Design, synthesis and biological evaluation of tetracyclic azafluorenone derivatives with topoisomerase I inhibitory properties as potential anticancer agents

Tsung-Chih Chen; Dah-Shyong Yu; Shiag-Jiun Chen; Chun-Liang Chen; Chia-Chung Lee; Ying-Yu Hsieh; Lien-Cheng Chang; Jih-Hwa Guh; Jing-Jer Lin; Hsu-Shan Huang

doi:10.1016/j.arabjc.2016.06.014

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Original article

12 (

); 4348-4364

doi:

10.1016/j.arabjc.2016.06.014

Design, synthesis and biological evaluation of tetracyclic azafluorenone derivatives with topoisomerase I inhibitory properties as potential anticancer agents

Tsung-Chih Chen^{^a,^b,^c}, Dah-Shyong Yu^{^b,^d}, Shiag-Jiun Chen^{^c}, Chun-Liang Chen^{^a,^b}, Chia-Chung Lee^{^a,^b}, Ying-Yu Hsieh^{^c}, Lien-Cheng Chang^{^e,^f}, Jih-Hwa Guh^{^g}, Jing-Jer Lin^{^e,⁎}, Hsu-Shan Huang^{^a,^b,^c,⁎}

a Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan

b Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan

c School of Pharmacy, National Defense Medical Center, Taipei 114, Taiwan

d Uro-Oncology Laboratory, Division of Urology, Department of Surgery, Tri-Service General Hospital and Institute of Preventive Medicine, National Defense Medical Center, Taipei 114, Taiwan

e Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei 100, Taiwan

f Food and Drug Administration, Ministry of Health and Welfare, Taipei 115, Taiwan

g School of Pharmacy, National Taiwan University, Taipei 100, Taiwan

⁎Corresponding authors at: Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan. Fax: +886 2 66387537 (H.-S. Huang). Fax: +886 2 2391 5295 (J.-J. Lin). jingjerlin@ntu.edu.tw (Jing-Jer Lin), huanghs99@tmu.edu.tw (Hsu-Shan Huang)

Received: 2015-12-31, Accepted: 2016-6-20, Published: 2016-12-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Several 9-chloro-11H-indeno[1,2-c]quinolin-11-one derivatives have been designed which is replacing side chains with different groups containing oxygen, nitrogen or sulfur atoms. Substitution of C-6 on the starting structure, 6,9-dichloro-11H-indeno[1,2-c]quinolin-11-one, using apposite nucleophilic group with a suitable base or acid could be obtained 28 novel tetracyclic azafluorenone derivatives. The cytotoxic activity of these analogues was examined in cancer cell lines by MTT assay and compounds 4, 5, 13, and 26 were selected to evaluate in topoisomerase I drug screening assay, respectively. At the same time, 17 compounds were selected for NCI-60 anticancer drug screen to prevent the narrower concept of an in vitro screening model. Its worth to find that 9-chloro-6-(piperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (12) showed greater cytotoxicity than another azafluorenone derivatives with an average GI₅₀ of 10.498 μM over 60 cell lines. We also found that another analogue, 9-chloro-6-(2-methylpiperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (13), exhibited preferential growth inhibition effect toward cancer cell lines and showed a significant inhibitory effect on topoisomerase I.

Keywords

Indenoquinolinone

Topoisomerase I

Azafluorenone

Cytotoxicity

NCI-60 anticancer drug screen

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1 Introduction

Camptothecin (CPT) is a cytotoxic quinoline alkaloid, as well as TAS-103 is a quinoline derivative which has been reported to be potent topoisomerase (topo) I and/or topo II poison (Aoyagi et al., 2000; Fujimoto, 2007; Yoshida et al., 2008; Shah et al., 2010 ). Two clinically anticancer drugs, irinotecan and topotecan, are derivatives of the CPT family that are used in colorectal cancer, either as single agents or in combination with radiotherapy and/or other chemotherapy drugs (Kehrer et al., 2001; Alagoz et al., 2012). Despite clinical success, they are the only Topo I-targeted anticancer drugs which still have several problems with CPT-derived anticancer agents (Teicher, 2008). Ongoing research aims to the lactone and A-D planar ring in CPT and its analogues and derivatives which suffer from major limitation and application drawback of non-mechanism related toxicity and poor solubility profile (Ulukan and Swaan, 2002; Kiselev et al., 2012; Perzyna et al., 2003 ). Because the direct target of above small molecules remained unclear, we tried structural hybridization of some preclinical and clinical anticancer drugs to design tetracyclic azafluorenone-derived small molecules as potential anticancer drugs (Fig. 1).

Structural hybridization design of tetracyclic quinoline derivatives.

The anthracycline antibiotics (e.g. doxorubicin, daunorubicin, mitoxantrone and ametantrone) have been shown to provide significant antiproliferative (or cytostatic) properties (Chen et al., 2013, 2015) and inhibit topoisomerase II (Suzuki et al., 1995) as well as intercalate into the minor groove of double-strand DNA base pairs via the core planar pharmacophore group (Wang et al., 1994). In addition, DMMA (6,8-dihydroxy-7-methoxy-1-methyl-3-azafluorenone), an active compound of the azafluorenone, was isolated from Polyalthia cerasoides in 2010 (Pumsalid et al., 2010) which exhibits cytotoxic to various cancer cell lines (Banjerdpongchai et al., 2013). Moreover, recent studies illustrated related indenoquinoline skeleton incorporate ring system and various substituents had been found as potential dual topo I/II inhibitors and anticancer candidates (Tseng et al., 2013, 2010, 2009, 2008 ). There are also several studies have found that azafluorenone-derived compounds had potent anticancer (Coothankandaswamy et al., 2010; McLaughlin, 2008; Pan et al., 2011 ), cytotoxic (Pumsalid et al., 2010), antimicrobial (Koyama et al., 2005; Addla et al., 2012), antitubercular (Yempala et al., 2012), anti-inflammatory (Chuang et al., 2008; Rojano et al., 2007) and antiprotozoal activities (Waechter et al., 1999).

Based on above considerations, azafluorenones can be considered as an attractive scaffold for the development of anticancer reagents. In the past, several studies had found that DNA top IB can regulate DNA topological structures by sequential breakage and is involved in DNA transcription, replication, and recombination (Shen et al., 2010; Stewart et al., 1998). Toward supporting the aforementioned hypothesis, we recently disclosed evidence as well as an in-depth continuous investigation toward synthesis of fluorenone analogs (Lee et al., 2013) led to discovery of azafluorenone-derived scaffold small molecules for the polypharmacology. With this motivation, we herein also describe efficient synthetic procedures for the preparation of a series of non-nucleoside fused tetracyclic compounds comprising benzene moiety and an azafluorenone backbone. We attempted to identify additional potent enzyme inhibitors and work toward some azafluorenone-derived structural modifications in order to develop some potential anticancer structures leads which could be compared to parent molecules in cancer chemotherapy.

2 Chemistry

2.1

2.1 Materials and instruments

The Pfitzinger synthetic reaction of isatin and 4-chlorophenylacetic acid was stirred at 200 °C with sodium acetate (NaOAc) as a basic catalyst for 3 h to obtain compound 1 (52%) as a key intermediate. An isatin was reacted with NaOAc as base by the amide bond hydrolysis to obtain 2-(2-aminophenyl)-2-oxoacetic acid. A reactant with a ketone group reacted with the aniline to give the imine and the enamine. The enamine cyclized and dehydrated to give the desired quinoline-4-carboxylic acids (1). Treatment of 1 with phosphoryl trichloride (POCl₃) afforded 6,9-dichloro-11H-indeno[1,2-c]quinolin-11-one (2). A series of 6-substituted 9-chloro-11H-indeno[1,2-c]quinolin-11-one homologues can be synthesized and the preparation involved various synthetic routes with approximate yields (overall 17–95%) in all steps: (i) reaction of isatin with 4-chlorophenylacetic acid and NaOAc; (ii) reaction of 1 with POCl₃; and (iii) reaction of compound 2 with a series of apposite primary or secondary amines, 2-mercaptoethanol, conc. hydrochloride (HCl), or sodium methoxide yielded the corresponding side chain compounds 3–29, respectively (Scheme 1). The quantity of the isolated products was dependent on substrate and reaction condition. All the crude mixtures were purified through tedious recrystallization from ethyl acetate/n-hexane and/or ethanol. The molecular weight of all synthetic compounds was determined by HRMS. The protons and carbons from tetracyclic azafluorenone structures were also obvious from the ¹H NMR and ¹³C NMR spectra.

Overall synthetic routes of the tetracyclic azafluorenone derivatives. Reagents and conditions: (i) 1: NaOAc, 200 °C, 2 h; (ii) 2: POCl3, 150 °C, reflux, 48 h; (iii) 3–26: appropriate amines, DMF, pyridine, miniclave, 150 °C, 2 h; 27: mercaptoethanol, K2CO3; 28: DMF, conc. HCl, reflux, 24 h; 29: NaOMe, MeOH, reflux, 16 h.

2.2

2.2 Synthesis of target compounds 1–29

The synthetic methods and physical data of compounds 1–29 were described and all compounds were tested and compared for their growth inhibition, cytotoxicity and topoisomerase activities. In this study, we synthesized and dedicated on the role of our systematic, tetracyclic, and heterocyclic pharmacophore by introducing a series of side chains linked to the 9-chloro-11H-indeno[1,2-c]quinolin-11-one moiety.

All reactions were monitored through a TLC (silica gel 60 F₂₅₄) plate with a 254-nm UV lamp. ¹H NMR and ¹³C NMR were measured on Varian GEMINI-300 (300 MHz) or Agilent 400 MR DD2 (400 MHz); δ values are in ppm relative to TMS (tetramethylsilane) as an internal standard. Multiplicities are recorded as s (singlet), d (doublet), t (triplet), q (quartet), quin (quintuplet), dd (doublet of doublets), dt (triplet of doublets), td (doublet of triplets), m (multiplet), and br (broadened). Mass spectra: High resolution electrospray ionization (HRESI): Finnigan MAT 95S (Instrumentation Center, National Taiwan University, Taipei, Taiwan) and High resolution electron impact ionization (HREI): Finnigan MAT MAT-95XL (Instrumentation Center, National Tsing Hua University, Hsinchu, Taiwan). Melting points of synthetic compounds were determined with a Büchi B-545 melting point apparatus. Typical experiments illustrating the synthetic procedures for the preparation of the tetracyclic small molecules are described below. These compounds were synthesized, starting from isatin and 4-chloro-phenylacetic acid. All the reagents and solvents required for synthesis were purchased from either Merck Chemical Company or Sigma–Aldrich Chemical Company without further purification.

2.2.1

2.2.1 Synthetic procedure i: preparation of compound 1

A mixture of isatin (3.14 g, 21 mmol), 4-chloro-phenylacetic acid (3.41 g, 20 mmol), and sodium acetate (1.00 g) was heated at 200 °C for 2 h (TLC monitoring). After cooling, 100 mL acetic acid was added to the mixture. The precipitate was filtrated and washed with acetic acid and n-hexane, and then collected the obtained orange compound.

2.2.2

2.2.2 Synthetic procedure ii: preparation of compound 2

A suspension of compound 1 (3.03 g, 10.1 mmol) and POCl₃ (20 mL) was stirred and heated at 150 °C for 48 h. After cooling, the mixture was poured into ice-water (300 mL) at 0 °C cautiously. The resulting precipitate that separated was collected by filtration. The filtered cake was suspended in 10% NaHCO₃ solution (300 mL) with vigorous stirring for 1 h. The resulting precipitate was collected and washed with H₂O. The crude was recrystallized from dichloromethane to give an orange product.

2.2.3

2.2.3 General procedure iii: preparation of compounds 3–26

Compound 2, primary or secondary amine (10 mmol) and N,N-diisopropylethylamine (DIPEA) (2 mmol) were dissolved in miniclave containing DMF (10 mL) and stirred at 150 °C for 4 h. The reaction was poured into ice-water (100 mL). The resulting precipitate was collected by filtration and purified by crystallization from ethanol to afford desired compound.

2.2.4

2.2.4 Synthetic procedure iv: preparation of compound 27

Compound 2, 2-mercaptoethanol (10 mmol) and N,N-diisopropylethylamine (DIPEA) (2 mmol) were dissolved in miniclave containing DMF (10 mL) and stirred at room 150 °C for 4 h. The reaction was poured into ice-water (100 mL). The resulting precipitate was collected by filtration and purified by crystallization from ethanol to afford desired compounds.

2.2.5

2.2.5 Synthetic procedure v: preparation of compound 28

A mixture of compound 2, conc. HCl (2 mL) and DMF (10 mL) was refluxed at 120 °C. After 4 h, the conc. HCl was added into the reaction again (TLC monitored). The mixture was evaporated in vacuum or dean-stark trap, treated with H₂O (20 mL), and filtered. The crude solid was washed with EtOH to give 28 as a red solid.

2.2.6

2.2.6 Synthetic procedure vi: preparation of compound 29

A methanol solution (20 mL) containing sodium methoxide (1.08 g, 20 mmol) was slowly added into the suspension of compound 2 in MeOH (10 mL) for 10 min. The reaction was refluxed at 100 °C for 16 h (TLC monitored). After cooled, the solvent was removed by rotary evaporator vacuum, filtrated and washed with ethanol and n-hexane to collect an orange solid.

2.3

2.3 Physical data

2.3.1

2.3.1 3-(4-Chlorophenyl)-2-hydroxyquinoline-4-carboxylic acid (1)

The pure compound was obtained as an orange solid (yield 80%). Mp 310–311 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 3234 (NH), 1637 (CO). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 6.60 (s, 1H, —OH), 6.91 (td, J = 7.2 Hz, 0.6 Hz, 1H, Ar—H), 7.12 (d, J = 8.4 Hz, 1H, Ar—H), 7.51 (d, J = 8.7 Hz, 2H, Ar—H), 7.53–7.59 (m, 2H, Ar—H), 7.74 (d, J = 8.7 Hz, 2H, Ar—H), 9.83 (s, 1H, —COOH). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 117.12, 120.66, 121.83, 122.20, 126.31, 128.86, 129.15, 132.67, 134.22, 135.21, 136.23, 139.59, 182.29 (CO). HRMS (ESI) m/z calcd for C₁₆H₁₀NO₃Cl⁺ [M]⁺ 299.0349, found [M+H]⁺: 300.0424, [M−H]⁻: 298.0238.

2.3.2

2.3.2 6,9-Dichloro-11H-indeno[1,2-c]quinolin-11-one (2)

Product 2 was cyclized from compound 1 using POCl₃ at 150 °C for 48 h. The red solid material was isolated in 30% yield. (R_f = 0.70 at CH₂Cl₂). Mp 241–243 °C. FT-IR (KBr; ν cm⁻¹): 1719 (C⚌O). ¹H NMR (300 MHz, CDCl₃) δ (ppm): 7.52 (dd, J = 8.25, 1.8 Hz, 1H, Ar—H), 7.62–7.68 (m, 2H, Ar—H), 7.70–7.76 (m, 1H, Ar—H), 7.97–8.01 (dt, J = 7.5, 0.6 Hz, 1H, Ar—H), 8.10 (d, J = 7.8 Hz, 1H, Ar—H), 8.77–8.80 (m, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃) δ (ppm): 122.97, 124.59, 125.30, 125.69, 129.13, 130.32, 131.70, 135.06, 135.14, 136.19, 136.74, 136.80, 140.15, 145.25, 150.48, 192.80 (CO). HRMS (ESI) m/z calcd for C₁₆H₇NOCl₂⁺, [M]⁺: 298.9905, found [M+H]⁺: 299.9965 (100), 301.9947 (65), 303.9917 (10).

2.3.3

2.3.3 9-Chloro-6-(methylamino)-11H-indeno[1,2-c]quinolin-11-one (3)

Product 3 was prepared from compound 2 and methylamine. The red solid material was isolated in 75% yield (R_f = 0.51 at CH₂Cl₂: n-hexane = 2:1). Mp 189–191 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1716 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 3.01 (s, 3H, N—CH₃), 7.41–7.47 (m, 2H, Ar—H), 7.57–7.62 (m, 3H, Ar—H), 7.84 (d, J = 8.4 Hz, 1H, Ar—H), 8.68 (d, J = 8.1 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 42.22, 120.81, 124.29, 124.93, 124.99, 127.02, 128.13, 130.44, 131.60, 134.33, 135.16, 135.24, 136.59, 141.85, 149.69, 158.14, 194.48 (CO). HRMS (ESI) m/z calcd for C₁₇H₁₁N₂OCl⁺ [M]⁺: 294.0560, found [M+H]⁺: 295.0634.

2.3.4

2.3.4 9-Chloro-6-(dimethylamino)-11H-indeno[1,2-c]quinolin-11-one (4)

Product 4 was prepared from compound 2 and dimethylamine. The red solid material was isolated in 74.5% yield (R_f = 0.51 at CH₂Cl₂: n-hexane = 1:1). Mp 193–195 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 3407 (NH), 1718 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 3.04 (s, 6H, —N—(CH₃)₂), 7.41–7.48 (m, 2H, Ar—H), 7.57–7.63 (m, 3H, Ar—H), 7.86 (d, J = 8.7 Hz, 1H, Ar—H), 8.68–8.71 (m, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 42.19, 120.80, 124.27, 124.91, 124.96, 126.99, 128.13, 130.40, 131.57, 134.30, 135.14, 135.22, 136.54, 141.84, 149.70, 158.13, 194.45 (CO). HRMS (EI) m/z calcd for C₁₈H₁₃N₂OCl⁺ [M]⁺: 308.0716, found 308.0708.

2.3.5

2.3.5 6-(2-(Diethylamino)ethylamino)-9-chloro-11H-indeno[1,2-c]quinolin-11-one (5)

Product 5 was prepared from compound 2 and N¹,N¹-diethylethane-1,2-diamine. The red solid material was isolated in 17.5% yield (R_f = 0.46 at CH₂Cl₂: n-hexane = 2:1). Mp 160–161 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 3317 (NH), 1712 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 1.11 (t, J = 7.2 Hz, 6H, —CH₃), 2.66 (q, J = 7.1 Hz, 4H, —NCH₂—), 2.84 (t, J = 5.7 Hz, 2H, —CH₂N—), 3.71–3.73 (m, 2H, —NHCH₂—), 6.14 (br, 1H, NH), 7.28–7.33 (m, 1H, Ar—H), 7.41–7.55 (m, 3H, Ar—H), 7.60 (d, J = 1.5 Hz, 1H, Ar—H), 7.70 (d, J = 8.7 Hz, 1H, Ar—H), 8.60 (m, J = 8.1 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 12.23, 38.81, 46.92, 51.64, 119.00, 122.48, 124.42, 124.95, 125.40, 126.94, 127.86, 130.53, 134.07, 134.90, 135.08, 135.45, 141.24, 150.68, 152.96, 194.65 (CO). HRMS (ESI) m/z calcd for C₂₂H₂₂N₃OCl⁺ [M]⁺: 379.1451, found[M+H]⁺: 380.1510.

2.3.6

2.3.6 9-Chloro-6-(pyrrolidin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (6)

Product 6 was prepared from compound 2 and pyrrolidine. The red solid material was isolated in 43.6% yield (R_f = 0.51 at CH₂Cl₂: n-hexane = 2:1). Mp 149–150 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1718 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 1.98 (quin, J = 3.6 Hz, 4H, pyrrolidine-H), 3.56 (t, J = 6.6 Hz, 4H, pyrrolidine-H), 7.33–7.42 (m, 3H, Ar—H), 7.54 (td, J = 7.5, 1.5 Hz, 2H, Ar—H), 7.76 (d, J = 8.4 Hz, 1H, Ar—H), 8.63 (dd, J = 8.4, 0.9 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 25.07, 50.37, 120.39, 124.25, 124.80, 124.96, 126.29, 127.78, 130.33, 130.40, 134.10, 134.83, 135.27, 136.46, 142.50, 149.78, 155.73, 194.65 (CO). HRMS (ESI) m/z calcd for C₂₀H₁₅N₂OCl⁺ [M]⁺: 334.0873, found [M+H]⁺: 335.0952.

2.3.7

2.3.7 9-Chloro-6-(piperidin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (7)

Product 7 was prepared from compound 2 and piperidine. The red solid material was isolated in 43% yield (R_f = 0.63 at CH₂Cl₂: n-hexane = 2:1). Mp 191–192 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1717 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 1.71 (br, 2H, piperidine-H), 1.83–1.86 (m, 4H, piperidine-H), 3.33 (br, 4H, piperidine-H), 7.43–7.48 (m, 2H, Ar—H), 7.58–7.66 (m, 3H, Ar—H), 7.90 (d, J = 8.1 Hz, 1H, Ar—H), 8.70 (d, J = 8.7 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 224.37, 26.01, 51.30, 120.81, 124.22, 124.41, 124.83, 127.09, 128.29, 130.24, 131.88, 134.24, 135.01, 135.17, 136.35, 142.05, 149.79, 158.35, 194.38 (CO). HRMS (ESI) m/z calcd for C₂₁H₁₇N₂OCl ⁺ [M]⁺: 348.1029, found [M+H]⁺: 349.1106.

2.3.8

2.3.8 9-Chloro-6-(4-methylpiperidin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (8)

Product 8 was prepared from compound 2 and 4-methylpiperidine. The brown solid material was isolated in 25.4% yield (R_f = 0.61 at CH₂Cl₂: n-hexane = 2:1). Mp 190–192 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1718 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 1.07 (d, J = 6 Hz, 3H, —CH₃), 1.46–1.60 (m, 3H, —CH₂—, —CH—), 1.84–1.87 (m, 2H, —CH₂—), 2.96 (t, J = 11.6 Hz, 2H, —NCH₂—), 3.67–3.71 (m, 2H, —NCH₂—), 7.44–7.46 (m, 2H, Ar—H), 7.58 (m, 3H, Ar—H), 7.82–7.85 (m, 1H, Ar—H), 8.66–8.69 (m, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 22.07, 30.87, 34.43, 50.69, 120.88, 124.27, 124.46, 124.91, 127.15, 128.31, 130.32, 131.98, 134.33, 135.07, 135.23, 136.44, 142.13, 149.83, 158.25, 194.52 (CO). HRMS (ESI) m/z calcd for C₂₂H₁₉N₂OCl⁺ [M]⁺: 362.1186, found [M+H]⁺: 363.1260.

2.3.9

2.3.9 6-(Azepan-1-yl)-9-chloro-11H-indeno[1,2-c]quinolin-11-one (9)

Product 9 was prepared from compound 2 and azepane. The red solid material was isolated in 36% yield (R_f = 0.69 at CH₂Cl₂: n-hexane = 2:1). Mp 146–147 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1712 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 1.72–1.75 (m, 4H, —CH₂—), 1.86 (br, 4H, —CH₂—), 3.64 (t, J = 5.6 Hz, 4H, —NCH₂—), 7.40–7.46 (m, 2H, Ar—H), 7.54–7.60 (m, 3H, Ar—H), 7.78–7.80 (m, 1H, Ar—H), 8.68 (dd, J = 8.4, 0.6 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 27.96, 28.40, 52.91, 120.48, 124.24, 124.76, 124.89, 126.60, 127.98, 130.30, 131.09, 134.13, 134.97, 135.15, 136.84, 142.54, 149.74, 157.83, 194.62 (CO). HRMS (ESI) m/z calcd for C₂₂H₁₉N₂OCl⁺ [M]⁺: 362.1186, found [M+H]⁺: 363.2000.

2.3.10

2.3.10 9-Chloro-6-morpholino-11H-indeno[1,2-c]quinolin-11-one (10)

Product 10 was prepared from compound 2 and morpholine. The red solid material was isolated in 47% yield (R_f = 0.54 at CH₂Cl₂: n-hexane = 2:1). Mp 207–208 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1712 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 3.41 (t, J = 4.5 Hz, 4H, —CH₂—), 3.98 (t, J = 4.5 Hz, 4H, —CH₂—), 7.48 (td, J = 8.1, 2.1 Hz, 2H, Ar—H), 7.59–7.65 (m, 3H, Ar—H), 7.87 (d, J = 8.7 Hz, 1H, Ar—H), 8.69–8.72 (dt, J = 8.1, 0.9 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 50.56, 66.98, 121.18, 124.35, 124.43, 125.23, 127.65, 128.51, 130.59, 131.46, 134.42, 135.21, 135.55, 136.80, 141.66, 149.81, 157.31, 194.14 (CO). HRMS (ESI) m/z calcd for C₂₀H₁₅N₂O₂Cl⁺ [M]⁺: 350.0822, found [M+H]⁺: 351.0898.

2.3.11

2.3.11 9-Chloro-6-thiomorpholino-11H-indeno[1,2-c]quinolin-11-one (11)

Product 11 was prepared from compound 2 and thiomorpholine. The orange solid material was isolated in 74% yield (R_f = 0.33 at CH₂Cl₂: n-hexane = 2:1). Mp 228–230 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1711 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 2.91 (t, J = 5.1 Hz, 4H, —CH₂—), 2.69–2.73 (br, 4H, —CH₂—), 7.45–7.50 (td, J = 7.8, 1.8 Hz, 2H, Ar—H), 7.57–7.64 (m, 3H, Ar—H), 7.85 (d, J = 9.0 Hz, 1H, Ar—H), 8.68–8.71 (dd, J = 8.25, 1.2 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 27.38, 52.36, 121.07, 124.29, 125.19, 127.66, 128.48, 129.44, 130.57, 131.59, 134.40, 135.10, 135.54, 136.87, 141.70, 149.70, 157.66, 194.17 (CO). HRMS (ESI) m/z calcd for C₂₀H₁₅N₂OSCl ⁺ [M]⁺: 366.0594, found [M+H]⁺: 367.0664.

2.3.12

2.3.12 9-Chloro-6-(piperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (12)

Product 12 was prepared from compound 2 and piperazine. The red solid material was isolated in 48% yield (R_f = 0.43 at CH₂Cl₂: n-hexane = 2:1). Mp 180–181 °C. FT-IR (KBr; ν cm⁻¹): 3341 (NH), 1718 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 3.16 (t, J = 4.8 Hz, 4H, —CH₂—), 3.36 (br, 4H, —CH₂—), 7.46–7.49 (m, 2H, Ar—H), 7.62–7.66 (m, 3H, Ar—H), 7.87 (d, J = 8.7 Hz, 1H, Ar—H), 8.71 (d, J = 8.7 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 46.17, 51.51, 121.05, 124.31, 124.54, 125.11, 127.45, 128.46, 130.48, 131.69, 134.39, 135.16, 135.40, 136.68, 141.88, 149.84, 157.83, 194.37 (CO). HRMS (ESI) m/z calcd for C₂₀H₁₆N₃OCl⁺ [M]⁺: 349.0982, found [M+H]⁺: 350.1063.

2.3.13

2.3.13 9-Chloro-6-(2-methylpiperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (13)

Product 13 was prepared from compound 2 and 3-methylpiperazine. The red solid material was isolated in 18% yield (R_f = 0.49 at CH₂Cl₂: n-hexane = 4:1). Mp 199–200 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 3222 (NH), 1719 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 1.17 (d, J = 6.3 Hz, 3H, —CH₃), 2.70 (t, 1H, —CH₂—), 3.03–3.07 (m, 1H, —NCH—), 3.15–3.19 (m, 3H, —CH₂—, —CH₂NH—), 3.60–3.65 (d, J = 12.6 Hz, 2H, —NHCH₂—), 7.44–7.48 (m, 2H, Ar—H), 7.58–7.62 (m, 3H, Ar—H), 7.86 (d, J = 8.4 Hz, 1H, Ar—H), 8.69 (d, J = 7.8 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 19.96, 45.98, 50.63, 50.75, 57.67, 120.98, 124.29, 124.47, 125.09, 127.39, 128.41, 130.47, 131.63, 134.37, 135.15, 135.36, 136.65, 141.89, 149.82, 157.56, 194.36 (CO). HRMS (ESI) m/z calcd for C₂₁H₁₈N₃OCl⁺ [M]⁺: 363.1138, found [M+H]⁺: 364.1201.

2.3.14

2.3.14 9-Chloro-6-(4-methylpiperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (14)

Product 14 was prepared from compound 2 and 1-methylpiperazine. The red solid material was isolated in 51% yield (R_f = 0.4 at CH₂Cl₂: n-hexane = 2:1). Mp 205–207 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 3462 (NH), 1720 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 2.42 (s, 3H, N-CH₃), 2.70 (br, 4H, —CH₂—), 3.43 (br, 4H, N—CH₂—), 7.44–7.47 (m, 2H, Ar—H), 7.58–7.61 (m, 3H, Ar—H), 7.84 (d, J = 8.4 Hz, 1H, Ar—H), 8.67 (d, J = 8.1 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 46.38, 49.97, 55.17, 121.00, 124.28, 124.54, 125.07, 127.38, 128.46, 130.45, 131.51, 134.35, 135.13, 135.38, 136.63, 141.84, 149.80, 157.37, 194.29 (CO). HRMS (ESI) m/z calcd for C₂₁H₁₈N₃OCl⁺ [M]⁺: 363.1138, found [M+H]⁺: 364.1222.

2.3.15

2.3.15 9-Chloro-6-(4-ethylpiperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (15)

Product 15 was prepared from compound 2 and 1-ethylpiperazine. The red solid material was isolated in 20% yield (R_f = 0.43 at CH₂Cl₂: n-hexane: MeOH = 2:1: 0.5). Mp 182–184 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1710 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 1.19 (t, 3H, J = 7.2 Hz, —CH₃), 2.57 (q, 2H, J = 7.4 Hz, —NCH₂—), 3.03 (br, 4H, —CH₂—), 3.46 (br, 4H, —CH₂—), 7.43–7.48 (m, 2H, Ar—H), 7.57–7.60 (m, 3H, Ar—H), 7.85 (d, J = 8.4 Hz, 1H, Ar—H), 8.69 (dd, J = 8.25, 0.9 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 12.06, 49.99, 52.69, 52.85, 121.02, 124.30, 124.59, 125.07, 127.36, 128.49, 130.43, 131.53, 134.36, 135.19, 135.38, 136.64, 141.89, 149.84, 15,740, 194.32 (CO). HRMS (ESI) m/z calcd for C₂₂H₂₀N₃OCl⁺ [M]⁺: 377.1295, found [M+H]⁺: 378.1380.

2.3.16

2.3.16 9-Chloro-6-(4-cyclopentylpiperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (16)

Product 16 was prepared from compound 2 and 1-cyclopentylpiperazine. The red solid material was isolated in 37% yield (R_f = 0.46 at CH₂Cl₂: n-hexane: MeOH = 2:1: 0.5). Mp 183–184 ^oC (EtOH). FT-IR (KBr; ν cm⁻¹): 1716 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 1.19 (t, 3H, J = 7.2 Hz, —CH₃), 2.57 (q, 2H, J = 7.4 Hz, —NCH₂—), 3.03 (br, 4H, —CH₂—), 3.46 (br, 4H, —CH₂—), 7.43–7.48 (m, 2H, Ar—H), 7.57–7.63 (m, 3H, Ar—H), 7.85 (d, J = 8.7 Hz, 1H, Ar—H), 8.69 (d, J = 8.1 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 24.32, 30.67, 50.12, 52.36, 67.87, 120.99, 124.28, 124.61, 125.05, 127.31, 128.50, 130.40, 131.56, 134.36, 135.19, 135.32, 136.58, 141.94, 149.85, 157.48, 194.40 (CO). HRMS (ESI) m/z calcd for C₂₅H₂₄N₃OCl⁺ [M]⁺: 417.1608, found [M+H]⁺: 418.1689.

2.3.17

2.3.17 9-Chloro-6-(4-(piperidin-1-yl)piperidin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (17)

Product 17 was prepared from compound 2 and 1-(piperidin-4-yl)piperidine. The red solid material was isolated in 57% yield. (R_f = 0.51 at CH₂Cl₂: n-hexane: MeOH = 2:1: 0.5). Mp 174–175 °C. FT-IR (KBr; ν cm⁻¹): 1718 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 1.48–1.50 (m, 2H, —CH₂—), 1.63–1.65 (m, 2H, —CH₂—), 1.72–1.85 (m, 4H, —CH₂—), 2.08 (d, J = 11.4 Hz, 2H, —CH₂—), 2.38–2.46 (m, 1H, —CH₂—), 2.60 (s, 4H, —CH—), 2.91–3.02 (m, 2H, —CH₂—), 3.76 (d, J = 12.3 Hz, 2H, —CH₂—), 7.41–7.45 (m, 2H, Ar—H), 7.55–7.61 (m, 3H, Ar—H), 7.82 (d, J = 8.4 Hz, 1H, Ar—H), 8.66 (d, J = 7.8 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 24.99, 26.61, 28.77, 50.17, 50.85, 62.51, 120.98, 124.27, 124.42, 124.96, 127.26, 128.36, 130.34, 131.86, 134.37, 135.08, 135.32, 136.45, 141.99, 149.80, 157.82, 194.39 (CO). HRMS (ESI) m/z calcd for C₂₆H₂₆ClN₃O⁺ [M]⁺: 431.1764, found [M+H]⁺: 432.1822.

2.3.18

2.3.18 9-Chloro-6-(4-phenylpiperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (18)

Product 18 was prepared from compound 2 and 1-phenylpiperazine. The red solid material was isolated in 52% yield (R_f = 0.91 at CH₂Cl₂: n-hexane: MeOH = 3:1:0.5). Mp 193–194 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1714 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 3.47 (br, 4H, —CH₂—), 3.57 (br, 4H, —CH₂—), 6.93 (t, J = 7.2 Hz, 1H, Ar—H), 7.04 (d, J = 7.8 Hz, 2H, Ar—H), 7.30–7.36 (m, 2H, Ar—H), 7.45–7.51 (m, 2H, Ar—H), 7.58–7.70 (m, 3H, Ar—H), 7.88 (d, J = 7.8 Hz, 1H, Ar—H), 8.72 (d, J = 8.1 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 49.44, 50.16, 116.66, 120.53, 121.14, 124.32, 124.52, 125.21, 127.60, 128.49, 129.57, 130.56, 131.60, 134.43, 135.16, 135.49, 136.68, 141.74, 149.78, 151.65, 157.41, 194.30 (CO). HRMS (ESI) m/z calcd for C₂₆H₂₀N₃OCl⁺ [M]⁺: 425.1295, found [M+H]⁺: 426.1370.

2.3.19

2.3.19 6-(4-Benzylpiperazin-1-yl)-9-chloro-11H-indeno[1,2-c]quinolin-11-one (19)

Product 19 was prepared from compound 2 and 1-benzylpiperazine. The red solid material was isolated in 41% yield (R_f = 0.37 at CH₂Cl₂: n-hexane = 2:1). Mp 178–180 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1718 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 2.73 (br, 4H, —CH₂—), 3.41 (br, 4H, —CH₂—), 3.65 (s, 2H, —CH₂—) 7.28–7.47 (m, 7H, Ar—H_8,10, Ar—H), 7.57–7.63 (m, 3H, Ar—H_2,3,4), 7.84 (d, J = 8.7 Hz, 1H, Ar—H₇), 8.68 (d, J = 7.05 Hz,1H, Ar—H₁). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 50.11, 53.21, 63.34, 120.97, 124.26, 124.60, 125.04, 127.34, 127.50, 128.42, 128.64, 129.40, 130.42, 131.61, 134.35, 135.11, 135.30, 136.53, 138.46, 141.83, 149.78, 157.55, 194.38 (CO). HRMS (ESI) m/z calcd for C₂₇H₂₂N₃OCl⁺ [M]⁺: 439.1451, found [M+H]⁺: 440.1503.

2.3.20

2.3.20 9-Chloro-6-(4-(2-fluorophenyl)piperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (20)

Product 20 was prepared from compound 2 and 1-(2-fluorophenyl)piperazine. The red solid material was isolated in 41% yield (R_f = 0.46 at CH₂Cl₂: n-hexane = 2:1). Mp 182–183 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1715 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 3.37 (br, 4H, —CH₂—), 3.59 (br, 4H, —CH₂—), 7.00–7.12 (m, 4H, Ar—H), 7.44–7.50 (m, 2H, Ar—H), 7.59–7.68 (m, 3H, Ar—H), 7.88 (d, J = 8.1 Hz, 1H, Ar—H), 8.71 (d, J = 8.1 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 50.27, 50.63, 116.47, 116.75, 119.48, 121.09, 123.06, 123.17, 124.31, 124.53, 124.80, 124.86, 125.17, 127.54, 128.49, 130.54, 131.55, 134.40, 135.16, 135.45, 141.76, 149.78, 157.37, 194.29 (CO). HRMS (ESI) m/z calcd for C₂₆H₁₉ClN₃O⁺ [M]⁺: 443.1201, found [M+H]⁺: 444.1269.

2.3.21

2.3.21 9-Chloro-6-(4-(2-methoxyphenyl)piperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (21)

Product 21 was prepared from compound 2 and 1-(2-methoxyphenyl)piperazine. The red solid material was isolated in 37% yield (R_f = 0.38 at CH₂Cl₂: n-hexane = 2:1). Mp 129–131 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1714 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 3.36 (br, 4H, —CH₂—), 3.60 (br, 4H, —CH₂—), 3.90 (s, 3H, —OCH₃), 6.91–7.07 (m, 4H, Ar—H), 7.47 (t, J = 7.5 Hz, 2H, Ar—H), 7.59–7.63 (m, 2H, Ar—H), 7.68 (d, J = 7.8 Hz, 1H, Ar—H), 7.87 (d, J = 8.1 Hz, 1H, Ar—H), 8.70 (d, J = 8.4 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 50.39, 50.75, 55.75, 112.15, 118.68, 121.03, 121.49, 123.55, 124.28, 124.61, 125.09, 127.40, 128.47, 130.46, 131.58, 134.36, 135.15, 135.36, 136.61, 141.66, 141.85, 149.81, 152.86, 157.53, 194.38 (CO). HRMS (ESI) m/z calcd for C₂₇H₂₂ClN₃O₂⁺ [M]⁺: 455.1401, found [M+H]⁺: 456.1473.

2.3.22

2.3.22 9-Chloro-6-(4-(3-methoxyphenyl)piperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (22)

Product 22 was prepared from compound 2 and 1-(3-methoxyphenyl)piperazine. The red solid material was isolated in 86% yield (R_f = 0.43 at CH₂Cl₂: n-hexane = 2:1). Mp 189–191 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1723 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 3.46 (br, 4H, —CH₂—), 3.55 (br, 4H, —CH₂—), 3.83 (s, 3H, —OCH₃), 6.50 (m, J = 8.1 Hz, 1H, Ar—H), 6.57 (s, 1H, Ar—H), 6.65 (d, J = 8.4 Hz, 1H, Ar—H), 7.22 (d, J = 8.1 Hz, 1H, Ar—H), 7.44–7.51 (m, 2H, Ar—H), 7.60–7.68 (m, 3H, Ar—H), 7.88 (d, J = 8.1 Hz, 1H, Ar—H), 8.71 (d, J = 7.2 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 49.36, 50.08, 55.46, 103.25, 105.29, 109.41, 121.14, 124.31, 124.50, 125.18, 127.60, 128.49, 130.25, 130.56, 131.56, 134.42, 135.15, 135.49, 136.68, 141.70, 149.77, 153.00, 157.35, 161.17, 194.23 (CO). HRMS (ESI) m/z calcd for C₂₇H₂₂ClN₃O₂⁺ [M]⁺: 455.1401, found [M+H]⁺: 456.1464.

2.3.23

2.3.23 9-Chloro-6-(4-(1-methylpiperidin-4-yl)piperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (23)

Product 23 was prepared from compound 2 and 1-(1-methylpiperidin-4-yl)piperazine. The red solid material was isolated in 31% yield (R_f = 0.90 at CH₂Cl₂: n-hexane: MeOH = 2:1: 0.5). Mp 208–209 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1710 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 1.64–1.72 (m, 2H, —CH₂—), 1.88 (d, J = 10.5 Hz, 1H, —CH₂—), 1.95–2.03 (m, 2H, —CH₂—), 2.29 (s, 4H, —CH—, —CH₃), 2.82 (br, 4H, —CH₂—), 2.95 (d, J = 9.6 Hz, 1H, Ar—H), 3.40 (br, 4H, —CH₂—), 6.47–6.50 (m, 1H, Ar—H), 6.57 (s, 1H, Ar—H), 6.65 (d, J = 8.4 Hz, 1H, Ar—H), 7.22 (d, J = 8.1 Hz, 1H, Ar—H), 7.42–7.47 (m, 2H, Ar—H), 7.57–7.61 (m, 3H, Ar—H), 7.84 (d, J = 8.4 Hz, 1H, Ar—H), 8.68 (d, J = 7.8 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 28.54, 46.34, 49.24, 50.49, 55.62, 61.83, 120.96, 124.27, 124.61, 125.04, 127.31, 128.45, 130.40, 131.54, 134.33, 135.15, 135.31, 136.55, 141.89, 149.81, 157.49, 194.38 (CO). HRMS (ESI) m/z calcd for C₂₆H₂₇ClN₄O ⁺ [M]⁺: 446.1873, found, [M+H]⁺: 447.1944.

2.3.24

2.3.24 9-Chloro-6-(4-(1,4-dioxa-8-azaspiro[4,5]dec-8-yl)-11H-indeno[1,2-c]quinolin-11-one (24)

Product 24 was prepared from compound 2 and 1,4-dioxa-8-azaspiro[4,5]dec-8-yl. The red solid material was isolated in 56% yield (R_f = 0.34 at CH₂Cl₂: n-hexane = 2:1). Mp 218–219 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1718 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 1.98 (t, J = 5.7 Hz, 4H, —CH₂—), 3.51 (br, 4H, —NCH₂—), 4.04 (s, 4H, —OCH₂—), 7.46 (td, J = 8.7, 2.1 Hz, 2H, Ar—H), 7.57–7.62 (m, 3H, Ar—H), 7.83 (d, J = 8.7 Hz, 1H, Ar—H), 8.69 (dd, J = 8.4, 0.9 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 35.00, 48.35, 64.66, 107.31, 120.98, 124.25, 125.06, 127.32, 128.41, 130.42, 131.65, 134.38, 135.09, 135.36, 136.57, 141.97, 149.74, 157.34, 194.43 (CO). HRMS (ESI) m/z calcd for C₂₃H₁₉ClN₂O₃ ⁺ [M]⁺: 406.1084, found [M+H]⁺: 407.1154.

2.3.25

2.3.25 9-Chloro-6-(4-((piperazin-1-yl)(piperidin-1-yl)methanone)-11H-indeno[1,2-c]quinolin-11-one (25)

Product 25 was prepared from compound 2 and (piperazin-1-yl)(piperidin-1-yl)methanone. The red solid material was isolated in 47% yield (R_f = 0.17 at CH₂Cl₂: n-hexane = 2:1). Mp 266–267 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1647, 1718 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 1.63 (s, 2H, —CH₂—), 3.28–3.47 (m, 16H, —CH₂—), 7.42–7.49 (m, 2H, Ar—H), 7.57–7.61 (m, 3H, Ar—H), 7.82 (d, J = 8.4 Hz, 1H, Ar—H), 8.69 (d, J = 7.8, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 24.86, 25.97, 47.12, 47.99, 49.99, 121.16, 124.32, 124.47, 125.20, 127.65, 128.39, 130.58, 131.62, 134.49, 135.09, 135.54, 136.68, 141.56, 149.69, 157.36, 164.84 (CO), 194.20 (CO). HRMS (ESI) m/z calcd for C₂₃H₁₉ClN₂O₃ ⁺ [M]⁺: 460.1666, found [M+H]⁺: 461.1739.

2.3.26

2.3.26 9-Chloro-6-(4-(3-(piperidin-4-yl)propyl)piperidin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (26)

Compound 26 was prepared from compound 2 and 4-(3-(piperidin-4-yl)propyl)piperidine. The pure product was obtained as red powder (yield 8.4%) (R_f = 0.41 at CH₂Cl₂: n-hexane = 2:1). Mp 149–151 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1718 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 1.07–1.46 (m, 12H, —CH₂—, —CH—), 1.66–1.91 (m, 4H, —CH₂—), 2.54–2.62 (m, 2H, —NCH_2(axial)—), 2.90–2.94 (m, 2H, —NCH_2(axial)—), 3.06 (d, J = 12 Hz, 2H, —NCH_{2(equatorial)}—), 3.70 (d, J = 12.3 Hz, 2H, —NCH_{2(equatorial)}—), 7.41–7.47 (m, 2H, Ar—H), 7.56–7.61 (m, 3H, Ar—H), 7.84 (d, J = 8.4 Hz, 1H, Ar—H), 8.68 (d, J = 8.1, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 23.81, 32.58, 33.95, 35.88, 36.49, 37.04, 37.65, 47.07, 50.75, 120.89, 124.27, 124.44, 124.92, 127.15, 128.31, 130.32, 131.96, 134.33, 135.08, 135.23, 136.43, 142.12, 149.83, 158.22, 194.52 (CO). HRMS (ESI) m/z calcd for C₂₉H₃₂ClN₃O ⁺ [M]⁺: 473.2234, found [M+H]⁺: 474.2318.

2.3.27

2.3.27 6-(2-Hydroxyethylthio)-9-chloro-11H-indeno[1,2-c]quinolin-11-one (27)

Compound 27 was prepared from compound 2 and 2-mercaptoethanol. The pure product was obtained as red powder (yield 95%) (R_f = 0.23 at CH₂Cl₂: n-hexane = 2:1). Mp 169–170 °C (EtOH). FT-IR (KBr; ν cm⁻¹): 1718 (C⚌O). ¹H NMR (300 MHz, CDCl₃): δ (ppm) 3.66 (t, J = 5.4 Hz, 2H, —SCH₂—), 4.11 (t, J = 5.25 Hz, 2H, —CH₂OH), 4.34 (br, 1H, —OH), 7.47 (dd, J = 8.4, 2.1 Hz, 1H, Ar—H), 7.53 (td, J = 8.4, 1.5 Hz, 1H, Ar—H), 7.61 (d, J = 2.1 Hz, 1H, Ar—H), 7.65 (td, J = 8.4, 1.5 Hz, 1H, Ar—H), 7.87 (d, J = 8.1 Hz, 1H, Ar—H), 7.92 (d, J = 8.1 Hz, 1H, Ar—H), 8.72 (d, J = 8.4 Hz, 1H, Ar—H). ¹³C NMR (75 MHz, CDCl₃): δ (ppm) 34.05, 63.23, 121.54, 124.64, 125.36, 125.83, 127.90, 128.79, 131.14, 134.11, 134.61, 135.02, 136.08, 136.70, 140.76, 149.91, 155.11, 193.79 (CO). HRMS (EI) m/z calcd for C₁₈H₁₂ClNO₂S ⁺[M]⁺: 341.0277, found 341.0287.

2.3.28

2.3.28 6-Hydroxy-9-chloro-11H-indeno[1,2-c]quinolin-11-one (28)

The pure product was obtained as red solid (yield 57%) (R_f = 0.24 at ethyl acetate: n-hexane = 3:2). Mp 384 °C (dec.). ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 7.30 (t, J = 7.6 Hz, 1H, Ar—H), 7.40 (d, J = 8.0 Hz, 1H, Ar—H), 7.56 (t, J = 7.6 Hz, 1H, Ar—H), 7.61 (s, 1H, Ar—H), 7.63 (d, J = 6.8 Hz, 1H, Ar—H), 7.93 (d, J = 7.6 Hz, 1H, Ar—H), 7.37 (d, J = 8.4 Hz, 1H, Ar—H), 12.42 (br, 1H, —OH). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 115.12, 116.39, 123.91, 124.41, 125.02, 131.62, 133.69, 134.33, 134.82, 135.68, 136.68, 140.78, 141.10, 159.20, 194.44 (C⚌O). HRMS (ESI) calcd for C₁₆H₈NO₂Cl [M]⁺ 281.0244; found [M+H]⁺ 282.0322, [M−H]⁻ 280.0178.

2.3.29

2.3.29 6-Methoxy-9-chloro-11H-indeno[1,2-c]quinolin-11-one (29)

The pure product was obtained as orange powder (yield 60%) (R_f = 0.52 at CH₂Cl₂: n-hexane = 1:1). Mp 259–261 °C (EtOH). ¹H NMR (400 MHz, CDCl₃): δ (ppm) 4.24 (3H, s, —OCH₃), 7.44 (1H, dd, J = 8.0 Hz, 2.0 Hz, Ar—H), 7.47 (1H, td, J = 7.6 Hz, 1.2 Hz, Ar—H), 7.59 (1H, d, J = 2.0 Hz, Ar—H), 7.62 (1H, td, J = 8.0 Hz, 1.6 Hz, Ar—H), 7.75 (1H, d, J = 7.6 Hz, Ar—H), 7.85 (1H, d, J = 8.4 Hz, Ar—H), 8.67 (1H, dd, J = 8.0 Hz, 1.2 Hz, Ar—H). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 53.92, 120.66, 124.09, 124.86, 125.03, 126.68, 127.48, 129.08, 130.25, 134.31, 134.51, 135.16, 136.14, 140.25, 148.79, 158.13, 193.88 (C⚌O). HRMS (ESI) calcd for C₁₇H₁₀NO₂Cl [M]⁺ 295.0400; found [M+H]⁺ 296.0482.

2.4

2.4 Cell culture and MTT assay

All of the synthesized compounds (1–29) were tested against renal CAKI-1 cell line by MTT assay. Moreover, we selected four compounds 4, 5, 13, and 26 to evaluate the topo I inhibitory activity. Simultaneously, seventeen of our structures (1, 2, 3, 4, 5, 8, 10, 11, 12, 14, 16, 18, 22, 25, 27, 28 and 29) were selected by NCI and were investigated against a panel of 60 human tumor cell lines. According to the primary screening, compounds 1, 5 and 12 were chosen for further cell growth inhibition screening for the GI₅₀ (50% growth inhibitory concentration), TGI (the total growth inhibition), and LC₅₀ (the 50% lethal concentration) by NCI.

A 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) (Sigma, USA) assay was performed to determine the cell viability and IC₅₀ values of our synthetic compounds against the MCF-7 (Mosmann, 1983; Denizot and Lang, 1986) and CAKI-1 cell lines. MCF-7 (3000 cells/well) and CAKI-1 (5000 cells/well) cells were seeded in 96-well microplates with RPMI-1641 supplemented with 10% FBS and treated with various concentrations of compounds for 72 h. After treatment, each well was washed with phosphate-buffered saline (PBS) three times, 100 μL of the MTT solution (0.5 mg/mL final concentration in the medium) was added to each well, and cells were incubated at 37 °C for 1 h. MTT is converted to blue formazan crystals by mitochondrial succinate dehydrogenase. The plates were then washed with PBS and solubilized in 100 μL of dimethyl sulfoxide (DMSO) per well. The absorbances at 540 nm were determined using an enzyme-linked immunosorbent assay (ELISA) microplate reader. Effects of our synthetic compounds on cell viability were demonstrated as the relative activity (relative to the DMSO control group).

2.5

2.5 DNA topoisomerase I assay

The topoisomerase I assay kit (TopoGEN, Inc., USA) was performed as described previously with modifications (Shchekotikhin et al., 2011). The activity of DNA topoisomerase I was determined by evaluating the relaxation of supercoiled DNA pHOT. The selected synthetic compounds and camptothecin were dissolved in DMSO at 10 mM as stock solution. A mixture containing 0.25 μg of the plasmid PHOT DNA and 1–2 units of recombinant human DNA topoisomerase I (TopoGEN INC., USA) was incubated with the prepared 0.5% DMSO (negative control), camptothecin 100 μM (positive control) or compounds in the buffer (10 mM Tris-HCl, pH 7.9; 1 mM EDTA; 0.15 M NaCI; 0.1% BSA; 0.1 mM Spermidine; 5% glycerol) at 37 °C for 45 min. The reaction was quenched by the addition of sodium dodecyl sulfate (final 1% concentration) and proteinase K (final 50 μg/mL concentration) at 37 °C for 15 min. To the reaction mixtures, the loading buffer containing 0.25% bromophenol blue and 50% glycerol was added 0.1 volume in reactions mixtures. These samples were electrophoresed on 1% agarose gel at 60 V for 1.5 h with TAE (Tris-acetate-EDTA) as the buffer. The gels were stained with ethidium bromide for 10 min and destained with water for 20 min after electrophoresis.

2.6

2.6 NCI in vitro 60-cell drug screening experiments

Eight of our synthesized compounds were selected by the NCI, and their anticancer activities at a single dose of 10 μM were determined by a sulforhodamine B (SRB) colorimetric assay according to previous protocols (Sikic, 1991; Monks et al., 1997; Kandeel et al., 2015 ). Cells (3000–5000 per well) were seeded into 96-well microtiter plates for 24 h at 37 °C, with 5% CO₂, 95% air, and 100% relative humidity. Two plates of each cell line were fixed with trichloroacetic acid (TCA) as a control of the cell population for each cell line at the time of drug exposure (T₀). After additional incubation with the vehicle (DMSO) or the test compounds for 48 h, cells were fixed with cold 50% (w/v) TCA (final concentration, 10% TCA) and then incubated for 60 min at 4 °C. Plates were then washed with tap water, and cells were treated with 100 μL of the SRB solution at 0.4% (w/v) in 1% acetic acid for 10 min at room temperature. After staining, the plates were washed with 1% acetic acid to remove any unbound dye, and SRB-bound cells were solubilized with 0.01 M Trizma base. The absorbance was measured using a spectrophotometer at a wavelength of 515 nm. Using the absorbance measurements, including time zero (T₀), control growth (C), and test growth in the presence of a drug (T_X), the percentage growth was calculated for each compound as follows: 100 − [(T_X − T₀)/(C − T₀)] × 100 for which concentrations in which T_X ⩾ T₀.

3 Results and discussion

The synthetic methods of 6-substituted 9-chloro-11H-indeno[1,2-c]quinolin-11-ones are depicted in Scheme 1. The intermediate 3-(4-Chlorophenyl)-2-hydroxyquinoline-4-carboxylic acid (1) was obtained from the reaction of isatin with 4-chlorophenylacetic acid via the Pfitzinger reaction (Pfitzinger, 1886, 1888). It is a common reaction to provide quinoline-4-carboxylic acids under basic condition. The following is the starting material compound 2, 6,9-dichloro-11H-indeno[1,2-c]quinolin-11-one, and it was produced from intermediate (1) in POCl₃ and carried out in an open system round-bottle flask with a condenser. The synthetic strategy of compounds 3–26 was accomplished via the reaction of an appropriate amine with compound 2 in DMF, and then treatment with pyridine to afford the desired compounds, which were purified by recrystallization. It was also worthy to note that compound 2 could react with HCl to obtain 28 or with sodium methoxide (NaOMe) to afford 29, respectively. All the synthetic methods are illustrated in the procedure Scheme 1.

The effects of the synthesized indenoquinolone derivatives 2–29 on cell viability of the MCF-7 breast cancer cell line and CAKI-1 renal cancer cell line were experimentally assessed by performing a MTT assay. Results are summarized in Table 1 and are expressed as IC₅₀ (μM) values. From the obtained results, we observed that some indenoquinolones exhibited interesting activities on the tested cancer cell lines. We found that the most potent compounds against CAKI-1 cells were 12, 13, 23 and 27 with IC₅₀ values less than 2 μM. In addition, the potent compounds against MCF-7 cells were 5 (2.13 ± 0.57), 12 (2.20 ± 0.33), 13 (2.40 ± 0.43), and 23 (2.31 ± 0.35) which showed low IC₅₀ values of cell viability. Of the compounds analyzed, we observed that introduction of substituted piperazinyl groups at the 6-position of the indenoquinolone scaffold could modulate the inhibition of cell viability of MCF-7 and CAKI-1 compared to the indenoquinolone 2 which possess two chloro atoms. However, the IC₅₀ values of compounds 14–22 containing a N-substituted piperazinyl group decreased potency significantly. Further, the micromolar IC₅₀ values of some potent compounds are presented in Table 1.

Table 1 Effect of synthetic compounds on cell viability against renal CAKI-1 cells and breast MCF-7 cells.

Compound	IC₅₀ (μM)^a ± SD^b		Compound	IC₅₀ (μM) ± SD
	CAKI-1	MCF-7		CAKI-1	MCF-7
1	>30	>30	16	19.66 ± 4.75	>30
2	>30	>30	17	5.83 ± 0.37	3.07 ± 0.36
3	23.26 ± 5.74	3.28 ± 0.88	18	3.59 ± 1.40	3.51 ± 1.4
4	13.52 ± 2.79	3.06 ± 0.77	19	>30	>30
5	3.85 ± 0.56	2.13 ± 0.57	20	10.80 ± 2.91	>30
6	>30	>30	21	>30	>30
7	>30	>30	22	12.96 ± 3.16	>30
8	>30	>30	23	1.81 ± 1.10	2.31 ± 0.35
9	29.64 ± 2.55	>30	24	5.95 ± 0.03	>30
10	7.19 ± 0.35	>30	25	>30	>30
11	9.07 ± 3.86	4.0 ± 0.55	26	21.83 ± 2.74	>30
12	1.52 ± 0.21	2.2 ± 0.33	27	1.99 ± 0.03	>30
13	1.66 ± 0.11	2.4 ± 0.433	28	>30	>30
14	8.18 ± 0.69	>30	29	>30	>30
15	9.66 ± 1.91	3.24 ± 0.66	CPT^c	–^d	1.24 ± 0.72

a IC₅₀ is the concentration of drug (μM) required to inhibit cell growth by 50% of the mean (N = 3).

b SD: standard derivation, all experiments were independently performed at least three times.

c CPT (camptothecin) as a reference drug.

d –: not determined.

According to the antiproliferative activities and the similar side chains, compounds 4, 5, 13, and 26 were selected to evaluate the topoisomerase I inhibition. In Fig. 2, the selected compounds showed various inhibitory effects against topoisomerase I at 25 and 100 μM. Among them, the synthetic compound 13 not only exhibited more potent inhibitory activity than compounds 4, 5, 26, and CPT, but also completely blocked topoisomerase I-mediated DNA relaxation at 25 μM. Compound 7 was chosen for further testing against topoisomerase I in a concentration-dependent manner doses at 3.125, 6.25, 12.5, and 25 μM (Fig. 3).

Structures and the inhibition of topoisomerase I relaxation activities of compounds 4, 5, 13, and 26. (A) Structure of compounds 4, 5, 13, and 26. (B) Effect of compounds 4, 5, 13, and 26 on topoisomerase I mediated supercoiled pHOT DNA relaxation. Lane 1: untreated supercoiled pHOT DNA. Lane 2: the pHOT DNA treated with topoisomerase I in the absence of drugs. Lanes 3–4 (compound 4), 5–6 (compound 5), 7–8 (compound 13) and 9–10 (compound 26) are the pHOT DNA treated with topoisomerase I in the present of drugs at 25 or 100 μM, respectively. Lane 11 is the pHOT DNA treated with topoisomerase I in the presence of CPT at 100 μM.

Effect of compound 13 on topoisomerase I mediated supercoiled pHOT DNA relaxation. Lanes 1: untreated supercoiled pHOT DNA. Lanes 2: the pHOT DNA treated with topoisomerase I in the absence of drugs. Lanes 3–6 are the pHOT DNA treated with topoisomerase I in the presence of compound 13 at 3.125, 6.25, 12.5 and 25 μM.

The effects of sixteen compounds 1 (NSC763972), 2 (NSC763969), 3 (NSC772856), 4 (NSC771781), 5 (NSC772864), 8 (NSC772860), 10 (NSC771782), 12 (NSC772862), 14 (NSC771783), 16 (NSC772859), 18 (NSC772861), 22 (NSC772863), 25 (NSC772858), 27 (NSC763971), 28 (NSC763970), and 29 (NSC765596) on cell viability were evaluated against the NCI-60 human tumor cell lines at 10 μM in vitro using the SRB protein-binding dye (Sikic, 1991; Monks et al., 1997). As shown in Table 2, this class of compounds bearing ethanolthiol group (27), hydroxyl group (28), methoxy group (29), and several N-substituted piperazinyl groups revealed no significant activities for all the cancer cell lines which respond to the previous in vitro consequences. Three synthetic compounds 1, 5, and 12 were selected for an advanced 60-cell panel assay at five logarithm concentrations (10⁻², 10⁻¹, 10⁰, 10¹ and 10² μM) and the results were indicated with GI₅₀, TGI and LC₅₀, because of their significant cytotoxicity when tested using MTT viability assay.

Table 2 Cytotoxicity of selected compounds in the NCI in vitro 60-cell drug screen program.

Panel/cell line	Compounds/growth percent^a
Compd. No.	1	2	3	4	5	8	10	11	12	14	16	18	22	25	27	28	29
NCI No.	NSC763972	NSC763969	NSC772856	NSC771781	NSC772864	NSC772860	NSC771782	NSC772857	NSC772862	NSC771783	NSC772859	NSC772861	NSC772863	NSC772858	NSC763971	NSC763970	NSC765596
Leukemia
CCRF-CEM	22.62	64.86	101.50	92.13	42.00	104.33	88.85	105.04	−35.45	92.44	95.17	93.52	93.78	109.87	71.56	105.78	89.25
HL-60(TB)	3.18	102.00	102.75	95.49	94.33	98.84	97.09	94.50	−38.36	101.15	103.04	105.45	99.09	109.78	91.75	102.31	108.77
K-562	12.16	102.16	90.99	88.81	45.29	103.57	89.01	100.89	−65.71	92.13	99.26	95.17	94.25	95.70	63.81	97.48	108.53
MOLT-4	43.74	70.83	93.26	88.92	34.63	94.41	88.74	93.29	−43.22	92.90	98.39	98.13	95.46	104.77	59.49	95.10	110.33
RPMI-8226	20.68	92.83	91.82	88.05	81.94	97.99	83.40	95.20	−43.57	85.92	94.89	95.33	93.63	104.31	62.28	90.60	90.30
SR	29.86	65.85	91.67	86.12	46.92	98.51	85.94	99.53	−55.19	75.13	97.09	95.93	95.05	105.15	61.70	77.02	N.T.

Non-small cell lung cancer
A549/ATCC	11.94	108.98	96.83	98.85	51.79	101.69	101.47	104.35	18.70	96.43	101.56	90.89	101.22	99.12	76.42	104.88	99.70
EKVX	N.T.^b	79.27	N.T.	N.T.	N.T.	N.T.	N.T.	N.T.	N.T.	N.T.	N.T.	N.T.	N.T.	N.T.	30.15	72.14	N.T.
HOP-62	29.61	94.89	87.10	78.02	24.86	64.18	78.71	78.17	−61.80	61.42	108.83	N.T.	90.22	96.39	91.46	91.26	96.01
HOP-92	N.T.	71.79	N.T.	44.26	N.T.	N.T.	66.08	N.T.	N.T.	38.50	N.T.	N.T.	N.T.	N.T.	N.T.	N.T.	77.75
NCI-H226	66.09	91.86	92.19	84.79	80.86	94.59	87.21	94.22	49.94	86.17	92.80	79.43	89.19	96.06	80.52	85.66	89.22
NCI-H23	58.72	79.30	86.83	82.62	80.63	90.42	90.40	94.33	49.19	86.27	92.80	83.34	87.22	95.41	66.22	81.56	93.26
NCI-H322M	89.67	95.38	80.85	78.83	69.83	107.08	91.28	92.29	35.89	87.41	98.50	98.63	91.93	94.59	73.47	90.72	99.80
NCI-H460	79.73	98.68	84.38	94.17	25.15	101.21	102.14	93.32	−66.36	91.83	101.46	97.04	101.76	98.85	79.22	83.32	98.27
NCI-H522	1.78	89.37	90.50	88.97	77.39	98.45	91.11	92.76	18.08	96.62	98.72	80.10	79.75	99.28	68.89	92.22	95.89

Colon cancer
COLO 205	14.63	99.18	66.47	100.97	45.69	104.79	100.71	96.30	−81.49	93.65	104.54	102.19	106.63	98.52	87.39	98.25	108.91
HCC-2998	88.12	98.52	57.51	81.50	88.65	102.76	107.23	103.16	−55.83	103.21	106.04	106.23	105.79	106.23	96.27	102.24	104.46
HCT-116	35.26	105.87	88.31	98.57	36.96	102.51	93.64	89.08	−83.19	92.78	91.34	78.18	93.02	101.67	77.94	103.43	88.60
HCT-15	21.44	114.88	66.20	80.49	50.38	97.78	80.29	97.88	−78.92	92.83	89.64	80.71	100.39	99.35	80.10	108.54	94.98
HT29	81.42	104.27	99.44	94.89	9.75	104.84	91.34	95.52	−80.89	93.76	93.22	82.40	95.93	108.65	84.03	102.38	111.60
KM12	79.30	111.92	66.82	94.44	53.42	94.80	109.99	97.97	−47.09	99.06	99.33	98.34	105.95	101.58	84.62	102.73	98.35
SW-620	66.13	98.29	96.25	96.35	43.72	88.89	103.71	91.88	−73.67	92.76	93.06	93.14	91.16	92.51	86.99	95.26	98.89

CNS cancer
SF-268	84.54	117.27	97.27	100.26	65.88	96.49	103.58	96.13	34.33	92.35	96.39	85.45	83.89	107.51	96.53	106.64	113.64
SF-295	N.T.	92.80	95.71	93.22	N.T.	96.18	102.43	93.33	23.75	90.01	96.87	60.91	90.05	97.83	73.28	92.48	N.T.
SF-539	104.63	110.08	92.08	87.80	85.04	90.22	96.24	92.93	36.91	95.23	101.34	83.17	83.97	92.98	100.05	105.79	91.49
SNB-19	93.45	101.30	95.95	104.19	94.02	103.87	100.01	113.43	67.25	85.41	95.06	88.40	105.27	108.66	89.19	99.28	96.35
SNB-75	74.33	82.91	79.70	69.12	71.52	84.42	81.55	72.13	22.34	70.00	86.36	33.20	37.76	80.84	66.06	80.33	59.75
U251	20.22	100.63	96.03	98.98	N.T.	N.T.	95.72	111.96	N.T.	85.86	N.T.	N.T.	N.T.	98.15	78.04	96.46	97.67

Melanoma
LOX IMVI	33.38	84.23	93.40	85.45	N.T.	N.T.	91.20	100.04	N.T.	87.17	N.T.	N.T.	N.T.	103.19	68.41	86.91	116.91
MALME-3M	73.87	102.06.	114.83	111.50	52.55	98.31	136.93	101.05	−32.28	106.59	92.48	100.05	92.79	102.62	107.23	103.37	107.54
M14	72.55	109.79	112.68	113.63	76.44	96.60	106.99	101.69	−56.24	106.31	105.34	97.52	105.07	101.18	98.89	108.50	96.50
MDA-MB-435	N.T.	100.46	107.24	97.33	92.72	104.53	106.62	107.70	−6.57	98.43	99.93	99.55	100.75	102.57	95.15	100.35	100.31
SK-MEL-2	110.64	117.11	N.T.	95.09	N.T.	N.T.	104.29	N.T.	N.T.	104.36	N.T.	N.T.	N.T.	N.T.	95.85	117.12	109.44
SK-MEL-28	73.75	90.08	114.89	113.43	87.40	105.21	113.81	110.84	−91.38	110.81	107.58	104.82	103.93	107.03	86.90	100.02	106.24
SK-MEL-5	66.21	86.30	80.96	94.89	82.51	98.52	99.19	89.52	59.18	97.67	97.48	99.23	96.87	92.50	68.56	84.87	98.51
UACC-257	38.15	127.99	107.69	101.11	92.18	106.47	103.31	107.44	−39.24	95.34	98.37	98.40	93.79	104.23	106.91	106.31	98.20
UACC-62	86.86	94.45	77.41	78.16	99.56	93.36	88.33	94.90	−81.71	96.67	95.06	87.17	86.44	93.92	84.47	95.17	74.08

Ovarian cancer
IGROV1	87.42	92.82	54.41	83.38	43.43	94.36	86.47	72.45	3.23	75.03	85.84	65.13	80.87	81.63	69.73	91.28	96.92
OVCAR-3	82.98	119.93	84.91	103.48	73.13	97.59	102.34	96.58	22.86	93.41	99.54	98.07	104.27	103.33	89.42	117.80	111.89
OVCAR-4	40.23	94.13	72.83	84.28	52.50	97.11	96.67	98.19	35.59	94.70	94.20	90.32	87.08	98.31	73.19	80.74	N.T.
OVCAR-5	104.97	137.00	81.54	89.14	89.34	104.92	90.30	94.37	39.14	99.31	106.41	88.56	98.22	102.33	112.54	126.77	96.81
OVCAR-8	16.01	101.08	94.33	84.55	61.36	96.38	95.25	96.11	−46.09	87.51	96.55	75.82	83.98	104.73	90.21	107.63	98.75
NCI/ADR-RES	43.83	96.78	92.22	92.57	69.55	90.28	94.32	102.85	−4.00	88.19	100.39	97.88	92.82	107.13	73.58	97.00	94.29
SK-OV-3	N.T.	N.T.	92.62	99.12	84.20	99.78	107.26	84.60	57.81	87.98	96.85	78.58	86.02	95.59	N.T.	N.T.	119.79

Renal cancer
786-0	103.15	112.40	103.34	108.01	74.13	102.73	103.71	99.53	25.31	89.51	97.90	81.41	81.67	105.05	100.58	114.55	103.51
A498	N.T.	N.T.	82.03	96.00	88.98	87.55	93.88	99.15	59.79	72.81	88.64	97.48	104.53	104.44	N.T.	N.T.	93.51
ACHN	51.33	111.67	88.46	87.55	55.86	94.75	81.66	86.42	−83.29	88.10	100.03	68.58	93.51	95.59	88.12	104.75	90.83
CAKI-1	N.T.	75.67	73.84	84.65	64.35	83.89	94.99	82.68	17.84	83.81	93.83	80.23	86.59	90.50	41.86	71.62	90.65
RXF 393	82.62	112.80	97.15	101.32	51.90	97.51	114.61	106.09	20.51	94.00	95.60	72.95	78.38	107.22	79.39	99.93	114.49
SN12C	48.72	88.03	94.82	95.38	61.60	99.08	96.95	93.10	15.89	83.57	99.08	87.90	88.17	101.12	84.58	96.69	89.48
TK-10	94.62	138.45	65.31	91.84	95.55	134.79	101.98	121.79	47.07	99.56	N.T.	115.61	133.34	131.97	99.82	126.63	N.T.
UO-31	15.98	80.62	56.91	48.63	25.86	76.25	45.74	65.33	−40.61	47.60	72.57	65.02	86.64	82.74	56.23	70.50	55.69

Prostate cancer
PC-3	32.02	92.73	89.44	82.55	41.01	90.64	79.64	87.77	−58.51	82.93	86.19	79.79	72.83	93.07	58.35	93.44	74.44
DU-145	89.22	113.91	106.88	106.50	65.48	103.47	119.02	106.04	−41.83	102.37	110.73	102.96	98.98	114.04	92.27	111.80	121.06

Breast cancer
MCF7	50.00	74.25	5.24	23.99	12.70	64.65	52.31	49.61	−100.00	68.30	75.18	53.31	77.46	97.24	67.82	68.09	71.98
MDA-MB-231/ATCC	41.97	87.66	83.87	76.68	25.16	90.96	79.28	93.08	−10.45	62.53	82.96	73.80	79.81	93.54	72.56	86.98	74.61
HS 578T	84.98	91.01	85.28	72.84	84.70	98.38	88.93	87.68	60.88	70.47	99.40	N.T.	71.75	102.06	58.28	105.98	92.74
BT-549	91.18	119.62	101.88	102.55	87.48	95.52	93.95	92.91	58.35	82.89	102.74	89.83	101.57	105.34	92.88	120.08	92.95
T-47D	−30.98	86.97	23.53	44.88	43.28	67.68	88.81	83.86	−31.54	76.27	92.20	75.93	86.68	95.99	63.89	67.23	78.97
MDA-MB-468	45.23	107.92	−31.61	0.60	33.81	97.12	59.47	97.71	−35.66	95.71	103.99	95.37	101.04	106.42	77.36	80.08	99.51

Mean	55.93	98.17	84.15	87.15	62.40	96.20	93.66	94.75	−14.37	88.15	96.53	87.29	92.15	100.53	79.52	96.60	96.23
Delta	86.91	33.31	115.76	86.55	52.65	32.02	47.92	45.14	85.63	49.65	23.96	54.09	54.39	19.69	49.37	29.37	40.54
Range	141.62	73.59	146.50	113.03	89.81	70.61	91.19	72.18	167.25	72.31	38.16	82.41	95.58	51.13	82.39	59.54	65.37

a Data obtained from NCI in vitro 60-cell drug screen program at 10 μM.

b N.T. = no test.

Compound 1, the average concentration required to inhibit GI₅₀ was 8.982 μM with a range of 0.238 μM (non-small cell lung cancer: NCI-H522) to >39.0 μM (colon cancer: HT-29). With 5, the average concentration required to inhibit GI₅₀ was 14.36 μM with a range of 0.44 μM (breast cancer: MCF-7) to >100 μM (colon cancer: HCC-2998). Furthermore, the average GI₅₀ concentration of 12 was 10.498 μM, with a range of 2.52 μM (ovarian cancer: IGROV1) to >100 μM (melanoma: SK-MEL-28). Compounds 1, 5, and 12 exhibited dose-dependent inhibition of proliferation in all 60 cancer cell lines. Compound 1 is active against most of the cancer cell lines with GI₅₀ values <1 μM for 12% (7/58) of the cell lines. With initial assessment at relative doses, 12 is more potent than 5. Compound 12 exhibited highly inhibitory effect against most of the cancer cell lines with GI₅₀ values <5 mM for 36.8% of the 57 cell lines. The GI₅₀, TGI and LC₅₀ values of the active compounds within each series are given in Table 3.

Table 3 In vitro anticancer activity by selected compounds 1 (NSC763972), 5 (NSC772864), 12 (NSC772862) in the NCI’s 60 human cancer cell lines.

Panel/cell line (μM)	1 (NSC763972)					5 (NSC772864)					12 (NSC772862)
	GI₅₀			TGI	LC₅₀	GI₅₀			TGI	LC₅₀	GI₅₀			TGI	LC₅₀
		Subpanel MID^b	Selectivity ratio				Subpanel MID^b	Selectivity ratio				Subpanel MID^b	Selectivity ratio
Leukemia
CCRF-CEM	1.42	1.114	8.063	15.5	>100	8.24	9.696	1.475	3.50	7.80	6.32	11.000	0.954	>100	>100
HL-60(TB)	1.06			4.28	>100	N.T.			N.T.	N.T.	36.9			>100	>100
K-562	0.315			>100	>100	8.75			3.61	7.46	7.80			>100	>100
MOLT-4	1.97			15.3	>100	6.24			3.43	7.91	4.41			>100	>100
RPMI-8226	0.549			>100	>100	20.20			4.31	9.73	7.48			>100	>100
SR	1.37			43.4	>100	5.05			4.06	11.60	3.09			>100	>100

Non-small cell lung cancer
A549/ATCC	2.95	5.840	1.538	>100	>100	15.20	11.819	1.210	3.51	6.72	4.87	6.477	1.621	>100	>100
EKVX	N.T.			N.T.	N.T.	N.T.			N.T.	N.T.	N.T.			N.T.	N.T.
HOP-62	3.84			>100	>100	4.51			2.83	5.74	12.3			>100	>100
HOP-92	1.21			13.4	>100	3.08			5.80	25.10	N.T.			N.T.	N.T.
NCI-H226	11.6			>100	>100	1.88			–	–	7.77			>100	>100
NCI-H23	3.44			63.6	>100	2.63			4.00	8.35	6.65			>100	>100
NCI-H322M	16			92.3	>100	34.50			13.10	36.20	5.62			>100	>100
NCI-460	7.44			86.3	>100	5.15			3.20	6.74	4.15			>100	>100
NCI-H522	0.238			0.644	3.57	27.60			3.80	7.23	3.98			>100	>100

Colon cancer
COLO 205	3.77	9.560	0.939	15.8	39.8	2.41	18.400	0.778	–	–	12.2	9.763	1.075	>100	>100
HCC-2998	11.0			>100	>100	>100			4.08	7.94	30.9			>100	>100
HCT-116	0.807			>100	>100	6.19			3.19	6.06	5.42			>100	>100
HCT-15	0.716			>100	>100	4.37			3.51	7.50	4.34			>100	>100
HT29	39.0			>100	>100	4.23			3.16	6.28	7.84			>100	>100
KM12	4.62			>100	>100	6.48			3.11	5.96	2.50			>100	>100
SW-620	7.01			>100	>100	5.12			3.06	6.67	5.14			>100	>100

CNS cancer
SF-268	4.28	14.364	0.625	>100	>100	17.70	14.875	0.962	3.27	6.52	8.71	8.053	1.304	>100	>100
SF-295	10.8			87.9	>100	17.80			4.60	9.85	7.79			>100	>100
SF-539	23.1			64.2	>100	20.90			3.15	5.86	14.2			>100	>100
SNB-19	30.9			>100	>100	18.20			3.42	–	8.27			>100	>100
SNB-75	N.T.			N.T.	>100	3.25			2.35	4.86	4.53			>100	>100
U251	2.74			>100	>100	11.40			3.06	5.88	4.82			>100	>100

Melanoma
LOX IMV1	0.64	10.637	0.844	57.00	>100	6.04	15.654	0.914	3.04	6.02	4.74	19.894	0.528	>100	>100
MALME-3M	8.32			42.20	>100	17.30			3.83	7.46	8.87			>100	>100
M14	3.75			>100	>100	16.50			5.48	19.30	7.49			>100	>100
MDA-MB-435	3.97			>100	>100	20.20			3.86	7.62	7.01			>100	>100
SK-MEL-2	17.10			50.20	>100	37.70			4.73	10.40	7.36			>100	>100
SK-MEL-28	33.00			>100	>100	6.00			3.21	5.83	>100			>100	>100
SK-MEL-5	7.36			22.50	56.5	1.15			2.61	–	4.10			>100	>100
UACC-257	6.39			65.10	>100	21.20			3.39	6.37	35.1			>100	>100
UACC-62	15.20			88.40	>100	14.80			3.31	6.68	4.38			>100	>100

Ovarian cancer
IGROV1	14.3	13.649	0.658	>100	>100	11.20	17.448	0.820	3.62	7.82	5.94	13.591	0.772	>100	>100
OVCAR-3	4.46			95.1	>100	N.T.			N.T.	N.T.	12.8			>100	>100
OVCAR-4	8.83			21.9	49.30	2.99			2.79	5.68	6.39			>100	>100
OVCAR-5	16.1			66.1	>100	21.50			3.32	6.52	34.5			>100	>100
OVCAR-8	1.49			>100	>100	13.80			3.63	7.76	8.66			>100	>100
NCI/ADR-RES	2.26			25.2	>100	19.30			3.67	7.41	22.5			>100	>100
SK-OV-3	48.1			>100	>100	35.90			5.57	17.20	4.35			>100	>100

Renal cancer
786-O	23.4	10.988	0.817	>100	>100	26.80	12.733	1.124	3.29	6.01	23.5	6.370	1.648	>100	>100
A498	6.75			71.7	>100	8.76			1.78	50.80	2.63			>100	>100
ACHN	4.2			>100	>100	6.44			3.04	5.89	3.14			>100	>100
CAKI-1	7.92			>100	>100	14.80			3.26	6.80	2.52			>100	>100
RXF 393	17.9			63.6	>100	N.T.			N.T.	N.T.	4.55			>100	>100
SN12C	3.24			>100	>100	4.12			2.87	6.16	5.42			>100	>100
TK-10	22.7			68.9	>100	24.60			3.70	6.38	N.T.			N.T.	N.T.
UO-31	1.79			35.3	>100	3.61			2.88	6.48	2.83			>100	>100

Prostate cancer
PC-3	0.606	2.968	3.026	>100	>100	12.50	15.650	0.914	3.82	8.98	5.93	5.360	1.959	>100	>100
DU-145	5.33	2.968	3.026	>100	>100	18.80	15.650	0.914	2.94	5.43	4.79	5.360	1.959	>100	>100

Breast cancer
MCF7	7.73	7.280	1.234	>100	>100	0.44	12.345	1.159	2.60	5.54	5.42	6.813	1.541	>100	>100
MDA-MB-231/ATCC	2.44			13.8	75.1	2.32			2.71	5.70	7.23			>100	>100
HS 578T	20.3			>100	>100	19.30			10.00	46.10	4.77			>100	>100
BT-549	7.55			>100	>100	32.50			9.50	45.50	10.6			>100	>100
T-47D	0.84			2.86	8.96	17.80			3.56	7.19	8.44			>100	>100
MDA-MB-468	4.82			29.4	>100	1.71			3.07	–	4.42			>100	>100

MID^a	8.982					14.306					10.498

MID^a = Average sensitivity of all cell lines in μM.

MID^b = Average sensitivity of all cell lines of a particular subpanel in μM.

Selectivity ratio = MID^a:MID^b.

N.T. = no test.

4 Conclusion

Because of the anticancer potential demonstrated by the indenoquinolone scaffold, an approach for synthesizing 6-substituted-9-chloro-indenoquinolones was developed, and the inhibition activities of the synthetic compounds on cell viability were evaluated. Based on our biological results, it was envisioned that introduction of piperazinyl groups with a 4-substituted side chain at the 6-position of the indenoquinolone scaffold decreased the cell viability of the breast cancer cell line MCF-7 and renal cancer cell line CAKI-1. Among the synthesized compounds, 12 (with a piperazinyl group) and 13 (with a 2-methylpiperazinyl group) were the most-active compound exhibiting potent inhibitory activity on the cell viability of MCF-7 and CAKI-1 cells. Through a series of promising in vitro experiments, we found that 6-substituted-9-chloro-indenoquinolone derivatives, especially compound 13, not only exhibited preferential growth inhibition effects toward cancer cell lines but also showed the inhibitory effect on topoisomerase I. Based on our results and structure–activity relationships (SARs) studies, compounds 12 and 13 could be potent antibreast cancer candidates and promising lead compounds that warrants further structure optimization.

Acknowledgments

The present study was supported by Ministry of Science and Technology, Taiwan (MOST104-2113-M-038-001), Taipei Medical University (TMUTOP103003-1) and National Defense Medical Center (TMU-NDMC-104-02), respectively. We are grateful to thank NIH-NCI for their supports.

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Introduction

Chemistry

Materials and instruments
Synthesis of target compounds 1–29
Physical data
Cell culture and MTT assay
DNA topoisomerase I assay
NCI in vitro 60-cell drug screening experiments

Results and discussion

Conclusion

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[1] Addla D., . Design, synthesis and antimicrobial evaluation of novel 1-benzyl 2-butyl-4-chloroimidazole embodied 4-azafluorenones via molecular hybridization approach. Rev. Bioorg. Med. Chem. Lett.. 2012;22(24):7475-7480.
[CrossRef] [Google Scholar]

[2] Alagoz M., . DNA repair and resistance to topoisomerase I inhibitors: mechanisms, biomarkers and therapeutic targets. Rev. Curr. Med. Chem.. 2012;19(23):3874-3885.
[Google Scholar]

[3] Aoyagi Y., . Establishment and characterization of 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one dihydrochloride (TAS-103)-resistant cell lines. Rev. Jpn. J. Cancer Res.. 2000;91(5):543-550.
[Google Scholar]

[4] Banjerdpongchai R., . 6,8-Dihydroxy-7-methoxy-1-methyl-azafluorenone induces caspase-8- and -9-mediated apoptosis in human cancer cells. Rev. Asian Pac. J. Cancer Prev.. 2013;14(4):2637-2641.
[Google Scholar]

[5] Chen T.C., . Structure-based hybridization, synthesis and biological evaluation of novel tetracyclic heterocyclic azathioxanthone analogues as potential antitumor agents. Rev. Eur. J. Med. Chem.. 2015;103:615-627.
[CrossRef] [Google Scholar]

[6] Chen T.C., . Structure-based design, synthesis and biological evaluation of novel anthra[1,2-d]imidazole-6,11-dione homologues as potential antitumor agents. Rev. Eur. J. Med. Chem.. 2013;69C:278-293.
[CrossRef] [Google Scholar]

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Design, synthesis and biological evaluation of tetracyclic azafluorenone derivatives with topoisomerase I inhibitory properties as potential anticancer agents

Abstract

Keywords

Indenoquinolinone

Topoisomerase I

Azafluorenone

Cytotoxicity

NCI-60 anticancer drug screen

1 Introduction

2 Chemistry

2.1 Materials and instruments

2.2 Synthesis of target compounds 1–29

2.2.1 Synthetic procedure i: preparation of compound 1

2.2.2 Synthetic procedure ii: preparation of compound 2

2.2.3 General procedure iii: preparation of compounds 3–26

2.2.4 Synthetic procedure iv: preparation of compound 27

2.2.5 Synthetic procedure v: preparation of compound 28

2.2.6 Synthetic procedure vi: preparation of compound 29

2.3 Physical data

2.3.1 3-(4-Chlorophenyl)-2-hydroxyquinoline-4-carboxylic acid (1)

2.3.2 6,9-Dichloro-11H-indeno[1,2-c]quinolin-11-one (2)

2.3.3 9-Chloro-6-(methylamino)-11H-indeno[1,2-c]quinolin-11-one (3)

2.3.4 9-Chloro-6-(dimethylamino)-11H-indeno[1,2-c]quinolin-11-one (4)

2.3.5 6-(2-(Diethylamino)ethylamino)-9-chloro-11H-indeno[1,2-c]quinolin-11-one (5)

2.3.6 9-Chloro-6-(pyrrolidin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (6)

2.3.7 9-Chloro-6-(piperidin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (7)

2.3.8 9-Chloro-6-(4-methylpiperidin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (8)

2.3.9 6-(Azepan-1-yl)-9-chloro-11H-indeno[1,2-c]quinolin-11-one (9)

2.3.10 9-Chloro-6-morpholino-11H-indeno[1,2-c]quinolin-11-one (10)

2.3.11 9-Chloro-6-thiomorpholino-11H-indeno[1,2-c]quinolin-11-one (11)

2.3.12 9-Chloro-6-(piperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (12)

2.3.13 9-Chloro-6-(2-methylpiperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (13)

2.3.14 9-Chloro-6-(4-methylpiperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (14)

2.3.15 9-Chloro-6-(4-ethylpiperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (15)

2.3.16 9-Chloro-6-(4-cyclopentylpiperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (16)

2.3.17 9-Chloro-6-(4-(piperidin-1-yl)piperidin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (17)

2.3.18 9-Chloro-6-(4-phenylpiperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (18)

2.3.19 6-(4-Benzylpiperazin-1-yl)-9-chloro-11H-indeno[1,2-c]quinolin-11-one (19)

2.3.20 9-Chloro-6-(4-(2-fluorophenyl)piperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (20)

2.3.21 9-Chloro-6-(4-(2-methoxyphenyl)piperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (21)

2.3.22 9-Chloro-6-(4-(3-methoxyphenyl)piperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (22)

2.3.23 9-Chloro-6-(4-(1-methylpiperidin-4-yl)piperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (23)

2.3.24 9-Chloro-6-(4-(1,4-dioxa-8-azaspiro[4,5]dec-8-yl)-11H-indeno[1,2-c]quinolin-11-one (24)

2.3.25 9-Chloro-6-(4-((piperazin-1-yl)(piperidin-1-yl)methanone)-11H-indeno[1,2-c]quinolin-11-one (25)

2.3.26 9-Chloro-6-(4-(3-(piperidin-4-yl)propyl)piperidin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (26)

2.3.27 6-(2-Hydroxyethylthio)-9-chloro-11H-indeno[1,2-c]quinolin-11-one (27)

2.3.28 6-Hydroxy-9-chloro-11H-indeno[1,2-c]quinolin-11-one (28)

2.3.29 6-Methoxy-9-chloro-11H-indeno[1,2-c]quinolin-11-one (29)

2.4 Cell culture and MTT assay

2.5 DNA topoisomerase I assay

2.6 NCI in vitro 60-cell drug screening experiments

3 Results and discussion

4 Conclusion

Acknowledgments

References

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