Synthesis , Characterization , and Antimicrobial Studies of Novel Series of 2 , 4-Bis ( hydrazino )-6-substituted-1 , 3 , 5-triazine and Their Schiff Base Derivatives

The present work represents the synthesis, characterization, and antimicrobial studies of novel series of 2,4-bis(hydrazino)-6substituted-1,3,5-triazine and their Schiff base derivatives. IR, NMR (1H and 13C), elemental analysis, and LC-MS characterized the prepared compounds. The biological activity of the target products was evaluated as well. Twenty-two of the prepared compounds were selected according to their solubility in aqueous DMSO. Only eight compounds showed good activity against the selected pathogenic bacteria and did not show antagonistic effect against fungus Candida albicans. Two compounds 4k and 5g have widerange effect presently in Gram-positive and Gram-negative bacteria while other compounds (4f, 4i, 4m, 5d, 6i, and 6h) showed specific effect against theGram-negative orGram-positive bacteria.Theminimum inhibitory concentration (MIC,μg/mL) of 4f, 4i, 4k, and 6h compounds against Streptococcus mutans was 62.5 μg/mL, 100 μg/mL, 31.25 μg/mL, and 31.25 μg/mL, respectively. The MIC of 4m, 4k, 5d, 5g, and 6h compounds against Staphylococcus aureus was 62.5 μg/mL, 31.25 μg/mL, 31.25 μg/mL, 100 μg/mL, and 62.5 μg/mL, respectively. TheMIC of 4k, 5g, and 6i compounds against Salmonella typhimurium was 31.25 μg/mL, 100 μg/mL, and 62.5 μg/mL, respectively. The MIC of 6i compound against Escherichia coli was 62.5 μg/mL.


Introduction
Hydrazones are considered as extraordinary class of compounds in the Schiff bases family, characterized by the presence of -C=N-N-.The two connected nitrogen atoms are unlike nature and the C-N double bond that is conjugated with a lone pair of the terminal nitrogen atom is mainly responsible for their physical and chemical properties [1][2][3][4][5][6][7].This class of compounds is usually used as polydentate chelating agents that can form a variety of complexes with a range of transition and inner transition metals which have attracted the attention of many researchers [8,9].Huge derivatives of hydrazones and their metal complexes have been studied and used in different applications, such as metal ions extraction and microdetermination of metal ion [10,11].

Experimental Section
2.1.Chemistry 2.1.1.Materials.The solvents used were of analytical reagent grade.NMR ( 1 H and 13 C) spectra were recorded on a JEOL 400, 600 MHz spectrometer at room temperature.The chemical shifts were measured using internal standard  = 0 ppm.Elemental analyses were performed on Perkin-Elmer 2400 elemental analyzer, and the values found were within ±0.3% of the theoretical values.Melting points were recorded on a Mel-Temp apparatus in an open capillary and are uncorrected.Fourier transform infrared spectroscopy (FTIR) spectra were recorded on Nicolet 560 spectrometer from KBr discs.The reaction was followed up and the purity was checked using TLC (silica gel-protected aluminum sheets type 60 GF254, Merck).2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) (2 mg/mL) was purchased from Sigma and used for the bacterial growth test.

General Method for the Synthesis of 2,4-Dihydrazino-6substituted-1,3,5-triazine Derivatives a-e. Compounds a-e
were prepared according to the reported procedure with some modifications [21,44,45].Hydrazine hydrate (80%) in 20 mL acetonitrile was added to a solution of a-e (20 mmol) in acetonitrile (50 mL) at room temperature.The reaction mixture refluxed for 3 h, and then the excess of hydrazine and solvent was removed under vacuum and the crude white precipitate was filtered off, washed with acetonitrile and ether, and then dried to afford the desired products with high yields and purities.The products were used directly in the next step.

Antimicrobial Activity.
The antibacterial activity of the prepared compounds was assessed against two selected bacteria groups: Gram-positive bacteria, namely, Streptococcus mutans (wild strain) and Staphylococcus aureus (ATCC 29213), and Gram-negative bacteria, namely, Escherichia coli (ATTC 25922) and Salmonella typhimurium (ATCC 14028), maintained in Brain Heart Infusion medium at −20 ∘ C. One mL of each targeted grown bacterial culture was added to 100 mL of Brain Heart Infusion broth and incubated at 37 ∘ C ± 1 ∘ C for subculture of all targeted bacterial cultures.Using of sterile physiological solution, concentration of bacterial inoculum was determined to be 10 8 CFU/mL (0.5 McFarland turbidity standards) for antimicrobial tests.In case of fungus Candida albicans, the PDA was used as a medium in antagonistic activity against the tested fungi.

Primary Screening of Synthetic Organic Compounds for
Bioactivity by Agar Diffusion Disc.The tested compounds were dissolved in DMSO and prepared at concentration 2500 g/mL.The sterilized Mueller Hinton agar plates seeded with pathogenic bacteria were prepared; 6 mm paper discs with 100 g of the tested compounds were placed in the plates seeded with tested pathogenic bacteria.The inoculated plates were kept at 37 ∘ C for 24 h for bacteria while, in case of fungi, the used medium in antagonistic activity against tested fungi is PDA.Antimicrobial activity was determined by the inhibition zone [46].

Minimum Inhibitory Concentration (MIC) Determination.
The bioactivity of the tested compounds was determined by using a microdilution method, using Mueller-Hinton broth.The inoculum was prepared as described previously and the tested compounds were stocked in 0.25% of DMSO (concentration of each compound 1.25 mg/mL).After that, serial dilutions were done by addition of 100 L of culture broth which was poured in microplate and 25 L of each concentration of tested compound in the first line of microplate to reach concentrations ranging from 125 to 0.973 g/mL in presence of the positive and negative control.The microplates were vaccinated with 5 L of a bacterial inoculum (10 8 CFU/mL) and were kept for 24 h at 37 ∘ C in triplicate tests.After incubation period, we added 20 L INT (0.5 mg/mL) to achieve bacterial growth.The INT containing microplates were incubated at 37 ∘ C for 30 min and the collected data were expressed in micrograms per milliliters [47].

Chemistry. The first chlorine atom of cyanuric chloride
was replaced by benzylamine, N-methylbenzylamine, morpholine, piperidine, or methoxy, while two hydrazine groups replaced the second and third chlorine atoms.Accordingly, 2,4-dihydrazino-6-substituted-1,3,5-triazine derivatives a-e were prepared by displacement of the two chlorine atoms by two hydrazine groups.
Cyanuric chloride was first reacted with different amines such as benzylamine, N-methylbenzylamine, morpholine, piperidine, or methanol at 0 ∘ C for 1-2 h to afford the products a-e (Scheme 1); the spectral data agreed with the reported data [44,45].The dichloro derivatives a-e were reacted with hydrazine hydrate (80%) under reflux using acetonitrile as a solvent to render the products of 2,4-dihydrazino-6-substituted-1,3,5-triazine derivatives a-e which were used directly in the next step without further purification (Scheme 1).
The productswere prepared by condensation of the hydrazine derivatives a-e with different aldehydes or Scheme 1: Synthesis of 6-substituted-s-triazine-Schiff base derivative.
substituted acetophenone in ethanol as solvent and in the presence of drops of acetic acid (Scheme 1).The 1 H NMR spectrum of b (Figure 1) as a prototype for the a-m series displayed two doublet peaks at  4.54 (J = 6.4 Hz) and 7.04 ppm (J = 8.0), attributed to resonances of protons of CH 2 N and 2H-6 respectively, doublet of doublet at  6.77 ppm which integrated two protons for 2H-4, two triplets at  7.22 (J = 8.0 Hz) and 7.32 ppm (J = 7.6) for H-4  , 2H-5, and H-3  , H-5  , and four singlets at  7.08, 7.38, 8.06, and 9.54 ppm for 2H-2, H-2  combined H-6  , CH, and OH, respectively, while the NH proton appeared as abroad singlet at  10.82 ppm.The 13 C NMR spectrum also reveals the symmetry of b, as indicated by the appearance of fourteen distinct carbon peaks, among which are four peaks at  112.9, 116.8, 118.3, 126.9, 127.6, 128.6, 130.1, 136.8, 140.7, and 157.9 ppm related to the aromatic carbons, in addition to four peaks at  43.8, 143.0, 164.6, and 166.6 ppm belonging to (CH 2 NH), and (C=N), respectively.The 1 H NMR spectrum of b as a prototype for the a-i series exhibited three singlets at  2.33, 3.10, and 4.87 ppm, which were assigned to protons of 2CH The 1 H NMR spectrum of i as a prototype for a-i series showed three singlet peaks at  1.55, 1.64, and 3.84 ppm for the 2CH 2 , CH 2 , and 2NCH 2 (piperidine moiety), while it showed a singlet at  2.31 ppm for the methyl group of the acetophenone moiety and two broad singlets at  9.90 and 11.0 ppm for OH and NH, respectively, beside two doublets forming AB system at  6.82 (J = 8.0 Hz) and 7.70 ppm (J = 7.2 Hz) for the aromatic proton H-3, H-5 and H-2, H-6, respectively.The 13 C NMR spectrum of i exhibited absorption peak at  14.4 related to methyl group, three peaks for the piperidine residue at  24.6, 26.

Bioactivity of Tested Compounds against the Pathogenic
Bacteria and Fungus Candida albicans.In the present study, twenty-two compounds from the above serieswere tested for bioactivities against the selected bacteria and fungi; only eight compounds have antiactivities of target pathogenic bacteria.The results in Table 1 showed that the tested compounds, namely, f, k, l, m, d, g, i, and  h, have bioactivity against the target pathogenic bacteria as antimicrobial agents and do not have antagonistic effect against fungus Candida albicans.The present study also showed that compounds k and g have wide-range effect presently in Gram-positive and Gram-negative bacteria, while compounds i and h were specific in the effect against the Gram-negative and Gram-positive bacteria, respectively.Table 1 also showed that compounds f and l showed activity against Streptococcus mutans, while compounds m and d showed activity against Staphylococcus aureus.
A lowest concentration of chemical compound that inhibits the bacterial growth is the minimum inhibitory concentration (MIC, g/mL).The MIC of compound k that inhibited all the tested pathogenic bacteria was 31.25 g/mL, while the MIC of compound f that inhibited the Streptococcus mutans was 62.5 g/mL, the MIC of compound l that inhibited Streptococcus mutans was 100 g/mL, and the MIC of compound m that inhibited Staphylococcus aureus was 62.50 g/mL.The MIC of compound d that inhibited Staphylococcus aureus was 31.25 g/mL, the MIC of compound g that inhibited Staphylococcus aureus and Salmonella typhimurium was 100 g/mL, the MIC of compound i that inhibited Escherichia coli and Salmonella typhimurium was 62.50 g/mL, and the MIC of compound h that inhibited Streptococcus mutans and Staphylococcus aureus was 31.25 and 62.50 g/mL, respectively (Table 2).It is obvious from Table 1 that small structural variations in s-triazine ring may induce an effect on antibacterial activity, such as in series of benzyl derivatives ( series) and the N-methylbenzyl derivatives ( series); the benzyl derivatives showed more activity than the N-benzyl derivatives.The same was observed when the benzyl was substituted by methoxy ( series); the benzyl derivatives showed more activity.Also the substituent effect on the benzylidene moiety has a great effect on the antibacterial activity as shown in case of series and series, and it is interesting to note that in most of the cases the fluorine derivatives showed higher activity than the chlorine and bromine derivatives as in case of series.On the other hand, not all the tested compounds showed any antifungal activity; this observation agreed with the reported data for s-triazine derivatives [48,49].

Conclusion
The present work represents the synthesis, characterization, and biological activity of novel series of the s-triazine bis-Schiff base.Only twenty-two compounds from the above series a-m, a-i, a-i, a-i, and a-g were tested against Gram-positive and Gram-negative bacteria according to their solubility in aqueous DMSO.Only eight compounds k, f, l, m, d, g, i, and h showed bioactivity against the target pathogenic bacteria as antimicrobial agents and did not show antagonistic effect against fungus Candida albicans.Two compounds k and g have wide-range effect presently in Gram-positive and Gram-negative bacteria, while compounds i and h have specific effect against the Gram-negative and Gram-positive bacteria, respectively.
In addition, compounds f and l showed activity against Streptococcus mutans while compounds m and d showed activity against Staphylococcus aureus.
It is clear that the substituent in the benzylidene as well as the triazine ring may have great effect on the antimicrobial activity.Further investigation is being run in our lab to get a clear depiction on the mode of action and the relation between the biological activity and substituent effect on the s-triazine moiety.
0, and 44.3 ppm related to (CH 2 ), (2CH 2 ), and (2CH 2 N), respectively, and absorption peaks at  154.2 and 160.0 ppm related to (C=N), besides four peaks at 115.6, 128.2, 131.1, and 159.1 ppm for the aromatic carbons.The LC-MS of compound i (Supporting Information, Figure S42 in Supplementary Data) using buffer A: 0.1% formic acid in H 2 O and buffer B: 0.1% formic acid in CH 3 CN in 30 min showed one peak at   14.08 min with the expected mass [M + 2H] 463.2 (m/z calcd.460.53).The 1 H NMR spectrum of b as a prototype for the a-g series revealed the resonances of protons methyl group at  2.33 ppm (s, 2CH 3 ) and methoxy group at  3.89 ppm (s, OCH 3 ), in addition to the two doublets that appeared at  7.25 (d, 4H, J = 7.2 Hz) and 7.56 ppm (d, 4H, J = 7.2 Hz) representing of 2H-2, 2H-3, and 2H-5, 2H-6 (AB system) while singlet (integrated for one proton) appearing at  8.14 ppm was attributed to CH.Finally, the proton of NH group appeared as broad singlet at  11.28 ppm.The 13 C NMR spectrum of b showed signal at  21.5 for the methyl carbon while methoxy carbon appeared at  54.4 ppm and the aromatic carbons signals at  127.1, 129.8, 132.4,and 139.6 ppm.The three signals at 144.3, 165.5, and 166.2 ppm in the later spectrum were assigned to (C=N).

Table 1 :
Bioactivities of the synthetic organic compounds against some pathogenic bacteria and unicellular fungus Candida albicans at 100 g/paper disc (6 mm).

Table 2 :
Minimum inhibitory concentration (MIC, g/mL) of bioactive synthetic compounds against target pathogenic bacteria.