Synthesis and Antiprotozoal Activity of Azabicyclo-Nonane Pyrimidine Hybrids

2,4-Diaminopyrimidines and (dialkylamino)azabicyclo-nonanes possess activity against protozoan parasites. A series of fused hybrids were synthesized and tested in vitro against pathogens of malaria tropica and sleeping sickness. The activities and selectivities of compounds strongly depended on the substitution pattern of both ring systems as well as on the position of the nitrogen atom in the bicycles. The most promising hybrids of 3-azabicyclo-nonane with 2-aminopyrimidine showed activity against P. falciparum NF54 in submicromolar concentration and high selectivity. A hybrid with pyrrolidino substitution of the 2-azabicyclo-nonane as well as of the pyrimidine moiety exhibited promising activity against the multiresistant K1 strain of P. falciparum. A couple of hybrids of 2-azabicyclo-nonanes with 2-(dialkylamino)pyrimidines possessed high activity against Trypanosoma brucei rhodesiense STIB900 and good selectivity.


Introduction
Malaria and human African trypanosomiasis (HAT, sleeping sickness) are both tropical diseases transmitted 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 Plasmodium falciparum, the deadliest malaria parasite. A lot of strains of Plasmodium falciparum have become resistant to previous generations of medicines [1]. In recent years even resistance to recommended artemisinin-based therapies has become prevalent across an expanding area of Southeast Asia [2,3]. There is growing evidence that resistant strains have reached regions in Africa [1]. Therefore, there is still a necessity to develop new compounds with distinct activity against Plasmodium falciparum.
Human African trypanosomiasis is caused by Trypanosoma brucei parasites. After continued control efforts the number of reported cases dropped below 1000 in 2019. However, as history has shown it may re-emerge and the estimated population at risk is 65 million people. In case of T. b. gambiense infections, the disease is chronic, generally lasting several years without any major signs or symptoms. However, infections with T. b. rhodesiense cause the acute form of the disease, lasting from a few weeks to several months and are lethal if untreated [4]. This East African sleeping sickness is difficult to treat considering the toxicity and complex administration of the drugs currently in use. For the treatment of T. b. rhodesiense infections of the central nervous system, melarsoprol is the only effective drug. Unfortunately, this arsenic-containing medication may cause encephalopathy, killing show antiplasmodial activity in submicromolar concentration. Their antitrypanosomal activity requires low micromolar concentrations [12,13].
In drug development against plasmodia the combination of different pharmacophores in one hybrid molecule is a potential option to bypass drug resistance, to improve pharmacokinetics or to achieve multistage antimalarial activity [14]. Recently, we reported about the impact of the linkage of tetrazole or sulfonamide cores to antiprotozoal active (dialkylamino)azabicyclo-nonanes [15]. A series of new quinoline-pyrimidine hybrids and their biological activities have been published in the last decade [16][17][18][19][20]. This paper presents the preparation of fused hybrids of (dialkylamino)azabicyclo-nonanes with the 2,4-diaminopyrimidine pharmacophore of pyrimethamine and selected analogues.

R1+R2
R3 R4+RS The structures of all newly synthesized compounds were clarified by one-and twodimensional NMR spectroscopy. Successful N-arylation was detected by typical shifts of resonances and remarkable signal broadening, particularly of the bicyclic ring atoms in the vicinity of the new bonding sites. The most striking change was a 17-19 ppm upfield shift of C-5 of the pyrimidine moiety due to the mesomeric effect of the new nitrogen substituent. In consequence of restricted rotation, the signals of each of the two protons H-2 and H-4 of the 3-azabicyclo-nonanes were strongly broadened. The corresponding signals of H-1 and one of the two H-3 protons of 2-azabicyclo-nonanes were even invisible in the 1 H NMR spectrum. The signals of the adjacent ring carbon atoms of both azabicyclo-nonanes were massively broadened and typically shifted about 1.5-5.5 ppm to lower frequencies. Their chemical shifts were usually deduced from 2D spectra, and 1 H-and 13 C-nmr spectra of the new compounds are given in the supplementary material.

Antiprotozoal Activity and Cytotoxicity
Compounds were tested for their activities against Plasmodium falciparum NF54 (sensitive to chloroquine) and Trypanosoma brucei rhodesiense STIB900, via serial dilution assays. Furthermore, selected compounds were tested against the K1 strain of Plasmodium falciparum (resistant against chloroquine, sulfadoxine and pyrimethamine). Their cytotoxicity was determined with rat skeletal myoblasts (L-6 cells). Chloroquine, melarsoprol and podophyllotoxin served as standards; additional data for pyrimethamine are included (Table 1).
The antitrypanosomal activity of the parent azabicyclo-nonanes 1-4 was moderate (T.b.r. IC 50 = 1.00-6.57 µM). Their activity was changed by the substitution with the pyrimidine substituent, but the impact of the substitution of the pyrimidine ring was varying. Compounds with a 2-azabicyclo-nonane skeleton were in general more active than their 3-azabicyclo-nonane analogues, and pyrrolidino substitution of the bridgehead atom furnished higher activity compared to piperidino substitution. The 2-(6-methylpyrimidin-4-yl)-5-pyrrolidino-2-azabicyclo[3.2.2]nonanes 6, 18, 22 showed the highest activity (T.b.r. IC 50 = 0.095-0.161 µM) and selectivity (SI = 62.90-158.4). All the other test compounds were by far less selective (SI ≤ 42.63). (Table 1) The molecular targets and the mechanisms of the antiplasmodial and the antitrypanosomal action of azabicyclo-nonanes are not yet known. The interpretation of test results relies on phenotypic screening, considering that inhibitors act against their antitrypanosomal targets in approximately physiological circumstances.

Instrumentation and Chemicals
IR spectra were recorded using a Bruker Alpha Platinum ATR FTIR spectrometer; frequencies are reported in cm −1 . NMR spectra: Varian Unity Inova 400 (298 K) 5 mm tubes, TMS as internal standard; 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra are reported in ppm; 1 H-and 13 C-resonances were assigned using 1 H, 1 H-; and 1 H, 13

General Procedure for the Syntheses of 5-12
The corresponding azabicyclo[3.2.2]nonane 1, 2, 3, 4, the 4-chloro-6-methylpyrimidin-2-amine, respectively, 6-chloropyrimidine-2,4-diamine and DIPEA were suspended in butan-1-ol. The reaction batch was refluxed for 48 h at 145 • C in an atmosphere of Ar. After cooling to room temperature, the mixture was diluted with diethyl ether or CH 2 Cl 2 and alkalized with 2N NaOH. The combined organic phases were washed with water, dried over anhydrous sodium sulfate, filtered and finally the solvent was removed in vacuo giving crude products which were purified by column chromatography.

General Procedure for the Synthesis of 17-20
The respective bicyclononane derivate 19-22 was mixed with excess pyrrolidine and acetonitrile and was refluxed at 105 • C for 20 h. The reaction progress was monitored by thin-layer chromatography (silica, CH 2 Cl 2 + MeOH = 19 + 1 and 9 + 1). When the reaction was completed, the mixture was diluted with CH 2 Cl 2 and alkalized with 2N NaOH. The organic phase was washed with water, dried over anhydrous sodium sulfate, filtered and finally the solvent was removed in vacuo giving crude products which were purified by column chromatography. The reaction of 0.120 g 14 (0.25 mmol) and 0.178 g pyrrolidine (2.50 mmol) in 8mL acetonitrile gave a crude product which was purified by column chromatography (silica, CH 2 Cl 2 + MeOH = 29 + 1) yielding 0.072 g 18 (56%, 0.14 mmol) as amorphous solid. IR

General Procedure for the Synthesis of 21-24
The respective bicyclononane derivate 13-16 was mixed with excess piperidine and acetonitrile and was refluxed at 105 • C for 48 h. The reaction progress was monitored by thin-layer chromatography (silica, CH 2 Cl 2 + MeOH = 9+1). When the reaction was completed, the mixture was diluted with CH 2 Cl 2 and alkalized with 2N NaOH. The organic phase was washed with water, dried over anhydrous sodium sulfate, filtered and finally the solvent was removed in vacuo giving crude products which were purified by column chromatography. The reaction of 0.125 g 13 (0.26 mmol) and 0.219 g piperidine (2.57 mmol) in 7 mL acetonitrile gave a crude product which was purified by column chromatography (silica, The IC 50 values were calculated by linear regression [29] from the sigmoidal dose inhibition curves using SoftmaxPro software (Molecular Devices Cooperation, Sunnyvale, CA, USA). Podophyllotoxin (Sigma P4405) was used as control.

Conclusions
The synthesis and the evaluation of the structure-activity relationships of the antiprotozoal activities of azabicyclo-nonane pyrimidine hybrids were reported. A number of structure-activity relationships were revealed. A couple of 1-piperidino-3-azabicyclononanes with a 2-aminopyrimidine moiety exhibited in vitro activity against P. falciparum NF54 in submicromolar concentration and high selectivity. A 2-azabicyclo-nonane with a pyrrolidino substituent on both the bicyclic ring system as well as on the pyrimidine ring even possessed improved activity against the multiresistant K1 strain of P. falciparum. Three 5-pyrrolidino-2-azabicyclo-nonanes have shown good trypanocidal activity and selectivity. Further modifications of the substitution pattern of the pyrimidine moiety will be investigated in a future project.

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