Schiff bases are widely studied and used in the fields of organic synthesis and metal ion complex [1,2] for a number of reasons: their physiological and pharmacological activities [3-5] their use in ion selective electrodes [6-11], in the determination of heavy metals ions in environmental samples [12], in the extraction of metals ions [13,14] and their many catalytic applications e.g. for epoxidation of olefins, alkene cyclopropanation [15,16] trimethylsily-lcyanation of ketones [17] asymmetric oxidation of methyl phenyl sulfide enantioselective epoxidation of silylenol [18] and ring-opening Polymerization of lactide [19]. Hydrazones are special group of compounds in the Schiff bases family. They are characterized by the presence of (C=NN= C). the presence of two inter-linked nitrogen atoms was separated from imines, oximes etc. Hydrazone Schiff bases of acyl, aroyl and heteroacroyl compounds have additional donor sites like C=O. The additional donor sites make them more flexible and versatile. This versatility has made hydrazones good polydentate chelating agents that can form a variety of complexes with various transition and inner transition metals and have attracted the attention of many researchers. Various hydrazones are obtained depending on the experimental conditions; which have application as biologically active compounds [20] and as analytical reagents [21]. As biologically active compounds, hydrazones find applications in the treatment of diseases such as anti-tumor [22], tuberculosis [22], leprosy and mental disorder [23]. Tuberculostatic activity is attributed to the formation of stable chelates with transition metals present in the cell. Thus many vital enzymatic reactions catalyzed by these transition metals cannot take place in the presence of hydrazones [24,25]. Hydrazones also act as herbicides, insecticides, nematocides, rodenticides and plant growth regulators.

Reagents and apparatus

All the chemicals used were of Analytical grade and procured from Sigma-Aldrich and Fluka. Metal salts were purchased from E. Merck and were used as received. The elements C, H, and N were analyzed on a Carlo-Erba 1106 elemental analyzer. The IR spectra were recorded on Jusco 300 instrument in KBr pellets. ¹H NMR spectra of ligands in CDCl₃ solution were recorded on a Bruker DT-400 MHz spectrometer, and chemical shifts are reported in ppm. Mass spectra were recorded using a KRATOS MS50TC spectrometer.

AA 929 Unicam Spectrometer was used for FAAS measurements with an air-acetylene flame. A pH meter (Metrohm –691 pH Meter) was also used. All extraction experiments were performed by using a mechanical flask agitator in 50 cm³ stoppered glass flasks, M.P Apparatus Digital (32-300ºC).

Method

Synthesis of Dimethyl isophthalate (I): Isophthalic acid (1.66 gm, 0.1 mmol) in super dry methanol (60 mL) containing 2-3 drops of concentrated H₂SO₄ (AR) was refluxed till it dissolved. Then, the reaction mixture was poured onto ice cold water, immediately a solid started separating from the clear solution. To this a solution of sodium bicarbonate was added till the effervescence seized. The ester thus obtained was filtered and washed with water for several times (M.P: 64-67ºC) [26].

Synthesis of dihydrazide of isophthalic acid (II): A mixture of dimethyl ester of isophthalic acid (2.22 gm) and hydrazine hydrate (98%, 2 cc) in methanol was refluxed for 4-5 hours. The reaction mixture was allowed to cool to room temperature then, the cooled solution was poured on to ice cold water. The dihydrazide of isophthalic acid thus obtained was filtered and recrystallized from ethanol [27].

Yield: (85%), M.P=241ºC, Empirical formula: (C₈H₁₀N₄O₂), M.Wt: (194 gm) (Scheme 1).

Scheme 1: Synthesis of dihydrazide of isophthalic acid(II).

Synthesis of Dimethyl terephthalate (III): Terephthalic acid (1.66 gm, 0.1 mmol) in super dry methanol (60 mL) containing 2-3 drops of concentrated H₂SO₄ (AR) was refluxed till it dissolved. Then, the reaction mixture was poured onto ice cold water, immediately a solid started separating from the clear solution. To this a solution of sodium bicarbonate was added till the effervescence seized. The ester thus obtained was filtered and washed with water for several times (M.P: 138-140ºC) [26].

Synthesis of dihydrazide of terephthalic acid (IV): A mixture of dimethyl ester of terephthalic acid (2.22 gm) and hydrazine hydrate (98%, 2 cc) in methanol was refluxed for 4-5 hours. The reaction mixture was allowed to cool to room temperature then the cooled solution was poured on to ice cold water. The dihydrazide of terephthalic acid thus obtained was filtered and recrystallized from ethanol [28].

Yield: (80%), M.P>300ºC, Empirical formula: (C₈H₁₀N₄O₂), M.Wt: (194 gm) (Scheme 2).

Scheme 2: Synthesis of dihydrazide of terephthalic acid.

Synthesis of 1,6-bis (2-formylphenyl)hexane (V): To a stirred solution of salicylaldehyde (24.4 gm, 0.2 mol) and K₂CO₃ (13.8 gm, 0.1 mol) in DMF (100 mL), was added drop wise 1,6-dibromo hexane (12.2 gm, 0.01 mol) in DMF (40 mL). The reaction was continued for 4 hours at 150- 155ºC and then for 4 hours at room temperature. Then, 200 mL distilled water was added and the mixture was kept in refrigerator. After 1 hour, the precipitate was filtered and washed with 500 ml water. It was dried in air and recrystallized from EtOH and filtered under vacuum [29].

Yield: 85%, M.P: 75°C, Empirical formula: (C₂₀H₂₂O₄), M.Wt: (326 gm) (Scheme 3).

Scheme 3: Synthesis of 1,6-bis (2-formylphenyl)hexane(V).

Synthesis of 1,7,8,14,15,21,22-hepta aza-10,12,24,26-di phenyl- 9,13,23,27-tetra oxo-cyclo hepta icozane-1,6,15,20-tetriene (VI): The macrocyclic Schiff base (VI) was prepared by drop wise addition of a solution of the dihydrazide of isophthalic acid (II) (0.388 gm, 0.002 mol) in DMF (40 mL) to a stirred solution of glutaraldehyde (0.200 gm, 0.002 mol) in DMF (60 mL) containing a few drops of concentrated HCl. The reaction mixture was heated to reflux for 5 hours, where yellow precipitate was formed after cooling. On cooling, 200 ml distilled water was added and the mixture was kept in a refrigerator. After 2 hours, the precipitate was filtered and washed with 200 mL water. The solid obtained was collected and recrystallized from mixture DMF, EtOH (9:1) as white crystals. A white colored precipitate was washed with water, ethanol, CHCl₃ and diethyl ether, respectively. Then the precipitate was dried in air (Scheme 4).

Scheme 4: Synthesis of 1,7,8,14,15,21,22-hepta aza-10,12,24,26-di phenyl-9,13,23,27-tetra oxo-cyclo hepta icozane-1,6,15,20-tetriene.

Yield: 80%, M.P >300°C, Anal. Calc. (C₂₆H₂₈N₈O₄): C: 60.45, H: 5.46, N: 21.69, O: 12.39, Found: C: 60.42, H: 5.29, N: 22.02, O: 12.27%, Mass spectrum (LCMS): m/z= 516 (C₂₆H₂₈N₈O₄), IR (KBr disk): 3431.4 cm^-1 (CO-NH-), 2946.0 cm^-1 (C-H, aliphatic), 1727.5 cm^-1 (C=O), 1688.4 cm^-1 (C=N), 1599.2-1619.6 cm^-1 (C=C, aromatic), 1227.2 cm^-1(C-O, aromatic).

¹H-NMR(CDCl₃-400MHz) δ=11.213 (s,2H,CO-NH-), 8.813 (s,2H,CH=N), 7.165-8.043 (m,8H,Ar), 1.274-2.981 (m,12 ,-CH₂CH₂CH₂), 4.459 (Solvent organic).

Synthesis of 1,16-di aza-3,4,13,14,19,22-tri phenyl-18,23-di oxo- 5,12-di oxa-cyclo tetra icozane-1,15-diene (VII): The macrocyclic Schiff base (VII) was prepared by drop wise addition of a solution of the dihydrazide of terephthalic acid (IV) (0.388 gm, 0.002 mol) in DMF (40 mL) to a stirred solution of 1,6-bis (2-acetylphenyl) hexane (V) (0.652 gm, 0.002 mol) in DMF (60 mL) containing a few drops of concentrated HCl. The reaction mixture was heated to reflux for 5 hours, where yellow precipitate was formed after cooling. On cooling, 200 ml distilled water was added and the mixture was kept in a refrigerator. After 2 hours, the precipitate was filtered and washed with 200 mL water. The solid obtained was collected and recrystallized from mixture DMF, EtOH (9:1) as white crystals. A white colored precipitate was washed with water, ethanol, CHCl₃ and diethyl ether, respectively. Then the precipitate was dried in air (Scheme 5).

Scheme 5: Synthesis of 1,16-di aza-3,4,13,14,19,22-tri phenyl-18,23-di oxo-5,12-di oxa-cyclo tetra icozane-1,15-diene.C

Yield: 85%, M.P >300°C, Anal. Calc. (C₂₈H₂₈N₄O₄): C: 69.41, H: 5.82, N: 11.56, O: 13.21, Found: C: 69.51, H: 5.76, N: 11.53, O: 13.20%, Mass spectrum (LCMS): m/z= 484 (C₂₈H₂₈N₄O₄).

IR (KBr disk): 3237.1-3476.7 cm^-1 (CO-NH-), 2867.0-2942.7 cm^-1 (C-H, aliphatic), 1723.2 cm^-1 (C=O), 1635.64 cm^-1 (C=N), 1600.5 cm^-1(C=C, aromatic), 1281.7-1249.7 cm^-1 (C-O, aromatic).

¹H-NMR(CDCl₃-400 MHz) δ =12.513 (s,2H,CO-NH-), 8.901 (s,2H,CH=N), 7.021- 8.126 (m,12H, Ar-H), 4.336-4.345 (s,4H ,-OCH _2-), 2.508-3.361 (m,8H,-CH₂-CH₂-CH₂-CH₂-).

Synthesis of 1,18-di aza-3,5,14,16,21,24-tri phenyl-20,25-di oxo- 6,13-di oxa-cyclo hexa icozane-1,17-diene (VIII): The macrocyclic Schiff base (VIII) was prepared by drop wise addition of a solution of the dihydrazide of terephthalic acid (IV) (0.388 gm, 0.002 mol) in DMF (40 mL) to a stirred solution of 1,6-bis (2-acetylphenyl) hexane (V) (0.692 gm, 0.002 mol) in DMF (60 mL) containing a few drops of concentrated HCl. The reaction mixture was heated to reflux for 5 hours, where yellow precipitate was formed after cooling. On cooling, 200 ml distilled water was added and the mixture was kept in a refrigerator. After 2 hours, the precipitate was filtered and washed with 200 mL water. The solid obtained was collected and recrystallized from mixture DMF, EtOH (9:1) as white crystals. A white colored precipitate was washed with water, ethanol, CHCl₃ and diethyl ether, respectively. Then the precipitate was dried in air (Scheme 6).

Scheme 6: Synthesis of 1,18-di aza-3,5,14,16,21,24-tri phenyl-20,25-di oxo-6,13-di oxa-cyclo hexa icozane-1,17-diene.

Yield: 82%, M.P =241ºC, Anal. Calc. (C₃₀H₂₄N₄O₄): C: 69.41, H: 5.82, N: 11.56, O: 3.21, Found: C: 69.67, H: 5.68, N: 11.48, O: 13.17%, Mass spectrum (LCMS): m/z= 484 (C₂₈H₂₈N₄O₄).

IR (KBr disk): 3140.11- 3278.99 cm^-1 (CO-NH-), 3072.7 cm^-1, 2843.07-2993.52 cm^-1 (C-H, aliphatic), 1705.07 cm^-1 (C=O), 1600.92 cm^-1 (C=N), 1577.77 cm^-1 (C=C, aromatic), 1242.16 cm^-1 (C-O, aromatic).

¹H-NMR (CDCl₃-400 MHz) δ=10.509 (s,2H,CO-NH-), 8.940- 8.977 (s,2H,CH=N), 6.958-8.138 (m,12H, Ar-H), 4.009-4.018 (s,4H ,-O-CH_2-),1.276-2.198 (m,8H,-CH₂-CH₂-CH₂-CH₂-), 2.383-3.743 (Solvents organic).

Biological activity

The prepared compounds were tested for their antimicrobial activity against four species of bacteria (Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Salmonella typhi) using filter paper disc method [38]. The screened compounds were dissolved individually in DMSO (dimethyl sulfoxide) in order to make up a solution of 50, 100, and 200 μg/ml concentration for each of these compounds. Filter paper discs (Whitman No.1 filter paper, 5 mm diameter) were saturated with the solution of these compounds. The discs were placed on the surface of solidified Nutrient agar dishes seeded by the tested bacteria. The diameters of inhibition zones (mm) were measured at the end of an incubation period, which was 24 hours at 37ºC for bacteria. Discs saturated with DMSO are used as solvent control. Ciprofloxacin 100 μg/ml was used as reference substance for bacteria [30].

Synthesis

The prepared macrocyclic hydrazone (VI, VII, VIII) were synthesized by condensation of intermediate compounds: 1,6- bis (2-formylphenel) hexane, and glutaraldehyde with both dihydrazide of isophthalic acid and dihydrazide of terephthalic acid in the molar ratio (2:2) in DMF. The reactions proceeded smoothly, producing the corresponding Schiff bases ligands in good yield. The ligands are soluble in common organic solvent but insoluble in water. The structures of the ligands were elucidated by elemental analyses, MS, FTIR, electronic absorption, and ¹H- NMR spectra, which help in elucidating their empirical formula (Table 1).

Table 1: Color, molecular weight and melting point of macrocyclichydrazone (V, VI, VII).

Elemental analyses of macrocyclic hydrazone (VI, VII, VIII)

The results of elemental analyses macrocyclic hydrazone (VI, VII,VIII), as shown in Table (2), are in good agreement with those required by the proposed formulae.

Table 2: Elemental analysis data ofmacrocyclichydrazone (VI, VII,VIII).

IR spectra analysis

Compound (VI): A strong band at 1688.4 and 1727.5 cm^-1 in the IR spectrum of the Schiff base (Figure (1)) are assigned to υ(C=N) of azomethine and carbonyl υ(C=O) vibrations, respectively. An intense band at 3431.4 cm^-1 is due to the -NH- vibrations of the hydrazine group. The band in the spectra at 1619.6 - 1599.2 cm^-1 is due to (C=C) of aromatic rings, while the band at 2946.0 cm^-1 is attributed to (CH, aliphatic). Also, the band at 3051.39 cm^-1 is attributed to (C-H, aromatic) [31-35].

Figure 1:IR spectrum ofSchiff base(VI).

Compound (VII): A strong band at 1637.9 and 1723.2 cm^-1 in the IR spectrum of the Schiff base (Figure (2)) are assigned to υ(C=N) of azomethine and carbonyl υ(C=O) vibrations, respectively. An intense band at 3476.7 -3237.1 cm^-1 is due to the -NH- vibrations of the hydrazine group. The band in the spectra at 1600.5 cm^-1 is due to (C=C) of aromatic rings, while the band at 2942.7 - 2867 cm^-1 are attributed to (C-H, aliphatic). Also, the band at 3027.1 cm^-1 is attributed to (C-H, aromatic) [31-35].

Figure 2:IR spectrum ofSchiff base(VII).

Compound (VIII): A strong band at 1600.92 and 1705.07 cm^-1 in the IR spectrum of the Schiff base (Figure (3)) are assigned to υ(C=N) of azomethine and carbonyl υ(C=O) vibrations, respectively. An intense band at 3278.99-3140.11 cm^-1 is due to the -NH- vibrations of the hydrazine group. The band in the spectra at 1577.77 cm^-1 is due to (C=C) of aromatic rings, while the band at 2993.52-2843.07 cm^-1 are attributed to (C-H, aliphatic). Also, the band at 3055.24 cm^-1 is attributed to (C-H, aromatic) [26-30]. However, in the IR spectra of Schiff bases this bands (C=O) disappears and a new vibration bands for azomethine (-HC=N-), indicating that complete condensation takes place [36,37] (Table 3).

Figure 3:IR spectrum ofSchiff base(VIII).

Table 3: IR spectral data (cm^-1) of macrocyclichydrazone (VI, VII, VIII).

¹H-NMR Spectra of macrocyclic hydrazone (VI, VII, VIII)

Compound (VI): The ¹H-NMR spectrums (Figure (4)) of the Schiff base (VI) showed that in the region 2.981-1.274 ppm was assigned to protons of methyl groups in two different environments [38]. The signals at 11.213 and 8.813 ppm were assigned to the protons of amide CONH and imine -CH=N groups respectively. Signals in the region 8.043-7.165 ppm were assigned to the aromatic protons.

Figure 4:¹H-NMR spectrum ofSchiff base(VI).

Compound (VII): The ¹H-NMR spectrums (Figure (5) of the Schiff base (VII) showed that in the region 2.449 - 1.278 ppm was assigned to protons of methyl groups in two different environments [38]. The signals at 10.510 and 8.901 ppm were assigned to the protons of amide CONH and imine -CH=N groups respectively. Signals in the region 8.126-7.021 ppm were assigned to the aromatic protons. While the singlet signals at 4.345 - 4.336 ppm were assigned to the protons (-OCH ₂-) group.

Figure 5:¹H-NMR spectrum ofSchiff base(VII).

Compound (VIII): The ¹H-NMR spectrums (Figure (6)) of the Schiff base (VIII) showed that in the region 2.198 - 1.276 ppm was assigned to protons of methyl groups in two different environments [38]. The signals at 10.509 and 8.977 ppm were assigned to the protons of amide CONH and imine -CH=N groups respectively. Signals in the region 8.138-6.958 ppm were assigned to the aromatic protons. While the singlet signals at 4.018-4.009 ppm assigned to the protons (-O-CH₂- ) group.

Figure 6:¹H-NMR spectrum ofSchiff base(VIII).

The other obtained values for ¹H-NMR chemical shifts of the compounds are given in the experimental section.

The ¹H-NMR spectral data of the new compounds are in good agreement with those previously reported for similar compounds. These results strongly suggest that the proposed compounds have been formed [36,37].

Biological activity

During the last two or three decades, attention has been increasingly paid to the synthesis of macrocyclic hydrazone (VI, VII, VIII), which exhibits various biological activities including antibacterial, fungicidal, tuberculostatic and plant growth regulative properties [39]. It was judicious to investigate the synthesis of various new types of Schiff base and studied their antibacterial activity against four strains of bacteria (Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Salmonella typhi). The concentrations used for the screened compounds are 50, 100, and 200 μg/ml. Ciprofloxacin was used as reference standard while DMSO as control and inhibition zones is measured in mm. The new compounds were tested against one strain each of a gram positive and two gram negative. The test results presence in Table 4, a new compound was active against tested and another compound are no active.

Table 4: Effect of new macrocyclichydrazone (VI, VII,VIII) on the growth of tested bacteria (conc.200 μg/ml).

All compounds are not active when used in the concentration of 50, 100 μg/ml but active in the concentration of 200 μg/ml (Table 5 and Figure 7).

Figure 7: Antibacterial activity (Gram positive, negative) of synthesized compounds.

Table 5: ¹H-NMR Spectra ofmacrocyclichydrazone (VI, VII,VIII).

The compounds are new and were prepared for the first time. The new compounds were identified by melting point, elemental analyses ¹HNMR, IR, LC-MS, spectral methods. The prepared compounds have been biologically screened i.e. studying their effects against two gram-positive, two gram-negative bacteria. The results show that their activities were found to vary from moderate to very strong.

The authors are grateful to the Department of Chemistry, Faculty of Science, University of Damascus, Damascus, Syria.