Reactivity of tetrazolo[1,5-a]pyrimidines in click chemistry and hydrogenation

Herein, we explore the synthetic potential of tetrazolo[1,5-a]pyrimidines to obtain new pyrimidine derivatives by click chemistry and hydrogenation. Click chemistry reactions of the trifluoromethyltetrazolo[1,5-a]pyrimidines with terminal acetylenes produced unprecedented trifluoromethylated triazolylpyrimidines in excellent yields (84-98 %) in which one of them was active against all tested microorganisms, presenting moderate MIC values (62.5-15.62 g/ml). Hydrogenation was carried out using Pd/C-H2 in MeOH under conventional, photochemical, and pressure (5 bar)


Introduction
Pyrimidines and their derivatives, such as tetrazolo-[1,5-a]pyrimidines, are extremely important heterocycles and have received special attention from researchers due to their significant biological and pharmaceutical properties [1,2]. Tetrazolopyrimidines were reported for the first time in the 1960s and 1970s, including an azide-tetrazole equilibrium. At the same time, tetrazoles and azides were reported to have different physical and chemical properties [3][4][5][6][7][8][9][10][11]. Therefore, pharmacokinetics and biological properties may arise from the differences in the chemical structure, and because of this, the azide-tetrazole equilibrium is of great interest from a pharmacological point of view. Recently, we published a study on the synthesis of trifluoromethyl tetrazolo [1,5-a]pyrimidine/2-azidopyrimidines and demonstrated the effects of substituents on regiochemistry and equilibrium [2]. Our findings revealed that when 3 precursor compounds (α,β-unsaturated ketones) were trifluoromethyl-or trichloromethyl-substituted, tetrazolo [1,5-a]pyrimidines were formed in high regioselectivity. When precursor compounds are substituted with aryl or methyl, it leads to a mixture of compounds, tetrazolo [1,5-a]pyrimidines (R in the 5-position of the ring) and 2-azidopyrimidines (R in the 4-position of the ring), which was attributed to an equilibrium of azide-tetrazole. In that study, we demonstrated that tetrazolo [1,5-a]pyrimidines reacted with terminal alkynes in a 1,3-dipolar cycloaddition catalyzed by copper salts (CuAAC) [12][13][14], forming 1,2,3-triazole and confirming that an azide intermediate is formed in solution.
Dihydro-or tetrahydropyrimidines are crucial for drug discovery due to transitions from aromatic to more flexible and three-dimensional structures. Synthetic pathways for these structures are less common than their aromatic analogs, and because of this, N-heterocycle hydrogenation has been an age-old concern. In fact, tetrazolopyrimidine hydrogenation is particularly rare, and only one example is found in the literature.
Desenko et al. [15] prepared 4,7-dihydrotetrazolo[1,5-a]pyrimidines and hydrogenated them using NaBH4, and the produced tetrahydrotetrazolopyrimidines were obtained in 10-75 % yields and formed as one stereoisomer, although the presence of two chiral centers in the molecules suggests the possible formation of a mixture of diastereoisomers. Reducing aminopyrimidines is more commonly found in the literature, for example, Baskaran et al. [16] reduced 2-aminopyrimidines using triethylsilane (TES) in trifluoroacetic acid (TFA). 2-Aminodihydro pyrimidine formation occurred at lower temperatures, and aminotetra hydropyrimidines were observed 4 when the reaction was run in refluxing TFA for 24 h. In 2014, Shaw et al. [17] reduced 2-arylaminopyrimidines using palladium on carbon (Pd/C) in MeOH, forming 2-arylamino-tetrahydro pyrimidines in excellent yields (71-98 %). Asymmetric hydrogenation of pyrimidines is an efficient method of synthesizing chiral dihydro-or tetrahydropyrimidines despite the asymmetric hydrogenation of pyrimidines being a novel theme in organic synthesis, and only recently have papers been published on this topic [18][19][20]. Asymmetric hydrogenation of 2-arylpyrimidine-4-substitution using [IrCl(cod)]2-Josiphos-I] as a catalytic system and the addition of Yb(OTf)3 allowed a broad range of pyrimidines to be converted into the corresponding tetrahydropyrimidines with a remarkable improvement in stereoselectivity and high yields (68-99 %) [18]. In the sequence, palladium-catalyzed asymmetric hydrogenation of 2-hydroxypyrimidines to corresponding tetrahydropyrimidines was developed with up to 99 % of ee. The catalytic system works for mono-, di-, and trisubstituted 2-hydroxypyrimidines [19]. In 2019, Chirik et al. developed a catalyst (rhodium precatalysts) for asymmetric hydrogenation of N-heterocycles, and a diverse array of unsubstituted N-heteroarenes including pyridine, pyrrole, and pyrazine, traditionally challenging substrates for hydrogenation, were successfully hydrogenated using the organometallic precatalysts. The hydrogenation of polyaromatic N-heteroarenes exhibited uncommon chemoselectivity, although only one pyrimidine was reduced in 53 % yield [20]. Nevertheless, advances in pyrimidine synthesis have been made, including asymmetric hydrogenation [18][19][20], and pyrazolopyrimidine hydrogenation still remains challenging. Thermodynamic stability, kinetic inertia of the heteroaromatic ring, highly coordinative nitrogen atoms, and the presence of weak C−H bonds adjacent to the nitrogen atoms, which promote deleterious side reactions, have so far remained unresolved goals [21][22][23].

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The pattern of pyrimidine substitution in relation to hydrogenation reaction is limited to 2-amino-, 2-hydroxy-, and 2-arylpyrimidines, and sparse examples of these substrates with other substituted positions can be found in the literature. Synthesizing 2-amino-trifluoromethylpyrimidines and their respective hydrogenated dihydro-and tetrahydropyrimidines from hydrogenation reactions are practically inexistent in the literature or have very low yields [16]. To the best of our knowledge, hydrogenating trifluoromethyl-substituted pyrazolopyrimidines has yet to be performed.
Considering the recent and growing interest in click chemistry of the versatile 1,2,3triazoles and the lack of hydrogenation methods for trifluoromethyl-substituted pyrazolopyrimidines, this study aimed to extend knowledge on the reactivity of trifluoromethyl-substituted tetrazolo [1,5-a]pyrimidines in the heterocyclic chemistry context. The scope and goals of this paper are given in Scheme 1.
Our findings showed that the electronic nature of the R and R 1 groups did not influence the formation of the product, and 1,2,3-triazolylpyrimidine formation confirms that although the azide form detection depends on solution conditions, even when it is not detectable, it is still present, and in the presence of acetylene, the tetrazolopyrimidine is converted into azide by chemical equilibrium displacement, forming the product of click chemistry in high yields.
The synthesis of compounds 6a-f, 7a-c,e, and 8a was confirmed by 1 H, 13  In the 13  respectively. X-ray diffractometry verified the regiochemistry of these compounds, and the ORTEP ® of compound 7b is illustrated in Figure 1.

Hydrogenation reaction
The hydrogenation of 5-aryl-7-trifluorometiltetrazolo[1,5-a]pyrimidines 1a-h to obtain di-or tetrahydropyrimidines was explored using three methods. First, the hydrogenation reaction was carried out using compound 1a and reacting with Pd/C-H2 in MeOH for 16 h. The 5-phenyl-7-trifluoromethyltetrazolo[1,5-a]pyrimidine 1a was reduced and the 2-amino-4-phenyl-6-trifluoromethylpyrimidine 9a formed in 97 % yield. Subsequently, this method was carried out for the other substituents 1b-h. The reaction time ranged from 16 to 24 h and yields of up to 97 % were obtained. In the case of 1c,g, in addition to the pyrimidine ring reduction, dehalogenation also occurred (loss of chlorine and bromine atoms), and tetrahydropyrimidine 10a was formed ( Table 2). This result deviates from Baskaran et al. [16], who reduced 2aminopyridines to 2-aminodihydropyrimidines using TES in TFA, in which the bromine was retained during hydrogenation. However, this is an expected result considering the hydrogenation reaction was done in the presence of palladium [26][27][28] and hydride sources such as H2, which is a well-known condition for aryl halide hydrodehalogenation.
Having established the conditions, 1b-h was submitted to the same conditions, forming 2-amino-6-aryl-4-trifluoromethylpyrimidines 9b-f,h ( Table 2). The products were obtained in moderate (43 %) to high yields (78 %). Nonetheless, the photochemical hydrogenation was the fastest and the yields were the lowest.
Non-trifluoromethyl-substituted tetrazolo[1,5-a]pyrimidines can also exist as an equilibrium with their azide form and were therefore submitted to hydrogenation.
a Yield of isolated product. b No product was observed.
14 When R = 4-OMe-Ph (11e), tetrahydropyrimidine (14e) was formed in excellent yield (85 %) ( Table 3). The photochemical condition also was tested. After 24 h, a reduction of 11a led to the formation of 2-aminipyrimidine 13a in low yield (45 %). No product was obtained during attempts of photochemical reduction of 11b-e,g ( Table   3). The third condition tested was Pd/C-H2 under high pressure (5 bar

Antimicrobial activity
Considering the biological and pharmacological potential of heterocycles reported herein, some of them (6a-f, 7a-c,e, 8a, 9e, 13a, and 14a-b,e) were screened against Gram-positive and Gram-negative bacteria and yeast using the well diffusion test (Table 4). The antimicrobial screening results involved measuring the average diameter of the inhibition zones (in mm), and we found that only compound 6c had growth inhibition compared to the respective positive control. The remaining compounds exhibited inhibition zones of 5 mm as the negative control (DMSO) and no growth inhibition for each microorganism tested.
Additionally, compound 6c was submitted to the microdilution method against the Gram-positive bacterial strains, and the MICs were evaluated (

Experimental
The reagents and solvents used were obtained from commercial suppliers without further purification. 1 [29]. Molecular graphs were prepared using ORTEP for Windows [30]. Data collection and structure refinement for the structures of 7b, 9c, and 14a are given in Table S1 in the Supporting Information File 1. High-resolution mass spectrometry (HRMS) was performed using HPLC/MICROTOF ESI-MS equipment. Additional information regarding the experimental data for the synthesized compounds is presented in the Supporting Information File 1.

General procedure to synthesize tetrazolo[1,5-a]pyrimidines 1a-h
The tetrazolo[1,5-a]pyrimidines 1a-h were synthesized from the reaction of 5aminotetrazole with 1,1,1-trifluor-4-metoxi-4-aril-3-alquen-2-onas according to the method developed in our laboratory [31]. MeOH, was initially deoxygenated for 1 h using nitrogen gas (or argon). Then, H2 was added and the mixture was stirred at room temperature for 16-24 h. After the reaction time, the source of H2 gas was removed and the resulting mixture was deoxygenated again using nitrogen gas (or argon). The resulting mixture was then filtered under reduced pressure using celite, and the solvent was removed under reduced pressure. The products were obtained in pure form.

General procedure to synthesize 2-amino-6-aryl-4-trifluormethylpyrimidines 9af,h, 2-amino-6-aryl-4-(trifluoromethyl)-1,4,5,6-tetrahydropyrimidines 10a, and 2amino-4-arylpyrimidines 13a using photochemical reactor
The mixture of compounds 1a-h (1 mmol) or 11a-e,g (1 mmol) in EtOH was initially deoxygenated for 1 h using nitrogen gas (or argon). Then, the mixture was subjected to photochemical irradiation using a photochemical reactor equipped with 16 lamps (254 nm) for 3.5-5 h. After the reaction time, the resulting mixture was deoxygenated again using nitrogen gas (or argon). The solvent was removed using reduced pressure. All compounds synthesized using this method needed to be purified using a hexane eluent:ethyl acetate (8:2) preparative plate. MeOH, was initially deoxygenated for 1 h using nitrogen gas (or argon). Then, in a closed system, H2 was added up to 5 bar of pressure and the mixture was stirred at room temperature for 16-24 h. After the reaction time, the source of H2 gas was removed, and the resulting mixture was deoxygenated again using nitrogen gas (or argon). The resulting mixture was then filtered under reduced pressure using celite, and the solvent was removed under reduced pressure. The products were obtained in pure form.
General procedure to obtain single crystals

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The single crystals were obtained by slow evaporation of the solvents at 25 °C.

Microbial strains
The in vitro antimicrobial study was done using Gram-positive bacteria (S. aureus

Microdilution method
The MIC of the compounds was determined using the two-fold serial broth microdilution assay. The compounds were dissolved in DMSO and diluted with Mueller-Hinton broth medium at concentrations ranging from 500 to 0.488 µg/mL.

Supporting Information
Supporting Information File 1 NMR spectra of the compounds and crystallographic data of new structures reported.