Fused Heterocyclic Systems with an s-Triazine Ring. 34. Development of a Practical Approach for the Synthesis of 5-Aza-isoguanines

Purine isosteres present excellent opportunities in drug design and development. Using isosteres of natural purines as scaffolds for the construction of new therapeutic agents has been a valid strategy of medicinal chemistry. Inspired by the similarity to isoguanine, we attempted to develop a practical method for the preparation of 5-aza-isoguanines. Several synthetic approaches were explored to establish a robust general protocol for the preparation of these compounds. The significant difference in the reactivity of the C-5 and C-7 electrophilic centers of 1,2,4-triazolo[1,5-a][1,3,5]triazines (5-azapurines) towards nucleophiles was demonstrated. The most practical and general method for the preparation of 5-aza-isoguanines involved a regioselective reaction of ethoxycarbonyl isothiocyanate with a 5-aminotriazole. The intramolecular ring closure of the resulted product followed by the S-methylation afforded 7-methylthio-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one, which could be effectively aminated with various amines. The resulted 5-aza-isoguanines resemble a known purine nucleoside phosphorylase inhibitor and could be interesting for further investigations as potential anticancer agents.

To further explore the trichloromethyl chemistry in the synthesis of 7-amino-substituted 1,2,4-triazolo [1,5-a] [1,3,5]triazin-5-ones 6, we subjected 1-guanyl-1,2,4-triazole 7, prepared as reported previously [28], to the reaction with trichloroacetonitrile (Scheme 3). Similar to triazolylguanidine 1 (X = N), the reaction pathway of 7 was solvent dependent. In ethanol, diamine 8, identical to that reported in reference [29], was formed, whereas 1,2,4-triazolo[1,5-a][1,3,5]triazine 9 was isolated exclusively when the reaction was carried out in toluene. In the 1 H-NMR spectrum of 9, two protons of the amino group gave independent signals appearing at rather low field: 9.22 and 9.65 ppm. These observations can be attributed to the prominent delocalisation of an electron pair on the amino group nitrogen directly attached to the highly electron-deficient 1,3,5-triazine ring. The electron-withdrawing trichloromethyl group further decreased electron density on the triazine ring, enhancing the partial double bond character of the C-NH 2 bond and restricting the rotation around it. The activation energy (∆G ‡ ) for the hindered rotation around this bond was estimated using dynamic 1 H-NMR spectroscopy and was equal 68.8 kJ at the coalescence temperature 343 K.
The trichloromethyl group of 9 was hydrolytically removed using aqueous solution of sodium carbonate to afford 6a. The substitution of trichloromethyl group of 9 with nucleophiles (particularly amines) was more problematic in comparison to the aminolysis of the isomeric structure 2. No reaction was observed when similar reaction conditions were applied. Moreover, unexpected results were obtained after prolonged heating with excess of amine depending on the reaction conditions and amine structure (Scheme 4). Replacement of the amino group in position 7 of the 1,2,4-triazolo[1,5-a] [1,3,5]triazine ring was observed instead of the proposed substitution of the trichloromethyl group of 9 (e.g., in the synthesis of 10) or both the processes took place together (e.g., in the preparation of 11). It was demonstrated earlier that compounds with identical leaving groups in positions 5 and 7 of the 1,2,4-triazolo[1,5-a] [1,3,5]triazine ring were suitable for the sequential nucleophilic substitution with amines: first in position 7, then in position 5 [30]. This strategy was successfully applied for the synthesis of bioactive 1,2,4-triazolo[1,5-a] [1,3,5]triazines [31,32]. However, to the best of our knowledge, no examples on transaminations at the position 7 of 1,2,4-triazolo[1,5-a] [1,3,5]triazines have been reported. For similar pyrazolo[1,5-a] [1,3,5]triazines, the replacement of a N-methylanilino substituent in the corresponding position with other amines was effectively employed in drug-discovery programs to prepare a variety of compounds with a diverse substitution pattern [33][34][35][36][37][38][39][40]. The hydrolysis of 7-amino substituted 10 can be applied to generate 6k. Nevertheless, difficulties with controlling reaction outcome in the synthesis of analogues of 10 and a long reaction time led us to design an intermediate with a leaving group more accessible for the nucleophilic substitution by amines than 9. This strategy was realized by the preparation of 1,2,4-triazolo[1,5-a] [1,3,5]triazine (15) (Scheme 5). Aminotriazole 12 reacted with carbon disulfide in DMF in the presence of potassium hydroxide followed by the selective S-methylation affording 13. Treatment of 13 with trichloroacetonitrile resulted in the formation of 15. Trichloroacetonitrile provided a C-N fragment for the construction of the triazine ring. We propose that the reaction involved an initial addition to the triple bond of trichloroacetonitrile followed by the intramolecular ring closure of intermediate 14 via nucleophilic substitution at the methyl dithiocarboxylate moiety. Surprisingly, hydrogen sulfide instead of methylthiol played the role of a leaving group in this reaction. The methylthio group of 15 can be readily replaced by stoichiometric quantity of amines, e.g., 4-methoxybenzylamine, providing 10. However, a long reaction time at the hydrolysis step arising from the low aqueous solubility of 10 and the necessity of chromatographic purification limited the practicality of this approach. Considering the smooth replacement of the methylthio group at position 7 of the 1,2,4-triazolo[1,5-a] [1,3,5]triazine with amines, we implemented an alternative synthetic approach for the preparation of 6.
The key intermediate in this protocol was 16, prepared from 5-amino-3-phenyl-1,2,4-triazole (12) (Scheme 6). The selective addition of 12 to ethoxycarbonyl isothiocyanate was challenging due to the presence of several competing nucleophilic centers on 12. It was found that regioselective addition occurs only at the endocyclic N-1 atom of 16 when the reaction was carried out under kinetic control in cold acetone for not more than 15 min. Extension of the reaction time or increasing temperature led to the formation of more thermodynamically stable N-carbethoxy-N -(1,2,4-triazol-5-yl)thiourea, which existed in solution as a mixture of two tautomers 17 and 17 (K T = 0.74) crystalizing in the form of the predominant tautomer 17 ( Figure 1) [41].  In solutions, 16 readily underwent rearrangement to 17 even at room temperature. Therefore, instant preparation of sufficiently pure 16 was critical, as changes in the product structure during purification were unavoidable. The alkali-induced cyclocondensation of 16 followed by S-methylation afforded 7-methylthio-2-phenyl-1,2,4-triazolo[1,5-a] [1,3,5]triazin-5-one (18).
The nucleophilic replacement of the methylthio group of 18 proceeded smoothly, with a variety of amines affording 6 with reasonable yields and high purity (Scheme 7). The reaction remained chemoselective even when aqueous solutions of fairly basic amines (synthesis of 6a-c) were used. The structure of the prepared compounds 6 was confirmed by NMR spectroscopy, clearly showing that the signal of methylthio group of 18 was replaced by signals of the corresponding amines in spectra of 6. The X-ray data for a representative example 6l also confirmed the proposed structure ( Figure 2) [42].  The synthetic route described in Schemes 6 and 7 was not placed on the top priority at the initial design of the synthetic routes due to the relatively low yield of the intermediate 16 and the uncertainty involved in the preparation of a pure regioisomer 16 as a result of the addition reaction between ethoxycarbonyl isothiocyanate to 5-amino-3-phenyl-1,2,4-triazole (12). However, this synthetic route unexpectedly became the most practical and versatile method that could be applied to the synthesis of a library of 7-amino substituted 2-phenyl-1,2,4-triazolo[1,5-a] [1,3,5]triazin-5-ones (6a-p).

Conclusions
In conclusion, several synthetic approaches to the synthesis of 5-aza-isoguanines 6 were explored to develop a practical method for their preparation. The significant difference in the reactivity of the C-5 and C-7 electrophilic centers of 1,2,4-triazolo[1,5-a] [1,3,5]triazines towards nucleophiles was demonstrated. The most practical and general method for the preparation of 5-aza-isoguanines 6 involved a regioselective addition of 5-amino-3-phenyl-1,2,4-triazole (12) to ethoxycarbonyl isothiocyanate followed by an intramolecular ring closure and S-methylation to afford 18, which could be effectively aminated with various amines. The prepared compound 6 resemble a known purine nucleoside phosphorylase inhibitor and could be interesting for further investigations as potential anticancer agents.