Amodiaquine Analogs Are Potent Inhibitors of Interleukin-6 Production Induced by Activation of Toll-Like Receptors Recognizing Pathogen Nucleic Acids

Abstract

Excessive inflammatory responses to viral infections, known as cytokine storms, are caused by overactivation of endolysosomal Toll-like receptors (TLRs) (TLR3, TLR7, TLR8, and TLR9) and can be lethal, but no specific treatment is available. Some quinoline derivatives with antiviral activity were tried during the recent coronavirus disease 2019 (COVID-19) pandemic, but showed serious toxicity, and their efficacy for treating viral cytokine storms was not established. Here, in order to discover a low-toxicity quinoline derivative as a candidate for controlling virally induced inflammation, we synthesized a series of derivatives of amodiaquine (ADQ), a quinoline approved as an antimalarial, and tested their effects on TLRs-mediated production of inflammatory cytokines and cell viability in vitro. In J774.1 murine macrophages, ADQ inhibited interleukin-6 (IL-6) production induced by TLR3 agonist poly(I:C), TLR7 agonist imiquimod, and TLR9 agonist cytosine-phosphate-guanosine oligodeoxynucleotide (CpG ODN) with IC₅₀ values of 2.43, 3.48, and 0.0359 µM, respectively, indicating that ADQ has a high inhibitory selectivity for TLR9 signaling. A structure–activity relationship study revealed that an appropriately substituted amino group on the phenol moiety and a halogen substituent on quinoline are important for potent anti-inflammatory activity and low cytotoxicity. ADQ analogs bearing N-butylethyl, N-3-fluoropiperidinyl, and N-4-fluoropiperidinyl groups in place of the N-diethyl group exhibited more potent activity and lower cytotoxicity than ADQ. ADQ and its analogs appear to inhibit the activity of TLRs recognizing pathogen nucleic acids via alkalinization of endolysosomes. Our results suggest that ADQ analogs are promising candidates as therapeutic agents for cytokine storms mediated by TLRs recognizing pathogen nucleic acid with reduced side effects.

INTRODUCTION

Sepsis is a life-threatening condition caused by an uncontrolled excessive production of inflammatory cytokines by immunocompetent cells (cytokine storm) in response to pathogen invasion, potentially leading to thrombus formation, multi-organ failure, and septic shock.¹⁾ It is a leading cause of death worldwide, with a fatality rate exceeding 20% and over 10 million deaths annually.²⁾ The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in large numbers of cases of sepsis, as defined according to the 2016 sepsis-3 criteria, and many deaths.^3,4) This has highlighted the need for new drugs to treat sepsis caused by emerging pathogens.

It is well known that when pathogens invade the body, pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns (PAMPs), and induce the production of inflammatory cytokines in response. Each TLR recognizes different PAMPs: TLR4 recognizes lipopolysaccharide (LPS), a component of the bacterial outer membrane; TLR3 or TLR7 recognizes viral double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA), respectively; and TLR9 recognizes the unmethylated cytosine-phosphate-guanosine (CpG) motif present in pathogen single-stranded DNA (ssDNA). The TLRs recognizing pathogen nucleic acids are expressed on endosomes/endolysosomes, whereas TLR4 is expressed mainly on the plasma membrane and partly on the endosome membrane. Overactivation of TLRs plays a key role in the induction of cytokine storms, suggesting that regulation of TLRs would be a promising therapeutic target.^5,6) However, a clinical trial of a TLR4 antagonist as a therapeutic agent for bacterial sepsis was unsuccessful, probably because numerous inflammatory signals are activated in a complex manner in septic patients.^5,7) In addition, the overproduction of some inflammatory cytokines induced by TLR activation are involved in the pathogenesis and severity of cytokine storm during sepsis. Among them, interleukin-6 (IL-6) is one of the inflammatory cytokines that is most deeply involved in the pathogenesis and severity of cytokine storms, as the amplification loop of IL-6 production mediated by nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3) is thought to be the main culprit of cytokine storms.⁸⁾ Though anti-human IL-6 monoclonal antibodies show a certain therapeutic effect on cytokine storms during coronavirus disease 2019 (COVID-19), it is difficult to consider their effect to be sufficient.⁹⁾ Therefore, we considered that regulating IL-6 production via multiple TLRs simultaneously might be more effective.

Many quinoline derivatives have been reported and some have been approved for clinical use as anticancer¹⁰⁾ and antiviral agents.¹¹⁾ Typical examples of quinoline-based drugs include chloroquine (CQ), hydroxychloroquine (HCQ), mefloquine (MQ), primaquine and amodiaquine (ADQ), which have been approved by U.S. Food and Drug Administration (FDA) as antimalarial agents. It was also reported that CQ, HCQ, and MQ inhibit SARS-CoV-2 infection by blocking endosome acidification.¹²⁾ CQ and HCQ were found to have anti-inflammatory effects, and are now being employed to treat several inflammatory diseases.¹³⁾ It has also been shown that ADQ exhibits antiviral activity against various RNA viruses such as Ebola virus,^14,15) severe fever with thrombocytopenia syndrome virus¹⁶⁾ and SARS-CoV-2,^17–20) though the results of clinical trials are awaited. Thus, drug discovery focused on quinoline derivatives is considered a promising approach, though toxicity remains an issue.

We have designed and synthesized a series of ADQ derivatives²¹⁾ and tested their inhibitory effect on the production of IL-6 in macrophages stimulated with poly(I:C) (a TLR3 agonist), LPS (a TLR4 agonist), imiquimod (a TLR7 agonist), and CpG ODN (a TLR9 agonist), as well as examining their cytotoxicity. Here, we describe the synthesis, IL-6 production-inhibitory activity and cytotoxicity of the selected compounds listed in Fig. 1. Our findings suggest that ADQ derivatives are promising candidates for anti-inflammatory drugs to treat sepsis.

Fig. 1. Chemical Structures of IL-6 Production Inhibitors Synthesized in This Work

MATERIALS AND METHODS

General Information

All reagents and solvents were of the highest commercial quality and were used without further purification. Anhydrous CH₂Cl₂ and N,N-dimethylformamide (DMF) were prepared by distillation from calcium hydride. Anhydrous CH₃CN was prepared by distillation from phosphorus(V) oxide. Polyinosinic-polycytidylic acid (poly(I:C)) (a TLR3 agonist) was purchased from Tocris Bioscience (Minneapolis, MN, U.S.A.). LPS from Escherichia coli O55:B5 (a TLR4 agonist) was purchased from Sigma-Aldrich Co., LLC (St. Louis, MO, U.S.A.). Imiquimod (a TLR7 agonist) was purchased from Chemscene (Monmouth Junction, NJ, U.S.A.). CpG oligodeoxynucleotide (ODN; 1826) (a TLR9 agonist) was purchased from Novus Biologicals (Centennial, CO, U.S.A.). All aqueous solutions were prepared using deionized water. Melting points were measured on a Yanaco micro melting point apparatus. ¹H (300 and 400 MHz) and ¹³C (75 and 100 MHz) NMR spectra were recorded on JEOL Always 300 (JEOL, Tokyo, Japan) and JNM-ECZ400S (JEOL) spectrometers. Tetramethylsilane (TMS) was used as an internal reference for ¹H-NMR measurements in CDCl_3, CD₃OD and dimethyl sulfoxide (DMSO)-d₆. IR spectra were recorded on a Perkin-Elmer FTIR Spectrum 100 (ATR) (PerkinElmer, Inc., Waltham, MA, U.S.A.). MS measurements were performed on a Sciex X500R QTOF (AB SCIEX, Framingham, MA, U.S.A.) spectrometer. Elemental analyses were performed on a Perkin-Elmer CHN 2400 analyzer (PerkinElmer, Inc.). TLC and silica gel column chromatography was performed using Merck Silica gel 60 F₂₅₄ plates (Merck KGaA, Darmstadt, Germany) and Fuji Silica Chemical FL-100D (Fuji Silysia Chemical, Aichi, Japan), respectively. The microwave irradiation used in the organic reactions was performed in a reaction chamber, Discover (CEM Corporation, Stallings, NC, U.S.A.).

4-(Quinolin-4-ylamino)phenol HCl (YMSA-0366·HCl): A mixture of 4-chloroquinoline (1a) (144 mg, 0.88 mmol) and 4-aminophenol (2) (99 mg, 0.90 mmol) in EtOH (7 mL) was reacted in microwave synthesizer (Discover, CEM) at 50 W and 80 °C for 1 h. The reaction mixture was cooled and the precipitates were collected by filtration, washed with Et₂O, and recrystallized from MeOH to afford YMSA-0366·HCl as yellow crystals (170 mg, 70%). Mp. 264–267 °C (dec). IR (ATR): ν = 3302, 3010, 2789, 1618, 1591, 1543, 1511, 1444, 1363, 1234, 1221, 1169, 824, 755, 660, 497 cm⁻¹. ¹H-NMR (300 MHz, DMSO-d₆/TMS): δ = 10.81 (d, J = 3.4 Hz, 1H), 9.93 (d, J = 3.3 Hz, 1H), 8.76–8.74 (m, 1H), 8.46–8.43 (m, 1H), 8.07–7.98 (m, 2H), 7.77 (dd, J = 8.1, 7.9 Hz, 1H), 7.26 (d, J = 8.6 Hz, 1H), 6.96 (d, J = 8.6 Hz, 1H), 6.62 (d, J = 6.9 Hz, 1H) ppm. ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 157.0, 155.6, 142.4, 138.1, 133.8, 122.9, 127.3, 126.9, 123.6, 120.1, 116.8, 116.5, 99.4 ppm. High resolution (HR)-MS (FAB⁺): Calcd for [M + H]⁺, C₁₅H₁₃N₂O: 237.1022; Found 237.1022. Anal. Calcd (%) for C₁₅H₁₃ClN₂O: C 66.05, H 4.80, N 10.27; Found: C 65.49, H 4.46, N 10.12. It should be noted that YMSA-0366 was synthesized just only as an intermediate for the synthesis of YMSA-0998 and hence its inhibition activity against IL-6 production was not evaluated.

4-((7-Chloroquinolin-4-yl)amino)phenol·HCl salt (KUMB-0002): A mixture of 4,7-dichloroquinoline (1b) (290 mg, 1.47 mmol) and 4-aminophenol (2) (161 mg, 1.48 mmol) in EtOH (12 mL) was reacted in microwave synthesizer (Discover, CEM) at 50 W and 80 °C for 1 h. The reaction mixture was cooled and the precipitates were collected by filtration, washed with Et₂O, and recrystallized from MeOH to afford KUMB-0002·HCl as yellow crystals (338 mg, 85%). Mp. 241–244 °C (dec.). IR (ATR): ν = 2160, 1616, 1590, 1514. 1429, 1367, 1270, 1209, 1168, 1099, 880, 826, 790, 648, 595, 508 cm⁻¹. ¹H-NMR (300 MHz, DMSO-d₆/TMS): δ = 8.84 (d, J = 9.2 Hz, 1H), 8.46 (d, J = 7.2 Hz, 1H), 8.16 (d, J = 1.8 Hz, 1H), 7.83 (dd, J = 9.2, 1.8 Hz, 1H), 7.26 (d, J = 8.9 Hz, 2H), 6.96 (d, J = 8.9 Hz, 2H), 6.63 (d, J = 6.9 Hz, 1H) ppm. ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 157.1, 155.4, 142.9, 139.0, 138.3, 127.6, 127.1, 126.0, 119.1, 116.4, 115.6, 99.9 ppm. HR-MS (FAB⁺): Calcd for [M + H]⁺, C₁₅H₁₁³⁵ClN₂O: 271.0633; Found 271.0630. Anal. Calcd (%) for C₁₅H₁₁Cl₂N₂O: C 58.65, H 3.94, N 9.12; Found: C 58.04, H 3.70, N 8.95.

4-((7-Chloroquinolin-4-yl)amino)-2-((methyl(pentyl)amino)methyl)phenol (YMSA-0203): N-Methylpentylamine (3a) (101.4 μL, 0.739 mmol) and 37% HCHO aqueous solution (77.8 μL, 0.960 mmol) were added to a solution of KUMB-0002·HCl (98.5 mg, 0.364 mmol) in EtOH (1 mL). The reaction mixture was refluxed for 24 h, then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane/AcOEt = 1 : 0 to 3 : 1 + 1% Et₃N). Recrystallization from MeCN afforded YMSA-0203 as pale yellow crystals (18.3 mg, 13%). Mp 146–148 °C. IR (ATR): ν = 2929, 2856, 1610, 1566, 1541, 1493, 1449, 1423, 1372, 1329, 1256, 1196, 1152, 1110, 1082, 1053, 1015, 978, 906, 878, 855, 814, 795, 761, 726, 660, 642, 606, 553, 528, 478, 461, 428, 409 cm⁻¹. ¹H-NMR (400 MHz, CDCl₃/TMS): δ = 8.48 (d, J = 5.6 Hz, 1H), 8.00 (d, J = 2.0 Hz, 1H), 7.81 (d, J = 9.2 Hz, 1H), 7.43 (dd, J = 9.0, 2.2 Hz, 1H), 7.10 (dd, J = 8.6, 2.6 Hz, 1H), 6.92 (d, J = 2.8 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.63 (d, J = 5.2 Hz, 1H), 6.49 (s, 1H), 3.71 (s, 2H), 2.50 (t, J = 7.2 Hz, 2H), 2.31 (s, 3H), 1.62–1.52 (m, 2H), 1.40–1.25 (m, 4H), 0.92 (t, J = 6.8 Hz, 3H) ppm. ¹³C-NMR (100 MHz, CDCl₃/TMS) δ = 156.5, 152.0, 149.6, 149.2, 135.1, 129.9, 129.0, 125.7, 125.6, 125.3, 123.2, 120.9, 117.4, 117.1, 101.3, 61.3, 57.1, 41.2, 29.3, 26.9, 26.6, 22.5, 14.0 ppm. HR-MS (electrospray ionization (ESI)⁺): Calcd for [M + H]⁺, C₂₂H₂₇³⁵ClN₃O: 384.1837; Found 384.1833. Anal. Calcd (%) for C₂₂H₂₆ClN₃O; C 68.83, H 6.83, N 10.95; Found: C 68.53, H 6.22, N 10.85.

2-((4-Fluoropiperidin-1-yl)methyl)-4-(quinolin-4-ylamino)phenol (YMSA-0998): 4-Fluoropiperidine·HCl (3b) (62.8 mg, 0.450 mmol), Et₃N (100 μL, 0.715 mmol) and 37% HCHO aqueous solution (126 μL, 1.56 mmol) were added to a solution of YMSA-0366·HCl (70.9 mg, 0.300 mmol) in EtOH (2.0 mL). The reaction mixture was refluxed for 21 h, then concentrated under reduced pressure and extracted with CHCl₃ from sat. NaHCO₃ aq. The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (AcOEt/MeOH = 10 : 1) to afford YMSA-0998·HCl as a pale yellow powder (22.6 mg, 21%). Mp 208 °C (dec). IR (ATR): ν = 2939, 2823, 1572, 1539, 1492, 1471, 1455, 1439, 1389, 1338, 1312, 1250, 1173, 1156, 1143, 1126, 1100, 1091, 1100, 1038, 1002, 985, 942, 924, 908, 891, 880, 836, 815, 767, 717, 651, 633, 591, 568, 556, 530, 508, 471, 449, 440, 426 cm⁻¹. ¹H-NMR (400 MHz, CDCl₃/TMS): δ = 8.49 (d, J = 5.2 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.70 (td, J = 8.4, 0.8 Hz, 1H), 7.48 (td, J = 7.6, 0.8 Hz, 1H), 7.12 (dd, J = 8.0, 2.8 Hz, 1H), 6.94 (d, J = 2.8 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 6.65 (d, J = 5.6 Hz, 1H), 6.55 (s, 1H), 4.90–4.65 (m, 1H), 3.72 (s, 2H), 2.65 (br s, 4H), 1.99–1.94 (m, 4H) ppm. ¹³C-NMR (100 MHz, CDCl₃/TMS): δ = 156.0, 150.8, 149.2, 148.8, 130.6, 130.0, 129.4, 126.7, 125.8, 125.5, 125.2, 122.5, 119.4, 119.0, 117.3, 101.1, 61.5, 48.8, 31.3, 31.1 ppm. HR-MS (ESI⁺): Calcd for [M + H]⁺, C₂₁H₂₃FN₃O: 352.1820; Found 352.1819. Anal. Calcd (%) for C₂₁H₂₂FN₃O; C 71.77, H 6.31, N 11.96; Found: C 71.08, H 6.06, N 10.95.

4-((7-Chloroquinolin-4-yl)amino)-2-((4,4-difluoropiperidin-1-yl)methyl)phenol (YMSA-0990): 4,4-Difluoropiperidine·HCl (3c) (47.3 mg, 0.300 mmol), Et₃N (100 μL, 0.715 mmol) and 37% HCHO aqueous solution (126 μL, 1.56 mmol) were added to a solution of KUMB-0002 (81 mg, 0.30 mmol) in EtOH (4 mL). The reaction mixture was refluxed for 44 h, then concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (hexane/AcOEt = 5 : 1 to 4 : 1) to afford YMSA-0990 as a pale yellow powder (17.3 mg, 14%). Mp. 204 °C (dec.). IR (ATR): ν = 2923, 2849, 2161, 1724, 1611, 1567, 1543, 1494, 1448, 1423, 1395, 1330, 1313, 1253, 1208, 1155, 1140, 1109, 1083, 1021, 1002, 976, 958, 936, 906, 876, 856, 808, 768, 662, 644, 607, 573, 557, 532, 505, 495, 468, 453, 427 cm⁻¹. ¹H-NMR (400 MHz, CDCl₃/TMS): δ = 8.46 (d, J = 5.6 Hz, 1H), 7.99 (d, J = 1.6 Hz, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.42 (dd, J = 8.4, 1.6 Hz, 1H), 7.13 (dd, J = 8.8, 2.0 Hz, 1H), 6.95 (s, 1H), 6.90 (d, J = 8.8 Hz, 1H), 6.61 (d, J = 5.6 Hz, 1H), 6.56 (br s, 1H), 3.77 (s, 2H), 2.72 (br s, 4H), 2.12–2.06 (m, 4H) ppm. ¹³C-NMR (100 MHz, CDCl₃/TMS): δ = 155.9, 151.7, 149.3, 135.4, 130.5, 128.8, 126.0, 126.0, 124.5, 122.2, 121.1, 117.5, 101.4, 60.7, 51.0, 50.0, 33.9, 29.8, 18.5 ppm. HR-MS (FAB+): Calcd for [M + H]⁺, C₂₁H₂₁³⁵ClF₂N₃O: 404.1341; Found 404.1341. Anal. Calcd (%) for C₂₁H₂₀ClF₂N₃O; C 62.46, H 4.99, N 10.40; Found: C 62.48, H 5.39, N 8.62.

2-((4-Fluoropiperidin-1-yl)methyl)-4-nitrophenol (5a): A mixture of 4-fluoropiperidine·HCl (3b) (71.8 mg, 0.309 mmol), Et₃N (150 µL, 1.08 mmol), and 2-hydroxy-5-nitrobenzyl bromide (4) (71.7 mg, 0.309 mmol) in tetrahydrofuran (THF) (3 mL) was refluxed for 1.5 h, then concentrated under reduced pressure and extracted with AcOEt from sat. NaHCO₃ aq. The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexanes/AcOEt = 3 : 1) to afford 5a as a yellow solid (63.6 mg, 91%). Mp 94–96 °C. IR (ATR): ν = 2964, 2824, 2514, 2196, 1619, 1589, 1523, 1480, 1463, 1450, 1375, 1336, 1276, 1177, 1148, 1132, 1121, 1110, 1087, 1033, 1014, 979, 966, 928, 903, 846, 831, 802, 788, 767, 750, 729, 660, 633, 542, 508, 495, 484, 461, 426 cm⁻¹. ¹H-NMR (300 MHz, CDCl₃/TMS): δ = 8.10 (dd, J = 9.0, 2.7 Hz, 1H), 7.95 (d, J = 2.7 Hz, 1H), 6.86 (d, J = 9.0 Hz, 1H), 4.95–4.65 (m, 1H), 3.81 (s, 2H), 2.70 (br s, 4H), 2.04–2.00 (m, 4H) ppm. ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 164.6, 140.2, 125.5, 124.8, 121.2, 116.6, 61.0, 49.0, 48.5, 31.1, 30.8 ppm. HR-MS (ESI⁺): Calcd for [M + H]⁺, C₁₂H₁₆FN₂O₃: 255.1139; Found 255.1138. Anal. Calcd (%) for C₁₂H₁₅FN₂O₃; C 56.69, H 5.95, N 11.02; Found: C 56.72, H 5.81, N 10.78.

2-((3-Fluoropiperidin-1-yl)methyl)-4-nitrophenol (5b): A mixture of 3-fluoropipreidine·HCl (3d) (53.0 mg, 0.208 mmol), Et₃N (105 µL, 0.751 mmol), and 4 (106.1 mg, 0.457 mmol) in THF (2.0 mL) was refluxed for 1 h, then concentrated under reduced pressure and extracted with AcOEt from sat. NaHCO₃ aq. The combined organic layer was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexanes/AcOEt = 4 : 1) to afford 5b as a yellow solid (26.6 mg, 50%). Mp 82.0–83.0 °C. IR (ATR): ν = 2940, 2841, 2555, 1620, 1589, 1516, 1470, 1457, 1406, 1391, 1368, 1334, 1306, 1279, 1192, 1137, 1087, 1044, 1024, 1005, 992, 964, 921, 896, 883, 827, 781, 771, 749, 722, 700, 656, 631, 553, 539, 513, 479, 444, 436, 410 cm⁻¹. ¹H-NMR (400 MHz, CDCl₃/TMS): δ = 8.10 (dd, J = 9.2, 2.8 Hz, 1H), 7.94 (d, J = 2.8 Hz, 1H), 6.88 (d, J = 9.2 Hz, 1H), 4.88–4.68 (m, 1H), 3.81 (s, 2H), 2.91–2.43 (m, 4H), 1.98–1.60 (m, 4H) ppm. ¹³C-NMR (100 MHz, CDCl₃/TMS) δ = 164.6, 140.2, 125.5, 124.8, 121.0, 116.8, 87.5, 85.8, 77.43, 77.1, 76.8, 60.9, 56.8, 56.6, 52.5, 20.8 ppm. HR-MS (ESI⁺): Calcd for [M + H]⁺, C₁₂H₁₆FN₂O₃: 255.1139; Found 255.1139. Anal. Calcd (%) for C₁₂H₁₅FN₂O₃; C 56.69, H 5.95, N 11.02; Found: C 56.71, H 5.88, N 10.66.

2-((Butyl(ethyl)amino)methyl)-4-nitrophenol (5c): A mixture of N-ethylbutylamine (3e) (49 µL, 0.36 mmol), Et₃N (50 µL, 0.36 mmol), and 4 (70.8 mg, 0.305 mmol) in THF (1.5 mL) was refluxed for 1.5 h, then concentrated under reduced pressure and extracted with AcOEt from sat. NaHCO₃ aq. The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexanes/AcOEt = 1 : 1) to afford 5c as a yellow solid (71.1 mg, 92%). Mp 38.0–39.0 °C. IR (ATR): ν = 3091, 3060, 2933, 2957, 2855, 2421, 2163, 1928, 1585, 1528, 1482, 1463, 1443, 1391, 1379, 1332, 1285, 1266, 1183, 1157, 1128, 1118, 1081, 1063, 1030, 990, 960, 939, 907, 840, 781, 752, 658, 631, 548, 498, 456, 443, 428 cm⁻¹. ¹H-NMR (400 MHz, CDCl₃/TMS): δ = 8.08 (dd, J = 9.2, 2.8 Hz, 1H), 7.93 (d, J = 2.8 Hz, 1H), 6.81 (d, J = 9.2 Hz, 1H), 3.86 (s, 2H), 2.67 (q, J = 7.2 Hz, 2H), 2.58 (t, J = 7.6 Hz, 2H), 1.59–1.51 (m, 2H), 1.38–1.29 (m, 2H), 1.14 (t, J = 7.2 Hz, 3H), 0.93 (t, J = 7.2 Hz, 3H) ppm. ¹³C-NMR (100 MHz, CDCl₃/TMS) δ = 165.7, 139.7, 125.4, 124.6, 121.8, 116.6, 57.1, 52.6, 46.8, 28.3, 20.6, 14.0, 10.8 ppm. HR-MS (ESI⁺): Calcd for [M + H]⁺, C₁₃H₂₁N₂O₃: 253.1547; Found 253.1545. Anal. Calcd (%) for C₁₃H₂₀N₂O₃; C 61.88, H 7.99, N 11.10; Found: C 61.92, H 7.85, N 10.97.

2-((Isopentyl(methyl)amino)methyl)-4-nitrophenol (5d): A mixture of N-isopentylmethylamine·HCl (3f) (77.6 mg, 0.564 mmol), Et₃N (96 µL, 0.69 mmol), and 4 (106.1 mg, 0.457 mmol) in THF (4.0 mL) was refluxed for 1 h, then concentrated under reduced pressure and extracted with AcOEt from sat. NaHCO₃ aq. The combined organic layer was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexanes/AcOEt = 1 : 1) to afford 5d as a yellow oil (84.6 mg, 73%). IR (ATR): ν = 2957, 2870, 1618, 1588, 1521, 1473, 1386, 1369, 1331, 1279, 1165, 1124, 1085, 1053, 1010, 970, 928, 904, 859, 824, 801, 772, 752, 730, 661, 635, 538, 509, 443, 420 cm⁻¹. ¹H-NMR (400 MHz, CDCl₃/TMS): δ = 8.07 (dd, J = 9.2, 2.8 Hz, 1H), 7.91 (d, J = 2.8 Hz, 1H), 6.81 (d, J = 8.8 Hz, 1H), 3.78 (s, 2H), 2.53 (t, J = 7.2 Hz, 2H), 2.31 (s, 3H), 1.64–1.55 (m, 1H), 1.45 (q, J = 7.2 Hz, 2H), 0.89 (d, J = 6.8 Hz, 6H) ppm. ¹³C-NMR (100 MHz, CDCl₃/TMS) δ = 165.4, 139.8, 125.5, 124.6, 121.7, 116.5, 60.8, 55.2, 41.2, 35.6, 26.2, 22.6 ppm. HR-MS (ESI⁺): Calcd for [M + H]⁺, C₁₃H₂₁N₂O₃: 253.1547; Found 253.1546.

4-Nitro-2-((4-phenylpiperidin-1-yl)methyl)phenol (5e): A mixture of 4-phenylpiperidine (3g) (49.3 mg, 0.306 mmol), Et₃N (95 µL, 0.680 mmol), and 4 (52.2 mg, 0.255 mmol) in THF (2.0 mL) was refluxed for 1 h, then concentrated under reduced pressure and extracted with AcOEt from sat. NaHCO₃ aq. The combined organic layer was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexanes/AcOEt = 1 : 1) to afford 5e as a yellow solid (75.1 mg, 97%). Mp. 172–174 °C. IR (ATR): ν = 2959, 2209, 1593, 1550, 1505, 1492, 1473, 1439, 1421, 1373, 1342, 1284, 1170, 1156, 1128, 1108, 1085, 1043, 950, 921, 909, 839, 827, 815, 776, 758, 738, 699, 645, 617, 573, 548, 534, 511, 491, 467, 442, 420, 406 cm⁻¹. ¹H-NMR (400 MHz, CDCl₃/TMS): δ = 8.09 (dd, J = 9.2, 2.8 Hz, 1H), 7.95 (d, J = 2.8 Hz, 1H), 7.31 (t, J = 7.6 Hz, 2H), 7.25–7.20 (m, 3H), 6.85 (d, J = 9.2 Hz, 1H), 3.83 (s, 2H), 3.12 (d, J = 11.6 Hz, 2H), 2.64–2.58 (m, 1H), 2.32 (td, J = 12.0, 2.4 Hz, 2H), 1.95 (d, J = 13.6 Hz, 2H), 1.88–1.77 (m, 2H) ppm. ¹³C-NMR (100 MHz, CDCl₃/TMS) δ = 165.0, 145.0, 140.1, 128.7, 126.8, 126.7, 125.4, 124.8, 121.4, 116.6, 61.2, 53.7, 42.0, 33.2 ppm. HR-MS (ESI⁺): Calcd for [M + H]⁺, C₁₈H₂₁N₂O₃: 313.1547; Found 313.1546. Anal. Calcd (%) for C₁₈H₂₀N₂O₃; C 69.21, H 6.45, N 8.97; Found: C 69.14, H 6.12, N 8.92.

4-((7-Chloroquinolin-4-yl)amino)-2-((4-fluoropiperidin-1-yl)methyl)phenol (YMSA-0448): SnCl₂·2H₂O (97.5 mg, 0.432 mmol) was added to a solution of 5a (25.7 mg, 0.101 mmol) in EtOH (1.1 mL) and the reaction mixture was refluxed for 1 h. Then, 4,7-chloroquinoline (1b) (27.7 mg, 0.140 mmol) was added. The reaction mixture was further refluxed for 2 h, then concentrated under reduced pressure, and extracted with AcOEt from 4 N NaOH aq. The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The remaining residue was purified by silica gel column chromatography (hexanes/AcOEt = 1 : 1) to afford YMSA-0448 as a pale yellow powder (32.3 mg, 83%). Mp 197–199 °C. IR (ATR): ν = 2951, 2830, 2157, 2036, 1611, 1567, 1540, 1492, 1250, 1422, 1393, 1372, 1328, 1310, 1261, 1244, 1199, 1164, 1154, 1103, 1082, 1039, 982, 929, 908, 876, 855, 839, 816, 767, 718, 710, 703, 689, 674, 660, 652, 643, 621, 607, 583, 553, 525, 513, 506, 484, 476, 468, 453, 446, 438, 430, 423, 416 cm⁻¹. ¹H-NMR (300 MHz, CDCl₃/TMS): δ = 8.48 (d, J = 5.1 Hz, 1H), 8.01 (d, J = 2.4 Hz, 1H), 7.81 (d, J = 8.7 Hz, 1H), 7.44 (dd, J = 8.7, 2.4 Hz, 1H), 7.12 (dd, J = 8.7, 2.4 Hz, 1H), 6.94 (d, J = 2.1 Hz, 1H), 6.89 (d, J = 8.7 Hz, 1H), 6.62 (d, J = 5.1 Hz, 1H), 6.44 (s, 2H), 4.90–4.65 (m, 1H), 3.74 (br s, 4H), 2.03–1.95 (m, 5H) ppm. ¹³C-NMR (100 MHz, CDCl₃/TMS): δ = 156.1, 152.0, 149.5, 149.4, 135.2, 130.3, 128.8, 125.5, 122.5, 121.2, 117.5, 117.3, 101.4, 61.4, 48.8, 31.3, 31.1 ppm. HR-MS (ESI⁺): Calcd for [M + H]⁺, C₂₂H₂₂³⁵ClFN₃O: 386.1430; Found 386.1429. Anal. Calcd (%) for C₂₁H₂₁ClFN₃O; C 65.37, H 5.49, N 10.89; Found: C 65.27, H 5.07, N 10.38.

2-((4-Fluoropiperidin-1-yl)methyl)-4-((7-iodoquinolin-4-yl)amino)phenol (YMSA-0381): SnCl₂·2H₂O (48.4 mg, 0.214 mmol) was added to a solution of 5a (12.9 mg, 0.0507 mmol) in EtOH (500 mL) and the reaction mixture was refluxed for 1 h. Then, 4-chloro-7-iodoquinoline (1c) (19.6 mg, 0.0677 mmol) was added. The reaction mixture was further refluxed for 1 h, then concentrated under reduced pressure, and extracted with AcOEt from 4 N NaOH aq. The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexanes/AcOEt = 1 : 1 to AcOEt/MeOH = 20 : 1) to afford YMSA-0381 as a pale yellow powder (20.7 mg, 86%). Mp 223–226 °C. IR (ATR): ν = 2955, 2924, 2832, 2159, 2018, 1924, 1735, 1620, 1601, 1571, 1539, 1495, 1451, 1420, 1367, 1341, 1329, 1307, 1273, 1259, 1241, 1208, 1195, 1171, 1156, 1122, 1104, 1091, 1071, 1043, 992, 980, 950, 939, 911, 920, 898, 884, 837, 805, 788, 770, 762, 690, 654, 634, 609, 575, 561, 525, 506, 474, 447, 429, 423 cm⁻¹. ¹H-NMR (300 MHz, CDCl₃/TMS): δ = 8.46 (d, J = 5.4 Hz, 1H), 8.43 (d, J = 1.8 Hz, 1H), 7.75 (dd, J = 8.7, 1.8 Hz, 1H), 7.59 (d, J = 9.0 Hz, 1H), 7.11 (dd, J = 8.4, 2.4 Hz, 1H), 6.93 (d, J = 2.7, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.64 (d, J = 5.4 Hz, 1H), 6.46 (s, 1H), 4.90–4.65 (m, 1H), 3.74 (s, 2H), 2.66 (br s, 4H), 2.01–1.95 (m, 4H) ppm. ¹³C-NMR (100 MHz, CDCl₃/TMS): δ = 156.3, 151.7, 129.9, 129.5, 139.0, 133.8, 130.4, 125.9, 125.6, 122.6, 121.1, 118.3, 117.4, 101.7, 95.6, 61.6, 48.9, 31.4, 31.2 ppm. HR-MS (ESI⁺): Calcd for [M + H]⁺, C₂₁H₂₂FIN₃O: 478.0786; Found 478.0784. Anal. Calcd (%) for C₂₁H₂₁FIN₃O; C 52.84, H 4.43, N 8.80; Found: C 53.80, H 4.43, N 8.25.

4-((7-Chloroquinolin-4-yl)amino)-2-((3-fluoropiperidin-1-yl)methyl)phenol (YMSA-0423): SnCl₂·2H₂O (65.4 mg, 0.290 mmol) was added to a solution of 5b (18.6 mg, 0.0732 mmol) in EtOH (1.5 mL) and the reaction mixture was refluxed for 30 min. Then, 1b (29.0 mg, 0.146 mmol) was added. The reaction mixture was further refluxed for 1 h, then concentrated under reduced pressure and extracted with AcOEt from 1 N NaOH aq. The combined organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexanes/AcOEt = 1 : 1) to afford YMSA-0423 as a pale yellow powder (17.4 mg, 62%). Mp 149–150 °C. IR (ATR): ν = 2948, 2820, 2162, 1612, 1566, 1542, 1492, 1448, 1419, 1372, 1328, 1308, 1258, 1218, 1198, 1166, 1155, 1139, 1104, 1083, 1043, 1026, 1006, 990, 970, 928, 905, 881, 856, 817, 797, 768, 720, 704, 642, 607, 557, 518, 476, 453, 429 cm⁻¹. ¹H-NMR (300 MHz, CDCl₃/TMS): δ = 8.49 (d, J = 5.1 Hz, 1H), 8.01 (d, J = 1.8 Hz, 1H), 7.81 (d, J = 8.7 Hz, 1H), 7.44 (dd, J = 9.0, 2.0 Hz, 1H), 7.12 (dd, J = 8.4, 3.0 Hz, 1H), 6.94–6.90 (m, 2H), 6.62 (d, J = 5.1 Hz, 1H), 6.44 (s, 1H), 4.83–4.68 (m, 1H), 3.77–3.48 (m, 2H), 2.77–2.48 (m, 4H), 1.95–1.60 (m, 7H). ¹³C-NMR (100 MHz, CDCl₃/TMS) δ = 156.2, 152.0, 149.6, 149.2, 135.2, 130.2, 129.1, 125.9, 125.5, 122.3, 121.0, 117.5, 101.4, 87.9, 86.2, 61.4, 57.0, 56.8, 52.6, 29.5, 21.3 ppm. HR-MS (ESI⁺): Calcd for [M + H]⁺, C₂₁H₂₁³⁵ClFN₃O: 386.1430; Found 386.1430. Anal. Calcd (%) for C₂₁H₂₀ClFN₃O; C 65.37, H 5.49, N 10.89; Found: C 65.21, H 5.48, N 10.53.

2-((Butyl(ethyl)amino)methyl)-4-((7-chloroquinolin-4-yl)amino)phenol (YMSA-0267): SnCl₂·2H₂O (163.8 mg, 0.726 mmol) was added to a solution of 5c (45.4 mg, 0.180 mmol) in EtOH (2.0 mL) and the reaction mixture was refluxed for 1 h. Then, 1b (43.2 mg, 0.218 mmol) was added. The reaction mixture was further refluxed for 1 h, then concentrated under reduced pressure and extracted with AcOEt from 4 N NaOH aq. The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was recrystallized with EtOH/H₂O to afford YMSA-0267 as pale yellow crystals (48.0 mg, 70%). Mp 150–151 °C. IR (ATR): ν = 2957. 2932, 2851, 2034, 1612, 1566, 1540, 1494, 1421, 1389, 1370, 1327, 1263, 1249, 1216, 1186, 1162, 1108, 1083, 1063, 989, 954, 928, 903, 880, 854, 818, 785, 766, 719, 640, 609, 576, 556, 506, 478, 450, 431, 422, 409 cm⁻¹. ¹H-NMR (300 MHz, CDCl₃/TMS): δ = 8.48 (d, J = 5.7 Hz, 1H), 8.00 (d, J = 1.8 Hz, 1H), 7.81 (d, J = 7.81 Hz, 1H), 7.43 (dd, J = 9.2, 1.8 Hz, 1H), 7.09 (dd, J = 8.4, 2.7 Hz), 6.92 (d, J = 2.4 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 6.62 (d, J = 5.1 Hz, 1H), 6.47 (s, 1H), 3.78 (s, 2H), 2.64 (q, J = 7.2 Hz, 2H), 2.56 (t, J = 7.2 Hz, 2H), 1.60–1.50 (m, 2H), 1.41–1.29 (m, 2H), 1.13 (t, J = 7.5 Hz, 3H), 0.932 (t, J = 7.2 Hz, 3H) ppm. ¹³C-NMR (100 MHz, CDCl₃/TMS) δ = 155.0, 151.8, 149.5, 133.7, 130.4, 127.6, 125.4, 124.7, 124.6, 124.2, 124.1, 117.7, 116.03, 100.4, 55.3, 52.3, 46.4, 28.2, 20.0, 13.8, 10.9 ppm. HR-MS (ESI⁺): Calcd for [M + H]⁺, C₂₂H₂₇³⁵ClN₃O_: 384.1837; Found 384.1833. Anal. Calcd (%) for C₂₂H₂₆ClN₃O; C 68.83, H 6.83, N 10.95; Found: C 68.30, H 6.36, N 10.60.

4-((7-Chloroquinolin-4-yl)amino)-2-((isopentyl(methyl)amino)methyl)phenol (YMSA-0454): SnCl₂·2H₂O (448.7 mg, 1.99 mmol) was added to a solution of 5d (135.5 mg, 0.537 mmol) in EtOH (5 mL) and the reaction mixture was refluxed for 1 h. Then, 1b (131.3 mg, 0.663 mmol) was added. The reaction mixture was refluxed for 1 h, then concentrated under reduced pressure and extracted with AcOEt from 1N NaOH aq. The combined organic layer was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was recrystallized from EtOH/H₂O to afford YMSA-0454 as pale yellow crystals (82.7 mg, 40%). Mp 149–150 °C. IR (ATR): ν = 2955, 2868, 2160, 2037, 1610, 1567, 1541, 1492, 1450, 1425, 1413, 1371, 1328, 1258, 1218, 1165, 1109, 1083, 1051, 1013, 973, 906, 879, 855, 814, 764, 642, 610, 574, 551, 516, 475, 454, 427 cm⁻¹. ¹H-NMR (300 MHz, CDCl₃/TMS): δ = 8.48 (d, J = 5.4 Hz, 1H), 8.00 (d, J = 2.1 Hz, 1H), 7.81 (d, J = 9.0 Hz, 1H), 7.43 (dd, J = 8.7, 2.1 Hz, 1H), 7.01 (dd, J = 8.4, 3.0 Hz, 1H), 6.92 (d, J = 2.1 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.44 (s, 1H), 3.71 (s, 1H), 2.52 (t, J = 7.5 Hz, 2H), 2.31 (s, 3H), 1.65–1.56 (m, 1H), 1.51–1.43 (m, 2H), 0.92 (d, J = 6.6 Hz, 6H) ppm. ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 164.0, 158.1, 154.7, 151.9, 149.5, 144.0, 133.7, 130.4, 127.6, 125.6, 124.8, 124.6, 124.2, 123.9, 116.0, 100.4, 58.8, 58.0, 54.6, 41.1, 35.4, 25.6, 22.5 ppm. HR-MS (ESI⁺): Calcd for [M + H]⁺, C₂₂H₂₇³⁵ClN₃O: 384.1837; Found 384.1835. Anal. Calcd (%) for C₂₂H₂₆ClN₃O; C 68.83, H 6.83, N 10.95; Found: C 68.53, H 6.22, N 10.85.

4-((7-Iodoquinolin-4-yl)amino)-2-((isopentyl(methyl)amino)methyl)phenol (YMSA-0455): SnCl₂·2H₂O (479.3 mg, 2.12 mmol) was added to a solution of 5d (133.9 mg, 0.531 mmol) in EtOH (5 mL) and the reaction mixture was refluxed for 30 min. Then, 4-chloro-7-iodoquinoline (1c) (180.1 mg, 0.622 mmol) was added. The reaction mixture was further refluxed for 1 h, then concentrated under reduced pressure and extracted with AcOEt from 1N NaOH aq. The combined organic layer was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was recrystallized from EtOH/H₂O to afford YMSA-0455 as pale yellow crystals (102.1 mg, 40%). Mp = 175–177 °C, IR (ATR): ν = 2955, 1602, 1570, 1493, 1444, 1420, 1365, 1328, 1254, 1169, 1107, 1069, 1011, 971, 880, 832, 812, 761, 634, 574, 548, 462, 445, 422 cm⁻¹. ¹H-NMR (300 MHz, CDCl₃/TMS): δ = 8.40 (d, J = 1.2 Hz, 1H), 8.35 (d, J = 5.7 Hz, 1H), 7.73–7.63 (m, 2H), 7.10 (dd, J = 8.3, 2.1 Hz, 1H), 6.94 (d, J = 2.7 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.58 (d, J = 5.4 Hz, 1H), 5.16 (br s, 1H), 3.72 (s, 2H), 2.53 (t, J = 7.5 Hz, 2H), 2.31 (s, 3H), 1.67–1.56 (m, 1H), 1.47 (q, J = 8.4 Hz, 2H), 0.92 (d, J = 6.6 Hz, 6H) ppm. ¹³C-NMR (75 MHz, CDCl₃/TMS): δ = 156.5, 151.6, 149.8, 149.3, 138.9, 133.5, 129.8, 125.6, 125.3, 123.2, 120.8, 118.1, 117.1, 101.5, 95.2, 61.2, 55.3, 41.3, 35.8, 26.1, 22.6 ppm. HR-MS (FAB⁺): Calcd for [M + H]⁺, C₂₂H₂₇IN₃O, 476.1199; Found 476.1200. Anal. Calcd (%) for C₂₂H₂₆IN₃O: C 55.59, H 5.51, N 8.84; Found: C 55.28, H 5.26, N 8.65.

4-((7-Chloroquinolin-4-yl)amino)-2-((4-phenylpiperidin-1-yl)methyl)phenol (YMSA-0995): SnCl₂·2H₂O (97.5 mg, 0.432 mmol) was added to a solution of 5e (49.4 mg, 0.158 mmol) in EtOH (3 mL) and the reaction mixture was refluxed for 30 min. Then, 1b (64.4 mg, 0.325 mmol) was added. The reaction mixture was refluxed for 1 h, then concentrated under reduced pressure and extracted with AcOEt from 1 N NaOH aq. The combined organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/AcOEt = 1 : 1) and by NH silica gel (CHROMATOREX, NH-DM1020, Fuji Silysia Chemical Ltd.) column chromatography (hexanes/AcOEt = 2 : 1 to 1 : 1) to afford YMSA-0995 as a pale yellow powder (49.2 mg, 70%). Mp 191–193 °C. IR (ATR): ν = 2931, 2813, 2159, 1977, 1607, 1566, 1538, 1492, 1453, 1419, 1370, 1325, 1261, 1247, 1142, 1106, 1082, 1026, 991, 967, 930, 903, 880, 854, 815, 783, 754, 702, 642, 611, 556, 539, 479, 451, 422, 413 cm⁻¹. ¹H-NMR (300 MHz, CDCl₃/TMS): δ = 8.47 (d, J = 5.1 Hz, 1H), 8.00 (d, J = 2.1 Hz, 1H), 7.84 (d, J = 9.0 Hz, 1H), 7.41 (dd, J = 8.7, 2.1 Hz, 1H), 7.35–7.30 (m, 2H), 7.26–7.20 (m, 3H), 7.11 (dd, J = 8.4, 2.4 Hz, 1H), 6.95 (d, J = 2.1 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.63 (brd, J = 5.4 Hz, 2H), 3.75 (s, 2H), 3.16 (d, J = 11.7 Hz, 2H), 2.63–2.54 (m, 1H), 2.26 (td, J = 11.7, 2.4 Hz, 2H), 1.96–1.77 (m, 4H) ppm. ¹³C-NMR (75 MHz, CDCl₃/TMS): δ = 156.5, 152.1, 149.7, 149.5, 145.5, 135.4, 130.3, 129.1, 128.8, 127.0, 126.6, 126.0, 125.9, 125.6, 122.9, 121.3, 117.6, 117.4, 101.6, 61.8, 54.0, 42.2, 33.5 ppm. HR-MS (ESI⁺): Calcd for [M + H]⁺, C₂₇H₂₇³⁵ClN₃O: 444.1837; Found 444.1836. Anal. Calcd (%) for C₂₇H₂₆ClN₃O; C 73.04, H 5.90, N 9.46; Found: C 72.92, H 5.51, N 9.40.

X-Ray Data Collection and Refinement of YMSA-0998

Crystalline YMSA-0998 were obtained by recrystallization from n-hexane/CHCl₃. The X-ray diffraction images were obtained using an XtaLAB synergy-S system (RIGAKU, Tokyo, Japan) and analyzed by Olex2 software Ver. 2-1. 3 (OlexSys, Durham, U.K.).²²⁾ The crystal structure was solved by the SHELXT program and refined by the SHELXL program.^23,24) Crystal data for YMSA-0998 (C₂₁H₂₂FN₃O, M_r = 351.42), Monoclinic, P2₁/n, a = 9.2920 (3), b = 19.0301 (6), c = 10.4853 (3) Å, V = 1833.80 (10) ų, α = 90°, β = 98.484°, γ = 90°, Z = 4, ρ_calc = 1.273 g·cm⁻³, R = 0.0645 (3021 reflections), R_w = 0.2187 (4875 reflections), GOF = 1.070. The crystal data in this manuscript can be obtained free of charge from The Cambridge Crystallographic Data Centre via https://www.ccdc.cam.ac.uk/structures/? (CCDC 2354891).

Cell Culture

Cell culture was performed as described previously.^25,26) J774.1 cells (RIKEN BioResource Center, Ibaraki, Japan) were grown in RPMI 1640 medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Waltham, MA, U.S.A.), 100 U/mL penicillin and 100 µg/mL streptomycin in a humidified atmosphere of 5% CO₂ in air at 37 °C.

Enzyme-Linked Immunosorbent Assay (ELISA)

The concentration of IL-6 was measured by ELISA, as described previously.^25,26) J774.1 cells were pre-incubated with ADQ or its derivatives for 30 min, then incubated with poly(I:C) (5 µg/mL), LPS (0.1 ng/mL), imiquimod (1 µg/mL) or CpG ODN (200 ng/mL) for 6 h in an atmosphere of 5% CO₂ in air at 37 °C.

Culture supernatant was harvested, and IL-6 was measured by ELISA. 96-well plates were coated with purified anti-mouse IL-6 monoclonal antibody (mAb) (1 : 2000) (eBioscience, San Diego, CA, U.S.A.). The plates were incubated at 4 °C overnight, washed with PBS containing 0.05% Tween-20, and blocked with PBS containing 1% bovine serum albumin (BSA). The plates were incubated for 1 h at room temperature, washed again, and then incubated overnight at 4 °C with culture supernatant and recombinant mouse IL-6 (BioLegend, San Diego, CA, U.S.A.) in order to obtain a standard curve. The plates were washed again, incubated with anti-mouse biotin-conjugated IL-6 mAb (1 : 2000) (eBioscience, San Diego, CA, U.S.A.) for 1 h at room temperature, then washed and incubated with avidin-horseradish peroxidase (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 10 min at room temperature. The plates were washed again, and 3,3′,5,5′-tetramethylbenzidine (TMB) (FUJIFILM Wako Pure Chemical Corporation) was added. When an appropriate color reaction was observed, 2.5 M H₂SO₄ was added to stop the reaction. The absorbance at 450 nm was measured with a Wallac 1420 ARVO Fluoroscan (Wallac, Turku, Finland).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay

Cell viability was evaluated by MTT assay, as described previously.^25,26) After removal of the culture supernatant to measure cytokine production, MTT reagent suspended in RPMI-1640 supplemented with 10% FBS was added to J774.1 cells in the 96-well plates. Alternatively, J774.1 cells were incubated with ADQ or its analogs for 24 h, then MTT reagent was added. After the color reaction associated with the reduction of MTT in the control group was observed to be sufficient, the stopping solution was added. The absorbance of each well was read on a WALLAC 1420 ARVO Fluoroscan (570 nm).

Real-Time RT-PCR

The level of IL-6 mRNA expression was measured by real-time RT-PCR, as described previously.²⁵⁾ RT²-qPCR^® primer assay for mouse IL-6 was purchased from Qiagen (Venlo, The Netherlands). J774.1 cells were pre-incubated with ADQ or its derivatives for 30 min, then incubated with CpG ODN (200 ng/mL) for 3 h in an atmosphere of 5% CO₂ in air at 37 °C. Total RNA was extracted from J774.1 cells using a ReliaPrep™ RNA Cell Miniprep System (Promega, Madison, WI, U.S.A.), and first-strand cDNA was synthesized with PrimeScript Reverse Transcriptase (TaKaRa Bio Inc., Shiga, Japan). The cDNA was used as a template for real-time PCR analysis: reactions were performed in a CFX Connect Real-Time System (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was determined as a positive control. Each sample was assayed in a 20 µL amplification reaction mixture, containing cDNA, primers and 2× KAPA SYBR^® FAST qPCR Master Mix (Kapa Biosystems, Wilmington, MA, U.S.A.). The amplification program included 40 cycles of two steps, (95 °C and 60 °C, respectively). Fluorescent products were detected at the last step of each cycle. The obtained values were normalized to GAPDH mRNA expression.

Lysosomal Activity Inhibition Assay

J774.1 cells were seeded in 12-well plates and incubated for 24 h in an atmosphere of 5% CO₂ in air at 37 °C. The cells were pretreated with HCQ, ADQ and ADQ derivatives (0.1–10 µM) for 30 min, and LysoTracker^® Red Lysosomal Probe (50 nM, Lonza, Basel, Switzerland) were added for 1 h. After the incubation, the cells were collected by trypsin treatment, washed with RPMI-1640 based buffer, and assessed with a Gallios flow cytometer (Beckman Coulter, Inc., Brea, CA, U.S.A.). Data analysis was done with Flow Jo software (FlowJo LCC, Ashland, OR, U.S.A.).

Calculation of IC₅₀ and 50% Cytotoxic Concentration (CC₅₀)

The values of the IC₅₀ or the CC₅₀ were calculated as previously described.²⁶⁾ The IL-6 levels were calculated from the standard curve obtained as described above, and the inhibitory ratio in each sample with respect to the control group was calculated. Cell viability was calculated from the ratio of the absorbance measured in MTT assay of each sample to that of the control group. The values of IC₅₀ and CC₅₀ were determined by linear interpolation from the two adjacent points in dose-response plots of the mean values of data obtained in three or more independent experiments.

Statistics

Values are given as the mean ± standard error (S.E.). Multiple groups were compared using one-way ANOVA followed by Dunnett’s test (control group vs. drug-treated group) or two-way ANOVA followed by Dunnett’s test (control group vs. drug-treated group) as implemented in GraphPad Prism version 9.0 statistical package (GraphPad Software, San Diego, CA, U.S.A.). The criterion of significance was set at p < 0.05.

RESULTS

Synthesis of Inhibitors of IL-6 Production in Macrophages

The candidate IL-6 production inhibitors were synthesized as shown in Figs. 2 and 3. First, 4-chloroquinoline (1a) and 4,7-dichloroquinoline (1b) were reacted with 4-aminophenol (2) in a microwave reactor to afford YMSA-0366 and KUMB-0002, respectively (Fig. 2). The Mannich reaction of YMSA-0366 with 4-fluoropiperidine (3b) gave YMSA-0988 and the similar reaction of KUMB-0002 with N-methylpentylamine (3a) and 4,4-difluoropiperidine (3c) afforded YMSA-0203 and -0990, respectively. We chose to introduce fluorine into the piperidine rings of YMSA-0998 and 0990 because it is reported to have a significant influence on the physical and chemical properties such as metabolic stability and cell membrane permeability of drugs.^27,28)

Fig. 2. Synthesis of YMSA-0203, 0998, and 0990

Fig. 3. Synthesis of YMSA-0267, 0448, 0381, 0423, 0454, 0455, and 0995

Because the chemical yields in the Mannich reactions of YMSA-0366 and KUMB-0002 described in Fig. 2 were unsatisfactory, the synthesis of other compounds was carried out as shown in Fig. 3. For the synthesis of YMSA-0267, 0448, 0381, 0423, 0454, 0455, and 0995, 2-(bromomethyl)-4-nitrophenol (4) was reacted with 3b and 3d–g to afford 5a–e, respectively, whose nitro groups were reduced with SnCl₂. The resulting amino compounds were reacted with the corresponding 4-chloroquinoline units 1a–c to obtain the corresponding compounds in moderate to high chemical yields.

X-Ray Crystal Structure of YMSA-0998

Recrystallization of some of the derivatives synthesized above was conducted to confirm their structures. Fine crystals of YMSA-0998 were obtained by recrystallization from hexanes/CHCl₃ and the X-ray crystal structure was solved (Fig. 4), establishing that the 6-membered piperidine adopts chair form and the fluorine atom at the 4-position resides in the axial position, possibly due to dipole-dipole repulsion (or dipole minimization) between the N-H bond and a lone pair of nitrogen in the piperidine ring.^27,28) It is very likely that the conformation of the 4-fluoropiperidine ring of YMSA-0998 is almost same as those of YMSA-0448 and 0381, which will be presented as potent inhibitors of IL-6 production below.

Fig. 4. ORTEP Drawing of the X-Ray Crystal Structure of YMSA-0998 (with 50% Probability Ellipsoids) Obtained by Recrystallization from Hexanes/CHCl₃ (CCDC No.: 2354891)

Each sphere represents an individual atom (white color: C; white small sphere: H; blue: N; red: O; green color: F).

Amodiaquine Potently Suppresses IL-6 Production in Murine Macrophages Induced by Agonists of TLRs Recognizing Pathogen Nucleic Acids

First, we evaluated the effect of ADQ on IL-6 production induced by TLR agonists in murine macrophages. ADQ significantly suppressed IL-6 release induced by the TLR3 agonist poly(I:C) (Fig. 5A) or the TLR7 agonist imiquimod (Fig. 5B) in a concentration-dependent manner in J774.1 cells. Interestingly, ADQ inhibited IL-6 release induced by the TLR9 agonist CpG ODN in a lower concentration range (Fig. 5C), whereas it had had no significant effect on IL-6 release induced by the TLR4 agonist LPS (Fig. 5D). ADQ was found to inhibit IL-6 production via TLR3, TLR7, and TLR9 activation with IC₅₀ values of 2.43, 3.48, and 0.0359 µM, respectively (Figs. 5E–H, Table 1). These results indicate that ADQ suppresses inflammatory responses induced via TLRs recognizing pathogen nucleic acids, with a high selectivity for response via TLR9. Meanwhile, KUMB-0002 lacking the diethylaminomethyl group of ADQ showed lower inhibitory activity towards IL-6 production induced by agonists of TLR3, TLR7, and TLR9 (Figs. 5E–H, Table 1), but higher activity towards IL-6 production induced by LPS. These results indicate that the dialkylaminomethyl group is an important structure for the inhibition of IL-6 production mediated by TLRs recognizing pathogen RNA or DNA.

Fig. 5. The Effect of ADQ and KUMB-0002 on IL-6 Production Induced by TLR Agonists in Murine Macrophages

J774.1 cells were pre-incubated with ADQ or KUMB-0002 (0.1–10 µM or 0.0001–10 µM) for 0.5 h, then incubated with poly(I:C) (5 µg/mL) (A, E), imiquimod (1 µg/mL) (B, F), CpG ODN (200 ng/mL) (C, G), or LPS (0.1 ng/mL) (D, H) for 6 h. IL-6 was measured by means of ELISA. Error bars indicate ± S.E. (ADQ: n = 18–36, KUMB-0002: n = 6–12, three or more independent experiments). (A–D) Statistical analysis was performed by one-way ANOVA followed by Dunnett’s test. Significant differences between control and test groups are indicated by *** (p < 0.001) or **** (p < 0.0001). ADQ: amodiaquine.

Table 1. Inhibitory Activity of ADQ and Its Derivatives towards IL-6 Production Mediated by TLRs and Effect on Cell Viability

The IC₅₀ values for inhibition of IL-6 production and the CC₅₀ values were determined from the dose-response curves. IC₅₀: 50% inhibitory concentration. CC₅₀: 50% cytotoxic concentration.

The Effect of Amodiaquine Analogs on IL-6 Production Induced by TLR Agonists in Murine Macrophages

Next, the effect of substituents on the amino group of ADQ on the anti-inflammatory activity was examined (Fig. 6, Table 1). YMSA-0203 bearing an n-methylpentyl substituent showed higher inhibitory activity than ADQ against TLR3-, TLR4-, and TLR7-mediated IL-6 production, but showed similar activity against TLR9-mediated IL-6 production. YMSA-0267 bearing an N-butylethyl-substituted amino group showed generally higher inhibitory activity against TLRs-mediated IL-6 production, as well as a higher ratio of IC₅₀ for IL-6 production via TLR9 relative to that via other TLRs (IC₅₀ value for TLR9-mediated IL-6 production = 0.00636 µM). YMSA-0454 and -0455, which possess an N-isopentylmethylamino group, showed more potent activity against TLR3-, TLR4-, and TLR7 mediated IL-6 production, while only YMSA-0454 showed lower activity against TLR9-mediated IL-6 production. These results indicate that the alkyl chain on the aminomethyl group and the halogen substituent on the quinoline ring are critical structures that can affect the IL-6 production-inhibitory activity of ADQ. Furthermore, substitution with highly hydrophobic linear alkyl chains tends to increase the activity.

Fig. 6. Inhibition by ADQ and Its Analogs of IL-6 Production Induced by TLR Agonists

(A–L) J774.1 cells were pre-incubated with ADQ or its analogs (0.1–10 µM or 0.0001–10 µM) for 0.5 h, and then incubated with poly(I:C) (5 µg/mL) (A, E, I), imiquimod (1 µg/mL) (B, F, J), CpG ODN (200 ng/mL) (C, G, K) or LPS (0.1 ng/mL) (D, H, L) for 6 h. IL-6 was measured by ELISA. The inhibitory ratio of each compound relative to the control is shown. (M) Cells were pre-incubated with ADQ, YMSA-0448, or KUMB-0002 (100 nM) for 0.5 h, and then incubated with CpG ODN (200 ng/mL) for 3 h. Expression of IL-6 mRNA was analyzed by real-time RT-PCR. Error bars indicate ± S.E. (A–L: n = 6–36, M: n = 3, three or more independent experiments). (M) Statistical analysis was performed by two-way ANOVA followed by Dunnett’s test. Significant differences between control and test groups are indicated by * (p < 0.05). ADQ: amodiaquine.

Subsequently, we evaluated the activity of analogs bearing a piperidine ring. YMSA-0423, which has a racemic 3-fluoropiperidinyl group, showed slightly lower activity against TLR3-mediated IL-6 production, but similar activity against TLR7-mediated, and higher activity against TLR4-mediated IL-6 production. YMSA-0448, which has a 4-fluoropiperidyl group, showed similar activity to ADQ against TLR3-, and TLR7-mediated IL-6 production, and very weak activity against TLR4-mediated IL-6 production. Interestingly, both YMSA-0423 and -0448 had potent activity against TLR9-mediated IL-6 production (IC₅₀ = 0.00346, and 0.00693 µM, respectively). These results indicate that replacement of the diethyl group at the nitrogen atom with a monofluoropiperidinyl group led to higher inhibitory selectivity for TLR9 signaling.

YMSA-0381 bearing a 7-iodoquinoline moiety showed markedly lower activity than ADQ or YMSA-0448 against IL-6 production induced by TLR3, TLR7, and TLR9 agonists. YMSA-0998, which has no halogen substituent on quinoline, showed similar activity to YMSA-0448 against TLR3-mediated IL-6 production, although lower activity against TLR7- and TLR9-mediated IL-6 production. These results suggest that the chlorine atom at the 7-position of the quinoline is required for potent anti-inflammatory activity.

On the other hand, YMSA-0990, which has a 4,4-difluoropiperidinyl group, showed much lower or no activity against TLRs-mediated IL-6 production. YMSA-0995, which has a 4-phenylpiperidinyl group, showed more potent inhibitory activity than ADQ towards IL-6 production induced by agonists of TLR3, TLR4 and TLR7, but lower activity towards that induced by the TLR9 agonist. These results suggest that substituents on the piperidine ring may be important for retaining the activity or controlling the inhibitory selectivity.

We then investigated the effect of ADQ and its derivatives on inflammatory cytokine gene transcription induced by the TLR9 agonist. The increased IL-6 mRNA expression induced by CpG ODN was significantly suppressed by pretreatment with ADQ and YMSA-0448, but not KUMB-0002 (Fig. 6M). This result indicates that ADQ and its analogs inhibit TLRs-mediated transcription of inflammatory cytokines.

Evaluation of Cytotoxicity of Amodiaquine and Its Analogs in Murine Macrophages

In clinical use, ADQ can induce side effects, such as granulocytopenia and hepatotoxicity,²⁹⁾ and it decreases the viability of mononuclear leukocytes in vitro.³⁰⁾ ADQ did not significantly reduce viability of J774.1 cells under the conditions used to evaluate the effects on IL-6 production (Figs. 7A–C), and also had no effect at the concentration of 50 µM (data not shown). However, 24 h of ADQ treatment induced a marked decrease in the viability of J774.1 cells (CC₅₀ = 16.5 µM). Therefore, in this study, the CC₅₀ value of each compound at 24 h was calculated (Figs. 7D–G, Table 1). The CC₅₀ values of the derivatives with 50% or more reduced cell viability at concentrations of less than 50 µM, were calculated by Figs. 7D–F, while the CC₅₀ values of the derivatives that did not show such a decrease were calculated by Fig. 7G, which was examined additionally using concentrations more than 50 µM.

Fig. 7. Cytotoxicity of ADQ and Its Analogs

(A–C) J774.1 cells were pre-incubated with ADQ or its analogs (0.1–10 µM) for 0.5 h, and then incubated with poly(I:C) (5 µg/mL) for 6 h. (D–F) Cells were incubated with ADQ or its analogs (0.1–50 µM) for 24 h. (G) Cells were incubated with selected ADQ analogs (10–100 µM) for 24 h. Cell viability was evaluated by MTT assay. Error bars indicate ± S.E. (A–C: n = 9–27, D–G: n = 9, three independent experiments). ADQ: amodiaquine.

KUMB-0002 showed no apparent cytotoxicity (CC₅₀ > 100 µM), suggesting that the aminomethyl group on the phenol moiety may be involved in the cytotoxicity of ADQ. YMSA-0454 and -0455 showed similar cytotoxicity to ADQ, while YMSA-0203 and -0995 showed more potent cytotoxicity, and YMSA-0267 was less cytotoxic (CC₅₀ = 26.6 µM). The compounds with a monofluoropiperidinyl group or a difluoropiperidinyl group were less cytotoxic. The CC₅₀ value of YMSA-0448 was 42.5 µM, while those of YMSA-0423, -0381, -0998, and -0990 were >50 µM. These results indicate that some ADQ analogs such as YMSA-0267 or those with a fluoropiperidinyl group, may have reduced side effects compared with ADQ.

Amodiaquine and Its Analogs May Inhibit IL-6 Production Mediated by TLRs Recognizing Pathogen Nucleic Acids through Impairing Endolysosomal Activity

HCQ, an antimalarial aminoquinoline that inhibits TLRs recognizing pathogen nucleic acids, is known to work by impairing endolysosomal activity.¹³⁾ A comparison of the effects of ADQ and HCQ showed that HCQ had slightly stronger inhibitory activity (Figs. 8A–D, Table 1). On the other hand, HCQ had more potent cytotoxicity than ADQ towards J774.1 cells (CC₅₀ = 4.67 µM) (Figs. 8E, F).

Fig. 8. The Effect of ADQ and HCQ on IL-6 Production Induced by TLR Agonists in Murine Macrophages

(A–C) J774.1 cells were pre-incubated with ADQ or HCQ (0.1–10 µM or 0.0001–1 µM) for 0.5 h, then incubated with poly(I:C) (5 µg/mL) (A), imiquimod (1 µg/mL) (B), or CpG ODN (200 ng/mL) (C) for 6 h. (D, E) Cells were pre-incubated with ADQ or HCQ (1–20 µM) for 30 min, then incubated with LPS (0.1 ng/mL) for 6 h. (F) Cells were incubated with ADQ or HCQ (0.1–50 µM) for 24 h. (A–D) IL-6 was measured by means of ELISA. The inhibitory ratio of each compound relative to the control is shown. (E, F) Cell viability was evaluated by MTT assay. Error bars indicate ± S.E. (n = 9, three independent experiments). ADQ: amodiaquine. HCQ: hydroxychloroquine.

Next, the effect of treatment with ADQ and its derivatives on endolysosomal activity was investigated by monitoring the fluorescence intensity of LysoTracker^® red lysosomal probe. ADQ, although less potent than HCQ, significantly impaired the fluorescence intensity of LysoTracker^® at concentrations of 1 µM or more, and KUMB-0002 strongly attenuated the activity (Fig. 9). YMSA-0267, -0423, and -0448 showed similar endolysosomal inhibition activity to ADQ, while YMSA-0990 had weaker activity (Fig. 9). The correlation between IL-6 inhibitory activity and lysosomal inhibitory activity suggests that the inhibitory activity of these compounds towards TLRs-mediated IL-6 production may be a consequence of inhibition of endolysosomal activity.

Fig. 9. The Effect of ADQ and ADQ Analogs on Lysosomal Activity in Murine Macrophages

J774.1 cells were pre-incubated with HCQ, ADQ, or ADQ analogs (0.1–10 µM) for 0.5 h, then treated with LysoTracker^® Red Lysosomal Probe (50 nM) for 1 h. The fluorescence intensity of the LysoTracker^® was measured by flow cytometry. Statistical analysis was performed by one-way ANOVA followed by Dunnett’s test. Error bars indicate ± S.E. (n = 3, three independent experiments). Significant differences between control and test groups are indicated by * (p < 0.05), ** (p < 0.01), or **** (p < 0.0001). Histograms of fluorescence intensity on several compound treatments are shown (A: HCQ, B: ADQ, C: KUMB-0002, D: YMSA-0448, gray: control, green: 0.1 µM, blue: 1 µM, and red: 10 µM of each compound treatment). ADQ: amodiaquine. HCQ: hydroxychloroquine.

DISCUSSION

The antimalarial drugs CQ and HCQ have a wide range of pharmacological activities, and are also used to treat autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus.¹³⁾ However, attempts to use them for COVID-19 therapy were unsuccessful due to serious side effects.^31,32) Here, we aimed to create novel candidate compounds for treating viral sepsis by focusing on the anti-inflammatory effects of ADQ, a CQ derivative that has not yet been approved for the treatment of any disease other than malaria. Our data indicate that ADQ and its analogs regulate viral inflammatory signaling by impairing endolysosomal activity. ADQ and its derivatives inhibited TLR9-mediated IL-6 production more potently than IL-6 production mediated by other TLRs. Structure–activity relationship analysis revealed that the alkyl chains on the amino group and the substituent at the 7-position of the quinoline ring are key factors for increased activity and reduced cytotoxicity. Notably, we identified some ADQ analogs with high anti-inflammatory activity and low cytotoxicity towards macrophages. These analogs may provide new options for the treatment of viral sepsis without serious side effects.

This is the first study to show that ADQ suppresses inflammatory cytokine production induced by stimulation of TLRs recognizing pathogen nucleic acids. It has been shown that CQ/HCQ inhibit activation of these TLRs by endolysosomal pH elevation.¹³⁾ Treatment with bafilomycin A1, which alkalinizes endolysosomes, also inhibited CpG DNA-induced endosomal maturation.³³⁾ In this study, ADQ was shown to have somewhat weaker inhibitory activities than HCQ on IL-6 production induced by activation of nucleic acid-recognizing TLRs and on the fluorescence intensity of LysoTracker^®, which localizes to lysosomes as it targets their acidic contents. KUMB-0002 was attenuated both inhibitory activities, suggesting that the basicity of the aminomethyl group may be important for inhibiting endolysosomal TLR signaling. However, since our experiments were designed to assess lysosomal activity in the absence of TLR agonists, the effect on endolysosomal maturation will require further investigation.

ADQ and all its derivatives were shown to inhibit IL-6 production induced by the TLR9 agonist much more potently than that induced by the TLR3 or the TLR7 agonist. In this respect, it is similar to HCQ, in accordance with a previous report on CQ/HCQ.³⁴⁾ It was reported that activation of TLR9 signaling is involved in malaria exacerbation by the malaria pigment hemozoin,³⁵⁾ and antimalarial drugs such as ADQ and CQ inhibit this mechanism.³⁶⁾ The TLR9 antagonist E6446 was shown to improve cerebral malaria.³⁷⁾ Thus, it is possible that all conventional antimalarials regulate TLR9 signaling. However, since acidification of endolysosomes is a prerequisite for activation of all endolysosomal TLRs, the effect on pH alone would not be enough to explain why ADQ has high selectivity for TLR9. Several recent reports indicate that direct binding of CQ to CpG DNA thereby inhibiting the interaction between CpG DNA and TLR9 occurred at a lower concentration of CQ than the action of elevating pH.^38–40) Although it is not clear whether ADQ binds directly to the TLR9 ligand, it seems likely that the potent inhibitory activity of ADQ towards TLR9 signaling involves also direct inactivation of the ligand in a similar manner to other antimalarials. Moreover, the known pharmacological activities of ADQ and CQ, such as downregulation of C-X-C chemokine receptor type 4 (CXCR4) expression, high mobility group box 1 (HMGB1) inhibition, inhibition of thromboxane A2 production via phospholipase A2,¹³⁾ nuclear receptor subfamily 4a2 (Nr4a2) activation⁴¹⁾ and histamine N-methyltransferase inhibition,⁴²⁾ might explain ADQ’s high selectivity of TLR9. Among them, it was reported that TLR9 agonist induces the upregulation of CXCR4 expression as well as enhance the biological activities mediated by CXCR4.^43,44) It was experimentally shown that HMGB1 interacts with TLR9 in the endoplasmic reticulum-Golgi intermediate compartment and promotes the distribution of TLR9 to early endosomes in response to CpG DNA.⁴⁵⁾ It has not been demonstrated whether TLR3 and TLR7 have a similar mechanism. Furthermore, a recent report showed that granulin is involved in the production of inflammatory cytokines via TLR9, but does not seem to be involved in that via TLR3, TLR4, or TLR7.⁴⁶⁾ As described above, the known pharmacological activities of CQ and ADQ and the specific signaling pathway of TLR9 could explain the high selectivity of ADQ for TLR9, but further investigation is required.

ADQ did not significantly suppress IL-6 production induced by LPS. As regards the effects of CQ/HCQ, it was reported that 20 µM CQ treatment only suppressed LPS-induced IL-6 production by less than 50% in RAW264.7 murine macrophage cells.⁴⁷⁾ The three quinolines may only inhibit the activation of TLR4 expressed on endosomal membrane, but not on plasma membrane. On the other hand, ADQ acts as an agonist of Nr4a2, improving depression-like behavior in LPS-treated mice.⁴⁸⁾ ADQ administration to Propionibacterium acnes-primed and LPS-treated hepatitis model mice improved hepatitis pathology by inhibiting histamine N-methyltransferase.⁴²⁾ Meanwhile, LPS and ADQ may synergistically induce liver damage.⁴⁹⁾ Thus, it appears that ADQ can exert an enhancing or protective effect on LPS-induced inflammation, depending on the cells, organs, and other factors. KUMB-0002 inhibited TLR4-mediated IL-6 production more potently than ADQ, suggesting that the hydroxyl group might be the active moiety in this case, whereas the tertiary amine structure may rather cause steric hindrance, weakening the activity.

Replacement of the N-diethyl group of ADQ with other N-substituents had a significant effect on TLRs-mediated inflammatory cytokine production. As shown in Figs. 6A, B and D, substitution with longer linear alkyl groups instead of the diethyl group tended to increase the inhibition of IL-6 production via TLR3, TLR4, and TLR7. It is considered that endolysosomal alkalinization may be efficiently inhibited owing to the increased basicity of this moiety. However, this cannot explain the effect on the TLR9-mediated response. As shown in Fig. 6C, YMSA-0267 showed increased activity against TLR9, so it may be important that both alkyl chains should exceed a certain length in order to exert an inhibitory effect on pathogen ssDNA. Thus, the basicity and/or nucleophilicity of the amino group may be important for inhibiting the endolysosomal TLRs-mediated inflammatory response.

Heterocyclic modification has become an important approach in contemporary small molecule drug discovery, and piperidine ring modification is one of the most common approaches.⁵⁰⁾ Our analysis in Fig. 6G showed that substitution with a monofluoropiperidinyl group instead of the diethylamino group enhanced the inhibitory activity towards TLR9-mediated IL-6 production. Stabilization due to the ring modification may result in more efficient inhibition, as the modified compound may be less susceptible to metabolism or further modification. On the other hand, as shown in Figs. 6I–L, YMSA-0990 failed to retain the activity. The introduction of two fluorine atoms may lead to steric hindrance or may impair the nucleophilicity and/or basicity of the nitrogen atom. Phenylpiperidinyl group-substituted YMSA-0995 showed increased inhibitory activity towards TLR3-, TLR4-, and TLR7-mediated responses, but not against TLR9-mediated response. It seems likely that substitution with an electron-withdrawing group increases the selectivity for TLR9, while substitution with an electron-donating group increases the selectivity for TLR3, TLR4, and TLR7. Thus, piperidine substituents may be able to modulate the selectivity for inhibition of inflammatory signaling.

Halogen substitution is a commonly used approach in drug development because it affects the interaction of a compound with its target.⁵¹⁾ For example, the electronegativity of the 7-position halogen of the quinoline in ADQ derivatives was inversely proportional to the anti-Ebola activity.¹⁵⁾ In our study, as shown in Figs. 6A–D, YMSA-0455 showed almost equal inhibitory activity to YMSA-0454 towards TLR3- or TLR7-mediated IL-6 production, but showed higher activity against TLR9-mediated IL-6 production. On the other hand, as shown in Figs. 6E–H, YMSA-0381 showed a significant decrease of inhibitory activity towards IL-6 production mediated by any of the TLRs, as compared with YMSA-0448. In addition, YMSA-0998 showed slightly higher inhibitory activity than YMSA-0381 towards IL-6 production via TLR3 and TLR7, though it was less potent than YMSA-0448, while it showed weaker activity towards IL-6 production via TLR9 than either of the other two compounds. Given these results, further study will be needed to optimize the 7-position substituent of the quinoline.

Creating ADQ derivatives with lower cytotoxicity was one of the objectives of this study, because ADQ itself can cause serious side effects.²⁹⁾ We found that ADQ showed significant cytotoxicity after treatment for 24 h at higher concentrations than the IC₅₀. Short-term ADQ exposure induced inhibition of IL-6 production with weak cytotoxicity. The aminomethyl group seems to be involved in the cytotoxicity of ADQ, since KUMB-0002 was not cytotoxic to J774.1 cells. Compared to ADQ, as shown in Figs. 7D and E, YMSA-0203 was more cytotoxic, while YMSA-0267 was less cytotoxic, and YMSA-0454 and -0455 showed similar cytotoxicity. These results suggest that the substituents on the amino group affect the cytotoxicity, whereas the halogen at the 7-position of the quinoline has little effect on the cytotoxicity. It is known that the major metabolites of ADQ, ADQ quinoneimine and desmethyl-ADQ, are important contributors to the toxicity,³⁰⁾ so the lower cytotoxicity of YMSA-0267 might be explained by a reduced level of oxidation of the hydroxyl group or dealkylation at the alkylamino group. Since the derivatives with a fluoropiperidinyl group showed significantly lower cytotoxicity as shown in Figs. 7E–G, the ring structure of this substituent may reduce the toxicity. YMSA-0995 showed more potent cytotoxicity, suggesting that the introduction of an electron-donating group is unfavorable. Judging from these results, YMSA-0267, -0423 and -0448, which have lower cytotoxicity without attenuation of IL-6 production-inhibitory activity, may be candidates as TLR9 antagonists for the treatment of sepsis and other diseases associated with TLR9-mediated inflammation. YMSA-0381, having slightly lower activity and lower cytotoxicity, could also be the next candidate.

ADQ analogs have the potential to become drugs that can control the inflammatory response caused in various infectious diseases. ADQ analogs may have potent antimalarial activity by inhibiting TLR9 activation induced by hemozoin.³⁵⁾ Also, they may be useful as a drug that inhibits inflammation induced by DNA viruses such as herpes simplex virus.⁵²⁾ Notably, the injection of TLR9 antagonists such as CQ and TLR9 knockout could improve survival rate via suppression of organ failures such as heart failure and acute kidney injury in bacterial sepsis models induced by cecal ligation and puncture (CLP).^53–55) Thus, ADQ analogs may be effective in treating cytokine storms caused also by bacterial sepsis. On the other hand, since ADQ analogs can inhibit the inflammatory responses via TLR3 and TLR7, they may also be useful for controlling inflammation during infection with RNA viruses such as influenza and coronaviruses.^6,52) Furthermore, because they can suppress inflammatory reactions caused by various pathogens, ADQ analogs may be valuable anti-inflammatory drugs that can be administered even when the causative pathogen of the sepsis patient is unidentified.

The potential clinical use of ADQ analogs may not be limited to diseases caused by infection. Some previous reports suggest that ADQ has therapeutic effects on inflammatory diseases such as rheumatoid arthritis,⁵⁶⁾ depression,⁴⁸⁾ hepatitis,⁴²⁾ and Parkinson’s disease.^57,58) Since TLRs are activated in these inflammatory diseases,^59–61) ADQ might be effective. Recently, a TLR9 antagonist, E6446, was found to have therapeutic effects on erythema nodosum leprosum,⁶²⁾ heart failure^63,64) and pulmonary hypertension.⁶⁵⁾ Other recent findings suggest that the anti-inflammatory effects of ADQ may include direct regulation of T cell activation.^66,67) Therefore, drug discovery based on ADQ derivatives might lead to therapeutic strategies for several inflammatory diseases.

In conclusion, our results indicate that ADQ and its analogs significantly suppress IL-6 production via pathogen nucleic acid recognition TLRs in macrophages. ADQ has potent inhibitory activity against TLR9-mediated inflammatory cytokine production activated by pathogen ssDNA. The diethylaminomethyl group and a halogen bonded to the quinoline ring are important structures for the inhibition of TLRs-mediated response and also for cytotoxicity. We identified several candidate agents such as YMSA-0267, 0423, and -0448 with more potent inhibitory activity against IL-6 production and lower cytotoxicity in macrophages, though toxicity tests in vivo of these compounds will be necessary. Overall, ADQ analogs may be useful as low-toxicity inhibitors of inflammation mediated by endolysosomal TLRs, especially TLR9.

Acknowledgments

This work was supported in part by a Tokyo University of Science Grant for the president’s research promotion (to MT, SA). This work was also supported in part by JST SPRING, Grant Number: JPMJSP2151, and JSPS KAKENHI Grant number: 23KJ1966 (JSPS Research Fellowship) (to YT).

We gratefully acknowledge to Prof. Mika Okamoto and Prof. Masanori Baba (Division of Infection Control Research, Center for Advanced Science Research and Promotion, Kagoshima University) for providing us with KUMB-0002 and some related compounds. We also gratefully acknowledge funding in the form of Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (Nos. 22390005, 24659011, and 24640156 for SA) and the TUS (Tokyo University of Science) fund for strategic research areas, from the Japan Agency for Medical Research and Development (AMED), Japan (20fk0108273h0001 and 22fk0108555h0001 for SA), and from Shionogi Infectious Disease Research Promotion Foundation, Japan. We wish to thank Ms. Fukiko Hasegawa, Ms. Noriko Sawabe, Ms. Yayoi Yoshimura, Ms. Yuki Honda, and Ms. Hitomi Isoda (Faculty of Pharmaceutical Sciences, Tokyo University of Science and Research Equipment Center, Tokyo University of Science) for conducting MS spectrometry, NMR, and the elemental analyses, respectively. We also thank Prof. Masanori Kitamura (Matsuyama University) for helpful discussion regarding X-ray crystal structure analysis.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Hotchkiss RS, Moldawer LL, Opal SM, Reinhart K, Turnbull IR, Vincent J-L. Sepsis and septic shock. Nat. Rev. Dis. Primers, 2, 16045 (2016).
• 2) Rudd KE, Johnson SC, Agesa KM, et al. Global, regional, and national sepsis incidence and mortality, 1990–2017: analysis for the Global Burden of Disease Study. Lancet, 395, 200–211 (2020).
• 3) Shappell C, Rhee C, Klompas M. Update on sepsis epidemiology in the era of COVID-19. Semin. Respir. Crit. Care Med., 44, 173–184 (2023).
• 4) Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, Bellomo R, Bernard GR, Chiche J-D, Coopersmith CM, Hotchkiss RS, Levy MM, Marshall JC, Martin GS, Opal SM, Rubenfeld GD, van der Poll T, Vincent J-L, Angus DC. The third international consensus definitions for sepsis and septic shock (Sepsis-3). JAMA, 315, 801–810 (2016).
• 5) Chen F, Zou L, Williams B, Chao W. Targeting Toll-like receptors in sepsis: from bench to clinical trials. Antioxid. Redox Signal., 35, 1324–1339 (2021).
• 6) Manik M, Singh RK. Role of toll-like receptors in modulation of cytokine storm signaling in SARS-CoV-2-induced COVID-19. J. Med. Virol., 94, 869–877 (2022).
• 7) Opal SM, Laterre P-F, Francois B, et al. Effect of eritoran, an antagonist of MD2-TLR4, on mortality in patients with severe sepsis: the ACCESS randomized trial. JAMA, 309, 1154–1162 (2013).
• 8) Hirano T. IL-6 in inflammation, autoimmunity and cancer. Int. Immunol., 33, 127–148 (2021).
• 9) Gordon AC, Mouncey PR, Al-Beidh F, et al. Interleukin-6 receptor antagonists in critically Ill patients with covid-19. N. Engl. J. Med., 384, 1491–1502 (2021).
• 10) Afzal O, Kumar S, Haider MR, Ali MR, Kumar R, Jaggi M, Bawa S. A review on anticancer potential of bioactive heterocycle quinoline. Eur. J. Med. Chem., 97, 871–910 (2015).
• 11) Kaur R, Kumar K. Synthetic and medicinal perspective of quinolines as antiviral agents. Eur. J. Med. Chem., 215, 113220 (2021).
• 12) Shionoya K, Yamasaki M, Iwanami S, Ito Y, Fukushi S, Ohashi H, Saso W, Tanaka T, Aoki S, Kuramochi K, Iwami S, Takahashi Y, Suzuki T, Muramatsu M, Takeda M, Wakita T, Watashi K. Mefloquine, a potent anti-severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) drug as an entry inhibitor in vitro. Front. Microbiol., 12, 651403 (2021).
• 13) Niemann B, Puleo A, Stout C, Markel J, Boone BA. Biologic functions of hydroxychloroquine in disease: From COVID-19 to cancer. Pharmaceutics, 14, 2551 (2022).
• 14) DeWald LE, Johnson JC, Gerhardt DM, Torzewski LM, Postnikova E, Honko AN, Janosko K, Huzella L, Dowling WE, Eakin AE, Osborn BL, Gahagen J, Tang L, Green CE, Mirsalis JC, Holbrook MR, Jahrling PB, Dyall J, Hensley LE. In vivo activity of amodiaquine against Ebola virus infection. Sci. Rep., 9, 20199 (2019).
• 15) Sakurai Y, Sakakibara N, Toyama M, Baba M, Davey RA. Novel amodiaquine derivatives potently inhibit Ebola virus infection. Antiviral Res., 160, 175–182 (2018).
• 16) Baba M, Toyama M, Sakakibara N, Okamoto M, Arima N, Saijo M. Establishment of an antiviral assay system and identification of severe fever with thrombocytopenia syndrome virus inhibitors. Antivir. Chem. Chemother., 25, 83–89 (2017).
• 17) Bocci G, Bradfute SB, Ye C, Garcia MJ, Parvathareddy J, Reichard W, Surendranathan S, Bansal S, Bologa CG, Perkins DJ, Jonsson CB, Sklar LA, Oprea TI. Virtual and in vivo antiviral screening revive therapeutic drugs for COVID-19. ACS Pharmacol. Transl. Sci., 3, 1278–1292 (2020).
• 18) Seliem IA, Panda SS, Girgis AS, Moatasim Y, Kandeil A, Mostafa A, Ali MA, Nossier ES, Rasslan F, Srour AM, Sakhuja R, Ibrahim TS, Abdel-Samii ZKM, Al-Mahmoudy AMM. New quinoline-triazole conjugate: synthesis, and antiviral properties against SARS-CoV-2. Bioorg. Chem., 114, 105117 (2021).
• 19) Si L, Bai H, Rodas M, et al. A human-airway-on-a-chip for the rapid identification of candidate antiviral therapeutics and prophylactics. Nat. Biomed. Eng., 5, 815–829 (2021).
• 20) Zhao J, Zhang Y, Wang M, Liu Q, Lei X, Wu M, Guo S, Yi D, Li Q, Ma L, Liu Z, Guo F, Wang J, Li X, Wang Y, Cen S. Quinoline and quinazoline derivatives inhibit viral RNA synthesis by SARS-CoV-2 RdRp. ACS Infect. Dis., 7, 1535–1544 (2021).
• 21) Baba M, Okamoto M, Toyama M, Aoki S, Tanaka T. PCT/JP2021/018602 (WO/2021/235390) (2021).
• 22) Dolomanov OV, Bourhis LJ, Gildea RJ, Howard JAK, Puschmann H. OLEX2: a complete structure solution, refinement and analysis program. J. Appl. Cryst., 42, 339–341 (2009).
• 23) Sheldrick GM. SHELXT-integrated space-group and crystal-structure determination. Acta Crystallogr. A Found. Adv., 71, 3–8 (2015).
• 24) Sheldrick GM. Crystal structure refinement with SHELXL. Acta Crystallogr. C Struct. Chem., 71, 3–8 (2015).
• 25) Takenaka Y, Tanaka R, Kitabatake K, Kuramochi K, Aoki S, Tsukimoto M. Profiling differential effects of 5 selective serotonin reuptake inhibitors on TLRs-dependent and -independent IL-6 production in immune cells identifies fluoxetine as preferred anti-inflammatory drug candidate. Front. Pharmacol., 13, 874375 (2022).
• 26) Takenaka Y, Tanaka R, Kitabatake K, Uchiumi F, Aoki S, Kuramochi K, Tsukimoto M. Structure–activity relationship analysis of fluoxetine for suppression of inflammatory cytokine production. Biol. Pharm. Bull., 47, 946–954 (2024).
• 27) Nairoukh Z, Strieth-Kalthoff F, Bergander K, Glorius F. Understanding the conformational behavior of fluorinated piperidines: the origin of the axial-F preference. Chemistry, 26, 6141–6146 (2020).
• 28) Hu X-G, Hunter L. Stereoselectivity fluorinated N-heterocycles: a brief survey. Beilstein J. Org. Chem., 9, 2696–2708 (2013).
• 29) Neftel KA, Woodtly W, Schmid M, Frick PG, Fehr J. Amodiaquine induced agranulocytosis and liver damage. BMJ, 292, 721–723 (1986).
• 30) Winstanley PA, Coleman JW, Maggs JL, Breckenridge AM, Park BK. The toxicity of amodiaquine and its principal metabolites towards mononuclear leucocytes and granulocyte/monocyte colony forming units. Br. J. Clin. Pharmacol., 29, 479–485 (1990).
• 31) Agarwal A, Hunt B, Stegemann M, et al. A living WHO guideline on drugs for covid-19. BMJ, 370, m3379 (2020).
• 32) Gagnon LR, Sadasivan C, Yogasundaram H, Oudit GY. Review of hydroxychloroquine cardiotoxicity: lessons from the COVID-19 pandemic. Curr. Heart Fail. Rep., 19, 458–466 (2022).
• 33) Ahmad-Nejad P, Häcker H, Rutz M, Bauer S, Vabulas RM, Wagner H. Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur. J. Immunol., 32, 1958–1968 (2002).
• 34) Lee J, Chuang T-H, Redecke V, She L, Pitha PM, Carson DA, Raz E, Cottam HB. Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of Toll-like receptor 7. Proc. Natl. Acad. Sci. U.S.A., 100, 6646–6651 (2003).
• 35) Coban C, Ishii KJ, Kawai T, Hemmi H, Sato S, Uematsu S, Yamamoto M, Takeuchi O, Itagaki S, Kumar N, Horii T, Akira S. Toll-like receptor 9 mediates innate immune activation by the malaria pigment hemozoin. J. Exp. Med., 201, 19–25 (2005).
• 36) Combrinck JM, Mabotha TE, Ncokazi KK, Ambele MA, Taylor D, Smith PJ, Hoppe HC, Egan TJ. Insights into the role of heme in the mechanism of action of antimalarials. ACS Chem. Biol., 8, 133–137 (2013).
• 37) Franklin BS, Ishizaka ST, Lamphier M, Gusovsky F, Hansen H, Rose J, Zheng W, Ataíde MA, de Oliveira RB, Golenbock DT, Gazzinelli RT. Therapeutical targeting of nucleic acid-sensing Toll-like receptors prevents experimental cerebral malaria. Proc. Natl. Acad. Sci. U.S.A., 108, 3689–3694 (2011).
• 38) Manzel L, Strekowski L, Ismail FM, Smith JC, Macfarlane DE. Antagonism of immunostimulatory CpG-oligodeoxynucleotides by 4-aminoquinolines and other weak bases: mechanistic studies. J. Pharmacol. Exp. Ther., 291, 1337–1347 (1999).
• 39) Rutz M, Metzger J, Gellert T, Luppa P, Lipford GB, Wagner H, Bauer S. Toll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner. Eur. J. Immunol., 34, 2541–2550 (2004).
• 40) Kužnik A, Bencina M, Svajger U, Jeras M, Rozman B, Jerala R. Mechanism of endosomal TLR inhibition by antimalarial drugs and imidazoquinolines. J. Immunol., 186, 4794–4804 (2011).
• 41) Munoz-Tello P, Lin H, Khan P, de Vera IMS, Kamenecka TM, Kojetin DJ. Assessment of NR4A ligands that directly bind and modulate the orphan nuclear receptor Nurr1. J. Med. Chem., 63, 15639–15654 (2020).
• 42) Yokoyama A, Mori S, Takahashi HK, Kanke T, Wake H, Nishibori M. Effect of amodiaquine, a histamine N-methyltransferase inhibitor, on, Propionibacterium acnes and lipopolysaccharide-induced hepatitis in mice. Eur. J. Pharmacol., 558, 179–184 (2007).
• 43) Jang SH, Shim JS, Kim J, Shin EG, Yoon JH, Lee LE, Kwon HK, Song JJ. Mitochondria activity and CXCR4 collaboratively promote the differentiation of CD11c⁺ B cells induced by TLR9 in lupus. Immune Netw., 24, e25 (2024).
• 44) Xu L, Zhou Y, Liu Q, Luo JM, Qing M, Tang XY, Yao XS, Wang CH, Wen ZK. CXCR4/SDF-1 pathway is crucial for TLR9 agonist enhanced metastasis of human lung cancer cell. Biochem. Biophys. Res. Commun., 382, 571–576 (2009).
• 45) Ivanov S, Dragoi AM, Wang X, Dallacosta C, Louten J, Musco G, Sitia G, Yap GS, Wan Y, Biron CA, Bianchi ME, Wang H, Chu WM. A novel role for HMGB1 in TLR9-mediated inflammatory responses to CpG-DNA. Blood, 110, 1970–1981 (2007).
• 46) Park B, Buti L, Lee S, Matsuwaki T, Spooner E, Brinkmann MM, Nishihara M, Ploegh HL. Granulin is a soluble cofactor for toll-like receptor 9 signaling. Immunity, 34, 505–513 (2011).
• 47) Cao Y, Chen J, Ren G, Zhang Y, Tan X, Yang L. Punicalagin prevents inflammation in LPS-Induced RAW264.7 macrophages by inhibiting FoxO3a/autophagy signaling pathway. Nutrients, 11, 2794 (2019).
• 48) He Y, Wang Y, Yu H, Tian Y, Chen X, Chen C, Ren Y, Chen Z, Ren Y, Gong X, Cheng K, Liu X, Zhong L, Guo Y, Xie P. Protective effect of Nr4a2 (Nurr1) against LPS-induced depressive-like behaviors via regulating activity of microglia and CamkII neurons in anterior cingulate cortex. Pharmacol. Res., 191, 106717 (2023).
• 49) Niknahad H, Heidari R, Firuzi R, Abazari F, Ramezani M, Azarpira N, Hosseinzadeh M, Najibi A, Saeedi A. Concurrent inflammation augments antimalarial drugs-induced liver injury in rats. Adv. Pharm. Bull., 6, 617–625 (2016).
• 50) Frolov NA, Vereshchagin AN. Piperidine derivatives: recent advances in synthesis and pharmacological applications. Int. J. Mol. Sci., 24, 2937 (2023).
• 51) Hernandes MZ, Cavalcanti SMT, Moreira DRM, de Azevedo Junior WF, Leite ACL. Halogen atoms in the modern medicinal chemistry: hints for the drug design. Curr. Drug Targets, 11, 303–314 (2010).
• 52) Lin GL, McGinley JP, Drysdale SB, Pollard AJ. Epidemiology and immune pathogenesis of viral sepsis. Front. Immunol., 9, 2147 (2018).
• 53) Hu D, Yang X, Xiang Y, Li H, Yan H, Zhou J, Caudle Y, Zhang X, Yin D. Inhibition of Toll-like receptor 9 attenuates sepsis-induced mortality through suppressing excessive inflammatory response. Cell. Immunol., 295, 92–98 (2015).
• 54) Fattahi F, Russell MW, Malan EA, Parlett M, Abe E, Zetoune FS, Ward PA. Harmful roles of TLR3 and TLR9 in cardiac dysfunction developing during polymicrobial sepsis. BioMed Res. Int., 2018, 4302726 (2018).
• 55) Yasuda H, Leelahavanichkul A, Tsunoda S, Dear JW, Takahashi Y, Ito S, Hu X, Zhou H, Doi K, Childs R, Klinman DM, Yuen PS, Star RA. Chloroquine and inhibition of Toll-like receptor 9 protect from sepsis-induced acute kidney injury. Am. J. Physiol. Renal Physiol., 294, F1050–F1058 (2008).
• 56) Kersley GD, Palin AG. Amodiaquine and hydroxychloroquine in rheumatoid arthritis. Lancet, 2, 886–888 (1959).
• 57) Sellnow RC, Steece-Collier K, Altwal F, Sandoval IM, Kordower JH, Collier TJ, Sortwell CE, West AR, Manfredsson FP. Striatal Nurr1 facilitates the dyskinetic state and exacerbates levodopa-induced dyskinesia in a rat model of Parkinson’s disease. J. Neurosci., 40, 3675–3691 (2020).
• 58) Kim C-H, Han B-S, Moon J, Kim D-J, Shin J, Rajan S, Nguyen QT, Sohn M, Kim W-G, Han M, Jeong I, Kim K-S, Lee E-H, Tu Y, Naffin-Olivos JL, Park C-H, Ringe D, Yoon HS, Petsko GA, Kim K-S. Nuclear receptor Nurr1 agonists enhance its dual functions and improve behavioral deficits in an animal model of Parkinson’s disease. Proc. Natl. Acad. Sci. U.S.A., 112, 8756–8761 (2015).
• 59) Figueroa-Hall LK, Paulus MP, Savitz J. Toll-Like receptor signaling in depression. Psychoneuroendocrinology, 121, 104843 (2020).
• 60) Heidari A, Yazdanpanah N, Rezaei N. The role of Toll-like receptors and neuroinflammation in Parkinson’s disease. J. Neuroinflammation, 19, 135 (2022).
• 61) Mencin A, Kluwe J, Schwabe RF. Toll-like receptors as targets in chronic liver diseases. Gut, 58, 704–720 (2009).
• 62) Dias AA, Silva CO, Santos JPS, Batista-Silva LR, Acosta CCD, Fontes ANB, Pinheiro RO, Lara FA, Machado AM, Nery JAC, Sarno EN, Pereira GMB, Pessolani MCV. DNA sensing via TLR-9 constitutes a major innate immunity pathway activated during erythema nodosum leprosum. J. Immunol., 197, 1905–1913 (2016).
• 63) Ueda H, Yamaguchi O, Taneike M, Akazawa Y, Wada-Kobayashi H, Sugihara R, Yorifuji H, Nakayama H, Omiya S, Murakawa T, Sakata Y, Otsu K. Administration of a TLR9 inhibitor attenuates the development and progression of heart failure in mice. JACC Basic Transl. Sci., 4, 348–363 (2019).
• 64) Yoshida K, Abe K, Ishikawa M, Saku K, Shinoda-Sakamoto M, Ishikawa T, Watanabe T, Oka M, Sunagawa K, Tsutsui H. Inhibition of TLR9-NF-κB-mediated sterile inflammation improves pressure overload-induced right ventricular dysfunction in rats. Cardiovasc. Res., 115, 658–668 (2019).
• 65) Ishikawa T, Abe K, Takana-Ishikawa M, Yoshida K, Watanabe T, Imakiire S, Hosokawa K, Hirano M, Hirano K, Tsutsui H. Chronic inhibition of Toll-like receptor 9 ameliorates pulmonary hypertension in rats. J. Am. Heart Assoc., 10, e019247 (2021).
• 66) Oh S, Shin JH, Jang EJ, Won HY, Kim HK, Jeong M-G, Kim KS, Hwang ES. Anti-inflammatory activity of chloroquine and amodiaquine through p21-mediated suppression of T cell proliferation and Th1 cell differentiation. Biochem. Biophys. Res. Commun., 474, 345–350 (2016).
• 67) Won HY, Shin JH, Oh S, Jeong H, Hwang ES. Enhanced CD25⁺Foxp3⁺ regulatory T cell development by amodiaquine through activation of nuclear receptor 4A. Sci. Rep., 7, 16946 (2017).