Synthesis, Characterization, and Tautomerism of 1,3-Dimethyl Pyrimidine-2,4,6-Trione s-Triazinyl Hydrazine/Hydrazone Derivatives

1School of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa 2Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia 3School of Laboratory Medicine and Medical Sciences, University of KwaZulu-Natal, Durban 4001, South Africa 4Department of Organic Chemistry, University of Barcelona, 08028 Barcelona, Spain 5CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, 08028 Barcelona, Spain 6School of Chemistry and Physics, University of KwaZulu-Natal, Durban 4001, South Africa 7Chemistry Department, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia, Alexandria 12321, Egypt

Prompted by such facts it is worthy to envisage that combination of such bioactive moieties (Figure 1) may form new biologically active agents.Herein, we report the synthesis of novel class of s-triazine-pyrimidinetrione hydrazone derivatives and their tautomeric behavior as well.and/or DMSO-d 6 using internal standard  = 0 ppm.Elemental analysis were performed on Perkin-Elmer 2400 elemental analyzer.Melting points were determined on a Mel-Temp apparatus and are uncorrected.Fourier transform infrared spectroscopy (FTIR) spectra were recorded on Nicolet 6700 spectrometer from KBr discs.Ultrasonic bath was purchased from Selecta (Barcelona, Spain).Highresolution mass spectrometric data were obtained using a Bruker microTOF-Q II instrument operating at room temperature and a sample concentration of approximately 1 ppm.All compounds were named by using ChemBioDraw Ultra version 14.0, Cambridge Soft Corporation (Cambridge, MA, USA).

Synthesis of 2-Chloro-4,6-disubstituted-s-triazine Derivatives 2a-h.
The target chloroderivatives were prepared following the reported method with slight modification [31].-h).The hydrazine derivatives were prepared according to the reported method with slight modification [12,32].Hydrazine hydrate (10 mL, 80%) was added in portion to a solution of 2-chloro-4,6-disubstituted-1,3,5-triazine 2a-h (20 mmol) in 50 mL acetonitrile at room temperature and then the reaction mixture was sonicated for 60 min at 60 ∘ C. Acetonitrile and excess hydrazine were removed under vacuum and then excess diethylether was added to afford the product as a white solid in yield >90% and used directly without further purification for the condensation reaction with 4.

Synthesis of 1,3-Dimethyl-5-acetyl Barbituric Acid (4).
Compound 4 was prepared following the reported method with slight modification [30,33]: 1,3-dimethyl barbituric acid (50 mmol) was suspended in very small amount of water (5-10 mL) and a concentrated water solution of sodium bicarbonate (NaHCO 3 , 50 mmol) was added.Acetic anhydride was added after the gas evolution ceased.The white precipitate was formed after about 5 minutes and the mixture was stirred overnight at room temperature.The white precipitate was filtered and dissolved in 15-20% ammonium hydroxide (NH 4 OH).To neutralise, hydrochloric acid (HCl) was added under cold conditions (as the reaction is exothermic) until pH was below 1.The increase in temperature was witnessed followed by formation of precipitate which was filtered and allowed to dry at room temperature.The product obtained as white solid from ethanol in yield 83%; mp 94-96 ∘ C;

Results and Discussion
The general method for the preparation of triazine derivatives is nucleophilic displacement of chlorine present in the inexpensive commercially available cyanuric chloride (1, 2,4,6trichloro-1,3,5-triazine) because of the reactivity of its chlorine atoms toward nucleophiles in presence of hydrochloride acceptor like sodium carbonate, bicarbonate, hydroxide, or tertiary amines [34][35][36].Here, we report synthesis of 2chloro-4,6-disubstituted-s-triazine derivatives using 1.Secondary amines like morpholine, diethylamine, and piperidine were chosen due to their known pharmacological properties [1,37].Taking advantage of the different reactivity in front of the nucleophiles of the tri-, di-, and monochloroderivatives of s-triazine, products 2a-i (Scheme 1) were obtained through one pot reaction [12].Thus, 1 was reacted at 0 ∘ C for 2 h with the first amine (1 equiv.) in acetone-water media or methanol in the presence of NaHCO 3 (1 equiv.)as hydrogen chloride scavengers.The second amine was added dropwise followed by addition of NaHCO 3 (1 equiv.)and the reaction temperature was raised gradually to room temperature and kept under stirring for 12 h (Scheme 1) to afford the target product.In case of the methoxy derivative 2h, the reported method [33] was used for its preparation and then reacted with the amine as mentioned in Scheme 1. Finally, the hydrazine derivatives 3a-h were obtained by treatment of 2a-h with hydrazine hydrate (80%) in acetonitrile and sonicated for 1 h [12] to afford the products in excellent yields and purities above 90%.(Scheme 1) which was used directly with 5-acetyl-1,3-dimethylbarbituric acid 4 without further purification.
The products 5a-h were obtained by reaction of the 2hydrazino-4,6-disubstituted-s-triazine derivatives 3a-h with 1,3 dimethyl-5-acetylpyrimidine-2,4,6-trione 4, which was obtained as previously described [33] in ethanol in the presence of drops of glacial acetic acid (Scheme 1) to afford the target products in excellent yields and purities as observed from their spectral data.
Compound 4 may exist in three tautomeric forms 4A, 4B, and 4C as shown in Figure 2. In solution, the NMR spectra in CDCl 3 showed that it exists in the enol form 4C which agreed with our previous reported data [35], rather than keto form 4A (Figure 2).The 1 H NMR spectra for compound 4 showed a singlet at  17.24; this peak is related to the OH group of the enol form 4C and it appears at low field due to the strong hydrogen bond between the OH and the carbonyl carbon of the acetyl group.On the other hand, the expected peak at  4.3 related to the CH flanked between the two carbonyl groups (-CO-CH-CO-) in the keto form 4A was not observed.The 1 H NMR showed that two singlet peaks at  3.29 and 3.34 related to the two N-CH 3 which agreed with structure 4C rather than 4B.The 13 C NMR for compound 4 also confirmed the enol form 4C rather than the keto form or the enol form 4B, where 13 C NMR showed peaks at  24.8 (CH 3 ), two peaks at 27.9 and 28.1 for the 2 N-CH 3 , 95.9 (C4=C-OH), 150.5 (C1), 161.0 (C5), 169.5 (acetyl C=O), and 196.2 (C=C3-OH) (Supporting Information Figure S7 in Supplementary Material).This observation is in agreement with the reported data by Sharma et al. [38] and Giziroglu et al. [39].Once it is established that 4 exists only in an enol form, the structure of 5a-h was studied, in particular respect to the enhydrazine-hydrazone tautomerism.In this case, the hydrazine moiety incorporates two NH.Thus in addition to the NH (in green, Figure 3), which can stabilizes the molecule (enhydrazine form) through a six-member ring as happens in the case of the enol form 4B or 4C (Figure 2), which is not the case for the hydrazone form 5b-A.The NMR data supported the structure 5b-B more than the others.
Taking as example compound 5b, it may exist in three tautomeric structures 5b-A, 5b-B, and 5b-C as indicated in Figure 3. 1 H NMR in DMSO-d 6 showed multiple peaks at  1.54-1.63for the three methylene groups (-CH 2 -) 3 of the piperidine moiety, singlet at  2.74 for the methyl group (N=C-CH 3 ), singlet at  3.29 for the two methyl group (N-CH 3 ), triplet for the two methylene groups (-CH 2 N) 2 of piperidine ring, and a singlet peak at  14.The NMR data for all the target products showed that 5a-h exist in the enhydrazine form B rather than the hydrazone form A or C as shown in Figure 3, where the two peaks in range of  90.0 and 198.0 ppm are related to the two carbon similar to those of the enhydrazine form (C=C-NH-NH-).In addition, the hydrogen bond in the enhydrazine form stabilizes the structure 5b-B more than the hydrazone isomer 5b-A as shown in Figure 3.This is also in agreement with the reported data for aroylhydrazine derivatives reported by Giziroglu et al. [39].
To expand the scope of this work, compound 5b was modelled using molecular mechanics MM2 calculations.Quantum mechanical calculations were carried out using Gaussian98 suite of programs.Geometry optimization was done by DFT method using B3LYP/6-31G * * basis set to assess the relative stability of the diastereomeric species (Figure 4).The calculated relative energy of hydrazone (5b-A) is −963917.8kcal/mole while that of enhydrazine (5b-B) is −963940.7 kcal/mole, while the calculated relative energy of 5b-C geometry was optimized and found to be −963939.1561kcal/mol, which shows higher energy level when compared with 5b-A and 5b-B.Hence on comparison 5b-C is found to be least stable amongst all the three diastereomers.From the energy values it can be seen that theoretically 5b-B is more favorable compared to 5b-A and 5b-C.From the energy values it can be found that the difference in energy level is 22.9 kcal/mol.Hence, explaining the stability of the enhydrazine (5b-B) form over hydrazone form (5b-A).
The antibacterial screening for all the products against gram positive [S.aureus (29213) and B. subtilis (6051) and gram negative E. coli (25822) and P. aeruginosa (27583)] showed no activity; this might be due to the poor solubility and precipitation during the dilution process.

Conclusion
Reaction of 2-hydrazino-2,6-disubstituted-1,3,5-triazine with 5-acetyl-1,3-dimethyl barbituric acid in ethanol affords the hydrazine derivatives in the enhydrazine form as a pure isomer rather than the hydrazone form as observed from the spectral data.The geometry optimization was done by DFT method using B3LYP/6-31G * * to calculate the relative energy of the three structures 5b-A, 5b-B, and 5b-C and indicated that the enhydrazine 5b-B is the most stable structure while 5b-A and 5b-C are less stable which agrees with the NMR spectral data.Although no significant activity of the first family of these compounds has been found as antibacterial, more derivatives are being prepared for overcoming the solubility problems, which are believed to be the cause of the poor biological activity.
3 drops of acetic acid, 2-hydrazino-4,6-disubstituted-1,3,5triazine 3a-h (10 mmol) was added and the reaction mixture was stirred under reflux for 3 h.The solvent was reduced under vacuum and the precipitated product was filtered off and dried at room temperature.The products were collected and recrystallized from ethylacetate.