SYNTHESIS , CHARACTERIZATION AND ANTIBACTERIAL ACTIVITY OF MACROCYCLIC SCHIFF BASES BASED ON 1 , 3-DOCARBONYL PHENYL DIHYDRAZIDE

Four new Macrocyclic Hydrazone Schiff bases were synthesized by condensation of intermediate compounds: 1,6bis (2formylphenel) hexane(III),1,6-bis (2-acetylphenyl)hexane(IV), α,α’-bis(2-carboxyaldehyde phenoxy) xylene(V), and isophthal aldehyde with dihydrazide of isophthalic acid. Identification of these macrocyclic Schiff bases ligands (VI, VII, VIII, IX).The Schiff bases were checked by different spectral technique (LC-MS, H-NMR, IR, elemental analyses). The new Macrocyclic Hydrazone Schiff Bases were studied for antibacterial activities against (Bacillus subtilis and Staphylococcus aureus) are Gram positive and (Salmonella typhi and Escherichia coli) are Gram negative. The compounds ligands were exhibited a variable activity of inhibi tion on the growth of the bacteria.


Introduction
Schiff bases are widely studied and used in the fields of organic synthesis and metal ion complexation [1,2] for a number of reasons: their physiological and pharmacological activities [3][4][5] their use in ion selective electrodes [6][7][8][9][10][11] in the determination of heavy metals ions in environmental samples [12] and 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 enantioselectiveepoxidation 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=N-N=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.
In the context of the above applications we have reported here the synthesis and characterization of novel macrocyclic hydrazone Schiffbases. All these compounds have been characterized byelementa analyses, LC-MS, IR, 1 H NMR spectra data.A Survey of the literature reveal that no work has been carried outon the synthesis of mcrocyclic hydrazone Schiff bases derived from1,3-Docarbonyl phenyl dihydrazide, and in the present study, we synthesized hydrazone Schiff bases and used it in studying antibacterial activity

2.1.Reagents and Apparatus.
All the chemicals used were of AnalaR grade and procured from Sigma-Aldrich and Fluka. Metal salts were purchased from E. Merck and were used as received. The C, H, and N were analyzed on a Carlo-Erba 1106 elemental analyzer. The IR spectra was recorded on Jusco 300 instrument in KBr pellets. 1-H NMR spectra of ligands in CDCl 3 solution were recorded on a Bruker DT-400 MHz spectrometer, and chemical shifts are indicated in ppm relative to tetra methyl silane. Mass spectra were recorded using a KRATOS MS50TC spectrometer. , M.P Apparatus Digital (32-300 0 C).

2.2.1.Synthesis of Dimethyl isophthalate(I)
Isophthalic acid (1.66 g, 0.1mmol) in super dry methanol (60 mL)containing 2-3 drops of concentrated H 2 SO 4 (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 (mp 64-67°C) [26].

2.2.4.Synthesis of 1,6-bis (2-acetylphenyl)hexane(IV).
To a stirred solution of 2-hydroxyl acetophenone (13.6 g, 0.1 mol) and K 2 CO 3 (6.9 g, 0.05mol) in DMF (50 mL), was added drop wise 1,6-dibromo hexane (6.1 g, 0.05 mol) in DMF (20 mL). The reaction was continued for 4 h at 150-155°C and then for 4 h at room temperature. Then, 100 mL distilled water was added and the mixture was kept in refrigerator. After 1 h, the precipitate was filtered and washed with 250 ml water. It was dried in air and recrystallized from EtOH and filtered under vacuum.. [29]. To a stirred solution of salicylaldehyde (24.4 g, 0.2 mol) and K 2 CO 3 (13.8 g, 0.1 mol) in DMF (100 mL), was added drop wise α,α'-Dichlor-p-xylene (17.4 g, 0.1mol) in DMF (40 mL). The reaction was continued for 4 hrs at 150-155°C and then for 4 h at room temperature. Then, 200 mL distilled water was added and the mixture was kept in refrigerator. After 1 h, the precipitate was filtered and washed with 500 mL water. It was dried in air and recrystalized from EtOH and filtered under vacuum.. [29].  The macrocyclic Schiff base (VII) was prepared by dropwise addition of a solution of the dihydrazide of isophthalic acid(II) ( 0.388 g, 0.002 mol) in DMF (40 mL) to a stirred solution of 1,6-bis (2-acetylphenyl)hexane (IV) (0.708 g, 0.002 mol) in DMF (60 mL) containing a few drops of concentrated HCl. The reaction mixture was heated to reflux for 5 h, 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 h, 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 dried in air. The macrocyclic Schiff base (VIII) was prepared by dropwise addition of a solution of the dihydrazide of isophthalic acid (II) ( 0.388 g, 0.002 mol) in DMF (40 mL) to a stirred solution of α,α'-bis(2-carboxyaldehyde phenoxy) xylene (V) (0.692 g, 0.002 mol) in DMF (60 mL) containing a few drops of concentrated HCl. The reaction mixture was heated to reflux for 5 h, 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 h, 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 dried in air.   Synthesis of 1,7,8,14,15,21,22-hepta aza-3,5,10,12,17, 19, 24,27-tetra phenyl-9,13 The macrocyclic Schiff base (IX) was prepared by dropwise addition of a solution of the dihydrazide of isophthalic acid (II) ( 0.388 g, 0.002 mol) in DMF (40 mL) to a stirred solution of isophthal aldehyde (0.268 g, 0.002 mol) in DMF (60 mL) containing a few drops of concentrated HCl. The reaction mixture was heated to reflux for 5 h, 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 h, 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 dried in air.

Biological Activity
The prepared compounds were tested for their antimicrobial activity against four speices of bacteria (Bacillus subtilis, Escherichia coli , Staphylococcus aureus, Salmonella typhi) using filter paper disc method [30]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,5mm 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 h at 37C for bacteria. Discs saturated with DMSO are used as solvent control. Ciprofloxacin 100 μg/ml was used as reference substance for bacteria. [30].

Compound (VII):
A strong band at 1651.0and 1726.6cm -1 in the IR spectrum of the Schiff base( figure  5) are assigned to υ(C=N) of azomethine and carbonyl υ(C=O) vibrations, respectively. An intense band at 3415.9 -3224.3cm -1 is due to the -NH-vibrations of the hydrazine group The band in the spectra at 1599.0 -1578.5cm -1 is due to (C=C) of aromatic rings. while the band at 2939.3 -2866.0 cm -1 are attributed to (C-H aliph). Also, the band at 3066.2cm -1 are attributed to (C-H ar). [31][32][33][34][35]. in the IR spectrum of the Schiff base( figure 11) are assigned to υ(C=N) of azomethine and carbonyl υ(C=O) vibrations, respectively. An intense band at 3439.9 -3192.1cm -1 is due to the -NH-vibrations of the hydrazine group The band in the spectra at 1522.70cm -1 is due to (C=C) of aromatic rings.Also, the band at 3052.3cm -1 are attributed to (C-H ar). [31][32][33][34][35] However, in the IR spectra of Schiff bases this bands (C=O) disappears and a new vibration bands for azomethine(-HC=N-).

Compound (VIII):
Indicating that complete condensation takes place.All IR spectral data of the synthesized compounds showed in theTable 3 [36][37].

Compound (VI):The
1 H NMR spectrum(figure 3)of the Schiff base (VI), showed that in the region 2.223 -1.873 ppm were assigned to protons of methyl groups in two differentenvironments [38]. The signals at 12.511 and 8.954ppm were assigned to the protons of amide CONH and imine -CH=N groups respectively. Signals in the region 8.391 -7.121 ppm were assigned to the aromatic protons.While the singlet signal at 4.147 ppm assigned to the protons (-O-CH 2 -) group.

Compound (VII):
The 1H NMR spectrum (figure 6)of the Schiff base (VII), showed that in the region 2.868 -1.305ppm were assigned to protons of methyl groups in        two differentenvironments [38]. The signals at 10.923ppm were assigned to the protons of amide CONH. Signals in the region 7.664 -6.978ppm were assigned to the aromatic protons.While the singlet signal at 4.126 -4.211 ppm assigned to the protons (-O-CH 2 -) group.
Compound (VIII):The 1 H NMR spectrum (figure 9)of the Schiff base (VIII), showed that in the signals at 13.133 and 9.035 ppm were assigned to the protons of amide CONH and imine -CH=N groups respectively. Signals in the region 7.923 -6.079 ppm were assigned to the aromatic protons.While the singlet signal at 3.982 -3.723 ppm assigned to the protons (-O-CH 2 -) group.. [38] Compound (IX):The 1 H NMR spectrum (figure 12) of the Schiff base (IX), showed that in the signals at 10.128 and 8.596 ppm were assigned to the protons of amide CONH and imine -CH=N groups respectively. Signals in the region 8.200 -6.711 ppm were assigned to the aromatic protons. [38] The other obtained values for 1-H-NMR chemical shifts of the compounds are given in the experimental section. The 1-HNMR spectral data of the new compoundsshowed in theTable 4. These data 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, IX), 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, 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 are 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 (3.11), a new compound was active against tested and another compounds are no active. All compounds are no active where used 50, 100 μg/ml but active in the concentrations 200 μg/ml see Table 5.

Conclusion
1-The compounds are new and were prepared for the first time. 2-The new compounds were identified by melting point, elementalanalyses 1 HNMR, IR, LC-MS, spectral methods. 3-The prepared compounds have been biologically screened i.e. studying their effects against two grampositive, two gram-negative bacteria. The results show that their activities were found to vary from moderate to very strong.