New 2-aminopyrimidine derivatives and their antitrypanosomal and antiplasmodial activities

Novel 2-aminopyrimidine derivatives were prepared from acyclic starting materials, benzylidene acetones and ammonium thiocyanates, via 5 steps, including ring closure, aromatization, S-methylation, oxidation to methylsulfonyl compounds, and formation of guanidines with suitable amines. The prepared compounds differ from each other by the substitutions of their amino group and of their phenyl ring. The 2-aminopyrimidines were tested by use of microplate assays for their in vitro activities against a causative organism of sleeping sickness, Trypanosoma brucei rhodesiense, as well as against a causative organism of malaria, Plasmodium falciparum NF54. Their cytotoxic properties were determined with L-6 cells (rat skeletal myoblasts). Some of the compounds exhibited quite good antitrypanosomal activity, and others showed excellent antiplasmodial activity. The influence of the structural modifications on these activities is discussed.


Introduction
In the past two decades, over two billion of the world's poorest people have been affected by neglected tropical diseases (NTDs). One of the 11 major NTDs studied is human African trypanosomiasis (HAT) [1]. HAT or sleeping sickness is caused by protozoa of the genus Trypanosoma like Trypanosoma brucei gambiense (Tbg) and Trypanosoma brucei rhodesiense (Tbr). The vector is the tsetse fly. Only one drug, melarsoprol, is available for the late-stage Tbr infection treatment [2]. This toxic arsenic compound causes severe side effects including a deadly encephalopathy in more than 5% of the patients [3]. Therefore it is an urgent Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s0070 6-020-02674 -7) contains supplementary material, which is available to authorized users. 1 3 need to develop new efficient antitrypanosomal compounds with less side effects.
In 2018 malaria globally affected 228 million people and caused 405,000 deaths [4]. The emergence and spread of resistance in Plasmodium falciparum malaria to artemisinin combination therapies in the Greater Mekong subregion poses a major threat to malaria control and elimination [5]. Since the last defense line, the artemisinines, might fall possibly, there is a great demand for antiplasmodial compounds with alternative mechanism of action.

Results and discussion
Starting from benzylidene acetones 1a-1d pyrimidinethiones 2a-2d were prepared by reaction with ammonium thiocyanate in refluxing benzene/cyclohexanol [14]. Aromatization of the heterocyclic ring took place in boiling xylene in the presence of sulfur giving compounds 3a-3d. Subsequently the SH group was methylated using methyl iodide in chloroform yielding 4a-4d. The next step was an oxidation to the methylsulfonyl compounds 5a-5d with m-chloroperbenzoic acid in dichloromethane. The final formation of the target compounds 6-10 took place in dioxane or tetrahydrofuran in the presence of the various amines under microwave irradiation at 120 °C or under reflux. Structural modifications were restricted to the amino substituent including amino, a (pyrrolidin-1-yl) and (4-methylpiperazin-1-yl) groups of compounds 6-8. Moreover, partial structures of chloroquine were used as substituents for compounds 9 and 10. The chiral aliphatic amine moiety was connected with the pyrimidine core giving compounds 9 as racemates. In a further step, the quinoline residue was attached. Similar compounds, bearing an additional ester function have already been investigated [15]. Further variations concerned the substitution pattern of a phenyl ring (Scheme 1).
Scheme 1. Syntheses of compounds 2-10. Reagents and conditions: (i) benzene, cyclohexanol, water separator, reflux, 6 h, (ii) S, xylene, reflux, overnight, (iii) CH 3 I, CHCl 3 , r.t., overnight to 3 days, (iv) m-chloroperbenzoic acid, CH 2 Cl 2 , 0-20 °C, 2 h, (v) NH 3 conc. or amine, dioxane or THF, 85 °C, reflux overnight or microwave 120 °C 2-13 h. For the aromatization from 2a-2d to 3a-3d we observed the disappearance of the proton signal at 4.9 ppm of the CH group attached to the aromatic moiety in 1 H NMR spectra as well as the shift of the signal of the olefinic proton from 4.7 ppm to 7.3 ppm. Furthermore, the signals of the NH protons at 8.8 and 9.5 ppm disappeared and a new signal was observed for the SH group at 13.5 ppm. In 13 C spectra, the signal of the carbon attached to the aromatic moiety shifted from 54 to 165 ppm due to aromatization. S-Methylation to compounds 4a-4d caused appearance of an additional signal at 2.6 ppm for the methylthio group in 1 H NMR spectra and at 13.8 ppm in 13 C spectra. The subsequent oxidation to the methylsulfonyl group in 5a-5d shifted the signal of the attached methyl group from 2.6 ppm to 3.4 ppm in 1 H NMR spectra and from 13.8 ppm to 39 ppm in 13 C spectra. The replacement of the methylsulfonyl group of compunds 5a-5d by amino substituents shifted the signal for the C-2 2-6 ppm to lower frequencies. Moreover, we observed long-range couplings from protons of the amino substituent to C-2 in HMBC spectra of compounds 6-10 which confirmed the attachment of the amino groups to this ring position.
All 2-aminopyrimidine derivatives 6-10 were tested for their antiplasmodial activities against P. falciparum NF54 and for their antitrypanosomal potencies against Trypanosoma brucei rhodesiense STIB 200 as well as for their cytotoxicity against rat skeletal myoblasts (L-6 cells) in microplate assays. The results are presented in Table 1.

Conclusion
Several new methyl-aryl-substituted 2-aminopyrimidines with differing amino and phenyl substitution have been prepared. The antitrypanosomal and antiplasmodial activities of the new compounds were determined. The most active antitrypanosomal compounds (IC 50 = 0.41, 1.03 µM) exhibited the same side chain as chloroquine. Compounds possessing the 7-chloroquinoline partial structure of chloroquine showed excellent activity against P. falciparum NF54 (IC 50 = 0.04-0.14 µM). The most  active compound was additionally tested against the multiresistant K 1 strain of P. falciparum and showed twice the activity of chloroquine against this strain. Therefore, this compound could be a lead for further optimization.

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 ppm) 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 ppm). Some spectra were acquired in DMSO-d 6 . Here the proton signal at 2.49 ppm served as internal reference as well as the central peak of the DMSOd 6 signal at 39.7 ppm. Abbreviations: aromatic H, ArH; aromatic C, ArC, quaternary aromatic C, ArC q . Signal multiplicities are abbreviated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; q, quartet; 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  ; the substances were detected in UV light at 254 nm. Unless otherwise stated silica gel was used for separations (CC, TLC). Microwave-assisted reactions were carried out in a CEM Discover/Explorer system in sealed 10 cm 3 standard vessels with temperature control. Syntheses of compounds 2a, 2b, and 2d were described previously [14]. Compund 3a was prepared following a reported procedure [17]. Its melting point (201-205 °C) corresponded well with the reported one (202-204 °C) [18]. The synthesis of 4a was already described and the melting point (214 °C) corresponded well with the reported one (213 °C) [19].

6-Methyl-4-(4-methylphenyl)-3,4-dihydropyrimidine-2(1H)-thione (2c, C 12 H 14 N 2 S)
The reaction of 21.27 g of 1c (132.76 mmol) with 8.26 g of ammonium thiocyanate (108.5 mmol) was carried out in 400 cm 3 of toluene in the presence of 4.12 g of cyclohexanol (41.16 mmol). The mixture was heated for 18 h at 160 °C oil bath temperature using a water separator filled with molecular sieves 4 Å. After cooling to r.t., the orange precipitate was collected by filtration, washed with ether and ethanol. Then it was dissolved in a mixture of hot ethanol/isopropanol (3:1), treated with charcoal and filtered. The filtrate was concentrated in vacuo. Thereafter it was allowed to stand overnight at r.t. to complete crystallization. The product was collected by filtration, washed with ethanol, and dried. Yield: 11.85 g of 2c (41%) as white crystals.

Preparation of pyrimidine-2-thiols 3b-3d
The aromatization of dihydropyrimidine-2(1H)-thiones 2b, 2c, and 2d took place overnight in refluxing xylene in the presence of sulfur. The solution was then allowed to cool to r.t.. A precipitate was formed which was collected by filtration. The solid was stirred with 1 N NaOH and filtered. The filtrate was acidified with 2 N HCl. The precipitate was collected by filtration, washed with water and recrystallized.

Preparation of (methylsulfanyl)pyrimidine hydroiodides 4b-4d
To a stirred suspension of 3b, 3c, or 3d in CHCl 3 methyliodide (CH 3 I) was added dropwise. Stirring was continued at r.t. for up to 3 days. Then the formed solid was collected by filtration. The filtrate was evaporated to dryness and the residue was crystallized by treatment with ethyl acetate to give a second portion of the product. The solids were combined and dried.

Preparation of (methanesulfonyl) pyrimidines 5a-5d
The methylthio-pyrimidine hydroiodides 4a, 4b, 4c, or 4d were dissolved in CH 2 Cl 2 . The resulting solution was cooled down to 0 °C and 3-chloroperoxybenzoic acid (77%) was added slowly with stirring and cooling. The color of the solution turned to cerise, the ice bath was removed and the reaction mixture was stirred for 2 h at r.t.. During the reaction the color of the solution turned to violet due to the oxidation of iodide to iodine. The organic layer was washed once with saturated NaHCO 3 solution, once with an aqueous Na 2 S 2 O 3 solution, and finally with brine. Then it was dried over anhydrous Na 2 SO 4 and filtered. The solvent was evaporated in vacuo giving white solids which were recrystallized with a small amount of ethyl acetate giving colorless needles.

Preparation of pyrimidin-2-amines 6a-6d
The pyrimidin-2-amines were prepared similar to a reported procedure [20]. Compounds 5a, 5b, 5c, or 5d were suspended in dioxane and concentrated aqueous NH 3 was added. The reaction mixture was subjected to microwave irradiation at 120 °C. The solvents were evaporated in vacuo to dryness. Water was added and the mixture was extracted 5 times with CH 2 Cl 2 . The combined organic layers were washed once with water, dried over anhydrous Na 2 SO 4 , and filtered. The solvent was evaporated in vacuo giving a crystalline residue. The crude products were purified by means of sublimation at reduced pressure yielding the products as white needles. -6-phenylpyrimidin-2-amine (6a, C 11 H 11 N 3

Preparation of (pyrrolidin-1-yl)pyrimidines 7a-7d
The compounds 5a, 5b, 5c, or 5d were dissolved in dry THF and pyrrolidine was added. The reaction mixture was refluxed at 85 °C overnight or subjected to microwave irradiation. Water was added and the mixture was extracted five times with diethyl ether. The combined organic layers were washed neutral with water, dried over anhydrous Na 2 SO 4 , and filtered. The solvent was evaporated in vacuo giving pure compounds 7a-7d as white to yellowish needles. For analytical purposes they were recrystallized giving white needles.

Preparation of 2-(4-methylpiperazin-1-yl) pyrimidines 8a-8d
The compounds 5a, 5b, 5c, or 5d were dissolved in dry THF and 1-methylpiperazine was added. The reaction mixture was refluxed at 100 °C overnight or subjected to microwave irradiation. Water was added and the mixture was extracted five times with diethyl ether. The combined organic layers were washed neutral with water, dried over anhydrous Na 2 SO 4 and filtered. The solvent was evaporated in vacuo giving a resin which was further purified. -1-yl)

Preparation of N-[5-(diethylamino) pentan-2-yl]pyrimidin-2-amines 9a-9d
The compounds 5a, 5b, 5c, or 5d were dissolved in dry THF and 2-amino-5-(diethylamino)pentane was added. The reaction mixture was subjected to microwave irradiation. Water was added and the mixture was extracted five times with diethyl ether. The combined organic layers were washed neutral with water, dried over anhydrous Na 2 SO 4 and filtered. The solvent was evaporated in vacuo giving residues which were further purified.