Discovery of fast-acting dual-stage antimalarial agents by profiling pyridylvinylquinoline chemical space via copper catalyzed azide-alkyne cycloadditions

Abstract

To identity fast-acting, multistage antimalarial agents, a series of pyridylvinylquinoline-triazole analogues have been synthesized via CuAAC. Most of the compounds display significant inhibitory effect on the drug-resistant malarial Dd2 strain at low submicromolar concentrations. Among the tested analogues, compound 60 is the most potent molecule with an EC₅₀ value of 0.04 ± 0.01 μM. Our current study indicates that compound 60 is a fast-acting antimalarial compound and it demonstrates stage specific action at the trophozoite phase in the P. falciparum asexual life cycle. In addition, compound 60 is active against both early and late stage P. falciparum gametocytes. From a mechanistic perspective, compound 60 shows good activity as an inhibitor of β-hematin formation. Collectively, our findings suggest that fast-acting agent 60 targets dual life stages of the malarial parasites and warrant further investigation of pyridylvinylquinoline hybrids as new antimalarials.

Graphical Abstract

1. Introduction

Malaria is a tropical parasitological disease, with estimated 228 million clinical cases worldwide and nearly 405,000 deaths in 2018, primarily in children under 5 years old.^[1] This disease arises from five species of parasitic protozoa of genus Plasmodium, among which P. falciparum is the deadliest species. Drug therapy has long been the mainstay for malaria management, either as chemo-prophylactic agents or in the treatment regimen. Unfortunately, current available drugs are quickly losing efficacy due to the rapid emergence of resistance. Even ACTs used worldwide as first-line treatments are showing an alarming spread of resistance.^[2] Therefore, this underscores an urgent need to develop new, safe and effective antimalarial leads, ideally possessing fast-acting and transmission-blocking properties.

The heme detoxification pathway of P. falciparum is an important target for antimalarials due to its essential role in parasite survival. CQ (1, Fig.1) and related 4-aminoquinolines such as AQ (2, Fig.1) and FQ (3, Fig.1) are known to intervene this pathway by restraining the formation of crystalline hemozoin in the DV.^{[3, 4]} Existing SAR study on 4-aminoquinolines reveals that the presence of electro-negative group at C7 position and diamino side chain at C4 position of quinoline core are prerequisite for the antiplasmodial activity.^{[5, 6]} Further optimization of the C4 substituent has brought potent antimalarial agents that are under preclinical and clinical development^{[7, 8]} Given the efficacy and safety profiles of 4-aminoquinolines, it is no surprise that this chemical scaffold is still a privileged structure for the development of new antimalarial drugs.

Fig.1.

Structures of 4-aminoquinoline antimalarials (CQ 1, AQ 2 and FQ 3).

Molecular hybridization as a drug discovery strategy involves the rational design of new chemical entities by covalent fusion of two or more existing pharmacophores.[7] To explore the 4-aminoquinoline chemical space, incorporation of 1,2,3-triazole pharmacophore to this scaffold or profiling other pharmacophore to it through a 1,2,3-triazole linkage seems particularly attractive. First, 1,2,3-triazole derivatives display a wide spectrum of pharmacological properties including anticancer,[9-12] antibacterial,[13-16] antitubercular,[17-19] and antimalarial activities,[20-24] possibly owing to their strong dipole moments that are capable of participating in hydrogen bonding, dipole-dipole attractions as well as non-covalent interactions.[23] The application of 1,2,3-triazole motif is exemplified by clinical drugs such as rufinamide (4, Fig.2), cefatrizine (5, Fig.2) and tazobactum (6, Fig.2). Second, the Lewis basic nature of the nitrogen atom could enable triazole compounds to accumulate in the acidic DV of malaria parasites,[25, 26] and a similar phenomenon is also observed in aminoquinoline family compounds. In the view of these, many 4-aminoquinolines based 1,2,3-triazole hybrids have been designed and developed as new antimalarial agents, as depicted in compounds 7,[27] 8,[28] 9,[29] 10,[30] 11,[26] 12,[31] 13,[32] 14,[33] 15,[34] and 16[35] (Fig.2). However, none of these triazole-linked quinoline derivatives have been reported to demonstrate transmission blocking potential, except compound 11 with moderate inhibitory activity against mature gametocytes (IC₅₀ = 8.50 μM). In addition, rare of them are disclosed as the fast-acting inhibitors.

Fig.2.

Selected examples of 1,2,3-triazole scaffold in commercial drugs and antimalarial molecules.

Styrylquinolines are known to possess diverse biological activity including anticancer,^[36] anti-HIV,^[37] and anti-leishmanicidal activity.^[38] In 2017, our groups reported that nitrostyrylquinoline derivative UCF501 (17, Fig.3), a fast-acting parasitocidal agent, exhibited potent antimalarial activity and good selectivity.^[39] A more recent study has demonstrated that quinolines bearing a 4-pyridylvinyl group, a scaffold that possesses good biological activity,^[40-42] display overall better inhibitory effects on CQR Dd2 strain of P. falciparum as compared to the nitrostyrylquinoline counterparts.^[43] In light of the good antiplasmodial potential of 4-aminoquinoline and 1,2,3-triazole derivatives as mentioned above and in conjugation with the potentially improved pKa values (drug accumulation) and solubility of 4-pyridylvinylquinolines relative to styrylquinolines, we envision that incorporation of 1,2,3-triazole moiety and other biologically active functionalities into 4-pyridylvinylquinoline scaffold will lead to new hybrid molecules that are endowed with desirable antimalarial properties.

Fig.3.

Chemical structure of styrylquinoline UCF501 (17).

Herein, as a continuation of our research on the development of quinoline-based antimalarial agents, we report the synthesis of a series of 4-pyridylvinylquinoline-triazole conjugates via CuAAC and the evaluation of their in vitro antiplasmodial activity and cytotoxicity. Additionally, we describe the preliminary SAR, physicochemical properties, microsomal stability, and the in-silico pharmacokinetics of these compounds. Furthermore, the stage specific action and speed-of-action profiles along with mechanistic study of the most active compound 60 are discussed.

2. Results and discussion

2.1. Chemistry

Given the importance of the basicity for antiplasmodial activity (possibly assist in compound accumulation via pH trapping^[44, ^45] and in the light of piperidine, pyrrolidine, morpholine and piperazine as important nitrogen-containing heterocycles embodied in a large number of pharmaceutical compounds (e.g. antimalarial drugs quinine and mefloquine), pyridylvinylquinolines 26-31 were devised. In addition, phthalimide as a privileged motif has been shown potentials for the discovery of antimalarial agents,^{[46, 47]} and thus pyridylvinylquinoline 32 bearing phthalimide moiety was designed and evaluated the in vitro antiparasitic activity. The general route for the preparation of target compounds 26-32 is outlined in Scheme 1. Quinoline 18, prepared in two steps from commercially available 4-chloroaniline, was reacted with sodium azide to provide azide 19, which was subjected to CuAAC to generate triazoles 20-25 using the method as described by Yan et al.^[48] Subsequent treatment of 20-25 with isonicotinaldehyde via condensation reaction in the presence of p-TsNH₂^[49] afforded pyridylvinylquinolines 26-31. Because only trace amount of triazole 32 was obtained by this route, an alternative synthetic sequence was adopted to prepare pyridylvinylquinoline 32. First, condensation reaction between methylquinoline 18 and isonicotinaldehyde was carried out under microwave irradiation condition^[43] or using p-TsNH₂ as catalyst at 130 °C for 4 h to afford pyridylvinylquinoline 33. Second, intermediate 33 was transformed to azide 34 via nucleophile substitution. Then, under the standard reaction conditions, triazole 32 was obtained by CuAAC.

Scheme 1.

Reagents and conditions: a) NaN₃, DMF, 80 °C , 12 h, 84%; b) propargyl bromide (80% w/v in toluene), THF-H₂O, Et₃N, amines, rt, 1 h; then azide 19, CuI, 70 °C, 2 h, 27-47%; c) isonicotinaldehyde, p-TsNH₂, xylene, 130 °C, 12 h, 50-86%; d) isonicotinaldehyde, TfNH₂, DMF, 140 °C, 20 min, MW, 46% or isonicotinaldehyde, p-TsNH₂, xylene, 130 °C, 4 h, 42%; e) CuI, alkyne 49, THF-H₂O,70 °C, 2 h, 81%.

The designed compounds 51-65 with different linker lengths were synthesized as depicted in Scheme 2. Chloroquinoline 18 was reacted with amino alcohols at 130 °C for 48 h to give aminoquinolines 35-36, which underwent olefination to afford pyridylvinylquinolines 37-38. Subsequent azidonation of alcohols 37-38 was accomplished in two steps, namely methanesulphonylation and nucleophilic substitution. Finally, the target compounds 51-65 were prepared by CuAAC between azides 41-42 and alkynes 43-50.

Scheme 2.

Reagents and conditions: a) 2-aminoethanol (for 35) or 4-aminobutanol (for 36), EtOH, 130 °C, 48 h, 93-97%; b) isonicotinaldehyde, p-TsNH₂, xylene, 130 °C, 6 h, 89-92%; c) MsCl, Et₃N, THF, 0 °C, 45 min, 57-64%; d) NaN3, DMF, 95 °C, overnight; e) CuI, alkynes 43-50, THF-H₂O,70 °C, 2 h, 55-86%.

As illustrated in Scheme 3, the synthesis of 69-70 was commenced from commercially available 2,4-thiazolidinedione 66, which was converted to 67 via substitution, followed by a Knoevenagel-like condensation to furnish alkyne 68.^[50] Then, pyridylvinylquinoline-triazoles 69-70 were prepared through CuAAC between azides 41-42 and precursor 68.

Scheme 3.

Reagents and conditions: a) propargyl bromide (80% w/v in toluene), K₂CO₃, acetone, reflux, 4 h, 89%; b) benzaldehyde, piperidine, HOAc, MeOH, reflux, 3 h, 36%; c) CuI, azides 41-42, THF-H₂O, 70 °C, 2 h, 66-68%.

2.2. Biology

2.2.1. Antiplasmodial activity of pyridylvinylquinoline-triazoles

The in vitro inhibitory activities of all synthesized compounds were determined against CQR Dd2 strain of P. falciparum by SYBR Green I-based fluorescent assay.^[39] CQ was used as a positive control. As shown in Table 1, when the triazole ring was directly linked to C4 position of pyridylvinylquinoline, incorporation of pyrrolidine, piperidine, 2-(piperazin-1-yl)ethanol and diethylamine functionality at the triazole ring resulted in good activity with EC₅₀ values in low micromolar range, as demonstrated by compounds 26, 27, 30 and 31, respectively. However, hybrids containing other groups such as morpholine, N-methylpiperazine and isoindoline-1,3-dione exhibited diminished activity against Dd2 strain (28-29 and 32, EC₅₀ > 8 μM).

Table 1.

In vitro antiplasmodial activity and cytotoxicity of pyridylvinylquinoline-triazoles

Compd	n	R	Antiplasmodial activity EC50(μM)a			Cytotoxicity EC50 (μM)a		LogP
			Dd2	3D7	RIb	HepG2	SIC
26	-		1.65 ± 0.20	nt	-	nt	-	3.82
27	-		1.24 ± 0.15	nt	-	nt	-	4.38
28	-		> 10	nt	-	nt	-	3.23
29	-		> 10	nt	-	nt	-	3.01
30	-		1.66 ± 0.08	nt	-	nt	-	2.46
31	-		1.32 ± 0.05	nt	-	nt	-	4.15
32	-		8.79 ± 0.82	nt	-	nt	-	3.98
51	1		2.96 ± 0.10	nt	-	nt	-	4.39
52	1		0.55 ± 0.03	0.47 ± 0.05	1.17	13.48 ± 3.84	24.51	4.78
53	1		1.85 ± 0.06	nt	-	nt	-	3.64
54	1		> 10	nt	-	nt	-	3.62
55	1		4.27 ± 0.15	nt	-	nt	-	3.05
56	1		1.32 ± 0.05	nt	-	nt	-	4.72
57	1		1.07 ± 0.18	nt	-	nt	-	4.52
58	1		1.18 ± 0.12	nt	-	nt	-	4.26
59	2		0.69 ± 0.06	0.52 ± 0.10	1.32	> 25	> 36.23	5.11
60	2		0.04 ± 0.01	0.07 ± 0.01	0.57	5.49 ± 0.98	137.25	5.45
61	2		0.17 ± 0.01	0.23 ± 0.02	0.74	> 25	147.06	4.21
62	2		0.16 ± 0.02	0.16 ± 0.03	1.00	15.83 ± 0.38	98.94	4.33
63	2		0.67 ± 0.06	0.64 ± 0.13	1.05	> 25	37.31	3.73
64	2		1.00 ± 0.18	nt	-	nt	-	4.93
65	2		0.35 ± 0.03	0.28 ± 0.02	1.25	21.24 ± 2.67	60.69	4.75
69	1		0.24 ± 0.01	0.44 ± 0.04	0.55	5.73 ± 0.27	23.88	5.13
70	2		0.60 ± 0.12	0.33 ± 0.01	1.82	> 25	41.67	5.77
CQ	-	-	0.17 ± 0.02	0.02 ± 0.01	8.50	10.43 ± 0.86	61.35	-

^aEC₅₀ values were calculated from at least three independent experiments.

^bRI = [EC₅₀ (Dd2) / EC₅₀ (3D7)].

^cSI = [EC₅₀ (HepG2) / EC₅₀ (Dd2)]. nt: not tested.

Optimization of the spacing parameter between pyridylvinylquinoline and triazole was accomplished by evaluating the antiplasmodial activity of compounds 51-58. Except compounds 54 and 55, these compounds showed comparable or better activity than the counterparts with no spacer. For instance, compound 53 (EC₅₀ = 1.85 ± 0.06 μM) showed above 5-fold improved activity over analogue 28, and compound 57 (EC₅₀ = 1.07 ± 0.18 μM was almost 8-fold more potent than compound 32. In this series, compound 52 was the most potent analogue with an EC₅₀ value of 0.55 ± 0.03 μM, which was 2-fold more potent than the counterpart 27 (EC₅₀ = 1.24 ± 0.15 μM). On basis of these results, we hypothesize that longer methylene linker might be beneficial for improving antiparasitic activity. To test this hypothesis, the tetramethylene linker was introduced between pyridylvinylquinoline core and triazole ring. We were delighted to find out that all these compounds (n = 2) exhibited significantly increased antiplasmodial activity as compared to their counterparts (n =1) with EC₅₀ values in the nanomolar ranges, except for compound 64. For instance, compound 61 showed approximately 11-fold improvement of antimalarial activity relative to compound 53, and compound 62 (EC₅₀ = 0.16 ± 0.02 μM) also displayed much stronger inhibitory activity compared to 54 (EC₅₀ > 10 μM). It is worth noting that compound 60 was the most active analogue against Dd2 strain with an EC₅₀ value of 0.04 ± 0.01 μM, which was 4-fold more potent than the positive control, CQ. Within the scope of our screening, it appears that the tetramethylene linker between the pyridylvinylquinoline and triazole moieties is optimal for antimalarial potency of the hybrids.

Considering that 2,4-thiazolidinedione and related heterocycles such as thiazolidinedione-triazoles and 5-benzylidene thiazolidinediones (5-benzylidene fragment conjugation with the carbonyl group at C4 position of thiazolidinedione scaffold makes such compounds reactive owing to possible Michael addition of nucleophilic protein residues to the exocyclic double bond) have been extensively used for the development of antidiabetic,^{[51, 52]} anticancer,^{[50, 53]} anti-inflammatory,^[54] and especially for antimalarial compounds,^[55-57] we turned our attention to the synthesis and antiplasmodial evaluation of hybrid molecules 69 and 70. Indeed, compound 69 showed potent activity against Dd2 strain (EC₅₀ = 0.24 ± 0.01 μM). Subsequent replacement of the ethylene linker in compound 69 with tetramethylene linker led to a less potent analogue 70 (EC₅₀ = 0.60 ± 0.12 μM).

Selected pyridylvinylquinoline-triazole conjugates with good antiparasitic potency (EC₅₀ < 1.0 μM) against CQR Dd2 strain were also evaluated for the in vitro inhibitory activity toward CQS 3D7 strain (Table 1). All compounds exhibited submicromolar activity toward 3D7 strain, with EC₅₀ values ranging from 0.07 ± 0.01 to 0.64 ± 0.13 μM. Another noteworthy observation was that most compounds showed similar or even better inhibitory activity toward CQR strain than that toward CQS strain (RI ≤ 1), e.g. compounds 60 (RI = 0.57), 61 (RI = 0.74) and 69 (RI = 0.55), suggesting no cross-resistance induced by pyridylvinylquinoline-triazole hybrids.

The lipophilicity of the synthesized compounds, measured by LogP, was estimated by using ALOGPS 2.1 software.^{[58, 59]} As illustrated in Table 1, the increased linker length led to the increase in LogP estimation for each group of pyridylvinylquinoline hybrids, and in some of these groups, the increased lipophilicity (linker length from 0 or 2 to 4) was correlated to the improved antiplasmodial activity, e.g. R = piperidine, morpholine and pyrazole. As for 5-benzylidene thiazolidinedione group, the increase in calculated LogP values correlated with the improved inhibitory potency against CQS 3D7 stain, whereas the opposite trend was observed for CQR Dd2 stain.

2.2.2. Cytotoxicity profiles of selected pyridylvinylquinoline-triazoles

Cytotoxicity of selected compounds were evaluated against HepG2 cells. As presented in Table 1, the cytotoxicity trend of compounds tested appeared to correlate with their antiplasmodial activity profiles, with the exception of compound 69. Additionally, we found that the majority of compounds show low cytotoxicity (EC₅₀ > 10 μM) and the selectivity indices of two most potent compounds 60 and 61 are greater than 100.

2.2.3. Physicochemical properties and in vitro microsomal stability study

A subset of compounds was evaluated to determine their physicochemical properties (Table 2). Tested compounds were typically selected based on having demonstrated good potency in the antiplasmodial activity assay. Predicted lipophilicity of all compounds was relatively high (LogP > 3.5). In addition, we observed that compounds 69 and 70 containing thiazolidinedione moiety showed very poor aqueous solubility. However, replacement of the thiazolidinedione motif by a pyrazole moiety or piperidine, morpholine and piperazine groups led to good aqueous solubility without compromising the antimalarial potency as seen with hybrids 60-63. It should be noted that the aqueous solubility of compound 60 with tetramethylene linker was approximately 1.6-fold better than 52 carrying dimethylene linker, although the lipophilicity of 60 was higher than that of 52. Meanwhile, another noteworthy observation was that compound 70 showed slightly improved solubility over compound 69. In addition, compound 60 exhibited better solubility than compound 63, albeit with higher lipophilicity (LogP = 5.45) relative to that of compound 63 (LogP = 3.73). These results could indicate that lipophilicity data of the compounds expressed by their LogP values, are not a good predictor for kinetic aqueous solubility in our case. On the other hand, all these compounds exhibited low permeability (Pe < 1.50 ×10⁻⁶ cm/s) by PAMPA at pH 7.4. Among them, compounds 60 and 63 presented relatively higher Pe.

Table 2.

Physicochemical properties and in vitro microsomal stability of selected pyridylvinylquinoline-triazoles

Compd	LogP	Solubility (μM)a pH= 7.4	PAMPAa pH= 7.4 (Pe × 10−6nm/s)	CLint (μL/min/mg)	t1/2 (min)
52	4.78	67.16	<0.15	nt	nt
60	5.45	106.06	0.68	116.86	11.86
61	4.21	113.42	< 0.08	173.90	7.97
62	4.33	94.96	0.34	135.75	10.21
63	3.73	85.41	0.75	nt	nt
65	4.75	31.96	c	150.49	9.21
69	5.13	< 0.31	c	116.67	11.88
70	5.77	1.98	c	nt	nt

^aKinetic aqueous solubility using chemiluminescent nitrogen detection.

^bPAMPA using chemiluminescent nitrogen detection.

^cCompound was not determined in the acceptor compartment (limit of quantitation, LOQ = 0.5 μM) and precipitated in donor compartment. nt: not tested

Five most active compounds were then assessed their propensity to be metabolized using in vitro mouse liver microsome assay. Table 2 showed the intrinsic clearance (CLint) and half-life (t_1/2) values for compounds 60-62, 65 and 69. In the microsomal stability experiments, extensive degradation of these compound was observed after 15 minutes, with less than 50% of the parent compound remaining. Among them, the most potent compound 60 (EC₅₀ = 0.04 ± 0.01 μM) bearing piperidine moiety had a half-life time comparable to that of compound 69 containing 2,4-thiazolidinedione moiety. However, morpholine, piperazine and pyrazole analogues turned out to be less stable upon enzyme degradation. On the basis of these analysis, 60 was chosen for further biological investigation, primarily due to its strong potency, high SI value, good solubility and relatively higher Pe.

2.2.4. Effects of compound 60 on P. falciparum asexual life cycle

To gain mechanistic insights of pyridylvinylquinoline-triazole hybrids in P. falciparum, we evaluated the stage-specificity of the most promising compound 60 by studying its effects on intraerythrocytic development of the malaria parasites.^{[39, 60]} The tightly synchronized P. falciparum Dd2 cultures were treated with 5 × EC₅₀ concentration of 60 at 6 (ring stage), 18 (trophozoite stage), 30 (late trophozoite/early schizont stage) and 42 (late schizont/segmenters) HPI of the merozoites into erythrocytes and subsequently monitored the parasite cell cycle progress at different post invasion time points (12 h intervals). As can be seen from Fig. 4A, the control culture matured through the developmental cycle, transitioning from ring to trophozoite to schizont to segmenter, and ring again. Exposure of the culture to 60 at 6, 18, and 30 HPI prevented the parasite progression to the next stage of the cell cycle. In contrast, the addition of the compound 60 at a later stage (42 h) did not affect the schizont maturation and reinvasion of new merozoites (Fig. 4A). The absence of morphological changes in the first 12 h post compound exposure when added at the ring stage (6 h), suggested that 60 acts more at the trophozoite than the ring stage. In addition to the morphological changes, the parasite DNA content (as indicative of cell cycle progression and number of parasites) was analyzed in YOYO-1-stained parasites using flow cytometric analysis. Histograms in Fig. 4B showed the inhibition in the cell cycle progression in parasites when exposed to 60. Parasites treated at 6, 18, and 30 HPI had a significant lower DNA content compared to the untreated control at the end of the incubation period. However, the content was slightly lower than the control when the parasites were exposed to 60 at 42 HPI, corroborating the less effectiveness of 60 on the growth of the parasite at the schizont stage.

Fig.4.

Stage-specific inhibition of P. falciparum growth by compound 60. Tightly synchronized Dd2 culture was treated at 6, 18, 30 and 42 h post-invasion (HPI) with compound 60 at 5 × EC₅₀ concentration. (A) Microscopic evaluation of Giemsa-stained thin smears (B) Histogram plot of YOYO-1 labeled cells from flow cytometric analysis. Results shown are representative of three independent biological replicates.

Large vacuoles were noted in parasites exposed to 60 (Fig. 4A). These enlarged food vacuoles were possibly due to an accumulation of undegraded hemoglobin and had been observed previously in parasite treated with inhibitors targeting protease in the parasite food vacuole.^[61] The presence of this phenotype and the fact that compound acts on the trophozoite (when most hemoglobin degradation occurs), suggested that the hemoglobin digestion could be a potential cellular target of 60.

2.2.5. Identification of compound 60 as a fast-acting parasitocidal agent

To investigate whether pyridylvinylquinoline-triazole hybrids exhibited their antiplasmodial effect through parasitocidal or parasitostatic mechanism, we used 60 to conduct kill kinetic experiments. The P. falciparum Dd2 strain was cultured in the presence of compound 60 at a 5 × EC₅₀ concentration for 6, 12, 24, 48 h followed by removing the compound and continued monitoring the growth of parasites.^[39] The untreated parasites and standard antimalarials DHA and atovaquone were used as control references. As shown in Fig.5A-B, parasitemia decreased markedly when the cultures were exposed to 60 for 6 h and 12 h as compared to the slow-acting antimalarial atovaquone, although a slight increase of parasitemia was observed at 96 h after removal of the inhibitor 60. Remarkably, longer exposure to 60 for 24 and 48 h caused a complete loss of parasite viability, and its inhibitory activity seemed to be comparable to DHA (Fig.5C-D). Therefore, the results indicate that 60 is a good fast-acting parasitocidal agent.

Fig.5.

Compound 60 is a fast-acting parasitocidal agent. Asynchronous Dd2 parasite cultures were exposed to 5 × EC₅₀ concentrations of test compounds for (A) 6 h, (B) 12 h, (C) 24 h, and (D) 48 h followed by washed with RPMI, resuspended in culture media, and monitored for parasite growth daily for 96 h. Parasitemia was determined by microscopy of Giemsa stained smear.

2.2.6. Effect of compound 60 on P. falciparum at the gametocyte stage

Only a handful compounds are effective against P. falciparum gametocytes, especially for the late stage (IV-V) gametocytes.^[62] The challenge lies in that IV-V gametocytes are more resistant to antimalarial drugs relative to early stage (I-III) gametocytes and asexual stage parasites. Currently, primaquine is the only approved antimalarial drug with good activity toward P. falciparum IV-V gametocytes, but with significant adverse effects such as causing acute haemolysis in patients with glucose-6-phosphate dehydrogenase deficiency. Therefore, we explored whether 60 possessed any inhibitory activity against early or late stage gametocytes. As shown in Fig. 6A, compound 60 exhibited promising activity towards early stage (I-III) gametocytes with an EC₅₀ value of 0.95 ± 0.08 μM and notably against late stage (IV-V) gametocytes (EC₅₀ = 0.89 ± 0.09 μM, Fig. 6B). These results confirm that 60 is a promising lead with good dual-stage (blood and gametocyte) activity.

Fig.6.

Activity of compound 60 against gametocyte stages. The viability of gametocytes after the treatment of compound 60 was determined on early (A) and late (B) gametocytes stages of 3D7 expressing luciferase. Results showed are representative of three independent biological replicates.

2.2.7. Inhibition of β-hematin formation

CQ and other 4-aminoquinoline analogs are known to exhibit their antimalarial activity by inhibiting the formation of crystalline hemozoin in the DV of the parasite. To determine whether this is a potential mechanism for pyridylvinylquinoline hybrids, we tested the ability of the most active compound 60 to inhibit β-hematin formation. As seen in Fig.7A-B, compound 60 demonstrated a concentration-dependent inhibition of β-hematin, albeit with lower activity than that of CQ. Therefore, the potent antimalarial activity of hybrid 60 is at least partially attributed to its inhibition of β-hematin formation.

Fig.7.

Effect of compound 60 on the β-hematin crystal formation. (A) Images of β-hematin crystals after incubation of 100 μM hemin, propionate buffer, phosphatidylcholine, and various concentrations of 60 for 16 h at 37 °C. Images were taken using a Nikon Eclipse TE200 optical microscope. (B) Free hemin, as indicative of β-hematin crystal formation, was determined using a linear calibration curve. Data are mean ± SEM.

2.2.8. In-silico pharmacokinetic properties

The pharmacokinetics parameters of compounds 52, 57, 60-65 and 69-70 (EC₅₀ ≤ 1.0 μM) were predicted using PreADMET server (https://preadmet.bmdrc.kr/), and the data generated were listed in Table S1. All the 11 compounds were predicted to demonstrate low permeability for in vitro MDCK cells and moderate permeability for in vitro Caco-2 cells. The permeability of the potent compounds (52 and 60-63) was measured by PAMPA assay, which was similar with that towards MDCK cells. For an orally active drug, it should have good gastrointestinal absorption. All of them were estimated to have high human intestinal absorption (> 95%), albeit with predicated low to moderate permeability. However, human fraction absorption could be less than 80% on the basis of Pe values determined (< 1.5 × 10⁻⁶ nm/s). In addition, it should be noted that these compounds could undergo extensive first pass metabolism based upon our in vitro microsomal stability data obtained and thus it is not clear whether the metabolism components were factored into the predicted human intestinal absorption. Plasma protein binding is another important factor in determining drug disposition and efficacy. Generally speaking, plasma protein binding reduces the free drug concentration, and thus it has a significant influence on the drug-target interactions. Under this prediction, compounds 57, 58, 65, 69 and 70 were found to bind strongly to plasma proteins, while compounds 52, 59-63 were shown to bind weakly to plasma proteins. P-gp is a transmembrane, ATP-dependent drug efflux pump that extrudes chemotherapeutic drugs out of cells. Therefore, inhibition of P-gp can potentially increase intracellular drug concentrations and overcome drug resistance. Interestingly, all these compounds were estimated to be P-gp inhibitors.

2.2.9. Toxicity prediction

To determine a possible toxic effect of newly synthesized compounds, we used ProTox-II,^[63] a webserver for the prediction of small molecules oral toxicity. The LD₅₀, the median lethal dose, is one of the most important parameters for toxicity expression, meaning the dose level at which 50% of animal tested die upon exposure to a target molecule. The LD₅₀ of pyridylvinylquinoline-triazole hybrids in this work were predicted to be above 500 mg/kg and less than 1600 mg/kg (Table S2). Notably, all these compounds showed at least 3-fold higher LD₅₀ than CQ with the exception of compound 57. Additionally, all of them came under the toxicity category of Class IV. Thus, this toxicity prediction study indicated that pyridylvinylquinoline-triazole hybrids can act as the lead molecules that deserved further investigation.

3. Conclusion

In conclusion, a series of pyridylvinylquinoline-triazole hybrids have been synthesized via CuAAC and their antimalarial activities were evaluated against CQR Dd2 strain. Our SAR analysis reveals that the length of alkyl linker between pyridylvinylquinoline scaffold and triazole motif have significant impacts on the antimalarial potency. Some of the newly synthesized compounds exhibit excellent antimalarial activity in the low submicromolar concentration in both CQR and CQS strains, among which the most active analogue 60 has an EC₅₀ value of 40 nM. Besides, compound 60 demonstrated good in-silico pharmacokinetic properties and aqueous solubility (106.06 μM at pH = 7.4). It is to be noted that compound 60 demonstrates promising activity against both early and late stage gametocytes, and it is a fast-acting agent that blocks the trophozoite stage of P. falciparum asexual life cycle. Further mechanic studies indicate that pyridylvinylquinoline-triazole 60 shows good inhibitory activity towards β-hematin formation. Altogether, compound 60 holds the potential as a good lead compound for further development as a partner drug in artemisinin combination therapy.

4. Experimental

4.1. Chemistry

Reagents and solvents were purchased from commercial vendors and used without further purification unless otherwise noted. Analytical TLC was performed with Silicycle silica gel 60 F254 plates. Melting points were determined by open capillary using a Stuart SMP20 melting point apparatus and were uncorrected. ¹H NMR and ¹³C NMR spectra were performed using a Bruker AV-400 spectrometer (¹H NMR at 400 MHz and ¹³C NMR at 100 MHz). High resolution mass was taken on an Agilent 6230 TOF LCMS instrument.

4.1.1. 4-Azido-6-chloro-2-methylquinoline (19)

A mixture of 4,6-dichloro-2-methylquinoline (18) (840 mg, 3.96 mmol) and NaN₃ (800 mg, 12.3 mmol) in DMF (8.0 mL) was stirred at 80 °C under a nitrogen atmosphere for 12 h. TLC analysis indicated complete consumption of the starting material, and the cooled reaction mixture was poured onto ice/water. The resulting solids were collected by filtration, washed with water, and dried to give 19 (730 mg, 84% yield) as a faint brown solid. ¹H NMR (400 MHz, CDCl₃) δ 7.99 (d, J = 2.4 Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H), 7.63 (dd, J = 9.0, 2.4 Hz, 1H), 7.05 (s, 1H), 2.73 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 159.8, 147.6, 145.7, 132.0, 131.7, 130.4, 121.6, 121.0, 110.2, 25.8.

4.1.2. Typical procedure for the synthesis of 2-methyl-4-(1H-1,2,3-triazolyl)quinoline (20-25)

A mixture of triethylamine (260 μL, 1.85 mmol), corresponding amines (1.11 mmol) and propargyl bromide (1.1 mmol) in THF-H₂O (6.0 mL, V/V = 1:1) was stirred at room temperature under a nitrogen atmosphere for 1 h. Azide 2 (110 mg, 0.50 mmol) and CuI (10 mg, 0.05 mmol) was then added slowly to the reaction mixture. Then, the resultant mixture was heated at 70 °C under nitrogen atmosphere for additional 2 h. Once the reaction was completed, as monitored by TLC, the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography using methanol/methylene chloride (0 → 1:33) as eluent to obtain triazoles 20-25, respectively.

4.1.2.1. 6-Chloro-2-methyl-4-(4-(pyrrolidin-1-ylmethyl)-1H-1,2,3-triazol-1-yl)quinoline (20)

Yellow oil, yield: 46.0%. ¹H NMR (400 MHz, CDCl₃) δ 8.06 (s, 1H), 8.04 (d, J = 9.0 Hz, 1H), 7.92 (d, J = 2.2 Hz, 1H), 7.69 (dd, J = 9.0, 2.2 Hz, 1H), 7.41 (s, 1H), 4.00 (s, 2H), 2.78 (s, 3H), 2.76 (t, J = 5.8 Hz, 4H), 1.88 – 1.85 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 159.9, 148.2, 146.2, 140.5, 133.9, 131.9, 131.2, 124.7, 122.2, 121.6, 118.0, 54.6, 50.9, 25.7, 23.9; HR-MS (ESI) m/z calcd for C₁₇H₁₉ClN₅ [M+H]⁺: 328.1329, found: 328.1325.

4.1.2.2. 6-Chloro-2-methyl-4-(4-(piperidin-1-ylmethyl)-1H-1,2,3-triazol-1-yl)quinoline (21)

Yellow oil, yield: 47%. ¹H NMR (400 MHz, CDCl₃) δ 8.03 (s, 1H), 7.97 (d, J = 9.0 Hz, 1H), 7.87 (d, J = 2.2 Hz, 1H), 7.63 (dd, J = 9.0, 2.2 Hz, 1H), 7.36 (s, 1H), 3.81 (s, 2H), 2.72 (s, 3H), 2.60 – 2.53 (m, 4H), 1.63 – 1.59 (m, 4H), 1.45 – 1.43 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 159.6, 147.8, 144.8, 140.2, 133.5, 131.6, 130.8, 125.0, 121.9, 117.6, 54.4, 54.0, 25.5, 25.3, 23.8; HR-MS (ESI) m/z calcd for C₁₈H₂₁ClN₅ [M+H]⁺: 342.1485, found: 342.1438.

4.1.2.3. 4-((1-(6-Chloro-2-methylquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)morpholine (22)

Yellow oil, yield: 41%. ¹H NMR (400 MHz, CDCl₃) δ 8.03 (d, J = 9.0 Hz, 1H), 7.96 (s, 1H), 7.89 (d, J = 2.2 Hz, 1H), 7.69 (dd, J = 9.0, 2.2 Hz, 1H), 7.40 (s, 1H), 3.81(s, 2H), 3.73 (t, J = 6.4 Hz, 4H), 2.78 (s, 3H), 2.60 (t, J = 6.4 Hz, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 159.9, 148.2, 145.5, 140.5, 133.9, 132.0, 131.2, 124.7, 122.1, 121.5, 117.9, 67.2, 25.7; HR-MS (ESI) m/z calcd for C₁₇H₁₉ClN₅O [M+H]⁺: 344.1278, found: 344.1275.

4.1.2.4. 6-Chloro-2-methyl-4-(4-((4-methylpiperazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl) quinoline (23)

Yellow oil, yield: 45%. ¹H NMR (400 MHz, CDCl₃) δ 8.06 (d, J = 9.0 Hz, 1H), 7.98 (s, 1H), 7.91 (d, J = 2.2 Hz, 1H), 7.71 (dd, J = 9.0, 2.2 Hz, 1H), 7.43 (s, 1H), 3.89 (s, 2H), 2.80 (s, 3H), 2.78 – 2.74 (m, 4H), 2.69 – 2.65 (m, 4H), 2.39 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 159.9, 148.2, 145.3, 140.5, 133.9, 132.0, 131.2, 124.8, 122.1, 121.5, 118.0, 55.1, 53.3, 52.8, 45.9, 25.7; HR-MS (ESI) m/z calcd for C₁₈H₂₂ClN₆ [M+H]⁺: 357.1594, found: 357.1592.

4.1.2.5. 2-(4-((1-(6-Chloro-2-methylquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methyl) piperazin-1-yl)ethan-1-ol (24)

Yellow oil, yield: 43%. ¹H NMR (400 MHz, CDCl₃) δ 8.07 (d, J = 9.0 Hz, 1H), 7.99 (s, 1H), 7.92 (d, J = 2.2 Hz, 1H), 7.72 (dd, J = 9.0, 2.2 Hz, 1H), 7.44 (s, 1H), 3.88 (s, 2H), 3.65 (t, J = 5.4 Hz, 2H), 2.81 (s, 3H), 2.74 – 2.65 (m, 8H), 2.61 (t, J = 5.4 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 160.0, 148.2, 145.5, 140.5, 133.9, 132.0, 131.2, 124.8, 122.1, 121.6, 118.0, 59.6, 58.0, 53.4, 53.3, 53.1, 25.7; HR-MS (ESI) m/z calcd for C₁₉H₂₄ClN₆O [M+H]⁺: 387.1700, found: 387.1686.

4.1.2.6. N-((1-(6-chloro-2-methylquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)-N-ethylethanamine (25)

Yellow oil, yield: 27%. ¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 8.04 (d, J = 9.0 Hz, 1H), 7.92 (s, 1H), 7.68 (d, J = 9.0 Hz, 1H), 7.43 (s, 1H), 4.01 (s, 2H), 2.94 – 2.62 (m, 7H), 1.20 (t, J = 6.4 Hz, 6H); HR-MS (ESI) m/z calcd for C₁₇H₂₁ClN₅ [M+H]⁺: 330.1485, found: 330.1445.

4.1.3. (E)-4,6-dichloro-2-(2-(pyridin-4-yl)vinyl)quinoline (33)

Method A: A mixture of 2-methylquinoline 18 (170 mg, 0.80 mmol), isonicotinaldehyde (102 mg, 0.96 mmol) and trifluoromethanesulphonamide (125 mg,0.84 mmol) was dissolved in DMF (0.6 mL) in a microwave tube. Then, the reaction mixture was irradiated in a Discover SP microwave reactor at 140 °C for 20 min. After cooling, the reaction mixture was concentrated and purified by silica gel column chromatography using ethyl acetate/hexane (1:4 → 1:2.2) as eluent to give vinylquinoline 5 (110 mg, 46% yield) as a yellow solid.

Method B: A mixture of 2-methylquinoline 18 (170 mg, 0.80 mmol), isonicotinaldehyde (430 mg, 4.0 mmol) and p-TsNH₂ (137 mg, 0.80 mmol) in xylene (6 mL) was heated at 130 °C for 4 h. Upon completion, the cooled mixture was directly loaded and purified on the silica gel column chromatography using ethyl acetate/hexane (1:4 → 1:2.2) as eluent to provide vinylquinoline 33 (100 mg, 42% yield) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.67 (d, J = 5.8 Hz, 2H), 8.20 (d, J = 2.4 Hz, 1H), 8.05 (d, J = 9.0 Hz, 1H), 7.77 (s, 1H), 7.73 (dd, J = 8.6, 1.8 Hz, 1H), 7.65 (d, J = 16.4 Hz, 1H), 7.49 (d, J = 2.8 Hz, 2H), 7.46 (d, J = 16.4 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 155.2, 150.7, 147.7, 143.7, 142.4, 134.2, 133.2, 132.3, 132.2, 131.7, 123.5, 121.8, 120.9.

4.1.4. (E)-4-azido-6-chloro-2-(2-(pyridin-4-yl)vinyl)quinoline (34)

A mixture of vinylquinoline 33 (190 mg, 0.63 mmol) and NaN₃ (127 mg, 1.95 mmol) in DMF (2.0 mL) was stirred at 80 °C under a nitrogen atmosphere for 12 h. After cooling, the reaction was quenched with water and the resultant mixture was extracted with methylene chloride. The combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. After the solvent was removed in vacuo, the residue was purified by silica gel chromatography using ethyl acetate/hexane (1:10 → 1:7) as eluent to give azide 34 (115 mg, 59% yield) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.65 (d, J = 6.0 Hz, 2H), 7.99 (d, J = 2.4 Hz, 1H), 7.97 (d, J = 9.0 Hz, 1H), 7.67 (dd, J = 8.6, 1.8 Hz, 1H), 7.64 (d, J = 16.4 Hz, 1H), 7.48 (d, J = 2.4 Hz, 2H), 7.45 (d, J = 16.4 Hz, 1H), 7.31 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 155.3, 150.7, 147.9, 146.2, 143.7, 133.0, 132.7, 132.5, 132.3, 131.1, 121.8, 108.2.

4.1.5. Typical procedure for the synthesis of 2-pyridylvinyl-4-triazolylquinoline (26-31)

A mixture of 2-methyl-4-triazolylquinolines (20-25) (0.1 mmol), isonicotinaldehyde (54 mg, 0.5 mmol) and p-TsNH₂ (17 mg, 0.1 mmol) in xylene (3.0 mL) was heated at 130 °C for 12 h. Upon completion, the cooled resultant content was directly loaded and purified on the gel chromatography using methanol/methylene chloride (1:50 → 1:14) as eluent to give pyridylvinylquinoline-triazoles 26-31, respectively.

4.1.5.1. (E)-6-chloro-2-(2-(pyridin-4-yl)vinyl)-4-(4-(pyrrolidin-1-ylmethyl)-1H-1,2,3-triazol-1-yl)quinoline (26)

Brown solid, yield: 65%, m.p. 198–200 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.66 (d, J = 5.2 Hz, 2H), 8.25 (s, 1H), 8.15 (d, J = 9.4 Hz, 1H), 8.01 (d, J = 2.2 Hz, 1H), 7.83 (s, 1H), 7.77 (d, J = 6.4 Hz, 1H), 7.72 (d, J = 16.4 Hz, 1H), 7.54 (d, J = 16.4 Hz, 1H), 7.47 (d, J = 6.0 Hz, 2H), 4.14 (s, 2H), 2.94 – 2.87 (m, 4H), 1.97 – 1.93 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 155.7, 150.8, 148.6, 143.4, 140.9, 135.0, 133.9, 132.5, 131.9, 129.5, 129.3, 125.6, 122.4, 121.8, 115.8, 54.7, 50.8, 23.9; HR-MS (ESI) m/z calcd for C₂₃H₂₂ClN₆ [M+H]⁺: 417.1594, found: 417.1610.

4.1.5.2. (E)-6-chloro-4-(4-(piperidin-1-ylmethyl)-1H-1,2,3-triazol-1-yl)-2-(2-(pyridin-4-yl) vinyl)quinoline (27)

Brown solid, yield: 86%, m.p. 215–217 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.60 (d, J = 5.4 Hz, 2H), 8.11 (s, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.91 (d, J = 2.2 Hz, 1H), 7.72 (s, 1H), 7.71 – 7.69 (m, 1H), 7.64 (d, J = 16.4 Hz, 1H), 7.47 (d, J = 16.4 Hz, 1H), 7.41 (d, J = 6.0 Hz, 2H), 3.87 (s, 2H), 2.64 – 2.59 (m, 4H), 1.67 – 1.61 (m, 4H), 1.48 – 1.43 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 155.2, 150.5, 148.2, 144.7, 143.1, 140.7, 134.6, 133.4, 132.2, 131.6, 131.5, 125.2, 122.1, 121.5, 115.5, 54.4, 53.6, 25.4, 23.8; HR-MS (ESI) m/z calcd for C₂₄H₂₄ClN₆ [M+H]⁺: 431.1751, found: 431.1735.

4.1.5.3. (E)-4-((1-(6-chloro-2-(2-(pyridin-4-yl)vinyl)quinolin-4-yl)-1H-1,2,3-triazol-4-yl)-methyl)morpholine (28)

Brown solid, yield: 81%, m.p. 230–233 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.65 (s, 2H), 8.08 (d, J = 9.0 Hz, 1H), 7.99 (s, 1H), 7.87 (d, J = 2.2 Hz, 1H), 7.73 – 7.68 (m, 2H), 7.63 (d, J = 16.4 Hz, 1H), 7.50 – 7.38 (m, 3H), 3.81 (s, 2H), 3.73 – 3.69 (m, 4H), 2.62 – 2.56 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 155.2, 150.5, 148.2, 145.3, 143.0, 140.7, 134.7, 133.5, 132.3, 131.6, 131.5, 124.5, 122.1, 121.9, 115.6, 66.8, 53.6; HR-MS (ESI) m/z calcd for C₂₃H₂₂ClN₆O [M+H]⁺: 433.1544, found: 433.1595.

4.1.5.4. (E)-6-chloro-4-(4-((4-methylpiperazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-2-(2-(pyridin-4-yl)vinyl)quinoline (29)

Brown solid, yield: 72%, m.p. 104–106 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.60 (d, J = 4.6 Hz, 2H), 8.08 (d, J = 9.0 Hz, 1H), 7.98 (s, 1H), 7.89 (d, J = 2.2 Hz, 1H), 7.73 (s, 1H), 7.71 (dd, J = 9.0, 2.2 Hz, 1H), 7.66 (d, J = 16.4 Hz, 1H), 7.48 (d, J = 16.4 Hz, 1H), 7.42 (d, J = 5.8 Hz, 2H), 3.86 (s, 2H), 2.73 – 2.66 (m, 4H), 2.62 – 2.52 (m, 4H), 2.33 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 155.3, 150.5, 148.2, 144.9, 143.1, 140.7, 134.6, 132.2, 131.6, 124.5, 122.1, 122.0, 121.5, 115.6, 54.8, 52.9, 52.3, 45.6; HR-MS (ESI) mz calcd for C₂₄H₂₅ClN₇ [M+H]⁺: 446.1860, found: 446.1868.

4.1.5.5. (E)-2-(4-((1-(6-chloro-2-(2-(pyridin-4-yl)vinyl)quinolin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)ethan-1-ol (30)

Brown solid, yield: 77%, m.p. 120–122 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.65 (d, J = 5.8 Hz, 2H), 8.14 (d, J = 9.0 Hz, 1H), 8.08 (s, 1H), 7.94 (d, J = 2.2 Hz, 1H), 7.81 (s, 1H), 7.76 (d, J = 9.0 Hz, 1H), 7.73 (d, J = 16.4 Hz, 1H), 7.71 (s, 1H), 7.54 (d, J = 16.4 Hz, 1H), 7.48 (d, J = 6.2 Hz, 2H), 3.92 (s, 2H), 3.71 (t, J = 5.4 Hz, 2H), 3.11 – 2.99 (m, 2H), 2.82 – 2.74 (m, 8H), 2.70 (t, J = 5.4 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 155.6, 150.8, 148.5, 145.2, 143.5, 140.9, 135.0, 133.8, 132.5, 131.9, 131.9, 125.0, 122.4, 122.3, 121.8, 116.0, 59.7, 57.8, 53.2, 53.1, 52.7; HR-MS (ESI) m/z calcd for C₂₅H₂₇ClN₇O [M+H]⁺: 476.1966, found: 476.1943.

4.1.5.6. (E)-N-((1-(6-chloro-2-(2-(pyridin-4-yl)vinyl)quinolin-4-yl)-1H-1,2,3-triazol-4-yl)-methyl)-N-ethylethanamine (31)

Brown solid, yield: 50%, m.p. 114–117 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.60 (d, J = 5.2 Hz, 2H), 8.20 (s, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.92 (d, J = 2.2 Hz, 1H), 7.73 (s, 1H), 7.71 (dd, J = 9.0, 2.2 Hz, 1H), 7.65 (d, J = 16.4 Hz, 1H), 7.48 (d, J = 16.4 Hz, 1H), 7.42 (d, J = 6.0 Hz, 2H), 4.03 (s, 2H), 2.75 (q, J = 7.2 Hz, 4H), 1.17 (t, J = 7.2 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 155.3, 150.5, 148.2, 143.1, 140.6, 134.7, 133.5, 132.2, 131.6, 122.1, 122.0, 121.5, 115.5, 47.5, 46.7, 11.0; HR-MS (ESI) m/z calcd for C₂₃H₂₄ClN6 [M+H]⁺: 419.1751, found: 419.1750.

4.1.6. Typical procedure for the synthesis of aminoalcohols (35-36)

A mixture of 4,6-dichloroquinoline (18) (840 mg, 3.96 mol) and aminoalkanols (99.4 mol) in EtOH (1.0 mL) was heated at 130 °C for 48 h. After cooling, the reaction was poured into water, and the resulting precipitate was collected by filtration and washed with water. Then the solid obtained was dried to give aminoalcohols 35-36, respectively.

4.1.6.1. 2-((6-Chloro-2-methylquinolin-4-yl)amino)ethan-1-ol (35)

Off-white solid, yield: 97%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.24 (d, J = 2.4 Hz, 1H), 7.65 (d, J = 9.0 Hz, 1H), 7.51 (dd, J = 9.0, 2.4 Hz, 1H), 7.02 (t, J = 5.2 Hz, 1H), 6.37 (s, 1H), 4.82 (t, J = 5.6 Hz, 1H), 3.61 (q, J = 5.8 Hz, 2H), 3.28 (q, J = 5.8 Hz, 2H), 2.40 (s, 3H).

4.1.6.2. 4-((6-Chloro-2-methylquinolin-4-yl)amino)butan-1-ol (36)

Off-white solid, yield: 93%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.28 (d, J = 2.4 Hz, 1H), 7.63 (d, J = 9.0 Hz, 1H), 7.50 (dd, J = 9.0, 2.4 Hz, 1H), 7.04 (t, J = 5.2 Hz, 1H), 6.34, (s, 1H), 4.41 (t, J = 5.2 Hz, 1H), 3.44 – 3.40 (m, 2H), 3.23 – 3.18 (m, 2H), 2.41 (s, 3H), 1.68 – 1.63 (m, 2H), 1.54 – 1.48 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 160.1, 150.2, 147.4, 131.2, 129.7, 128.2, 121.6, 119.1, 99.5, 61.3, 43.1, 30.9, 26.0, 25.3.

4.1.7. General procedure for the synthesis of pyridylvinylquinolines (37-38)

To a stirred solution of isonicotinaldehyde (18.0 mmol) and p-TsNH₂ (514 mg, 3.0 mmol) in xylene (12 mL) was added 2-methylquinolines 35-36 (2.0 mmol). The reaction mixture was heated at 130 °C for 6 h. Upon completion, the resulting solution was directly loaded and purified on the silica gel column chromatography using methanol/methylene chloride (1:50 → 1:12.5) as eluent to give pyridylvinylquinolines 37-38, respectively.

4.1.7.1. (E)-2-((6-chloro-2-(2-(pyridin-4-yl)vinyl)quinolin-4-yl)amino)ethan-1-ol (37)

Yellow solid, yield: 89%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.61 (d, J = 5.8 Hz, 2H), 8.39 (d, J = 2.2 Hz, 1H), 7.82 (d, J = 9.0 Hz, 1H), 7.73 (d, J = 16.2 Hz, 1H), 7.67 (d, J = 6.0 Hz, 2H), 7.64 (dd, J = 9.0, 2.2 Hz, 1H), 7.59 (d, J = 16.2 Hz, 1H), 7.29 (t, J = 5.0 Hz, 1H), 6.89 (s, 1H), 4.88 (t, J = 5.5 Hz, 1H), 3.74 (q, J = 5.7 Hz, 2H), 3.49 – 3.44 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 155.5, 150.7, 150.6, 147.2, 144.2, 134.5, 131.4, 130.7, 130.2, 128.9, 121.8, 121.6, 119.6, 98.6, 59.3, 45.8.

4.1.7.2. (E)-4-((6-chloro-2-(2-(pyridin-4-yl)vinyl)quinolin-4-yl)amino)butan-1-ol (38)

Yellow solid, yield: 92%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.55 (d, J = 5.8 Hz, 2H), 8.35 (d, J = 2.2 Hz, 1H), 7.76 (d, J = 9.0 Hz, 1H), 7.68 (d, J = 16.2 Hz, 1H), 7.61 (d J = 6.0 Hz, 2H), 7.58 (dd, J = 8.6, 1.8 Hz, 1H), 7.54 (d, J = 16.2 Hz, 1H), 6.79 (s, 1H), 4.46 (t, J = 7.0 Hz, 1H), 3.45 (t, J = 6.6 Hz, 2H), 3.31 (t, J = 6.6 Hz, 2H), 1.75 – 1.68 (m, 2H), 1.59 – 1.51 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 155.6, 151.0, 150.9, 147.3, 144.4, 134.6, 131.5, 131.1, 130.6, 129.2, 122.1, 121.9, 119.9, 98.9, 61.3, 43.3, 30.9, 25.3.

4.1.8. General procedure for the synthesis of methanesulfonates (39-40)

To a solution of alcohols 37-38 (1.68 mmol) and triethylamine (465 μL, 3.36 mmol) in anhydrous THF (8 mL) at the ice-salt bath was added methanesulfonyl chloride (194 μL, 2.52 mmol) dropwise under a nitrogen atmosphere. After complete addition, the resultant mixture was stirred at 0 °C for a further 45 min. Then, the reaction was quenched by the addition of saturated aqueous NaHCO₃ and saturated NaCl solution. The reaction contents were then transferred to a separatory funnel, diluting with ethyl acetate. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic extracts were then dried over Na₂SO₄, filtered, and evaporated. The residue was purified on the gel chromatography using methanol/methylene chloride (1:25 → 1:23) as eluent to give methanesulfonates 39-40, respectively.

4.1.8.1. (E)-2-((6-chloro-2-(2-(pyridin-4-yl)vinyl)quinolin-4-yl)amino)ethyl methane sulfonate (39)

Yellow solid, 64% yield. ¹H NMR (400 MHz, CDCl₃ + DMSO-d₆) δ 8.63 (d, J = 5.8 Hz, 2H), 7.92 (d, J = 9.0 Hz, 1H), 7.77 (d, J = 2.2 Hz, 1H), 7.58 (dd, J = 8.6, 1.8 Hz, 1H), 7.59 (d, J = 16.2 Hz, 1H), 7.46 (d, J = 5.6 Hz, 2H), 7.40 (d, J = 16.2 Hz, 1H), 6.69 (s, 1H), 4.61 (t, J = 6.6 Hz, 2H), 3.77 (q, J = 5.4 Hz, 2H), 3.11 (s, 3H); ¹³C NMR (100 MHz, CDCl₃ + DMSO-d₆) δ 155.7, 150.7, 149.1, 147.4, 144.2, 133.9, 131.9, 131.5, 131.3, 130.9, 121.7, 119.5, 98.4, 67.1, 42.9, 38.2.

4.1.8.2. (E)-4-((6-chloro-2-(2-(pyridin-4-yl)vinyl)quinolin-4-yl)amino)butyl methane sulfonate (40)

Yellow solid, 57% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.62 (d, J = 5.8 Hz, 2H), 7.90 (d, J = 9.0 Hz, 1H), 7.76 (d, J = 2.2 Hz, 1H), 7.58 (d, J = 16.2 Hz, 1H), 7.55 (dd, J = 6.8, 1.8 Hz, 1H), 7.46 (d, J = 4.0 Hz, 2H), 7.40 (d, J = 16.2 Hz, 1H), 6.62 (s, 1H), 4.34 (t, J = 6.6 Hz, 2H), 3.42 (q, J = 5.6 Hz, 2H), 3.04 (s, 3H), 1.96 – 1.93 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 155.5, 150.6, 149.8, 147.1, 144.3, 133.9, 131.6, 131.4, 130.9, 130.7, 121.8, 119.5, 119.4, 98.5, 69.8, 43.0, 37.9, 27.2, 25.2.

4.1.9. General procedure for the synthesis of azides (41-42)

A mixture of methanesulfonates 39-40 (1.19 mmol) and NaN₃ (271 mg, 4.17 mmol) in DMF (3.0 mL) was stirred at 95 °C overnight. Upon completion, the resultant mixture was cooled to room temperature, and water was added. The resulting precipitate was collected by filtration, washed with water, and dried to give azides 41-42. The crude products were taken for the next reaction without further purification.

4.1.10. 3-(Prop-2-yn-1-yl)thiazolidine-2,4-dione (67)

To a stirred suspension of thiazolidine-2,4-dione (66) (586 mg, 5.0 mmol) and potassium carbonate (829 mg, 6.0 mmol) in acetone (12 mL) was added propargyl bromide (593 μL, 5.5 mmol) slowly at room temperature. The resultant mixture was refluxed for 4 h. After cooling, the solvent was evaporated to dryness. The residue was dissolved in ethyl acetate and washed with water and brine. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were then dried over Na₂SO₄, filtered, and evaporated under reduced pressure to give thiazolidinedione 67 (690 mg, 89% yield) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.30 (d, J = 2.6 Hz, 2H), 3.98 (s, 2H), 2.23 (t, J = 2.6 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 171.0, 170.6, 76.5, 72.5, 34.2, 31.0.

4.1.11. (Z)-5-benzylidene-3-(prop-2-yn-1-yl)thiazolidine-2,4-dione (68)

A mixture of thiazolidinedione 31 (320 mg, 2.06 mmol), benzaldehydealdehyde (219 mg, 2.06 mmol), glacial acetic acid (2-4 drops) and piperidine (0.103 mmol) in anhydrous methanol (3.0 mL) was refluxed for 3 h. After cooling, the resultant solvent was evaporated to dryness. The residue was poured in crushed ice and the resulting solids were collected by filtration, washed with water, and dried. The crude product was purified by silica gel column chromatography using ethyl acetate/hexane (1:33→1:16) as eluent to give compound 68 (160 mg, 36% yield) as a colorless solid. ¹H NMR (400 MHz, CDCl₃) δ 7.95 (s, 1H), 7.52 – 7.46 (m, 5H), 4.51 (d, J = 2.6 Hz, 2H), 2.28 (t, J = 2.6 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 167.2, 165.4, 135.0, 133.4, 131.1, 130.6, 129.6, 121.3, 76.4, 72.6, 31.0.

4.1.12. General procedure for the synthesis of pyridylvinylquinoline-triazoles (32, 51-65 and 69-70)

To a stirred solution of appropriate alkynes (0.21 mmol) and azides (0.20 mmol) in THF-H₂O (6 mL, v/v = 1:1) was added CuI (10 mg, 0.05 mmol) under a nitrogen atmosphere. Then the reaction mixture was heated at 70 °C for 2 h. Upon completion, the resulting solution was filtered to remove the solid. The filter cake was rinsed with THF/MeOH. The combined filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel using methanol/methylene chloride (1:50 → 1:7) as eluent to give triazoles 32, 51-65 and 69-70, respectively.

4.1.12.1. (E)-2-((1-(6-chloro-2-(2-(pyridin-4-yl)vinyl)quinolin-4-yl)-1H-1,2,3-triazol-4-yl) methyl)isoindoline-1,3-dione (32)

Yellow solid, yield 81%. m.p. 182–184 °C ¹H NMR (400 MHz, CDCl₃) δ 8.62 (s, 2H), 8.09 (s, 1H), 8.07 (d, J = 9.0 Hz, 1H), 7.87 (d, J = 2.0 Hz, 1H), 7.84 (dd, J = 5.4, 3.0 Hz, 2H), 7.71 – 7.68 (m, 4H), 7.65 (s, 1H), 7.62 (d, J = 16.4 Hz, 1H), 7.46 (d, J = 16.4 Hz, 1H), 7.42 (s, 1H), 5.13 (s, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 167.7, 155.1, 150.4, 148.2, 143.7, 143.1, 140.5, 134.8, 134.3, 133.4, 132.3, 132.0, 131.6, 131.5, 124.8, 123.6, 122.1, 122.0, 115.6, 32.9; HR-MS (ESI) m/z calcd for C₂₇H₁₈ClN₆O₂ [M+H]⁺: 493.1180, found: 493.1153.

4.1.12.2. (E)-6-chloro-2-(2-(pyridin-4-yl)vinyl)-N-(2-(4-(pyrrolidin-1-ylmethyl)-1H-1,2,3-triazol-1-yl)ethyl)quinolin-4-amine (51)

Brown solid, yield: 55%, m.p. 117–119 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.45 (d, J = 4.2 Hz, 2H), 7.95 (d, J = 2.2 Hz, 1H), 7.81 (s, 1H), 7.71 (d, J = 9.0 Hz, 1H), 7.56 (d, J = 6.0 Hz, 2H), 7.52 – 7.47 (m, 2H), 7.33 (d, J = 16.4 Hz, 1H), 6.65 (s, 1H), 4.70 – 4.66 (m, 2H), 3.93 (t, J = 5.6 Hz, 2H), 3.78 (s, 2H), 2.53 – 2.48 (m, 4H), 1.65 – 1.61 (m, 4H); ¹³C NMR (100 MHz, CD₃OD) δ 155.5, 150.4, 149.2, 146.3, 145.1, 142.1, 133.3, 131.2, 130.2, 129.6, 125.2, 121.7, 120.2, 119.1, 96.3, 53.0, 49.1, 48.8, 42.2, 22.6; HR-MS (ESI) m/z calcd for C₂₅H₂₇ClN₇ [M+H]⁺: 460.2016, found: 460.2041.

4.1.12.3. (E)-6-chloro-N-(2-(4-(piperidin-1-ylmethyl)-1H-1,2,3-triazol-1-yl)ethyl)-2-(2-(pyridin-4-yl)vinyl)quinolin-4-amine (52)

Brown solid, yield: 57%, m.p. 123–125 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.43 (d, J = 5.8 Hz, 2H), 7.91 (d, J = 2.2 Hz, 1H), 7.72 (s, 1H), 7.68 (d, J = 9.0 Hz, 1H), 7.52 (d, J = 6.2 Hz, 2H), 7.47 (dd, J = 9.0, 2.2 Hz, 1H), 7.43 (d, J = 16.4 Hz, 1H), 7.28 (d, J = 16.4 Hz, 1H), 6.55 (s, 1H), 4.65 (t, J = 5.6 Hz, 2H), 3.89 (t, J = 5.6 Hz, 2H), 3.47 (s, 2H), 2.19 – 2.12 (m, 4H), 1.33 – 1.29 (m, 4H), 1.23 – 1.18 (m, 2H); ¹³C NMR (100 MHz, CD₃OD) δ 155.5, 150.3, 149.2, 146.4, 145.1, 142.5, 133.4, 131.0, 130.3, 130.1, 129.6, 125.2, 121.6, 120.2, 96.2, 53.2, 52.6, 49.2, 42.3, 24.8, 23.2; HR-MS (ESI) m/z calcd for C₂₆H₂₈ClN₇ [M+H]⁺: 474.2173, found: 474.2180.

4.1.12.4. (E)-6-chloro-N-(2-(4-(morpholinomethyl)-1H-1,2,3-triazol-1-yl)ethyl)-2-(2-(pyridin-4-yl)vinyl)quinolin-4-amine (53)

Brown solid, yield: 71%, m.p. 131–133 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.57 (d, J = 6.0 Hz, 2H), 8.06 (d, J = 2.2 Hz, 1H), 7.84 – 7.81 (m, 2H), 7.68 (d, J = 6.2 Hz, 2H), 7.63 (dd, J = 9.0, 2.2 Hz, 1H), 7.59 (d, J = 16.4 Hz, 1H), 7.43 (d, J = 16.4 Hz, 1H), 6.69 (s, 1H), 4.78 (t, J = 5.6 Hz, 2H), 4.03 (t, J = 5.6 Hz, 2H), 3.54 (s, 2H), 3.51 – 3.47 (m, 4H), 2.28 – 2.23 (m, 4H); ¹³C NMR (100 MHz, CD₃OD) δ 155.4, 150.4, 149.2, 146.2, 145.1, 143.1, 133.2, 131.2, 130.4, 130.3, 129.5, 125.0, 121.7, 120.2, 96.2, 66.1, 52.6, 49.2, 42.2; HR-MS (ESI) m/z calcd for C₂₅H₂₇ClN₇O [M+H]⁺: 476.1966, found: 476.2013.

4.1.12.5. (E)-6-chloro-N-(2-(4-((4-methylpiperazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-2-(2-(pyridin-4-yl)vinyl)quinolin-4-amine (54)

Brown solid, yield: 65%, m.p. 84–86 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.44 (d, J = 4.8 Hz, 2H), 7.94 (d, J = 2.2 Hz, 1H), 7.77 (s, 1H), 7.68 (d, J = 9.0 Hz, 1H), 7.53 (d, J = 5.7 Hz, 2H), 7.52 – 7.49 (m, 1H), 7.47 (d, J = 16.4 Hz, 1H), 7.28 (d, J = 16.4 Hz, 1H), 6.58 (s, 1H), 4.66 (t, J = 5.6 Hz, 2H), 3.91 (t, J = 5.6 Hz, 2H), 3.48 (s, 2H), 2.60 – 2.56 (m, 4H), 2.36 (s, 3H), 2.34 – 2.22 (m, 4H); ¹³C NMR (100 MHz, CD₃OD) δ 155.0, 150.7, 149.3, 145.6, 144.9, 143.1, 132.7, 131.5, 130.5, 129.0, 125.0, 121.7, 120.4, 96.3, 53.7, 50.5, 49.1, 43.4, 42.3; HR-MS (ESI) m/z calcd for C₂₆H₃₀ClN₈ [M+H]⁺: 489.2282, found: 489.2296.

4.1.12.6. (E)-2-(4-((1-(2-((6-chloro-2-(2-(pyridin-4-yl)vinyl)quinolin-4-yl)amino)ethyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)ethan-1-ol (55)

Brown solid, yield: 62%, m.p. 91–94 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.55 (d, J = 4.4 Hz, 2H), 8.24 (d, J = 2.2 Hz, 1H), 8.10 (s, 1H), 7.89 (d, J = 3.6 Hz, 1H), 7.85 (d, J = 16.4 Hz, 1H), 7.75 (dd, J = 6.8, 2.2 Hz, 1H), 7.68 (d, J = 6.0 Hz, 2H), 7.45 (d, J = 16.4 Hz, 1H), 6.95 (s, 1H), 4.19 (t, J = 5.8 Hz, 2H), 3.85 – 3.73 (m, 4H), 3.68 (s, 2H), 3.17 – 3.09 (m, 6H), 2.71 – 2.62 (m, 4H); ¹³C NMR (100 MHz, CD₃OD) δ 154.0, 151.6, 149.8, 149.5, 144.0, 142.9, 136.0, 133.3, 132.3, 127.1, 125.7, 124.4, 122.4, 121.8, 118.2, 96.8, 58.5, 55.8, 52.1, 51.6, 49.4, 49.1, 43.1; HR-MS (ESI) m/z calcd for C₂₇H₃₂ClN₈O [M+H]⁺: 519.2388, found: 519.2361.

4.1.12.7. (E)-6-chloro-N-(2-(4-((diethylamino)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-2-(2-(pyridin-4-yl)vinyl)quinolin-4-amine (56)

Brown solid, yield: 44%, m.p. 76–78 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.57 (d, J = 5.2 Hz, 2H), 8.06 (d, J = 2.2 Hz, 1H), 7.83 (d, J = 10.4 Hz, 2H), 7.68 (d, J = 5.8 Hz, 2H), 7.63 (dd, J = 9.0, 2.2 Hz, 1H), 7.59 (d, J = 16.4 Hz, 1H), 7.43 (d, J = 16.4 Hz, 1H), 6.70 (s, 1H), 4.78 (t, J = 5.6 Hz, 2H), 4.04 (t, J = 5.6 Hz, 2H), 3.78 (s, 2H), 2.30 (q, J = Hz, 4H), 0.96 (t, J = 7.2 Hz, 6H); ¹³C NMR (100 MHz, CD₃OD) δ 155.6, 150.3, 149.2, 146.4, 145.1, 141.5, 133.4, 131.1, 130.3, 130.2, 129.7, 125.5, 121.6, 120.2, 119.1, 96.2, 49.3, 46.1, 45.5, 42.1, 10.0; HR-MS (ESI) m/z calcd for C₂₅H₂₉ClN₇ [M+H]⁺: 462.2173, found: 462.2188.

4.1.12.8. (E)-2-((1-(2-((6-chloro-2-(2-(pyridin-4-yl)vinyl)quinolin-4-yl)amino)ethyl)-1H-1,2,3-triazol-4-yl)methyl)isoindoline-1,3-dione (57)

Yellow solid, yield: 77%, m.p. > 260 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.63 (s, br. 2H), 7.83 (d, J = 9.0 Hz, 1H), 7.77 – 7.64 (m, 7H), 7.52 – 7.47 (m, 3H), 7.31 (d, J = 16.2 Hz, 1H), 6.50 (s, 1H), 5.73 (t, J = 7.2 Hz, 1H), 4.96 (s, 2H), 4.76 – 4.71 (m, 2H), 3.97 – 3.92 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.0, 155.9, 151.0, 150.3, 144.3, 143.0, 135.3, 134.6, 132.3, 131.8, 131.3, 130.5, 129.4, 124.8, 124.0, 121.6, 119.9, 98.8, 48.8, 43.3, 33.6; HR-MS (ESI) m/z calcd for C₂₉H₂₃ClN₇O₂ [M+H]⁺: 536.1602, found: 536.1601.

4.1.12.9. (E)-N-(2-(4-((1H-pyrazol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-6-chloro-2-(2-(pyridin-4-yl)vinyl)quinolin-4-amine (58)

Yellow solid, yield: 83%, m.p. 213–215 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.57 (s, br, 2H), 8.19 (d, J = 2.4 Hz, 1H), 8.06 (s, 1H), 7.78 (d, J = 9.0 Hz, 1H), 7.68 (d, J = 16.2 Hz, 1H), 7.63 (d, J = 2.4 Hz, 2H), 7.60 (dd, J = 8.6, 1.8 Hz, 2H), 7.51 (d, J = 16.2 Hz, 1H), 7.36 (d, J = 2.4 Hz, 1H), 6.86 (s, 1H), 6.16 (t, J = 3.2 Hz, 1H), 5.33 (s, 2H), 4.66 (t, J = 6.0 Hz, 2H), 3.81 (d, J = 6.0 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 156.0, 151.0. 150.4, 147.5, 144.4, 143.6, 139.6, 134.7, 131.8, 131.3, 130.6, 130.5, 129.5, 125.2, 122.2, 121.7, 119.9, 106.2, 98.9, 48.7, 47.2, 43.2; HR-MS (ESI) m/z calcd for C₂₄H₂₂ClN₈ [M+H]⁺: 457.1656, found: 457.1635.

4.1.12.10. (E)-6-chloro-2-(2-(pyridin-4-yl)vinyl)-N-(4-(4-(pyrrolidin-1-ylmethyl)-1H-1,2,3-triazol-1-yl)butyl)quinolin-4-amine (59)

Brown solid, yield: 71%, m.p. 63–66 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.44 (d, J = 5.0 Hz, 2H), 8.05 (d, J = 2.2 Hz, 1H), 7.85 (s, 1H), 7.71 (d, J = 9.0 Hz, 1H), 7.57 (d, J = 6.0 Hz, 2H), 7.52 – 7.48 (m, 2H), 7.41 (d, J = 16.4 Hz, 1H), 6.74 (s, 1H), 4.42 (t, J = 6.8 Hz, 2H), 3.75 (s, 2H), 3.36 (t, J = 7.2 Hz, 2H), 2.61 – 2.55 (m, 4H), 2.02 – 1.97 (m, 2H), 1.71 – 1.68 (m, 4H), 1.66 – 1.62 (m, 2H); ¹³C NMR (100 MHz, CD₃OD) δ 155.4, 150.8, 149.2, 146.2, 145.2, 133.5, 130.9, 130.1, 129.3, 123.9, 121.7, 120.4, 119.3, 96.8, 53.2, 49.6, 49.2, 41.8, 27.5, 24.8, 22.8; HR-MS (ESI) m/z calcd for C₂₇H₃₁ClN₇ [M+H]⁺: 488.2329, found: 488.2325.

4.1.12.11. (E)-6-chloro-N-(4-(4-(piperidin-1-ylmethyl)-1H-1,2,3-triazol-1-yl)butyl)-2-(2-(pyridin-4-yl)vinyl)quinolin-4-amine (60)

Brown solid, yield: 74%, m.p. 98–100 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.50 (s, br, 2H), 8.12 (d, J = 2.2 Hz, 1H), 8.07 (s, 1H), 7.77 (d, J = 16.4 Hz, 1H), 7.65 (d, J = 9.0 Hz, 1H), 7.62 (d, J = 3.2 Hz, 2H), 7.58 (dd, J = 8.6, 1.8 Hz, 1H), 7.44 (d, J = 16.4 Hz, 1H), 6.83 (s, 1H), 4.52 (t, J = 6.8 Hz, 2H), 3.92 (s, 2H), 3.46 (t, J = 7.2 Hz, 2H), 2.77 – 2.72 (m, J = 4.0 Hz, 4H), 2.12 – 2.05 (m, 2H), 1.78 – 1.71 (m, 2H), 1.66 – 1.60 (m, 4H), 1.47 – 1.43 (m, 2H); ¹³C NMR (100 MHz, CD₃OD) δ 154.6, 151.7, 149.5, 145.1, 144.8, 132.1, 131.0, 130.8, 128.7, 128.3, 125.7, 122.1, 121,0, 119.2, 97.1, 53.4, 52.1, 50.0, 42.3, 27.8, 25.1, 24.3, 22.7; HR-MS (ESI) m/z calcd for C₂₈H₃₃ClN₇ [M+H]⁺: 502.2486, found: 502.2474.

4.1.12.12. (E)-6-chloro-N-(4-(4-(morpholinomethyl)-1H-1,2,3-triazol-1-yl)butyl)-2-(2-(pyridin-4-yl)vinyl)quinolin-4-amine (61)

Brown solid, yield: 78%, m.p. 106–108 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.52 (d, J = 5.0 Hz, 2H), 8.14 (d, J = 2.2 Hz, 1H), 7.91 (s, 1H), 7.78 (d, J = 9.0 Hz, 1H), 7.66 (d, J = 16.4 Hz, 1H), 7.64 (d, J = 9.0 Hz, 2H), 7.61 (dd, J = 8.6, 1.8 Hz, 1H), 7.47 (d, J = 16.4 Hz, 1H), 6.85 (s, 1H), 4.50 (t, J = 6.8 Hz, 2H), 3.61 – 3.57 (m, 6H), 3.47 (t, J = 7.0 Hz, 2H), 2.44 – 2.41 (m, 4H), 2.10 – 2.05 (m, 2H), 1.78 – 1.70 (m, 2H); ¹³C NMR (100 MHz, CD₃OD) δ 154.5, 151.9, 149.6, 145.1, 144.7, 132.3, 132.1, 131.1, 130.8, 128.2, 124.4, 122.1, 121.0, 119.2, 97.2, 66.5, 53.1, 52.9, 49.8, 42.3, 27.8, 25.0; HR-MS (ESI) m/z calcd for C₂₇H₃₁ClN₇O [M+H]⁺: 504.2279, found: 504.2255.

4.1.12.13. (E)-6-chloro-N-(4-(4-((4-methylpiperazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)butyl)-2-(2-(pyridin-4-yl)vinyl)quinolin-4-amine (62)

Brown solid, yield: 80%, m.p. 78–81 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.47 (d, J = 4.6 Hz, 2H), 8.12 (d, J = 2.2 Hz, 1H), 7.87 (s, 1H), 7.74 (d, J = 9.0 Hz, 1H), 7.64 (d, J = 16.4 Hz, 1H), 7.60 – 7.56 (m, 3H), 7.41 (d, J = 16.4 Hz, 1H), 6.83 (s, 1H), 4.43 (t, J = 6.8 Hz, 2H), 3.60 (s, 2H), 3.43 (t, J = 7.1 Hz, 2H), 2.81 – 2.72 (m, 4H), 2.58 – 2.50 (m, 4H), 2.46 (s, 3H), 2.04 – 1.98 (m, 2H), 1.71 – 1.63 (m, 2H); ¹³C NMR (100 MHz, CD₃OD) δ 153.5, 152.1, 149.4, 144.6, 142.9, 132.7, 132.4, 131.3, 130.8, 127.0, 124.1, 121.8, 120.9, 118.7, 96.9, 53.7, 51.6, 50.3, 49.5, 43.2, 42.1, 27.4, 24.7; HR-MS (ESI) m/z calcd for C₂₈H₃₄ClN₈ [M+H]⁺: 517.2595, found: 517.2598.

4.1.12.14. (E)-2-(4-((1-(4-((6-chloro-2-(2-(pyridin-4-yl)vinyl)quinolin-4-yl)amino)butyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)ethan-1-ol (63)

Brown gum, yield: 75%. ¹H NMR (400 MHz, CD₃OD) δ 8.56 (d, J = 5.4 Hz, 2H), 8.41 (d, J = 2.2 Hz, 1H), 8.15 (s, 1H), 8.10 (d, J = 16.4 Hz,1H), 7.96 (d, J = 9.0 Hz, 1H), 7.84 (dd, J = 6.8 Hz, 2.2 Hz, 1H), 7.77 (d, J = 6.2 Hz, 2H), 7.66 (d, J = 16.4 Hz, 1H), 7.27 (s, 1H), 4.56 (t, J = 7.0 Hz, 2H), 3.88 (t, J = 5.8 Hz, 2H), 3.84 (t, J = 5.8 Hz, 2H), 3.80 (s, 2H), 3.75 (t, J = 7.0 Hz, 2H), 3.20 (t, J = 5.8 Hz, 2H), 2.96 – 2.80 (m, 6H), 2.18 – 2.11 (m, 2H), 1.87 – 1.79 (m, 2H); ¹³C NMR (100 MHz, CD₃OD) δ 155.3, 149.8, 149.7, 149.5, 143.7, 137.2, 137.1, 134.2, 132.8, 125.0, 124.8, 122.7, 122.4, 121.9, 117.8, 97.9, 58.4, 55.5, 52.2, 51.7, 49.9, 49.3, 43.5, 27.7, 25.1; HR-MS (ESI) m/z calcd for C₂₇H₃₆ClN₈O [M+H]⁺: 547.2701, found: 547.2709.

4.1.12.15. (E)-2-((1-(4-((6-chloro-2-styrylquinolin-4-yl)amino)butyl)-1H-1,2,3-triazol-4-yl)methyl)isoindoline-1,3-dione (64)

Yellow solid, yield: 82%, m.p. 236–238 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.64 (s, br, 2H), 7.90 (d, J = 9.2 Hz, 1H), 7.81 (d, J = 3.0 Hz, 1H), 7.80 (d, J = 3.0 Hz, 1H), 7.75 (s, 1H), 7.69 (d, J = 3.2 Hz, 1H), 7.68 (d, J = 3.2 Hz, 1H), 7.66 (s, 1H), 7.61 (d, J = 16.0 Hz, 1H), 7.54 (dd, J = 8.0, 3.2 Hz, 2H), 7.51 (d, J = 8.0 Hz,1H), 7.42 (d, J = 16.0 Hz, 1H), 6.61 (s, 1H), 5.01 (s, 2H), 4.45 (t, J = 6.8 Hz, 2H), 3.40 (t, J = 6.8 Hz, 2H), 2.16 – 2.08 (m, 2H), 1.85 – 1.78 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 168.0, 155.5, 150.6, 149.8, 144.3, 143.4, 134.5, 133.8, 132.3, 131.5, 130.9, 130.7, 123.8, 123.4, 119.6, 119.4, 98.4, 50.1, 43.1, 33.4, 28.2, 25.9; HR-MS (ESI) m/z calcd for C₃₁H₂₇ClN₇O₂ [M+H]⁺: 564.1915, found: 564.1908.

4.1.12.16. (E)-N-(4-(4-((1H-pyrazol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)butyl)-6-chloro-2-(2-(pyridin-4-yl)vinyl)quinolin-4-amine (65)

Yellow solid, yield: 86%, m.p. 193–195 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.59 (d, J = 6.0 Hz, 2H), 8.24 (d, J = 2.2 Hz, 1H), 7.96 (s, 1H), 7.86 (d, J = 9.0 Hz, 1H), 7.76 (s, 1H), 7.72 – 7.68 (m, 4H), 7.54 (d, J = 16.4 Hz, 1H), 7.48 (d, J = 1.8 Hz, 1H), 6.96 (s, 1H), 6.29 (s, 1H), 5.43 (s, 2H), 4.53 (t, J = 7.0 Hz, 2H), 3.53 (t, J = 7.2 Hz, 2H), 2.14 – 2.07 (m, 2H), 1.81– 1.75 (m, 2H); ¹³C NMR (100 MHz, CD₃OD) δ 153.8, 152.0, 149.3, 144.7, 143.7, 143.2, 139.3, 132.5, 131.1, 130.7, 130.0, 127.3, 123.5, 121.8, 120.8, 105.6, 96.8, 49.5, 46.1, 42.0, 27.4, 24.7; HR-MS (ESI) m/z calcd for C₂₆H₂₆ClN₈ [M+H]⁺: 485.1969, found: 485.1970.

4.1.12.17. 5-((Z)-benzylidene)-3-((1-(2-((6-chloro-2-((E)-2-(pyridin-4-yl)vinyl)quinolin-4-yl)amino)ethyl)-1H-1,2,3-triazol-4-yl)methyl)thiazolidine-2,4-dione (69)

Yellow solid, 68% yield, m.p. 228–230 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.64 (s, br, 2H), 7.91 (d, J = 9.0 Hz, 1H), 7.76 (d, J = 4.2 Hz, 2H), 7.67 (d, J = 2.0 Hz, 1H), 7.51 (d, J = 16.2 Hz, 2H), 7.47 (d, J = 2.2 Hz, 1H), 7.44 – 7.41 (m, 5H), 7.34 (d, J = 16.2 Hz, 2H), 6.49 (s, 1H), 5.00 (s, 2H), 4.75 (t, J = 6.8 Hz, 2H), 3.95 (q, J = 5.6 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 167.8, 166.0, 155.5, 150.6, 149.1, 147.2, 144.2, 142.3, 134.8, 133.7, 133.3, 131.7, 131.6, 131.2, 131.1, 130.8, 130.6, 129.6, 125.1, 121.2, 119.5, 119.4, 98.0, 49.5, 43.5, 36.7; HR-MS (ESI) m/z calcd for C₃₁H₂₅ClN₇O₂S [M+H]⁺: 594.1479, found: 594.1528.

4.1.12.18. 5-((Z)-benzylidene)-3-((1-(4-((6-chloro-2-((E)-2-(pyridin-4-yl)vinyl)quinolin-4-yl)amino)butyl)-1H-1,2,3-triazol-4-yl)methyl)thiazolidine-2,4-dione (70)

Yellow solid, 66% yield, m.p. 215–217 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.63 (s, br, 2H), 7.91 (d, J = 9.0 Hz, 1H), 7.88 (s, 1H), 7.75 (d, J = 2.2 Hz, 1H), 7.69 (s, 1H), 7.61 (d, J = 16.4 Hz, 1H), 7.55 (d, J = 6.8 Hz, 1H), 7.49 (d, J = 5.0 Hz, 2H), 7.47 (d, J = 2.0 Hz, 1H), 7.46 (t, J = 5.0 Hz, 3H), 7.43 (d, J = 16.4 Hz, 2H), 6.63 (s, 1H), 5.07 (s, 2H), 4.46 (t, J = 6.8 Hz, 2H), 3.42 (d, J = 6.4 Hz, 2H), 2.16 – 2.12 (m, 2H), 1.86 – 1.82 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 167.9, 166.1, 155.4, 150.6, 149.9, 144.3, 142.2, 134.8, 133.7, 133.3, 131.5, 131.3, 131.1, 131.0, 130.8, 130.6, 129.6, 128.6, 123.8, 121.8, 121.4, 119.6, 119.4, 98.5, 50.2, 43.1, 36.9, 28.2, 25.9; HR-MS (ESI) m/z calcd for C₃₃H₂₉ClN₇O₂S [M+H]⁺: 622.1792, found: 622.1796.

4.2. In vitro antiplasmodial activity assay

Different dilutions of test compounds in DMSO were added to P. falciparum culture in 96-well plates (Santa Cruz Biotechnology, Dallas, TX) containing 1% parasitemia and 1% hematocrit. Maximum DMSO concentration was 0.1%. Following 72 h incubation at 37 °C in an atmosphere containing 5% CO₂, the plates were frozen at 80 °C. Subsequently, plates were thawed and 100 μL of lysis buffer (20 mM Tris-HCl, 0.008% (wt/vol) saponin, 5 mM EDTA, 0.08% Triton X-100 (vol/vol), and 0.01% SYBR Green I (vol/vol)) was added to each well. Following incubation in the dark at room temperature for 1 h, fluorescence emission from the plates were read at 485 nM excitation and 530 nM of emission using a Synergy Neo2 multi-mode reader (BioTek Instuments, Winooski, VT). The EC₅₀ values were calculated using non-linear dose-response curves generated in GraphPad Prism 7.0 software.

4.3. In vitro cytotoxic activity assay^[39]

Tested compounds were assessed for cytotoxicity in 384 well clear bottom plates (Santa Cruz Biotechnology) using HepG2 cell line. Plates were seeded with 2250 cells per well and incubated for 24 h at 37 °C in 5% CO₂. Serial dilutions of compounds were added starting at 25 μM and then plates were incubated for additional 48 h. 10 μL of MTS (CellTiter 96^® Aqueous One, Promega, Madison, WI) was added to each well and the plates were incubated for an additional 3 h at 37 °C. Cell viability was determined by measuring the absorbance at 490 nm using Synergy Neo2 multi-mode reader (BioTek).

4.4. Kinetic aqueous solubility assay

The kinetic aqueous solubility assay was performed at Analiza (Cleveland, Ohio, USA). In detail, test samples were supplied as DMSO dissolved stocks for analysis in 1 × PBS pH 7.4 buffer. A final DMSO concentration of 2.0% and maximum theoretical compound concentrations of 200 μM was achieved by diluting a 6 μL aliquot of DMSO stock with 294 μL of the appropriate buffer using Hamilton Starlet liquid handling and incubated directly in a Millipore solubility filter plate. Following 24 h incubation at ambient temperature (22.5-23.5 °C), the samples were vacuum filtered. The filtrates were injected into the nitrogen detector for quantification on Analiza’s Automated Discovery Workstation. The equimolar nitrogen response of the detector is calibrated using standards which span the dynamic range of the instrument from 0.08 to 4500 μg/mL nitrogen. The filtrates were quantified with respect to this calibration curve. The calculated solubility values are corrected for background nitrogen present in Analiza’s in house DMSO and 1 × PBS used to prepare the samples. All reported values were reported here in μM.

4.5. PAMPA assay

The PAMPA assay was performed at Analiza (Cleveland, Ohio, USA). Dilutions of the DMSO stocks were prepared in PBS 7.4 for a dose concentration of 200 μM in a volume of 300 μL directly in the Donor compartment of the Corning Gentest TM Pre-coated PAMPA plate. After preparation of the Donor plate, any precipitation was noted. The Acceptor compartment was filled with 1 × PBS (200 μL), pH 7.4. After careful assembly of the PAMPA plate, it was left to incubate for 5 h in the dark at ambient temperature. A sister plate is created (50 × dilution of 10 mM test articles was prepared in 1 × PBS, pH 7.4) directly in a Millipore solubility filter plate to measure the initial concentration of the sample in buffer (C0). Following incubation, the PAMPA plate was disassembled, and the samples were transferred from the Donor and Acceptor plates to 96-well plates for analysis by CLND. The C0 plate was filtered prior to analysis by CLND. The concentration values from the Donor and Acceptor compartment were used in the calculation of the effective permeability (Pe) of the compound.

4.6. Microsomal stability assay^[64]

The metabolic stability of compounds was determined in mouse liver microsomes using an NADPH-regenerating system. Sample stocks at 25 μM in DMSO were diluted to a final concentration of 1 μM with a mixture containing 0.5 mg/mL mouse liver microsomes, 1.3 mM NADP, 3.3 mM glucose 6-phoshate, 3.3 mM MgCl₂, glucose-6-phosphate dehydrogenase (0.4 U/mL) in 0.1 M potassium phosphate buffer (pH = 7.4). The mixture was incubated in a shaking platform at 37 °C, and aliquots were taken and quenched with the addition of an equal volume of cold acetonitrile at 60 min. Samples were centrifuged at 14,000 g for 10 min at 20 °C to remove debris. Sample quantification was carried out by LC/ MS. The stability of the compound is calculated as the percent remaining of the unchanged parent compound at each time point (T = 60 min) relative to the peak area at T = 0 min.

4.7. Stage specific action assay

Tightly synchronous P. falciparum Dd2 cultures were exposed to the compound at 5 × EC₅₀ at 6, 18, 30, and 42 h post invasion. After treatment, samples were taken at 12 h intervals to prepare Giemsa-stained thin smears for microscopic assessment of intraerythrocytic development. At the same time samples were collected and fixed with 0.0075% glutaraldehyde and 4% paraformaldehyde in PBS, permeabilized with 0.25% Triton X-100, treated with RNAse (50 μg/mL) and stained with YOYO-1 DNA binding dye (Invitrogen) at final concentration of 500 nM or flow cytometric cell cycle analysis.^[65] A total of 500,00 events were analyzed per sample. Flow data were acquired using the CytoFLEX flow cytometer (Beckman Coulter, Indianapolis, IN, USA) and analyzed with FlowJo software (FlowJo LLC, Ashland, OR, USA).

4.8. In Vitro killing profile assay

P. falciparum Dd2 asynchronous cultures at 1% parasitemia and 2% hematocrit were treated with compound 60 at 5 × EC₅₀ for 6, 12, 24, and 48 h. Parasites treated with 0.15% DMSO, atovaquone (6.6 nM), and DHA (50 nM) were included as negative, slow- and fast-acting controls, respectively. After treatment, cultures were washed 3 times with RPMI to remove the compound. Parasitemia was followed for 4 days after the addition of compounds in Giemsa-Stained thin smears.

4.9. Gametocyte inhibition assay

Plasmodium falciparum 3D7 line luc7 was used for the EC₅₀ determination.^[66] An established method of gametocyte induction was performed to obtain highly synchronous gametocyte cultures.^{[67, 68]} Briefly, gametocytes were induced by nutrient starvation, and then parasites were continually cultured in heparin treated medium to eliminate asexual stage parasites. Early stage (II-III) and late stage (IV-V) of gametocytes were purified by Percoll gradient centrifugation. 50,000 gametocytes in 100 μL of culture were added into each well in black 96-well plate, and exposed to compound 60 in a 19-point dose-response gradient (concentration range, 0.038 nM to 10 μM) for 72 h. Luciferase assay was performed using the ONE-Glo luciferase assay system (Promega) following the manufacturer instruction. The culture was incubated with 100 μL of reagent for 5 min, mix well before luciferase intensity detection by plate reader (PerkinElmer). EC₅₀ values were obtained using measured luciferase intensity and a nonlinear variable slope four-parameter regression curve fitting model in GraphPad Prism 7.0 software.

4.10. β-Hematin formation assay

β-hematin crystal growth inhibition was assessed under physiological conditions in the presence of lipid catalyst using a 96-well plate assay previously described.^[69] Briefly, a mixture of 10 μL of 2 mM hemin (Sigma-Aldrich, St. Louis, MO) dissolved in 0.1 M NaOH, 180 μL of propionate buffer at pH 5.2 and 10 μL of previously sonicated phosphatidylcholine (Sigma-Aldrich, St. Louis, MO) suspension (10 mg/mL) were incubated with different concentrations of the test compound at 37 °C with gentle shaking for 16 h. Samples treated with DMSO and CQ were used as controls. The reaction was stopped by the addition of 100 μL of a solution of SDS 7.5% (wt/vol) dissolved in 0.1M bicarbonate buffer (pH 9.1). The well contents were gently mixed and incubated at room temperature for 10 min. A 50 μL of aliquot from each well was transferred to a second plate pre-loaded with 200 μL/well of SDS solution (2.5% wt/vol in 0.1 M bicarbonate buffer), and the absorbance at 405 nm was read using Synergy Neo2 multi-mode reader (BioTek). Free hemin in solution was estimated using a linear calibration curve measured for each assay (hemin concentrations ranging from 0 to 16 μM freshly prepared in 2.5% SDS-0.1 M bicarbonate buffer pH 9.1). Images of crystals were taken before stopping the reaction with SDS using a Nikon Eclipse TE200 inverted microscope.

Statistical Analysis

Data were reported as means ± standard deviations (SD). One-way ANOVA followed by Student’s t-test was performed using Prism 7.0 (GraphPad, San Diego, CA, USA). P values ≤ 0.05 were considered statistically significant.

Highlights:

1. Pyridylvinylquinoline-triazole hybrids demonstrate potent antiplasmodial activity.

2. The fast-acting parasitocidal agent 60 kills asexual stage parasites at the trophozoite phase.

3. Compound 60 exhibits strong inhibitory activity against P. falciparum early and late stage gametocytes.

Abbreviations

CuAAC

copper-catalyzed azide-alkyne cycloaddition

CQ

chloroquine

AQ

amodiaquine

FQ

ferroquine

DV

digestive vacuole

ACTs

artemisinin-based combination therapies

SAR

structure-activity relationship

p-TsNH2

p-toluenesulfonamide

CQR

CQ-resistant

CQS

CQ-sensitive

LogP

logarithm of partition coefficient

RI

resistance index

SI

selectivity index

HepG2

hepatocellular carcinoma cells

PAMPA

parallel artificial membrane permeability assay

P-gp

P-glycoprotein

HPI

hours post invasion

DHA

dihydroartemisin

MTS

[(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium)