Modifications and hybrids of 1,2,3,4-tetrahydropyridinium salts and their antiprotozoal potencies

The antiprotozoal activity of 1-benzyltetrahydropyridin-4-yliden iminium salts is reported. This paper describes the preparation of a series of analogs from dihydropyridines or dihydrothiopyrans as educts. The new compounds were investigated for their activity against Plasmodium falciparum NF54, a causative organism of Malaria tropica and Trypanosoma brucei rhodesiense, the causative organism of Human African Trypanosomiasis (sleeping sickness). Several structure–activity relationships were detected. Both the substituents in ring positions 1 and 4 of the tetrahydropyridinium moiety had a strong impact on the antiprotozoal activities as well as on the cytotoxicity of compounds against L-6 cells (rat skeletal myoblasts). All new compounds were characterized using FT-IR spectroscopy, HRMS, and NMR spectroscopy.


Introduction
In 2019, an estimated 229 million cases of malaria, leading to estimated 409 000 deaths, occurred worldwide. Children aged under 5 years are the most vulnerable group affected by malaria. In 2019, they accounted for 67% of all malaria deaths worldwide. Plasmodium falciparum is the most prevalent malaria parasite [1]. There is currently a restricted arsenal of drugs [2] and the extension of Plasmodium falciparum resistance to existing antimalarial drugs is worrying. Faced with this problem, the search for new and effective compounds is necessary [3].
Human African Trypanosomiasis (HAT), also known as sleeping sickness is one of 20 neglected tropical diseases listed by the World Health Organization, which lead to death if left untreated [4]. This disease is caused by Trypanosoma brucei gambiense, which causes the chronic form of the 1 3 disease in western and central Africa, and by T. brucei rhodesiense (Tbr), which causes the acute form of the disease in eastern and southern Africa [5]. Currently, melarsoprol, an old arsenical drug, is the only drug available for the latestage Tbr infection treatment [6]. Unfortunately, it causes a deadly encephalopathy in more than 5% of the patients [7].
We already reported the antiprotozoal activities of tetrahydropyridin-4-ylidene ammoniumsalts [8]. Since, 1-benzyl substitution significantly enhanced the antiprotozoal activities, we initially focused our efforts on the optimization of these benzyl moieties [9,10]. To clear up the influence of the substituents attached to the ring nitrogen on the biological activities, we prepared compounds with smaller (methyl-) and larger (phenetyl-, indolylethyl-) residues at this position. Since, 4-chlorobenzyl derivatives showed enhanced activities [9,10], we prepared some derivatives with other exocyclic amino residues. In addition to that, we prepared some hybrid molecules bearing partial structures of chloroquine, the diethylaminopentyl-and the 7-chloroquinolin-4-yl residue on different positions. This paper reports the synthesis of a series of new tetrahydropyridin-4-ylidene ammonium salts and their activities against Plasmodium falciparum NF54 and Trypanosoma brucei gambiense.
The preparation of the base 1f started from the 6-sulfanylidenpiperidin-4-one 8 [11] which reacted with tryptamine to the corresponding 4-amino derivative 9 in a similar procedure as reported [8]. The sulfanylidene group was methylated. Desulfurization was achieved selectively with Raney nickel. The obtained dihydropyridin-4(1H)-imine 1f was alkylated with 4-chlorobenzyl chloride giving 10f (Scheme 2). The 1 H NMR spectrum of compound 10f showed two sets of signals belonging to the corresponding (E) and (Z) forms. NOE-experiments established the main component as (Z) form. For the (E) form a through-space coupling from H-3 to H-1′ was observed, whereas, for the (Z) form H-5 showed a through-space coupling to H-1′ (Fig. 1).
For the preparation of compounds with a partial structure of chloroquine, we used differing pathways: the chlorobenzyl derivative 10g exhibits the aminoalkyl side-chain of chloroquine in ring position 4. It was synthesized from its N,Ndimethyliminium analog 7c. Hydrolysis of the iminium salt yielded the respective dihydropyridin-4(1H)-one 11. Reaction of 11 with N 1 ,N 1 -diethylpentane-1,4-diamine gave compound 10g (Scheme 3).
For the synthesis of compounds with the 7-chloroquinolin-4-amine part of chloroquine, we started from the thiopyrane derivative 12 [12]. The 5,6-dihydropyridin-2(1H)thiones 13 and 16 were obtained by a aminolysis/Dimroth rearrangement sequence. Subsequent reaction of 13 with iodomethane yielded the methylsulfanyl derivative 14. Surprisingly, we were not able to remove the methylthio group via the usual reduction process with Raney nickel. Not a trace of 15 was found in the reaction mixture (Scheme 4).
All compounds were investigated for their antiplasmodial and antitrypanosomal activities against Plasmodium falciparum NF54 and Trypanosoma brucei rhodesiense, respectively. In addition, the cytotoxicity was determined using L-6 cells. The results are presented in Table 1.
Tetrahydropyridin-4-iminium halides 3-7 with lipophilic and bulky groups at the ring nitrogen showed antiplasmodial activity against Plasmodium falciparum NF54 in low concentration (IC 50 = 0.019-0.3 µM), whereas, their 1-methyl analogs 2a and 2b were practically ineffective (IC 50 = 2.80-3.22 µM). Due to their usually low cytotoxicity most of them showed very promising selectivity (SI PN = 268-9207). The most promising compound of this series had a 4-cyanobenzyl group in ring position 1 and a pyrrolidinium moiety. It shows high

Free-Wilson analysis
Free-Wilson analysis is a QSAR method to assign a contribution to the overall activity to each occurring substitution group in an SAR dataset using the following equation: BA i the biological activity of a series is expressed as the sum of the biological activity contributions a jk of the substituents R k in each position j, µ is referring to the overall average activity value for the series [13].
A Free-Wilson least squares model was calculated in Biovia's Pipeline Pilot with the script "Create Free-Wilson least squares model". The Free-Wilson predicted activity was based on a 17-compound subset (2a-10g) of the tested molecules that shared.
The Free-Wilson analysis based on the pIC 50 values for activities against Plasmodium falciparum NF54 led to a model with an R 2 of 0.906. For the individual groups, the contributions which were calculated are presented in Table 2.
The different contributions of substitutions to the total activity against Plasmodium falciparum NF54 in the R1 position show that the tetrahydropyridin-4-iminium halides

Scheme 5
Reagents and conditions: (i) pyrrolidine or piperidine, reflux, 4 h. show an increase in activity with a larger ring size with R1 B (compound 7e) yielding a higher activity contribution than R1 F and R1 A. Negative contributions are found for the R1 indol substitution (R1 C) and the N 1 -diethylpentane group (R1 E). The substitutions in the R2 position all showed a positive contribution, the strongest being observed in the chlorophenyl groups (R2 C/D) with the chlorobenzyl group yielding the best contribution (1.9352).

Conclusion
A number of tetrahydropyridin-4-yliden iminiumsalts and a few related compounds have been prepared in several steps from dihydropyridines or dihydrothiopyrans as starting compounds. The new compounds were tested for antiplasmodial activity against Plasmodium falciparum NF54 as well as for antitrypanosomal activity against Trypanosoma brucei rhodesiense. Furthermore, their cytotoxicity against L6-cells was determined. Some tetrahydropyridin-4-yliden iminium salts with large and lipophilic substituents at the tetrahydropyridine nitrogen atom showed high antiplasmodial activity  and selectivity. The most promising compound of this series has a 4-cyanobenzyl substituent in ring position 1 and a pyrrolidinium moiety in position 4. It showed activity in low concentration (IC 50 = 0.029 µM) and possessed excellent selectivity (SI PN = 9207). Noteworthy antitrypanosomal activity was observed for a tetrahydropyridin-4-yliden iminium salt with in total 3 benzyl substituents on the nitrogen atoms. However, due to its high cytotoxicity the selectivity index was quite low. Far better selectivity was observed for an analog with an additional methylsulfanyl group in ring position 2. The effect of an amino substituent in the same ring position was non-uniform. Further modifications at this ring position are in progress.

Experimental
Melting points were obtained on a digital melting point apparatus Electrothermal IA 9200. IR spectra: Bruker Alpha Platinum ATR FT-IR spectrometer (KBr discs). NMR spectra: Varian Inova 400 (300 K) 5 mm tubes, spectra were acquired in CDCl 3 containing 0.03% TMS. Chemical shifts were recorded in parts per million (ppm), for 1 H spectra TMS (0.00) was used as internal standard and for 13 C spectra the central peak of the CDCl 3 peak was used as the internal reference (77.0). Some spectra were acquired in DMSO-d 6 .
Here ; the substances were detected in UV light at 254 nm. If no stationary phase is mentioned (CC and TLC) the separation took place using silica gel. The preparation of the hydroiodides of compounds 1a-1e was reported earlier [8].
The bases were set free by shaking with 2 M NaOH and subsequent extraction with CHCl 3 . The preparation of compounds 7a [9] and 7b [10] was already reported. Compound 8 was prepared according to a reported procedure [11]. Its melting point (139 °C) corresponds well with the reported one (Ref. [11] 138 °C). Synthesis of compound 12 (m.p.: 198 °C) was done at our institute as already described [12] as well as the preparation of 17a (m.p.: 160 °C [14]) and 17b (m.p.: 188 °C [11]); the melting points are identical with the reported ones. Compounds 18a is described, the melting point (192 °C) does not correspond very well with the reported one (204 °C) [15] maybe due to the use of different solvents. The preparation of compound 18b is also described, but no melting point is given [15]. Therefore, we support full data for compounds 18a and 18b.

1-(4-Chlorobenzyl)-2,3-dihydro-2,2-dimethylpyridin-4(1H)-one (11, C 14 H 16 ClNO)
To a suspension of 1.12 g of 7c (2.72 mmol) in 20 cm 3 of benzene, 15 cm 3 of 2 M NaOH were added. The mixture was refluxed overnight and cooled to room temperature. The aqueous phase was diluted with 20 cm 3 of water and the phases were separated. The water phase was extracted three times with benzene. Then the organic layers were combined and washed with water. After drying (Na 2 SO 4 ) and filtration, the solvent was evaporated in vacuo giving 680 mg of 11 (98%) as a yellowish resin. 1

(RS)-()-4-[[1-(4-Chlorobenzyl)-2,2-dimethyl-1,2,3,4-tetrahydropyridin-4-yliden]amino]-N,N-dimethylpentanamin (10 g, C 23 H 36 ClN 3 )
The residue from co-distillation of 613 mg of 11 (2.45 mmol) with benzene was dissolved in 20 cm 3 of toluene. Then 778 mg of N 1 ,N 1 -diethylpentane-1,4-diamine (4.9 mmol) and 273 mg of glacial acetic acid (4.55 mmol) were added and the mixture was refluxed overnight using a Dean-stark apparatus, filled with 0.4 nm activated molecular sieves. The solvent was evaporated in vacuo and the residue was subjected to CC over basic aluminium oxide using (CH 2 Cl 2 :MeOH = 8:1) as eluent. The fractions containing pure products were combined and evaporated, giving 40 mg of 10g (4.2%). 1  . During the reaction, compressed air was passed through the reaction mixture. After 15 d the solvent was evaporated in vacuo and the residue crystallized from chloroform/ethyl acetate. The precipitate was dissolved in hot ethanol, treated with charcoal, and filtered and part of the solvent was evaporated. Crystallization took place overnight and the solid was sucked off and used for the synthesis of thione 16. The mother liquor was evaporated and the residue crystallized from acetone via stirring overnight at room temperature. It was sucked off and dried to get the desired compound 13. The solid was stirred with 2 M NaOH, extracted 3 times with chloroform and washed twice with water. Then it was dried (Na 2 SO 4 ), filtered, and evaporated to get the free base. This was dissolved in 50 cm 3 N-[1-[2-[(7-Chloroquinolin-4-yl) N-(2,2-Dimethyl-6-pyrrolidino-1,2,3,4

-tetrahydropyridin-4-ylidene)pyrrolidin-1-ium iodide (18a, C 15 H 26 IN 3 )
A suspension of 1.96 g of 17a (5.56 mmol) in 20 g of pyrrolidine (0.28 mol) was refluxed for 4 h on an oil bath. The pyrrolidine was removed by evaporation in vacuo and the residue was dissolved in the minimum amount of hot propan-2-ol. The product precipitated by addition of ethyl acetate, giving 1.

In vitro assays
The in vitro growth inhibition assay of Plasmodium falciparum NF54 and the in vitro growth inhibition assay of Trypanosoma b. rhodesiense, as well as the assay for the determination of cytotoxicity against L6-cells were performed as described earlier [16].
Funding Open access funding provided by University of Graz.
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