human neutrophil elastase inhibitors

Article abstract of DOI:10.3109/14756366.2015.1057718

Compounds that can effectively inhibit the proteolytic activity of human neutrophil elastase (HNE) represent promising therapeutics for treatment of inflammatory diseases. We present here the synthesis, structure-activity relationship analysis, and biolog

Full text of DOI:10.3109/14756366.2015.1057718

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–12
2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1057718
!
RESEARCH ARTICLE
Cinnoline derivatives as human neutrophil elastase inhibitors
1
Maria Paola Giovannoni , Igor A. Schepetkin , Letizia Crocetti , Giovanna Ciciani , Agostino Cilibrizzi ,
2
1
1
3
1
4,5
2
Gabriella Guerrini , Andrei I. Khlebnikov , Mark T. Quinn , and Claudia Vergelli
1
1
2
NEUROFARBA, Sezione di Farmaceutica e Nutraceutica, Universit a` degli Studi di Firenze, Sesto Fiorentino, Italy, Department of Immunology and
3
Infectious Diseases, Montana State University, Bozeman, MT, USA, Department of Chemistry, Imperial College London, South Kensington, London,
4
5
UK, Department of Biotechnology and Organic Chemistry, Tomsk Polytechnic University, Tomsk, Russia, and Department of Chemistry, Altai State
Technical University, Barnaul, Russia
Abstract
Keywords
Compounds that can effectively inhibit the proteolytic activity of human neutrophil elastase
(HNE) represent promising therapeutics for treatment of inflammatory diseases. We present
Cinnoline, human neutrophil elastase,
inhibitory activity
here the synthesis, structure–activity relationship analysis, and biological evaluation of a new
series of HNE inhibitors with a cinnoline scaffold. These compounds exhibited HNE inhibitory
activity but had lower potency compared to N-benzoylindazoles previously reported by us. On
the other hand, they exhibited increased stability in aqueous solution. The most potent
compound, 18a, had a good balance between HNE inhibitory activity (IC50 value ¼ 56 nM) and
chemical stability (t1/2 ¼ 114 min). Analysis of reaction kinetics revealed that these cinnoline
derivatives were reversible competitive inhibitors of HNE. Furthermore, molecular docking
studies of the active products into the HNE binding site revealed two types of HNE inhibitors:
molecules with cinnolin-4(1H)-one scaffold, which were attacked by the HNE Ser195 hydroxyl
group at the amido moiety, and cinnoline derivatives containing an ester function at C-4, which
is the point of attack of Ser195.
History
Received 11 March 2015
Revised 6 May 2015
Accepted 6 May 2015
Published online 21 July 2015
1
0,11
12
, acute respiratory distress syndrome (ARDS) , acute
Introduction
(CF)
lung injury (ALI) , as well as other inflammatory disorders, such
1
3
Human neutrophil elastase (HNE) is a serine protease belonging
to the chymotrypsin family and is stored in azurophilic granules of
neutrophils where, with other serine proteases, it participates in
the oxygen-independent pathway of intracellular and extracellular
pathogen destruction . HNE is a small, basic, and soluble
glycoprotein of about 30 kDa, containing 218 amino acid residues
and four disulfide bridges. HNE utilizes a catalytic triad
as psoriasis, dermatitis, atherosclerosis, and rheumatoid arth-
14–16
ritis
sion of various types of cancer
. HNE has also recently been implicated in the progres-
17,18
. In addition, HNE plays a
central role in both acute pathogenesis and chronic functional
1
,2
19
recovery after brain traumatic injury . Thus, it is evident that
compounds able to modulate the proteolytic activity of HNE
could represent promising therapeutic agents for the treatment of
inflammatory diseases involving excessive HNE activity
Many examples of peptide and non-peptide HNE inhibitors
have been reported in the literature
are currently available for clinical use : Prolastin (purified a1-
3
consisting of Ser195, His57, and Asp102 . Due to its proteolytic
activity against a variety of extracellular matrix proteins, such as
20,21
.
4
elastin, fibronectin, collagen, proteoglycans, laminin , and some
20,21
5
matrix metalloproteinases (i.e. MMP-2, MMP-3, and MMP-9) ,
. However, only two drugs
Õ
22
HNE plays an important role in many physiological processes,
such as blood coagulation, apoptosis, and inflammation, and is
6
able to modulate cytokine and growth factors expression . Under
AT), a peptide drug used for the treatment of a -antitripsin
1
23
deficiency (AATD) and Sivelestat (Elaspol 100), a low
Õ
molecular weight non-peptide selective HNE inhibitor with an
IC50 value of 44 nM (Figure 1). However, the use of Sivelestat for
ALI and ARDS is only approved in Japan and Korea
Additionally, the neutrophil elastase inhibitor AZD9668
Alvelestat) (Figure 1) is currently under evaluation in clinical
trials for patients with bronchiectasis . We recently discovered a
new class of HNE inhibitors with an N-benzoylindazole scaffold
(Figure 1, structure A). The most active compounds had IC50
values in the low nanomolar range, and studies on the mechanism
of action showed that these compounds were competitive and
pseudo-irreversible HNE inhibitors, with an appreciable selectiv-
physiological conditions, the proteolytic activity of HNE is
regulated by serpins, a family of endogenous inhibitors that
includes a -antitripsin (a -AT), elafin, and secretory leucocyte
24,25
.
1
1
7
,8
protease inhibitor (SLPI)
.
(
Alterations in the balance between HNE activity and its
regulatory inhibitors have been implicated in the development of a
variety of diseases affecting the pulmonary system, such as
26
9
chronic obstructive pulmonary disease (COPD) , cystic fibrosis
Address for correspondence: Maria Paola Giovannoni, NEUROFARBA,
Sezione Farmaceutica e Nutraceutica, Via Ugo Schiff 6, Sesto Fiorentino,
2
7,28
ity for HNE versus other proteases
. An essential requirement
for the activity of these compounds was a carbonyl group
at position 1.
50019 Firenze, Italy. Tel: +39 055 4573682. E-mail:
mariapaola.giovannoni@unifi.it

2
M. P. Giovannoni et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
O
anhydrous THF (23 mL) under nitrogen, 4.50 mmol of NaH
ꢀ
H
were added. The suspension was stirred at 80 C for 1 h, then a
N
OH
29
solution of 1 (2.95 mmol) in anhydrous THF (12 mL) was slowly
O
ꢀ
NH
O
added. The mixture was stirred at 80 C for 3 h. After cooling, a
saturated solution of NH Cl was added, and the mixture was
S
O
4
O
Sivelestat
extracted with ethyl acetate (3 ꢁ 15 mL). The organic layer was
O
evaporated in vacuo to obtain compound 2, which was purified by
column chromatography using cyclohexane/ethyl acetate 2:1 as
1
eluent. Yield ¼ 95ꢀ; oil. H NMR (CDCl ) ꢀ 1.21 (t, 3H,
3
OCH CH , J ¼ 6.8 Hz), 2.05–2.10 (m, 4H, 2 ꢁ CH pyrrolidine),
2
3
2
F3C
H3C
N
3.68–3.73 (m, 2H, CH pyrrolidine), 3.96–4.04 (m, 2H, CH2
2
pyrrolidine), 4.15 (q, 2H, OCH CH , J ¼ 6.8 Hz), 4.17 (s, 2H,
2
3
O
COCH CO), 7.18 (t, 1H, Ar, J ¼ 7.2 Hz), 7.44 (t, 1H, Ar,
2
J ¼ 6.8 Hz), 7.51 (d, 1H, Ar, J ¼ 7.6 Hz), 7.69 (d, 1H, Ar,
J ¼ 8.0 Hz). ESI-MS calcd. for C H N O , 289.33; found: m/z
N
O
O
S
CH3
Alvelestat (AZD9668)
15 19 3 3
H
N
O
+
290.05 [M + H] .
N
Ethyl 1-(cyclopropanecarbonyl)-4-oxo-1,4-dihydrocinnoline-
30
N
ꢀ
3
(
-carboxylate (4): To a cooled (0 C) suspension of 3
1.40 mmol) in anhydrous CH Cl (2 mL), Et N (0.1 mL) and
CH3
2
2
3
4
.2 mmol of cyclopropanecarbonyl 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 neutralized with 0.5 N NaOH. The
reaction mixture was extracted with CH Cl (3 ꢁ 15 mL), the
R2
N
CN
R3
N
N
CH3
2
2
N
solvent was dried over sodium sulfate, evaporated in vacuo, and
compound 4 was purified by column chromatography (eluent:
R1
O
O
ꢀ
cyclohexane/ethyl acetate 2:1). Yield ¼ 13ꢀ; mp ¼ 57–58 C
StructureA[27,28]
5b[28]
1
EtOH/H O). H NMR (CDCl ) ꢀ 1.23–1.28 (m, 2H, cyclopro-
(
pyl), 1.33–1.38 (m, 2H, cyclopropyl), 1.46 (t, 3H, OCH CH ,
2
3
2
3
Figure 1. Structures of selected HNE inhibitors.
J ¼ 7.2 Hz), 3.20–3.25 (m 1H, cyclopropyl) 4.52 (q, 2H,
OCH CH , J ¼ 7.2 Hz), 7.57 (t, 1H, Ar, J ¼ 7.6 Hz), 7.81 (t, 1H,
2
3
Further research in this field led us to investigate other Ar, J ¼ 7.2 Hz), 8.38 (d, 1H, Ar, J ¼ 9.6 Hz), 8.87 (d, 1H, Ar,
nitrogenous bicyclic scaffolds, including cinnolines, which contain J ¼ 8.8 Hz). ESI-MS calcd. for C15
H N O , 286.28; found: m/z
14 2 4
+
a pyridazine ring instead of a pyrazole. Using the connoline 287.10 [M + H] .
scaffold, we also evaluated those substituents that previously gave
as the best results for modification of the N-benzoylindazoles in A mixture of 0.23 mmol of 3 , 0.34 mmol of Na
order to define if the requirements were similar for the two systems. 1.15 mmol of CH I in 1 mL of anhydrous CH CN was refluxed for
h. After cooling, the precipitate was filtered, the solvent was
evaporated in vacuo, and compound 5 was obtained by crystal-
Ethyl 1-methyl-4-oxo-1,4-dihydrocinnoline-3-carboxylate (5):
3
0
CO , and
3
2
3
3
6
Materials and methods
ꢀ
1
lization with ethanol. Yield ¼ 66ꢀ; mp ¼ 185–190 C (EtOH). H
All melting points were determined on a B u¨ chi apparatus (New
Castle, DE) and are uncorrected. Extracts were dried over
Na SO , and the solvents were removed under reduced pressure.
NMR (DMSO-d ) ꢀ 1.31 (t, 3H, OCH CH , J ¼ 7.2 Hz), 4.16
6
2
3
(
s, 3H, CH ), 4.32 (q, 2H, OCH CH , J ¼ 7.2 Hz), 7.62 (t, 1H, Ar,
3
2
3
2
4
J ¼ 7.6 Hz), 7.89 (d, 1H, Ar, J ¼ 8.8 Hz), 7.95 (t, 1H, Ar,
Merck F-254 commercial plates (Merck, Durham, NC) were used
for analytical TLC to follow the course of reactions. Silica gel 60
13
J ¼ 8.4 Hz), 8.19 (d, 1H, Ar, J ¼ 8.0 Hz). C NMR (DMSO-d )
6
ꢀ
(
14.58 (CH ), 31.16 (CH), 44.94 (CH ), 61.47 (CH ), 117.85
3 3 2
(
Merck 70–230 mesh, Merck, Durham, NC) was used for column
CH), 125.28 (CH), 126.34 (C), 127.0 (CH), 134.88 (CH), 138.84
ꢂ1
1
13
chromatography. H NMR and C NMR spectra were recorded
on an Avance 400 instrument (Bruker Biospin Version 002 with
SGU, Bruker Inc., Billerica, MA). Chemical shifts (ꢀ) 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 TopSpin 1.3 software (Nicolet
Instrument Corp., Madison, WI) and are rounded to the nearest
ꢂ1
(CH), 141.24 (C). IR: 1590 cm
(C ¼ O ester), 1693 cm
12 2 3
(
2
C ¼ O). ESI-MS calcd. for C H N O , 232.24; found: m/z
1
2
+
33.09 [M + H] .
Ethyl 1-(3-methylbenzyl)-4-oxo-1,4-dihydrocinnoline-3-
carboxylate (6):
0.7 mmol), and 3-methylbenzyl chloride (0.80 mmol) in 2 mL
30
A
mixture of
3
(0.46 mmol), K CO3
2
(
ꢀ
of anhydrous DMF was stirred at 80 C for 1 h. After cooling, the
mixture was diluted with cold water, and the precipitate was
0
.1vHz. 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 performed with a Perkin–Elmer 260
elemental analyzer (PerkinElmer, Inc., Waltham, MA) for C, H,
and N, and the results were within ±0.4ꢀ of the theoretical
values, unless otherwise stated. Reagents and starting material
were commercially available.
ꢀ
recovered by filtration. Yield ¼ 61ꢀ: mp ¼ 100–102 C (EtOH).
1
H NMR (CDCl ) ꢀ 1.47 (t, 3H, OCH CH , J ¼ 7.2 Hz), 2.33
3
2
3
(
s, 3H, CH ), 4.52 (q, 2H, OCH CH , J ¼ 7.2 Hz), 5.68 (s, 2H,
3
2
3
CH ), 7.05 (s, 2H, Ar), 7.13 (d, 1H, Ar, J ¼ 6.4 Hz), 7.24 (d, 1H,
2
Ar, J ¼ 8.0 Hz), 7.46 (t, 2H, Ar, J ¼ 8.8 Hz), 7.66 (t, 1H, Ar,
13
J ¼ 7.2 Hz), 8.44 (d, 1H, Ar, J ¼ 8.0 Hz). C NMR (CDCl ) ꢀ
3
1
(
4.29 (CH ), 21.43 (CH ), 60.82 (CH ), 61.88 (CH ), 116.16
3 3 2 2
CH), 123.65 (CH), 126.14 (CH), 126.67 (CH), 127.13 (CH),
1
1
29.03 (CH), 129.23 (CH), 134.04 (CH), 134.68 (C), 139.04 (C),
ꢂ1
Chemistry
ꢂ1
40.50 (C). IR: 1717 cm (C ¼ O ester), 1632 cm (C ¼ O).
(
(
E)-Ethyl 3-oxo-3-(2-(pyrrolidin-1-yldiazenyl)phenyl)propanoate ESI-MS calcd. for C H N O , 322.36; found: m/z 323.14
19 18 2 3
+
2): To a suspension of diethylcarbonate (4.50 mmol) in [M + H] .

DOI: 10.3109/14756366.2015.1057718
Cinnoline derivatives as human neutrophil elastase inhibitors
3
Ethyl 1-(3-methylbenzoyl)-4-oxo-1,4-dihydrocinnoline-3-carb- (m, 1H, OCH-H), 4.02 (t, 1H, OCH-H, J ¼ 9.6 Hz), 4.80 (d, 1H,
ꢀ
oxylate (7): To a cooled (0 C) suspension of the appropriate CH-H, J ¼ 12.4 Hz), 4.90–5.00 (m, 2H, CH cC H O + CH-H),
5
9
3
0
substrate 3 (0.40 mmol) in anhydrous CH Cl (2 mL), Et N 7.39 (t, 1H, Ar, J ¼ 7.6 Hz), 7.53–7.58 (m, 1H, Ar), 7.70 (t, 1H,
2
2
3
(
0.1 mL), and 1.15 mmol of m-toluoyl chloride were added. The Ar, J ¼ 8.8 Hz), 8.29 (d, 1H, Ar, J ¼ 8.0 Hz). ESI-MS calcd. for
ꢀ
14 16 2 3
+
solution was stirred at 0 C for 2 h and then at room temperature C H N O , 260.29; found: m/z 261.04 [M + H] .
for 2 h. After evaporation of the solvent, the residue was mixed
with ice-cold water (20 mL) and neutralized with 0.5 N NaOH. methyl]cinnolin-4(1H)-one (13): Compound 13 was obtained
Compound was recovered by extraction with CH Cl following the same procedure performed for compound 7 but
3 ꢁ 15 mL) and was purified by column chromatography using starting from precursor 12. Compound 13 was recovered by
1-(3-Methylbenzoyl)-3-[(tetrahydro-2H-pyran-2-yloxy)-
7
2
2
(
cyclohexane/ethyl acetate 2:1 as eluent. Yield ¼ 39ꢀ; extraction with CH Cl (3 ꢁ 15 mL) and was purified by column
2
2
ꢀ
1
mp ¼ 81–83 C (EtOH). H NMR (CDCl ) ꢀ 1.34 (t, 3H, chromatography using toluene/ethyl acetate 8:2 as eluent.
3
1
OCH CH , J ¼ 7.2 Hz), 2.46 (s, 3H, CH ), 4.39 (q, 2H, Yield ¼ 55ꢀ; oil. H NMR (CDCl ) ꢀ 1.44–1.65 (m, 6H,
2
3
3
3
OCH CH , J ¼ 7.2 Hz), 7.41 (t, 1H, Ar, J ¼ 7.6 Hz), 7.48 cC H O), 2.46 (s, 3H, CH ), 3.45–3.50 (m, 1H, OCH-H), 3.85–
2
3
5
9
3
(
(
d, 1H, Ar, J ¼ 7.6 Hz), 7.60 (t, 1H, Ar, J ¼ 8.0 Hz), 7.68 3.90 (m, 1H, OCH-H), 4.70–4.80 (m, 3H, CH cC H O + CH ),
5
9
2
d, 1H, Ar, J ¼ 7.6 Hz), 7.74 (s, 1H, Ar), 7.83 (t, 1H, Ar, 7.37–7.47 (m, 2H, Ar), 7.56 (t, 1H, Ar, J ¼ 8.0 Hz), 7.58–7.68 (m,
J ¼ 7.5 Hz), 8.43 (t, 2H, Ar, J ¼ 8.0 Hz). ESI-MS calcd. for 2H, Ar), 7.81 (t, 1H, Ar, J ¼ 8.8 Hz), 8.38 (d, 1H, Ar, J ¼ 9.6 Hz),
+
C H N O , 336.34; found: m/z 337.11 [M + H] .
1
acid (8): A mixture of 7 (0.15 mmol) and 6 N NaOH (5 mL) was
8.53 (d, 1H, Ar, J ¼ 8.8 Hz). ESI-MS calcd. for C H N O ,
22 22 2 4
1
9
16
2
4
+
-(3-Methylbenzoyl)-4-oxo-1,4-dihydrocinnoline-3-carboxylic 378.42; found: m/z 379.16 [M + H] .
3-(Hydroxymethyl)-1-(3-methylbenzoyl)cinnolin-4(1H)-one
stirred at 100 C for 5 h. After cooling, the mixture was acidified (11d): A mixture of 13 (0.11 mmol), trifluoroacetic acid
ꢀ
with 6 N HCl, and the precipitate was recovered by suction and (0.3 mL), and CH Cl (1.7 mL) was stirred at room temperature
2
2
ꢀ
crystallized with ethanol. Yield ¼ 11ꢀ; mp ¼ 268–270 C dec for 3 h. Evaporation of the solvent resulted in compound 11d,
1
EtOH). H NMR (CDCl ) ꢀ 2.5 (s, 3H, CH ), 7.41 (m, 2H, Ar), which was purified by column chromatography using CH Cl /
(
3
3
2
2
1
7
.73 (m, 3H, Ar), 7.88 (d, 1H, Ar, J ¼ 8.4 Hz), 8.02 (t, 1H, Ar, MeOH 9.5:0.5 as eluent. Yield ¼ 62ꢀ; oil. H NMR (CDCl ) ꢀ
3
J ¼ 7.8 Hz), 8.27 (d, 1H, Ar, J ¼ 8.4 Hz). ESI-MS calcd. for 2.40 (s, 3H, CH ), 5.58 (s, 2H, CH ), 7.31–7.46 (m, 4H, Ar), 7.74
3 2
+
C H N O , 308.29; found: m/z 309.08 [M + H] .
(t, 1H, Ar, J ¼ 7.6 Hz), 7.91 (d, 2H, Ar, J ¼ 8.0 Hz), 8.34 (d, 1H,
3 3 2
1
7 12 2 4
ꢀ
13
General procedure for 10 b,c: To a cooled (0 C) suspension of Ar, J ¼ 8.8 Hz). C NMR (CDCl ) ꢀ 21.35 (CH ), 59.17 (CH ),
3
1
32
the appropriate substrate 9b or 9c (1.40 mmol) in conc. HCl 116.83(CH), 124.47 (CH), 124.94 (CH), 127.23 (CH), 128.24
1.2 mL), 2.24 mmol of NaNO in 1 mL of H O was slowly added. (CH), 130.34 (CH), 133.82 (CH), 134.09 (CH), 141.72 (C),
(
The mixture was stirred at 0 C for 2 h and then at room 171.20 (C). ESI-MS calcd. for C H N O , 294.30; found: m/z
2
2
ꢀ
1
7 14 2 3
+
temperature for 48 h. After concentration of the solvent, the 295.10 [M + H] .
solution was extracted with ethyl acetate (3 ꢁ 15 mL), and General procedure for 16a–c and 17a–c: Compounds 16a–c
the organic layer was washed with brine (5 mL). Evaporation of and 17a–c were obtained following the same procedure performed
30,34
the solvent resulted in final compounds 10b,c, which were for compound 7 but starting from precursors 15a–c .
purified by column chromatography using toluene/ethyl acetate Compounds 16a,c and 17a,c were recovered by extraction with
7
3
2
2
:3 (for 10b) or cyclohexane/ethyl acetate 3:1 (for 10c) as eluents. CH Cl (3 ꢁ 15 mL), while the crude 16b and 17b were recovered
2
2
-Cyclopropylcinnolin-4(1H)-one (10b): Yield ¼ 12ꢀ; mp ¼ 238– by vacuum filtration. Final 16a–c and 17a–c were purified by
ꢀ
1
45 C dec (EtOH). H NMR (CDCl ) ꢀ 0.95–1.04 (m, 4H, column chromatography using cyclohexane/ethyl acetate in the
3
ꢁ CH cyclopropyl), 2.62–2.68 (m, 1H, CH cyclopropyl), 7.37 following different ratios as eluents: 3:1 for 16a and 17a; 6:1 for
2
(
t, 2H, Ar, J ¼ 7.2 Hz), 7.67 (t, 1H, Ar, J ¼ 7.6 Hz), 8.33 (d, 1H, 16b and 17b; 5:1 for 16c and 17c. 3-Chloro-1-(3-methylbenzoyl)-
ꢂ1
ꢀ
Ar, J ¼ 8.4 Hz), 10.50 (exch br s, 1H, NH). IR ¼ 1718 cm
cinnolin-4(1H)-one (16a). Yield ¼ 33ꢀ; mp ¼ 128–135 C
1
(
1
C ¼ O). ESI-MS calcd. for C H N O, 186.21; found: m/z (EtOH). H NMR (CDCl ) ꢀ 2.48 (s, 3H, CH ), 7.43 (t, 1H, Ar,
1
1
10
2
3
3
+
87.02 [M + H] .
J ¼ 7.6 Hz), 7.49 (d, 1H, Ar, J ¼ 7.6 Hz), 7.59–7.65 (m, 2H, Ar),
General procedure for 11a–c: Compounds 11a–c were 7.70 (s, 1H, Ar), 7.85 (t, 1H, Ar, J ¼ 8.0 Hz), 8.43 (d, 1H, Ar,
ꢂ1
obtained by following the same procedure performed for J ¼ 8.0 Hz), 8.49 (d, 1H, Ar, J ¼ 9.2 Hz). IR ¼ 1663 cm (C¼O
3
3
ꢂ1
compound 7, starting from precursors 10a–c [10a ]. amide), 1723 cm (C¼O). ESI-MS calcd. for C H ClN O ,
1
6
11
2 2
+
298.72; found: m/z 300.05 [M + H] . 3-Chlorocinnolin-4-yl
Compounds 11a–c were recovered by extraction with CH Cl
2
2
ꢀ
(
3 ꢁ 15 mL) and were purified by column chromatography using 3-methylbenzoate
(17a): Yield ¼ 17ꢀ; mp ¼ 115–123 C
1
toluene/ethyl acetate 8:2 (for 11a,b) or cyclohexane/ethyl acetate (EtOH). H NMR (CDCl ) ꢀ 2.53 (s, 3H, CH ), 7.52 (t, 1H, Ar,
3
3
3
4
:1 (for 11c) as eluents. 1-(3-Methylbenzoyl)-3-phenylcinnolin- J ¼ 7.8 Hz), 7.60 (d, 1H, Ar, J ¼ 7.6 Hz), 7.83 (t, 1H, Ar,
ꢀ
1
(1H)-one (11a): Yield ¼ 22ꢀ; mp ¼ 119–121 C (EtOH).
H
J ¼ 7.6 Hz), 7.90–7.95 (m, 2H, Ar), 8.15 (s, 2H, Ar), 8.64
ꢂ1
NMR (CDCl ) ꢀ 2.48 (s, 3H, CH ), 7.42–7.55 (m, 4H, Ar), 7.54 (d, 1H, Ar, J ¼ 8.4 Hz). IR ¼ 1744 cm (C¼O ester). ESI-MS
3
3
+
(
d, 1H, Ar, J ¼ 7.6 Hz), 7.81 (t, 1H, Ar, J ¼ 7.6 Hz), 7.88–7.97 calcd. for C H ClN O , 298.72; found: m/z 300.05 [M + H] .
1
6
11
2 2
( General procedure for 18a–g: Compounds 18a–g were
MS calcd. for C H N O , 340.37; found: m/z 341.12 [M + H] . obtained following the same procedure as performed for com-
m, 2H, Ar), 8.03 (m, 4H, Ar), 8.71 (d, 1H, Ar, J ¼ 8.4 Hz). ESI-
+
2
2 16 2 2
3
pound 7 but starting from precursor 14 . Compounds 18a,e,g
0
3
-[(Tetrahydro-2H-pyran-2-yloxy)methyl]cinnolin-4(1H)-one
(
12): To a mixture of ammonium cerium(IV) nitrate (CAN) were recovered by extraction with CH Cl (3 ꢁ 15 mL), while
2
2
(0.09 mmol) in anhydrous CH CN (5 mL), 4.50 mmol of substrate crude 18b–d and 18f were recovered by vacuum filtration. Final
3
3
0d and 4.50 mmol of 3,4-dihydro-2H-pyran (commercially compounds 18a,b,e,g were purified by column chromatography
3
1
available) were added, and the suspension was stirred at room using cyclohexane/ethyl acetate 4:1 (for 18a) or 1:1 (for 18b and
temperature for 24 h. After evaporation of the solvent, 20 mL of 18g) or toluene/ethyl acetate 9:1 (for 18e) as eluents, or by
H O was added, and the mixture was extracted with CH Cl . The crystallization from ethanol (for 18c,d,f).
2
2
2
organic layer was evaporated in vacuo, resulting in crude 12,
1-(3-Methylbenzoyl)cinnolin-4(1H)-one (18a): Yield ¼ 55ꢀ;
ꢀ
3
1
which was purified by column chromatography using CH Cl / mp ¼ 138–139 C (EtOH). H NMR (CDCl ) ꢀ 2.47 (s, 3H,
2
2
1
MeOH 9:1 as eluent. Yield ¼ 42ꢀ; oil. H NMR (CDCl ) ꢀ 1.55– CH ), 7.40–7.50 (m, 2H, Ar), 7.56–7.66 (m, 3H, Ar), 7.76–7.86
3
3
1
.67 (m, 4H, cC H O), 1.76–1.86 (m, 2H, cC H O), 3.57–3.62 (m, 2H, Ar), 8.36 (d, 1H, Ar, J ¼ 9.6 Hz), 8.50 (d, 1H, Ar,
5
9
5 9

4
M. P. Giovannoni et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
1
3
J ¼ 9.2 Hz). C NMR (CDCl ) ꢀ 21.35 (CH ), 118.77 (CH), of inhibitor that caused 50ꢀ inhibition of the enzymatic
3
3
1
25.54 (CH), 126.82 (CH), 127.90 (CH), 128.24 (CH), 131.15 reaction (IC ) was calculated by plotting ꢀ inhibition versus
50
(
CH), 133.99 (CH), 134.61 (CH), 140.11 (CH), 171.15 (C), logarithm of inhibitor concentration (at least six points). The
ꢂ1
ꢂ1
1
ESI-MS calcd. for C H N O , 264.28; found: m/z 265.09 independent experiments with relative standard deviations
71.25 (C). IR ¼ 1645 cm (C¼O amide), 1709 cm (C¼O). data are presented as the mean values of at least three
16 12 2 2
+
[
M + H] .
4-Hydroxy-3,4-dihydrocinnolin-1(2H)-yl)(m-tolyl)meyhanone
19): Compound 18a (0.189 mmol) was subjected to catalytic values were determined using Dixon plots of three to four
of 515ꢀ.
(
For selected lead compounds, the inhibition constant (Ki)
(
reduction in EtOH (15 mL) for 4 h with a Parr instrument (Parr different concentrations of the substrate, as described previ-
3
9
Instrument Company, Moline, IL) using 0.094 mmol of 10ꢀ Pd/C ously . At each substrate concentration, rates were determined
as a catalyst and a constant pressure at 30 PSI. The catalyst was with five different inhibitor concentrations, and the inverse of
filtered, the solvent was evaporated under vacuum, and the crude the velocities was plotted against the final inhibitor concentra-
product was purified by column chromatography using cyclohex- tion. K was determined from the intersection of the plotted
i
ꢀ
ane/ethyl acetate 1:1 as eluent. Yield ¼ 42ꢀ; mp ¼ 187–190 C lines.
1
dec (EtOH). H NMR (DMSO-d ) ꢀ 2.35 (s, 3H, CH ), 2.95–3.01
Reactivation of the HNEꢂinhibitor complex was investigated,
6
3
4
0
m, 1H, CH), 3.08–3.15 (m, 1H, CH), 4.45 (s, 1H, C-H), 5.44 as described previously . Briefly, HNE (20 mU/mL) was
(
(
exch br s, 1H, NH), 5.71–5.76 (m, 1H, OH), 7.17 (t, 1H, Ar, incubated with excess inhibitor at a final concentration of
J ¼ 7.4 Hz), 7.24–7.31 (m, 3H, Ar), 7.41 (d, 2H, Ar, J ¼ 8.4 Hz), 50 mM in 1 mL of Tris bufer (200 mM Tris-HCl, pH 7.5). After
1
3
7
.49 (d, 1H, Ar, J ¼ 7.2 Hz), 7.83 (d, 1H, Ar, J ¼ 8.4 Hz).
C
20 min, a 50 mL aliquot was removed and assayed to verify
NMR (DMSO-d ) ꢀ 21.41 (CH ), 53.49 (CH ), 61.94 (CH), complete inhibition of HNE enzymatic activity. Excess inhibitor
6
3
2
1
(
1
22.08 (CH), 124.60 (CH), 125.80 (CH), 127.16 (CH), 127.76 was removed via Vivaspin 10 000 MWCO PES (Sartorius,
CH), 129.05 (CH), 130.34 (CH), 130.59 (CH), 130.76 (CH), Goettingen, Germany) filtration by centrifuging at 10 000 g for
ꢂ1
37.07 (C), 137.66 (C), 138.52 (C), 171.62 (C). IR ¼ 1644 cm
5 min, adding 0.5 mL of the Tris buffer to the retentate, and
ꢂ1
ꢂ1
(
C¼O amide), 3266 cm (NH), 3431 cm (OH). ESI-MS calcd. centrifuging again under the same conditions. This process was
+
for C H N O , 268.31; found: m/z 269.12 [M + H] .
repeated again, and the final retentate was suspended in 0.5 mL of
16 16 2 2
ꢀ
General procedure for 21a–d, 22, and 24: Compounds 21a–d, buffer A at 25 C. At the indicated times, 50 mL aliquots were
2, and 24 were obtained following the same procedure as for removed and added to microplate wells containing 25 mM of the
2
compound 7 but starting from precursors 20a–d
3
4–37
38
and 23 . fluorogenic HNE substrate dissolved in 50 mL of buffer A, and
Compounds 21a–d, 22, and 24 were recovered by extraction with enzymatic activity was monitored, as described above. A control
CH Cl (3 ꢁ 15 mL) and were purified by column chromatog- containing HNE (20 mU/mL) and 0.25ꢀ DMSO was run under
2
2
raphy using cyclohexane/ethyl acetate 3:1 (for 21a,b,d and 22), the same conditions.
2
:1 (for 21c), or 1:6 (for 24) as eluents.
-(3-Methylbenzoyl)-6-nitrocinnolin-4(1H)-one (21a): Yield ¼
1
ꢀ
1
Analysis of compound stability
1
8 ꢀ; mp ¼4300 C dec (EtOH). H NMR (CDCl ) ꢀ 2.48 (s, 3H,
3
CH ), 7.45 (t, 1H, Ar, J ¼ 7.6 Hz), 7.52 (d, 1H, Ar, J ¼ 7.6 Hz), Spontaneous hydrolysis of selected derivatives was evaluated at
3
ꢀ
7
8
1
.65 (d, 1H, Ar, J ¼ 8.0 Hz), 7.68 (s, 1H, Ar), 7.87 (s, 1H, Ar), 25 C in 0.05 M phosphate buffer, pH 7.3. Kinetics of hydrolysis
.58–8.64 (m, 2H, Ar), 9.20 (d, 1H, Ar, J ¼ 2.4 Hz). IR ¼ 1376– were monitored by measuring changes in absorbance spectra over
ꢂ1
ꢂ1
ꢂ1
525 cm (NO ), 1654 cm (C¼O amide), 1723 cm (C¼O). time using a SpectraMax Plus microplate spectrophotometer
2
ESI-MS calcd. for C H N O , 309.28; found: m/z 310.08 (Molecular Devices, Sunnyvale, CA). Absorbance (A ) at the
4
1
6
11
3
t
+
M + H] .
[
6-Nitrocinnolin-4-yl
ꢀ
3-methylbenzoate
1
(22): characteristic absorption maxima of each compound was
Yield ¼ 18ꢀ; mp ¼ 145–147 C (EtOH). H NMR (CDCl ) ꢀ measured at the indicated times until no further absorbance
3
4
1
2
.55 (s, 3H, CH ), 7.55 (t, 1H, Ar, J ¼ 7.0 Hz), 7.62 (d, 1H, Ar, decreases occurred (A ) . Using these measurements, we
0
t
3
1
J ¼ 7.6 Hz), 8.15 (s, 2H, Ar), 8.66 (d, 1H, Ar, J ¼ 9.6 Hz), 8.84 created semilogarithmic plots of log(A –A ) versus time, and k
1
(
d, 1H, Ar, J ¼ 9.6 Hz), 9.03 (s, 1H, Ar), 9.73 (s, 1H, Ar). values were determined from the slopes of these plots. Half-
ꢂ1 ꢂ1
0
2 1/2
IR ¼ 1376–1583 cm (NO ), 1752 cm (C¼O). ESI-MS calcd. conversion times were calculated using t ¼ 0.693/k , as
+
27,39
for C H N O , 309.28; found: m/z 310.08 [M + H] .
16 11 3 4
described previously
.
The stability of selected compounds in human serum and
buffer was also evaluated by analytical RP-HPLC. Human serum
was obtained by allowing fresh human blood to coagulate at
HNE inhibition assay
Compounds were dissolved in 100ꢀ DMSO at 5 mM stock room temperature for 1 h. To 500 mL of human serum or
concentrations. We confirmed that the final concentration of phosphate buffer (0.05 M, pH 7.3), 10 mL of the 10 mM
DMSO in the reactions (1ꢀ) had no effect on enzyme activity. compound stock solution (in DMSO) was added, and the mixture
ꢀ
The HNE inhibition assay was performed in black flat-bottom was incubated at 25 C. At the indicated times, aliquots (50 mL)
3
9
96-well microtiter plates, as described previously . Briefly, were removed, instantly mixed with 200 mL acetonitrile to
buffer A (200 mM Tris-HCl, pH 7.5, 0.01ꢀ bovine serum precipitate proteins in serum samples, and centrifuged for
albumin, and 0.05ꢀ Tween-20) plus 20 mU/mL of HNE 5 min at 10 000g. The samples were analyzed on an automated
(Calbiochem, New Orleans, LA) was added to wells containing HPLC system (Shimadzu, Torrance, CA) with a Phenomenex
different concentrations of each compound. After a 5-min pre- Jupiter C18 300 A column (Phenomenex Inc., Torrance, CA)
incubation, the reaction was initiated by addition of 25 mM (5 mm, 25 cm ꢁ0.46 cm) eluted with acetonitrile/water (65ꢀ/35ꢀ,
elastase substrate (N-methylsuccinyl–Ala–Ala–Pro–Val-7-amino- v/v) containing 0.1ꢀ (v/v) TFA at a flow rate of 1 mL/min at
ꢀ
methyl-coumarin; Calbiochem) in a final reaction volume of 40 C. The elution was monitored using a diode array detector
1
00 mL/well. Kinetic measurements were obtained every 30 s (Shimadzu SPD-M10A VP, Shimadzu Corporation, Kyoto,
ꢀ
for 10 min at 25 C using a Fluoroskan Ascent FL fluores- Japan) set to spectrum max plot (wavelength at which the
cence microplate reader (Thermo Electron, Franklin, MA) highest absorbance occurs) in the region of 200ꢂ300 nm. The
with excitation and emission wavelengths at 355 and 460 nm, amount of compound that remained in serum was calculated
respectively. For all compounds tested, the concentration from the area under the eluted peak.

DOI: 10.3109/14756366.2015.1057718
Cinnoline derivatives as human neutrophil elastase inhibitors
5
Molecular modeling
Keeping m-methylbenzoyl at position N-1, we next inserted
various substituents at position 3 of cinnoline, as shown in
Initial structures of the compounds were generated with
HyperChem 8.0 (Shimadzu Corporation, Kyoto, Japan) and
optimized by the semi-empirical PM3 method. Docking of the
molecules was performed with the use of Molegro Virtual Docker
31,32
Scheme 2. Starting from precursors 9a–d
, which were
46
synthesized following the Sonogoshira reaction , we performed
the cyclization to 4-oxo-1,4-dihydrocinnoline with HCl and
33
NaNO2 to form intermediates 10a–d [10a,d ]. Compounds
(
CLC Bio, København, Denmark) (MVD), version 4.2.0 (CLC
2
8
10a–c were then treated with m-toluoyl chloride and Et
N in
3
Bio, København, Denmark), as described previously . The
structure of HNE complexed with a peptide chloromethyl
2
ketone inhibitor was used for the docking study (1HNE entry
CH Cl to obtain 11a–c. To obtain 11d, it was necessary to
2
2
4
protect 10d with 3,4-dihydro-2H-pyran (12), insert the benzoyl
fragment at N-1 (13), and finally remove the protecting group
with trifluoroacetic acid (11d).
of the Protein Data Bank). The search area for docking poses was
˚
defined as a sphere with 10 A radius centered at the nitrogen atom
The introduction of halogens at position 3 is shown in Scheme
30
. 1H-cinnolin-4-one [14 ] was treated with N-chlorosuccini-
in the five-membered ring of the peptide chloromethyl ketone
inhibitor. After removal of this peptide and co-crystallized water
molecules from the program workspace, we set side chain
flexibility for the 42 residues closest to the center of the search
area (His40, Phe41, Cys42, Gly43, Ala55, Ala56, His57, Cys58,
Val59, Ala60, Tyr94, Pro98, Asn99A, Leu99B, Asp102, Trp141,
Gly142, Leu143, Leu167, Arg177, Val190, Cys191, Phe192,
Gly193, Asp194, Ser195, Gly196, Ser197, Ala213, Ser214,
Phe215, Val216, Arg217A, Gly218, Gly219, Cys220, Ser222,
Leu223, Tyr224, Asp226, Ala227, and Phe228). Fifteen docking
runs were performed for each compound, with full flexibility of a
ligand around all rotatable bonds and side chain flexibility of the
above-mentioned residues of the enzyme. Parameters used within
Docking Wizard of Molegro program were as described
3
mide (NCS), N-iodosuccinimide (NIS), or bromine to obtain 15a–
30,34
c
, which, in turn, were reacted with m-toluoyl chloride. The
presence of a halogen at position 3 shifts the prototropic tautomer
composition, and in the reaction mixture it is possible to recover
in analog ratio the amide derivatives 16a–c and the ester
derivatives 17a–c from the attack at N-1 and at OH of position
4
, respectively. Scheme 3 also shows synthesis of the 3-
unsubsituited cinnolines 18a–g, which were obtained following
the same procedure used for 16a–c. The 4-carbonyl group of 18a
was further reduced to obtain compound 19.
Scheme 4 shows the synthetic routes for the 3-substituted
34–37
derivatives 21a–d, 22, and 24. The intermediates 20a–d
were
2
8
treated with m-toluoyl chloride following the same conditions as
described previously to obtain compounds 21a–d. The potent
electron withdrawing effect of the nitro group in compound 20a,
making possible the existence of the two tautomers, led to a
mixture of the 1-amido 21a and the 4-ester derivative 22 in a 1:1
previously .
The docking poses corresponding to the lowest-energy binding
mode of each inhibitor were evaluated for the ability to form a
Michaelis complex between the hydroxyl group of Ser195 and the
carbonyl group in the amido moiety of an inhibitor. For this
34
ratio. The 6-nitro compound 20a was also transformed into the
38
corresponding 6-amino 23 by reduction with ammonium formate
purpose, values of d [distance O(Ser195)ꢃC between the Ser195
1
hydroxyl oxygen atom and the inhibitor carbonyl carbon atom
closest to O(Ser195)] and a [angle O(Ser195)ꢃꢃꢃC¼O, where C¼O
is the carbonyl group of an inhibitor closest to O(Ser195)] were
and Pd/C as a catalyst. Further treatment of 23 with m-toluoyl
chloride resulted in a double addition final compound 24.
4
3
determined for each docked compound . In addition, we
estimated the possibility of proton transfer from Ser195 to Structure–activity relationship analysis and kinetic
Asp102 through His57 (the key catalytic triad of serine proteases) features of cinnoline derivatives
by calculating distances d between the NH hydrogen in His57
2
2
8
All compounds were evaluated for their ability to inhibit HNE,
and the results are reported in Tables 1–3. Since this new series
was designed as an elaboration of the potent N-benzoylindazoles
and carboxyl oxygen atoms in Asp102, as described previously .
The distance between the hydroxyl proton in Ser195 and the
pyridine-type nitrogen in His57 is also important for proton
transfer. However, because of easy rotation of the hydroxyl about
27,28
previously synthesized by our research group
, the choice of
substituents that were inserted into the cinnoline nucleus was
made with the goal of evaluating the similarity and differences
between the two scaffolds. Thus, we inserted the functional
groups and substituents that produced the best results in the
previous series, such as nitro and bromine at position 6, ethyl ester
at position 3, and m-methylbenzoyl at position 1. We also
evaluated if COOH and H at position 3 resulted in a similar loss of
activity, as was observed for the N-benzoylindazoles. Moreover,
we synthesized two compounds lacking the amide function at
the C–O bond in Ser195, we measured distance d between the
3
oxygen in Ser195 and the basic nitrogen atom in His57. The
effective length L of the channel for proton transfer was calculated
as L ¼ d + min (d ).
3
2
Results and discussion
Chemistry
All final compounds were synthesized as reported in Schemes position 1 in order to understand if, like the N-benzoylindazole
–4, and the structures were confirmed on the basis of the derivatives, the carbonylic group at N-1 was also the point of
analytical and spectral data. Scheme 1 depicts the synthetic attack of Ser195 for this series.
1
pathway for the 3-carbethoxy compounds 4–7 with different
Among the 3-carbethoxy derivatives (Table 1), only com-
substitutions at N-1. Compound 3, which was previously pounds 4 and 7 containing an amide group at position 1 exhibited
3
0
described , is the key intermediate for final compounds 4–7. It submicromolar HNE inhibitory activity (IC50 ¼ 0.58 and 0.21 mM,
2
9
was synthesized starting from precursor 1 , which was treated respectively). As expected, elimination of the carbonyl group led
with diethylcarbonate and NaH (2) and then transformed into the to completely inactive derivatives (5 and 6), confirming that the
4
acetic acid. According to the literature, the 4-oxo tautomer is also evident in our previous studies with indazole-based HNE
-oxo-1,4-dihydro-cinnoline 3 (30) by cyclization with trifluor- C¼O at N-1 was essential for HNE inhibitory activity, which was
4
4,45
predominant for the 4-hydroxycinnoline nucleus
. In any case, inhibitors. While keeping the m-methylbenzoyl fragment, which
3
0
28
treatment of intermediate 3 with different halides led to the was found to be the best substituent for the series of indazoles ,
corresponding N-1 derivatives (products 4–7). Furthermore, we modified and eliminated the groups at position 3. The
hydrolysis of the ethyl ester of compound 7 with NaOH resulted introduction of a phenyl (compound 11a), hydroxymethyl (com-
in compound 8.
pound 11d), or COOH (compound 8) group led to the loss

6
M. P. Giovannoni et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
O
O
O
O
O
CH3
OCH CH3
2
OCH CH3
2
a
b
N
N
N
N
H
N
N
N
N
3
1
2
c
O
N
O
O
O
d
OH
OCH2CH3
N
N
for 7
N
R
CH3
O
4-7
8
Comp.
R
4
5
6
7
COcC H5
3
CH3
CH -m-CH -Ph
2
3
CO-m-CH -Ph
3
Reagents and conditions: (a) (C H O) CO, anhydrous THF, NaH, reflux, 4h; (b) CF COOH, 0°C; rt, 2h; (c) for 4: cyclopropancarbonyl
2
5
2
3
chloride, Et N, anhydrous CH Cl , 0°C, 2h; rt, 2h; for 5: CH I, anhydrous CH CN, Na CO , reflux, 8h; for 6: 3-methylbenzyl chloride,
3
2
2
3
3
2
3
K CO , anhydrous DMF, 80°C, 1h; for 7: m-toluoyl chloride, Et N, anhydrous CH Cl , 0°C, 2h; rt, 2h; (d) NaOH 6N, 100°C, 5h.
2
3
3
2
2
Scheme 1. Synthesis of cinnoline derivatives 4–8.
R
O
O
O
R
a
c
O
N
N
NH2
N
H
for 10d
N
H
9a-d
10a-d
12
b
b
for 10a-c
O
O
N
O
R
9
-11
R
d
O
N
N
a
b
c
Ph
N
cC H5
3
CH3
CH3
O
O
C H
3
7
d
CH OH
2
13
11a-d
Reagents and conditions: (a) HCl conc., NaNO solution, 0°C, 2h; rt, 48h; (b) m-toluoyl chloride, Et N, anhydrous CH Cl , 0°C,
2
3
2
2
2
h; rt, 2h; (c) 3,4-dihydro-2H-pyran, (NH ) Ce(NO ) , anhydrous CH CN, rt, 24h; (d) CF COOH/CH Cl 1:6, rt, 3h.
4 2 3 6 3 3 2 2
Scheme 2. Synthesis of cinnoline derivatives 11a–d.

DOI: 10.3109/14756366.2015.1057718
Cinnoline derivatives as human neutrophil elastase inhibitors
7
O
O
O
R
a
b
N
N
N
X
N
H
N
H
N
1
5a-c
O
1
4
1
8a-g
b
c
CH3
for 18a
O
R
H
OH
O
O
N
N
N
R
NH
N
CH3
O
N
CH3
O
1
7a-c
1
6a-c
1
9
1
8
X
1
5-17
R
a
m-CH3-Ph
CH3
b
c
d
e
f
a
b
c
Cl
Br
I
C H
2
5
7
9
C H
3
C H
4
cC H
3
5
9
g
cC H
5
Reagents and conditions: (a) 15a,c: NCS or NIS, anhydrous DMF, 60°C, 3h; 15b: Br , CH COOH, CH COONa,
2
3
3
reflux, 1h; (b) R-COCl, Et N, CH Cl , 0°C, 2h; rt, 2h; (c) H , Pd/C, EtOH abs., 30PSI (Parr), 4h.
3
2
2
2
Scheme 3. Synthesis of cinnoline derivatives 16a–c, 17a–c, and 19.
of activity. The activity was also very low for compound 11c the amide derivatives. Finally, the 3,4-dihydrocinnoline 19 was
IC50 ¼ 8.10 mM) containing a propyl group at position 3. completely devoid of activity.
In contrast, HNE inhibitory activity was maintained with Based on our SAR analysis, we can highlight the similarities
(
the insertion of an halogen (compounds 16a–c) or (cyclo)alkyl and differences between the new scaffold and N-benzoylindazoles
group (11b) (IC50 ¼ 0.43–1.69 mM). Starting from this result of the previous series. Considering the cinnoline core, our data
and maintaining an unsubstituted position 3, we evaluated suggest that the carbonyl group at N-1 is the one involved in the
effects of the replacement at position 1 of the phenyl with Ser195 attack, and the best substituent at this position is 3-
2
8
(
cyclo)alkyl fragments (compounds 18b–g) (Table 1), but methylbenzoyl, which was observed for the N-benzoylindazoles .
the activity of this series was one order of magnitude lower Position 3 of the cinnolines should not be substituted because
than for 18a. inclusion of various groups or atoms led to a decrease in activity,
We next introduced at position 6 (R ) of 18a a variety of which was not observed with the indazole series. The same effect
6
substituents that were found to be favorable for activity of the on activity occurred for position 6, probably because the insertion
2
8
reference N-benzoylindazoles . However, none of these substitu- of a carbonyl to expand the pentatomic ring influenced the
ents led to increased potency, and these compounds had IC50 interaction of the molecule with the catalytic site. In addition, the
values in the micromolar/submicromolar range (compounds aromatic compounds showed an appreciable HNE inhibitory
2
1a–d and 24, IC50 value ¼ 0.26–6.30 mM) (Table 2).
In Table 3, we report HNE inhibitory activity of compounds catalysis.
7a–c and 22, which are the ester isomers of the N-1 benzoyl Inhibition constants (K ) were determined for the two com-
activity, suggesting that the carbonyl ester may be involved in the
1
i
derivatives of 16a–c and 21a. Interestingly, all compounds, pounds with the highest HNE inhibitory activity (18a and 18e).
although lacking the amidic function at N-1, exhibited moderate Although compounds 18a and 18e were quite potent, with Ki
inhibitory activity. Moreover, compounds 17c (R ¼ I) and 22 values of 75 and 110 nM, respectively, their activity was lower
3
(
1
R ¼ NO ) had IC values comparable with their amidic isomers that our reference N-benzoylindazole HNE inhibitor (K ¼ 27 nM),
6
2
50
i
6c and 21a (IC values ¼ 2.70 and 1.30 mM for 17c and 16c, which was previously reported as compound 5b (1-(3-methyl-
5
0
2
8
respectively, and IC50 values ¼ 0.59 and 0.26 mM, for 22 and 21a, benzoyl)-1H-indazole-3-carbonitrile) . In addition, double-recip-
respectively). Thus, these results suggest that ester derivatives rocal Lineweaver–Burk plots of substrate hydrolysis by HNE in
1
7a–c and 22 probably interact with the enzyme differently from the absence and presence of compounds 18a and 18e showed that

8
M. P. Giovannoni et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
O
O
N
O
R
O N
2
R
a
a
N
N
N
N
for 20a
for 20b-d
N
H
CH3
CH3
O
O
2
0a-d
2
1a
21b-d
b
for 20a
O
N
O
O
O N
H N
20,21
R
2
2
CH3
N
N
a
b
c
NO2
N
H
Cl
Br
I
23
2
2
d
a
CH3
O
O
HN
N
N
O
CH3
24
Reagents and conditions: (a) m-toluoyl chloride, Et N, anhydrous CH Cl , 0°C, 2h; rt, 2h; (b) HCOONH , Pd/C, EtOH abs., 80°C, 2h.
3
2
2
4
Scheme 4. Synthesis of cinnoline derivatives 21a–d, 22. and 24.
they were competitive inhibitors (Figure 2 shows a representative buffer analyzed by this method was consistent with the results
plot for 18a).
obtained by conventional spectrophotometry (Figure S1). We
found that following 5 min of incubation in human serum,53ꢀ of
the initial amounts remained. After 10 min in serum, there were
essentially no detectable compounds (Figure S2).
Compound and HNE-inhibitor complex stability
The most active cinnoline derivatives with an IC 51 mM were
Two relatively stable compounds (11b and 18b) were
evaluated for chemical stability in aqueous buffer using spectro- evaluated for reversibility of HNE inhibition over time in
5
0
2
8
photometry to assess compound hydrolysis. As an example, the comparison with HNE inhibitor 5b (Figure 1). As shown in
hydrolysis of compound 21a is shown in Figure S1. The Figure S3, an inhibition was maximal during the first 2 h after
absorbance maxima at 325 nm decreased over time, indicating treatment with 10 mM 18b, but inhibition was soon reversed and
that this compound was hydrolyzed almost completely after recovery of HNE activity was observed. Although HNE inhibitory
60 min in aqueous buffer with a t1/2 of 33 min. The other activity of 11b was lower than reference compound 5b, both
compounds had t1/2 values from 38.5 to 233 min (Table 4), compounds were equally active over the 10-h incubation period
indicating that the tested cinnoline derivatives were more stable (Figure S3).
than our previously described HNE inhibitors with the
The stability of HNE–inhibitor complex was also evaluated by
. Indeed, cinnoline derivatives treating the most potent cinnoline derivatives (18a and 18e) at a
2
7,28
N-benzoylindazole scaffold
possess an endocyclic carbonyl group, which participates in the relatively high concentration (25 mM), removing free inhibitor by
total conjugation system of the molecule. Acceptor character of ultrafiltration of the enzyme/inhibitor mixture, and then monitor-
this group causes destabilization of the carbocation intermediate ing recovery of HNE activity over time. We found that inhibition
that emerges on protonation of the amide oxygen atom and may of HNE by the selected inhibitors was fully reversible, whereas the
lead to lowered hydrolysis rates of cinnolines as compared to their reference compound 5b completely inhibited activity of HNE even
indazole analogues.
To test compound stability in serum, the two most potent
cinnoline HNE inhibitors (18a and 18e) were incubated in human
serum for various times at 25 C, and then the samples were
analyzed by analytical RP-HPLC to quantify the percent of initial The geometric characteristics of molecule orientations and
compound remaining. Note that compound stability in phosphate arrangements in the catalytic triad (Ser195, His57, and
after a 6-h incubation (data not shown).
Molecular modeling
ꢀ

DOI: 10.3109/14756366.2015.1057718
Cinnoline derivatives as human neutrophil elastase inhibitors
9
Table 1. HNE inhibitory activity of cinnolone derivatives 4–8, 11a–d, Table 3. HNE inhibitory activity of cinnoline derivatives 17a–c, 22,
6a–c, and 18a–g. and 19.
1
O
CH3
R3
H
OH
NH
N
N
O
O
N
R1
N
R6
R3
CH3
O
N
Comp
R
1
R
3
IC50 (mM)*
1
7a-c,22
19
4
5
6
7
8
COcC
CH
CH -m-CH
3
H
5
COOEt
COOEt
COOEt
COOEt
COOH
Ph
0.58 ± 0.16
NA
NA
0.21 ± 0.23
NA
NA
0.43 ± 0.013
8.10 ± 1.3
NA
0.97 ± 0.23
1.69 ± 0.52
1.30 ± 0.37
0.056 ± 0.017
0.93 ± 0.37
2.20 ± 0.14
0.25 ± 0.05
0.15 ± 0.04
0.20 ± 0.014
0.44 ± 0.14
3
2
3
-Ph
CO-m-CH
CO-m-CH
CO-m-CH
CO-m-CH
CO-m-CH
CO-m-CH
CO-m-CH
CO-m-CH
CO-m-CH
CO-m-CH
3
3
3
3
3
3
3
3
3
3
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
Comp
R3
R6
IC50 (mM)*
1
1
1
2
1
7a
7b
7c
2
Cl
Br
I
H
–
H
H
H
NO
–
4.80 ± 1.6
4.26 ± 1.18
2.70 ± 0.9
0.59 ± 0.15
NA
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1a
1b
1c
1d
6a
6b
6c
8a
8b
8c
8d
8e
8f
3
cC H
5
3 7
C H
CH OH
2
2
9
Cl
Br
I
H
H
H
H
H
H
H
NA: no inhibitory activity was found at the highest concentration of
compound tested (50 mM).
IC50 values are presented as the mean ± SD of three independent
experiments.
*
COCH
3
COC
COC
COC
2
3
4
H
5
H
H
7
9
0.05
[
I] = 200 nM
COcC
COcC H
3
H
5
Compound 18a
8g
5
9
0
0
0
.04
.03
.02
NA: no inhibitory activity was found at the highest concentration of
compound tested (50 mM).
IC50 values are presented as the mean ± SD of three independent
experiments.
*
[
I] = 100 nM
[
I] = 50 nM
Table 2. HNE Inhibitory activity of cinnolone derivatives 21a–d and 24.
[
I] = 25 nM
I] = 0 nM
O
[
R6
0.01
0.00
N
N
0.00
0.02
0.04
0.06
/[S]
0.08
0.10
CH3
O
1
Figure 2. Kinetics of HNE inhibition by cinnoline derivative 18a.
Representative double-reciprocal Lineweaver–Burk plot of substrate
hydrolysis by HNE in the absence and presence of the compounds 18a
is shown. The representative plot is from three independent experiments.
Comp
R
6
IC50 (mM)*
21a
21b
21c
21d
24
NO
Cl
Br
I
2
0.26 ± 0.05
3.60 ± 1.1
3.00 ± 0.35
0.74 ± 0.012
6.30 ± 1.4
Table 4. Half-life (t1/2) for the spontaneous hydrolysis of selected
cinnolinone derivatives.
Comp
t
1/2 (min)
l
max (nm)*
3
NHCO-m-CH -Ph
4
86.3
172
38.5
46.8
114
75.4
121
118
233
116
240
235
255
270
260
260
260
260
240
240
260
240
260
*
IC50 values are presented as the mean ± SD of three independent
experiments.
1
1
1
1
1
1
1b
6a
7c
8a
8b
8d
Asp102) were in general agreement with the HNE inhibitory
activities of the compounds (Table 5). The key residues were 18e
oriented in a fashion favorable for proton transfer within the 18f
1
2
2
2
8g
1a
1d
2
oxyanion hole and formation of the Michaelis complex import-
ant for inhibition. Both cinnoline derivative 18a and reference
33.2
104
41.9
2
N-benzoylindazole 5b (Figure 1) were H-bonded with Gly193
8
and Ser195, but had somewhat different poses within the
receptor cavity (Figures 3A and S4). Particularly, the poses *Absorption maximum used for monitoring spontaneous hydrolysis.

1
0
M. P. Giovannoni et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Table 5. Geometric parameters of the enzyme–inhibitor complexes predicted by molecular docking.
Comp
d
1
a
d
3
d
2
Ly
1
1
1
5
6b
3.442
3.628
3.545
3.448
83.4
89.5
82.2
105.2
2.838
2.964
2.838
3.142
1.798; 3.354
1.879; 3.322
1.810; 3.391
2.181; 3.755
4.636
4.843
4.648
5.323
7b*
8a
2
b
8
*
According to the docking results, a Michaelis complex with Ser195 is formed with participation of the ester
carbonyl group.
yLength of the channel for proton transfer calculated as d
3 2
+ min(d ).
Figure 3. Superimposed docking poses of peptide chloromethyl ketone and novel small-molecule HNE inhibitors. Co-crystallized peptide
2
8
˚
chloromethyl ketone inhibitor is shown in yellow. Residues within 5 A of this ligand are visible. Panel (A) Docking poses of reference compound 5b
dark-green) and compound 18a (blue). Panel (B) Docking poses of compounds 16b (violet) and 17b (brown).
(

DOI: 10.3109/14756366.2015.1057718
Cinnoline derivatives as human neutrophil elastase inhibitors 11
O
compounds, showed that this series is more stable than the N-
benzoylindazoles, with 18f (IC50 value ¼ 0.2 mM) being the most
stable (t1/2 ¼ 233 min). Finally, molecular modeling studies show
that we synthesized two different types of HNE inhibitors: (1)
compounds with a cinnolin-4(1H)-one scaffold, where Ser195-
OH attacks the N1 C¼O and (2) cinnoline derivatives bearing an
ester function at C-4, which is the attack point of Ser195-OH.
Both types of the nucleophilic attack of Ser195 on a carbonyl
carbon atom can be accompanied by proton transfer from the
serine hydroxyl group via His57 to Asp102. Our results indicate
that the geometric features of docking poses and orientations of
side chains for the catalytic triad (Ser195, His57, and Asp102) are
favorable for accomplishment of this mechanism. Thus, the novel
HNE inhibitors reported here represent potential starting points
for future manipulation and optimization.
O
O
N
N
Asp102
O
α
H
N
N
H
O
Ser195
His57
Declaration of interest
This work was supported in part by NIH IDeA Program COBRE Grant
GM110732 (MTQ) and a USDA National Institute of Food and
Agriculture Hatch project, and the Montana State University
Agricultural Experiment Station.
Figure 4. Hypothetical model for the nucleophilic attack of Ser195 at the
carbonyl group of cinnoline derivative (18a) accompanied by synchron-
ous proton transfer from Ser195 to Asp102 via the catalytic triad. The key
angle a is indicated (see text for details). The model is based on the
proposed mechanism of synchronous proton transfer from the oxyanion
4
0,47
hole in serine proteases
.
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Supplementary material available online
Supplementary material, Figures S1–S6.