8-Amino-6-Methoxyquinoline—Tetrazole Hybrids: Impact of Linkers on Antiplasmodial Activity

A new series of compounds was prepared from 6-methoxyquinolin-8-amine or its N-(2-aminoethyl) analogue via Ugi-azide reaction. Their linkers between the quinoline and the tert-butyltetrazole moieties differ in chain length, basicity and substitution. Compounds were tested for their antiplasmodial activity against Plasmodium falciparum NF54 as well as their cytotoxicity against L-6-cells. The activity and the cytotoxicity were strongly influenced by the linker and its substitution. The most active compounds showed good activity and promising selectivity.


Introduction
Malaria, a vector-borne parasitic disease, is still one of the most dangerous infectious diseases worldwide. With more than 200 million cases in 2019 and 400.000 deaths malaria is a huge burden for mostly sub-Saharan African, South American, Southeast Asian countries [1]. Mostly children under the age of five are prone to a critical and fatal course of disease. Malaria is caused by one of five human pathogens of the genus Plasmodium with Plasmodium falciparum and Plasmodium vivax being responsible for most of the infections. P. falciparum is responsible for a majority of fatal outcomes and is therefore considered the deadliest parasite of these five pathogens [2]. Especially P. falciparum's rapid development of resistances against established drugs is of concern. Even against artemisinin derivatives, which are usually combined with other drugs in the first-line treatment of malaria, first resistances are emerging [3,4]. Because of the threat of potentially untreatable malaria infections new active compounds are urgently needed.
The discovery of chloroquine in the 1960s was an essential early turning point to improve malaria treatment. Unfortunately, first resistances were discovered fairly soon. However, chloroquine with its quinoline moiety is still an attractive pharmcophore, possessing limited toxicity, simple cost-effective synthesis and high clinical efficacy [5][6][7]. A promising strategy for developing new antimalarials and circumventing resistances is quinoline hybridization [8,9] Pandey et al. published a series of compounds where a 4-aminochinoline moiety was linked with a tetrazole ring. In this case an aminophenyl motive functioned as a linker. These compounds possessed antimalarial activities in the submicromolar range. Especially the tetrazole ring was supposed to be important for complexing heme [10]. Recently we published a series of 7-chloroquinoline derivatives that were linked with a tetrazole ring as well as different lipophilic aliphatic and aromatic side chains. A piperidine ring was used as a linker instead of the aminophenyl moiety. These compounds showed distinct antimalarial activities [11].
The focus of the current work was the synthesis of novel active compounds by using a hybridization approach. The 8-amino-6-methoxyquinoline pharmacophore is part of antimalarials like primaquine and tafenoquine and was therefore used as a central element of the new derivatives. (Figure 1) It was linked to a tetrazole ring via different linkers. Furthermore, lipophilic side chains were varied. All new compounds were characterized and tested in vitro for their activities against P. falciparum. The results were compared to those of formerly prepared analogues and of drugs in use.
Molecules 2021, 26, x FOR PEER REVIEW 2 of 13 ring as well as different lipophilic aliphatic and aromatic side chains. A piperidine ring was used as a linker instead of the aminophenyl moiety. These compounds showed distinct antimalarial activities [11].
The focus of the current work was the synthesis of novel active compounds by using a hybridization approach. The 8-amino-6-methoxyquinoline pharmacophore is part of antimalarials like primaquine and tafenoquine and was therefore used as a central element of the new derivatives. (Figure 1) It was linked to a tetrazole ring via different linkers. Furthermore, lipophilic side chains were varied. All new compounds were characterized and tested in vitro for their activities against P. falciparum. The results were compared to those of formerly prepared analogues and of drugs in use.
Compounds 13-22 were obtained by an Ugi-azide reaction of amine 2 with tert-butyl isocyanide, trimethylsilyl azide and various aldehydes to investigate the influence of different side chains adjacent to the newly formed tetrazole group [14].
In case of compounds 7-12 an additional ethyl linker was integrated into the molecule to examine how a greater distance between the 8-amino group of the quinoline moiety and the tetrazole ring impacts the activity. A straightforward nucleophilic substitution reaction of compound 2 with 2-chloroethyl-1-amine hydrochloride to amine 6 did not give any product. Therefore, we looked for a different approach and the linker was synthesized via multiple steps. At first compound 2 reacted with chloroacetyl chloride to an amide 3 [15,16]. The terminal chloro group of the newly formed compound was subsequently substituted by an azide group using sodium azide in DMF as solvent [16]. This azide group of 4 was converted to an amine in a Staudinger reaction by using triphenylphosphine [16]. In a final reaction with LiAlH4 the amide group of 5 was reduced yielding amine 6 [17]. Different synthesis methods for compounds 3, 5 and 6 are known, but without any NMR data given for these substances [18][19][20][21].
Afterwards compound 6 was converted to compounds 7-12 via Ugi-azide reactions. For this reaction the aldehyde component was varied to obtain compounds with different aliphatic and aromatic side chains close to the tetrazole moiety (Scheme 1) [14].
Compounds 13-22 were obtained by an Ugi-azide reaction of amine 2 with tert-butyl isocyanide, trimethylsilyl azide and various aldehydes to investigate the influence of different side chains adjacent to the newly formed tetrazole group [14].
In case of compounds 7-12 an additional ethyl linker was integrated into the molecule to examine how a greater distance between the 8-amino group of the quinoline moiety and the tetrazole ring impacts the activity. A straightforward nucleophilic substitution reaction of compound 2 with 2-chloroethyl-1-amine hydrochloride to amine 6 did not give any product. Therefore, we looked for a different approach and the linker was synthesized via multiple steps. At first compound 2 reacted with chloroacetyl chloride to an amide 3 [15,16]. The terminal chloro group of the newly formed compound was subsequently substituted by an azide group using sodium azide in DMF as solvent [16]. This azide group of 4 was converted to an amine in a Staudinger reaction by using triphenylphosphine [16]. In a final reaction with LiAlH 4 the amide group of 5 was reduced yielding amine 6 [17]. Different synthesis methods for compounds 3, 5 and 6 are known, but without any NMR data given for these substances [18][19][20][21].
Afterwards compound 6 was converted to compounds 7-12 via Ugi-azide reactions. For this reaction the aldehyde component was varied to obtain compounds with different aliphatic and aromatic side chains close to the tetrazole moiety (Scheme 1) [14]. The structures of all newly synthesized compounds were clarified by one-and twodimensional NMR spectroscopy. Products of the Ugi-azide reaction showed characteristic signals in the NMR spectra of compounds 7-22. Their tert-butyl group gave an intense proton resonance at low frequencies as well as carbon resonances at about 30 and 61.5 ppm. A longrange coupling was observed from their new methine proton to the tetrazole carbon which gave resonances at 155-157 ppm depending on substitution of the methine carbon atom. 1 H-and 13 C-NMR-spectra are given in the Supplementary Materials.

Antiplasmodial Activity and Cytotoxicity
All compounds were tested for their antiplasmodial activity against the chloroquinesensitive strain NF54 of P. falciparum. Further on their cytotoxicity was determined using skeletal myoblasts (L-6 cells). As standards served chloroquine and podophyllotoxin (Table 1).
The prepared compounds showed heterogeneous activities against P. falciparum NF54. The structures of all newly synthesized compounds were clarified by one-and twodimensional NMR spectroscopy. Products of the Ugi-azide reaction showed characteristic signals in the NMR spectra of compounds 7-22. Their tert-butyl group gave an intense proton resonance at low frequencies as well as carbon resonances at about 30 and 61.5 ppm. A longrange coupling was observed from their new methine proton to the tetrazole carbon which gave resonances at 155-157 ppm depending on substitution of the methine carbon atom. 1 H-and 13 C-NMR-spectra are given in the Supplementary Materials.

Antiplasmodial Activity and Cytotoxicity
All compounds were tested for their antiplasmodial activity against the chloroquinesensitive strain NF54 of P. falciparum. Further on their cytotoxicity was determined using skeletal myoblasts (L-6 cells). As standards served chloroquine and podophyllotoxin ( Table 1).
The prepared compounds showed heterogeneous activities against P. falciparum NF54.  In the case of compounds 7-12 the least active compound was 7 (Pf NF54 IC 50 = 15.98 µM) with an ethyl side chain. A more lipophilic phenyl ring as side chain improved antiplasmodial activity in general, which is apparent in compounds 8-12 (Pf NF54 IC 50 = 7.05-2.51 µM). In this series the most active compounds were 11 and 12 (Pf NF54 IC 50 = 2.92-2.51 µM) with a 4-bromophenyl and a 2-(trifluoromethyl)phenyl side chain, respectively. Replacement of the bromine atom by a hydrogen, a fluorine or a chlorine atom led to less active compounds 8-10 (Pf NF54 IC 50 = 7.05-5.34 µM).
Compounds 13-22 with the methyl linker were generally more active. The least active compound 13 (Pf NF54 IC 50 = 23.60 µM) was again the one with the ethyl side chain. Its more lipophilic phenyl analogue 14 (Pf NF54 IC 50 = 5.12 µM) showed improved, but still weak activity. Substitution in ring position 4 of the phenyl ring with a fluorine atom, a methyl or an isopropyl group as well as its replacement with a 1-naphthyl moiety further increased the antiplasmodial activity (
2-Chloro-N-(6-methoxyquinolin-8-yl)acetamide (3): 6-Methoxyquinolin-8-amine 2 (0.523 g (3.00 mmol)) was dissolved in in dry CH 2 Cl 2 (15 mL) and cooled to 0 • C with an ice bath. Triethylamine (2.079 mL (15.00 mmol)) was added and the mixture was stirred for 10 min. Chloroacetyl chloride (0.477 mL (6.00 mmol)) in dry CH 2 Cl 2 (15 mL) was added dropwise via a dropping funnel. The ice bath was removed and the reaction mixture stirred at 25 • C for 20 h. Then, the reaction was quenched with 2N NaOH at 0 • C and the mixture was basified to a pH of 10-11. The aqueous and organic phases were separated and the aqueous phase was extracted with CH 2 Cl 2 . The combined organic phases were washed with 0.1% aqueous NaHCO 3 and dried over anhydrous sodium sulfate and filtered. The solvent was removed in vacuo and the obtained raw product purified by column chromatography (silica gel, diethyl ether) to yield compound 3 as off-white amorphous solid (0.700 g (93%)). 2-Azido-N-(6-methoxyquinolin-8-yl)acetamide (4): Compound 3 (0.702 g (2.80 mmol)) was dissolved in dry DMF (30 mL) and cooled to 0 • C with an ice bath. Sodium azide (0.364 g (5.60 mmol)) was added in small portions and after that the ice bath was removed and the reaction mixture stirred at 25 • C for 48 h. Then, the reaction was quenched with 0.1% aqueous NaHCO 3 (50 mL). The aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with 0.1% aqueous NaHCO 3 and dried over anhydrous sodium sulfate and filtered. The solvent was removed in vacuo and the obtained raw product purified by column chromatography (silica gel, ethyl acetate/ethanol 6:1) to yield compound 4 as off-white amorphous solid (0.634 g (88%)). 1 (5): Compound 4 (0.630 g (2.45 mmol)) was dissolved in THF/H 2 O 6:1 (40 mL) at room temperature. Triphenylphosphine (1.26 g (4.90 mmol)) was added in small portions. Followed by that the brown reaction mixture was refluxed for 20 h. The mixture was cooled to room temperature and the solvent removed in vacuo. The residue was adsorbed on a column with silica gel and excess triphenylphosphine and triphenylphospine oxide were removed by elution with diethyl ether. Elution of the product with CH 2 Cl 2 /MeOH 1:1 afforded a raw product which was further purified by column chromatography (silica gel, ethyl acetate/ethanol 6:1) to yield compound 5 as orange amorphous solid (0.504 g (89%)). 1  N 1 -(6-Methoxyquinolin-8-yl)ethan-1,2-diamine (6): Compound 5 (0.509 g (2.20 mmol)) was dissolved in dry diethyl ether (30 mL) and cooled to 0 • C with an ice bath. Pulverized LiAlH 4 (0.167 g (4.40 mmol)) was added slowly in small portions and the reaction mixture was stirred for 30 min at 0 • C. Then the mixture was refluxed for 20 h. The suspension was cooled to 0 • C and quenched with 2N NaOH and was basified to a pH of 10-11. The aqueous and organic phases were separated and the aqueous phase was extracted with diethyl ether. The combined organic phases were dried over anhydrous sodium sulfate and filtered. The solvent was removed in vacuo and the obtained raw product purified by column chromatography (silica gel, CH 2 Cl 2 /MeOH 5:1) to yield compound 6 as off-white amorphous solid (0.277 g (58%)). 1  The general procedure for the Ugi-azide reaction (7-22) is as follows: 6-Methoxyquinoline-8amine 2 (0.75 mmol) or compound 6 (0.75 mmol) were dissolved in dry methanol (5 mL). The corresponding aldehyde (0.75 mmol) was added and the mixture stirred at room temperature for 1 h under an argon atmosphere. Trimethylsilyl azide (0.75 mmol) and tert-butyl isocyanide (0.75 mmol) were added dropwise and the reaction mixture was stirred for 20-120 h. After that, the solvent was evaporated in vacuo and the residue was dissolved in CH 2 Cl 2 . The solution was washed several times with 30% aqueous sodium disulfite followed by 0.1% aqueous NaHCO 3 . The organic phase was dried over anhydrous sodium sulfate and filtered. The solvent was removed in vacuo, yielding the raw products 7-22, which were further purified by column chromatography.

Conclusions
A series of 8-amino-6-methoxyquinoline hybrids was prepared via Ugi-azide reaction. The compounds exhibit different linkers between a 6-methoxyquinolin-8-amine moiety and a tetrazole ring. Compounds with a short and non-basic linker with lipophilic substitution showed the highest antiplasmodial activity. Six of the new derivates have promising selectivity due to their low cytotoxity. The optimum linker length will be investigated in a future project.

Data Availability Statement:
The data presented in this study are available in this article.