Based on what stated above and to pursue our research on the synthesis of novel DHA- Schiff and their biological properties, herein we have reported the synthesis, characterisation and biological activity of novel DHA- Schiff derivativres. 

Experimental

DHA was purchased from E-Merck Germany, 4-amino phenol, 2-aminopyridine, 2-amino-6-nitrobenzothiazole, 2-amino-5-chlorobenzophenone and 2-amino-4,6-dimethylpyrimidine were obtained from Avra and Acros Organics chemicals. The solvents were dried and distilled before use as per reported procedure [11]. Elemental (C, H and N) analysis was carried out on Perkin Elmer CHN Analyser (2400). The electronic spectra were recorded on Shimadzu UV–VIS Spectrophotometer UV1700, in the range of 200–500 nm at our research center. The IR spectra of Schiff’s bases were recorded on Perkin Elmer (1430) FTIR spectrophotometer in the range 4000 to 666 cm^-1 by KBr pellet method and on alpha Brucker at our research centre RajarshiShahuMahavidyalaya (Autonomous) Latur. ¹HNMR spectra were recorded on Brucker FT 300/400/500 MHz NMR spectrophotometer in CDCl₃ solvent using TMS as reference. The biological activities (antibacterial and antifungal) of synthesized Schiff bases were tested against Escherichia coli (E.coli) (ATCC2331), Staphylococcus aureus (S.aureus) (NCIM-2079) and Bacillus subtilis (B.subtilis) (NCIM-2063) as bacterial strains and Candida albicans (C.albicans) (MTCC-227) and Aspergillus niger (A.niger) (NCIM-545) as fungal strains as per the procedure [12, 14] from our Biotechnology Research Centre.

Synthesis of schiffbases

DHA Schiff bases were synthesized by the addition of ethanolic solution of individual primary amine (0.05 mol) into hot ethanolic solution of DHA (Dehydroacetic acid) (0.05 mol) as per the procedure [13, 14] [Figure 1].

Figure 1. Synthesis of DHA Schiff bases

Results and Discussion

Elemental analysis

All the synthesized DHA Schiff bases are yellow, orange to brown coloured solids, stable to air and non-hygroscopic. They are insoluble in water and soluble in hot ethanol. Their physical characteristics and elemental analysis data are summarized in Table 1.

Table1. Elemental analysis

 Spectral characterization

Electronic spectra

All the synthesized DHA Schiff bases exhibited weak absorption bands λ_max in the region 45871 to 42918 cm^-1, which was assigned to conjugated azomethine π→π* transition appear for >C=N.

The absorption band λ_max in the region 40000 - 37453 cm^-1 appeared due to π→π*and n→π* transitions of >C=N in a heterocyclic ring of amine moiety i.e pyridine at 38785 cm^-1, thiazole 38934 cm^-1 and Pyrimidine at 38979 cm^-1. Benzene ring also showed absorption at 40000 cm^-1 [15].

All the DHA Schiff bases showed one more absorption band λ_max in the region 33167 to 31655 cm^-1 assigned to lactone carbonyl group >C=O, auxochrome –OH attached to homoannular conjugated diene, which was closer to calculated value 31948 cm^-1 [16].

Conjugated Lactone displayed absorption in the region of 50000-41666 cm^-1. The extended conjugation and auxochrome produce a bathochromic shift [15].

FTIR analysis

The absorption peak pattern in IR spectra exhibited complex nature due to various vibrational modes. However, with few objectives, only characteristic peaks which were specific to all Schiff bases related to enolic O-H, aromatic C=C, azomethine >C=N, aryl azomethine, lactone carbonyl C=O and enolic C-O/C=O of Schiff bases were taken into consideration for characterization.

In all the synthesized DHA Schiff bases the characteristic O-H stretching frequencies were observed as broad weak band at 3454 to 3368 cm^-1 due to strong intermolecular hydrogen bonding between enolic O-H and N of azomethine group.

Azomethine >C=N stretching frequency is dependent on its substituent and mostly causes resonance interaction and H-bonding. In the present work azomethine (>C=N-) depicted strong absorption stretching vibration band in the region 1644-1634 cm^-1.

The peak in the region 1708 to 1685 cm^-1 appeared due to>C=O lactone carbonyl stretching vibrations; peak in the region 1388 to 1333 cm^-1appeared due toaryl/aliphatic (C-N) stretching vibrations and the peak in the region 1256 to 1214 cm^-1appeared due to (C-O) enolic stretching vibrations.[15,16,17] IRCharacteristic IR frequencies are summarized in Table 2.

Table 2. Characteristic IR frequencies (cm^-1) of Schiff bases

¹HNMR Spectra

¹HNMR spectra of all the compounds were recorded in CDCl₃ at room temperature. The following signals, chemical shift value δ (ppm) relative to TMS as internal standard were observed:

Singlet signal at δ value 2.1—2.28 for the methyl group at C₆, singlet signal at δ value 14.72 —16.00 of enolic O-H group which is highly deshielded, singlet signal at δ value 5.00—5.94 belonging to H atom at C₅ and singlet signal at δ value 2.57—2.75 for the methyl group attached to azomethine C atom of DHA moiety and different signals of amine moiety.[17]

L₁ =¹HNMR (500 MHz, CDCl₃, δ, ppm : 2.26 (3H, s, C₆–CH₃), 15.88 (1H, s, O –H), 5.92 (1H, s, C₅–H), 2.58 (3H, s, N=C-CH₃,H bonded to ‘C’ azomethine) for DHA moiety,7.4 (2H, d, Ar–H), 7.6 (2H, d, Ar–H), 9.6 (1H, s, Broad) for phenol moiety.

L₂ = ¹HNMR (400 MHz CDCl₃, δ, ppm: 2.2 (3H, s, C₆–CH₃), 16.00 (1H, s, O –H), 5.8(1H, s, C₅–H), 2.6(3H, s, N=C-CH_3­, H bonded to ‘C’ azomethine) for DHA moiety, 7.7-8.5 (4H, m, Ar)  for pyridine  moiety.

L₃ = ¹HNMR (400 MHz CDCl₃, δ, ppm: 2.1(3H, s, C6 –CH₃), 15.85(1H, s, O–H), 5.9(1H, s, C5 –H), 2.6(3H, s, N=C-CH₃, H bonded to ‘C’ azomethine) for DHA moiety, 7.7 (1H, m, C₅-H), 6.9-7.2 (2H, d, C₇& C₈-H),  for benzothiazole moiety.

L₄= ¹HNMR (300 MHz CDCl₃, δ, ppm: 2.2 (3H, s, C₆ –CH₃), 15.54 (1H, s, O–H), 5.75(1H, s, C₅–H), 2.7(3H, s, N=C-CH₃,H bonded to ‘C’ azomethine) for DHA moiety, 2.3(3H, s, ring –CH₃), 7.4 -7.6 (5H, s, Ar.)  7.6 -7.8 (3H, m, Ar.) for benzophenone moiety.

L₅=¹HNMR (300 MHz CDCl₃, δ, ppm: 2.11 (3H, s, C₆–CH₃), 14.82(1H, s, O–H), 5.7(1H, s, C₅–H), 2.75(3H, s, N=C-CH₃, H bonded to ‘C’ azomethine) for DHA moiety, 2.34 (6H, s,  two CH₃ to Ar), 7.6 (1H, s,), for pyrimidine moiety.

Screening for bioactivity

In vitro antibacterial and antifungal activities of the compounds were screened by considering zone of inhibition of growth. The synthesized DHA Schiff bases were screened with their different concentrations with standard antibiotics such as streptomycin (1 mg/mL) and griseofulvin (1 mg/mL).

The DHA Schiff bases have shown harmonious antibacterial and antifungal action. All the DHA Schiff bases were found to be biologically active against E.coli with maximum zone of inhibition 21 mm by L₅. L₁, L₂ and L₃ showed moderate zone of inhibition 17 to 18 mm and L₄ has showed minimum zone of inhibition 15 mm againstE.coli.  L₅ showed 16.4 mm, maximum zone of inhibition against S. aureus.  L₂ and L₄, were found to be moderate in growth inhibition while L₃ showed low activity with maximum zone of inhibition 9.00 mm against the same species. All the compounds were found to be moderate in growth with 11.3 mm zone of inhibition by L₂ while Schiff base L₄ low activity with 4.5 mm zone against B. subtilis. All the DHA Schiff bases have shown harmonious antifungal action.  All compounds   showed about 13 to 16 mm zone of inhibition and L₅ depicted maximum zone of inhibition 19 mm against A. niger. All compounds showed about 13 to 16 mm zone of inhibition, while L₂ showed 21 mm maximum zone of inhibition, along with the compound L₁ low zone of inhibition with 11.3 mm against human opportunistic pathogen, C. albicans.  The bioactivity data is summarized in Table 3.

Table 3. Summarized bioactivity data

B1 - E. coli, B2 -S. aureus, B3- B. subtilis, F1-C. albicans, F2- A. niger, NA-Not applicable

Figure 2. Graphical presentation of bioactivity