Introduction

Malaria is a major disease and public health concern. It is mainly prevalent in Africa, South-East Asia and the Eastern Mediterranean. Malaria is caused by Plasmodium parasite. The main transmission vector is the female Anopheles mosquito [1, 2]. Mosquitoes taking a blood meal on infected individuals ingest parasites into the midgut. Subsequently, the parasites move into the mosquito’s salivary glands and subsequently infect a human host [3,4,5,6]. Humans can be infected with one or more of the following five strains: P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi [4,5,6,7]. Currently, P. falciparum and P. vivax are the prevalent protozoan species causing high mortality, P. falciparum being the particular most virulent species [2]. The clinical symptoms of malaria are mainly caused by schizont rupture and destruction of erythrocytes. Major symptoms of malaria are fever, chills, headaches, and diaphoresis [8].

Currently, the commercially available antimalarial drugs are quinoline derivatives, artemisinin derivatives, antifolates and some antimicrobial agents [9]. Antimalarial agents principally aim to inhibit one or two stages in the life cycle of the parasite. Among these drugs, quinine and chloroquine are the oldest drugs and the first choice for treating all species of malaria [10]. An artemisinin-based combination therapy based regimen could be applied where chloroquine is ineffective in eradiacating malaria [10]. However, the emergence of drug resistance to available antimalarial drugs is a serious problem [11]. Two of the five human malaria parasite species, P. falciparum and P. Vivax, are frequently resistant. The incidence of chloroquine resistance of P. falciparum is a major problem for malarial control. Both artemisinin and its partner drugs are also threatened, especially in Africa [2]. On the other hand, P. malariae or P. ovale remain unknown for current drug resistance [4,5,6]. Genetic mutations or genetic malfunction on the major pathways of drug resistance, i.e., enzymes or transporters, are the main factors leading to drug resistance [12]. The examples of multidrug resistant strains of P. falciparum are Dd2, K1 and W2 [13]. The most common cause of chloroquine resistance is derived from mutation in the Pfmdr1 and Pfcrt genes in P. falciparum [14, 15].

To afford better malaria control, many researchers have sought new compounds or to modify the molecular structure of available current drugs such as chloroquine [10, 16]. The mechanism of chloroquine is inhibition of hemozoin formation. Normally, the parasites digest hemoglobin to release free heme which is toxic to the parasites themselves. However, parasites have a self-protective mechanism, crystallizing heme to hemozoin which is insoluble and harmless for the parasites [17, 18]. Chloroquine is able to prevent the formation of hemozoin. In particular, the current proposed mechanism is accumulation of chloroquine in acidic digestive vacuoles of intraerythrocytic trophozoites by a weak base mechanism [19]. At physiological pH, chloroquine can exist in three forms: unprotonated, monoprotonated, and diprotonated forms. Only uncharged chloroquine can permeate the erythrocyte membrane and finally reach the vacuole. In this vacuole, displaying acid condition chloroquine molecules become protonated and, since membranes are not permeable to charged species, the drug accumulates into the acidic digestive vacuole [20, 21]. Another mechanism of chloroquine, the inhibition of heme polymerase in the food vacuole of the malaria trophozoite, results in a lack of essential amino acids and leads to parasite death. There have been several studies on synthesis of chloroquine derivatives including their metal complexes to optimize the bioactivity of synthesized compounds [10]. The substituted chloroquine derivatives were reported as having interesting antimalarial activity. Azoles are an important class of heterocyclic compounds containing nitrogen with various biological activities [22, 23]. The linker of azoles with the core quinoline structure was known to increase its antimalarial activities [10]. A plausible reason for this was heme complexation through azoles and presented antimalarial activity [23].

The aim of this study is to find new compounds with antimalarial properties, particularly the ability to tackle the chloroquine resistant Plasmodium strains. Naturally, we maintained the core structures which preserve nitrogen atoms in the ring, similar to quinoline, but we decided to alter the position of nitrogen atoms probing the antimalarial activity of isoquinolines and attempt to synthesize quinolines containing a phenyl or triazole ring as shown in Fig. 1. We decorated the isoquinoline scaffold with two reactions, namely (i) the Suzuki–Miyaura reaction and (ii) the Sharpless–Fokin reaction to afford two series; isoquinoline-phenyl and isoquinoline-triazole (Fig. 1).

Fig. 1
figure 1

Concept of structural modification

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The modification of the two series is displayed in Fig. 2. All synthesized compounds were programmed to examine biological activity in two strains, the chloroquine-sensitive strain (3D7) and the chloroquine resistant strain (K1) Plasmodium falciparum. We determined the resistance index (Ri) as the ratio of the IC50 value of the resistant strain to that of the sensitive strain. The resistance indices of K1 and 3D7 were calculated and eventually assessed for any relationship between structures and activities of compounds.

Fig. 2
figure 2

Modified structures of the two drug series a isoquinoline-phenyl and b isoquinoline-triazole

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Results and discussion

Chemical synthesis

To generate new compounds, we rationalized the structure of the isoquinoline core structure and modified it in two series (Fig. 2), isoquinoline phenyl derivatives 1–13 and isoquinoline triazole derivatives 14–32. Isoquinoline phenyl derivatives were prepared using the Suzuki–Miyaura reaction (Scheme 1). This cross-coupling reactions were in which commercially available 4-bromoisoquinoline and different aryl boronic acid in the presence of palladium as a catalyst under basic conditions. For isoquinoline triazole derivatives, we proceeded to synthesize by using the click reaction (Scheme 2). The archetypical click chemistry is a two-component reaction in which an alkyne and an azide react in the presence of copper(I) catalyst to give rise to a 1,4-disubstituted 1,2,3-triazole. We therefore use different azide compounds bearing the desired triazoles. The azide compounds were prepared as described in Utsintong et al. [24].

Scheme 1
scheme 1

Synthesis of compounds 1–13 via the Suzuki–Miyaura reaction

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Scheme 2
scheme 2

Synthesis of compounds 14–32 via the click reaction

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The average percentage yields of isoquinoline phenyl derivatives, compounds 1–13 were more than 70 (Table 1). Compound 2 obtained the lowest percentage yield (20%) possibly due to the presence of an unprotected hydroxyl group. In the series of isoquinoline triazole derivatives, compounds 14–32, the 1,2,3-triazole ring was easily obtained via azide-alkyne cycloaddition in the presence of a small catalytic amount of copper (I) giving the desired products with percentage yields ranging from 19 to 88% (Table 2). All synthesized compounds were purified by column chromatography and characterization by FTIR and 1H-NMR. Full characterization was performed on the compounds most active against P. falciparum. The typical aromatic ring band at 3100–3000, 1600–1450 cm−1 in IR and signal at around δ9.20–8.00 ppm in 1H NMR spectra of all derivatives confirm the presence of isoquinoline ring. The presence of traizole ring in compounds 14–32 was confirmed by the observed 1H singlet at δ 8.3 ppm in 1H NMR spectra and the signal at around δ 133 ppm in 13C NMR. Compound 6 conatins m-OCH3 and is confirmed by signal at δ 3.85 ppm (3H, singlet) in 1H NMR along with signals at δ 55.3 ppm in 13C NMR. In the mass spectrum of compound 6 [M+H]+ peak was observed confirming the corresponding structure. The complete spectral data along with the assignments are provided in the Experimental section and partial spectra data are provided in the Supplementary section.

Table 1 Percentage yield and antimalarial activity of isoquinoline phenyl derivatives against chloroquine sensitive (3D7) and chloroquine resistant (K1) strains of P. falciparum and resistance index (Ri)
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Table 2 Percentage yield and antimalarial activity of isoquinoline triazole derivatives against 3D7 and K1 strains of P. falciparum and resistance index (Ri)
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Antimalarial activity of synthetic compounds

The IC50 values against P. falciparum of individual synthesized compounds and four standard antimalarial drugs are summarized in Tables 1–3. All synthesized compounds were active against the 3D7 strain of P. falciparum, with IC50 from 2.31 to 364.93 µM. The activity of all synthesized compounds against the K1 strain of P. falciparum with IC50 values from 1.91 to 1104.51 µM. Compound 6 was the most active of all 32 compounds with an IC50 value of 2.31 ± 0.33 µM and 1.91 ± 0.21 µM against 3D7 and K1, respectively. The less potent compounds were compound 16 with an IC50 value of 364.93 ± 16.79 µM against 3D7 and compound 24 with an IC50 value of 1104.51 ± 133.17 µM against K1.

Table 3 Antimalarial activity of four current drugs against 3D7 and K1 strains of P. falciparum and resistance index (Ri)
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Fidock and Kalra categorized antimalarial activity into three classes: potent activity if the IC50 is <1 µM, moderately potent if the IC50 is between 1 and 5 µM, and weak activity if the IC50 is >5 µM [25, 26]. Under this classification, only compound 6 was a moderate antimalarial agent against both strains, while compound 15 was as moderate antimalarial agent against the resistant strain.

In addition, the resistance indices (Ri) of K1 and 3D7 were calculated by finding the ratio between the IC50 of the resistant strain (K1) to that of the sensitive strain (3D7). A high index value represents the resistance of the tested compound in the sensitive strain. One-third of the thirty-two compounds were equipotent against both 3D7 and K1 strains. Compounds 12, 15, 16 and 25 displayed lower Ri values than reference standards (compound 12: Ri 0.35, 15: Ri 0.12, 16: Ri 0.26, 25: Ri 0.24) (Tables 1–3). Compound 15 was the most potent against K1 strains, with an IC50 of 4.55 ± 0.1 µM (Fig. 3).

Fig. 3
figure 3

Resistance index and chemical structures of some synthesized compounds

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Regarding the isoquinoline-phenyl series, the results displayed that the position of substituted groups on the phenyl ring connected to the isoquinoline was very important. Compared with compound 1, substitution with one donor group (–OCH3) in the meta position on phenyl ring as compound 6 resulted in a 10 and 25-fold increment in activity against P.falciparum 3D7 and K1 respectively. In contrast, the potency of analogues substituted with donor groups (–OCH3, –OH, –SH) or a withdrawing group (–SO2CH3) in the ortho/para position displayed a distinct reduction of activity. This substituition showed improper orientation resulting in activity decrement. In addition, disubstition of methoxy groups at meta orientation resulted in a 10–20-fold decrement in antiplasmodial activity. This study demonstrated the possible potential of modification on the phenyl ring in increasing antimalarial activity.

When considering the isoquinoline-triazole series without a methylene bridge between the 1,2,3-triazole and the phenyl ring, the change of substituted position from para- to meta- on the phenyl ring had a significant effect on antiplasmodial activity against both 3D7 and K1 strains as shown in the resistant index values (Table 2). The results show that the para-substituted chlorine in compound 15 was the preferred group on phenyl ring but meta-substitution in compound 16 resulted in a tenfold decrement in activity against both 3D7 and K1 strains. Furthermore, the isoquinoline-triazole series with one methylene bridge and substitution at p-position of the phenyl ring still maintained activity. Compound 22 with tert-butyl was the most active for antimalarial activity on both strains.

Finally, to understand the importance of the isoquioline ring, this ring was replaced with a naphthyl group. Compounds 27, 28 and 29 were compared in activity to similar compounds (22, 15 and 25). All modified derivatives demonstrated obviously decrease antimalarial activity in both strains. Furthermore, we attempted to mimic CQ structure by replacing the isoquinoline ring with a 7-chloroquinoline ring as presented in compound 30, 31 and 32. The results exhibited the effect of the 7-chloroquinoline ring in decreasing their corresponding activity in both strains.

Parasite growth and morphological change cells following exposure to compounds

The morphology change of 3D7 and K1 P. falciparum following exposure to selected compounds (compounds 6 and 15) at IC90 were determined. CQ and DHA were used as a positive control and evaluated at IC100. The experiment with no compound exposure was determined as a negative control. The effect of compounds on parasite growth was observed during a 60–72 h incubation period (Fig. 4). Compound 6 inhibited parasite growth and multiplication in both 3D7 and K1 strains since h12 at the ring stage. On the other hand, under negative control, parasites still developed to the trophozoite stage. Parasite shrinkage and death were observed after 12 h incubation. Compound 15 had markedly different antimalarial activity against 3D7 and K1 strains with higher specific activity against K1 than 3D7. In addition, compound 15 was able to delay parasite growth since h24, with early schizonts observed in the control sample, whereas the compound-exposed parasite remained at the trophozoite stage and gradually developed to schizonts at h60 and h72 in K1 and 3D7 respectively. Furthermore, compound 15 may have affected hemozoin formation because abnormal hemozoin crystals were found since h36 (Fig. 4, shown with arrows).

Fig. 4
figure 4

Morphology of Plasmodium falciparum after compound exposure 6. Two standard antimalarials, chloroquine (CQ) and dihydroartemisinin (DHA), were used as the positive control. Red arrows pointed to abnormal hemozoin crystals found in K1 after 36 h exposure

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The results displayed the effects of parasite exposure to compounds 6 and 15 for 60 h. Both compounds 6 and 15 could inhibit Plasmodium falciparum time-dependent growth compared to the negative control. However, compound 15 was less potent than CQ and DHA. Furthermore, compound 6 and CQ completely inhibited growth of the 3D7 strain while DHA completely inhibited parasite growth in the K1 strain at 6 h of testing exposure (data not presented in chart) (Fig. 5).

Fig. 5
figure 5

Percentage growth of Plasmodium falciparum after exposure to compound 6 at 212 µM concentration and 15 at 163 µM concentration for 60 h. Two standard antimalarial drugs, chloroquine (CQ) at 100 µM concentration and dihydroartemisinin (DHA) at 50 nM concentration were used as a positive control. The vehicle was used as a negative control (CTRL)

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Conclusion

In this study, the antimalarial activity of novel synthesized isoquinoline derivatives was evaluated. Compound 6 exhibited antiplasmodial activity compared to reference standard antimalarial drugs. Although compound 6 only displayed moderate antimalarial activity, it was active against both chloroquine sensitive and chloroquine resistant strains. In addition, compound 15 showed interesting selective activity against resistant strains over sensitive strains. Overall this work provides knowledge for further study about the discovery of antimalarial agents, which selectively work on resistant strains.

Experimental section

Chemistry

Unless otherwise noted, all reagents, chemicals and catalysts were purchased from commercial sources (Sigma-Aldrich; USA, Fluorochem; UK). Some solvents had to be dried by distillation under nitrogen and used as freshly prepared solvents. All other solvents were used as supplied without further purification.

Instrumentation

1H NMR and 13C NMR spectra were recorded at 500 MHz and 125 MHz, respectively (Bruker Biospin, USA). 1H NMR chemical shifts are referenced to TMS or CDCl3 (0; 7.26 ppm). 13C NMR was referenced to CDCl3 (77.0 ppm). Mass spectra and high-resolution mass spectra (HRMS) were measured using the electrospray ionization (ESI) technique (Bruker MicrOTOF, USA). Infrared spectra were performed by Fourier Transformed infrared spectrometer (Simadzu, Japan). Purification was carried out on column chromatography using silica gel 60 (Merck, 230–400 mesh). Chemical shifts are reported in part per million (ppm) and spectral data are represented in the following order: chemical shift; multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, td = triplet of doublets, m = multiplet); coupling constant (J, Hz); number of protons. Column chromatography was performed on silica gel Merck Kieselgel 70–230 mesh ASTM. Thin layer chromatography (TLC) was carried out on 5 × 20 cm plates with a layer thickness of 0.25 mm (Merck Silica gel 60 F254). Elemental Analysis (C, H, N) of the target compounds are within ±0.4% of the calculated values unless otherwise noted.

4-(3-methoxyphenyl)isoquinoline (6)

To a solution of commercially available 4-bromoisoquinoline (0.080 g, 0.38 mmol) and 3-methoxyphenylboronic acid (0.087 g, 0.57 mmol) in 4 ml of DMF, the tetrakis(triphenylphosphine) palladium (0.022 g, 0.019 mmol) and an aqueous solution of sodium carbonate (0.12 g 1.14 mmol) were added and stirred. The reaction was then warmed at 65 °C for overnight. The mixture was diluted with water and extracted with ethyl acetate (×3). The combined organic fractions were washed with brine (×1), dried over sodium sulfate and concentrated under vacuum. The crude product was purified by column chromatography by using hexane/ethyl acetate (8:2) as eluent, to give 0.065 g of a colorless liquid (73%); Rf = 0.59 (Hexane/EtOAc 6:4); yield 73%; IR (KBr) 3053, 2937, 1602, 1484, 1226, 1160, 886 cm−1; 1H-NMR (300 MHz, CDCl3, 25 °C) δ 9.23 (s, 1H, H-1), 8.48 (s, 1H, H-3), 8.02 (d, J = 5.7 Hz, 1H, H-8), 7.93 (d, J = 6.0 Hz, 1H, H-5), 7.63 (m, 2H, H-6, H-7), 7.42 (t, J = 5.7 Hz, 1H, H-5′), 7.09–6.99 (m, 3H, H-2′, H-4′, H-6′), 3.85 (s, 3H, O-CH3); 13C-NMR (75 MHz, CDCl3, 25 °C) δ 159.7 (C-3′), 152.0 (C-1), 142.7 (C-1′), 138.3 (C-3), 134.2 (C-6), 130.6 (C-4), 129.6 (C-5′), 128.4 (C-4a), 127.9 (C-8), 127.2 (C-7), 124.8 (C-8a), 122.6 (C-5), 115.8 (C-6′), 113.4 (C-4′), 108.4 (C-2′), 55.3 (O-CH3); HRMS (ESI) m/z calculated: C16H13NO: 235.0997 found: 236.1076 (M+H)+.

4-(4-(azepan-1-ylmethyl)phenyl)isoquinoline (12)

To a solution of commercially available 4-bromoisoquinoline (0.070 g, 0.34 mmol) and 4-(homopiperidine)methyl)phenylboronic acid (0.127 g, 0.40 mmol) in 4 ml of DMF, the tetrakis(triphenylphosphine) palladium (0.039 g, 0.017 mmol) and an aqueous solution of sodium carbonate (0.11 g 1.01 mmol) were added and stirred. The reaction was then warmed at 65 °C for overnight. The mixture was diluted with water and extracted with ethyl acetate (×3). The combined organic fractions were washed with brine (×1), dried over sodium sulfate and concentrated under vacuum. The crude product was purified by column chromatography by using hexane/ ethyl acetate (7:3) as eluent, to give 0.083 g of a yellow liquid (78%); Rf = 0.29 (Hexane/EtOAc 8:2); IR (KBr) 3090, 2986, 2822, 1622, 1587, 1397, 1234 cm−1; 1H-NMR (300 MHz, CDCl3, 25 °C) δ 9.26 (s, 1H, H-1), 8.51 (s, 1H, H-3), 8.01 (d, J = 7.9, 1H, H-8), 7.66 (m, 3H, H-5, H-6, H-7), 7.50 (m, 4H, H-2′, H-3′), 3.77 (s, 2H, -Ph-CH2-N), 2.72 (m, 4H, H-2″), 1.68 (m, 8H, CH2-CH2); 13C-NMR (75 MHz, CDCl3, 25 °C) δ 151.9 (C-1), 142.8 (C-4′), 134.2 (C-3), 132.2 (C-6), 132.0 (C-4), 130.9 (C-4a), 130.5 (C-3′, C-5′), 130.0 (C-7), 129.2 (C-2′, C-6′), 128.4 (C-8), 127.9 (C-8a), 127.2 (C-1′), 124.9 (C-5), 62.3 (CH2-N), 55.6 (C-1″), 27.8 (C-2″), 27.1 (C-3″); HRMS (ESI) m/z calculated: 316.1939 found: 317.2042 (M+H)+.

4-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)isoquinoline (15)

To a solution of 4-ethynylisoquinoline (0.05 g, 0.33 mmol) and 1-azido-4-chlorobenzene (0.05 g, 0.33 mmol) in a mixture of tert-butanol (1:1, 0.5 mL). Sodium ascorbate (6.5 mg, 0.033 mmol) and copper sulfate pentahydrate (0.8 mg, 0.0033 mmol) were added. The solution was stirred at room temperature for 48 h. The mixture was diluted with water and extracted with ethyl acetate (×3). The combined organic fractions were washed with brine (×1), dried over sodium sulfate and concentrated under vacuum. The crude product was purified by column chromatography by using hexane/ ethyl acetate (8:2) as eluent, to give 0.042 g of a slightly yellow solid (42%); Rf = 0.15 (Hexane/EtOAc 7:3); m.p. 240–243 °C; IR (KBr) 3082, 3044, 1503, 1383, 1092, 990, 777 cm−1; 1H-NMR (300 MHz, CDCl3, 25 °C) δ 9.32 (s, 1H, H-1), 8.80 (s, 1H, H-3), 8.62 (d, J = 8.5, 1H, H-8), 8.33 (S, 1H, H-5’), 8.09 (d, J = 8.1, 1H, H-5), 7.86–7.83 (m, 3H, H-6, H-2″, H-6″), 7.72 (m, 1H, H-7), 7.60 (m, 2H, H-3″, H-5″); 13C-NMR (75 MHz, CDCl3, 25 °C) δ 153.4 (C-1), 143.0 (C-1′), 135.4 (C-3), 134.9 (C-1″), 133.7 (C- 4″), 133.6 (C- 5′), 131.4 (C-4), 130.1 (C-6), 128.5 (C-4a), 128.1 (C-3″, C-5″), 127.7 (C-8), 124.4 (C-7), 121.8 (C-8a), 121.4 (C-2″, C-6″), 120.5 (C-5); HRMS (ESI) m/z calculated: 306.0672 found: 307.0750 (M+H)+.

4-(1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)isoquinoline (16)

To a solution of 4-ethynylisoquinoline (0.05 g, 0.33 mmol) and 1-azido-3-chlorobenzene (0.05 g, 0.33 mmol) in a mixture of tert-butanol (1:1, 0.5 mL). Sodium ascorbate (6.5 mg, 0.033 mmol) and copper sulfate pentahydrate (0.8 mg, 0.0033 mmol) were added. The solution was stirred at room temperature for overnight. The mixture was diluted with water and extracted with ethyl acetate (×3). The combined organic fractions were washed with brine (×1), dried over sodium sulfate and concentrated under vacuum. The crude product was purified by column chromatography by using hexane/ ethyl acetate (8:2) as eluent, to give 0.073 g of a yellow solid (73%); Rf = 0.09 (Hexane/EtOAc 8:2); m.p. 236–240 °C; IR (KBr) 3119, 1624, 1599, 1499, 1057, 798 cm−1; 1H-NMR (300 MHz, CDCl3, 25 °C) δ 9.32 (s, 1H, H, H-1), 8.81 (s, 1H, H-3), 8.61 (d, J = 8.6 Hz, 1H, H-8), 8.35 (s, 1H, H-5′), 8.10 (d, J = 8.1, 1H, H-5), 7.94 (m, 1H, H-2″), 7.86–7.79 (m, 2H, H-6, H-6″), 7.72 (m, 1H,H-7), 7.58 (t, J = 7.6, 1H, H-5″), 7.52 (m, 1H, H-4″); 13C-NMR (75 MHz, CDCl3, 25 °C) δ 153.4 (C-1), 145.4 (C-1′), 143.0 (C-3), 137.7 (C-1″), 135.8 (C-3″), 133.6 (C-5′), 131.4 (C-4), 131.0 (C-5″), 129.1 (C-6), 128.6 (C-4a), 128.1 (C-4″), 127.7 (C-8), 124.8 (C-7), 121.3 (C-6″), 120.9 (C-2″), 120.5 (C-8a), 118.6 (C-5); HRMS (ESI) m/z calculated: 306.0672 found:307.0749 (M+H)+.

3-(4-(isoquinolin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile (19)

To a solution of 4-ethynylisoquinoline (0.05 g, 0.33 mmol) and 3-azidobenzonitrile (0.047 g, 0.33 mmol) in a mixture of tert-butanol (1:1, 0.5 mL). Sodium ascorbate (6.5 mg, 0.033 mmol) and copper sulfate pentahydrate (0.8 mg, 0.0033 mmol) were added. The solution was stirred at room temperature for overnight. The mixture was diluted with water and extracted with ethyl acetate (×3). The combined organic fractions were washed with brine (×1), dried over sodium sulfate and concentrated under vacuum. The crude product was purified by column chromatography by using hexane/ethyl acetate (8:2) as eluent, to give 0.085 g of a white solid (88%); Rf = 0.17 (Hexane/EtOAc 7:3); m.p. 247–249 °C; IR (KBr) 3057, 2228, 1595, 1420, 1045, 798 cm−1; 1H-NMR (300 MHz, CDCl3, 25 °C) δ 9.34 (s, 1H, H-1), 8.82 (s, 1H, H-3), 8.59 (d, J = 8.5 Hz, 1H, H-8) 8.40 (s, 1H, H-5′), 8.24 (m, 1H, H-6″), 8.20 (m, 1H, H-4″), 8.11 (d, J = 8.1 Hz, 1H, H-5), 7.81–7.88 (m, 2H, H-6, H-2″), 7.79 (d, J = 7.8 Hz, 1H, H-5″) 7.74 (m, 1H, H-7); 13C-NMR (75 MHz, CDCl3, 25 °C) δ 153.5 (C-1), 145.8 (C-1′), 142.9 (C-3), 137.4 (C-6″), 133.2 (C-2″), 132.4 (C-4″), 131.6 (C-5′), 131.1 (C-6), 128.6 (C-4), 128.2 (C-5″), 127.8 (C-4a), 124.8 (C-1″), 124.6 (C-8), 123.7 (C-7), 121.3 (C-8a), 120.3 (C-5), 117.3 (C≡N), 114.3 (C-3″); MS (ESI) m/z calculated: 297.1014 found: 298.1089 (M+H)+.

4-(1-benzhydryl-1H-1,2,3-triazol-4-yl)isoquinoline (25)

To a solution of 4-ethynylisoquinoline (0.05 g, 0.33 mmol) and (azidomethylene)dibenzene (0.068 g, 0.33 mmol) in a mixture of tert-butanol (1:1, 0.5 mL). Sodium ascorbate (6.5 mg, 0.033 mmol) and copper sulfate pentahydrate (0.8 mg, 0.0033 mmol) were added. The solution was stirred at room temperature for overnight. The mixture was diluted with water and extracted with ethyl acetate (×3). The combined organic fractions were washed with brine (×1), dried over sodium sulfate and concentrated under vacuum. The crude product was purified by column chromatography by using hexane/ethyl acetate (7:3) as eluent, to give 0.046 g of a white solid (39%); Rf = 0.20 (Hexane/EtOAc 7:3); m.p. 139–143 °C; IR (KBr) 3088, 3030, 1495, 1227, 1057, 901, 750 cm−1; 1H-NMR (300 MHz, CDCl3, 25 °C) δ 9.25 (S, 1H, H-1), 8.68 (s, 1H, H-3), 8.62 (d, J = 8.5 Hz, 1H, H8), 8.04 (d, J = 8.0 Hz, 1H, H-5), 7.84–7.74 (m, 2H, H-6, H-5′), 7.68 (dd, J = 8.0, 1.1 Hz, 1H, H-7), 7.57–7.32 (m, 6H, H-3″, H-4″, H-5″), 7.57–7.32 (m, 5H, H-2″, H-6″, CH); 13C-NMR (75 MHz, CDCl3, 25 °C) δ 152.4 (C-1), 144.1 (C-1′), 141.9 (C-3), 138.0 (C-1″), 133.7 (C-6), 131.6 (C-5′), 129.1 (2, C-3″, C4a), 128.8 (C-4a), 128.1 (2, C-8, C-2″), 127.8 (C-7), 125.2 (C-4″), 122.7 (C-8a), 122.2 (C-5), 68.4 (CH); MS (ESI) m/z calculated: 362.1531 found: 363.1609 (M+H)+.

1-(4-(tert-butyl)benzyl)-4-(naphthalen-1-yl)-1H-1,2,3-triazole (27)

To a solution of 4-ethynylisoquinoline (0.02 g, 0.131 mmol) and 1-(azidomethyl)-4-(tert-butyl)benzene (0.025 g, 0.131 mmol) in a mixture of tert-butanol (1:1, 0.5 mL). Sodium ascorbate (2.6 mg, 0.013 mmol) and copper sulfate pentahydrate (0.3 mg, 0.0013 mmol) were added. The solution was stirred at room temperature for overnight. The mixture was diluted with water and extracted with ethyl acetate (×3). The combined organic fractions were washed with brine (×1), dried over sodium sulfate and concentrated under vacuum. The crude product was purified by column chromatography by using hexane/ethyl acetate (8:2) as eluent, to give 0.015 g of an amorphous white solid (33%); Rf = 0.33 (Hexane/EtOAc 8:2); m.p. 158–160 °C; IR (KBr) 3119, 2963, 1458, 1360, 1221, 1053, 980, cm−1; 1H-NMR (300 MHz, CDCl3, 25 °C) δ 8.36 (m, 1H, H-5), 7.85 (m, 2H, H-1, 8), 7.71–7.66 (m, 2H, H-5’, H-3), 7.54–7.47 (m, 3H, H-2, 6, 7), 7.41 (d, J = 8.0 Hz, 2H, H-3″, H-5″), 7.29 (d, J = 8.0 Hz, 2H, H-2″, H-6″), 5.58 (s, 2H, CH2), 1.30 (s, 9H, CH3); 13C-NMR (75 MHz, CDCl3, 25 °C) δ 152.0 (C-4″), 147.3 (C-1′), 133.9 (C-4), 131.6 (C-8a), 131.1 (C-1″), 128.9 (C-5a), 128.4 (C-5′), 128.0 (C-8), 127.2 (C-5), 126.6 (C-1), 126.1 (C-7), 126.0 (C-2″), 125.5 (C-6), 125.3 (C-2), 124.4 (C-3″), 122.4 (C-3), 54.0 (CH2), 34.7 (CH), 31.3 (CH3); MS (ESI) m/z calculated: 341.1892 found:364.1785 (M+Na)+.

1-(4-chlorophenyl)-4-(naphthalen-1-yl)-1H-1,2,3-triazole (28)

To a solution of 1-ethynylnaphthalene (0.02 g, 0.128 mmol) and 1-azido-4-chlorobenzene (0.02 g, 0.128 mmol) in a mixture of tert-butanol (1:1, 0.5 mL). Sodium ascorbate (2.5 mg, 0.013 mmol) and copper sulfate pentahydrate (0.3 mg, 0.0013 mmol) were added. The solution was stirred at room temperature for overnight. The mixture was diluted with water and extracted with ethyl acetate (×3). The combined organic fractions were washed with brine (×1), dried over sodium sulfate and concentrated under vacuum. The crude product was purified by column chromatography by using hexane/ethyl acetate (8:2) as eluent, to give 0.032 g of a yellow solid (83%); Rf = 0.40 (Hexane/EtOAc 8:2); m.p. 149–151 °C; IR (KBr) 3125, 3063, 1593, 1500, 1233, 1049, 829 cm−1; 1H-NMR (300 MHz, CDCl3, 25 °C) δ 8.40 (m, 1H, H-5), 8.21 (s, 1H, H-5′), 7.90 (m, 2H, H-1, H-8), 7.79 (m, 3H, H-3, H-2”), 7.55 (m, 4H, H-6, H-7, H-3″, H-5″); 13C-NMR (75 MHz, CDCl3, 25 °C) δ 147.8 (C-1′), 135.6 (C-4), 134.6 (C-1″), 133.9 (C-4″), 131.1 (C-8a), 130.0 (C-4a), 129.3 (C-5’), 128.6 (C-3″), 127.4 (C-5), 126.9 (C-1), 126.2 (C-8), 125.4 (C-7), 125.3 (C-6), 123.8 (C-2), 121.7 (C-3), 120.4 (C-2″); MS (ESI) m/z calculated: 305.0720 found: 328.0626 (M+Na)+.

1-benzhydryl-4-(naphthalen-1-yl)-1H-1,2,3-triazole (29)

To a solution of 1-ethynylnaphthalene (0.024 g, 0.154 mmol) and (azidomethylene)dibenzene (0.032 g, 0.154 mmol) in a mixture of tert-butanol (1:1, 0.5 mL). Sodium ascorbate (3.1 mg, 0.015 mmol) and copper sulfate pentahydrate (0.4 mg, 0.0015 mmol) were added. The solution was stirred at room temperature for overnight. The mixture was diluted with water and extracted with ethyl acetate (×3). The combined organic fractions were washed with brine (×1), dried over sodium sulfate and concentrated under vacuum. The crude product was purified by column chromatography by using hexane/ethyl acetate (9:1) as eluent, to give 0.017 g of a white solid (30%); Rf = 0.39 (Hexane/EtOAc 8:2); m.p. 161–163 °C; IR (KBr) 3088, 3057, 1599, 1498, 1450, 1051 cm−1; 1H-NMR (300 MHz, CDCl3, 25 °C) δ 8.37 (m, 1H, H-5), 7.86 (m, 2H, H-1, H-8), 7.68 (m, 2H, H-2, H-3), 7.48 (m, 3H, H-1′, H-6, H-7), 7.39 (m, 6H, H-3″, H-4″, H-5″), 7.21 (m, 5H, H-2″, CH); 13C-NMR (75 MHz, CDCl3, 25 °C) δ 146.7 (C-1′), 138.2 (C-4), 133.9 (C-1′), 130.1 (C-8a), 129.0 (C-3″), 128.9 (C-5′), 128.7 (C-4a), 128.5 (C-5), 128.2 (C-2′), 127.8 (C-8), 127.3 (C-1), 126.7 (C-6), 126.0 (C-7), 125.5 (C-4′), 125.3 (C2), 122.3 (C-3), 68.3 (CH); MS (ESI) m/z calculated: 361.1579 found: 384.1488 (M+Na)+.

4-(1-(4-(tert-butyl)benzyl)-1H-1,2,3-triazol-4-yl)-7-chloroquinoline (30)

To a solution of 7-chloro-4-ethynylquinoline (0.08 g, 0.49 mmol) and 1-(azidomethyl)-4-(tert-butyl)benzene (0.092 g, 0.49 mmol) in a mixture of tert-butanol (1:1, 0.5 mL). Sodium ascorbate (9.7 mg, 0.049 mmol) and copper sulfate pentahydrate (1.2 mg, 0.0049 mmol) were added. The solution was stirred at room temperature for overnight. The mixture was diluted with water and extracted with ethyl acetate (×3). The combined organic fractions were washed with brine (×1), dried over sodium sulfate and concentrated under vacuum. The crude product was purified by column chromatography by using hexane/ ethyl acetate (9:1) as eluent, to give 0.068 g of a colorless liquid; (39%); Rf = 0.17 (Hexane/EtOAc 9:1); IR (KBr) 3121, 2965, 1605, 1458, 1219, 1047, 991, 723 cm−1; 1H-NMR (300 MHz, CDCl3, 25 °C) δ 8.79 (d, J = 4.4 Hz, 1H, H-2), 8.55 (d, J = 9.1 Hz, 1H, H-5), 8.05 (s, 1H, H-8), 7.90 (s, 1H, H-5′), 7.46 (d, J = 4.4 Hz, 1H, H-6), 7.44–7.33 (m, 3H, H-3, H-3″, H-5″), 7.25 (d, J = 8.1 Hz, 2H, H-2″, H-6″), 5.57 (s, 2H, CH2), 1.26 (s, 9H, 3xCH3); 13C-NMR (75 MHz, CDCl3, 25 °C) δ 152.3 (C-2), 150.8 (C-4″), 149.1 (C-8a), 144.9 (C-1′), 136.3 (C-4), 135.6 (C-7), 131.1 (C-1″), 128.5 (C-5’), 128.1 (2) (C-5, C-8), 127.5 (C-6), 126.3 (C-2″), 123.9 (C-3″), 123.5 (C-4a), 120.3 (C-3), 54.2 (CH2), 34.7 (CH), 31.2 (CH3); MS (ESI) m/z calculated: 376.1455 found: 377.1537 (M+H)+.

7-chloro-4-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)quinoline (31)

To a solution of 7-chloro-4-ethynylquinoline (0.08 g, 0.49 mmol) and 1-azido-4-chlorobenzene (0.075 g, 0.49 mmol) in a mixture of tert-butanol (1:1, 0.5 mL). Sodium ascorbate (9.7 mg, 0.049 mmol) and copper sulfate pentahydrate (1.2 mg, 0.0049 mmol) were added. The solution was stirred at room temperature for overnight. The mixture was diluted with water and extracted with ethyl acetate (×3). The combined organic fractions were washed with brine (×1), dried over sodium sulfate and concentrated under vacuum. The crude product was purified by column chromatography by using hexane/ ethyl acetate (9:1) as eluent, to give 0.066 g of an orange solid (39%); Rf = 0.20 (Hexane/EtOAc 7:3); m.p. 161–164 °C;IR (KBr) 3125, 2957, 1593, 1499, 1391, 1230, 1047, 991, 827 cm−1; 1H-NMR (300 MHz, DMSO-d6, 25 °C) δ 9.61 (s, 1H, H-5″), 9.06 (d, J = 4.4 Hz, 1H, H-2), 8.90 (d, J = 9.1 Hz, 1H, H-5), 8.18 (s, 1H, H-8), 8.09 (d, J = 8.7 Hz, 2H, H-2″, H-6″), 7.94 (d, J = 4.4 Hz, 1H, H-6), 7.74 (m, 3H, H-3, H-3″, H-5″); 13C-NMR (75 MHz, CDCl3, DMSO-d6, 25 °C) δ 150.7 (C-2), 150.0 (C-1′), 149.9 (C-8a), 139.4 (C-4), 136.0 (C-7), 135.5 (C-1″), 134.7 (C-4″), 129.9 (C-5′), 128.9 (C-5), 128.3 (C-8), 127.8 (C-3″), 125.3 (C-6), 122.6 (C-2″), 121.7 (C-4a), 120.3 (C-3); MS (ESI) m/z calculated: 340.0283 found: 341.0363 (M+H)+.

4-(1-benzhydryl-1H-1,2,3-triazol-4-yl)-7-chloroquinoline (32)

To a solution of 7-chloro-4-ethynylquinoline (0.08 g, 0.49 mmol) and (azidomethylene)dibenzene (0.102 g, 0.49 mmol) in a mixture of tert-butanol (1:1, 0.5 mL). Sodium ascorbate (9.7 mg, 0.049 mmol) and copper sulfate pentahydrate (1.2 mg, 0.0049 mmol) were added. The solution was stirred at room temperature for overnight. The mixture was diluted with water and extracted with ethyl acetate (×3). The combined organic fractions were washed with brine (×1), dried over sodium sulfate and concentrated under vacuum. The crude product was purified by column chromatography by using hexane/ ethyl acetate (9:1) as eluent, to give 0.062 g of an yellow solid (32%); Rf = 0.20 (Hexane/EtOAc 7:3); m.p. 168–170 °C; IR (KBr) 3092, 3045, 2955, 1587, 1493, 1049, 878 cm−1; 1H-NMR (300 MHz, CDCl3, 25 °C) δ 8.88 (d, J = 4.3 Hz, 1H, H-2), 8.65 (d, J = 9.1 Hz, 1H, H-5), 8.13 (s, 1H, H-8), 7.83 (s, 1H, H-1′), 7.53 (m, 2H, H-3, H-6), 7.39 (m, 6H, 2 H-3″, 2 H-4″, 2 H-5″), 7.20 (m, 5H, CH, 2 H-2″, 2 H-6″); 13C-NMR (75 MHz, CDCl3, 25 °C) δ 149.9 (C-2), 148.2 (C-8a), 144.4 (C-1′), 137.6 (C-4), 137.2 (C-1″), 136.4 (C-7), 129.2 (C5′), 129.0 (C-3″), 128.7 (C-5), 128.1 (C-8), 127.9 (C-6), 127.8 (C-2″), 124.0 (C-4″), 123.8 (C-4a), 120.2 (C-3), 68.6 (CH); MS (ESI) m/z calculated: 396.1142 found: 397.1230 (M+H)+.

Biological assay

Assessment of in vitro antimalarial activity of synthetic compounds

All synthesized compounds were evaluated for their in vitro antiplasmodial activity against the P. falciparum chloroquine-sensitive (3D7) and chloroquine-resistant (K1) laboratory strains. The parasite was briefly cultured according to the method laid out in [27], in O+ human erythrocytes suspended in an RPMI culture medium supplemented with 10% human B serum, 25 mM HEPES and 15 mg/ml gentamicin at 37 °C in a 5% CO2, 5% O2, and 90% N2 atmosphere. The parasite was maintained at 5% parasitemia and subcultured every 48 h. Antimalarial activity of the synthetic compounds was assessed using SYBR Green I assay [28, 29]. The parasites were synchonized by 5% sorbitol to obtain the ring stage of P. falciparum. The highly synchronous ring stage parasite culture was prepared to a parasitemia of 2% and hematocrit of 1%. The tested compound was dissolved in DMSO (vehicle) and diluted for 50 times with the complete media to obtain the first working concentration containing 2% DMSO. The 50 μL of parasite mixture was inoculated into the 50 μL pre-dosed compounds in 96-well plates. The 100 µL final volume contained a parasite with a parasitemia of 1% and hematocrit of 0.5%, and maximum DMSO (vehicle) at 1% for the highest compound concentration. The parasite mixture was exposed to the following eight levels of concentration as testing compounds: 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 μM (at 37 °C in a 5% CO2, 5% O2, and 90% N2 atmosphere) for 48 h. The parasite DNA content was stained by SYBR green I diluted in lysis buffer, and fluorescence intensity was measured using a microplate reader at excitation and emission wavelengths of 485 nm and 530 nm, respectively. The experiment was repeated three times and triplicated for each experiment. The IC50 values (concentrations that inhibit the parasite growth by 50%) were calculated from a log-dose–response curve plotted using the CalcusynTM version 1.1 (BioSoft, Cambridge, UK) and used as indicators of antimalarial activity. Four standard antimalarial drugs (chloroquine, CQ; quinine, Q; mefloquine, MQ and dihydroartemisinin, DHA) were used as a positive control.

Observation of morphological change in cells following exposure to the selected synthetic compounds

Synchronized P. falciparum 3D7 and K1 strains were used in the experiment. The parasites were exposed to the separated selected synthetic compounds and two standard antimalarial drugs, chloroquine and DHA, which were used as a positive control at IC100. The treated parasites were incubated in a 5% CO2, 5% O2, and 90% N2 atmosphere at 37 °C for 72 h. Blood films were prepared at the following time points: 6, 12, 24, 36, 48, 60, 72 h and stained with Giemsa (Biotechnical Thai, Bangkok, Thailand). The morphology of the resulting parasites was observed and captured under a light microscope (×100, Leica Microsystems, Hesse, Germany) [30].