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. Author manuscript; available in PMC: 2017 Sep 1.
Published in final edited form as: Drug Dev Res. 2016 Jul 30;77(6):285–299. doi: 10.1002/ddr.21323

Synthesis and pharmacological evaluation of indole derivatives as deaza analogues of potent human neutrophil elastase inhibitors (HNE)

Letizia Crocetti a, Igor A Schepetkin b, Giovanna Ciciani a, Maria Paola Giovannoni a,*, Gabriella Guerrini a, Antonella Iacovone a, Andrei I Khlebnikov c, Liliya N Kirpotina b, Mark T Quinn b, Claudia Vergelli a
PMCID: PMC5062748  NIHMSID: NIHMS818634  PMID: 27474878

Abstract

A number of N-benzoylindoles were designed and synthesized as deaza analogues of our previously reported potent and selective HNE inhibitors with an indazole scaffold. The new compounds containing substituents and functions that were most active in the previous series were active in the micromolar range (the most potent had IC50=3.8 µM) or inactive. These results demonstrated the importance of N-2 in the indazole nucleus. Docking studies performed on several compounds containing the same substituents but with an indole or an indazole scaffold, respectively, highlight interesting aspects concerning the molecule orientation and H-bonding interactions, which could help to explain the lower activity of this new series.

Keywords: indoles, synthesis, human neutrophil elastase (HNE), inhibitors

INTRODUCTION

Neutrophil serine proteases (NSPs) are granule-associated enzymes mainly known for their role in the intracellular killing of pathogens and are stored into acidic granules tightly bound with proteoglycans [Reeves et al., 2002]. Their extracellular release following neutrophil activation is traditionally considered the main cause for tissue damage at sites of inflammation [Pham, 2006]. This protease family consists of neutrophil elastase, proteinase 3, cathepsin G and the recently discovered NSP4 [Perera et al., 2012].

Human neutrophil elastase (HNE) is a small, soluble protein of about 30 kDa. It contains 218 amino acid residues and is stabilized by four disulfide bridges [Sihna et al., 1987]. HNE is a basic glycoprotein with a catalytic triad, consisting of Ser195, His57, and Asp102 [Bode et al., 1989]. HNE is currently considered a multifunctional enzyme involved in the killing of pathogens and in the regulation of inflammation and tissue homeostasis [Pham, 2006] through its proteolytic action against a variety of extracellular matrix proteins, such as elastin, collagen, fibronectin, laminin, proteoglycans [Chua and Laurent, 2006], and some matrix metalloproteinases [Geraghty et al., 2007]. HNE seems to play an important role in chemotaxis and migration of neutrophils to inflammatory mediators by the splitting of adhesion molecules at cell junctions [Cepinskas et al., 1999; Hermant et al., 2003]. The serpin family of endogenous inhibitors, including α-1 antitripsin (α1-AT), α2-macroglobuline, elafin, and secretory leucocyte protease inhibitor (SLPI), is able to reduce the tissue damage of HNE under physiological conditions to regulate inflammatory processes [Potempa et al., 1994; Tremblay et al., 2003; Heutinck et al., 2010]. Alteration of the balance between HNE and serpin activity can contribute to certain pathologies, especially in the lung, where the excess of HNE activity leads to hydrolysis of elastin and other extracellular matrix proteins, such as inflammatory mediators, cell surface receptors, and lung surfactants. Moreover, HNE can cause the activation of other proteases and cytokines, resulting in a massive amplification of the inflammatory response [Heutinck et al., 2010; Korkmaz and Moreau, 2008; Korkmaz and Horwitz, 2010]. The main pulmonary diseases involving HNE are chronic obstructive pulmonary disease (COPD) [O’Donnell et al., 2004; Hogg et al., 2004], acute respiratory distress syndrome [Wang et al., 2009], and acute lung injury [Kawabata et al., 2002]. Additionally, cystic fibrosis is a very serious genetic disease where excessive mucus production is associated with a massive influx of neutrophils and HNE release [Voynow et al., 2008; Gifford and Chalmers, 2014]. HNE is also involved in other inflammatory disorders, such as psoriasis [Meyer-Hoffert et al., 2004], dermatitis, atherosclerosis [Henrisken and Sallenave, 2008], rheumatoid arthritis [Hilbert et al., 2002], and various types of cancer [Sato et al., 2006; Moroy et al., 2012]. Lastly, HNE plays a central role both in acute pathogenesis and chronic functional restoration after brain traumatic injury [Semple et al., 2015].

Taking into account the evidence summarized above, it is clear that HNE represents an important therapeutic target [Groutas et al., 2011; Henriksen, 2014]. In the literature, the classification of HNE inhibitors is based on their structure (peptide and non-peptide) or on their mechanism of action (mechanism-based inhibitors [Zhong and Groutas, 2004], acylating-enzyme inhibitors [Lucas et al., 2011], transition-state analogues, and noncovalent inhibitors [Sjö, 2012]). At the present, only two drugs are available for clinical use: Prolastin® (purified α1-AT) for the treatment of α1-antitripsin deficiency [Bayer Corp, 2002] and Sivelestat (Elaspol® 100, Figure 1) marketed in Japan and Korea for the treatment of acute lung injury associated with systemic inflammatory response syndrome [Iwata et al., 2010]. Moreover, two HNE inhibitors are currently in clinical trials: AZD9668 (Alvelestat) for bronchiectasis and COPD [Stockley et al., 2013; Vogelmeier et al., 2012] and BAY 85-8501, a novel dihydropyrimidinone compound for the treatment of pulmonary disease (Figure 1) [Von Nussbaum et al., 2015].

Figure 1.

Figure 1

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HNE inhibitors.

We recently discovered novel HNE inhibitors belonging to different chemical classes [Crocetti et al., 2011; Crocetti et al., 2013; Giovannoni et al., 2015]. These compounds are competitive, pseudo-irreversible HNE inhibitors and show an appreciable selectivity toward HNE versus the other kinases tested. The most interesting HNE inhibitors had an N-benzoylindazole scaffold (structure A, Figure 2) and exhibited activity in the low nanomolar range (IC50 = 7–80 nM). The essential requirement for activity was shown to be the carbonyl group at position 1, and we demonstrated that it is the point of attack of Ser195 in the active site [Crocetti et al., 2013].

Figure 2.

Figure 2

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Indazole derivatives (A) and indole derivatives (B) as deaza analogues.

In the present paper, we report the synthesis of a new series of indole derivatives (B) as deaza analogues of the previously described potent indazoles in order to evaluate the importance of the nitrogen at position 2 for HNE inhibitory activity (Figure 2). In the indole scaffold, we inserted the substituents and functions which in the previous series afforded the best results, as well as additional modifications. The results of these studies provide new information on the importance of various substructures in the development of new synthetic HNE inhibitors.

METHODS AND MATERIALS

Chemistry

All new compounds were synthesized as reported in Figure 3–5, and the structures were confirmed on the basis of analytical and spectral data. Figure 3 shows the synthetic pathway used to obtain the final compounds bearing an ester function (2a–g and 3a,b), a cyano group (4a,b) [Wang and Chuang, 1997], or a phenylamide (5a–g) at position 3, respectively. The starting compounds 1a–d were synthesized following previously reported procedures [Shahidul et al.,2006; Spinks et al., 2003; Yuen et al., 2013; Veale et al., 2015]. The introduction at N-1 of the benzoyl meta or para substitution was performed by treatment with the suitable benzoyl chloride either with sodium hydride in anhydrous tetrahydrofuran (THF) at room temperature (compounds 2c–g, 3a,b, 4a,b or with a catalytic amount of triethylamine in dichloromethane (compounds 5a–e). The latter method, using the appropriate sulfonylchloride, was used for the sulfonamide derivatives 5f,g. Compounds 2a and 2b, lacking the amide function at position 1, were synthesized by a cross-coupling reaction (2a) with 3-methylphenylboronic acid, using copper acetate as catalyst, and triethylamine in dichloromethane or with 3-methylbenzyl chloride in anhydrous acetonitrile and K2CO3 (2b).

Figure 3.

Figure 3

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Synthesis of the final compounds 2a–g, 3a, b, 4a, b, and 5a–g. Reagents and conditions: a) for 2a: 3-methylphenyl boronic acid, (CH3COO)2Cu, Et3N, anhydrous CH2Cl2, r.t., 24 h; for 2b: 3-methylbenzylbenzylchloride, K2CO3, anhydrous CH3CN, 90 °C, 3h; for 2c–g, 3a,b and 4a,b: NaH, Ar-COCl, anhydrous THF, r.t., 24 h; for 5a–e: Ar-COCl, Et3N, anhydrous CH2Cl2, 0 °C, 2h; r.t., 1–8 h; for 5f,g: Ar-SO2Cl, Et3N, anhydrous CH2Cl2, 0 °C, 2h; r.t., 3–6 h.

Figure 5.

Figure 5

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Synthesis of the final compounds 11a–d. Reagents and conditions: a) NaH, m- o p-toluoyl chloride, anhydrous THF, r.t, 24 h.

We next inserted a bromine or a nitro at positions 5 and 6 of indole nucleus, as shown in Figure 4. Starting from precursors 6a-c, synthesized as described previously [Tantak et al., 2013; Li et al., 2012; DeGraw and Goodman, 1964], we obtained the final compounds 7a–e using the same procedure as described in Figure 3. The 5-NO2 derivative 7e was then converted by catalytic reduction with a Parr instrument into the corresponding 5-amino compound 8, which, in turn, was treated with acetyl chloride in dichloromethane and trimethylamine, resulting in the final compound 9.

Figure 4.

Figure 4

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Synthesis of the final compounds 7a–e, 8 and 9. Reagents and conditions: a) NaH, m- o p-toluoyl chloride, anhydrous THF, r.t., 24 h; b) H2, Pd/C, EtoH 96%, 30 PSI (Parr), 2 h; c) CH3COCl, Et3N, anhydrous CH2Cl2, 0 °C, 2h; r.t., 2h.

Starting from compounds 10a,b [Panatur et al., 2013; Sudhakara et al., 2009] and following the same procedures reported in Figures 3 and 4, we obtained the desired 11a–d as isomers of 2c,d (5-unsubstituted) and 7d,e (5-NO2).

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 commercial 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 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) reported in Hz were calculated using TopSpin 1.3 software (Nicolet Instrument Corp., Madison, WI) and are rounded to the nearest 0.1 Hz. 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 are within ±0.4% of the theoretical values, unless otherwise stated. Reagents and starting material were commercially available.

Ethyl 1-(m-tolyl)-1H-indole-3-carboxylate (2a)

A mixture of dry CH2Cl2 (8 mL), 1a (0.53 mmol), 3-methylphenylboronic acid (1.06 mmol), Cu(Ac)2 (0.79 mmol), and Et3N (1.06 mmol) was stirred at room temperature for 24 h. The solution was first washed with water (3 × 20 mL), and then with 33% aqueous ammonia (3 × 5 mL). The organic phase was dried over sodium sulfate, and the solvent was evaporated in vacuo to obtain the final compound 2a, which was purified by column chromatography using cyclohexane/ethyl acetate (5:1) as eluent. Yield = 19%; oil. 1H NMR (CDCl3) δ 1.46 (t, 3H, OCH2CH3, J = 7.2 Hz), 2.48 (s, 3H, CH3), 4.44 (q, 2H, OCH2CH3, J = 7.2 Hz), 7.26–7.36 (m, 5H, Ar), 7.45 (t, 1H, Ar, J = 8.2 Hz), 7.53 (d, 1H, Ar, J = 8.0 Hz), 8.04 (d, 1H, Ar, J = 2.4 Hz), 8.28 (d, 1H, Ar, J = 8.4 Hz). 13C NMR (CDCl3) δ 14.60 (CH3), 21.42 (CH3), 59.85 (CH2), 67.00 (C), 105.00 (C), 111.08 (CH), 120.90 (CH), 121.86 (CH), 122.38 (CH), 123.30 (CH), 125.44 (CH), 128.55 (CH), 129.57 (CH), 130.01 (C), 134.19 (CH), 137.05 (C), 138.30 (C), 140.33 (C). ESI-MS calcd. for C18H17NO2, 279.33; found: m/z 280.13 [M + H]+. Anal. C18H17NO2 (C, H, N).

Ethyl 1-(3-methylbenzyl)-1H-indole-3-carboxylate (2b)

A mixture of ethyl 1H-indole-3-carboxylate 1a (0.47 mmol), K2CO3 (0.94 mmol) and 3-methylbenzyl chloride (0.71 mmol) in 2 mL of anhydrous acetonitrile was stirred at reflux for 3 h. After cooling, the mixture was concentrated in vacuo, diluted with ice-cold water (10 mL), and extracted with ethyl acetate (3 × 15 mL). The organic phase was dried over sodium sulfate, and the solvent was evaporated in vacuo to obtain the final compound 2b, which was purified by column chromatography using toluene/ethyl acetate (9.5:0.5) as eluent. Yield = 66%; oil. 1H NMR (CDCl3) δ 1.45 (t, 3H, OCH2CH3, J = 7.0 Hz), 2.33 (s, 3H, CH3), 4.42 (q, 2H, OCH2CH3, J = 7.0 Hz), 5.31 (s, 2H, CH2), 6.97–7.02 (m, 2H, Ar), 7.13 (d, 1H, Ar, J = 7.2 Hz), 7.22–7.36 (m, 4H, Ar), 7.88 (s, 1H, Ar), 8.23 (dd, 1H, Ar, J = 6.8 Hz, J = 1.2 Hz). 13C NMR (CDCl3) δ 13.60 (CH3), 21.20 (CH3), 59.10 (CH2), 61.80 (CH2), 102.05 (C), 111.07 (CH), 120.14 (CH), 121.03 (CH), 122.00 (CH), 126.24 (CH), 126.30 (CH), 128.05 (CH), 128.10 (C), 128.31 (CH), 129.97 (CH), 137.60 (C), 137.73 (C), 139.00 (C), 167.05 (C). ESI-MS calcd. for C19H19NO2, 293.36; found: m/z 294.14 [M + H]+. Anal. C19H19NO2 (C, H, N).

General procedure for compounds (2c–g)

To a suspension of the substrate 1a (0.53 mmol) in 10 mL of anhydrous THF, 1.06 mmol of sodium hydride and 0.64 mmol of appropriate benzoyl 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 crystallization from ethanol.

Ethyl 1-(3-methylbenzoyl)-1H-indole-3-carboxylate (2c)

Yield = 23%; mp = 74–76 °C (EtOH). 1H NMR (CDCl3) δ 1.43 (t, 3H, OCH2CH3, J = 7.2 Hz), 2.49 (s, 3H, CH3), 4.42 (q, 2H, OCH2CH3, J = 7.2 Hz), 7.42–7.50 (m, 4H, Ar), 7.55 (d, 1H, Ar, J = 6.8 Hz), 7.60 (s, 1H, Ar), 8.02 (s, 1H, Ar), 8.22 (d, 1H, Ar, J = 8.4 Hz), 8.39 (d, 1H, Ar, J = 8.0 Hz). 13C NMR (CDCl3) δ 13.60 (CH3), 20.50 (CH3), 59.10 (CH2), 102.00 (C), 111.06 (CH), 120.13 (CH), 121.08 (CH), 122.01 (CH), 124.00 (CH), 126.75 (CH), 128.02 (C), 128.96 (CH), 130.43 (CH), 135.00 (CH), 136.11 (C), 136.64 (C), 138.19 (C), 167.11 (C), 190.01 (C). ESI-MS calcd. for C19H17NO3, 307.34; found: m/z 308.12 [M + H]+. Anal. C19H17NO3 (C, H, N).

Ethyl 1-(4-methylbenzoyl)-1H-indole-3-carboxylate (2d)

Yield = 74%; mp = 109–111 °C (EtOH). 1H NMR (CDCl3) δ 1.40 (t, 3H, OCH2CH3, J = 7.0 Hz), 2.48 (s, 3H, CH3), 4.39 (q, 2H, OCH2CH3, J = 7.0 Hz), 7.36 (d, 2H, Ar, J = 7.6 Hz), 7.40–7.45 (m, 2H, Ar), 7.66 (d, 2H, Ar, J = 8.0 Hz), 8.02 (s, 1H, Ar), 8.19 (d, 1H, Ar, J = 6.4 Hz), 8.34 (d, 1H, Ar, J = 6.4 Hz). 13C NMR (CDCl3) δ 14.45 (CH3), 21.71 (CH3), 60.50 (CH2), 102.02 (C), 111.06 (CH), 116.13 (CH), 121.71 (CH), 124.85 (CH), 125.56 (CH), 128.00 (CH), 128.10 (C), 129.57 (CH), 129.73 (CH), 133.52 (CH), 133.70 (C), 136.04 (C), 143.65 (C), 167.01 (C), 190.00 (C). ESI-MS calcd. for C19H17NO3, 307.34; found: m/z 308.12 [M + H]+. Anal. C19H17NO3 (C, H, N).

Ethyl 1-(4-chlorobenzoyl)-1H-indole-3-carboxylate (2e)

Yield = 89%; mp = 123–125 °C (EtOH). 1H NMR (CDCl3) δ 1.44 (t, 3H, OCH2CH3, J = 7.2 Hz), 4.42 (q, 2H, OCH2CH3, J = 7.2 Hz), 7.45–7.50 (m, 2H, Ar), 7.58 (d, 2H, Ar, J = 8.4 Hz), 7.74 (d, 2H, Ar, J = 8.4 Hz), 7.97 (s, 1H, Ar), 8.10 (d, 2H, Ar, J = 8.8 Hz). 13C NMR (CDCl3) δ 14.11 (CH3), 60.90 (CH2), 108.62 (C), 115.66 (CH), 119.83 (CH), 121.81 (CH), 124.35 (CH), 124.39 (CH), 126.36 (C), 129.30 (CH), 129.38 (CH), 131.10 (C), 131.33 (CH), 131.37 (CH), 135.72 (C), 140.10 (C), 162.54 (C), 167.71 (C). ESI-MS calcd. for C18H14ClNO3, 327.76; found: m/z 329.06 [M + H]+. Anal. C18H14ClNO3 (C, H, N).

Ethyl 1-(3-chlorobenzoyl)-1H-indole-3-carboxylate (2f)

Yield = 30%; mp = 89–91 °C (EtOH). 1H NMR (CDCl3) δ 1.44 (t, 3H, OCH2CH3, J = 7.2 Hz), 4.43 (q, 2H, OCH2CH3, J = 7.2 Hz), 7.45–7.50 (m, 2H, Ar), 7.54 (t, 2H, Ar, J = 8.0 Hz), 7.63–7.68 (m, 2H, Ar), 7.78 (s, 1H, Ar), 7.95 (s, 1H, Ar), 8.06 (d, 1H, Ar, J = 7.6 Hz). 13C NMR (CDCl3) δ 14.19 (CH3), 60.95 (CH2), 108.66 (C), 115.60 (CH), 119.89 (CH), 121.84 (CH), 124.31 (CH), 124.35 (CH), 126.36 (C), 129.20 (CH), 129.98 (CH), 130.60 (CH), 131.93 (C), 134.67 (CH), 134.82 (C), 135.70 (C), 162.54 (C), 167.73 (C). ESI-MS calcd. for C18H14ClNO3, 327.76; found: m/z 329.06 [M + H]+. Anal. C18H14ClNO3 (C, H, N).

Ethyl 1-(4-fluorobenzoyl)-1H-indole-3-carboxylate (2g)

Yield = 89%; mp = 123–125 °C (EtOH). 1H NMR (CDCl3) δ 1.49 (t, 3H, OCH2CH3, J = 7.2 Hz), 4.45 (q, 2H, OCH2CH3, J = 7.2 Hz), 7.25 (t, 2H, Ar, J = 8.0 Hz), 7.48–7.53 (m, 2H, Ar), 7.70–7.75 (m, 2H, Ar), 8.01 (s, 1H, Ar), 8.15–8.20 (m, 2H, Ar). 13C NMR (CDCl3) δ 14.12 (CH3), 60.94 (CH2), 108.62 (C), 115.61 (CH), 116.03 (CH), 116.07 (CH), 119.55 (CH), 121.89 (CH), 124.33 (CH), 124.39 (CH), 126.38 (C), 128.60 (C), 131.53 (CH), 131.57 (CH), 135.72 (C), 162.50 (C), 167.74 (C), 168.71 (C). ESI-MS calcd. for C18H14FNO3, 311.31; found: m/z 312.10 [M + H]+. Anal. C18H14FNO3 (C, H, N).

General procedure for compounds (3a,b)

Compounds 3a,b were obtained following the same procedure performed for compounds 2c–g and starting from intermediate 1b. The final compounds 3a,b were purified by column chromatography using cyclohexane/ethyl acetate (6:1) as eluent.

Ethyl 2-(1-(3-methylbenzoyl)-1H-indol-3-yl)acetate (3a)

Yield = 14%; oil. 1H NMR (CDCl3) δ 1.24 (t, 3H, OCH2CH3, J = 7.2 Hz), 2.44 (s, 3H, CH3), 3.68 (s, 2H, CH2), 4.16 (q, 2H, OCH2CH3, J = 7.2 Hz), 7.30 (s, 1H, Ar), 7.33–7.40 (m, 4H, Ar), 7.48–7.58 (m, 3H, Ar), 8.39 (d, 1H, Ar, J = 8.0 Hz). 13C NMR (CDCl3) δ 14.19 (CH3), 21.38 (CH3), 31.17 (CH2), 61.08 (CH2), 114.50 (C), 116.58 (CH), 119.03 (CH), 123.86 (CH), 125.22 (CH), 126.10 (CH), 126.17 (CH), 128.42 (CH), 129.59 (CH), 130.51 (C), 132.60 (CH), 134.60 (C), 136.24 (C), 138.63 (C), 168.72 (C), 170.79 (C). ESI-MS calcd. for C20H19NO3, 321.37; found: m/z 322.14 [M + H]+. Anal. C20H19NO3 (C, H, N).

Ethyl 2-(1-(4-methylbenzoyl)-1H-indol-3-yl)acetate (3b)

Yield = 37%; mp = 63–65 °C (EtOH). 1H NMR (CDCl3) δ 1.25 (t, 3H, OCH2CH3, J = 7.0 Hz), 2.46 (s, 3H, CH3), 3.69 (s, 2H, CH2), 4.17 (q, 2H, OCH2CH3, J = 7.0 Hz), 7.31–7.41 (m, 5H, Ar), 7.57 (d, 1H, Ar, J = 7.6 Hz), 7.64 (d, 2H, Ar, J = 8.0 Hz), 8.38 (d, 1H, Ar, J = 8.0 Hz). 13C NMR (CDCl3) δ 14.20 (CH3), 21.65 (CH3), 31.13 (CH2), 61.09 (CH2), 114.31 (C), 116.51 (CH), 119.00 (CH), 123.78 (CH), 125.16 (CH), 126.18 (CH), 129.26 (CH), 129.27 (CH), 129.37 (CH), 129.38 (CH), 130.45 (C), 131.67 (C), 136.25 (C), 142.59 (C), 168.57 (C), 170.85 (C). ESI-MS calcd. for C20H19NO3, 321.37; found: m/z 322.14 [M + H]+. Anal. C20H19NO3 (C, H, N).

General procedure for compounds (5a-g)

To a cooled (0 °C) suspension of the substrate 1d (0.40 mmol) in anhydrous CH2Cl2 (2 mL), Et3N (0.10 mmol) and 1.15 mmol of the appropriate benzoyl chloride (for compounds 5a–e) or sulfonyl chloride (for compounds 5f,g) were added. The solution was stirred at 0 °C for 2 h and then at room temperature for 2 h. After evaporation of the solvent, ice-cold water (20 mL) was added, and the mixture was neutralized with 0.5 N NaOH. Compounds 5a,b,e were recovered by extraction with CH2Cl2 (3 × 15 mL), while all other compounds were recovered by vacuum filtration. The final compounds 5a-g were purified by crystallization from ethanol.

1-(4-chlorobenzoyl)-N-phenyl-1H-indole-3-carboxamide (5a)

Yield = 60%; mp = 171–173 °C (EtOH). 1H NMR (CDCl3) δ 7.18 (t, 1H, Ar, J = 7.4 Hz), 7.39 (t, 2H, Ar, J = 7.8 Hz), 7.45–7.50 (m, 2H, Ar), 7.55 (d, 2H, Ar, J = 8.8 Hz), 7.64 (d, 2H, Ar, J = 7.6 Hz), 7.72 (d, 2H, Ar, J = 8.8 Hz), 7.88 (s, 1H, Ar), 8.08–8.13 (m, 1H, Ar), 8.33–8.38 (m, 1H, Ar). 13C NMR (CDCl3) δ 112.00 (C), 115.66 (CH), 119.83 (CH), 121.61 (CH), 121.67 (CH), 121.80 (CH), 124.35 (CH), 124.39 (CH), 126.36 (C), 128.03 (CH), 128.90 (CH), 128.95 (CH), 129.34 (CH), 129.39 (CH), 131.11 (C), 131.31 (CH), 131.36 (CH), 135.72 (C), 137.93 (C), 140.10 (C), 164.77 (C), 167.70 (C). ESI-MS calcd. for C22H15ClN2O2, 374.82; found: m/z 376.08 [M + H]+. Anal. C22H15ClN2O2 (C, H, N).

1-(3-chlorobenzoyl)-N-phenyl-1H-indole-3-carboxamide (5b)

Yield = 58%; mp = 161–163 °C (EtOH). 1H NMR (CDCl3) δ 7.18 (t, 1H, Ar, J = 7.2 Hz), 7.40 (t, 2H, Ar, J = 7.2 Hz), 7.49–7.55 (m, 3H, Ar), 7.60–7.65 (m, 4H, Ar), 7.73 (exch br s, 1H, NH), 7.78 (s, 1H, Ar), 7.88 (s, 1H, Ar), 8.11 (d, 1H, Ar, J = 8.0 Hz), 8.40 (d, 1H, Ar, J = 7.2 Hz). 13C NMR (CDCl3) δ 112.02 (C), 115.60 (CH), 119.85 (CH), 121.63 (CH), 121.68 (CH), 121.80 (CH), 124.34 (CH), 124.37 (CH), 126.31 (C), 128.03 (CH), 128.92 (CH), 128.96 (CH), 129.24 (CH), 129.99 (CH), 130.61 (CH), 131.91 (C), 134.66 (CH), 134.82 (C), 135.73 (C), 137.90 (C), 164.77 (C), 167.71 (C). ESI-MS calcd. for C22H15ClN2O2, 374.82; found: m/z 376.08 [M + H]+. Anal. C22H15ClN2O2 (C, H, N).

1-(4-methylbenzoyl)-N-phenyl-1H-indole-3-carboxamide (5c)

Yield = 35%; mp = 150–151 °C (EtOH). 1H NMR (CDCl3) δ 2.51 (s, 3H, CH3), 7.19 (t, 1H, Ar, J = 7.4 Hz), 7.30–7.40 (m, 4H, Ar), 7.45–7.50 (m, 2H, Ar), 7.60–7.75 (m, 5H, 4H Ar + 1H NH), 7.96 (s, 1H, Ar), 8.08–8.13 (m, 1H, Ar), 8.34–8.39 (m, 1H, Ar). 13C NMR (CDCl3) δ 21.30 (CH3), 112.05 (C), 115.64 (CH), 119.83 (CH), 121.62 (CH), 121.68 (CH), 121.85 (CH), 124.35 (CH), 124.38 (CH), 126.31 (C), 128.04 (CH), 128.90 (CH), 128.96 (CH), 129.54 (CH), 129.59 (CH), 129.81 (CH), 129.85 (CH), 130.06 (C), 135.72 (C), 137.95 (C), 144.20 (C), 164.75 (C), 167.71 (C). ESI-MS calcd. for C23H18N2O2, 354.40; found: m/z 355.14 [M + H]+. Anal. C23H18N2O2 (C, H, N).

1-(4-fluorobenzoyl)-N-phenyl-1H-indole-3-carboxamide (5d)

Yield = 77%; mp = 218–220 °C (EtOH). 1H NMR (CDCl3) δ 7.19 (t, 1H, Ar, J = 7.2 Hz), 7.28 (t, 2H, Ar, J = 7.8 Hz), 7.39 (t, 2H, Ar, J = 8.0 Hz), 7.46–7.52 (m, 2H, Ar), 7.65 (d, 2H, Ar, J = 8.0 Hz), 7.71 (exch br s, 1H, NH), 7.80–7.85 (m, 2H, Ar), 7.92 (s, 1H, Ar), 8.10–8.15 (m, 1H, Ar), 8.34–8.39 (m, 1H, Ar). 13C NMR (CDCl3) δ 112.03 (C), 115.65 (CH), 116.03 (CH), 116.07 (CH), 119.88 (CH), 121.65 (CH), 121.69 (CH), 121.88 (CH), 124.31 (CH), 124.34 (CH), 126.30 (C), 128.06 (CH), 128.64 (C), 128.92 (CH), 128.96 (CH), 131.53 (CH), 131.56 (CH), 135.75 (C), 137.96 (C), 164.75 (C), 167.71 (C), 168.70 (C). ESI-MS calcd. for C22H15FN2O2, 358.37; found: m/z 359.12 [M + H]+. Anal. C22H15FN2O2 (C, H, N).

1-(3-fluorobenzoyl)-N-phenyl-1H-indole-3-carboxamide (5e)

Yield = 30%; mp = 164–167 °C (EtOH). 1H NMR (CDCl3) δ 7.19 (t, 1H, Ar, J = 7.2 Hz), 7.40 (t, 3H, Ar, J = 7.6 Hz), 7.50–7.60 (m, 5H, Ar), 7.64 (d, 2H, Ar, J = 8.0 Hz), 7.71 (exch br s, 1H, NH), 7.89 (s, 1H, Ar), 8.09–8.14 (m, 1H, Ar), 8.39–8.44 (m, 1H, Ar). 13C NMR (CDCl3) δ 112.05 (C), 114.75 (CH), 115.63 (CH), 119.87 (CH), 121.38 (CH), 121.65 (CH), 121.69 (CH), 121.80 (CH), 124.33 (CH), 124.38 (CH), 126.35 (C), 126.76 (CH), 128.04 (CH), 128.93 (CH), 128.97 (CH), 130.83 (CH), 132.16 (C), 135.70 (C), 137.95 (C), 163.45 (C), 164.71 (C), 167.70 (C). ESI-MS calcd. for C22H15FN2O2, 358.37; found: m/z 359.12 [M + H]+. Anal. C22H15FN2O2 (C, H, N).

N-phenyl-1-tosyl-1H-indole-3-carboxamide (5f)

Yield = 64%; mp = 219–221 °C (EtOH). 1H NMR (CDCl3) δ 2.39 (s, 3H, CH3), 7.19 (t, 1H, Ar, J = 7.2 Hz), 7.30–7.45 (m, 5H, Ar), 7.60–7.70 (m, 3H, Ar), 7.85 (d, 2H, Ar, J = 7.2 Hz), 8.02 (d, 1H, Ar, J = 8.0 Hz), 8.12 (d, 1H, Ar, J = 7.6 Hz), 8.15 (s, 1H, Ar). 13C NMR (CDCl3) δ 21.35 (CH3), 112.00 (C), 114.54 (CH), 119.89 (CH), 121.63 (CH), 121.69 (CH), 121.85 (CH), 124.95 (CH), 126.38 (C), 127.31 (CH), 128.04 (CH), 128.20 (CH), 128.26 (CH), 128.94 (CH), 128.99 (CH), 130.01 (CH), 130.05 (CH), 134.86 (C), 135.82 (C), 137.95 (C), 139.40 (C), 164.75 (C). ESI-MS calcd. for C22H18N2O3S, 390.45; found: m/z 391.11 [M + H]+. Anal. C22H18N2O3S (C, H, N).

1-((4-chlorophenyl)sulfonyl)-N-phenyl-1H-indole-3-carboxamide (5g)

Yield = 67%; mp = 216–218 °C (EtOH). 1H NMR (CDCl3) δ 7.20 (t, 1H, Ar, J = 7.4 Hz), 7.40–7.50 (m, 7H, Ar), 7.60–7.70 (m, 2H, 1H Ar + 1H NH), 7.89 (d, 2H, Ar, J = 8.8 Hz), 8.01 (d, 1H, Ar, J = 7.6 Hz), 8.13 (s, 2H, Ar). 13C NMR (CDCl3) δ 112.01 (C), 114.53 (CH), 119.80 (CH), 121.61 (CH), 121.66 (CH), 121.85 (CH), 124.97 (CH), 126.35 (C), 127.31 (CH), 128.05 (CH), 128.91 (CH), 128.96 (CH), 129.74 (CH), 129.79 (CH), 129.81 (CH), 129.85 (CH), 135.86 (C), 135.92 (C), 137.95 (C), 139.30 (C), 164.71 (C). ESI-MS calcd. for C21H15ClN2O3S, 410.87; found: m/z 412.05 [M + H]+. Anal. C21H15ClN2O3S (C, H, N).

General procedure for compounds 7a–e

Compounds 7a–e were obtained following the same procedure performed for compounds 2c–g and 3a,b, starting from intermediates 6a–c. The final compounds 7a–e were purified by crystallization from ethanol.

Ethyl 5-bromo-1-(3-methylbenzoyl)-1H-indole-3-carboxylate (7a)

Yield = 53%; mp = 136–137 °C (EtOH). 1H NMR (CDCl3) δ 1.40 (t, 3H, OCH2CH3, J = 7.2 Hz), 2.46 (s, 3H, CH3), 4.40 (q, 2H, OCH2CH3, J = 7.2 Hz), 7.42–7.56 (m, 5H, Ar), 7.98 (s, 1H, Ar), 8.23 (d, 1H, Ar, J = 8.8 Hz), 8.33 (d, 1H, Ar, J = 2.0 Hz). 13C NMR (CDCl3) δ 14.10 (CH3), 23.90 (CH3), 60.90 (CH2), 108.70 (C), 113.36 (CH), 117.03 (C), 121.10 (CH), 121.21 (CH), 124.40 (CH), 126.95 (CH), 128.32 (C), 129.20 (CH), 130.23 (CH), 130.50 (C), 134.71 (C), 134.80 (CH), 138.90 (C), 166.01 (C), 167.81 (C). ESI-MS calcd. for C19H16BrNO3, 386.24; found: m/z 387.03 [M + H]+. Anal. C19H16BrNO3 (C, H, N).

Ethyl 6-bromo-1-(3-methylbenzoyl)-1H-indole-3-carboxylate (7b)

Yield = 62%; oil. 1H NMR (CDCl3) δ 1.38 (t, 3H, OCH2CH3, J = 7.2 Hz), 2.46 (s, 3H, CH3), 4.37 (q, 2H, OCH2CH3, J = 7.2 Hz), 7.41–7.55 (m, 5H, Ar), 7.94 (s, 1H, Ar), 8.02 (d, 1H, Ar, J = 8.4 Hz), 8.57 (d, 1H, Ar, J = 1.6 Hz). 13C NMR (CDCl3) δ 14.42 (CH3), 21.38 (CH3), 60.58 (CH2), 113.32 (C), 119.30 (CH), 120.09 (C), 122.83 (CH), 126.55 (CH), 128.21 (CH), 128.76 (CH), 129.92 (CH), 132.91 (C), 133.44 (CH), 133.64 (CH), 136.94 (C), 139.10 (C), 144.00 (C), 163.67 (C), 168.59 (C). ESI-MS calcd. for C19H16BrNO3, 386.24; found: m/z 387.03 [M + H]+. Anal. C19H16BrNO3 (C, H, N).

Ethyl 6-bromo-1-(4-methylbenzoyl)-1H-indole-3-carboxylate (7c)

Yield = 44%; mp > 300 °C (EtOH). 1H NMR (CDCl3) δ 1.39 (t, 3H, OCH2CH3, J = 7.2 Hz), 2.48 (s, 3H, CH3), 4.38 (q, 2H, OCH2CH3, J = 7.0 Hz), 7.37 (d, 2H, Ar, J = 8.0 Hz), 7.52 (dd, 1H, Ar, J = 8.6 Hz, J = 1.8 Hz), 7.65 (d, 2H, Ar, J = 8.0 Hz), 7.97 (s, 1H, Ar), 8.04 (d, 1H, Ar, J = 8.4 Hz), 8.57 (d, 1H, Ar, J = 1.2 Hz). 13C NMR (CDCl3) δ 14.43 (CH3), 21.75 (CH3), 60.68 (CH2), 113.20 (C), 119.27 (CH), 119.43 (C), 122.85 (CH), 126.54 (C), 128.22 (CH), 129.67 (CH), 129.76 (CH), 129.99 (C), 130.65 (CH), 133.45 (CH), 133.70 (CH), 137.02 (C), 143.99 (C), 163.79 (C), 168.43 (C). ESI-MS calcd. for C19H16BrNO3, 386.24; found: m/z 387.03 [M + H]+. Anal. C19H16BrNO3 (C, H, N).

Ethyl 1-(3-methylbenzoyl)-5-nitro-1H-indole-3-carboxylate (7d)

Yield = 93%; mp = 153–155 °C (EtOH). 1H NMR (CDCl3) δ 1.44 (t, 3H, OCH2CH3, J = 7.2 Hz), 2.47 (s, 3H, CH3), 4.44 (q, 2H, OCH2CH3, J = 7.2 Hz), 7.46–7.55 (m, 3H, Ar), 7.59 (s, 1H, Ar), 8.15 (s, 1H, Ar), 8.32 (dd, 1H, Ar, J = 9.2 Hz, J = 2.4 Hz), 8.46 (d, 1H, Ar, J = 9.2 Hz), 9.08 (d, 1H, Ar, J = 2.0 Hz). 13C NMR (CDCl3) δ 14.42 (CH3), 21.41 (CH3), 61.13 (CH2), 113.97 (C), 116.54 (CH), 118.17 (CH), 120.77 (CH), 126.74 (CH), 127.71 (C), 128.97 (CH), 130.11 (CH), 132.24 (C), 134.28 (CH), 135.79 (CH), 139.25 (C), 139.38 (C), 145.16 (C), 163.11 (C), 167.51 (C). ESI-MS calcd. for C19H16N2O5, 352.34; found: m/z 353.11 [M + H]+. Anal. C19H16N2O5 (C, H, N).

Ethyl 1-(4-methylbenzoyl)-5-nitro-1H-indole-3-carboxylate (7e)

Yield = 95%; mp = 176–178 °C (EtOH). 1H NMR (CDCl3) δ 1.44 (t, 3H, OCH2CH3, J = 7.2 Hz), 2.50 (s, 3H, CH3), 4.44 (q, 2H, OCH2CH3, J = 7.2 Hz), 7.40 (d, 2H, Ar, J = 8.0 Hz), 7.69 (d, 2H, Ar, J = 8.0 Hz), 8.17 (s, 1H, Ar), 8.31 (dd, 1H, Ar, J = 9.2 Hz, J = 2.4 Hz), 8.43 (d, 1H, Ar, J = 9.2 Hz), 9.08 (d, 1H, Ar, J = 2.4 Hz). 13C NMR (CDCl3) δ 14.12 (CH3), 21.31 (CH3), 60.93 (CH2), 108.60 (C), 112.04 (CH), 115.07 (CH), 116.00 (CH), 124.34 (CH), 127.21 (C), 129.50 (CH), 129.81 (CH), 130.04 (C), 132.28 (C), 134.25 (CH), 135.70 (CH), 141.89 (C), 144.25 (C), 162.50 (C), 167.71 (C). IR = 1332–1558 cm−1 (NO2), 1690 cm−1 (C=O amide), 1710 cm−1 (C=O ester). ESI-MS calcd. for C19H16N2O5, 352.34; found: m/z 353.11 [M + H]+. Anal. C19H16N2O5 (C, H, N).

Ethyl 5-amino-1-(4-methylbenzoyl)-1H-indole-3-carboxylate (8)

Compound 7e (1.42 mmol) was reduced in Parr instrument (27 mL EtOH, 320 mg 10% Pd/C, 30 psi, 2 h). The catalyst was filtered off, and the solvent was evaporated under vacuum, resulting in the final compound, which was purified by column chromatography using cyclohexane/ethyl acetate (1:1) as eluent. Yield = 26%; mp = 152–154 °C (EtOH). 1H NMR (DMSO-d6) δ 1.26 (t, 3H, OCH2CH3, J = 7.2 Hz), 2.40 (s, 3H, CH3), 4.23 (q, 2H, OCH2CH3, J = 7.2 Hz), 5.21 (exch br s, 2H, NH2), 6.69 (d, 1H, Ar, J = 8.4 Hz), 7.24 (s, 1H, Ar), 7.39 (d, 2H, Ar, J = 8.0 Hz), 7.63–7.68 (m, 3H, Ar), 7.93 (d, 1H, Ar, J = 8.6 Hz). 13C NMR (DMSO-d6) δ 14.10 (CH3), 21.31 (CH3), 60.95 (CH2), 104.60 (CH), 108.64 (C), 107.37 (CH), 111.90 (CH), 124.34 (CH), 125.71 (C), 126.90 (C), 127.61 (C), 129.54 (CH), 129.88 (CH), 134.25 (CH), 135.73 (CH), 143.99 (C), 144.25 (C), 162.56 (C), 167.70 (C). IR = 1689 cm−1 (C=O amide), 1710 cm−1 (C=O ester), 3356–3460 cm−1 (NH2). ESI-MS calcd. for C19H18N2O3, 322.36; found: m/z 323.14 [M + H]+. Anal. C19H18N2O3 (C, H, N).

Ethyl 5-acetamido-1-(4-methylbenzoyl)-1H-indole-3-carboxylate (9)

Compound 9 was obtained starting from intermediate 8 and following the same procedure performed for compounds 5a−e using acetyl chloride as reagent. Compound 9 was recovered by vacuum filtration and was purified by column chromatography using cyclohexane/ethyl acetate (1:2) as eluent. Yield = 15%; mp = 187–189 °C dec. (EtOH). 1H NMR (DMSO-d6) δ 1.40 (t, 3H, OCH2CH3, J = 6.8 Hz), 2.22 (s, 3H, CH3CONH), 2.47 (s, 3H, CH3), 4.37 (q, 2H, OCH2CH3, J = 6.4 Hz), 7.35 (d, 2H, Ar, J = 7.6 Hz), 7.44 (exch br s, 1H, NH), 7.64 (d, 3H, Ar, J = 7.2 Hz), 7.99 (s, 1H, Ar), 8.23–8.28 (m, 2H, Ar). 13C NMR (DMSO-d6) δ 14.11 (CH3), 21.30 (CH3), 24.05 (CH3), 60.90 (CH2), 108.63 (C), 109.94 (CH), 111.37 (CH), 112.61 (CH), 124.30 (CH), 126.31 (C), 127.60 (C), 128.95 (CH), 129.51 (CH), 129.84 (CH), 130.11 (CH), 131.38 (C), 133.85 (C), 144.23 (C), 162.50 (C), 167.75 (C), 168.96 (C). ESI-MS calcd. for C21H20N2O4, 364.39; found: m/z 365.15 [M + H]+. Anal. C21H20N2O4 (C, H, N).

General procedure for compound (11a–d)

Compounds 11a–d were obtained starting from intermediates 10a,b and following the same procedure performed for compounds 2c–g, 3a,b, and 7a–e. The final compounds 11a–d were purified by column chromatography using toluene/ethyl acetate (9:1) (for 11a) or cyclohexane/ethyl acetate (6:1) (for 11b–d) as eluents.

Ethyl 1-(3-methylbenzoyl)-1H-indole-2-carboxylate (11a)

Yield = 4%; oil. 1H NMR (CDCl3) δ 1.08 (t, 3H, OCH2CH3, J = 7.2 Hz), 2.38 (s, 3H, CH3), 3.97 (q, 2H, OCH2CH3, J = 7.2 Hz), 7.27–7.41 (m, 5H, Ar), 7.46 (d, 1H, Ar, J = 7.6 Hz), 7.56 (s, 1H, Ar), 7.70 (d, 1H, Ar, J = 8.0 Hz), 7.77 (d, 1H, Ar, J = 8.4 Hz). 13C NMR (CDCl3) δ 14.10 (CH3), 20.95 (CH3), 60.90 (CH2), 108.42 (CH), 115.66 (CH), 119.83 (CH), 123.58 (CH), 124.31 (CH), 126.20 (C), 126.35 (C), 128.12 (CH), 129.16 (CH), 130.13 (CH), 130.40 (C), 134.81 (CH), 138.94 (C), 140.69 (C), 160.11 (C), 167.70 (C). ESI-MS calcd. for C19H17NO3, 307.34; found: m/z 308.12 [M + H]+. Anal. C19H17NO3 (C, H, N).

Ethyl 1-(4-methylbenzoyl)-1H-indole-2-carboxylate (11b)

Yield = 8%; oil. 1H NMR (CDCl3) δ 1.10 (t, 3H, OCH2CH3, J = 7.2 Hz), 2.41 (s, 3H, CH3), 3.99 (q, 2H, OCH2CH3, J = 7.2 Hz), 7.23–7.30 (m, 3H, Ar), 7.35–7.40 (m, 2H, Ar), 7.61 (d, 2H, Ar, J = 8.4 Hz), 7.68–7.73 (m, 2H, Ar). 13C NMR (CDCl3) δ 14.10 (CH3), 21.35 (CH3), 60.92 (CH2), 108.40 (CH), 115.68 (CH), 119.81 (CH), 123.55 (CH), 124.30 (CH), 126.25 (C), 126.31 (C), 129.52 (CH), 129.86 (CH), 130.03 (C), 130.40 (CH), 134.85 (CH), 140.64 (C), 144.20 (C), 160.10 (C), 167.75 (C). ESI-MS calcd. for C19H17NO3, 307.34; found: m/z 308.12 [M + H]+. Anal. C19H17NO3 (C, H, N).

Ethyl 1-(3-methylbenzoyl)-5-nitro-1H-indole-2-carboxylate (11c)

Yield = 33%; mp = 123–125 °C (EtOH). 1H NMR (CDCl3) δ 1.12 (t, 3H, OCH2CH3, J = 7.2 Hz), 2.40 (s, 3H, CH3), 4.03 (q, 2H, OCH2CH3, J = 7.0 Hz), 7.35 (t, 1H, Ar, J = 7.6 Hz), 7.40–7.45 (m, 3H, Ar), 7.55 (s, 1H, Ar), 7.79 (d, 1H, Ar, J = 9.2 Hz), 8.26 (dd, 1H, Ar, J = 9.2 Hz, J = 2.4 Hz), 8.66 (d, 1H, Ar, J = 2.0 Hz). 13C NMR (CDCl3) δ 13.82 (CH3), 21.27 (CH3), 61.89 (CH2), 114.18 (CH), 114.81 (CH), 119.21 (CH), 121.81 (CH), 126.73 (CH), 128.14 (C), 128.92 (CH), 130.06 (CH), 131.50 (C), 134.29 (C), 134.87 (CH), 139.17 (C), 141.02 (C), 144.08 (C), 160.15 (C), 168.30 (C). ESI-MS calcd. for C19H16N2O5, 352.34; found: m/z 353.11 [M + H]+. Anal. C19H16N2O5 (C, H, N).

Ethyl 1-(4-methylbenzoyl)-5-nitro-1H-indole-2-carboxylate (11d)

Yield = 13%; mp = 109–111 °C (EtOH). 1H NMR (CDCl3) δ 1.13 (t, 3H, OCH2CH3, J = 7.2 Hz), 2.44 (s, 3H, CH3), 4.06 (q, 2H, OCH2CH3, J = 7.2 Hz), 7.28 (d, 2H, Ar, J = 8.0 Hz), 7.49 (s, 1H, Ar), 7.60 (d, 2H, Ar, J = 8.4 Hz), 7.73 (d, 1H, Ar, J = 9.2 Hz), 8.25 (dd, 1H, Ar, J = 9.2 Hz, J = 2.0 Hz), 8.66 (d, 1H, Ar, J = 2.0 Hz). 13C NMR (CDCl3) δ 13.85 (CH3), 21.84 (CH3), 61.88 (CH2), 114.03 (CH), 114.76 (CH), 119.26 (CH), 121.71 (CH), 126.60 (C), 129.76 (CH), 129.77 (CH), 129.80 (CH), 129.81 (CH), 131.49 (C), 133.80 (C), 141.00 (C), 143.96 (C), 145.39 (C), 160.15 (C), 168.32 (C). ESI-MS calcd. for C19H16N2O5, 352.34; found: m/z 353.11 [M + H]+. Anal. C19H16N2O5 (C, H, N).

Pharmacology

Compounds were dissolved in 100% DMSO at 5 mM stock concentrations. The final concentration of DMSO in the reactions was 1%, and this level of DMSO had no effect on enzyme activity. The HNE inhibition assay was performed in black flat-bottom 96-well microtiter plates. Briefly, a mixture of 200 mM Tris–HCl, pH 7.5, 0.01% bovine serum albumin (Fisher Scientific), 0.05% Tween-20, and 20 mU/mL of HNE (Calbiochem) was added to wells containing different concentrations of each compound. The reaction was 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 measurements 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 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%.

Molecular modeling

Initial structures of the compounds 2c, 4a, 7d, and A1–A3 (Tables 1,2) 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, version 4.2.0 (CLC Bio, København, Denmark), as described previously [Giovannoni et al., 2015]. The structure of HNE complexed with a peptide chloromethyl ketone inhibitor was used for the docking study (1HNE from the Protein Data Bank). The search area for docking poses was defined as a sphere with 10 Å radius centered at the nitrogen atom 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 as reported previously [Giovannoni et al., 2015]. These flexible residues included the catalytic triad of Ser195, His57, and Asp102. 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 the Molegro program were as described previously [Giovannoni et al., 2015].

Table 1.

HNE inhibitory activity of indole derivatives 2a–g, 3a,b, 4a,b, 5a–g.

graphic file with name nihms818634t1.jpg
Comp R3 X R IC50 (µM)a
2a COOEt - m-CH3 NAb
2b COOEt CH2 m-CH3 NAb
2c COOEt CO m-CH3 NAb
2d COOEt CO p-CH3 NAb
2e COOEt CO p-Cl 3.8 ± 0.4
2f COOEt CO m-Cl NAb
2g COOEt CO p-F 12.2 ± 2.3
3a CH2COOEt CO m-CH3 NAb
3b CH2COOEt CO p-CH3 NAb
4a CN CO m-CH3 NAb
4b CN CO p-CH3 10.1 ± 1.3
5a CONHPh CO p-Cl NAb
5b CONHPh CO m-Cl NAb
5c CONHPh CO p-CH3 45.3 ± 6.7
5d CONHPh CO p-F NAb
5e CONHPh CO m-F NAb
5f CONHPh SO2 p-CH3 NAb
5g CONHPh SO2 p-Cl NAb
A1c graphic file with name nihms818634t2.jpg 0.41 ± 0.11
A2d graphic file with name nihms818634t3.jpg 0.007 ± 0.0015
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a

IC50 values are presented as the mean ± SD of three independent experiments.

b

NA: no inhibitory activity was found at the highest concentration of compound tested (50 µM).

Table 2.

HNE inhibitory activity of indole derivatives 7a–e, 8 and 9.

graphic file with name nihms818634t4.jpg
Comp R R1 IC50 (µM)a
7a 5-Br m-CH3 21.6 ± 3.3
7b 6-Br m-CH3 NAb
7c 6-Br p-CH3 10.5 ± 1.7
7d 5-NO2 m-CH3 2.4 ± 0.4
7e 5-NO2 p-CH3 13.2 ± 1.8
8 5-NH2 p-CH3 NAb
9 5-NHCOCH3 p-CH3 NAb
A3c graphic file with name nihms818634t5.jpg 0.02 ± 0.028
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a

IC50 values are presented as the mean ± SD of three independent experiments.

b

NA: no inhibitory activity was found at the highest concentration of compound tested (50 µM).

RESULTS AND DISCUSSION

All compounds were evaluated for their ability to inhibit HNE, and the results are reported in Tables 1–3 together with activity values of some potent N-benzoylindazoles (A1–A3) that we previously synthesized [Crocetti et al., 2011; Crocetti et al., 2013]. From a first analysis of the biological data, it appears evident that the indole nucleus is not as effective as the indazole nucleus for HNE inhibitors, since the most active compounds had IC50 values in the micromolar range, and many of the synthesized compounds were inactive at the highest tested concentrations (50 µM). On the other hand, these results do provide new information on the importance of various substituents in developing compounds with HNE inhibitory activity.

Table 3.

HNE inhibitory activity of indole derivatives 11a–d.

graphic file with name nihms818634t6.jpg
Comp R R1 IC50 (µM)a
11a H m-CH3 NAb
11b H p-CH3 NAb
11c NO2 m-CH3 NAb
11d NO2 p-CH3 NAb
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a

IC50 values are presented as the mean ± SD of three independent experiments.

b

NA: no inhibitory activity was found at the highest concentration of compound tested (50 µM).

As shown in Table 1, the introduction of functions and groups (COOEt, CN, CONHPh) at position 3 led of the indazole series and resulted in several compounds with activity in the micromolar range (2e, 2g, 4b, and 5c with IC50 values of 3.8 µM, 12.2 µM, 10.1 µM, and 45.3 µM, respectively). All other compounds were inactive. Interestingly, we found opposite activities between the pairs of isomers 2e/2f and 4a/4b. In both pairs, the derivative with the benzoyl fragment substituted in the meta position was inactive (i.e., compounds 2f and 4a), whereas the corresponding para-substituted derivatives had appreciable activity. This trend was in contrast to that found for the indazole series, where both isomers were active [Crocetti et al., 2013]. Furthermore, inactive 4a is the deaza analogue of reference compound A2, which is the most potent HNE inhibitor we have synthesized to date (IC50 = 7 nM). Although compounds 2a,b and 5f,g were synthesized with the goal of confirming the importance of the carbonyl group at N-1, the lack of activity of these compounds suggests this molecular feature is not the point of attack of serine OH.

The introduction at position 5 or 6 of substituents that led to the best results in the previous series of compounds [Crocetti et al., 2013], such as nitro, bromine, and acetamido, led only to compounds with low HNE inhibitory activity (7a, IC50 = 21.6 µM; 7c, IC50 = 10.5 µM; 7d, IC50 = 2.4 µM; 7e, IC50 = 13.2 µM) or no activity (7b, 8 and 9) (Table 2). The same results were found by moving the carbethoxy function from position 3 to position 2 (Table 3).

Our docking studies showed that pairs of molecules differing in the presence or absence of pyrazole-type nitrogen atom in the five-membered heterocycle (A1 vs. 2c and A2 vs. 4a, respectively) exhibited differences in molecule orientation within the receptor cavity. However, molecules A3 and 7d had almost coinciding positions of the m-tolyl and heterocyclic moieties with the ester and amide fragments. According to the general catalytic mechanism of serine proteases, the ligand forms a Michaelis complex through attack of its electron-deficient atom (usually carbonyl carbon) at the Ser195 hydroxyl group. This complex formation is accompanied by the proton transfer from Ser195 to Asp102 via His57. Length L of the proton transfer channel is one of the factors influencing the interaction of a ligand with HNE. The other factor is the geometry of the Michaelis complex. It was reported earlier [Vergely et al., 1996; Peters and Merz, 2006] that the value of angle α between the C=O bond in the ligand and the Ser195…CO axis should lie within 80…120° for effective binding of the carbonyl group to Ser195 (Figure 6). Although docking poses are not identical for Michaelis complexes, geometry of the poses can be used for the evaluation of complexation possibilities. Specifically, the amide carbonyl group was located in the vicinity of HNE Ser195 for all of the compounds evaluated in the docking study except for inhibitor A1, which has an ester carbonyl group orientation favorable for interaction with the catalytic triad (Ser195, His57, and Asp102).

Figure 6.

Figure 6

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Geometric parameters important for formation of a Michaelis complex in the HNE active site [an example is shown for compounds 4a (X=CH) and A2 (X=N)]. Based on the model of synchronous proton transfer from the oxyanion hole in HNE [Vergely et al., 1996; Peters and Merz, 2006].

The geometric characteristics of pose orientations and arrangements of the triad were in general agreement with the inhibitory activities of the compounds towards HNE (Table 4). Thus, the proton transfer channel had significantly shorter lengths for active compounds A1 and A2 (L value is between 5 and 6 Å) compared to their inactive indole counterparts 2c and 4a, respectively (L > 8 Å). HNE inhibitor A3 had a geometry of the Ser195…C=O fragment more preferable for interaction with the catalytic triad than its less active indole analogue 7d, which had a large angle α of 147°. Although the docking pose of A3 was characterized by a slightly higher L value than the pose for 7d, the magnitude of α for A3 fell into the optimum interval (see Table 4).

Table 4.

Biological activities and geometric parameters of the enzyme–inhibitor complexes predicted by molecular docking

Comp IC50 (µM)a α d1 d2 d3 Lb
A1c 0.4 84.8 2.529 2.361, 3.688 3.502 5.863
2c NA 91.2 3.681 5.713, 5.989 2.454 8.167
A2 0.007 105.2 3.448 2.181, 3.755 3.142 5.323
4a NA 78.5 3.936 5.756, 5.919 2.480 8.236
A3 0.02 116.9 4.078 3.056, 4.281 3.119 6.175
7d 2.4 147.0 4.260 2.828, 3.920 2.219 5.747
Open in a new tab
a

HNE inhibitory activity.

b

The length of the proton transfer channel was calculated as L = d3 + min(d2).

c

According to the docking results, Michaelis complex with Ser195 is formed with participation of the ester carbonyl group.

We have found that the pyrazole-type nitrogen of indazoles forms H-bonds with Ser195 (inhibitor A1) or Gly193 (inhibitor A2) (see an example in Figure 7). The carbonyl group in A3 is strongly H-bonded to Val216, while the corresponding indole counterpart 7d did not form H-bonds with HNE. Perhaps, these H-bonding interactions play an important role in the proper anchoring of inhibitors within the HNE binding site and can be used to explain differences in inhibitory properties of indazole and indole derivatives.

Figure 7.

Figure 7

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Docking poses of HNE inhibitor A1 (panel A) and compound 2c (panel B). Ser195 of HNE is H-bonded with the pyrazole-type nitrogen of inhibitor A1 (light-blue dashed line). Residues within 4 Å of the pose are shown.

CONCLUSIONS

We found that the new indazole derivatives designed as deaza analogues of our potent N-benzoylindazoles are weak HNE inhibitors or even inactive. Thus, these results suggest the crucial role of the nitrogen at position 2 of the heterocyclic scaffold. The biological results are supported by docking studies, which highlighted the different orientation within the receptor cavity for indazoles and indoles: the former exhibiting H-bonding interactions favorable for interaction with the catalytic triad, while the latter were characterized by unfavorable anchoring.

Acknowledgments

Funding: This research was supported in part by National Institutes of Health IDeA Program COBRE Grant GM110732; a USDA National Institute of Food and Agriculture Hatch project; Montana University System Research Initiative: 51040-MUSRI2015-03; and the Montana State University Agricultural Experiment Station.

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