Ultrasonic Irradiation: Synthesis, Characterization, and Preliminary Antimicrobial Activity of Novel Series of 4,6-Disubstituted-1,3,5-triazine Containing Hydrazone Derivatives

1Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia 2Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia 3Department of Biotechnology and Food Technology, Thamar University, Dhamar, Yemen 4Chemistry Department, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia, Alexandria 12321, Egypt


Introduction
Ultrasound has been employed more and more frequently in organic synthesis [1,2], because it improved the reaction rate and could adjust the selectivity performance of the reaction [3].Comparing with traditional methods, ultrasonic irradiation is suitable and simply controlled and it considered as a green powerful synthetic technique in chemical processes.Ultrasonic irradiation has proven to be a particularly important tool for meeting goals of the green chemistry, which is minimization of waste, and decreasing of the energy required for the reaction [4].Applications of ultrasonic irradiation in organic synthesis are playing important role, especially in cases where traditional methods require drastic conditions or elongated reaction time [5][6][7][8][9][10].
Richards and Loomis first reported the chemical effects of ultrasound in 1927 [11].The effect of ultrasound during organic reaction is due to cavitation, which led to separation of molecules of liquids and then the collapse of the bubbles offers strong impulsions that generate shortlived regions with high pressure and temperature.Such localized hot spots act as microreactors in which the sound energy converted into beneficial chemical form [12][13][14].
Recently Schiff base hydrazone derivatives have attracted great attention in many applications [15][16][17].Compounds bearing hydrazone moiety exhibit a broad range of biological activities, including antifungal, antibacterial, antiviral, antimalarial, antiproliferative, anti-inflammatory, and antipyretic properties [18][19][20][21][22]. On the other hand, cyanuric chloride 1 is the most important reagent for s-triazine derivatives, because of the selective reactivity of its chlorine atoms toward nucleophiles.Cyanuric chloride 1 is commercially available and a very cheap reagent, which makes its applications even more attractive [23][24][25][26][27][28][29].The easy displacement of chlorine atoms in cyanuric chloride by various nucleophiles was controlled by temperature to run in a stepwise manner as shown in Scheme 1.
As part of our continuous research on s-triazine derivatives and their biological activities [40,41], we report here the synthesis and the preliminary antimicrobial activity of novel series of 4,6-disubstituted-s-triazine containing hydrazone derivatives.and/or DMSO-d 6 using internal standard  = 0 ppm.Elemental analysis was 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.The reaction was follow-up and checks of the purity using TLC on silica gel-protected aluminum sheets (Type 60 GF254, Merck) using a mixture of methanolchloroform (1 : 9) as an eluent.LC-MS (see Figures S11,  S13, and S18 in Supplementary Material) was performed on Shimadzu 2020 UFLC-MS using an YMC Triart C 18 (5 m, 4.6×150 mm) column, and data processing was carried out by the LabSolution software.Buffer A: 0.1% formic acid in H 2 O and buffer B: 0.1% formic acid in CH 3 CN in 30 min at  max 254 nm were used.High-resolution mass spectrometric data were obtained using a Bruker micrOTOF-Q II instrument operating at room temperature and a sample concentration of approximately 1 ppm.Ultrasonic bath was purchased from Selecta (Barcelona, Spain).All compounds were named by using ChemBioDraw Ultra version 14.0, Cambridge Soft Corporation (Cambridge, MA, USA).

Experimental Section
(i) General Method for the Synthesis of 2-Hydrazino-4,6-dimorpholinio or Dimethoxy-1,3,5-triazine (3 and 5).Hydrazine hydrate (10 mL, 80%) was added dropwise to a solution of 2-chloro-4,6-dimorpholino or dimethoxy-1,3,5-triazine 2 or 4 (20 mmol) in 50 mL ethanol at room temperature and then the reaction mixture was sonicated for 60 min at 60 ∘ C. Ethanol and excess hydrazine were removed under vacuum and then excess diethyl ether was added to afford the product as a white solid in yield >90%.The spectral data of the two products 3 and 5 were in good agreement with the reported data [40,42] and were used directly without further purification.
Method A: Conventional Method.2-Hydrazino-4,6-disubstituted-1,3,5-triazine 3 or 5 (10 mmol) was added to a solution of aldehyde or ketone 6 (10 mmol) in ethanol (30 mL) containing 2-3 drops of acetic acid and then the reaction mixture was stirred under reflux for 3 hours.After completion of the reaction, the solvent was reduced under vacuum and the precipitated product was filtered off, dried at room temperature, and then recrystallized from ethyl acetate to afford the target product (Table 1).
Method B: Ultrasound Assisted Method.A mixture of aldehyde or ketone 6 (10 mmol) in ethanol (30 mL), 2-hydrazino-4,6disubstituted-1,3,5-triazine 3 or 5 (10 mmol), and 2-3 drops of acetic acid in a flask was heated into a sonicator at 40 ∘ C for 30-60 min.After completion of reaction (TLC, methanolchloroform 1 : 9) UV lamp was used for spot visualization at  max 254; the solvent was removed under vacuum and the precipitated product was recrystallized from ethyl acetate to afford the target product (Table 1).

(E)-4-((2-(4,6-Dimorpholino-1,3,5-triazin-2-yl)hydrazono)methyl)-N,N-dimethylaniline (7n, Supporting Information Figure S16 in Supplementary Material
The microbial inoculation suspensions were prepared in sterile sodium chloride solution (0.89%) from activated microbial cultures.The optical densities of microbial suspensions were adjusted to 0.64 at 600 nm.A sterile swab was moistened with the microbial suspensions and inoculating the dried surface of the medium in a Petri dish.After inoculation, all Petri dishes were kept at 25 ∘ C for 5-10 minutes before dispensing the standard antibiotic agents and the prepared compounds discs on the surface of the media.
In the present work, tobramycin (10 g/desk), chloramphenicol (30 g/disk), fusidic acid (10 g/disk), augmentin (10 g/disk), cycloheximide (30 g/disk), canesten (10 g/disk), and caspofungin (10 g/disk) were used as standard antibacterial and antifungal agents.To prepare disks (4 mg/disk) from the tested compounds, 200 mg from each compound was dissolved in dimethylsulfoxide (DMSO) and then 20 L of the solution was added on the sterile filter disk (6 mm) and then allowed to dry at room temperature inside the safety biological cabinet.After 18-24 h of incubation at 37 ∘ C for bacteria and 48-72 h at 25 ∘ C for fungi, the minimum inhibitory concentration (MIC mg/mL) of the prepared compounds was measured [44].The microdilution assay was applied in 96-well plate (Corning Incorporated, USA) using twofold serial dilution.The original concentration was 4 mg/mL and the total volume was 200 L (1 : 1, chemical suspension: bacterial suspension); 4-iodonitrotetrazolium violet (Sigma, USA) reagent was added after the incubation to measure the bacterial growth through the emergence of violet color.

Results and Discussion
3.1.Chemistry.Compounds 2 and 4 were obtained using onestep reaction, where cyanuric chloride 1 was reacted with morpholine (2 equiv.) in acetone-water media or methanol (as a solvent) in the presence of NaHCO 3 as hydrogen chloride removal at 0 ∘ C for 2 h.In the case of the synthesis of dimorpholino derivative 2, the reaction temperature raised gradually to room temperature and kept under stirring for 12 h at the same temperature, while the reported method [40,42] was used for the preparation of the dimethoxy derivative 4 (Scheme 2).The products 2 and 4 were obtained in good yields and their spectral data agreed with the reported data [40,42].
The hydrazine derivatives 3 and 5 were obtained by treatment of 2 or 4 with hydrazine hydrate (80%) in ethanol for 60 min at 60 ∘ C employing ultrasonic irradiation (Scheme 2) to afford the products in excellent yields (>90%) and purity.
The products 7a-o were obtained by condensation of the hydrazine derivative 3 or 5 with substituted benzaldehyde or acetophenone 6 in ethanol containing 2-3 drops of glacial acetic acid using ultrasonic irradiation for 30-60 min at 40 ∘ C (Scheme 3) to afford the target products in excellent yields and purity as observed from LC-MS (see Supplementary Figures S11, S13, and S18).Ultrasonic irradiation gave the target products in high yields in shorter reaction time compared with the conventional heating as shown in Table 1.
The 1 H NMR spectrum of 7k as a prototype for the benzaldehyde derivatives showed a multiplet peak in the range at  3.71-3.74ppm related to 4 methylene groups (4 OCH 2 -), another multiplet peak in the range at  3.75-3.84ppm related to 4 methylene groups (4 NCH 2 -), doublet at  7.36 ppm for the two aromatic protons H-3 and H-5, doublet at  7.72 for the twoaromatic protons H-2 and H-6, singlet at  7.82 related to the CH (CH=N-group), and a broad singlet at  8.31 for the NH.The 13 C NMR spectrum of 7k showed absorption peak for the morpholine residue at  43.6, 43.7, 66.8, and 66.9 related to 2 CH 2 -N-CH 2 , and 2 CH 2 -O-CH 2 respectively, absorption peaks at  128.2, 128.9, 132.8, and 135.4 ppm related to the aromatic carbons, and absorption peaks at  142.5 and 165.4 ppm for C=N.
The LC-MS of compound 7k using buffer A, 0.1% formic acid in H 2 O, and buffer B, 0.1% formic acid in CH 3 CN, in Compound 7k as an example could adopt two different geometrical isomers (E, Z; Scheme 4).Therefore, 7k was demonstrated using molecular mechanics MM2 calculations.In addition, quantum chemical calculations were carried out with the GAUSSIAN 98 suite of programs.Geometry optimization was carried out using the DFT level (B3LYP/6-31G * * ) of theory to assess the relative stability of the E-Z isomeric species.Computed relative energies of 7k indicated that the E-isomer (total energy content 47.7837 kcal/mol) is more stable than the Z ones (total energy content 41.7295 kcal/mol) by 6.0542 kcal/mol.This observation agreed with our reported data in which the s-triazine hydrazone preferred the E-isomer rather than the Z-ones [41].
As a prototype for acetophenone derivatives, the 1 H NMR spectrum of 7o showed a singlet peak at  2.24 ppm for the methyl group of the acetophenone moiety, two multiplet peaks in the range  3.70-3.83ppm related to the 8 methylene groups of the two morpholino rings (4 -N-CH 2 -CH 2 -O), and a broad singlet at  8.01 ppm related to the NH, beside two doublets forming an AB system at  6.80 (J = 8.8 Hz) and 7.698 ppm (J = 8.46) for the aromatic protons H-3, H-5, and H-2, H-6 respectively.The 13 C NMR spectrum showed absorption peak at  12.7 related to methyl group and two peaks at  43.7 and 66.9 ppm related to the methylene groups (-NCH 2 and O-CH 2 , resp.); the absorption peaks at  156.8, 164.7, 165.2, and 165.4 ppm were assigned for carbons of  2. The results revealed that only two compounds 7i and 7k from all the tested compounds were biologically active with different spectrum activity; on the other hand, they did not show any antifungal activity (Table 2).The results in Table 2 showed that 7i had good biological activity against S. aureus, N. meningitides, and tobramycin resistant B. laterosporus, while it had poor biological activity against S. mutans (10 mm).Compound 7k showed a good biological activity against N. meningitides (17 mm) and a very weak activity (<8) against E. coli and S. typhimurium.The measured minimum inhibitory concentrations (MIC) of 7i that inhibited S. aureus, S mutans, N. meningitides, and B. laterosporus were 0.10, 0.20, 0.10 and 0.05 mg/mL, respectively, while the MIC of 7k that inhibited N. meningitides was 0.10 mg/mL.
It is obvious from Table 2 that a small structural variation in s-triazine ring may induce an effect on antibacterial activity.By observing the antibacterial activities of all the derivatives, it is interesting to note that the dimorpholino-4chlorophenyl-s-triazine derivatives 7k show good antibacterial activity against N. meningitides and weak activity against E. coli and S. typhimurium.The other derivatives in the same family did not show any biological activity; this observation agreed with the reported data for substituted s-triazine [45].The presence of pyridine ring (compound 7o) in place of the benzaldehyde ring (compound 7a) changed the biological activity significantly as observed from Table 2; on the other hand, all the tested compounds did not show any antifungal activity; this observation also agreed with the reported data for s-triazine derivatives [45].

Conclusion
The target products (7a-o) were prepared using conventional heating and ultrasonic irradiation.The ultrasonic irradiation affords the products in higher yields and purity in shorter reaction time.The biological activity for the prepared compounds showed that only two compounds 7i (pyridine derivative) and 7k (4-chlorobenzaldehyde derivative) have biological activities against some Gram-positive and Gramnegative bacteria, while all tested compounds did not show any antifungal activity.It is obvious that changing the substituent in the s-triazine ring as well as the benzylidene moiety may affect the antimicrobial activity.Further investigations are run in our lab to get insights into the mode of action and the relation between the biological activity and substituent effect in the pattern in s-triazine.

Table 1 :
Yield, reaction time, and melting point of 7a-o derivativesusing conventional method and ultrasonic irradiation.
Aspergillus niger (AUMC 8777), and Fusarium sp were used in the evaluation test.

Table 2 :
Antimicrobial activity (zones of inhibition, mm) and a minimum inhibitory concentration (MIC, mg/ml) of 7i and 7k comparing with several standard antimicrobial drugs.