Antiprotozoal Activity of Azabicyclo-Nonanes Linked to Tetrazole or Sulfonamide Cores

N-(Aminoalkyl)azabicyclo[3.2.2]nonanes possess antiplasmodial and antitrypanosomal activity. A series with terminal tetrazole or sulfonamido partial structure was prepared. The structures of all new compounds were confirmed by NMR and IR spectroscopy and by mass spectral data. A single crystal structure analysis enabled the distinction between isomers. The antiprotozoal activities were examined in vitro against strains of Plasmodium falciparum and Trypanosoma brucei rhodesiense (STIB 900). The most active sulfonamide and tetrazole derivates showed activities in the submicromolar range.


Introduction
Malaria and Human African Trypanosomiasis (HAT) both are tropical diseases transmitted to people by the bite of infected insects.
Malaria is caused by Plasmodium parasites. In 2020, there were about 241 million estimated cases of malaria and about 627 000 reported deaths [1]. There are five types of human malaria parasites, but the majority of infections are caused by P. falciparum, the most deadly malaria parasite [2]. Many strains of P. falciparum have become resistant to previous generations of medicines [1]. In recent years, resistance even to recommended artemisinin-based therapies has become prevalent across an expanding area of Southeast Asia [3][4][5][6]. Therefore, the development of new drugs for the fight against the most deadly strains of P. falciparum is absolutely essential.
HAT, also known as sleeping sickness, is caused by Trypanosoma parasites. After continued control efforts, the number of reported cases dropped below 1000 in 2019 [7]. However, transmitted by the tsetse fly, it puts 55 million people at risk and is fatal if untreated [8].
In the case of T. b. rhodesiense infections, the disease is acute, lasting from a few weeks to several months, while in case of T. b. gambiense infections, the disease is chronic, generally lasting several years without any major signs or symptoms. Sleeping sickness is difficult to treat considering the toxicity and complex administration of the drugs currently in use. Only a few drugs are available for the therapy of HAT: pentamidine, suramin, melarsoprol, nifurtimox, eflornithine and fexinidazole. For the treatment of T. b. rhodesiense infections of the central nervous system, melarsoprol is the only effective drug [7]. Unfortunately, melarsoprol causes an encephalopathy that kills 5% of the patients [9]. Therefore, the development of new drugs against Human African Trypanosomiasis is still required. This paper reports the synthesis and the antiprotozoal activities of azabicyclo[3.2.2]nonanes with terminal tetrazole or sulfonamido partial structure. The structures of all newly synthesized compounds were elucidated by 1D-and 2D-NMR spectroscopy. Their activities against strains of P. falciparum and against T. b. rhodesiense were investigated in vitro and the results compared to those of formerly prepared analogues and of drugs in use.
The syntheses of 2-azabicyclo[3.2.2]nonanes 1-3 and 3-azabicyclo[3.2.2]nonanes 4, 5 as starting materials were already described elsewhere [10,11]. They were refluxed with 2-chloroacetamide in EtOH yielding their carbamoylmethyl derivates, which were hydrogenated using LiAlH4, yielding the corresponding N-(2-aminoalkyl) analogues 6-10. Afterward, 6-10 were converted to tetrazoles 11-15 in moderate to good yields at mild conditions via the Ugi-azide reaction with diverse aldehydes, tert-butylisocyanide and trimethylsilylazide (Scheme 1).  The structures of all newly synthesized compounds were clarified by one-and twodimensional NMR spectroscopy. Successful alkylation of the ring nitrogen atom of 2azabicyclononanes was obvious from 7 ppm downfield shifts of the 13 C resonances of the adjacent ring atoms C-1 and C-3 of compounds 6-8. The same effect was observed in the 3-azabicyclononane series for the signals of the corresponding C-2 and C-4 atoms of compounds 9, 10. The sulfonylation of the ring nitrogen atom of the 2-azabicyclononane 3 led to small downfield shifts < 2 ppm of the resonances of C-1 and C-3 of 18; however, remarkable 0.5-1.0 ppm downfield shifts were observed for the signals of 1-H and 3-H in its proton nmr spectrum. Similarly, the sulfonylation of the ethanamine nitrogen of compounds 7, 8 shifted the resonances of the 2'-H and C-2' slightly to higher frequencies, which were detected in the spectra of sulfonamides 19, 20. The formation of the tetrazole derivates 11-15 was established by a number of changes in 1 H and 13 C nmr spectra. The N-alkylation of 9, 10 caused a downfield shift of 6-7 ppm for the C-2' signal. Moreover, we observed additional resonances for the newly introduced protons and carbons of the C-1 chain, the tetrazole moiety and their substituents. Evidence of structure was provided by the expected long-range couplings from 1"-H to C-2' and C-1"', which were detected in the HMBC spectra of compounds 11-15. The distinction between isomers 11-13 was enabled by a single crystal structure analysis of compound 12B (Figure 2). The crystal structure analysis of 12B confirmed the compound as N-[(1-tert-butyl-1H-tetrazol-5-yl)(phenyl)methyl]-2-[6,9-diphenyl-1-(piperidin-1-yl)-3-azabicyclo[3.2.2]nonan-3-yl]eth-an-1-amine. All atoms lie on general positions. Owing to the absence of heavier elements, the absolute structure of the chiral molecule could not be determined reliably from the diffraction data. Arbitrarily, the structure is described as the R,R,S enantiomer ( Figure 1). The tetrazole ring has adopted the orientation where the distance from N44 to the H atom of the NH group is smallest [N30···N44 2.841(4) Å]. Presumably, the intramolecular hydrogen bond is weak because of the small angle N30-H30···N44 of 104(2) • . CCDC 2,194  The structures of all newly synthesized compounds were clarified by one-and twodimensional NMR spectroscopy. Successful alkylation of the ring nitrogen atom of 2azabicyclononanes was obvious from 7 ppm downfield shifts of the 13 C resonances of the adjacent ring atoms C-1 and C-3 of compounds 6-8. The same effect was observed in the 3-azabicyclononane series for the signals of the corresponding C-2 and C-4 atoms of compounds 9, 10. The sulfonylation of the ring nitrogen atom of the 2-azabicyclononane 3 led to small downfield shifts < 2 ppm of the resonances of C-1 and C-3 of 18; however, remarkable 0.5-1.0 ppm downfield shifts were observed for the signals of 1-H and 3-H in its proton nmr spectrum. Similarly, the sulfonylation of the ethanamine nitrogen of compounds 7, 8 shifted the resonances of the 2´-H and C-2´ slightly to higher frequencies, which were detected in the spectra of sulfonamides 19, 20. The formation of the tetrazole derivates 11-15 was established by a number of changes in 1 H and 13 C nmr spectra. The N-alkylation of 9, 10 caused a downfield shift of 6-7 ppm for the C-2´ signal. Moreover, we observed additional resonances for the newly introduced protons and carbons of the C-1 chain, the tetrazole moiety and their substituents. Evidence of structure was provided by the expected long-range couplings from 1´´-H to C-2´ and C-1´´´, which were detected in the HMBC spectra of compounds 11-15. The distinction between isomers 11-13 was enabled by a single crystal structure analysis of compound 12B (Figure 2). The crystal structure analysis of 12B confirmed the compound as N-[(1-tert-butyl-1H-tetrazol-5yl)(phenyl)methyl]-2-[6,9-diphenyl-1-(piperidin-1-yl)-3-azabicyclo[3.2.2]nonan-3-yl]ethan-1-amine. All atoms lie on general positions. Owing to the absence of heavier elements, the absolute structure of the chiral molecule could not be determined reliably from the diffraction data. Arbitrarily, the structure is described as the R,R,S enantiomer ( Figure 1). The tetrazole ring has adopted the orientation where the distance from N44 to the H atom of the NH group is smallest [N30···N44 2.841(4) Å ]. Presumably, the intramolecular hydrogen bond is weak because of the small angle N30-H30···N44 of 104(2)°. CCDC  showed antiplasmodial activity against P.f. K 1 (P.f. K 1 IC 50 = 1.26; 1.30 µM) and antitrypanosomal (IC 50 = 2.65; 1.26 µM) activity in the low micromolar range [15]. Replacement of the 4-methyl by a 4-chloro substituent and the insertion of an aminomethyl linker between the bridged ring system and the sulfonyl group led to compounds 18-20 with slightly improved antitrypanosomal activities (T. b. r. IC 50 = 0.647-1.19 µM). Moreover, compounds 18-20 showed antiplasmodial activities against a sensitive strain in submicromolar concentration (P.f. NF54 IC 50 = 0.487-0.606 µM). Their selectivities (SI ≤ 18.7) were only moderate, unfortunately. Similarly, tetrazole derivates 11-15 showed activity against P. falciparum NF54 (IC 50 = 0.252-2.98 µM) and T. brucei rhodesiense (IC 50 = 0.329-6.61 µM) in the low micromolar range, but selectivity indices (SI ≤ 20.1) were unfavorable due to their comparably high cytotoxicity. The intermediates 6-9 showed similar antiplasmodial activity against the multiresistant strain but distinctly improved selectivity (SI = 14.5-93.3 µM). The most promising compound of this series was 7 exhibiting submicromolar antiplasmodial activity (P.f. K 1 IC 50 = 0.180 µM) and quite good selectivity (SI = 93.3 µM).

.2]nonan-3-yl]ethan-1-amines 9 and 10
The bicyclononane 1-5 was dissolved in 20 mL dry ethanol and cooled on an ice bath with stirring under an atmosphere of argon. A solution of chloroacetamide in 12 mL dry ethanol was added. The mixture was refluxed for 48 h at 100 • C, allowed to cool to room temperature, diluted with water and alkalized with 2N NaOH. It was then extracted 4 times with diethyl ether. The combined organic phases were washed with water, dried over anhydrous sodium sulfate, filtered and finally the solvent was removed in vacuo. The obtained crude products were purified, if necessary, by column chromatography giving the corresponding 2-substituted acetamide derivate as colorless resin. It was suspended in 20 mL dry diethyl ether under stirring and cooling in an ice bath. LiAlH 4 was added in portions and the mixture was refluxed overnight. The reaction was cautiously quenched with ice water; 2N NaOH was added and the mixture was extracted 5 times with CH 2 Cl 2 ; the combined organic phases were washed twice with water, dried over anhydrous sodium sulfate, filtered and finally the solvent was removed in vacuo giving colorless oils.

General Procedure for the Synthesis of N-[(1-tert-butyl-1H-tetrazol-5-yl)methyl]-2-[6,9-diphenyl-1-(piperidin-1-yl)-3-azabicyclo[3.2.2]nonan-3-yl]ethan-1-amines 11-15
2-(3-Azabicyclononan-3-yl)ethan-1-amine 10 was dissolved in dry MeOH in an atmosphere of Ar. The corresponding aldehyde was added and the reaction batch was stirred for 1 h at ambient temperature. Subsequently, tert-butylisocyanide and trimethylsilylazide were added and the reaction was stirred for an additional 20 h at ambient temperature in an atmosphere of Ar. Subsequently, the solvent was evaporated in vacuo yielding crude products 11-15, which were separated by column chromatography into their isomers (silica, diethyl ether/dioxane/MeOH = 25 + 1 + 1).  [17] and refined by full-matrix least-squares techniques against F 2 (SHELXL-2014/6) [18]. The nonhydrogen atoms were refined with anisotropic displacement parameters without any constraints. Owing to the absence of heavier elements, the absolute structure of the chiral molecule could not be determined reliably from the diffraction data and was chosen arbitrarily. The H atom bonded to N30 was taken from a difference Fourier map and refined without any positional constraints with an individual isotropic displacement parameter. The H atoms of the tertiary C-H groups were refined with individual isotropic displacement parameter and all X-C-H angles equal at a C-H distance of 1.00 Å. The H atoms of the CH 2 groups were refined with common isotropic displacement parameters for the H atoms of the same group and idealized geometry with approximately tetrahedral angles and C-H distances of 0.99 Å. The H atoms of the phenyl rings were put at the external bisectors of the C-C-C angles at C-H distances of 0.95 Å, and common isotropic displacement parameters were refined for the H atoms of the same ring. The H atoms of the methyl groups were refined with common isotropic displacement parameters for the H atoms of the same group and idealized geometries with tetrahedral angles, enabling rotations around the C-C bonds, and C-H distances of 0.98 Å. For 443 parameters, final R indices of R 1 = 0.0513 and wR 2

General Procedure for the Synthesis of Benzenesulfonamides 18-20
Azabicyclo-nonanes 3, 7, 8 were dissolved in dry CH 2 Cl 2 . Then, 4-DMAP or DIPEA and the respective aromatic sulfonyl chloride were added under stirring. The mixture was refluxed for 20 h at 50 • C. Subsequently, the reaction batch was shaken with 2 N aq NaOH, washed with water until the aqueous phase reacted neutral, dried over anhydrous sodium sulfate, filtered and finally the solvent was removed in vacuo giving crude products, which were purified by column chromatography yielding compounds 18-20. The reaction of 0.143 g 2-azabicyclo-nonane 7 (0.35 mmol), 0.137 g toluene-4-sulfonyl chloride (0.72 mmol) and 0.118 g DIPEA (0.91 mmol) in 5 mL CH 2 Cl 2 abs. gave a crude product, which was purified by column chromatography (aluminum oxide neutral, CH/EtAc = 5 + 1 → 1 + 1 finished by CH/EtAc/MeOH 1 + 1 + 0.1) yielding 0. Assays were performed in 96-well microtiter plates, each well containing 0.1 mL of RPMI 1640 medium supplemented with 1% L-glutamine (200 mM) and 10% fetal bovine serum and 4000 L-6 cells (a primary cell line derived from rat skeletal myoblasts, ATCC CRL-1458™) [25,26]. Serial drug dilutions of 11 three-fold dilution steps covering a range from 100 to 0.002 µg/mL were prepared. After 70 h of incubation, the plates were inspected under an inverted microscope to assure the growth of the controls and sterile conditions. Then, 0.01 mL resazurin solution (resazurin, 12.5 mg in 100 ml double-distilled water) was added to each well and the plates incubated for another 2 h. The plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. The IC 50 values were calculated by linear regression [23] from the sigmoidal dose inhibition curves using SoftmaxPro software (Molecular Devices Cooperation, Sunnyvale, CA, USA). Podophyllotoxin (Sigma P4405) was used as control.

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