β-Carboline and chloroquine hybrids as potent antiplasmodial agents: Design, synthesis and biological evaluation

INTRODUCTION

Malaria is a widespread parasitic disease caused by Plasmodium protozoa and transmitted by infected female Anopheles mosquitoes. Among the species capable of infecting humans (P. falciparum, P. vivax, P. ovale, P. knowlesi, and P. malariae), P. falciparum is responsible for the most malaria-related deaths worldwide, whereas P. vivax represents a major cause of malaria morbidity in Southeast Asia and South America (1, 2).

In 2024, 282 million malaria cases were reported worldwide, with 610,000 estimated deaths reflecting a persistent global increase in malaria incidence and burden observed since 2015. The reasons for this increase are driven by multiple factors, including population growth in endemic regions and disruptions of health services. Additionally, it is presumed that improvement in case detection may increase the number of reported malaria cases in the coming years (3). At the same time, emergence of resistant parasites to antimalarial drugs, along with increasing insecticide resistance in mosquito vectors continues to threaten malaria control efforts and drive the search for new antimalarial agents (2).

Recent advances in antimalarial drug discovery have identified several structurally diverse chemotypes active against P. falciparum, among which the compounds presented in Fig. 1 are currently in advanced phase of clinical development (4).

Cipargamin, a spiroindolone derivative structurally related to β-carboline, has demonstrated strong potency against P. falciparum by inhibiting the parasite P-type ATPase PfATP4, a Na⁺ efflux pump essential for ionic homeostasis, leading to intracellular Na⁺ accumulation and rapid parasite death (5–7).

Among quinoline-based antimalarial candidates in advanced clinical development, M5715 that is an elongator factor-2 inhibitor is currently being evaluated in combination with pyronaridine, whereas ferroquine, an inhibitor of hemozoin formation, is being investigated in combination with ZY19489 (4).

Ganaplacide is an imidazolopiperazine antimalarial candidate that exhibits potent multistage activity against Plasmodium parasites and is currently in late-stage clinical development in combination with lumefantrine and triple combination with cipargamin (8). Its mechanism of action is not fully elucidated but appears to involve disruption of the parasite intracellular secretory pathway, with resistance linked to mutations in the PfCARL locus (9).

Fig. 1. Cipargamin (A), M5715 (B), ferroquine (C), ganaplacide (D) and lumefantrine (E).

The continued emergence of new antimalarial chemotypes highlights the importance of exploring heterocyclic scaffolds (10, 11).

Quinoline-based antimalarials, including chloroquine, primaquine, and tafenoquine, continue to play a key role in malaria treatment by targeting different stages of the parasite life cycle through distinct mechanisms of action (12, 13). β-Carbolines represent another biologically active heteroaromatic scaffold with reported antiparasitic and DNA-interacting properties (14). In this context, quinoline derivatives represent a well-established antimalarial pharmacophore, while β-carbolines constitute biologically active heterocycles with reported antiparasitic activity, making their molecular hybridization an attractive strategy for the development of novel antiplasmodial agents.

While combination therapy is a cornerstone in the treatment of infectious diseases due to its ability to improve efficacy and limit resistance, molecular hybridization offers a promising alternative by incorporating multiple pharmacophores into a single molecule, enabling improved metabolic stability, reduced cytotoxicity, and activity against resistant parasites (15). Consequently, the molecular hybridization of β-carboline and quinoline motifs represents a rational strategy to combine two privileged antiplasmodial scaffolds within a single molecule, potentially enabling multitarget activity with the aim of antiplasmodial activity (16).

Encouraged by the exceptional antiplasmodial activity of the second generation harmiquins bridged at the nitrogen at the position 9 of the β-carboline ring with quinoline from our previous work (17), we have decided to explore additional functionalities on the β-carboline, while maintaining the preferred position for bridging quinoline moiety and the amide/triazole linker.

EXPERIMENTAL

Chemistry. General information

Melting points were determined on a Stuart Melting Point Apparatus (Barloworld Scientific, UK) in open capillaries and were uncorrected. FTIR-ATR spectra were recorded using a Fourier-Transform Infrared Attenuated Total Reflection UATR Two spectrometer (PerkinElmer, USA) in the range from 450 to 4000 cm^–1. ¹H and ¹³C NMR spectra were recorded on a Bruker Avance III HD operating at 300, 400 or 600 MHz for the ¹H and 75, 101 or 151 MHz for the ¹³C nuclei (Bruker, USA). Samples were measured in DMSO-d₆ solutions at 20 °C in 5 mm NMR tubes. Chemical shifts (δ) are reported in parts per million (ppm) using tetramethylsilane (TMS) as a reference in the ¹H and DMSO residual peak as a reference in the ¹³C spectra (39.52 ppm). Coupling constants (J) are reported in hertz (Hz). Mass spectra were recorded on Agilent 1200 Series HPLC coupled with Agilent 6400 Series Triple Quad (Agilent Technologies, USA). The mobile phase consisted of Milli Q water with 0.1 % formic acid as component A and MeOH (HPLC grade, J. T. Baker) as component B. Separation was performed on a Zorbax XDB C18 column (4.6 × 75 mm, 3.5 μm, Agilent Technologies, USA) at 25 °C. Gradient elution was used at a flow rate of 0.5 mL min^–1, and 5 μL of analyte solution was injected per analysis. The starting conditions and gradient steepness were adjusted according to the analyte polarity. A diode array detector was utilized, while the data were presented as a total wavelength chromatogram (TWC). Mass spectrometry conditions were as follows: electrospray ionization (ESI) in positive and negative mode was used. Capillary voltage and current were set to 4.0 kV and 20 nA, respectively. Nebulizer pressure was set to 15 psi, while the drying gas (nitrogen) temperature and flow were 300 °C and 11 L min^–1. For the MS data analysis Agilent MassHunter software (Agilent Technologies) was used. HPLC purity of the final compounds was determined by liquid chromatography with UV diode-array detection at 254 nm, with compound identity confirmed by LC-MS using electrospray ionization in positive and negative ion modes, and was ≥ 95 %. Microwave-assisted reactions were performed in a microwave reactor CEM Discover (CEM, USA) in a glass reaction vessel. All compounds were routinely checked by TLC with silica gel 60F-254 glass plates (Merck, Germany) using DCM/MeOH or cyclohexane/EtOAc/MeOH as the solvent system. Spots were visualized by UV light (λ = 254 nm; 365 nm). Column chromatography was performed on silica gel 0.063–0.200 mm (Sigma-Aldrich, USA) with the same eluents used for TLC. All chemicals and solvents were of analytical grade and purchased from commercial sources.

7-Chloroquinoline-based azide 1 and carboxylic acid 2, β-carboline phenol 5, β-carbolines and their respective alkynes and amines 11–13, 16, 20–21, 23, 25, 31, 33 and 34 were prepared according to procedures published by us or others (17–20).

General procedure for the synthesis of β-carbolines 6 and 7

To a stirred solution of β-carboline phenol 5 in anhydrous DMF (5 mL), under an argon atmosphere, Cs₂CO₃ was added. The resulting suspension was stirred at room temperature for 20 min, followed by the addition of an appropriate alkyl bromide. The reaction mixture was stirred at room temperature, then at 40 °C, cooled down, poured into water (50 mL) and extracted with ethyl acetate (3×50 mL). The collected organic layers were washed with water and brine, dried over anhydrous sodium sulfate, filtered, and evaporated under the reduced pressure. The crude product was purified by column chromatography (DCM:MeOH = 9:1) and triturated with diethyl ether.

6-Ethoxy-1-methyl-9H-pyrido[3,4-b]indole (6). – β-Carboline phenol 5: 0.567 g, 2.86 mmol; Cs₂CO₃: 1.305 g, 4.00 mmol, 1.4 equiv.; ethyl bromide: 0.747 g, 6.86 mmol, 2.4 equiv.; 2 h at 40 °C; yield: 0.336 g (52 %); mp > 200 °C; IR (ATR, ν/cm^–1) 2982, 1600, 1564, 1497, 1465, 1289, 1208, 1120, 1070, 1041, 985, 880, 850, 818, 702, 622, 579; ¹H NMR (DMSO-d₆) δ 11.36 (s, 1H), 8.15 (d, 1H, J = 5.3 Hz), 7.89 (d, 1H, J = 5.3 Hz), 7.73 (d, 1H, J = 2.5 Hz), 7.50 (d, 1H, J = 8.9 Hz), 7.17 (dd, 1H, J = 8.8, 2.5 Hz), 4.12 (q, 2H, J = 7.0 Hz), 2.74 (s, 3H), 1.39 (t, 3H, J = 7.0 Hz); ¹³C NMR (DMSO-d₆) δ 155.16, 144.81, 139.50, 137.89, 137.74, 129.43, 124.08, 120.93, 115.39, 107.07, 66.27, 23.05, 17.54; ESI-MS: m/z 227.1 (M + 1)⁺.

6-Isopropoxy-1-methyl-9H-pyrido[3,4-b]indole (7). – Phenol 5: 0.500 g, 2.52 mmol; Cs₂CO₃: 3.452 g, 10.594 mmol, 4.2 equiv.; isopropyl bromide: 2.482 g, 20.178 mmol, 8 equiv.; 23 h at room temperature; yield: 0.400 g (66 %); mp 161.5–168 °C; IR (ATR, ν/cm^–1) 3123, 2975, 1578, 1496, 1370, 1334, 1287, 1204, 1124, 1062, 987, 961, 877, 845, 744, 699, 626, 578; ¹H NMR (DMSO-d₆) δ 11.35 (s, 1H), 8.15 (d, 1H, J = 5.3 Hz), 7.90 (d, 1H, J = 5.3 Hz), 7.75 (d, 1H, J = 2.3 Hz), 7.49 (d, 1H, J = 8.8 Hz), 7.15 (dd, 1H, J = 8.8, 2.4 Hz), 4.65 (hept, 1H, J = 6.0 Hz), 2.74 (s, 3H), 1.31 (d, 6H, J = 6.0 Hz); ¹³C NMR (DMSO-d₆) δ 151.61, 142.57, 137.35, 135.80, 135.57, 127.18, 122.02, 119.99, 113.21, 113.13, 107.34, 70.70, 22.47, 20.87; ESI-MS: m/z 241.2 (M + 1)⁺.

General procedure for the synthesis of β-carboline-based alkynes 8, 9, 14, 15, 22, 26, 32

An appropriate β-carboline (6, 7, 11, 12, 20, 25, 30) was dissolved in dry DMF (1 mL/0.1 g; 3 mL). Under an argon atmosphere, 60 % dispersion of sodium hydride in mineral oil (60 % NaH) (2.66 equiv.; 1.5 equiv. for 26 and 32) or Cs₂CO₃ (2.88 equiv. for 22) was added, followed by a dropwise addition of 80 % solution of propargyl bromide in toluene (3 equiv.; 1.44 equiv. for 22; 1.5 equiv. for 26 and 32). The reaction was stirred at r.t. and under an argon atmosphere for 2 h. Upon completion, the reaction mixture was poured into 30 mL of water. The product was extracted with ethyl acetate (3×30 mL). Organic layers were collected and washed with water and brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by column chromatography (DCM:MeOH = 95:5 or 9:1) and triturated with diethyl ether/petroleum ether/MeOH.

6-Ethoxy-1-methyl-9-(prop-2-yn-1-yl)-9H-pyrido[3,4-b]indole (8). – Compound 6: 0.100 g, 0.44 mmol; 60 % NaH: 0.047 g, 1.18 mmol; 80 % solution of propargyl bromide in toluene: 148 μL, 0.197 g, 1.33 mmol; yield: 0.031 g (26 %); mp 187.0–188.0 °C; IR (ATR, ν/cm^–1) 3146, 2978, 2108, 1739, 1562, 1488, 1455, 1394, 1289, 1226, 1199, 1150, 1114, 1050, 987, 910, 847, 813, 797, 756, 692, 626, 615, 521; ¹H NMR (DMSO-d₆) δ 8.24 (d, 1H, J = 4.9 Hz), 7.99 (d, 1H, J = 4.8 Hz), 7.81 (s, 1H), 7.72 (d, 1H, J = 8.9 Hz), 7.21 (d, 1H, J = 8.6 Hz), 5.76 (d, 2H, J = 6.1 Hz), 4.13 (q, 2H, J = 6.5 Hz), 3.37 (s, 1H), 2.98 (s, 3H), 1.39 (t, 3H, J = 6.7 Hz); ¹³C NMR (DMSO-d₆) δ 153.51, 142.39, 138.27, 135.29, 134.59, 128.51, 121.99, 118.31, 113.07, 112.38, 104.52, 86.04, 63.64, 23.22, 14.79; ESI-MS: m/z 265.20 (M + 1)⁺.

6-Isopropoxy-1-methyl-9-(prop-2-yn-1-yl)-9H-pyrido[3,4-b]indole (9). – Compound 7: 0.160 g, 0.67 mmol; 60 % NaH: 0.070 g, 1.77 mmol; 80 % solution of propargyl bromide in toluene: 223 μL, 0.297 g, 2.00 mmol; yield: 0.108 g (58 %); mp 126.5–138.0 °C; IR (ATR, ν/cm^–1) 3173, 2988, 2935, 2125, 2110, 1584, 1564, 1485, 1451, 1384, 1332, 1288, 1198, 1122, 1110, 1032, 987, 921, 863, 816, 800, 737, 624, 594, 507; ¹H NMR (DMSO-d₆) δ 8.21 (d, 1H, J = 5.2 Hz), 7.99 (d, 1H, J = 5.3 Hz), 7.81 (d, 1H, J = 2.4 Hz), 7.69 (d, 1H, J = 9.1 Hz), 7.24 (dd, 1H, J = 8.9, 2.5 Hz), 5.42 (d, 2H, J = 2.3 Hz), 4.72–4.66 (m, 1H), 3.36 (t, 1H, J = 2.3 Hz), 3.05 (s, 3H), 1.32 (d, 6H, J = 6.0 Hz); ¹³C NMR (DMSO-d₆) δ 151.80, 141.69, 137.72, 135.79, 134.71, 119.46, 113.06, 111.23, 106.68, 96.35, 85.91, 70.11, 34.19, 22.49, 21.87; ESI-MS: m/z 279.20 (M + 1)⁺.

6-Methoxy-9-(prop-2-yn-1-yl)-1-(trifluoromethyl)-9H-pyrido[3,4-b]indole (14). – Compound 11: 0.150 g, 0.57 mmol; 60 % NaH: 0.060 g, 1.50 mmol; 80 % solution of propargyl bromide in toluene: 189 μL, 0.252 g, 1.70 mmol; yield: 0.104 g (61 %); mp 90.7–92.5 °C; IR (ATR, ν/cm^–1) 3278, 3183, 1592, 1490, 1458, 1416, 1345, 1288, 1238, 1193, 1178, 1147, 1101, 1082, 1039, 886, 834, 800, 771, 714, 657, 627, 595, 523; ¹H NMR (DMSO-d₆) δ 8.57 (d, 1H, J = 5.0 Hz), 8.52 (d, 1H, J = 5.0 Hz), 7.99 (d, 1H, J = 2.6 Hz), 7.78 (d, 1H, J = 9.0 Hz), 7.40 (dd, 1H, J = 9.0, 2.6 Hz), 5.30 (d, 1H, J = 2.4 Hz), 3.91 (d, 3H, J = 2.5 Hz), 3.28 (t, 1H, J = 2.3 Hz); ¹³C NMR (DMSO-d₆) δ 154.91, 137.20, 136.68, 132.78, 132.59, 128.66 (q, J = 41.1 Hz), 121.42, 121.14, 119.63, 119.19, 112.40, 103.93, 86.17, 75.05, 55.80, 34.89 (q, J = 5.3 Hz); ESI-MS: m/z 305.20 (M + 1)⁺.

9-(Prop-2-yn-1-yl)-1-(trifluoromethyl)-9H-pyrido[3,4-b]indole (15). – Compound 12: 0.120 g, 0.51 mmol; 60 % NaH: 0.054 g, 1.35 mmol; 80 % solution of propargyl bromide in toluene: 170 μL, 0.227 g, 1.52 mmol; yield: 0.062 g (44 %); mp 73.5–75.0 °C; IR (ATR, ν/cm^–1) 3047, 2967, 1624, 1490, 1468, 1452, 1441, 1422, 1366, 1349, 1331, 1308, 1293, 1193, 1166, 1147, 1135, 1101, 1077, 1066, 1054, 940, 908, 893, 857, 836, 778 , 750, 738, 729, 694, 623, 576, 552; ¹H NMR (DMSO-d₆) δ 8.59 (d, 1H, J = 2.1 Hz), 8.42 (d, 1H, J = 7.7 Hz), 7.84 (d, 1H, J = 8.4 Hz), 7.73 (t, 1H, J = 7.7 Hz), 7.46 (t, 1H, J = 7.4 Hz), 7.29–7.17 (m, 1H), 5.78 (d, 1H, J = 6.2 Hz), 3.36 (s, 1H); ¹³C NMR (DMSO-d₆) δ 141.79, 138.17, 132.66, 132.31, 130.04, 129.16 (q, J = 35.4, 34.9 Hz), 122.07, 121.78, 121.25, 120.52, 119.02, 111.74, 94.97 (q, J = 4.4 Hz), 85.98, 34.83 (q, J = 5.1 Hz); ESI-MS: m/z 275.2 (M + 1)⁺.

Methyl 1-methyl-9-(prop-2-yn-1-yl)-9H-pyrido[3,4-b]indole-3-carboxylate (22). – Compound 20: 0.150 g, 0.62 mmol; Cs₂CO₃: 0.586 g, 1.798 mmol, 2,88 equiv.; 80 % solution of propargyl bromide in toluene: 100 μL, 0.134 g, 0.899 mmol, 1,44 equiv.; yield: 0.132 g (76 %); mp 166.5–170.5 °C; IR (ATR, ν/cm^–1) 3220, 1709, 1433, 1350, 1272, 1237, 1128, 1060, 780, 743, 724, 601, 525, 514; ¹H NMR (DMSO-d₆) δ 8.82 (s, 1H), 8.43 (dt, 1H, J = 7.8, 1.0 Hz), 7.93–7.85 (m, 1H), 7.69 (ddd, 1H, J = 8.4, 7.1, 1.2 Hz), 7.38 (ddd, 1H, J = 7.9, 7.1, 0.9 Hz), 5.56 (d, 2H, J = 2.4 Hz), 3.92 (s, 3H), 3.46 (t, 1H, J = 2.4 Hz), 3.14 (s, 3H); ¹³C NMR (DMSO-d₆) δ 165.79, 141.83, 141.21, 136.62, 135.78, 128.93, 128.62, 122.07, 121.07, 120.97, 116.01, 110.79, 76.11, 52.01, 34.38, 22.66; ESI-MS: m/z 279.2 (M + 1)⁺.

7-Fluoro-1-methyl-9-(prop-2-yn-1-yl)-9H-pyrido[3,4-b]indole (26). – Compound 25: 0.180 g, 0.90 mmol; 60 % NaH: 0.054 g, 1.349 mmol, 1.5 equiv.; 80 % solution of propargyl bromide in toluene: 151 μL, 0.201 g, 1.349 mmol, 1.5 equiv.; yield: 0.140 g (65 %); mp 141.0–143.0 °C; IR (ATR, ν/cm^–1) 3121, 2106, 1628, 1565, 1494, 1440, 1401, 1351, 1334, 1298, 1248, 1167, 1129, 974, 935, 924, 820, 803, 732, 667, 647, 615, 593, 551; ¹H NMR (DMSO-d₆) δ 8.31–8.27 (m, 2H), 8.01 (d, 1H, J = 5.2 Hz), 7.74 (dd, 1H, J = 10.5, 2.1 Hz), 7.16 (td, 1H, J = 9.4, 2.2 Hz), 5.47 (d, 2H, J = 2.3 Hz), 3.40 (t, 1H, J = 2.3 Hz), 3.07 (s, 3H); ¹³C NMR (DMSO-d₆) δ 162.89 (d, J = 242.1 Hz), 141.87 (d, J = 12.9 Hz), 141.70, 138.86, 134.88 (d, J = 2.0 Hz), 128.41, 123.38, 117.55, 112.90, 108.52 (d, J = 24.6 Hz), 97.28 (d, J = 27.5 Hz), 75.84, 34.49, 22.50; ESI-MS: m/z 239.3 (M + 1)⁺.

1-Bromo-9-(prop-2-yn-1-yl)-9H-pyrido[3,4-b]indole (32). – Compound 30: 0.180 g, 0.729 mmol; 60 % NaH: 0.044 g, 1.093 mmol, 1.5 equiv.; 80 % solution of propargyl bromide in toluene: 122 μL, 0.163 g, 1.093 mmol, 1.5 equiv.; yield: 0.107 g (52 %); mp 137.5–139.0 °C; IR (ATR, ν/cm^–1) 3217, 1624, 1534, 1489, 1446, 1434, 1414, 1329, 1298, 1275, 1207, 1193, 1157, 1135, 1056, 927, 843, 834, 798, 769, 742, 724, 700, 624, 594, 553; ¹H NMR (DMSO-d₆) δ 8.34 (d, 1H, J = 7.8 Hz), 8.28 (d, 1H, J = 5.0 Hz), 8.22 (d, 1H, J = 5.0 Hz), 7.86 (d, 1H, J = 8.4 Hz), 7.72 (t, 1H, J = 7.7 Hz), 7.41 (t, 1H, J = 7.5 Hz), 5.68 (d, 2H, J = 1.9 Hz), 3.34 (t, 2H, J = 3.2 Hz); ¹³C NMR (DMSO-d₆) δ 141.40, 139.23, 132.88, 131.76, 129.43, 122.29, 121.97, 121.24 120.36, 115.13, 111.05, 75.68, 33.75; ESI-MS: m/z 285.0 (M + 1)⁺.

General procedure for the synthesis of β-carboline based Boc-protected amines 17 and 27

To a stirred solution of compounds 13 or 25 in anhydrous DMF (1 mL/0.1 g; 6 mL) at 90 °C under an argon atmosphere, 60 % NaH (8 equiv.) was added. The resulting suspension was stirred at 90 °C for 20 min, followed by the addition of 2-(Boc-amino)ethyl bromide (3–4 equiv.). The reaction mixture was stirred at 90 °C for 18 h, cooled down, poured into water (60 mL) and extracted with ethyl acetate (3×60 mL). The collected organic layers were washed with water and brine, dried over anhydrous sodium sulfate, filtered, and evaporated under the reduced pressure. The crude product was purified by column chromatography (DCM:MeOH = 9:1) and triturated with diethyl ether.

Tert-butyl (2-(1-methyl-9H-pyrido[3,4-b]indol-9-yl)ethyl)carbamate (17). – Compound 13: 0.300 g, 1.65 mmol; 60 % NaH: 0.527 g, 13.17 mmol; 2-(Boc-amino)ethyl bromide: 1.107 g, 4.94 mmol, 3 equiv.; yield: 0.258 g (48 %); mp 158.5–159.5 °C; IR (ATR, ν/cm^–1) 3215, 2984, 1700, 1621, 1548, 1448, 1408, 1379, 1364, 1312, 1278, 1253, 1201, 1174, 1129, 1036, 971, 910, 872, 823, 776, 752, 739, 630, 587, 520; ¹H NMR (DMSO-d₆) δ 8.25–8.20 (m, 1H), 7.99 (d, 1H, J = 5.2 Hz), 7.68 (d, 1H, J = 8.3 Hz), 7.59 (ddd, 1H, J = 8.3, 7.0, 1.2 Hz), 7.27 (t, 1H, J = 7.4 Hz), 7.06 (t, 1H, J = 6.0 Hz), 4.62 (t, 1H, J = 6.7 Hz), 3.32 (q, 1H, J = 6.6 Hz), 2.99 (s, 2H), 1.31 (s, 8H), 1.00 (s, 1H); ¹³C NMR (DMSO-d₆) δ 155.71, 141.30, 137.51, 134.60, 128.11, 128.04, 121.45, 120.68, 119.54, 112.94, 110.21, 77.87, 43.86, 40.24, 28.11, 23.26; ESI-MS: m/z 326.4 (M + 1)⁺.

Tert-butyl (2-(7-fluoro-1-methyl-9H-pyrido[3,4-b]indol-9-yl)ethyl)carbamate (27). – Compound 25: 0.188 g, 0.939 mmol; 60 % NaH: 0.300 g, 7.512 mmol; 2-(Boc-amino)ethyl bromide: 0.842 g, 3.756 mmol, 4 equiv.; yield: 0.181 g (56 %); mp 146.0–147.0 °C; IR (ATR, ν/cm^–1) 2979, 1698, 1632, 1570, 1532, 1495, 1446, 1409, 1366, 1348, 1308, 1277, 1245, 1174, 1126, 1082, 1011, 979, 949, 858, 825, 803, 793, 748, 682, 620, 602, 581, 547; ¹H NMR (DMSO-d₆) δ 8.29–8.19 (m, 2H), 7.98 (d, 1H, J = 5.2 Hz), 7.50 (dd, 1H, J = 10.6, 2.3 Hz), 7.10 (td, 1H, J = 9.1, 2.2 Hz), 7.00 (t, 1H, J = 6.0 Hz), 4.58 (t, 1H, J = 6.3 Hz), 3.33 (s, 1H), 2.96 (s, 1H), 1.26 (s, 8H), 0.94 (s, 1H); ¹³C NMR (DMSO-d₆) δ 162.71 (d, J = 241.4 Hz), 155.62, 142.36 (d, J = 12.7 Hz), 141.18, 138.04, 135.26, 127.99, 123.12 (d, J = 10.6 Hz), 117.37, 112.77, 107.86 (d, J = 24.6 Hz), 96.95 (d, J = 26.9 Hz), 77.81, 44.18, 40.29, 28.03, 23.17; ESI-MS: m/z 344.2 (M + 1)⁺.

General procedure for the synthesis of β-carboline based amines 18 and 28

A solution of the Boc-protected amine (17 or 27) and 4 mol L^–1 HCl in MeOH (10 equiv.) was stirred at 50 °C for 4 h (18) or 18 h (28). Upon completion, solvent was removed under the reduced pressure. The residue was dissolved in water, basified to pH 12 with 5 % NaOH, and extracted with ethyl acetate (5×20 mL). The collected organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The crude product was purified by column chromatography (DCM:MeOH = 9:1, 18) and/or triturated with diethyl ether.

2-(1-Methyl-9H-pyrido[3,4-b]indol-9-yl)ethan-1-amine (18). – Compound 17: 0.200 g, 0.615 mmol; 4 mol L^–1 HCl in MeOH: 0.1536 mL, MeOH: 3.75 mL; yield: 0.073 g (53 %); mp 114.5–118.5 °C; IR (ATR, ν/cm^–1) 3215, 2984, 1700, 1621, 1548, 1448, 1408, 1379, 1364, 1312, 1278, 1253, 1201, 1174, 1129, 1036, 971, 910, 872, 823, 776, 752, 739, 630, 587, 520; ¹H NMR (DMSO-d₆) δ 8.23 (d, 1H, J = 7.9 Hz), 8.21 (d, 1H, J = 5.2 Hz), 7.99 (d, 1H, J = 5.1 Hz), 7.76 (d, 1H, J = 8.4 Hz), 7.59 (t, 1H, J = 7.7 Hz), 7.26 (t, 1H, J = 7.4 Hz), 4.57 (t, 2H, J = 7.2 Hz), 3.00 (s, 2H), 2.92 (t, 2H, J = 7.2 Hz); ¹³C NMR (DMSO-d₆) δ 141.30, 137.31, 134.65, 127.86, 127.77, 121.27, 120.44, 119.30, 112.74, 110.44, 82.58, 47.24, 42.12, 23.31; ESI-MS: m/z 226.2 (M + 1)⁺.

2-(7-fluoro-1-methyl-9H-pyrido[3,4-b]indol-9-yl)ethan-1-amine (28). – Compound 27: 0.170 g, 0.495 mmol; 4 mol L^–1 HCl in MeOH: 0.1237 mL, MeOH: 3.19 mL; yield: 0.061 g (51 %); mp 148.5–151.5 °C; IR (ATR, ν/cm^–1) 1628, 1443, 1408, 1228, 1148, 1128, 952, 834, 805, 622, 550; ¹H NMR (DMSO-d₆) δ 8.25 (dd, 1H, J = 8.6, 5.7 Hz), 8.22 (d, 1H, J = 5.2 Hz), 7.98 (d, 1H, J = 5.2 Hz), 7.66 (dd, 1H, J = 10.7, 2.3 Hz), 7.10 (ddd, 1H, J = 9.5, 8.6, 2.3 Hz), 4.53 (t, 2H, J = 7.1 Hz), 2.98 (s, 3H), 2.92 (t, 2H, J = 7.1 Hz); ¹³C NMR (DMSO-d₆) δ 162.76 (d, J = 241.2 Hz), 142.33 (d, J = 12.9 Hz), 141.29, 137.94, 135.29 (d, J = 1.8 Hz), 127.74, 123.09 (d, J = 10.9 Hz), 117.16, 112.75, 107.81 (d, J = 24.7 Hz), 97.31 (d, J = 27.1 Hz), 47.46, 42.17, 23.29; ESI-MS: m/z 244.1 (M + 1)⁺.

General procedure for the synthesis of AT hybrids 35–37

A suspension of a 7-chloroquinoline-based carboxylic acid 2, appropriate amine (18, 28 or 34; 1.1 equiv.) and TEA (2 equiv.) in dry DMF (1 mL) was stirred at room temperature for 10 min, followed by the dropwise addition of T3P (≥ 50 % in ethyl acetate, 1 equiv.). The reaction mixture was stirred at room temperature for 18 h, or 72 h for 35. Afterwards, the reaction mixture was placed in an ultrasonic bath and 5 % NaOH was added dropwise until the formation of white precipitate was completed. In the synthesis of 35 and 37, the reaction mixture was extracted 7× (35) or 1× (37) with an appropriate amount of ethyl acetate. The collected organic layers were washed with water and brine, dried over anhydrous sodium sulfate, filtered, and evaporated under the reduced pressure. In the synthesis of 36, the formed precipitate was filtered off. The crude product was purified by column chromatography (DCM:MeOH = 85:15 for 35, DCM:MeOH = 8:2 for 36 and DCM:MeOH = 75:25 for 37) and trituration with diethyl ether/petroleum ether/MeOH.

2-((7-Chloroquinolin-4-yl)amino)-N-(2-(1-methyl-9H-pyrido[3,4-b]indol-9-yl)ethyl)acetamide (35). – Carboxylic acid 2: 0.067 g, 0.222 mmol; amine 18: 0.070 g, 0.311 mmol; TEA: 0.057 g, 0.566 mmol; T3P: 0.100 g, 0.283 mmol; yield: 0.020 g (19 %); 262.0–262.5 °C; IR (ATR, ν/cm^–1) 3294, 1657, 1582, 1563, 1448, 1407, 1349, 1286, 1237, 1204, 1148, 1082, 983, 879, 849, 793, 747, 731, 639, 597, 561, 545; ¹H NMR (DMSO-d₆) δ 8.37–8.29 (m, 2H), 8.28–8.18 (m, 3H), 8.00 (d, 1H, J = 4.1 Hz), 7.82 (s, 1H), 7.73 (d, 2H, J = 6.9 Hz), 7.60 (t, 1H, J = 7.0 Hz), 7.50 (d, 1H, J = 8.2 Hz), 7.28 (t, 1H, J = 6.8 Hz), 6.08 (d, 1H, J = 5.4 Hz), 4.64 (t, 1H, J = 7.1 Hz), 3.85 (d, 1H, J = 4.4 Hz), 3.52 (q, J = 6.8 Hz, 1H, 2'), 3.00 (s, 1H, 13); ¹³C NMR (DMSO-d₆) δ 169.47, 151.77, 150.19, 148.79, 141.36, 141.23, 137.65, 134.50, 133.54, 128.13, 128.10, 127.40, 124.37, 124.17, 121.54, 120.66, 119.66, 117.51, 112.97, 110.19, 98.98, 45.84, 43.35, 38.99, 23.24; ESI-MS: m/z 444.4 (M + 1)⁺; HPLC purity 98.8 %.

2-((7-Chloroquinolin-4-yl)amino)-N-(2-(7-fluoro-1-methyl-9H-pyrido[3,4-b]indol-9-yl)ethyl)acetamide (36). – Carboxylic acid 2: 0.044 g, 0.189 mmol; amine 28: 0.050 g, 0.206 mmol; TEA: 0.038 g, 0.374 mmol; T3P: 0.060 g, 0.187 mmol; yield: 0.020 g (11 %); mp 252.0–255.0 °C; IR (ATR, ν/cm^–1) 3288, 1656, 1630, 1585, 1447, 1408, 1370, 1351, 1306, 1266, 1253, 1211, 1173, 1151, 1133, 1078, 956, 878, 850, 828, 809, 762, 651, 623, 549; ¹H NMR (DMSO-d₆) δ 8.39 (t, 1H, J = 6.1 Hz), 8.34 (d, 1H, J = 5.5 Hz), 8.31–8.27 (m, 2H), 8.24 (d, 1H, J = 5.2 Hz), 7.99 (d, 2H, J = 5.2 Hz), 7.84 (d, 1H, J = 2.2 Hz), 7.59 (dd, 1H, J = 10.5, 2.3 Hz), 7.53 (dd, 1H, J = 9.0, 2.3 Hz), 7.12 (ddd, 1H, J = 9.4, 8.5, 2.2 Hz), 6.11 (d, 1H, J = 5.6 Hz), 4.59 (t, 2H, J = 7.1 Hz), 3.88 (d, 2H, J = 5.8 Hz), 3.51 (q, 2H, J = 6.8 Hz), 2.99 (s, 3H); ¹³C NMR (DMSO-d₆) δ 169.28, 162.78 (d, J = 241.7 Hz), 150.82, 150.76, 147.67, 142.17 (d, J = 12.8 Hz), 141.22, 138.12, 135.15 (d, J = 1.8 Hz), 134.02, 127.96, 126.49, 124.65, 124.38, 123.32 (d, J = 10.8 Hz), 117.33 (d, J = 11.1 Hz), 112.85, 108.08 (d, J = 24.6 Hz), 98.92, 96.95 (d, J = 27.1 Hz), 45.80, 43.61, 38.72, 23.07; ESI-MS: m/z 462.4 (M + 1)⁺.

N-(2-(9H-pyrido[3,4-b]indol-9-yl)ethyl)-2-((7-chloroquinolin-4-yl)amino) acetamide (37). – Carboxylic acid 2: 0.072 g, 0.306 mmol; amine 34: 0.071 g, 0.336 mmol; TEA: 0.062 g, 0.611 mmol; T3P: 0.097 g, 0.306 mmol; yield: 0.020 g (15 %); mp 263.0–265.5 °C; IR (ATR, ν/cm^–1) 3282, 1664, 1582, 1453, 1368, 1247, 1213, 1151, 849, 815, 747, 727, 550; ¹H NMR (DMSO-d₆) δ 9.04 (s, 1H), 8.40 (d, 1H, J = 5.2 Hz), 8.29 (t, 1H, J = 2.7 Hz), 8.28 (s, 1H), 8.20 (dd, 2H, J = 7.2, 1.7 Hz), 8.15 (dd, 1H, J = 5.2, 1.1 Hz), 7.81 (d, 1H, J = 6.5 Hz), 7.79 (d, 1H, J = 2.3 Hz), 7.70 (d, 1H, J = 8.3 Hz), 7.62 (ddd, 1H, J = 8.3, 7.0, 1.2 Hz), 7.48 (dd, 1H, J = 9.0, 2.2 Hz), 7.34–7.27 (m, 1H), 5.85 (d, 1H, J = 5.5 Hz), 4.58 (t, 2H, J = 6.1 Hz), 3.75 (d, 2H, J = 6.0 Hz), 3.58 (q, 2H, J = 6.0 Hz); ¹³C NMR (DMSO-d₆) δ 169.66, 151.48, 150.98, 148.45, 141.39, 138.99, 136.76, 134.25, 132.96, 128.78, 127.80, 127.22, 124.94, 124.72, 122.46, 120.94, 120.01, 117.76, 115.08, 110.41, 99.28, 46.11, 42.45, 38.59; ESI-MS: m/z 430.4 (M + 1)⁺; HPLC purity 98.2 %.

General procedure for the synthesis of TT hybrids 38–47

To a solution of an alkyne and the 7-chloroquinoline-based azide 1 (1.1 equiv.) in MeOH (4–5 mL), catalytic amount of Cu(OAc)₂ was added. The reaction mixture was stirred at rt for 24 h. Upon completion of the reaction, the solvent was removed under the reduced pressure. The residue was purified by column chromatography (with an additional Al₂O₃ layer to remove Cu-salts) with DCM:MeOH = 85:15, cyclohexane:ethyl acetate:methanol = 1:1:0.5 for 40 and 41 or DCM:MeOH = 75:25 for 46 as a mobile phase. The crude product was triturated with diethyl ether or MeOH.

7-Chloro-N-(2-(4-((6-ethoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl)methyl)-1H-1,2,3-triazol-1-yl)ethyl)quinolin-4-amine (38). – Alkyne 8: 0.045 g, 0.170 mmol; azide 1: 0.046 g, 0.187 mmol; yield: 0.022 g (25 %); mp > 200 °C; IR (ATR, ν/cm^–1) 2971, 1579, 1448, 1368, 1324, 1244, 1220, 1194, 1141, 1103, 1049, 986, 879, 841, 819, 807, 741, 693, 622, 533; ¹H NMR (DMSO-d₆) δ 8.32 (d, 1H, J = 5.3 Hz), 8.18 (d, 1H, J = 5.2 Hz), 8.03 (d, 1H, J = 9.0 Hz), 7.96 (d, 1H, J = 5.2 Hz), 7.94 (s, 1H), 7.80 (d, 1H, J = 2.0 Hz), 7.77 (d, 1H, J = 2.3 Hz), 7.63 (d, 1H, J = 9.0 Hz), 7.42 (dd, 1H, J = 9.0, 2.1 Hz), 7.35 (t, 1H, J = 5.6 Hz), 7.14 (dd, 1H, J = 8.9, 2.4 Hz), 6.44 (d, 1H, J = 5.4 Hz), 5.82 (s, 2H), 4.55 (t, 2H, J = 5.9 Hz), 4.12 (q, 2H, J = 6.9 Hz), 3.69 (q, 2H, J = 5.7 Hz), 2.99 (s, 3H), 1.40 (t, 3H, J = 6.9 Hz); ¹³C NMR (DMSO-d₆) δ 153.38, 152.26, 149.99, 149.41, 144.41, 142.22, 137.65, 136.27, 135.34, 133.87, 128.31, 127.96, 124.72, 124.22, 123.82, 121.62, 118.57, 117.80, 113.45, 111.88, 104.77, 99.18, 64.09, 48.30, 42.69, 23.65 15.27; ESI-MS: m/z 512.20 (M + 1)⁺; HPLC purity 100.0 %.

7-Chloro-N-(2-(4-((6-isopropoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl)methyl)-1H-1,2,3-triazol-1-yl)ethyl)quinolin-4-amine (39). – Alkyne 9: 0.026 g, 0.093 mmol; azide 1: 0.025 g, 0.103 mmol; yield: 0.046 g (41 %); mp 80.0–89.5 °C; IR (ATR, ν/cm^–1) 2971, 1579, 1489, 1448, 1324, 1292, 1244, 1220, 1194, 1141, 1103, 1049, 986, 909, 879, 841, 819, 807, 741, 622, 533; ¹H NMR (DMSO-d₆) δ 8.32 (d, 1H, J = 5.1 Hz), 8.17 (d, 1H, J = 5.1 Hz), 8.05 (d, 1H, J = 9.0 Hz), 7.97 (s, 1H), 7.96 (s, 1H), 7.80 (d, 1H, J = 2.2 Hz), 7.78 (d, 1H), 7.61 (d, 1H, J = 8.9 Hz), 7.44 (d, 2H, J = 7.0 Hz), 7.12 (dd, 1H, J = 8.9, 2.0 Hz), 6.45 (d, 1H, J = 5.4 Hz), 5.81 (s, 2H), 4.65 (hept, 1H, J = 5.8, 5.3 Hz), 4.55 (t, 2H, J = 5.8 Hz), 3.70 (q, 2H, J = 6.0 Hz), 2.99 (s, 3H), 1.31 (d, 6H, J = 6.0 Hz); ¹³C NMR (DMSO-d₆) δ 151.56, 151.35, 149.74, 148.43, 143.89, 141.62, 137.09, 135.84, 134.83, 133.56, 127.77, 127.07, 124.32, 123.77, 123.32, 121.18, 119.32, 117.19, 112.98, 111.31, 106.48, 98.63, 70.08, 47.78, 42.19, 23.07, 21.89; ESI-MS: m/z 526.20 (M + 1)⁺; HPLC purity 100.0 %.

7-Chloro-N-(2-(4-((6-methoxy-1-(trifluoromethyl)-9H-pyrido[3,4-b]indol-9-yl)methyl)-1H-1,2,3-triazol-1-yl)ethyl)quinolin-4-amine (40). – Alkyne 14: 0.060 g, 0.197 mmol; azide 1: 0.054 g, 0.217 mmol; yield: 0.026 g (24 %); mp 133.5–139.0 °C; IR (ATR, ν/cm^–1); 2919, 1579, 1489, 1345, 1288, 1190, 1136, 1111, 1065, 835, 720, 557; ¹H NMR (DMSO-d₆) δ 8.55 (d, 1H, J = 4.9 Hz), 8.49 (d, 1H, J = 5.0 Hz), 8.31 (d, 1H, J = 5.4 Hz), 8.02 (d, 1H, J = 9.1 Hz), 7.94 (d, 1H, J = 2.6 Hz), 7.78 (s, 2H), 7.60 (d, 1H, J = 9.0 Hz), 7.40 (dd, 1H, J = 9.0, 2.2 Hz), 7.37 (t, 1H, J = 5.6 Hz), 7.21 (dd, 1H, J = 9.0, 2.6 Hz), 6.41 (d, 1H, J = 5.5 Hz), 5.73 (s, 2H), 4.53 (t, 2H, J = 6.0 Hz), 3.88 (s, 3H), 3.68 (q, 2H, J = 5.9 Hz); ¹³C NMR (DMSO-d₆) δ 154.61, 151.53, 149.75, 148.65, 142.72, 136.91, 136.59, 133.59, 132.97, 132.37, 128.44 (q, J = 34.9 Hz), 127.26, 124.31, 123.80, 123.26, 121.50, 120.97, 119.34, 119.04, 117.28, 112.60, 103.68, 98.69, 55.75, 47.90, 44.48, 42.25; ESI-MS: m/z 552.4 (M + 1)⁺; HPLC purity 97.7 %.

7-chloro-N-(2-(4-((1-(trifluoromethyl)-9H-pyrido[3,4-b]indol-9-yl)methyl)-1H-1,2,3-triazol-1-yl)ethyl)quinolin-4-amine (41). – Alkyne 15: 0.062 g, 0.226 mmol; azide 1: 0.062 g, 0.249 mmol; yield: 0.030 g (41 %); mp 234.0–237.5 °C; IR (ATR, ν/cm^–1) 2922, 1613, 1584, 1429, 1347, 1323, 1295, 1193, 1142, 1120, 1068, 838, 803, 745, 726, 642, 557; ¹H NMR (DMSO-d₆) δ 8.58 (d, 1H, J = 5.0 Hz), 8.54 (d, 1H, J = 4.9 Hz), 8.39 (d, 1H, J = 7.8 Hz), 8.30 (d, 1H, J = 5.5 Hz), 8.05 (d, 1H, J = 9.0 Hz), 7.83 (s, 1H), 7.79 (d, 1H, J = 2.2 Hz), 7.70 (d, 1H, J = 8.4 Hz), 7.60 (ddd, 1H, J = 8.3, 7.0, 1.2 Hz), 7.46 (d, 1H, J = 5.9 Hz), 7.42 (dd, 1H, J = 9.0, 2.2 Hz), 7.40–7.36 (m, 1H), 6.42 (d, 1H, J = 5.5 Hz), 5.77 (s, 2H), 4.54 (t, 2H, J = 6.0 Hz), 3.69 (q, 2H, J = 5.9 Hz), 3.20 (s, 1H), 2.63 (s, 2H); ¹³C NMR (DMSO-d₆) δ 151.26, 149.95, 148.33, 142.61, 142.10, 137.22, 133.74, 132.71, 132.59, 129.73, 128.42 (q, J = 35.1 Hz), 127.01, 124.42, 123.87, 123.33, 121.78, 121.49, 121.18, 120.43, 119.00, 117.23, 111.64, 98.68, 47.91, 44.49, 42.27; ESI-MS: m/z 522.4 (M + 1)⁺; HPLC purity 97.6 %.

7-Chloro-N-(2-(4-((1-methyl-9H-pyrido[3,4-b]indol-9-yl)methyl)-1H-1,2,3-triazol-1-yl)ethyl)quinolin-4-amine (42). – Alkyne 16: 0.060 g, 0.272 mmol; azide 1: 0.074 g, 0.300 mmol; yield: 0.040 g (31 %); mp 178.0–181.0 °C; IR (ATR, ν/cm^–1) 2957, 1616, 1582, 1445, 1405, 1354, 1327, 1300, 1245, 1200, 1178, 1141, 1084, 1047, 999, 973, 911, 875, 840, 775, 750, 733, 688, 646, 584, 558, 519; ¹H NMR (DMSO-d₆) δ 8.25 (s, 1H), 8.23 (d, 1H, J = 7.9 Hz), 8.11 (d, 1H, J = 8.9 Hz), 8.01 (d, 1H, J = 4.8 Hz), 7.98 (s, 1H), 7.84 (s, 1H), 7.73 (d, 1H, J = 8.3 Hz), 7.65 (d, 1H, J = 7.0 Hz), 7.52 (t, 1H, J = 7.7 Hz), 7.46 (d, 1H, J = 9.0 Hz), 7.26 (t, 1H, J = 7.4 Hz), 6.51 (s, 1H), 5.86 (s, 2H), 4.56 (t, 2H, J = 6.0 Hz), 3.73 (q, 2H, J = 6.0 Hz), 3.00 (s, 3H); ¹³C NMR (DMSO-d₆) δ 150.47, 149.88, 143.93, 143.42, 141.76, 140.96, 137.70, 135.52, 134.20, 128.16, 128.11, 126.85, 126.34, 124.80, 124.08, 123.47, 121.47, 120.77, 119.81, 113.06, 110.54, 103.51, 47.88, 42.36, 23.21; ESI-MS: m/z 468.5 (M + 1)⁺; HPLC purity 100.0 %.

Methyl 9-((1-(2-((7-chloroquinolin-4-yl)amino)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1-methyl-9H-pyrido[3,4-b]indole-3-carboxylate (43). – Alkyne 22: 0.060 g, 0.216 mmol; azide 1: 0.059 g, 0.237 mmol; yield: 0.036 g (32 %); mp > 240 °C; IR (ATR, ν/cm^–1) 1713, 1578, 1430, 1350, 1273, 1240, 1140, 1050, 910, 875, 840, 786, 749, 730, 532; ¹H NMR (DMSO-d₆) δ 8.81 (s, 1H), 8.40 (d, J = 7.8 Hz, 1H), 8.31 (d, J = 7.7 Hz, 1H), 8.06 (d, J = 9.0 Hz, 1H), 8.03 (s, 1H), 7.81 (d, J = 8.1 Hz, 2H), 7.64 (s, 1H), 7.58 (t, J = 7.7 Hz, 1H), 7.46 (d, J = 8.9 Hz, 1H), 7.33 (t, J = 7.5 Hz, 1H), 6.47 (d, J = 5.3 Hz, 1H), 5.93 (s, 2H), 4.57 (t, J = 5.9 Hz, 2H), 3.91 (s, 3H), 3.73 (q, J = 5.9 Hz, 2H), 3.06 (s, 3H); ¹³C NMR (DMSO-d₆) δ 165.89, 150.47, 143.54, 141.89, 141.36, 136.14, 136.06, 134.18, 128.65, 128.17, 127.61, 126.33, 124.77, 124.03, 123.65, 121.89, 120.97, 120.76, 115.99, 111.01, 110.00, 98.72, 51.98, 47.91, 42.33, 23.29; ESI-MS: m/z 526.3 (M + 1)⁺; HPLC purity 100.0 %.

(9-((1-(2-((7-Chloroquinolin-4-yl)amino)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1-methyl-9H-pyrido[3,4-b]indol-3-yl)methanol (44). – Alkyne 23: 0.065 g, 0.260 mmol; azide 1: 0.071 g, 0.286 mmol; yield: 0.042 g (33 %); mp 159.5–166.5 °C; IR (ATR, ν/cm^–1) 3079, 1622, 1580, 1456, 1427, 1360, 1336, 1297, 1256, 1197, 1137, 1123, 1031, 1014, 983, 914, 842, 808, 762, 732, 643, 627, 580, 567; ¹H NMR (DMSO-d₆) δ 8.30 (d, 1H, J = 5.4 Hz), 8.21 (d, 1H, J = 7.8 Hz), 8.10 (d, 1H, J = 8.8 Hz), 8.01 (s, 1H), 8.00 (s, 1H), 7.79 (d, 1H, J = 2.2 Hz), 7.71 (d, 1H, J = 8.3 Hz), 7.51 (ddd, 1H, J = 8.3, 7.0, 1.2 Hz), 7.46 (d, 1H, J = 5.6 Hz), 7.42 (dd, 1H, J = 9.0, 2.3 Hz), 7.24 (t, 1H, J = 7.4 Hz), 6.43 (d, 1H, J = 5.5 Hz), 5.84 (s, 2H), 5.38 (s, 1H), 4.67 (s, 2H), 4.56 (, 2H t, J = 6.0 Hz), 3.69 (q, 2H, J = 6.0 Hz), 2.98 (s, 3H); ¹³C NMR (DMSO-d₆) δ 151.80, 150.24, 149.68, 148.96, 143.92, 141.31, 140.51, 133.48, 133.42, 129.03, 128.02, 127.47, 124.31, 123.98, 123.44, 121.37, 120.93, 119.67, 117.41, 110.52, 109.06, 98.73, 64.35, 47.89, 42.27, 23.08; ESI-MS: m/z 498.10 (M + 1)⁺; HPLC purity 99.0 %.

7-chloro-N-(2-(4-((7-fluoro-1-methyl-9H-pyrido[3,4-b]indol-9-yl)methyl)-1H-1,2,3-triazol-1-yl)ethyl)quinolin-4-amine (45). – Alkyne 26: 0.060 g, 0.252 mmol; azide 1: 0.069 g, 0.277 mmol; yield: 0.054 g (44 %); mp 234.5–250.5 °C; IR (ATR, ν/cm^–1) 3063, 1628, 1578, 1444, 1331, 1244, 1161, 1128, 1039, 927, 803, 764, 619, 549; ¹H NMR (DMSO-d₆) δ 8.31 (d, 1H, J = 5.5 Hz), 8.28–8.25 (m, 1H), 8.25–8.22 (m, 1H), 8.06 (d, 1H, J = 9.0 Hz), 8.03 (s, 1H), 7.98 (d, 1H, J = 5.3 Hz), 7.80 (d, 1H, J = 2.3 Hz), 7.66 (dd, 1H, J = 10.5, 2.3 Hz), 7.58 (t, 1H, J = 5.8 Hz), 7.43 (dd, 1H, J = 8.9, 2.2 Hz), 7.11 (td, 1H, J = 9.0, 2.3 Hz), 6.48 (d, 1H, J = 5.6 Hz), 5.84 (s, 2H), 4.58 (t, 2H, J = 6.0 Hz), 3.73 (q, 2H, J = 5.9 Hz), 3.01 (s, 3H); ¹³C NMR (DMSO-d₆) δ 162.74 (d, J = 241.8 Hz), 150.73, 150.29, 147.69, 143.52, 141.95 (d, J = 12.8 Hz), 141.72, 138.31, 135.12, 134.02, 127.98, 126.52, 124.63, 123.91, 123.58, 123.20 (d, J = 10.9 Hz), 117.46, 117.12, 112.81, 108.21 (d, J = 24.5 Hz), 98.71, 97.40 (d, J = 27.1 Hz), 47.87, 42.33, 39.10, 23.14; ESI-MS: m/z 486.4 (M + 1)⁺; HPLC purity 100.0 %.

N-(2-(4-((9H-pyrido[3,4-b]indol-9-yl)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-7-chloroquinolin-4-amine (46). – Alkyne 31: 0.027 g, 0.131 mmol; azide 1: 0.036 g, 0.144 mmol; yield: 0.026 g (44 %); mp 223.5–225.5 °C; IR (ATR, ν/cm^–1) 1578, 1452, 1328, 1033, 841, 746, 728, 557; ¹H NMR (DMSO-d₆) δ 9.17 (s, 1H), 8.40 (d, 1H, J = 5.2 Hz), 8.30 (d, 1H, J = 5.6 Hz), 8.26 (dt, 1H, J = 7.8, 1.0 Hz), 8.18 (s, 1H), 8.14–8.10 (m, 2H), 7.84–7.80 (m, 2H), 7.72 (s, 1H), 7.57 (ddd, 1H, J = 8.3, 7.1, 1.2 Hz), 7.46 (dd, 1H, J = 9.0, 2.2 Hz), 7.28 (ddd, 1H, J = 7.9, 7.0, 0.9 Hz), 6.49 (d, 1H, J = 5.7 Hz), 5.76 (s, 2H), 4.59 (t, 2H, J = 6.0 Hz), 3.74 (q, 2H, J = 6.0 Hz); ¹³C NMR (DMSO-d₆) δ 150.53, 150.45, 142.75, 140.46, 138.68, 135.79, 134.11, 133.02, 128.27, 127.41, 126.31, 124.72, 124.13, 123.92, 121.90, 120.92, 120.49, 119.69, 117.11, 114.56, 110.52, 98.72, 47.81, 42.36, 37.72; ESI-MS: m/z 454.4 (M + 1)⁺; HPLC purity 97.1 %.

N-(2-(4-((1-bromo-9H-pyrido[3,4-b]indol-9-yl)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-7-chloroquinolin-4-amine (47). – Alkyne 32: 0.060 g, 0.210 mmol; azide 1: 0.057 g, 0.231 mmol; yield: 0.009 g (8 %); mp 165.0–169.0 °C; IR (ATR, ν/cm^–1) 3295, 2921, 2852, 1612, 1579, 1536, 1487, 1450, 1434, 1354, 1330, 1194, 1141, 1051, 912, 842, 768, 749, 728, 639, 596, 556; ¹H NMR (DMSO-d₆) δ 8.30 (d, 1H, J = 7.8 Hz), 8.25 (d, 2H, J = 5.0 Hz), 8.20 (d, 1H, J = 10.0 Hz), 8.17 (d, 1H, J = 5.0 Hz), 8.00 (s, 1H), 7.88 (s, 1H), 7.78 (d, 1H, J = 8.4 Hz), 7.60 (t, 1H, J = 7.7 Hz), 7.53 (d, 1H, J = 9.0 Hz), 7.34 (t, 1H, J = 7.4 Hz), 6.52 (s, 1H), 6.07 (s, 2H), 4.60 (t, 2H, J = 5.9 Hz), 3.81 (q, 2H, J = 5.9 Hz); ¹³C NMR (DMSO-d₆) δ 152.11, 150.94, 144.66, 143.45, 141.54, 138.62, 135.42, 132.95, 131.31, 129.12, 125.47, 124.50, 123.65, 122.13, 121.75, 121.57, 121.15, 120.83, 120.12, 115.03, 111.26, 98.49, 47.87, 42.51, 39.01; ESI-MS: m/z 533.95 (M + 1)⁺.

Prediction in SwissADME tool

An open-access web page, SwissADME, was used as a tool for prediction of selected drug-like properties of the title compounds 35–47 (21).

Parasite culture and in vitro antiplasmodial activity against erythrocytic stages of P. falciparum

Parasite culture and the antiplasmodial activity of novel hybrids 35–47 was evaluated against two P. falciparum strains (3D7, CQ-sensitive, and Dd2, multi-drug resistant) as previously described, using the histidine-rich protein 2 (HRP2) assay (22, 23). 96-Well plates were pre-coated with test compounds in a three-fold serial dilution, after which ring-stage parasites were added in complete culture medium at a haematocrit of 1.5 % and a parasitaemia of 0.05 %. Plates were incubated for 72 h at 37 °C under an atmosphere of 5 % CO₂ and 5 % O₂, then frozen until analysis by HRP2-ELISA. All compounds were tested in duplicate in at least two independent experiments. IC₅₀ values were determined by non-linear regression analysis of log concentration–response curves using the drc package (v0.9.0) in R (v2.6.1) (24).

HepG2 culture and in vitro cytotoxicity in HepG2 cells

Human hepatocellular carcinoma cell line (HepG2) was maintained, and cytotoxicity assay was performed using the neutral red uptake assay as described in (22, 25). Briefly, cells were seeded in 96-well plates in complete culture medium; on the following day, serial dilutions of the test compounds were added. After 24 h of incubation, cytotoxicity was assessed by addition of neutral red, followed by cell lysis and measurement of absorbance at 540 nm using a plate reader (CLARIOstar, BMG Labtech, Germany). IC₅₀ values were determined as described for the antiplasmodial assay. The selectivity index (SI) was calculated as the ratio of IC₅₀ values for HepG2 cells and the P. falciparum 3D7 strain.

RESULTS AND DISCUSSION

Chemistry

The three amide-type (AT) (35–37) and ten triazole-type (TT) (38–47) β-carboline and CQ hybrids bearing various substituents at the positions 1, 3, 6 and/or 7 of the β-carboline ring (Fig. 2) were synthesized via two distinctive strategies.

Fig. 2. Structural variety of the β-carboline in the title compounds.

The quinoline azide 1 and carboxylic acid 2 (Fig. 3) were prepared according to previously described modified procedures (26–28).

Fig. 3. 7-chloroquinoline-based intermediates 1 and 2.

To this end, the following β-carbolines and β-carboline-based building blocks are reported herein for the first time: β-carbolines 6–7, their corresponding alkynes 8–9, alkynes 14, 15, 22, 26 and 32, Boc-protected amines 17 and 27 and their corresponding amines 18 and 28 (Schemes 1–5).

Scheme 1

β-carbolines 4, 13, 20 and 25 were obtained according to previously described procedures (19), while the β-carbolines 11–12 were synthesized following modified literature procedures (18, 19). Phenol 5 was prepared according to a modified previously described procedure (29). β-carboline-based building blocks 21 and 23 were synthesised according to procedures previously reported by our group (30), while 16 (31), 31 (32), 33 and 34 (33), although previously described, were prepared following procedures reported in our earlier work (17) (Schemes 1–5).

Scheme 2

Synthesis of β-carboline-based building blocks was conducted in multiple reaction steps. Alkylation of phenol 5 with ethyl bromide (2.4 equiv.) using cesium carbonate (1.4 equiv.) as a base afforded novel β-carboline 6. The reaction was initiated with 1.2 equiv. of ethyl bromide and stirred at room temperature. Heating the reaction mixture to 40 °C did not improve conversion, and additional 1.2 equiv. of ethyl bromide and continued stirring at 40 °C, resulted in the appearance of by-products, thus the reaction was therefore stopped. Alkylation of phenol 5 with isopropyl bromide (8 equiv.) using cesium carbonate (4.2 equiv.) as a base afforded novel β-carboline 7. The β-carboline 6 was obtained in 52 % yield, whereas β-carboline 7 was synthesized at room temperature over 23 h in 66 % yield.

Scheme 3

The corresponding alkynes were obtained in moderate to high yields (8: 26 % and 9: 58 %) by alkylation at N-9 with propargyl-bromide (3 equiv.) using 60 % NaH (2.66 equiv.) as a base at room temperature for 2 h. The same procedure was applied for the synthesis of alkynes 14 (61 %) and 15 (44 %) which were also obtained in moderate to high yields. Alkynes 26 and 32 were obtained in a similar way in moderate to high yields (26: 65 %; 32: 52 %) by alkylation of the corresponding β-carboline at N-9 with propargyl-bromide (1.5 equiv.) using 60 % NaH (1.5 equiv.) as a base at room temperature for 2 h. Alkyne 22 was synthesised in high yield (76 %) by reaction with propargyl bromide (1.44 equiv.) in the presence of cesium carbonate as the base (2.88 equiv.). The reaction was initially carried out using 1.44 equiv. of cesium carbonate. TLC monitoring after 2 h indicated incomplete conversion and the presence of a considerable amount of starting material; consequently, a further 1.44. equiv. of cesium carbonate was added. The reaction was completed within 24 h at room temperature.

Scheme 4

N-9 β-carboline-based amines 18 and 28 were synthesised via a two-step procedure. Initially, β-carbolines 13 and 25 were alkylated with 2-(Boc-amino)ethyl bromide (3–4 equiv.) using 60 % NaH (8 equiv.) at 90 °C for 18 h to afford Boc-protected intermediates 17 (48 %) and 27 (56 %). Subsequent acidic deprotection of the Boc group yielded the corresponding primary amines 18 and 28. The deprotection step was carried out at 50 °C for 4 h or 18 h, respectively, affording the desired amines in 53 % and 51 % yields (Schemes 2, 4 and 5).

Scheme 5

The AT hybrids (35–37) (Scheme 6) were obtained through a coupling reaction between the β-carboline-based amines (18, 28 and 34) and 7-chloroquinoline-based carboxylic acid 2 using propylphosphonic anhydride (T3P), which has shown to be an efficient coupling reagent, in the presence of triethylamine (TEA). The reactions proceeded smoothly at room temperature over 18 h, affording the desired products in low yields (11–19 %).

In contrast, the TT hybrids (38–47) (Scheme 6) were synthesized via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) also known as “click” reaction. The reactions were carried out using copper(II) acetate as a precatalyst in methanol at room temperature overnight, providing the corresponding hybrids in moderate to good yields (24–44 %). This methodology proved advantageous due to its operational simplicity, mild conditions, and tolerance toward various functional groups present in the β-carboline fragments.

Scheme 6

Comparison of the two synthetic approaches indicates that the CuAAC strategy generally afforded higher yields than the coupling route. Moreover, the triazole linker introduces additional rigidity and potential for interactions with the target, while maintaining metabolic stability, which may contribute to the enhanced biological activity observed for several triazole derivatives.

Both synthetic routes enabled efficient access to structurally diverse hybrids, allowing systematic variation of substituents on the β-carboline core and facilitating subsequent structure-activity relationship studies.

The synthesis of some intermediates, particularly alkynes and Boc-protected amines, also resulted in moderate to low yields, indicating incomplete conversions.

In contrast, the CuAAC approach was more robust and reproducible, resulting in the higher number of final compounds; however, it is limited to substrates bearing azide and alkyne functionalities, requiring additional synthetic steps for intermediates preparation. Overall, while the developed synthetic routes are versatile, they could be further optimized to improve yields and streamline access to more demanding derivatives.

Structures of AT and TT hybrids were confirmed by MS, IR, ¹H, and ¹³C NMR spectra. Analytical and spectral data are given in Tables I and II.

Table I. Analytical and MS data for AT and TT hybrids 35–47

^an.d. not determined

Table II. IR, ¹H and ¹³C NMR spectroscopic data for AT and TT hybrids 35–47

SwissADME prediction

Physicochemical descriptors of the title compounds 35–47 were assessed by using SwissADME tool. The determined parameters are outlined in Table III: molecular weight (MW), heavy atoms (HA), number of rotatable bonds (RB), number of hydrogen bond acceptors (HBA) and donors (HBD), topological polar surface area (TPSA), consensus partition coefficient (cLog P) and Lipinski’s (LV) and Veber’s violations (VV). The number of HBA and HBD and cLogP are in line with the Lipinski’s rule of five. However, some compounds (38–41, 43, 45–47) have molecular weight slightly above the limit of 500. TPSA represents the sum of the surface areas of all polar atoms in one molecule indicating its cellular permeability. All β-carboline and CQ hybrids have TPSA values within the suggested range (20–130 Ų). In addition, all compounds are in line with Veber’s rules (10 or less RB and TPSA equal to or less than 140 Ų or 12 or fewer HBD and HBA), which indicates high probability of their good oral bioavailability (34).

Table III. Drug-like properties based on Lipinski’s and Veber’s rules

MW, molecular weight; HA, number of heavy atoms; RB, number of rotatable bonds; HBA, number of H-bond acceptors; HBD, number of H-bond donors; TPSA, polar surface area; cLogP, partition coefficient; LV, number of violations of the Lipiski rules; VV, number of the violations of the Veber rules.

Antiplasmodial activity

The antiplasmodial activity of the synthesized hybrids (35–47) was evaluated against the two P. falciparum strains (3D7 and Dd2), and the results are summarized in Table IV. All compounds exhibited activity in the nanomolar to low micromolar range, with IC₅₀ values spanning from 1.0 to 365.5 nmol L^–1 for 3D7 and from 2.2 to 2415.3 nmol L^–1 for Dd2 strain. The most potent compounds against 3D7 strain were 36 (IC₅₀ = 1.0 ± 0.2 nmol L^–1), 45 (1.2 ± 0.2 nmol L^–1), and 42 (2.9 ± 0.1 nmol L^–1), showing superior activity compared to the reference drug chloroquine (CQ, IC₅₀ = 6.9 ± 0.0 nmol L^–1). Compounds 35 (4.6 ± 0.3 nmol L^–1) and 43 (11.9 ± 3.0 nmol L^–1) demonstrated potency comparable to CQ. Compounds 38–41, 43, 44, 46, and 47, were less active compared to CQ. In the case of multi-drug resistant P. falciparum strain Dd2, only three compounds were less active than CQ (37, 44 and 46), which strongly confirms the overall activity of the whole series, resulting in the resistance ratios significantly lower than for CQ.

Table IV. In vitro antiplasmodial activity of hybrids 35–47 against P. falciparum erythrocytic stage (Pf3D7 and PfDd2 strains) and resistance index (RI) calculated as ratio between multi-drug resistant (PfDd2) and CQ sensitive (Pf3D7) P. falciparum strains

^aIC₅₀, the concentration of the tested compound that is necessary for 50 % growth inhibition; ^b the results are expressed as mean ± SD (n ≥ 2); ^c CQ, chloroquine; ^d (20); ^e RI, resistance index expressed as ratio between IC₅₀ obtained for multi-drug resistant (Dd2) and CQ-sensitive (3D7) P. falciparum strain; IC₅₀(PfDd2)/IC₅₀(Pf3D7); ^fHBD/HBA, hydrogen bond donors/hydrogen bond acceptors present in the β-carboline.

By introducing various scaffolds in the β-carboline ring, we were once again able to establish SAR. Compounds 38 and 39, structurally resembling to the novel hybrids from our previous work, were compared to the compound 39 bearing O-methyl group at the position 6 of the β-carboline ring from the previous series (17). Substitution of methyl group with ethyl (38) or i-propyl (39) in the new series, led to reduction in activity against 3D7 and increase in activity against Dd2 strain.

The compounds could be divided in five subgroups based on their structural features:

Almost all compounds demonstrated resistance ratios better than CQ, with the exception of 42 and 44. Compounds 38, 39, 40, 43 and 47 were more active against multi-drug resistant P. falciparum strain Dd2, then against CQ sensitive 3D7, with resistance ratios spanning from 0.19 for 40 to 0.9 for 47, respectively.

In vitro cytotoxicity screening

Cytotoxicity of the title compounds was evaluated against the human HepG2 cell line. The results are presented in Table V. No significant cytotoxic effects were observed up to the highest tested concentration (IC₅₀ > 250 µmol L^–1) for all tested compounds. Consequently, selectivity indices (SI), defined as the ratio of IC₅₀ (HepG2) to IC₅₀ (P. falciparum 3D7), are reported as lower limits. All tested compounds displayed very high selectivity, with SI values exceeding 10³ for the most of compounds.

Table V. In vitro cytotoxicity screening of the novel compounds 35–47 against HepG2 and calculated selectivity indices

^aIC₅₀, the concentration of the tested compound that is necessary for 50 % growth inhibition; ^b the results are expressed as mean ± SD (n ≥ 2); ^c the precise IC₅₀ value couldn't be determined as the activity was only detectable at the highest concentration tested (20); ^d SI (selectivity index) = IC₅₀ (HepG2)/IC₅₀ (Pf3D7); ^e the precise Selectivity Index (SI) couldn't be obtained due to the inability to determine the exact IC₅₀ value.

Harmine, used as a reference, exhibited significantly weaker antiplasmodial activity (IC₅₀ = 8250 ± 2830 nmol L^–1), resulting in a markedly lower selectivity index (SI > 30). This finding highlights the importance of hybridization with the CQ derived 4-amino-7-chloroquinoline moiety for enhancing both potency and selectivity.

CONCLUSIONS

Overall, the obtained results demonstrate that the newly synthesized β-carboline and CQ hybrids combine outstanding antiplasmodial potency with low cytotoxicity toward human cells and favorable drug-like properties, highlighting compounds 36, 42, and 45 as the most promising candidates for further investigation with the aim of elucidating their mechanism of action.

Acronyms and abbreviations. – AT – amide-type, Boc – tert-butyloxycarbonyl, CQ – chloroquine, CuAAC – copper(I)-catalyzed azide-alkyne cycloaddition, DCM – dichloromethane, DMF – N,N-dimethylformamide, DMSO – dimethyl sulfoxide, ESI – electrospray ionization, FTIR-ATR – attenuated total reflectance Fourier transform infrared spectroscopy, HepG2 – human hepatocellular carcinoma cell line, HRP2 – histidine-rich protein 2, IC₅₀ – the concentration of the tested compound necessary for 50 % growth inhibition, MeOH – methanol, NMR – nuclear magnetic resonance, PfATP4 – P. falciparum P-type ATPase, a Na⁺ efflux pump essential for ionic homeostasis, Pf3D7 – chloroquine-sensitive strain of P. falciparum, PfDd2 – multidrug-resistant strain of P. falciparum, PfCARL – P. falciparum cyclic amine resistance locus, RI – resistance index, SI – selectivity index, T3P – propylphosphonic anhydride, TEA – triethylamine, TMS – tetramethylsilane, TT – triazole-type, TWC – total wavelength chromatogram.

Conflicts of interest. – The authors declare no conflict of interest

Funding. – This paper has been funded by the University of Zagreb (support for 2025) and the European Union (NextGenerationEU) under the National Recovery and Resilience Plan 2021 –2026 (NRRP), through the UNIZG Faculty of Pharmacy and Biochemistry institutional project “Synthesis and Biological Activity of Novel Hybrid Antimalarial Compounds Based on the β-Carboline Scaffold, Artemisinin, and Chloroquine” (NPOO25-FBF-6), approved by the Republic of Croatia Ministry of Science, Education and Youth (component C3.2, source 581). This work has been supported in part by project Strengthening the scientific research and innovation capacities of the Faculty of Pharmacy and Biochemistry, University of Zagreb (FarmInova; project number KK.01.1.1.02.0021), financed from the European Regional Development Fund, Operational Program Competitiveness and Cohesion for the period 2014–2020. The work of doctoral student A. Penava was fully supported by the Young researchers’ career development project – training of doctoral students of the Croatian Science Foundation, founded by the European Union through the European Social Fund.

Author’s contribution.– Conceptualisation, I.P. and Z.R.; methodology, A.P., L.P.d.C., J.H., and G.P.; writing, original draft preparation, I.P. and A.P.; writing, review and editing, I.P.; funding acquisition, Z.R. and I.P. All authors have read and agreed to the published version of the manuscript.

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