Further modifications of 1H-pyrrolo[2,3-b]pyridine derivatives as inhibitors of human neutrophil elastase

Article abstract of DOI:10.1002/ddr.21539

Human neutrophil elastase (HNE) is a potent protease that plays an important physiological role in many processes and is considered to be a multifunctional enzyme. HNE is also involved in a variety of pathologies affecting the respiratory system. Thus, compounds able to inhibit HNE proteolytic activity could represent effective therapeutics. We present here a new series of pyrrolo[2,3-b]pyridine derivatives of our previously reported potent HNE inhibitors. Our results show that position 2 of the pyrrolo[2,3-b]pyridine scaffold must be unsubstituted, and modifications of this position resulted in loss of HNE inhibitory activity. Conversely, the introduction of certain substituents at position 5 was tolerated, with retention of HNE inhibitory activity (IC₅₀ = 15–51 nM) after most substitutions, indicating that bulky and/or lipophilic substituents at position 5 probably interact with the large pocket of the enzyme site and allow Michaelis complex formation. The possibility of Michaelis complex formation between Ser195 and the ligand carbonyl group was assessed by molecular docking, and it was found that highly active HNE inhibitors are characterized by geometries favorable for Michaelis complex formation and by relatively short lengths of the proton transfer channel via the catalytic triad.

Full text of DOI:10.1002/ddr.21539

Received: 14 December 2018
DOI: 10.1002/ddr.21539
Revised: 13 March 2019
Accepted: 5 April 2019
R E S E A R C H A R T I C L E
Further modifications of 1H-pyrrolo[2,3-b]pyridine derivatives
as inhibitors of human neutrophil elastase
Maria P. Giovannoni1 | Niccolò Cantini1 | Letizia Crocetti1
Antonella Iacovone1 | Igor A. Schepetkin2 | Claudia Vergelli1
Andrei I. Khlebnikov3,4 | Mark T. Quinn2
| Gabriella Guerrini1
|
|
1NEUROFARBA, Pharmaceutical and
Abstract
Nutraceutical Section, University of Florence,
Sesto Fiorentino, Italy
Human neutrophil elastase (HNE) is a potent protease that plays an important physi-
ological role in many processes and is considered to be a multifunctional enzyme.
HNE is also involved in a variety of pathologies affecting the respiratory system.
Thus, compounds able to inhibit HNE proteolytic activity could represent effective
therapeutics. We present here a new series of pyrrolo[2,3-b]pyridine derivatives of
our previously reported potent HNE inhibitors. Our results show that position 2 of
the pyrrolo[2,3-b]pyridine scaffold must be unsubstituted, and modifications of this
position resulted in loss of HNE inhibitory activity. Conversely, the introduction of
certain substituents at position 5 was tolerated, with retention of HNE inhibitory
activity (IC50 = 15–51 nM) after most substitutions, indicating that bulky and/or lipo-
philic substituents at position 5 probably interact with the large pocket of the
enzyme site and allow Michaelis complex formation. The possibility of Michaelis com-
plex formation between Ser195 and the ligand carbonyl group was assessed by
molecular docking, and it was found that highly active HNE inhibitors are character-
ized by geometries favorable for Michaelis complex formation and by relatively short
lengths of the proton transfer channel via the catalytic triad.
2Department of Microbiology and
Immunology, Montana State University,
Bozeman, Montana
3Kizhner Research Center, Tomsk Polytechnic
University, Tomsk, Russia
4Scientific Research Institute of Biological
Medicine, Altai State University, Barnaul,
Russia
Correspondence
Letizia Crocetti, Dipartimento di
NEUROFARBA, Via Ugo Schiff 6, Sesto
Fiorentino 50019, Firenze, Italy.
Email: letizia.crocetti@unifi.it
Funding information
Ministry of Education and Science of the
Russian Federation, Grant/Award Number:
4.8192.2017/8.9; Montana University System
Research Initiative, Grant/Award Number:
51040-MUSRI2015-03; National Institutes of
Health IDeA Program COBRE, Grant/Award
Number: GM110732; Tomsk Polytechnic
University Competitiveness Enhancement
Program; USDA National Institute of Food and
Agriculture Hatch, Grant/Award Number:
1009546
K E Y W O R D S
human neutrophil elastase inhibitors, molecular docking, pyrrolo[2,3-b]pyridine
secondary or specific, tertiary or gelatinase, and secretory (Pham,
2006). Specifically, the azurophil granules contain myeloperoxidase,
1
| INTRODUCTION
Polymorphonuclear neutrophils represent a large percentage of the cir-
culating leukocyte population and are the most abundant type of white
blood cells in human blood. They are also known as granulocytes due
to the numerous granules present in their cytoplasm and are character-
ized by their nuclear morphology, which is segmented into three to five
lobes joined together by a thin membrane. These cells play a fundamen-
tal role in immune defense against pathogenic organisms and are the
first mediators in the inflammatory response (Korkmaz et al., 2010).
Neutrophil granules are classified into primary or azurophil,
some bactericidal proteins, and three serine proteases: proteinase
3, cathepsin G, and human neutrophil elastase (HNE), which is a
globular glycoprotein of about 30 kDa belonging to the chymotryp-
sin family. HNE consists of a single polypeptide chain of 218 amino
acids, with two asparagine-linked carbohydrate side chains at Asn95
and Asn144 (Lucas et al., 2011). HNE is stabilized by four disulfide
bridges and has 19 arginine residues, resulting in an isoelectric point
of 10–11. As with other neutrophil serine proteases, HNE plays an
important physiological role and is considered a multifunctional
Drug Dev Res. 2019;1–12.
wileyonlinelibrary.com/journal/ddr
© 2019 Wiley Periodicals, Inc.
1

2
GIOVANNONI ET AL.
enzyme that contributes to killing of pathogens, inflammatory pro-
cesses, and maintenance of tissue homeostasis (Pham, 2006). HNE
proteolytic activity depends on the catalytic triad consisting of
Ser195-His57-Asp102 (Fujinaga et al., 1996).
The proteolytic activity of serine proteases, which is essential for
the maintenance of important host functions, can also be harmful if
not properly regulated, and a number of regulatory mechanisms are
present (Von Nussbaum et al., 2016). For example, these proteases
are stored as inactive precursors in specialized compartments, such as
azurophil granules for serine proteases. Once active, serine protease
activity can be regulated by endogenous inhibitors, such as
α1-antitripsin (also known as α1-proteinase inhibitor [α1-PI]), elafin,
and secretory leukocyte protease inhibitor, which inhibit protease
activity (Von Nussbaum et al., 2015; Zhong et al., 2017). The involve-
ment of HNE and other neutrophil serine proteases in the develop-
ment of chronic pulmonary inflammatory diseases, such as chronic
obstructive pulmonary disease (COPD), acute lung injury, acute respi-
ratory distress syndrome (ARDS), and cystic fibrosis (CF) is widely
documented and correlated with an imbalance between HNE and
endogenous inhibitor activity (Polverino et al., 2017).
Recently, we identified and characterized a number of potent
HNE inhibitors with differing structural scaffolds (Crocetti et al.,
2011; Crocetti et al., 2013; Crocetti et al., 2016; Crocetti et al., 2018;
Giovannoni et al., 2016; Giovannoni et al., 2018; Schepetkin,
Khlebnikov, & Quinn, 2007; Vergelli et al., 2017). In the most recent
studies, we identified HNE inhibitors with a pyrrolo[2,3-b]pyridine
scaffold (Crocetti et al., 2018) and found that the most potent com-
pounds exhibited IC50 values in the low nanomolar range (14–87 nM)
(see Figure 1). Based on these studies, we performed further modifica-
tions of the pyrrolo[2,3-b]pyridine scaffold. While keeping the best
substituents identified for N-1 (m-toluoyl) and position 3 (CN group),
we introduced substituents with different features at position 5 to
screen for more potent HNE inhibitors. Additionally, we investigated
substitutions at position 2 of the scaffold by shifting CN from the 3 to
the 2 position or inserting other groups.
FIGURE 1 Further modifications of the pyrrolo[2,3-b]pyridine
scaffold
5-substituted-pyrrolo[2,3-b]pyridines 3a-c (Bhat et al., 2015; Chen
et al., 2015; Joydev et al., 2017) by the Duff reaction, was transformed
into the corresponding oxime by treatment with hydroxylamine hydro-
chloride and NaHCO3 at high temperature, resulting in compounds 5a-
c (5b, Bahekar et al., 2007). Further reaction with POCl3 resulted in the
2
| MATERIALS AND METHODS
2.1 | Chemistry
All final compounds were synthesized as reported in Figures 2–4, and
the structures were confirmed on the basis of analytical and spectral
data. To obtain the 2- or 2,3-disubstituted pyrrolo[2,3-b]pyridines (2a-e),
we followed the procedures shown in Figure 2. Starting from the previ-
ously synthesized compounds 1a-e (Bahekar et al., 2007; Baltus et al.,
2016; Pires et al., 2016; Sandham et al., 2009), we performed benzo-
ylation with m-toluoyl chloride and triethylamine in anhydrous dic-
hloromethane, resulting in final compounds 2a-e. The synthesis of
compounds with different substitutions at position 5 is shown in Fig-
ures 3 and 4. Figure 3 shows the synthesis of pyrrolo[2,3-b]pyridines
substituted with a bromine, chlorine, or nitro group at position 5. The
formyl group of intermediates 4a-c (Ermoli et al., 2009; Nirogi et al.,
2012; Xi & Li, 2014), obtained starting from the corresponding
1,2
a
R2
CH3
R3
H
b
CH2CH3
COOEt
CN
H
c
H
d
H
e
CH3
CN
FIGURE 2 Reagents and conditions: (a) m-Toluoyl chloride, Et3N,
dry DCM, 0 ꢀC, 2 hr; r.t., 2 hr

GIOVANNONI ET AL.
3
and 13C NMR spectra were recorded on an Avance 400 instrument
(Bruker Biospin Version 002 with SGU, Bruker Inc., Billerica, MA).
Chemical shifts (δ) are reported in ppm to the nearest 0.01 ppm using
the solvent as an internal standard. Coupling constants (J values) are
given in Hz and were calculated using TopSpin 1.3 software (Nicolet
Instrument Corp., Madison, WI) and are rounded to the nearest
0.1 vHz. Mass spectra (m/z) were recorded on an ESI-TOF mass spec-
trometer (Brucker Micro TOF, Bruker Inc., Billerica, MA), and reported
mass values are within the error limits of 5 ppm mass units. Micro-
analyses indicated by the symbols of the elements were performed
with a Perkin–Elmer 260 elemental analyzer (PerkinElmer, Inc., Wal-
tham, MA) for C, H, and N, and the results were within 0.4% of the
theoretical values, unless otherwise stated. Reagents and starting
materials were commercially available.
General procedure for compounds 2a-e. To a cooled (0 ꢀC) sus-
pension of the appropriate substrate 1a-e (Bahekar et al., 2007; Baltus
et al., 2016; Pires et al., 2016; Sandham et al., 2009) (0.56 mmol) in
anhydrous CH2Cl2 (2 mL), 0.72 mmol of Et3N, and 1.67 mmol of m-
toluoyl chloride were added. The mixture was stirred at 0 ꢀC for 2 hr
and then at room temperature for an additional 2 hr. The solvent was
evaporated, cold water was added, and the mixture was neutralized
with 0.5 N NaOH. The reaction mixture was extracted with CH2Cl2
(3 × 15 mL), and the solvent was dried over sodium sulfate and evapo-
rated in vacuum. The final compounds 2a-e were purified by column
chromatography using toluene/ethyl acetate 9.5:0.5 (for 2a,b) or
cyclohexane/ethyl acetate 2:1 (for 2c,d) or 5:1 (for 2e) as eluents.
(2-Methyl-1H-pyrrolo[2,3-b]pyridin-1-yl)(m-tolyl)methanone (2a).
Yield = 67%; oil. 1H-NMR (CDCl3-d1) δ 2.39 (s, 3H, m-CH3-Ph), 2.56
(s, 3H, CH3), 7.02–7.07 (m, 1H, Ar), 6.38 (s, 1H, Ar), 7.30 (t, 1H, Ar,
J = 8.0 Hz), 7.41 (d, 1H, Ar, J = 8.0 Hz), 7.50 (d, 1H, Ar, J = 8.0 Hz),
7.66 (s, 1H, Ar), 7.76 (d, 1H, Ar, J = 8.0 Hz), 8.05 (d, 1H, Ar, J = 4.4 Hz).
13C-NMR (CDCl3-d1) δ 15.15 (CH3), 21.33 (CH3), 104.52 (CH), 117.37
(CH), 121.44 (C), 127.52 (CH), 128.04 (CH), 128.16 (CH), 131.13 (CH),
134.19 (CH), 134.77 (C), 138.16 (C), 138.90 (C), 142.62 (CH), 149.53
(C), 169.70 (CO). ESI-MS calcd. For C16H14N2O, 250.30; found: m/z
251.11 [M+H]+. Anal. C16H14N2O (C, H, N).
3-7 R5
a
b
c
Br
Cl
NO2
FIGURE 3 Reagents and conditions: (a) HTMA, CH3COOH, reflux,
4 hr; (b) NH2OHÁHCl, H2O, 60 ꢀC, 30 min; NaHCO3, reflux, 4hr;
(c) POCl3, reflux, 1 hr; (d) m-Toluoyl chloride, NaH, dry THF, r.t., 24 hr
3-carbonitrile derivatives 6a-c (6a, Graczyk, Palmer, & Khan, 2004),
which were benzoylated at position 1 with m-toluoyl chloride and
sodium hydride in anhydrous tetrahydrofuran to obtain the
5-substituted pyrrolo[2,3-b]pyridines 7a-c (Figure 3).
Figure 4 shows the synthetic steps for insertion of (hetero)aro-
matic rings at position 5 to obtain final compounds 14a-g. In the first
step, the nitrogen at position 1 of intermediate 3a (Joydev et al.,
2017) was protected with benzensulfonyl chloride to obtain com-
pound 8 (Liu et al., 2016), which subsequently was treated with
tetrakis(triphenylphosphine)palladium(0), 2 M sodium carbonate solu-
tion, and the appropriate boronic acid in hot anhydrous toluene to
obtain the corresponding 5-pyrrolo[2,3-b]pyridine derivatives 9a-g.
The protecting group at position N-1 was then removed with
tetrabutylammonium fluoride (TBAF) in hot anhydrous tetrahydrofu-
ran, resulting in pyrrolo[2,3-b]pyridines 10a-g (10a, Laha et al., 2017;
10c and 10d, Ibrahim et al., 2007; 10f and 10g, Singh et al., 2017).
Reaction of these compounds with hexamethylenetetramine (HMTA)
in acetic acid at reflux resulted in the 3-formyl derivatives 11a-g (11g,
Ibrahim et al., 2007), which, when treated with hydroxylamine hydro-
chloride (12a-g), dehydrated with POCl3 (13a-g), and benzoylated at
position 1 with m-toluoyl chloride, led to final compounds 14a-g.
(2-Ethyl-1H-pyrrolo[2,3-b]pyridin-1-yl)(m-tolyl)methanone (2b).
Yield = 17%; oil. 1H-NMR (CDCl3-d1) δ 1.30 (t, 3H, CH2CH3, J = 7.2 Hz),
2.39 (s, 3H, m-CH3-Ph), 2.98 (q, 2H, CH2CH3, J = 7.2 Hz), 6.43 (s, 1H,
Ar), 7.03–7.08 (m, 1H, Ar), 7.30 (t, 1H, Ar, J = 7.6 Hz), 7.41 (d, 1H, Ar,
J = 7.6 Hz), 7.49 (d, 1H, Ar, J = 7.6 Hz), 7.66 (s, 1H, Ar), 7.78 (d, 1H,
Ar, J = 7.6 Hz), 8.05 (d, 1H, Ar, J = 4.4 Hz). 13C-NMR (CDCl3-d1) δ
12.82 (CH3), 21.44 (CH3), 21.81 (CH2), 102.58 (CH), 118.02 (CH),
121.33 (C), 128.08 (CH), 129.30 (CH), 131.13 (CH), 134.29 (CH),
134.78 (C), 138.20 (C), 142.76 (CH), 145.09 (C), 149.74 (C), 169.93
(CO). ESI-MS calcd. For C17H16N2O, 264.32; found: m/z 265.13 [M
+H]+. Anal. C17H16N2O (C, H, N).
2.2 | Experimental
All melting points were determined on a Büchi apparatus (New Castle,
DE) and are uncorrected. Extracts were dried over Na2SO4, and the
solvents were removed under reduced pressure. Merck F-254 com-
mercial plates (Merck, Durham, NC) were used for analytical TLC to
follow the course of reactions. Silica gel 60 (Merck 70–230 mesh,
Merck, Durham, NC) was used for column chromatography. 1H NMR
Ethyl 1-(3-methylbenzoyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate
(2c). Yield = 34%; oil. 1H-NMR (CDCl3-d1) δ 1.45 (t, 3H, OCH2CH3,
J = 7.2 Hz), 2.38 (s, 3H, m-CH3-Ph), 4.21 (q, 2H, OCH2CH3, J = 7.2 Hz),
7.16–7.21 (m, 1H, Ar), 7.29–7.34 (m, 2H, Ar), 7.42 (d, 1H, Ar, J = 7.6 Hz),
7.56 (d, 1H, Ar, J = 7.6 Hz), 7.71 (s, 1H, Ar), 8.04 (d, 1H, Ar, J = 8.0 Hz),
8.41 (d, 1H, Ar, J = 4.4 Hz). 13C-NMR (CDCl3-d1) δ 13.94 (CH3), 21.29

4
GIOVANNONI ET AL.
9-14
R5
Ph
a
b
c
d
e
f
m-CF3-Ph
m-CH3-Ph
m-OCH3-Ph
6-OCH3-naphthalene
3-thienyl
g
3-pyridyl
FIGURE 4 Reagents and conditions: (a) Ph-SO2Cl, Et3N, dry DCM, 0 ꢀC, 2 hr then r.t., 2 hr; (b) R5-B(OH)2, Pd(PPh3)4, Na2CO3 2 M, anhydrous
toluene, reflux, 4–5 hr; (c) TBAF, dry THF, reflux, 2–6 hr; (d) HMTA, CH3COOH, reflux, 6 hr; (e) NH2OHÁHCl, H2O, 60 ꢀC, 30 min; NaHCO3, 100 ꢀC,
4 hr; (f) POCl3, reflux, 2 hr; (g) m-Toluoyl chloride, NaH, dry THF, r.t., 24 hr
(CH3), 61.59 (CH2), 111.04 (CH), 118.64 (CH), 119.46 (C), 127.93 (CH),
128.56 (CH), 130.65 (C), 131.02 (CH), 131.18 (CH), 134.03 (C), 135.01
(CH), 138.61 (C), 147.70 (CH), 150.05 (C), 160.65 (C), 169.00 (CO). ESI-
MS calcd. For C18H16N2O3, 308.34; found: m/z 309.12 [M+H]+. Anal.
138.91 (C), 142.4 (CH), 146.36 (C), 167.70 (CO). ESI-MS calcd. For
C17H13N3O, 275.30; found: m/z 276.11 [M+H]+. Anal. C17H13N3O
(C, H, N).
General procedure for compounds 5a and 5c. A mixture of the
appropriate 1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde 4a (Xi & Li,
2014) or 4c (Ermoli et al., 2009) (0.78 mmol) and hydroxylamine
hydrochloride (2.35 mmol) in 2 mL of water was heated at 60 ꢀC for
30 min. NaHCO3 (2.35 mmol) was then added, and the reaction mix-
ture was heated under reflux for 4 hr at 100 ꢀC. After cooling at room
temperature, the solid was filtered, washed with the excess of ice-cold
water and dried. The crude product was recrystallized from hexane to
obtain the pure product.
C18H16N2O3 (C, H, N).
1-(3-Methylbenzoyl)-1H-pyrrolo[2,3-b]pyridine-2-carbonitrile (2d).
Yield = 41%; oil. 1H-NMR (CDCl3-d1)
δ 2.41 (s, 3H, m-CH3-Ph),
7.25–7.33 (m, 1H, Ar), 7.34–7.40 (m, 2H, Ar), 7.48 (d, 1H, Ar, J = 7.6 Hz),
7.58 (d, 1H, Ar, J = 7.6 Hz) 7.69 (s, 1H, Ar), 8.03 (dd, 1H, Ar, J = 8.0 Hz
and J = 1.2 Hz), 8.38 (d, 1H, Ar, J = 3.6 Hz). 13C-NMR (CDCl3-d1) δ
21.53 (CH3), 110.31 (C), 112.22 (C), 118.23 (CH), 119.60 (C), 120.03
(CH), 128.32 (CH), 128.59 (CH), 131.13 (CH), 131.61 (CH), 132.56 (C),
135.23 (CH), 138.52 (C), 148.37 (CH), 166.85 (CO). ESI-MS calcd. For
(E)-5-Bromo-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde
oxime
(5a). Yield = 94%; mp = 235–237 ꢀC (hexane). 1H-NMR (DMSO-d6) δ
7.81 (s, 1H, CH=N-OH), 8.21 (s, 1H, Ar), 8.33 (s, 1H, Ar), 8.38 (s, 1H,
Ar), 10.75 (exch br s, 1H, CH=N-OH), 12.16 (exch br s, 1H, NH). ESI-
MS calcd. For C8H6BrN3O, 240.06; found: m/z 240.97 [M+H]+. Anal.
C8H6BrN3O (C, H, N).
C
16H11N3O, 261.28; found: m/z 262.09 [M+H]+. Anal. C16H11N3O
(C, H, N).
2-Methyl-1-(3-methylbenzoyl)-1H-pyrrolo[2,3-b]pyridine-3-carbo-
nitrile (2e). Yield = 10%; oil. 1H-NMR (CDCl3-d1) δ 2.40 (s, 3H, m-CH3-
Ph), 2.74 (s, 3H, CH3), 7.21–7.26 (m, 1H, Ar), 7.35 (d, 1H, Ar), 7.48 (d,
2H, Ar, J = 7.6 Hz), 7.64 (s, 1H, Ar), 7.98 (d, 1H, Ar, J = 7.2 Hz), 8.19 (d,
1H, Ar, J = 4.0 Hz). 13C-NMR (CDCl3-d1) δ 9.43 (CH3), 20.91 (CH3),
101.37 (C), 115.65 (CH), 115.92 (C), 121.15 (CH), 128.10 (CH), 128.36
(CH), 129.14 (CH), 130.17 (CH), 130.49 (CH), 134.86 (CH), 137.33 (C),
(E)-5-nitro-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde oxime (5c).
Yield = 40%; mp = 193–195 ꢀC (hexane). 1H-NMR (DMSO-d6) δ 8.00
(s, 1H, Ar), 8.27 (s, 1H, CH=N-OH), 9.04 (s, 1H, Ar), 9.13 (s, 1H, Ar),
11.02 (exch br s, 1H, CH=N-OH), 12.70 (exch br s, 1H, NH). ESI-MS

GIOVANNONI ET AL.
5
calcd. For C8H6N4O3, 206.16; found: m/z 207.05 [M+H]+. Anal.
C8H6N4O3 (C, H, N).
General procedure for compounds 9a-g. To a suspension of inter-
mediate 8 (Liu et al., 2016) (0.34 mmol) in 3 mL of toluene, 0.051 mmol
of tetrakis(triphenylphosphine)palladium(0), 3 mL of Na2CO3 2 M solu-
tion, and 0.68 mmol of the appropriate hetero(phenyl)-boronic acid
were added. The mixture was stirred at reflux for 4 hr. After cooling,
ice-cold water (20 mL) was added, the suspension was extracted with
CH2Cl2 (3 × 15 mL), dried over sodium sulfate, and the solvent was
evaporated in vacuo to obtain compounds 9a-g, which were purified by
column chromatography using hexane/ethyl acetate 3:1 (for 9b,e),
hexane/acetone 4:1 (for 9a,c,d,f), or cyclohexane/ethyl acetate 1:5 (for
9g) as eluents.
General procedure for compounds 6b and 6c. A suspension of
intermediate 5b (Bahekar et al., 2007) or 5c (0.92 mmol) in 4 mL of
POCl3 was stirred at reflux for 2 hr. After cooling, ice-cold water
(20 mL) was slowly added, and the precipitate was filtered under vac-
uum and washed with abundant cold water to obtain the desired com-
pound, which was recrystallized from ethanol.
5-Chloro-1H-pyrrolo[2,3-b]pyridine-3-carbonitrile (6b). Yield = 90%;
mp = 291–294 ꢀC (EtOH). 1H-NMR (DMSO-d6) δ 8.27 (d, 1H, Ar,
J = 2.0 Hz), 8.40 (d, 1H, Ar, J = 2.4 Hz), 8.51 (d, 1H, Ar, J = 3.2 Hz), 13.05
(exch br s, 1H, NH). IR ѵ (cm−1): 2240 (CN). ESI-MS calcd. For C8H4ClN3,
177.59; found: m/z 179.01 [M+H]+. Anal. C8H4ClN3 (C, H, N).
5-Nitro-1H-pyrrolo[2,3-b]pyridine-3-carbonitrile (6c). Yield = 81%;
mp = 248–250 ꢀC (EtOH). 1H-NMR (DMSO-d6) δ 8.71 (s, 1H, Ar), 8.91
(s, 1H, Ar), 9.21 (s, 1H, Ar), 13.56 (exch br s, 1H, NH). IR ѵ (cm−1): 2220
(CN). ESI-MS calcd. For C8H4N4O2, 188.14; found: m/z 189.04 [M+H]+.
Anal. C8H4N4O2 (C, H, N).
5-Phenyl-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (9a).
Yield = 79%; mp = 123–125 ꢀC (EtOH). 1H-NMR (CDCl3-d1) δ 6.63
(d, 1H, Ar, J = 4.0 Hz), 7.36 (t, 1H, Ar, J = 7.2 Hz), 7.42–7.49 (m, 4H, Ar),
7.52–7.57 (m, 3H, Ar), 7.75 (d, 1H, Ar, J = 4.0 Hz), 7.98 (d, 1H, Ar,
J = 2.0 Hz), 8.22 (d, 2H, Ar, J = 7.2 Hz), 8.65 (d, 1H, Ar, J = 2.0 Hz).
ESI-MS calcd. For C19H14N2O2S, 334.39; found: m/z 335.08 [M+H]+.
Anal. C19H14N2O2S (C, H, N).
General procedure for compounds 7a-c. To a suspension of the
substrate 6a-c (6a, Graczyk et al., 2004) (0.53 mmol) in 10 mL of anhy-
drous THF, 1.06 mmol of sodium hydride and 0.64 mmol of m-toluoyl
chloride were added. The mixture was stirred at room temperature
overnight. The solvent was concentrated in vacuo to obtain a residue
that was purified by column chromatography using cyclohexane/ethyl
acetate 6:1 (for 7a,b) or 5:1 (for 7c) as eluents.
1-(Phenylsulfonyl)-5-(3-[trifluoromethyl]phenyl)-1H-pyrrolo[2,3-b]
pyridine (9b). Yield = 79%; mp = 145–148 ꢀC (EtOH). 1H-NMR
(CDCl3-d1) δ 6.66 (d, 1H, Ar, J = 4.0 Hz), 7.50 (t, 2H, Ar, J = 8.0 Hz),
7.56–7.61 (m, 2H, Ar), 7.63 (d, 1H, Ar, J = 7.6 Hz), 7.72 (d, 1H, Ar,
J = 7.6 Hz), 7.78 (d, 2H, Ar, J = 4.0 Hz), 8.02 (d, 1H, Ar, J = 2.0 Hz), 8.22
(d, 2H, Ar, J = 7.2 Hz), 8.63 (d, 1H, Ar, J = 2.0 Hz). ESI-MS calcd. For
C
C
20H13F3N2O2S, 402.39; found: m/z 403.07 [M+H]+. Anal.
5-Bromo-1-(3-methylbenzoyl)-1H-pyrrolo[2,3-b]pyridine-3-carbo-
nitrile (7a). Yield = 30%; mp = 156–159 ꢀC (EtOH). 1H-NMR (CDCl3-
d1) δ 2.44 (s, 3H, m-CH3-Ph), 7.41 (t, 1H, Ar, J = 7.6 Hz), 7.50–7.55 (m,
2H, Ar), 7.62 (s, 1H, Ar), 8.20–8.25 (m, 2H, Ar), 8.46 (s, 1H, Ar).
13C-NMR (CDCl3-d1) δ 21.52 (CH3), 90.31 (C), 127.78 (CH), 128.67
(CH), 131.11 (CH), 131.77 (C), 135.21 (CH), 136.23 (CH), 138.87
(C), 145.41 (C), 166.46 (CO). IR ѵ (cm−1): 2224 (CN). ESI-MS
calcd. For C16H10BrN3O, 340.17; found: m/z 341.00 [M+H]+.
Anal. C16H10BrN3O (C, H, N).
20H13F3N2O2S (C, H, N).
1-(Phenylsulfonyl)-5-(m-tolyl)-1H-pyrrolo[2,3-b]pyridine (9c).
Yield = 78%; mp = 140–143 ꢀC (EtOH). 1H-NMR (CDCl3-d1) δ 2.41 (s,
3H, m-CH3-Ph), 6.63 (d, 1H, Ar, J = 4.0 Hz), 7.19 (s, 1H, Ar), 7.30–7.35
(m, 3H, Ar), 7.48 (t, 1H, Ar, J = 8.0 Hz), 7.56 (t, 2H, Ar, J = 7.6 Hz), 7.75
(d, 1H, Ar, J = 4.0 Hz), 7.98 (d, 1H, Ar, J = 2.0 Hz), 8.22 (d, 2H, Ar,
J = 7.6 Hz), 8.64 (d, 1H, Ar, J = 2.0 Hz). ESI-MS calcd. For
C
20H16N2O2S, 348.42; found: m/z 349.10 [M+H]+. Anal. C20H16N2O2S
(C, H, N).
5-(3-Methoxyphenyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine
5-Chloro-1-(3-methylbenzoyl)-1H-pyrrolo[2,3-b]pyridine-3-carbo-
nitrile (7b). Yield = 10%; mp = 150–152 ꢀC (EtOH). 1H-NMR (CDCl3-
d1) δ 2.44 (s, 3H, m-CH3-Ph), 7.41 (t, 1H, Ar, J = 7.2 Hz), 7.50–7.55 (m,
2H, Ar), 7.62 (s, 1H, Ar), 8.10 (s, 1H, Ar), 8.24 (s, 1H, Ar), 8.38 (s, 1H,
Ar). 13C-NMR (CDCl3-d1) δ 21.32 (CH3), 112.77 (C), 127.79 (CH),
127.96 (CH), 128.48 (CH), 128.71 (C), 130.99 (CH), 131.01 (C), 135.06
(CH), 136.04 (CH), 138.82 (C), 145.11 (C), 145.63 (CH), 166.33 (CO).
IR ѵ (cm−1): 2223 (CN). ESI-MS calcd. For C16H10ClN3O, 295.72;
found: m/z 297.05 [M+H]+. Anal. C16H10ClN3O (C, H, N).
(9d). Yield = 78%; mp = 106–109 ꢀC (EtOH). 1H-NMR (CDCl3-d1) δ
3.85 (s, 3H, OCH3), 6.64 (d, 1H, Ar, J = 4.0 Hz), 6.92 (dd, 1H, Ar,
J = 2.0 Hz and J = 8.4 Hz), 7.07 (s, 1H, Ar), 7.12 (d, 1H, Ar, J = 7.6 Hz),
7.37 (t, 1H, Ar, J = 7.6 Hz), 7.50 (t, 2H, Ar, J = 8.0 Hz), 7.58 (t, 1H, Ar,
J = 7.6 Hz), 7.75 (d, 1H, Ar, J = 4.0 Hz), 8.00 (d, 1H, Ar, J = 2.0 Hz), 8.22
(d, 2H, Ar, J = 7.6 Hz), 8.65 (d, 1H, Ar, J = 2.0 Hz). ESI-MS calcd. For
C
20H16N2O3S, 364.42; found: m/z 365.09 [M+H]+. Anal. C20H16N2O3S
(C, H, N).
5-(6-Methoxynaphthalen-2-yl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]
1-(3-Methylbenzoyl)-5-nitro-1H-pyrrolo[2,3-b]pyridine-3-carbo-
nitrile (7c). Yield = 35%; mp = 152–154 ꢀC (EtOH). 1H-NMR (CDCl3-
d1) δ 2.45 (s, 3H, m-CH3-Ph), 7.43 (t, 1H, Ar, J = 7.6 Hz), 7.55 (d, 2H,
Ar, J = 7.6 Hz), 7.64 (s, 1H, Ar), 8.43 (s, 1H, Ar), 8.96 (d, 1H, Ar,
J = 2.4 Hz), 9.27 (d, 1H, Ar, J = 2.4 Hz). 13C-NMR (CDCl3-d1) δ 20.93
(CH3), 101.34 (C), 115.51 (C), 122.13 (C), 123.68 (CH), 128.16 (CH),
129.19 (CH), 130.42 (C), 130.57 (CH), 131.62 (C), 134.11 (CH),
137.65 (CH), 138.94 (C), 152.78 (C), 167.74 (CO). IR ѵ (cm−1): 2220
(CN). ESI-MS calcd. For C16H10N4O3, 306.28; found: m/z 307.08 [M
+H]+. Anal. C16H10N4O3 (C, H, N).
pyridine (9e). Yield = 91%; mp = 173–176 ꢀC (EtOH) dec. 1H-NMR
(CDCl3-d1) δ 3.93 (s, 3H, OCH3), 6.65 (d, 1H, Ar, J = 3.6 Hz), 7.15–7.21
(m, 2H, Ar), 7.49 (t, 2H, Ar, J = 8.0 Hz), 7.56 (d, 1H, Ar, J = 7.2 Hz), 7.64
(d, 1H, Ar, J = 8.0 Hz), 7.75–7.83 (m, 3H, Ar), 7.92 (s, 1H, Ar), 8.08 (s, 1H,
Ar), 8.24 (d, 2H, Ar, J = 7.6 Hz), 8.75 (s, 1H, Ar). ESI-MS calcd. For
C
24H18N2O3S, 414.48; found: m/z 415.11 [M+H]+. Anal. C24H18N2O3S
(C, H, N).
1-(Phenylsulfonyl)-5-(thiophen-3-yl)-1H-pyrrolo[2,3-b]pyridine (9f).
Yield = 55%; mp = 186–189 ꢀC (EtOH). 1H-NMR (CDCl3-d1)
δ

6
GIOVANNONI ET AL.
6.59–6.63 (m, 1H, Ar), 7.22–7.27 (m, 1H, Ar), 7.35–7.50 (m, 4H, Ar),
7.53–7.58 (m, 1H, Ar), 7.70–7.75 (m, 1H, Ar), 8.00 (s, 1H, Ar), 8.18–8.23
(m, 2H, Ar), 8.68 (s, 1H, Ar). ESI-MS calcd. For C17H12N2O2S2, 340.43;
found: m/z 341.04 [M+H]+. Anal. C17H12N2O2S2 (C, H, N).
8.52 (s, 1H, Ar), 8.63 (d, 1H, Ar, J = 2.0 Hz), 8.71 (d, 1H, Ar, J = 2.0 Hz),
9.45 (s, 1H, CHO), 12.65 (exch br s, 1H, NH). ESI-MS calcd. For
C15H9F3N2O, 290.24; found: m/z 291.07 [M+H]+. Anal. C15H9F3N2O
(C, H, N).
1-(Phenylsulfonyl)-5-(pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridine (9g).
Yield = 98%; mp = 148–152 ꢀC (EtOH). 1H-NMR (CDCl3-d1) δ 6.68 (d,
1H, Ar, J = 3.6 Hz), 7.49–7.62 (m, 4H, Ar), 7.82 (s, 1H, Ar), 8.03 (s, 2H,
Ar), 8.23 (d, 2H, Ar, J = 7.6 Hz), 8.62–8.67 (m, 2H, Ar), 8.85 (s, 1H, Ar).
ESI-MS calcd. For C18H13N3O2S, 335.38; found: m/z 336.08 [M+H]+.
Anal. C18H13N3O2S (C, H, N).
5-(m-Tolyl)-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde (11c).
Yield = 62%; mp = 162–165 ꢀC (EtOH). 1H-NMR (DMSO-d6) δ 2.38 (s,
3H, CH3), 7.19 (d, 1H, Ar, J = 7.6 Hz), 7.36 (t, 1H, Ar, J = 7.2 Hz), 7.48
(d, 1H, Ar, J = 7.6 Hz), 7.52 (s, 1H, Ar), 8.48 (s, 1H, Ar), 8.55 (d, 1H, Ar,
J = 2.0 Hz), 8.62 (d, 1H, Ar, J = 2.0 Hz), 9.93 (s, 1H, CHO), 12.75 (exch
br s, 1H, NH). ESI-MS calcd. For C15H12N2O, 236.27; found: m/z
237.10 [M+H]+. Anal. C15H12N2O (C, H, N).
General procedure for compounds 10b and 10e. To a solution of
intermediate 9b or 9e (0.29 mmol) in anhydrous THF (3 mL), 0.87 mmol
of TBAF was added, and the mixture was stirred at reflux for 4 hr. After
cooling, the solvent was evaporated, and cold-ice water (20 mL) was
added. The precipitate was recovered by vacuum filtration and purified
by crystallization from ethanol to obtain the desired compound 10b or
by column chromatography using cyclohexane/ethyl acetate 1:1 as elu-
ent for 10e.
5-(3-Methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde
(11d). Yield = 37%; mp = 200–203 ꢀC (EtOH). 1H-NMR (DMSO-d6) δ
3.90 (s, 3H, OCH3), 6.97 (d, 1H, Ar, J = 8.4 Hz), 7.17 (s, 1H, Ar), 7.23
(s, 1H, Ar), 7.42 (t, 1H, Ar, J = 8.0 Hz), 8.07 (s, 1H, Ar), 8.64 (s, 1H, Ar),
8.92 (s, 1H, Ar), 10.08 (s, 1H, CHO), 12.70 (exch br s, 1H, NH). ESI-
MS calcd. For C15H12N2O2, 252.27; found: m/z 253.09 [M+H]+. Anal.
C15H12N2O2 (C, H, N).
5-(3-[Trifluoromethyl]phenyl)-1H-pyrrolo[2,3-b]pyridine
(10b).
5-(6-Methoxynaphthalen-2-yl)-1H-pyrrolo[2,3-b]pyridine-3-carbal-
dehyde (11e). Yield = 84%; mp = 145–147 ꢀC (hexane). 1H-NMR
(DMSO-d6) δ 3.88 (s, 3H, OCH3), 7.20 (d, 1H, Ar, J = 8.8 Hz), 7.36 (s,
1H, Ar), 7.84–7.95 (m, 3H, Ar), 8.21 (s, 1H, Ar), 8.52 (s, 1H, Ar), 8.69 (s,
1H, Ar), 8.78 (s, 1H, Ar), 9.97 (s, 1H, CHO), 12.57 (exch br s, 1H, NH).
ESI-MS calcd. For C19H14N2O2, 302.33; found: m/z 303.11 [M+H]+.
Anal. C19H14N2O2 (C, H, N).
Yield = 97%; mp = 158–161 ꢀC (EtOH). 1H-NMR (CDCl3-d1) δ 6.65 (d,
1H, Ar, J = 3.2 Hz), 7.47 (d, 1H, Ar, J = 3.2 Hz), 7.59–7.67 (m, 2H, Ar),
7.80 (d, 1H, Ar, J = 7.2 Hz), 7.86 (s, 1H, Ar), 8.27 (d, 1H, Ar, J = 1.2 Hz),
8.54 (s, 1H, Ar), 10.12 (exch br s, 1H, NH). ESI-MS calcd. For
14H9F3N2, 262.23; found: m/z 263.08 [M+H]+. Anal. C14H9F3N2
(C, H, N).
5-(6-Methoxynaphthalen-2-yl)-1H-pyrrolo[2,3-b]pyridine (10e).
C
5-(Thiophen-3-yl)-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde
(11f). Yield = 37%; mp = 194–196 ꢀC (EtOH). 1H-NMR (DMSO-d6) δ
7.60–7.66 (m, 2H, Ar), 7.95 (s, 1H, Ar), 8.46 (s, 1H, Ar), 8.60 (s, 1H, Ar),
8.74 (s, 1H, Ar), 9.93 (s, 1H, CHO), 12.71 (exch br s, 1H, NH). ESI-MS
calcd. For C12H8N2OS, 228.27; found: m/z 229.04 [M+H]+. Anal.
C12H8N2OS (C, H, N).
Yield = 45%; mp = 247–250 ꢀC dec (EtOH). 1H-NMR (CDCl3-d1) δ
3.87 (s, 3H, OCH3), 6.50 (d, 1H, Ar, J = 1.6 Hz), 7.18 (dd, 1H, Ar,
J = 2.4 Hz and J = 8.4 Hz), 7.33 (s, 1H, Ar), 7.50 (d, 1H, Ar, J = 2.4 Hz),
7.82–7.90 (m, 3H, Ar), 8.16 (d, 1H, Ar, J = 4.0 Hz), 8.29 (d, 1H, Ar,
J = 3.6 Hz), 8.62 (d, 1H, Ar, J = 3.2 Hz), 11.71 (exch br s, 1H, NH). ESI-
MS calcd. For C18H14N2O, 274.32; found: m/z 275.11 [M+H]+. Anal.
General procedure for compounds 12a-g. Compounds 12a-g
were obtained following the same procedure reported for compounds
5a,c, but starting with intermediates 11a-g (11g, Ibrahim et al., 2007).
The crude products were purified by crystallization from ethanol
(12c,d,g) or by column chromatography using cyclohexane/ethyl ace-
tate 1:5 (for 12a,b), cyclohexane/ethyl acetate 1:2 (for 12e), or cyclo-
hexane/ethyl acetate 1:3 (for 12f) as eluents.
C
18H14N2O (C, H, N).
General procedure for compounds 11a-f. A mixture of the appro-
priate 1H-pyrrolo[2,3-b]pyridine derivative 10a-f (0.95 mmol) (10a,
Laha et al., 2017; 10c and 10d, Ibrahim et al., 2007; 10f, Singh et al.,
2017) and HMTA (1.43 mmol) in glacial acetic acid (3 mL) was heated
under reflux for 6 hr. The reaction mixture was cooled at room tem-
perature and diluted with cold water. The solid obtained was filtered,
washed with an excess of ice-cold water, and dried. The crude com-
pound was purified by crystallization from hexane (11b,e) or by col-
umn chromatography using cyclohexane/ethyl acetate 1:3 (for 11a,d),
dichloromethane/methanol 95:5 (for 11c), or cyclohexane/ethyl ace-
tate 1:5 (for 11f) as eluents.
5-Phenyl-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde oxime (12a).
Yield = 42%; mp = 224–226 ꢀC (EtOH). 1H-NMR (CDCl3-d1) δ 7.40 (d,
1H, Ar, J = 7.6 Hz), 7.46–7.52 (m, 3H, 2H Ar + 1H CH N-OH), 7.63
(d, 2H, Ar, J = 7.6 Hz), 8.33 (s, 1H, Ar), 8.59 (s, 1H, Ar), 8.69 (s, 1H, Ar),
10.31 (exch br s, 1H, CH N-OH), 11.95 (exch br s, 1H, NH). ESI-MS
calcd. For C14H11N3O, 237.26; found: m/z 238.09 [M+H]+. Anal.
C14H11N3O (C, H, N).
5-Phenyl-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde
(11a).
Yield = 56%; mp = 212–215 ꢀC (EtOH). 1H-NMR (CDCl3-d1) δ 7.42 (t,
1H, Ar, J = 7.2 Hz), 7.51 (t, 2H, Ar, J = 7.6 Hz), 7.67 (d, 2H, Ar,
J = 7.2 Hz), 8.10 (s, 1H, Ar), 8.68 (s, 1H, Ar), 8.89 (s, 1H, Ar), 10.08 (s,
1H, CHO), 11.82 (exch br s, 1H, NH). ESI-MS calcd. For C14H10N2O,
222.24; found: m/z 223.08 [M+H]+. Anal. C14H10N2O (C, H, N).
5-(3-[Trifluoromethyl]phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbal-
dehyde (11b). Yield = 80%; mp = 193–196 ꢀC (hexane). 1H-NMR
(DMSO-d6) δ 7.23 (d, 2H, Ar, J = 7.2 Hz), 8.04 (d, 2H, Ar, J = 8.4 Hz),
5-(3-[Trifluoromethyl]phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbal-
dehyde oxime (12b). Yield = 52%; mp = 247–250 ꢀC (EtOH). 1H-NMR
(DMSO-d6) δ 7.72 (d, 3H, Ar, J = 4.0 Hz), 7.82 (s, 1H, CH N-OH), 7.96
(s, 1H, Ar), 8.28 (s, 1H, Ar), 8.51 (d, 1H, Ar, J = 2.0 Hz), 8.64 (d, 1H, Ar,
J = 1.6 Hz), 10.74 (exch br s, 1H, CH N-OH), 12.10 (exch br s, 1H,
NH). ESI-MS calcd. For C15H10F3N3O, 305.25; found: m/z 306.08 [M
+H]+. Anal. C15H10F3N3O (C, H, N).

GIOVANNONI ET AL.
7
5-(m-Tolyl)-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde oxime (12c).
Yield = 51%; mp = 208–211 ꢀC (EtOH). 1H-NMR (DMSO-d6) δ 2.37 (s,
3H, CH3), 7.16 (s, 1H, CH N-OH), 7.35 (t, 1H, Ar, J = 7.6 Hz), 7.44 (t,
1H, Ar, J = 7.6 Hz), 7.50–7.56 (m, 1H, Ar), 7.78 (d, 1H, Ar, J = 2.0 Hz),
8.25–8.30 (m, 1H, Ar), 8.46 (d, 1H, Ar, J = 2.0 Hz), 8.54–8.59 (m, 1H, Ar),
10.71 (exch br s, 1H, CH N-OH), 12.00 (exch br s, 1H, NH). ESI-MS
calcd. For C15H13N3O, 251.28; found: m/z 252.11 [M+H]+. Anal.
5-(m-Tolyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonitrile (13c). Yield =
74%; mp = 194–197 ꢀC (EtOH). 1H-NMR (DMSO-d6) δ 2.38 (s, 3H,
CH3), 7.19 (d, 1H, Ar, J = 7.6 Hz), 7.36 (t, 1H, Ar, J = 7.6 Hz), 7.55 (d, 1H,
Ar, J = 7.6 Hz), 7.61 (s, 1H, Ar), 8.29 (d, 1H, Ar, J = 1.6 Hz), 8.46 (d, 1H,
Ar, J = 2.4 Hz), 8.67 (d, 1H, Ar, J = 2.0 Hz), 12.87 (exch br s, 1H, NH). IR
ѵ (cm−1): 2220 (CN). ESI-MS calcd. For C15H11N3, 233.27; found: m/z
234.10 [M+H]+. Anal. C15H11N3 (C, H, N).
C
15H13N3O (C, H, N).
5-(3-Methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonitrile (13d).
Yield = 94%; mp = 201–204 ꢀC (EtOH). 1H-NMR (CDCl3-d1) δ 3.83
(s, 3H, OCH3), 6.94 (d, 1H, Ar, J = 7.6 Hz), 7.30–7.35 (m, 2H, Ar), 7.38
(t, 1H, Ar, J = 7.2 Hz), 8.33 (d, 1H, Ar, J = 1.6 Hz), 8.47 (s, 1H, Ar), 8.69
(d, 1H, Ar, J = 1.6 Hz), 12.91 (exch br s, 1H, NH). IR ѵ (cm−1): 2220
(CN). ESI-MS calcd. For C15H11N3O, 249.27; found: m/z 250.09 [M
+H]+. Anal. C15H11N3O (C, H, N).
5-(3-Methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde
oxime (12d). Yield = 84%; mp = 191–194 ꢀC (EtOH). 1H-NMR
(DMSO-d6) δ 3.81 (s, 3H, OCH3), 6.91–6.96 (m, 1H, Ar), 7.17–7.22 (m,
2H, Ar), 7.35–7.40 (m, 2H, 1H Ar + 1H CH N-OH), 7.78 (s, 1H, Ar),
8.26 (s, 1H, Ar), 8.64 (s, 1H, Ar), 10.72 (exch br s, 1H, CH N-OH),
12.03 (exch br s, 1H, NH). ESI-MS calcd. For C15H13N3O2, 267.28;
found: m/z 268.10 [M+H]+. Anal. C15H13N3O2 (C, H, N).
5-(6-Methoxynaphthalen-2-yl)-1H-pyrrolo[2,3-b]pyridine-3-
carbonitrile (13e). Yield = 63%; mp = 193–196 ꢀC (EtOH). 1H-NMR
(DMSO-d6) δ 3.88 (s, 3H, OCH3), 7.14–7.19 (m, 1H, Ar), 7.35 (s, 1H,
Ar), 7.91–7.99 (m, 3H, Ar), 8.28 (s, 1H, Ar), 8.43–8.50 (m, 2H, Ar),
8.82 (s, 1H, Ar), 12.89 (exch br s, 1H, NH). IR ѵ (cm−1): 2221 (CN).
ESI-MS calcd. For C19H13N3O, 299.33; found: m/z 300.11 [M+H]+.
Anal. C19H13N3O (C, H, N).
5-(6-Methoxynaphthalen-2-yl)-1H-pyrrolo[2,3-b]pyridine-3-carbal-
dehyde oxime (12e). Yield = 37%; mp = 204–207 ꢀC dec (EtOH).
1H-NMR (DMSO-d6) δ 3.88 (s, 3H, OCH3), 7.19 (d, 1H, Ar, J = 8.4 Hz),
7.35 (s, 1H, Ar), 7.74–7.79 (m, 1H, Ar), 7.89–7.94 (m, 3H, Ar), 8.12 (s,
1H, CH N-OH), 8.29 (d, 1H, Ar, J = 6.8 Hz), 8.57 (s, 1H, Ar), 8.66–8.71
(m, 1H, Ar), 10.73 (exch br s, 1H, CH N-OH), 12.01 (exch br s, 1H,
NH). ESI-MS calcd. For C19H15N3O2, 317.34; found: m/z 318.12 [M
+H]+. Anal. C19H15N3O2 (C, H, N).
5-(Thiophen-3-yl)-1H-pyrrolo[2,3-b]pyridine-3-carbonitrile (13f).
Yield = 88%; mp = 165–167 ꢀC (EtOH). 1H-NMR (DMSO-d6)
δ
5-(Thiophen-3-yl)-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde oxime
(12f). Yield = 54%; mp = 239–242 ꢀC (EtOH). 1H-NMR (DMSO-d6) δ 7.50
(d, 1H, Ar, J = 4.8 Hz), 7.66 (s, 1H, Ar), 7.77 (d, 1H, Ar, J = 7.6 Hz), 8.25 (s,
1H, CH N-OH), 8.45 (s, 1H, Ar), 8.62–8.67 (m, 2H, Ar), 10.71 (exch br s,
1H, CH N-OH), 11.99 (exch br s, 1H, NH). ESI-MS calcd. For C12H9N3OS,
243.28; found: m/z 244.05 [M+H]+. Anal. C12H9N3OS (C, H, N).
7.67–7.73 (m, 2H, Ar), 8.05 (s, 1H, Ar), 8.39–8.44 (m, 2H, Ar), 8.80 (s,
1H, Ar), 12.86 (exch br s, 1H, NH). IR ѵ (cm−1): 2222 (CN). ESI-MS
calcd. For C12H7N3S, 225.27; found: m/z 226.04 [M+H]+. Anal.
C12H7N3S (C, H, N).
5-(Pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridine-3-carbonitrile
(13g).
Yield = 71%; mp = 285–287 ꢀC dec (EtOH). (DMSO-d6) δ 7.44–7.49
(m, 1H, Ar), 8.18 (d, 1H, Ar, J = 6.0 Hz), 8.40 (s, 1H, Ar), 8.47 (s, 1H,
Ar), 8.57 (s, 1H, Ar), 8.73 (s, 1H, Ar), 8.97 (s, 1H, Ar), 12.96 (exch br s,
1H, NH). IR ѵ (cm−1): 2220 (CN). ESI-MS calcd. For C13H8N4, 220.23;
found: m/z 221.08 [M+H]+. Anal. C13H8N4 (C, H, N).
5-(Pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde oxime
(12g). Yield = 67%; mp = 241–243 ꢀC (EtOH). 1H-NMR (DMSO-d6) δ
7.50 (d, 1H, Ar, J = 4.8 Hz), 7.79 (s, 1H, Ar), 8.07 (d, 1H, Ar, J = 6.4 Hz),
8.27 (s, 1H, CH N-OH), 8.49–8.65 (m, 3H, Ar), 8.81 (s, 1H, Ar), 10.74
(exch br s, 1H, CH N-OH), 12.10 (exch br s, 1H, NH). ESI-MS calcd.
General procedure for compounds 14a-g. Compounds 14a-g
were obtained following the same procedure reported for compounds
7a-c, but starting with intermediates 13a-g. The crude products were
purified by column chromatography using cyclohexane/ethyl acetate
5:1 (for 14a-e), cyclohexane/ethyl acetate 4:1 (for 14f), or cyclo-
hexane/ethyl acetate 1:3 (for 14g) as eluents.
For
C
13H10N4O, 238.24; found: m/z 239.09 [M+H]+. Anal.
C
13H10N4O (C, H, N).
General procedure for compounds 13a-g. Compounds 13a-g
were obtained following the same procedure reported for compounds
6b,c, but starting with intermediates 12a-g. The crude products were
purified by crystallization from ethanol.
1-(3-Methylbenzoyl)-5-phenyl-1H-pyrrolo[2,3-b]pyridine-3-carbo-
nitrile (14a). Yield = 17%; mp = 160–163 ꢀC (EtOH). 1H-NMR (CDCl3-
d1) δ 2.45 (s, 3H, m-CH3-Ph), 7.40–7.45 (m, 2H, Ar), 7.51 (t, 3H, Ar,
J = 7.2 Hz), 7.61 (t, 3H, Ar, J = 7.6 Hz), 7.68 (s, 1H, Ar), 8.24 (s, 1H, Ar),
8.26 (d, 1H, Ar, J = 2.0 Hz), 8.68 (d, 1H, Ar, J = 2.0 Hz). 13C-NMR
(CDCl3-d1) δ 20.93 (CH3), 101.18 (C), 115.32 (C), 123.82 (CH), 127.54
(CH), 128.17 (CH), 130.86 (CH), 134.56 (CH), 136.41 (C), 138.83 (C),
144.65 (C), 167.76 (CO). IR ѵ (cm−1): 2220 (CN). ESI-MS calcd. For
5-Phenyl-1H-pyrrolo[2,3-b]pyridine-3-carbonitrile (13a). Yield = 40%;
mp = 199–202 ꢀC (EtOH). 1H-NMR (CDCl3-d1) δ 7.47 (d, 1H, Ar,
J = 7.2 Hz), 7.53 (t, 2H, Ar, J = 7.2 Hz), 7.63 (d, 2H, Ar, J = 7.6 Hz), 7.97 (s,
1H, Ar), 8.48 (s, 1H, Ar), 8.71 (s, 1H, Ar), 12.65 (exch br s, 1H, NH). IR ѵ
(cm−1): 2220 (CN). ESI-MS calcd. For C14H9N3, 219.24; found: m/z 220.08
[M+H]+. Anal. C14H9N3 (C, H, N).
5-(3-[Trifluoromethyl]phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbo-
nitrile (13b). Yield = 85%; mp = 240–242 ꢀC (EtOH). 1H-NMR (CDCl3-
d1) δ 7.70 (t, 1H, Ar, J = 7.6 Hz), 7.76 (d, 1H, Ar, J = 7.6 Hz), 7.82 (d,
1H, Ar, J = 7.6 Hz), 7.88 (s, 1H, Ar), 8.09 (s, 1H, Ar), 8.62 (s, 1H, Ar),
8.71 (s, 1H, Ar), 12.75 (exch br s, 1H, NH). IR ѵ (cm−1): 2222 (CN).
ESI-MS calcd. For C15H8F3N3, 287.24; found: m/z 288.07 [M+H]+.
Anal. C15H8F3N3 (C, H, N).
C
22H15N3O, 337.37; found: m/z 338.12 [M+H]+. Anal. C22H15N3O
(C, H, N).
1-(3-Methylbenzoyl)-5-(3-[trifluoromethyl]phenyl)-1H-pyrrolo[2,3-
b]pyridine-3-carbonitrile (14b). Yield = 10%; mp = 155–157 ꢀC (EtOH).
1H-NMR (CDCl3-d1) δ 2.46 (s, 3H, m-CH3-Ph), 7.43 (t, 1H, Ar, J = 7.6 Hz),
7.52 (d, 1H, Ar, J = 8.0 Hz), 7.58–7.71 (m, 4H, Ar), 7.80 (d, 1H, Ar,

8
GIOVANNONI ET AL.
J = 7.6 Hz), 7.85 (s, 1H, Ar), 8.28 (s, 2H, Ar), 8.67 (d, 1H, Ar, J = 2.0 Hz).
13C-NMR (CDCl3-d1) δ 21.31 (CH3), 103.41 (C), 115.62 (C), 121.42
(CH), 124.75 (CH), 125.18 (CH), 126.74 (CH), 128.96 (CH), 129.56
(CH), 130.48 (CH), 130.92 (CH), 136.72 (C), 138.92 (C), 144.67 (C),
167.48 (CO). IR ѵ (cm−1): 2220 (CN). ESI-MS calcd. For C23H14F3N3O,
405.37; found: m/z 406.11 [M+H]+. Anal. C23H14F3N3O (C, H, N).
1-(3-Methylbenzoyl)-5-(m-tolyl)-1H-pyrrolo[2,3-b]pyridine-3-carbo-
nitrile (14c). Yield = 10%; mp = 115–118 ꢀC (EtOH). 1H-NMR (CDCl3-
d1) δ 2.45 (s, 6H, 2 × m-CH3-Ph), 7.24 (s, 1H, Ar), 7.38–7.43 (m, 4H, Ar),
7.51 (d, 1H, Ar, J = 7.2 Hz), 7.59 (d, 1H, Ar, J = 7.2 Hz), 7.67 (s, 1H, Ar),
8.23 (s, 1H, Ar), 8.25 (d, 1H, Ar, J = 2.0 Hz), 8.67 (d, 1H, Ar, J = 2.0 Hz).
13C-NMR (CDCl3-d1) δ 21.39 (CH3), 21.56 (CH3), 90.72 (C), 113.51 (C),
121.17 (C), 124.55 (CH), 126.66 (CH), 127.26 (CH), 127.83 (CH), 128.22
(CH), 128.45 (CH), 129.06 (CH), 129.18 (CH), 131.03 (CH), 132.07 (C),
134.31 (CH), 134.88 (CH), 137.30 (C), 138.75 (C), 139.03 (C), 146.04
(CH), 146.35 (C), 166.72 (CO). IR ѵ (cm−1): 2220 (CN). ESI-MS calcd. For
C23H17N3O, 351.40; found: m/z 352.14 [M+H]+. Anal. C23H17N3O
(C, H, N).
ѵ (cm−1): 2222 (CN). ESI-MS calcd. For C20H13N3OS, 343.40; found:
m/z 344.08 [M+H]+. Anal. C20H13N3OS (C, H, N).
1-(3-Methylbenzoyl)-5-(pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridine-3-
carbonitrile (14g). Yield = 32%; mp = 155–157 ꢀC (EtOH). 1H-NMR
(CDCl3-d1) δ 2.44 (s, 3H, m-CH3-Ph), 7.40–7.67 (m, 5H, Ar), 7.93 (d,
1H, Ar, J = 6.0 Hz), 8.27 (s, 2H, Ar), 8.67 (d, 2H, Ar, J = 7.6 Hz), 8.88 (s,
1H, Ar). 13C-NMR (CDCl3-d1) δ 21.32 (CH3), 90.71 (C), 113.22 (C),
121.28 (C), 123.57 (CH), 126.84 (CH), 126.98 (CH), 127.85 (CH),
128.47 (CH), 130.83 (C), 131.02 (CH), 131.92 (C), 133.19 (C), 134.83
(CH), 135.02 (CH), 138.81 (C), 145.54 (CH), 145.77 (CH), 146.76 (C),
149.27 (CH), 166.56 (CO). IR ѵ (cm−1): 2220 (CN). ESI-MS calcd. For
C
21H14N4O, 338.36; found: m/z 339.12 [M+H]+. Anal. C21H14N4O
(C, H, N).
2.3 | Pharmacology
Compounds were dissolved in 100% DMSO at 5 mM stock concentra-
tions. The final concentration of DMSO in the reactions was 1%, and this
level of DMSO had no effect on HNE activity. The HNE inhibition assay
was performed in black flat-bottom 96-well microtiter plates. Briefly, a
buffer solution containing 200 mM Tris–HCl, pH 7.5, 0.01% bovine
serum albumin, 0.05% Tween-20, and 20 mU/mL HNE (Calbiochem)
was added to wells containing different concentrations of each com-
pound. The reactions were initiated by addition of 25 μM elastase
substrate (N-methylsuccinyl-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin,
Calbiochem) in a final reaction volume of 100 μL/well. Kinetic measure-
ments were obtained every 30 s for 10 min at 25 ꢀC using a Fluoroskan
Ascent FL fluorescence microplate reader (Thermo Electron, MA) with
excitation and emission wavelengths at 355 and 460 nm, respectively.
For all compounds tested, the concentration of inhibitor that caused
50% inhibition of the enzymatic reaction (IC50) was calculated by
5-(3-Methoxyphenyl)-1-(3-methylbenzoyl)-1H-pyrrolo[2,3-b]pyr-
idine-3-carbonitrile (14d). Yield = 10%; mp = 152–155 ꢀC (EtOH).
1H-NMR (CDCl3-d1) δ 2.45 (s, 3H, m-CH3-Ph), 3.88 (s, 3H, OCH3),
6.97 (d, 1H, Ar, J = 8.0 Hz), 7.13–7.20 (m, 2H, Ar), 7.41 (d, 2H, Ar,
J = 7.6 Hz), 7.51 (d, 1H, Ar, J = 7.2 Hz), 7.60 (d, 1H, Ar, J = 7.2 Hz), 7.67
(s, 1H, Ar), 8.25 (d, 2H, Ar, J = 7.6 Hz), 8.67 (s, 1H, Ar). 13C-NMR
(CDCl3-d1) δ 21.18 (CH3), 55.76 (CH3), 102.47 (C), 113.48 (CH), 114.39
(CH), 115.49 (C), 119.82 (CH), 121.43 (C), 123.64 (CH), 126.12 (CH),
128.17 (CH), 129.32 (CH), 130.14 (CH), 131.18 (C), 134.56 (CH),
138.97 (C), 144.65 (C), 161.18 (C), 168.71 (CO). IR ѵ (cm−1): 2220 (CN).
ESI-MS calcd. For C23H17N3O2, 367.40; found: m/z 368.14 [M+H]+.
Anal. C23H17N3O2 (C, H, N).
5-(6-Methoxynaphthalen-2-yl)-1-(3-methylbenzoyl)-1H-pyrrolo
[2,3-b]pyridine-3-carbonitrile (14e). Yield = 22%; oil. 1H-NMR
(CDCl3-d1) δ 2.46 (s, 3H, m-CH3-Ph), 3.95 (s, 3H, OCH3), 7.18–7.25
(m, 2H, Ar), 7.43 (t, 1H, Ar, J = 7.6 Hz), 7.51 (d, 1H, Ar, J = 7.2 Hz),
7.61 (d, 1H, Ar, J = 7.2 Hz), 7.68–7.73 (m, 2H, Ar), 7.81–7.88 (m, 2H,
Ar), 8.00 (s, 1H, Ar), 8.25 (s, 1H, Ar), 8.36 (s, 1H, Ar), 8.79 (s, 1H, Ar).
13C-NMR (CDCl3-d1) δ 21.19 (CH3), 55.85 (CH3), 101.06 (C), 105.92
(CH), 113.49 (CH), 114.39 (C), 115.41 (C), 119.87 (CH), 121.43 (C),
123.68 (CH), 125.44 (CH), 126.15 (CH), 128.17 (CH), 129.35 (CH),
129.60 (CH), 130.14 (CH), 131.11 (C), 134.56 (CH), 134.82 (C),
138.91 (C), 144.62 (C), 157.28 (C), 167.70 (CO). IR ѵ (cm−1): 2220
(CN). ESI-MS calcd. For C27H19N3O2, 417.46; found: m/z 418.15 [M
+H]+. Anal. C27H19N3O2 (C, H, N).
TABLE 1 HNE inhibitory activity of 1H-pyrrolo[2,3-b]pyridine
derivatives 2a-e
R3
R2
N
N
O
2a-e
Compound
R2
R3
H
IC50 (μM)a
N.A.b
2a
CH3
CH2CH3
COOEt
CN
2b
H
N.A.b
2c
H
N.A.b
1-(3-Methylbenzoyl)-5-(thiophen-3-yl)-1H-pyrrolo[2,3-b]pyridine-
3-carbonitrile (14f). Yield = 40%; mp = 171–173 ꢀC (EtOH). 1H-NMR
(CDCl3-d1) δ 2.45 (s, 3H, m-CH3-Ph), 7.42 (d, 2H, Ar, J = 7.2 Hz),
7.47–7.52 (m, 2H, Ar), 7.54–7.59 (m, 2H, Ar), 7.66 (s, 1H, Ar), 8.22
(s, 1H, Ar), 8.25 (d, 1H, Ar, J = 2.0 Hz), 8.71 (d, 1H, Ar, J = 2.0 Hz).
13C-NMR (CDCl3-d1) δ 21.35(CH3), 90.66 (C), 113.42 (C), 121.22 (C),
121.75 (CH), 125.68 (CH), 126.07 (CH), 127.36 (CH), 127.80 (CH),
128.41 (CH), 129.05 (C), 130.99 (CH), 132.08 (C), 132.08 (CH), 135.33
(CH), 138.25 (C), 138.72 (C), 145.36 (CH), 146.09 (C), 166.64 (CO). IR
2d
H
3.5 1.2
10.8 2.2
0.015 0.004
0.050 0.020
2e
CH3
H
CN
CN
Xc
Sivelestat
aIC50 values are presented as the mean SD of three independent
experiment with a 6-point dilution series for each experiment.
bNA: no inhibitory activity was found at the highest concentration of
compound tested (50 μM).
cCrocetti et al., 2018.

GIOVANNONI ET AL.
9
structure of HNE co-crystallized with a peptide chloromethyl ketone
inhibitor (1HNE entry of the Protein Data Bank) (Navia et al., 1989)
was used for the docking study. The search area was chosen as a
sphere of 10 Å radius centered at the nitrogen atom in the five-
membered ring of the co-crystallized inhibitor. This peptide and co-
crystallized water molecules were removed from the 1HNE structure,
and side chain flexibility was set for the 42 residues closest to the
center of the sphere (Crocetti et al., 2013), including the following res-
idues surrounding the HNE binding site (i.e., located in the vicinity of
the co-crystallized inhibitor): His57, Cys58, Leu99B, Val190, Cys191,
Phe192, Gly193, Asp194, Ser195, Ala213, Ser214, Phe215, and
Val216. For each compound, 15 docking runs were performed with
full flexibility of the ligand around all rotatable bonds and side chain
flexibility of the above-mentioned residues of the enzyme. Docking
poses with the lowest docking score of each compound were checked
for the ability to form a Michaelis complex between the Ser195
hydroxyl group and the carbonyl moiety in a ligand. In brief, the d1
values [distance O(Ser195)Á Á ÁC between the Ser195 hydroxyl oxygen
atom and the ligand carbonyl carbon atom closest to O(Ser195)] and α
[angle O(Ser195)Á Á ÁCO, where CO is the carbonyl group of a ligand
closest to O(Ser195)] were measured for each docked compound
(Peters & Merz, 2006). The ability of proton transfer from Ser195 to
Asp102 via His57 (the key catalytic triad of serine proteases) was also
estimated by determining distances d2 between the NH nitrogen in
His57 and carboxyl oxygen atoms in Asp102, as described previously
(Crocetti et al., 2013). The distance between the pyridine-type nitro-
gen in His57 and the OH proton in Ser195 is also important for pro-
ton transfer. However, because of easy rotation of the Ser195 OH
group about the C O bond, we measured distance d3 between the
Ser195 oxygen atom and the basic nitrogen atom in His57. The effec-
tive length L of the proton transfer channel was determined as
L = d3 + min (d2).
TABLE 2 HNE inhibitory activity of 1H-pyrrolo[2,3-b]pyridine
derivatives 7a-c and 14a-g
CN
R5
N
N
O
7a-c
14a-g
Compound
7a
R5
IC50 (μM)a
Br
0.051 0.014
0.16 0.05
0.40 0.11
0.050 0.02
0.18 0.07
0.13 0.04
0.070 0.015
1.6 0.33
7b
Cl
7c
NO2
Ph
14a
14b
m-CF3Ph
14c
m-CH3Ph
14d
m-OCH3Ph
14e
6-OCH3-naphthalene
14f
3-thienyl
0.042 0.015
0.015 0.004
0.015 0.004
0.050 0.020
14g
3-pyridyl
Xb
H
Sivelestat
–
aIC50 values are presented as the mean SD of three independent
experiments with a 6-point dilution series for each experiment.
bCrocetti et al., 2018.
plotting
% inhibition versus logarithm of inhibitor concentration
(at least six points). The data are presented as the mean values of at
least three independent experiments with relative standard deviations
of <15%.
2.4 | Molecular modeling
Initial 3D structures of the compounds were generated with
ChemOffice Professional (Perkin Elmer, Waltham, MA) and refined by
the semiempirical PM3 method using HyperChem 8.0 (Shimadzu Cor-
poration, Kyoto, Japan). Docking computations were performed using
Molegro Virtual Docker (MVD), version 6.0 (CLC Bio, Copenhagen,
Denmark), as described previously (Crocetti et al., 2013). The
3
| RESULTS AND DISCUSSION
All new products were tested for their ability to inhibit HNE, and the
experiments were conducted in triplicate using N-methylsuccinyl-Ala-
Ala-Pro-Val-7-amino-4-cumarin as the substrate. The results are
reported in the Tables 1 and 2 and are compared with the Sivelestat
TABLE 3 Geometric parameters of the enzyme-inhibitor complexes predicted by molecular dockinga
Compound
IC50 (μM)
0.015
3.5
d1 (Å)
2.601
3.562
3.342
3.084
4.364
α (degree)
117.0
d3 (Å)
3.302
2.600
3.535
2.466
3.007
d2 (Å)
L (Å)b
6.520
5.077
8.890
6.430
5.952
Xc
3.218, 4.390
2.477, 3.562
5.355, 6.141
3.964, 5.421
4.143, 2.945
2d
41.1
2e
10.8
117.23
67.33
14c
14g
0.05
0.015
117.9
aThe geometric parameters important for formation of a Michaelis complex in the HNE active site are as reported previously (Crocetti et al., 2011), based
on the model of synchronous proton transfer from the oxyanion hole in HNE (Groutas et al., 1997). According to the docking results, a Michaelis complex
with Ser195 is formed with participation of the ester carbonyl group.
bLength of the channel for proton transfer calculated as d3 + min(d2).
cCrocetti et al., 2018.

10
GIOVANNONI ET AL.
and 1-(3-methylbenzoyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonitrile (com-
pound X), which we previously found to be a potent HNE inhibitor
(Crocetti et al., 2018). As shown in Table 1, the introduction of substitu-
ents at position 2 of the pyrrolo[2,3-b]pyridine scaffold was not favor-
able for HNE inhibitory activity. In fact, introduction of an alkyl group
(compounds 2a and 2b) or a carbethoxy group (2c) led to products
completely devoid of activity. In addition, shift of the carbonitrile group
from position 3 of compound X to position 2 (2d) also led to a signifi-
cant loss in HNE inhibitory activity (two orders of magnitude lower).
Likewise, the introduction of a methyl group at position 2 of compound
X was detrimental for activity, resulting in 2e with an IC50 of 10.8 μM,
further confirming that substituents at position 2 are not tolerated
without significant loss in HNE inhibitory activity.
geometry of the inhibitor-enzyme complex is favorable for the forma-
tion of a Michaelis complex when the oxygen atom of Ser195 attacks
the carbonyl carbon atom of compound X.
Although compound 2e has distance d1 and angle α suitable for
inhibitory activity, arrangement of the amino acid residues of the key
triad Ser195-His57-Asp102 becomes unfavorable because the length
L of the proton transfer channel is greatly increased (Table 3), which
may be the main reason for the low inhibitory activity of compound
2e, despite the formation of H-bonds with Ser195 and Gly193
In contrast, position 5 was more tolerant to substitutions, as most
of the 5-substitued pyrrolo[2,3-b]pyridines exhibited IC50 values in
the nanomolar range (0.015–0.070 μM) (see Table 2). The 5-bromine
derivative was about threefold more potent (7a, IC50 = 0.051 μM)
than the 5-chloro (7b, IC50 = 0.160 μM) derivative, suggesting that
lipophilicity (π = 0.86 for Br and 0.71 for Cl), steric bulk (Mr = 8.88 for
Br and 6.03 for Cl), or electron withdrawing ability could be important
requirements for activity. On the other hand, the introduction of a
nitro group (7c), which has an intermediate Mr between bromine and
chlorine (Mr = 7.36) but hydrophilic properties (π = −0.28) was about
twofold less potent than the 5-chloro derivative 7b (IC50 = 0.400 and
0.160 μM, respectively).
Insertion of bulky and lipophilic rings was favorable for HNE
inhibitory activity within certain limits, as demonstrated by the com-
parable potency of the 5-(substituted)phenyl derivatives 14a-d
(IC50 = 0.050–0.180 μM) and the low activity of compound 14e bear-
ing a 6-OCH3-naphtalen at position 5 (IC50 = 1.6 μM). Replacement of
the phenyl at position 5 in compound 14a with the isostere thienyl or
pyridyl rings resulted in the thienyl derivative 14f with comparable
potency to 14a (IC50 = 0.042 versus 0.050 μM, respectively) and the
pyridyl derivative 14g, which was about threefold more potent than
14a (IC50 = 0.015 versus 0.050 μM, respectively). Thus, the presence
of pyridine in compound 14g may be important not only for steric hin-
drance but also for the formation of an H-bond that involves a nitro-
gen atom, which is not found in 14a.
Taking into account these biological results and in particular the
observation that the most potent 5-substituted compound (14g)
exhibited the same IC50 as the 5-unsubstitued lead compound X, we
can conclude that the introduction of certain substituents at position
5 maintained HNE inhibitory activity, probably by interacting with the
large pocket of the enzyme site. On the other hand, we were not able
to improve the HNE inhibitory activity with respect the lead X.
The difference in activities of the test compounds can be
explained by the length L of the proton transfer channel, angle α, and
length d1. As reported previously, a nitrogen atom in the six-
membered pyridine moiety of the lead X forms an H-bond with
Asp194 (Crocetti et al., 2018). Additional H-bonding occurs with
Val216 due to the nitrogen atom of the cyano group in lead com-
pound X (Crocetti et al., 2018). The distance d1 for molecule X is
smaller than for other active compounds in Table 3. In general, the
FIGURE 5 Docking poses of inhibitors 2e and 14g based on the
pyrrolo[2,3-b]pyridine scaffold in the human neutrophil elastase
binding site (1HNE entry of Protein Data Bank). Panel (a) Docking
pose of molecule 2e. Ser195 is H-bonded to amide nitrogen atom,
while the cyano group forms a hydrogen bond with Gly193. Panel
(b) Docking pose of molecule 14g. The carbonyl oxygen atom forms
a hydrogen bond with Val216. Residues within 4 Å from each pose
are shown

GIOVANNONI ET AL.
11
involving two nitrogen atoms (Figure 5a). Although compound 2d has
a short length L, it has strong anchoring due to H-bonding of the car-
bonyl oxygen with Ser195 of the catalytic triad (not shown). Clearly,
significant fixation of the molecule at the binding site leads to unfa-
vorable activity (i.e., angle α is too acute for compound 2d, see
Table 3). This may cause difficulties in formation of the Michaelis
complex and, therefore, reduced activity of 2d. It should be noted that
the cyano group at position 2 does not form H-bonds with the
enzyme, in contrast with the same group at position 3 of molecule X.
As for the highly active HNE inhibitors 14c and 14g, the geometric
parameters of the docking poses, as well as the distance L, were favor-
able for the formation of Michaelis complexes and for proton transfer
via the catalytic triad (Table 3). It should be noted that the angle α for
compound 14c falls slightly out of the optimal interval 80–120ꢀ
(Groutas et al., 1997; Vergely et al., 1996), which can explain the
reduced inhibitory activity in comparison with inhibitor 14g. Mole-
cules 14c and 14g are fixed within the HNE binding site by H-bonds
in the above-mentioned orientations that are favorable for biological
activity (Figure 5b). Thus, the geometrical parameters of the docking
poses found with flexible side chains of the enzyme allow us to
explain the differences in inhibitory activity of the investigated
compounds.
and Science of the Russian Federation project No. 4.8192.2017/8.9, and
the Montana State University Agricultural Experiment Station.
ORCID
Letizia Crocetti
https://orcid.org/0000-0003-3473-2683
REFERENCES
Bahekar, R. H., Jain, M. R., Jadavav, P. A., Projapati, V. M., Patel, D. N.,
Gupta, A. A., … Modi, H. (2007). Synthesis and antidiabetic activity of
2,5-disubstituted-3-imidazol-2-yl-pyrrolo[2,3-b]pyridines. Bioorganic &
Medicinal Chemistry, 15(21), 6782–6795.
Baltus, C. B., Jorda, R., Marat, C., Berka, C., Bazgier, V., Krystal, V., … Viad-
Massuard, M. C. (2016). Synthesis, biological evaluation and molecular
modeling of a novel series of 7-azaindole based tri-heterocyclic com-
pounds as potent CDK2/cyclin E inhibitors. European Journal of Medici-
nal Chemistry, 108, 701–719.
Bhat, P. V., Dere, R. T., Ravikumar, S., Hindupur, R. M., & Pati, H. N. (2015).
Efficient and scalable process for synthesis of 5-nitro-7-azaindole.
Organic Process Research and Development, 19, 1282–1285.
Chen, G., Ren, H., Zhang, N., Lennox, W., Turpoff, A., Paget, S., … Gu, Z.
(2015). (Azaindol-2-yl)pyridine-3-sulfonamides as potent and selective
inhibitors targeting hepatitis C virus NS4B. Bioorganic & Medicinal
Chemistry Letters, 25, 781–786.
Crocetti, L., Giovannoni, M. P., Schepetkin, I. A., Quinn, M. T.,
Khlebnikov, A. I., Cantini, N., … Vergelli, C. (2018). 1H-pyrrolo[2,3-b]
pyridine: A new scaffold for human neutrophil elastase (HNE) inhibitor.
Bioorganic & Medicinal Chemistry, 26, 5583–5595
Crocetti, L., Giovannoni, M. P., Schepetkin, I. A., Quinn, M. T.,
Khlebnikov, A. I., Cilibrizzi, A., … Vergelli, C. (2011). Design, synthesis
and evaluation of N-benzoylindazole derivatives and analogues as
inhibitors of human neutrophil elastase. Bioorganic & Medicinal Chemis-
try, 19, 4460–4472.
Crocetti, L., Schepetkin, I. A., Ciciani, G., Giovannoni, M. P., Guerrini, G.,
Iacovone, A., … Vergelli, C. (2016). Synthesis and farmacological evalu-
ation of indole derivatives as deaza analogues of potent human neu-
trophil elastase inhibitors. Drug Development Research, 77(6), 285–289.
Crocetti, L., Schepetkin, I. A., Cilibrizzi, A., Graziano, A., Vergelli, C.,
4
| CONCLUSIONS
In the present study, we further investigated the pyrrolo[2,3-b]pyri-
dine scaffold, which was previously found to be suitable for the syn-
thesis of HNE inhibitors (IC50 = 14–87 nM) (Crocetti et al., 2018).
While maintaining the best substituents identified for N-1 and posi-
tion 3 (m-toluoyl and CN groups, respectively), we introduced substit-
uents with different features at position 5 in order to assess the
impact of these modifications on binding and HNE inhibition. More-
over, we also evaluated modification of the substituent at position
2 of the scaffold. The biological results clearly demonstrated that posi-
tion 2 of the pyrrolo[2,3-b]pyridine scaffold must be unsubstituted,
whereas the introduction of certain substituents at position
5 maintained HNE inhibitory activity comparable with that of refer-
ence compound X, probably by interacting with the large pocket of
the enzyme site. According to our molecular modeling, the most active
HNE inhibitors bind the enzyme in conformations suitable for the for-
mation of a Michaelis complex between the ligand C O group and
Ser195. Additionally, for the potent inhibitors, a mutual orientation of
the ligand with the catalytic triad is necessary for effective proton
transfer from the oxyanion hole.
Giomi, D.,
… Giovannoni, M. P. (2013). Optimization of N-
benzoylindazole derivatives as inhibitors of human neutrophil elastase.
Journal of Medicinal Chemistry, 56, 6259–6272.
Ermoli, A., Bargiotti, A., Brasca, M. G., Ciavolella, A., Colombo, N.,
Fachin, G., … Vanotti, E. (2009). Cell division cycle 7 kinase inhibitors:
1H-pyrrolo[2,3-b]pyridines, synthesis and structure–activity relation-
ships. Medicinal Chemistry, 52, 4380–4390.
Fujinaga, M., Chernaiaa, M. M., Halenbeck, R., Koths, K., & James, M. N. G.
(1996). The crystal structure of PR3, a neutrophil serine proteinase
antigen of Wegener's granulomatosis antibodies. Journal of Molecular
Biology, 261, 267–278.
Giovannoni, M. P., Schepetkin, I. A., Crocetti, L., Ciciani, G., Cilibrizzi, A.,
Guerrini, G., … Vergelli, C. (2016). Cinnoline derivatives as human neu-
trophil elastase inhibitors. Journal of Enzyme Inhibition and Medicinal
Chemistry, 31(4), 628–639.
Giovannoni, M. P., Schepetkin, I. A., Quinn, M. T., Cantini, N., Crocetti, L.,
Guerrini, G., … Vergelli, C. (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.
Graczyk, P., Palmer, V., & Khan, A. (2004). Preparation of 3-ethynyl or
3-cyano-1H-pyrrolo[2,3-b]pyridine derivatives as c-Jun N-terminal
kinase (JNK) inhibitors. From PCT Int Appl WO 2004101565.
ACKNOWLEDGMENTS
This research was supported in part by National Institutes of Health
IDeA Program COBRE Grant GM110732; USDA National Institute of
Food and Agriculture Hatch project 1009546; Montana University Sys-
tem Research Initiative: 51040-MUSRI2015-03; Tomsk Polytechnic Uni-
versity Competitiveness Enhancement Program; Ministry of Education

12
GIOVANNONI ET AL.
Groutas, W. C., Kuang, R., Venkataraman, R., Epp, J. B., Ruan, S., &
Prakash, O. (1997). Structure-based design of a general class of
mechanism-based inhibitors of the serine proteinases employing a
novel amino acid-derived heterocyclic scaffold. Biochemistry, 36,
4739–4750.
Ibrahim, P. N., Artis, D. R., Bremer, R., Habets, G., Mamo, S., Nespi, M., …
West, B. (2007). Pyrrolo[2,3-b]pyridine derivatives as protein kinase
inhibitors and their preparation, pharmaceutical compositions and use
in the treatment of diseases. From PCT Int Appl WO 2007002433.
Joydev, K. L., Rohan, A. B., & Mandeep Kaur, H. (2017). Intramolecular oxi-
dative arylations in 7-azaindoles and pyrroles: Revamping the synthe-
sis of fused N-heterocycle tethered fluorenes. Chemistry: A European
Journal, 23, 2044–2050.
Polverino, E., Rosales-Mayor, E., Dale, G. E., Dembowsky, K., & Torres, A.
(2017). The role of neutrophil Elastase inhibitors in lung diseases.
Chest, 152(2), 249–262.
Sandham, D. A., Adcock, C., Bala, K., Barker, L., Brown, Z., Dubois, G., …
Wilson, C. (2009). 7-Azaindole-3-acetic acid derivatives: Potent and
selective CRTh2 receptor antagonists. Bioorganic & Medicinal Chemis-
try, 19, 4794–4798.
Schepetkin, I. A., Khlebnikov, A. I.,
& Quinn, M. T. (2007). N-
benzoylpyrazoles are novel small-molecule inhibitors of human neutro-
phil elastase. Journal of Medicinal Chemistry, 50, 4928–4938.
Singh, U., Chashoo, G., Khan, S. U., Mahajan, P., Singh, A., Sharma, A., …
Singh, P. P. (2017). Design o novel 3-pyrimidinylazaindole CDK2/9
inhibitors with potent in vivo antitumor efficacy in a triple negative
breast cancer model. Journal of Medicinal Chemistry, 60(23), 9470–9489.
Vergelli, C., Schepetkin, I. A., Crocetti, L., Iacovone, A., Giovannoni, M. P.,
Guerrini, G., … Quinn, M. T. (2017). Isoxazol-5(2H)-one: A new scaffold
for potent human neutrophil elastase (HNE) inhibitors. Journal of
Enzyme Inhibition and Medicinal Chemistry, 32(1), 821–831.
Vergely, I., Laugaa, P., & Reboud-Ravaux, M. (1996). Interaction of human
leukocyte elastase with a N-aryl azetidinone suicide substrate: Confor-
mational analyses based on the mechanism of action of serine protein-
ases. Journal of Molecular Graphics, 14(3), 158–167.
Korkmaz, B., Horwitz, M. S., Jenne, D. E., & Gauthier, F. (2010). Neutrophil
elastase, proteinase 3, and cathepsin G as therapeutic targets in
human diseases. Pharmacological Reviews, 62, 726–759.
Laha, J. K., Bhimpuria, R. A., & Hunjan, M. K. (2017). Intramolecular oxida-
tive arylations in 7-azaindoles and pyrroles: Revamping the synthesis
of fused n-heterocycle tethered fluorenes. Chemistry: A European Jour-
nal, 23, 2044–2050. https://doi.org/10.1002/chem.201604192
Liu, N., Wang, Y., Huang, G., Ji, C., Fan, W., Li, H., … Tian, H. (2016).
Design, synthesis and biological evaluation of 1H-pyrrolo[2,3-b]pyri-
dine and 1H-pyrazolo[3,4-b]pyridine derivatives as c-Met inhibitors.
Bioorganic Chemistry, 65, 146–158.
Lucas, S. D., Costa, E., Guedes, R. C., & Moreira, R. (2011). Targeting
COPD: Advances on low-molecular-weight inhibitors of human neu-
trophil elastase. Medicinal Research Reviews, 33, E73–E101.
Navia, M. A., McKeever, B. M., Springer, J. P., Lin, T. Y., Williams, H. R.,
Fluder, E. M., … Hoogsteen, K. (1989). Structure of human neutrophil
elastase in complex with a peptide chloromethyl ketone inhibitor at
1.84 Å resolution. Proceedings of the National Academy of Sciences of
the United States of America, 86, 7–11.
Von Nussbaum, F., & Li, V. M. J. (2015). Neutrophil elastase inhibitors for
the treatment of (cardio)pulmonary diseases: Into clinical testing with
pre-adaptive pharmacophores. Bioorganic & Medicinal Chemistry Let-
ters, 25, 4370–4381.
Von Nussbaum, F., Li, V. M. J., Daniel Meibom, D., Anlauf, S., Bechem, M.,
Delbeck, M., … Schamberger, J. (2016). Potent and selective human
neutrophil elastase inhibitors with novel equatorial ring topology:
in vivo efficacy of the polar pyrimidopyridazine BAY-8040 in a pulmo-
nary arterial hypertension rat model. ChemMedChem, 11, 199–206.
Xi, N., & Li, X. (2014). Preparation of alkenyl-pyrrolo[2,3-b]pyridine com-
pounds as protein tyrosine kinase modulators useful for treatment of
proliferative disorders. From PCT Int Appl WO 2014193647.
Nirogi, R., Shinde, A., Daulatabad, A., Kambhampati, R., Gudla, P.,
Shaik, M., … Reballi, V. (2012). Design, synthesis, and pharmacological
evaluation of piperidin-4-yl amino aryl sulfonamides: novel, potent,
selective, orally active, and brain penetrant 5-HT6 receptor antago-
nists. Journal of Medicinal Chemistry, 55, 9255–9269.
Zhong, Q. Q., Wang, X., Li, Y. F., Peng, L. J., & Jiang, Z. S. (2017). Secretory leu-
kocyte protease inhibitor promising protective roles in obesity-associated
atherosclerosis. Experimental Biology and Medicine, 242(3), 250–257.
Peters, M. B., & Merz, K. M. (2006). Semiempirical comparative binding
energy analysis (SECOMBINE) of a series of trypsin inhibitors. Journal
of Chemical Theory and Computation, 2, 383–399.
Pham, C. T. (2006). Neutrophil serine proteases: Specific regulators of
inflammation. Nature Reviews. Immunology, 6, 541–550.
Pires, M. J. D., Poeira, D. L., Purificacao, S. I., & Marques, M. M. B. (2016).
Synthesis of substituted 4-, 5-, 6- and 7-azaindoles from aminopyridines
via a cascade C-N cross-coupling/heck reaction. Organic Letters, 18(13),
3250–3253.
How to cite this article: Giovannoni MP, Cantini N,
Crocetti L, et al. Further modifications of 1H-pyrrolo[2,3-b]
pyridine derivatives as inhibitors of human neutrophil elastase.
Drug Dev Res. 2019;1–12. https://doi.org/10.1002/ddr.21539