Synthesis, Characterization and Evaluate the Biological Activity of Novel Heterocyclic Derivatives from Azomethine Compounds

Introductıon

Azomethine compounds are the compounds containing (-HC=N-) group, the best and easiest method of azomethine synthesis is the condensation reaction between the carbonyl group (C=O) of ketones or aldehydes and the amino group (NH₂) of primary amines [1-4]. The equivalent rate 2:1 of 3- bromo benzaldehyde and 1,3- diamino propan- 2- ol gave the azomethine compound described in the following reaction [5].

Oxazepines are heterocyclic compounds of the seven-membered ring with two heteroatoms (O and N), the oxygen atom is located at position 1 and a nitrogen atom in positions -2, -3 or -4 [1]. To a solution of 0.01 mole of azomethine in dry toluene, a solution of maleic anhydride 0.02 mole in ethanolic solution was added dropwise with stirring then refluxed for 3-4 hours [6]. The reaction of the following azomethine compound with phthalic anhydride in dry benzene gave substituted oxazepine derivatives [7].

In this study, the synthesized compounds have been proven by TLC techneque and melting point assay. Their structures have been characterized by ¹H-NMR and FT-IR spectroscopic methods. The antifungal and antibacterial activity of the synthesized 1,3-oxazepine-dione derivatives were studied against Geotrichum sp., Escherichia coli, Klebsiella sp. and Staphylococcus aureus.

 Experimental

 Materials and Methods

Aldehydes, mines and other chemicals were purchased from Sigma Aldrich. Melting points were recorded on Electrothermal Melting point Apparatus (uncorrected). FT-IR spectra were recorded at room temperature from 4000-400cm^-1 with KBr disc on Infrared Spectrophotometer Model Tensor 27 Bruker Co., Germany. The ¹H-NMR spectra were recorded on Bruker Ac-300MHz spectrometer.

General Procedure for the synthesis of azomethine compounds R₁-R₄

Equimolar mixtures 0.01 mole of aromatic amines and 0.02 mole of aromatic aldehydes dissolved in 25 mL absolute ethanol was placed in a 100 mL round-bottom flask. 3-4 drops of glacial acetic acid was added as catalyst, the mixture was allowed to react at reflux temperature for 4 hours, and then let to cool down to the room temperature, the progress of the reaction and the purity of the compounds were monitored with TLC technique, whereby a crystalline solid was separated out and recrystallized from ethanol, the structural formula, names, melting points, colors and percentage of yields for the synthesized azomethine compounds R₁-R₄ are recorded [8-12].

General Procedure for the synthesis of 1,3-oxazepine-dione derivatives R₅-R₉

Equimolar mixtures 0.01 mole of azomethine compounds and 0.02 mole anhydride compounds dissolved in 30 mL of tetra hydro furan (THF) placed in a 100 mL round-bottom flask, the reaction mixture was refluxed for 3 hours, the progress of the reaction and the purity of the compounds were monitored with TLC technique then solid product was precipitated, filtered off and recrystallized from ethanol, the structural formula, names, melting points, colors and percentage of yields for the synthesized 1,3-oxazepine-dione derivatives R₅-R₉ were recorded [13, 14].

Anti-fungal and anti-bacterial activity of synthesized 1,3-oxazepine-dione derivatives R₅-R

Anti-microbial activity of the derivatives against Geotrichum sp., E. Coli, Staphylococcus aureus and Klebsiella sp. Using well diffusion method on Mueller Hinton Agar plates. The well diameter is 6 mm. The zone of inhibition was recorded as antimicrobial activity. About 6 µg well^-1 of chemicals in DMSO introduced into the bore wells on the agar using a sterile dropping pipette. The extracts were allowed to diffuse before inoculated with the yeast then incubated at 37 ^oC for 24 hour. The plates were examined for measuring any inhibition zone [15]. About 50 µg Gentamycine per well was used as a positive control and 50 µl DMSO was used as a negative control

Results and discussion

Chemistry

Tables 1 and 2 exhibited structural formula, nomenclature, the percentage of yield, melting point and the color of all prepared compounds. The best yield of the synthesized azomethine was for compounds R₁ 86% and R₃ 85%, while the lower yield was for compound R₂ 78% and the best yield of the synthesized 1,3-oxazepine-dione derivatives was for R₅ 89% while the lower yield was for R₇ 73%. The higher melting point for azomethine compounds was for compound R₃, the lower melting point was for compound R₄, while the higher melting point of the synthesized 1,3-oxazepine-dione derivatives was for compound R₉, the lower melting point was for compound R₅.  The different colors, melting points and the number with distance of spots in the TLC technique to the products compared with the raw material are initial evidence of interaction.

Table 1: Structural formulas, nomenclature, melting points, colors and % yields of azomethine compounds R1-R4. Click here to view table

Table 2: Structural formulas, nomenclature, melting points, colors and % yields of the synthesized 1,3-oxazepine-dione derivatives R5-R9. Click here to view table

 

Characterization of synthesized azomethine compounds R₁-R₄

Azomethine compounds were synthesized from commercially available aldehydes and primary amines. TLC technique was used to follow the chemical reaction, the synthesized azomethine identified by their melting points and FT-IR spectra. The FT-IR spectra showed the appearance of the stretching absorption bands of azomethine (C=N) at 1601-1628 cm^-1 indicative of the formation of the resulting azomethine compounds, (C-N) at 1154-1167 cm^-1, (C-S) at 687-692 cm^-1, (C-Cl) at 933-937 cm^-1 beside the characteristic bands of the residual groups in the structure [16].

The previous studies suggest that the aromatic aldehyde which contain such a -N(CH₃)₂ group at para position decreases the reaction speed when condensing with aromatic amine, while the reaction speed increases when such a -CF₃ group in the same site on the aromatic aldehyde ring because the drawing electrons group such a -CF₃ increase the the positive charge of the carbon of the carbonyle group (C=O) in aromatic aldehyde, while the donor electrons group such a -N(CH₃)₂ decrease the the positive charge of the carbon of the carbonyle group (C=O) in aromatic aldehyde, glacial acid is used as a catalyst to increase the electrophile of the carbonyl group (C=O) to accelerate the reaction and increase the precentage yield. See table 3 and FT-IR spectra of R₃ and R₄ compounds (figure 1).

Table 3: FT-IR of azomethine compounds R₁-R₄.

FT-IRa, n(cm-1)b
Compound	C=N	C-N	C=C	C-H	C-H Aliphatic		Others
			Aromatic	Aromatic	Asymmetric	symmetric
R1	1617	1166	1572	3028	—	—	C-S: 692
R2	1616	1154	1598	3052	2987	2937	O-H: 3500
R3	1601	1163	1574	3055	2977	2886	C-S: 687
R4	1628	1167	1578	3067	2989	2908	C-F: 1307

 

Figure 1: FT-IR spectra of R3 and of R4. Click here to view figure

 

The mechanism of azomethine compounds formation involves a nucleophile attack of the electron pair of NH₂ amine on the C=O of aldehyde to form a hemiaminal N-substituted medium that loses a water molecule to give the stable compound (azomethine). The reaction is believed to occur in the following mechanism [17]. See (figure 2).

Figure 2: Mechanism of azomethine compounds formation. Click here to view figure

 

Characterization of synthesized 1,3-oxazepine-dione derivatives R₅-R₉

Synthesis of 1, 3- oxazepine- dione derivatives was achieved by the reaction of azomethine group (C=N) with anhydride. TLC techneque used to follow the chemical reaction, the resulted products were identified by their melting points, FT-IR and ¹H-NMR spectra. The FT-IR spectra of the products showed disappearance of the absorption bands of the (C=N) of the azomethine compounds and the stretching absorption bands of two anhydride compounds and showed the appearance of the stretching absorption at 1608-1650cm^-1 indicative of C=O lactam bonds, stretching absorption at 1698-1728cm^-1 indicative of C=O lacton bonds, stretching absorption at 1277-1285cm^-1 indicative of C-O bonds, stretching absorption at 1185-1203cm^-1 indicative of C-N bonds, stretching absorption at 676-683 cm^-1 indicative of C-S bonds, stretching absorption at (1345-1363)-(1482-1533)cm^-1 indicative of NO₂ group, beside the characteristic bands of the residual groups in the structure [18]. See table 4 and FT-IR spectra of R₇ and R₈ compounds (figure 3).

Table 4: FT-IR of the 1,3-oxazepine-dione derivatives R₅-R₉.

FT-IRa (KBr), n(cm-1)b
Compound	C=C	C-O	C-H	C-N	C=O	C=O	C-H Aliphatic Asymmetric Symmetric		Others
	Aromatic		Aromatic		Lactam	Lacton
R5	1586	1277	3102	1203	1624	1698	2975	2886	C-S: 682
R6	1559	1280	3049	1187	1608	1701	2985	2920	O-H:3470
R7	1599	1285	3098	1185	1650	1728	2934	2897	C-S: 676
R8	1573	1278	3042	1196	1629	1698	2939	2891	C-F: 1307
R9	1562	1282	3054	1186	1614	1715	2988	2929	O-H:3480

 

Figure 3: FT-IR spectra of R7 and R8. Click here to view figure

 

The ¹H-NMR spectrum of R₅ in solvent DMSO showed chemical shifts (δ ppm) as folllows: the singlet at 1.98 indicates the presence 6H of the two groups of (CH₃), the singlet at 6.08 indicates the presence 2H of the two groups of (=CH), the singlet at 10.25 indicates the presence 2H of the two groups of (N-CH), multiplet at 7.24-7.95 indicates the presence 18H of the aromatic protons and the spectrum of R₆  showed chemical shifts (δ ppm) as follows: the singlet at 4.04 indicates the presence 2H of the group (CH₂), the singlet at 9.02 indicates the presence 2H of the two groups of (N-CH), the singlet at 13.16 indicates the presence 2H of two groups of (-OH), multiplet at 6.97-7.68 indicates the presence 20H of the aromatic protons [19]. Other chemical shifts (δ ppm) of compounds R₇-R₉ are given in (Table 5). See ¹H-NMR spectra of R₆ and R₉ compounds (figure 4).

Table 5: The ¹H-NMR Spectra of the 1,3-oxazepine-dione derivatives R₅-R₉ in DMSO.

Compound	Chemical Shift δ ppma
R5	Singlet in 1.98 for (6H, 2 CH3), singlet in 6.08 for (2H, 2 =CH), singlet in 10.25 for (2H, 2 N-CH), multiplet in  7.24-7.95 for (18H, aromatic protons).
R6	Singlet in 4.04 for (2H, CH2), singlet in 9.02 (2H, 2 N-CH), singlet in 13.16 (2H, 2 -OH), multiplet in 6.97-7.68 (20H,aromatic protons).
R7	Singlet in 3.03 for  (12H, 4 N-CH3), singlet in 9.67 for (2H, 2 N-CH), multiplet and doublet of doublet in 6.78-8.44 for (22H, aromatic protons).
R8	Singlet in 4.01 for (2H, CH2), singlet in 10.13 for (2H, 2 N-CH), multiplet and doublet of doublet in 6.69-8.71 for (20H, aromatic protons).
R9	Singlet in 3.09 for (6H, 2 CH3), singlet in 4.04 for (2H, CH2), singlet in 5.20 for (2H, 2 =CH), singlet in 9.02 for (2H, 2 N-CH), singlet in 13.16 for (2H, 2 -OH), multiplet in  6.97-7.68 for (14H, aromatic protons).

 

^a The references point (the chemical shift of tetramethylsilane (CH₃)₄Si).

Figure 4: 1H-NMR Spectra of R6 and R9. Click here to view figure

 

The both reactions of the synthesized azomethine compounds with two anhydrides are given by the following equations (figure 5).

Figure 5: Structure of the synthesized 1,3-oxazepine-dione derivatives from two types of anhydrides. Click here to view figure

 

It may be concluded that both reactions take place via interaction between HOMO orbital of anhydrides with LUMO orbital of (C=N) group by concerted dipolar cycloaddition mechanism as represented in the following reaction [20], see figure 6.

Figure 6: Mechanism of 1,3-oxazepine-dione derivatives formation for two types of anhydrides. Click here to view figure

 

The mechanism involves the addition of one σ-carbonyl to π-bond (C=N) to give 4- membered cyclic and 5-membered cyclic ring of anhydride in the same transition state [T.S.], which opens into 4-nitroisobenzofuran-1,3-dione and 3-methylfuran-2,5-dione anhydrides to give seventh-membered cyclic ring 1,3-oxazepine-dione derivatives [21].

The antifungal and antibacterial activity of 1,3-oxazepine-dione derivatives R₅-R₉

Table 6 exhibited that the higher zone of inhibition was 18.3 mm by compound R₈ against S. aureus, followed 14.6 and 14.0 mm by the same compound against Klebsiella sp. and E. coli, respectively. The lower zone of inhibition was 8.0 mm by compound R₉ against S. aureus and E. coli, and by compound R₆ toward E. coli. While, compound R₆ did not inhibit other microbes in this test. Also, the compounds R₅ and R₉ did not inhibit Geotrichum sp. and Klebsiella sp., respectively. The role of these compounds may be linked and destroyed the cell wall of microbes or stopped replication of microbial DNA [22, 23]. The differences in the inhibitory effect related to the chemical synthesis of each compound as above, see figures 7 and 8. Gentamycin showed the inhibition zone approx. 26 mg while the negative control (50 µl DMSO) did not exhibit any inhibition zone.

Table 6: Zone inhibition of the synthesized 1,3-oxazepine-dione derivatives R₅-R₉.

Pathogenic fungi and bacteria	Zone inhibition (mm)a					Gentamycinb 50 µg/well	DMSOc 50 µg/well
	R5	R6	R7	R8	R9
Geotrichum sp.	0	0	11.1	12	0	26	0
E. coli	9	8	11.6	14	8	27	0
S. aureus	9	0	11.1	18.3	8	26	0
Klebsiella sp.	9	0	10	14.6	0	25	0

 

^a Zone inhibition of the milimeter unit. ^b The positive control. ^c The negative control.

Figure 7: Antimicrobial activity of the synthesized 1,3-oxazepine-dione derivatives R5-R9 in DMSO against Geotrichum sp. and E. Coli. Click here to view figure

 

Figure 8: Antimicrobial activity of the 1,3-oxazepine-dione derivatives R5-R9 in DMSO against S. aureus and Klebsiella sp. Click here to view figure

 

Conclusions

The formation of stable seventh- membered heterocyclic derivatives as (dihydro-1,3-oxazepine-4,7-dione) and (dihydrobenzo [e] [1,3]oxazepine-1,5-dione) has been achieved by the interaction between HOMO orbital of anhydrides with LUMO orbital of (C=N). The results of FT-IR and ¹H-NMR showed that the target molecules were formed due to the least obstructive effect in all preparation processes. TLC technique identified the synthesized compounds; this makes for all the synthesized compounds with high purity. Generally, R₈ derivative is the best derivative that has significantly (p<0.01) recorded a stronger influence to inhibit the growth of all types of bacteria and fungi, while R₆ derivative has recorded inhibition against one of the types of bacteria and fungi. Slight variation in the structure of those derivatives can show the very dramatic effect on the efficiency of these compounds in their bio-activity. The present work may be helpful in designing more potent antifungal and antibacterial agents for therapeutic use in the future.

Acknowledgments

Special thanks to Anbar University’s President Professor Dr. Khalid Battal Najim and Dean of the Applied Sciences – Heet College Professor Dr. Tahseen Ali Zaidan for their continuous support to publishing the research in certified international journals. Grateful is for Dr. Mustafa Nadhim Owaid, Al-Mustafa laboratory for helping to complete the bioactivity tests.

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