Benzyl- and dibenzyl tetrahydropyridinylidene ammonium salts with antiplasmodial and antitrypanosomal activity

Several 1-benzyl and 1,3-dibenzyl derivatives of tetrahydropyridinylidene salts with differing electron withdrawing substituents at the aromatic residues have been prepared. In addition, the amine moiety in position 4 was varied. The new compounds were investigated for their antiplasmodial and antitrypanosomal activities as well as for their cytotoxicity. They were characterized using FT-IR, HRMS and NMR spectroscopy. Structure–activity relationships including reported compounds are discussed.


Introduction
Neglected Tropical Diseases (NTDs) and infectious illnesses, such as malaria, tuberculosis and Zika fever, represent a major public health concern in many countries and regions worldwide, especially in developing ones [1]. Most of the drugs available for therapy are toxic and have considerable adverse effects. A considerable number is obsolete, especially with respect to resistance.
Trypanosoma brucei is one of the protozoan parasites that penetrates the blood-brain barrier causing injury associated with toxic effects of parasite-derived molecules or with immune response against infection. Other protozoan parasites that can cause pathology in the brain tropism include Toxoplasma, Plasmodium, Amoeba and, eventually, other Trypanosomatids such as T. cruzi and Leishmania. Together, these parasites affect billions of people worldwide and are responsible for more than 500,000 deaths annually [2]. New drugs against these parasitic protozoa are urgently needed to counteract drug resistance, toxicity and the high cost of commercially available drugs [3].
Recently, we reported about the synthesis of 1-benzyl [4,5] and 1,3-dibenzyl derivatives of tetrahydropyridinylidene salts (THPS) [6]. Their activities against Trypanosoma brucei rhodesiense (T.b.r.), the protozoan pathogen of the East African form of sleeping sickness were investigated. Moreover, their activities against the sensitive NF54 strain and the multiresistant K 1 strain of Plasmodium falciparum 1 3 (P.falc.) were determined [6]. The most promising of these compounds were also investigated for their in vivo activity against Plasmodium berghei in a mouse model [4].
Due to the fact that compounds with electron withdrawing groups at the aromatic moiety show better and more selective activities, we introduced such groups in a series of new benzyl-and dibenzyl-THPS in order to reveal structure-activity relationships and to optimize the compounds regarding their activity and cytotoxicity.

Chemistry
The synthesis of the benzyl-THPS 2-5 starts from bases 1a-1c [5,7] by alkylation of the nitrogen atom of the dihydropyridine ring with benzyl halides as described earlier [4,5]. The obtained compounds 2-5 have differing substitution in ring positions 4 of the piperidine as well as of the phenyl ring. Particularly THPS with electron-withdrawing substituents showed promising activity against protozoan parasites whereas electron-donating substituents like 4-methoxy or 4-alkyl showed moderate potency. Bigger alkyl groups in position 4 causes enhanced cytotoxicity and 3,4,5-trimethoxy compounds have low activity [5,6]. Therefore, we prepared further analogues with cyano and nitro groups. In addition, the azepane moiety was chosen as a slightly more lipophilic amine component. Compounds 6-8 with an additional benzyl substituent in ring position 3 were afforded by reaction of benzyl-THPS 2-5 with benzyl halides in the presence of potassium carbonate. Analogues 9-12 with identical substitution in ring positions 1 and 3 were obtained by a one pot reaction of 1 with benzyl halides in the presence of potassium carbonate (Scheme 1).
The alkylation at the ring nitrogen in compounds 1 follows a S N 2 mechanism. The electron-pair of the ring nitrogen attacks the carbon of the aryl-alkyl halide and leads to 13, the typical transition state. Detachment of the halide ion causes a migration of double bonds giving the N-benzyl-THPS 2-5 (Scheme 2).
The formation of the 1,3-dibenzyl compounds 6-8 starts with a proton abstraction in position 3 of compounds 2-5 to intermediate 14 using potassium carbonate as base. With the aryl-alkyl halide a S N 2-type transition state 15 is supposed. By the leaving of the halide ion, 1,3-dibenzyl-THPS 6-8 are formed (Scheme 3).

Confirmation of structures
The formation of compounds 2-5 was verified by NMR spectroscopy. The successful alkylation was confirmed by the appearance of resonances of additional protons of the benzyl moiety between 4.77 and 4.95 ppm in 1 H NMR spectra. For those protons long range couplings to C-2 and C-6 of the dihydropyridine ring were observed in HMBC spectra to establish connectivity. The additional proton signals of the aromatic protons of the benzyl residues are observed too in 1 H NMR spectra. In 13 C NMR spectra the signals for the methylene group of the benzyl residues appear at around 53 ppm. The additional signals for the aromatic carbons were detected as well. The results of the HRMS measurements confirmed the proposed structures too. Finally, the structures of compounds 2-5 are also established by a single X-ray crystal analysis of a reported compound prepared in the same manner [4].
The methylene protons of the additional benzyl group of compounds 6-12 appear as two doubledoublets at around 2.5 ppm (J = 13 and 10 Hz) and around 3 ppm (J = 13 and 5 Hz). The coupling with the smaller coupling constant is due to the interaction with the remaining proton in position 3, respectively. Furthermore, additional cross-peaks to H-3 in the H,H-COSY spectra, as well as long range couplings to C-4 in the HMBC spectra were observed to verify the position of the substitution. In 13 C NMR spectra the signals for the methylene group attached to position 3 appear at around 35 ppm. The signal for C-3 shifted from around 39-45 ppm due to substitution and was detected in DEPT spectra as CH group instead of the former CH 2 group. HRMS measurements confirmed the proposed structures of 6-12 as well.

3
From a similar reported compound prepared by the same way, a single X-ray structure analysis is described [6].

Antiprotozoal activities
All new THPS were investigated for their activity against chloroquine sensitive P.falc. NF54 and T.b.r. as well as for their cytotoxicity against L-6 cells using microplate assays. The results are listed in Table 1. For comparative reasons known compounds were included. The most promising compounds with high antiplasmodial activity and high selectivity were additionally tested against P.falc. K 1 .

Structure-activity relationships
The above stated observation, that the presence of electron withdrawing substituents at the aromatic moiety increase the antiplasmodial activity was confirmed for compounds with only one benzyl residue attached to the ring nitrogen. Furthermore, due to the selection of 4-nitro and 4-cyano substituents, the selectivity was increased significantly due to the much lower cytotoxicity compared with 4-chloro substituted compounds. For the bis-benzyl substituted compounds we observed that the substitution pattern of 4-nitro and 4-cyano substituted benzyl residues in position 3 and at the same time unsubstituted benzyl residues at the ring nitrogen are advantageous for both, antiplasmodial activity and selectivity of compounds. As a result, compounds with outstanding activity and selectivity were yielded. The introduction of 4-nitro and 4-cyano substituted benzyl residues to both positions resulted in lower potent compounds.
The insertion of the bigger azepane ring as amino moiety in position 4 was in general advantageous, often the compounds with this substitution were the most active within their series.

Conclusion
To continue our studies about the antiprotozoal activities of tetrahydropyridinylidene ammonium salts, we prepared 1-substituted benzyl and 1,3-disubstituted dibenzyl derivatives with electron withdrawing substituents at the aromatic moieties, since such substituents were identified to be advantageous for antiplasmodial action. 4-Cyano and 4-nitro compounds show increased antiplasmodial potency and raised selectivity due to less cytotoxicity compared to 4-chloro compounds. In addition to that, the larger azepane ring was introduced as an amino substituent in ring position 4 giving highly active compounds. The most promising of the new compounds is the 1-benzyl-3-(4-nitrobenzyl) derivative 8b showing low cytotoxicity and antiplasmodial activity against a sensitive and a multiresistant strain of Plasmodium falciparum in low nanomolar concentration. The main goal, to improve the biological activity and to decrease cytotoxicity was reached. Physicochemical properties where calculated and a correlation between biological activities and lipophilicity of compounds was detected. Further investigations should provide insight into structure-activity relationships of these compounds and varying substitution pattern on the aromatic moieties.

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: Bruker Ascend 400, 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 ppm) was used as internal standard and for 13 C spectra the central peak of the CDCl 3 signal was used as the internal reference (77.0 ppm). Some spectra were acquired in DMSO-d 6 . In this case the central peaks of the DMSO-d 5 signal at 2.49 ppm in 1 H spectra and at 39.7 ppm in 13 C spectra served as internal reference. Abbreviations: aromatic H, ArH; aromatic C, ArC, quaternary aromatic C, ArC q . Signal multiplicities are abbreviated as follows: s, singlet; d, doublet; dd, doubledoublet; ddd, doubledoubledoublet; dt, doubletriplet; t, triplet; m, multiplet; br, broad. Coupling constants (J) are reported in Hertz (Hz). 1 H and 13 C resonances were assigned using 1 H, 1 H-and 1 H, 13 C-correlation spectra. 1  ; 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 benzyl compounds 2a, 2b, 4a, 4b, 5a, 5b is reported [4,5] as well as the synthesis of dibenzyl compounds 6a, 7a, 8a, 9a, 9b, 10a and 10b [6]. Benzyl-2,2-dimethyl-1,2,3,4-tetrahydropyridin-4-ylidene)azepan-1-ium bromide (2c, C 20 H 29 BrN 2 ) A solution of 1.159 g of 1c (5.62 mmol) and 1.604 g of benzyl bromide (9.38 mmol) in 25 cm 3 of CHCl 3 was stirred at r.t. for 20 h. A part of the solvent was evaporated in vacuo and cooled with an ice bath. Ethyl acetate was added until crystallisation seemed to be complete. The solid was sucked off and dried giving 1.540 g (73%) of 1c as bright yellow crystals.

4tetrahydropyridin-4-ylidene]pyrrolidin-1-ium bromide (11a, C 25 H 29 BrN 4 O 4 )
To a solution of 1 g of 1a (5.64 mmol) in 25 cm 3 of CHCl 3 2.03 g of 4-nitrobenzyl bromide (9.4 mmol) were added. It was stirred at r.t. for two days and ethyl acetate was added to the already turbid solution. The formed precipitate was sucked off giving 1.98 g (89%) of the monosubstituted product 4a. 950 mg of 4a (2.41 mmol) and 627 mg of 4-nitrobenzyl bromide (2.9 mmol) in 40 cm 3 of CHCl 3 were refluxed overnight in the presence of 2.7 g of K 2 CO 3 (19.5 mmol). Then 100 cm 3 of CHCl 3 were added and the mixture was treated with charcoal and filtered. The solvent was evaporated in vacuo and the residue was dissolved in acetone and ethyl acetate was added until the solution got turbid. Upon stirring on an ice bath, a precipitate was formed which was sucked off and washed with ethyl acetate. It was recrystallized three times from acetone giving bright brown needles containing acetone. They were sucked off and dissolved in CH 2 Cl 2 . The solvent was evaporated to yield 120 mg of 11a (4%) as a brownish foam.

(3RS)-(±)-N-[1,3-Bis(4-cyanobenzyl)-2,2-dimethyl-1,2,3,4tetrahydropyridin-4-ylidene]pyrrolidin-1-ium bromide (12a, C 27 H 29 BrN 4 )
A mixture of 1.615 g of 1a (9.06 mmol) and 3.919 g of 4-bromomethyl benzonitrile (19.99 mmol) in 50 cm 3 of CHCl 3 was stirred for 6 d at r.t. in the presence of 4.673 g of K 2 CO 3 (33.81 mmol). The reaction mixture was treated with charcoal, filtered and the solvent was evaporated in vacuo. The residue was dissolved in acetone and ethyl acetate was added until the first turbidity appeared. While stirring at r.t. and then on an ice-bath a precipitate was formed which was sucked of and recrystallized from acetone giving a mixture of bis-and monosubstituted products as white powder. In order to complete the reaction, the mixture was dissolved in 35 cm 3 of CHCl 3 and refluxed overnight in the presence of 2.215 g of K 2 CO 3 (16.03 mmol) and 996 mg 4-bromomethyl benzonitrile (5.08 mmol). Then 15 cm 3 of CHCl 3 were added and the mixture was treated with charcoal and filtered. The solvent was evaporated in vacuo and the residue was dissolved in acetone. Then ethyl acetate was added until the first turbidity appeared. While stirring at r.t. and then on an ice-bath a precipitate was formed which was sucked of, recrystallized from acetone and dried giving 0.817 g of 12a (18%) as white powder containing acetone. Therefore, it was dissolved in CHCl 3 and the solvent was evaporated to yield a white foam.

In vitro growth inhibition assay of Plasmodium falciparum NF54
In vitro activity against erythrocytic stages of P. falciparum was determined using a 3 H-hypoxanthine incorporation assay [8,9], using the drug sensitive NF54 strain (Schipol Airport, The Netherlands, [10]) and the standard drug chloroquine (Sigma C6628). Compounds were dissolved in DMSO at 10 mg/cm 3 and added to parasite cultures incubated in RPMI 1640 medium without hypoxanthine, supplemented with HEPES (5.94 g/dm 3 ), NaHCO 3 (2.1 g/dm 3 ), neomycin (100 U/cm 3 ), Albumax R (5 g/dm 3 ) and washed human red cells A + at 2.5% haematocrit (0.3% parasitaemia). Serial drug dilutions of eleven threefold dilution steps covering a range from 100 to 0.002 μg/cm 3 were prepared. The 96-well plates were incubated in a humidified atmosphere at 37 °C; 4% CO 2 , 3% O 2 , 93% N 2 . After 48 h 50 mm 3 of 3 H-hypoxanthine (= 0.5 μCi) was added to each well of the plate. The plates were incubated for a further 24 h under the same conditions. The plates were then harvested with a Betaplate ™ cell harvester (Wallac, Zurich, Switzerland), and the red blood cells were transferred onto a glass fibre filter and then washed with distilled water. The dried filters were inserted into a plastic foil with 10 cm 3 of scintillation fluid and counted in a Betaplate ™ liquid scintillation counter (Wallac, Zurich, Switzerland). IC 50 values were calculated from sigmoidal inhibition curves by linear regression [11] using Microsoft Excel. Chloroquine was used as control.

In vitro growth inhibition assay of Trypanosoma b. rhodesiense
Minimum Essential Medium (50 mm 3 ) supplemented according to [12] with 25 mM HEPES, 1 g/dm 3 additional glucose, 1% MEM non-essential amino acids (100x), 0.2 mM 2-mercaptoethanol, 1 mM Na-pyruvate and 15% heat inactivated horse serum was added to each well of a 96-well microtiter plate. Serial drug dilutions of eleven threefold dilution steps covering a range from 100 to 0.002 μg/cm 3 were prepared. Then 4 × 10 3 bloodstream forms of T. b. rhodesiense (STIB 900) in 50 mm 3 was added to each well and the plate incubated at 37 °C under a 5% CO 2 atmosphere for 70 h. 10 mm 3 Alamar Blue (resazurin, 12.5 mg in 100 cm 3 double-distilled water) was then added to each well and incubation continued for a further 2-4 h [13]. Then the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wave length of 536 nm and an emission wave length of 588 nm. The IC 50 values were calculated by linear regression [11] from the sigmoidal dose inhibition curves using SoftmaxPro software (Molecular Devices Cooperation, Sunnyvale, CA, USA). Melarsoprol was used as control.