Introduction

Increasing bacterial resistance to antibiotics associated with an increase in the mortality rate from infectious diseases. There is a crucial medical need for the progress of new antibacterial agents with new and more efficient mechanisms (Rice, 2006). Schiff bases supposed as promising antibacterial agents; Hugo Schiff first stated them in 1864. A Schiff’s base compound contains azomethine group, which is a functional group that contains a carbon-nitrogen double bond -C=N- with the nitrogen atom attached to an aryl or alkyl group. The general formula of Schiff’s bases is R¹R²C=NR³, where R side chain is an organic group resulted from condensation of ketones (or) aldehydes with primary amines (Xavier & Srividhya, 2014). Schiff bases are stated to have a wide range of pharmacological activities including antimicrobial, antibacterial, antifungal, antipyretic, anti-inflammatory and anticancer activities, which are largely due to distinguishing C=N group (Dhar & Taploo, 1982; Przybylski, Huczynski, Pyta, Brzezinski, & Bartl, 2009; Silva et al., 2011; Souza et al., 2007). Furthermore, heterocyclic compounds are widely used for the development of new and strong agents therapeutically important (Kumar & Honnalli, 2014). Heterocyclic compounds containing acyclic hydroxylated side chains, for example, acyclovir (ACV), are an essential class of antiviral acyclonucleosides (Schaeffer et al., 1978). Therefore, the design of heterocycles incorporates open-chain carbohydrate residues that takes a special interest in the spacious research objects that concentrate on modification of the acyclic side chain and/or heterocyclic base (Ashry, Abdel-Rahman, Rashed, & Rasheed, 1999). The purpose of this work was to synthesize and evaluate new series of acyclovir analogues bearing a Schiff base moiety.

Acyclovir is a selective and potent inhibitor of herpes viruses, and it extensively designated for the treatment of genital herpes, lips, and face cold sores, chicken pox, and shingles (Spruance & Kriesel, 2002). Normally, this drug undergo phosphorylation by viral thymidine kinase into its monophosphate form. Acyclovir used as oral, parenteral, and topical dosage forms for the treatment of herpes infections (Laskin, 1983). However, acyclovir have low skin permeability causes the topical dosage forms to be ineffective (Spruance & Crumpacker, 1982).

Materials and Methods

Materials

2-hdroxy-3-methoxy benzaldehyde, 4-hydroxy benzaldehyde, 2-hydroxy benzaldehyde, and ethanol obtained from Merck and Fluka. Ethyl acetate and nutrient agar obtained from LaB and Himedia. Analytical grade solvents from Merck used without further purification. The chemical liquids were distilled before use.

Laboratory investigations

The infrared spectra of all the compounds documented in the range 4000–400 cm–1 using a Shimadzu FTIR–8400 spectrophotometer applying the KBr disc technique. The elemental The 1H NMR spectra were carried out in the university of Tehran recorded by Spectrometer Inova 500MHz.

Schiff bases preparation 1 and 2

Synthesis of (E)-2- ((4-hydroxy benzylidene) amino) -9- ((2-hydroxyethoxy) methyl) -1, 9-dihydro -6H-purin-6-one 1

A hot stirred solution of acyclovir (0.001mol, 0.225g), 40ml of ethanol (96%) was added to 4-hydroxy benzaldehyde (0.001mol, 0.122g) were mixed in 250ml round bottom flask. To the reaction mixture (6 drops) of glacial acetic acid were added as a catalyst. The reaction was reflexed with stirrer for 12 hours, and the time reaction was tested by TLC. The mixture was cooled overnight. The precipitate was filtered and purified by recrystallization from methanol; its result is shown in Table 1.

Synthesis of (E)-2-amino-9-((2-hydroxyethoxy) methyl)-1, 9-dihydro-6H-purin-6-one 2

A hot stirred solution of acylovir (0.001mol. 0.225g), 40ml of ethanol (96%), was added to 2-hydroxy-4-methoxy benzaldehyde (0.001mol. 0.152g) were mixed in 250ml round bottom flask. To the reaction mixture (8drops) of glacial acetic acid were added as a catalyst. The reaction was reflexed with stirrer for 9 hours, and TLC tested the time reaction. Then, the mixture was cooled overnight. The precipitate was filtered and purified by recrystallization from methanol. Its result is shown in Table 1.

Procedure for measurement of antibacterial activities of Schiff base compounds

Kirby Bauer process was used with 6 mm diameter discs of filter paper, which were put in the autoclave apparatus at 100 °C for 1 hr for sterilization under 1 atm. And kept in clean and sterilized glass screw plugs. The plate agar was diffused using Muller-Hinton agar with slight modification by (M.H.A) medium and was sterilized at 121 °C for 15 minutes under 1 atm by autoclave apparatus and then poured after cooling into 11 cm sterilized Petri dishes.

The synthesized compounds tested for antibacterial activity using five microorganisms, including Gram-positive Bacillus and Gram-negative Escherichia coli (E- Coli), Proteus, Pseudomonas aeruginosa, and Klebsiella. Youth colonies of about 24hours of age obtained by streaking of the isolated bacteria on nutrient agar (N. A.). The discs saturated with the synthesized compounds (conc. 0.009 mg/ml) in DMSO (Dimethyl sulphoxide), added by clean forceps to (M.H.A.) medium, and formerly incubated for 24 hours at 37 °C. After that, the dishes observed for the presence or absence of bacterial growth and the diameter of the zone where there is no bacterial growth around each disc measured in millimeters and the averages of inhibition zone diameters measured using Hahn method. This method based on antimicrobial efficiency by measuring the agent’s zones of inhibition whose sizes are relative to the sensitivity of the organism to the particular antibiotic in the disc and the disc diffusion for testing chemical agents. Size of the inhibition zone correlated with the antimicrobial activity of the drug.

Results and Discussion

The new series of acyclovir analogues bearing a Schiff base moiety were prepared and studied in this research. Schiff bases 1 and 2 were formed by the condensation reaction of some aldehydes such as 4-hydroxy benzaldehyde, 2-hydroxy-4-methoxybenzaldehyde and 2-hydroxy benzaldehyde with acyclovir drug in absolute ethanol with the elimination of water at the end of the reaction. As shown in the Figure 1.

The mechanism of the reaction, as illustrated in the Figure 1, included the nucleophilic attack of an amine at the carbon atom of the carbonyl group.

(E)-2-((4-hydroxybenzylidene) amino)-9-((2-hydroxyethoxy) methyl)-1, 9-dihydro-6H-purin-6-one

FTIR (KBr) cm-1:3475 (OH alcohol) 3549 (OH phenol), 3178(NH), 1639(C=C), 2854(C-H aliphatic), 3024(C-H aromatic), 1719(C=O), 1660(C=N). NMR [CDCl3] 500MHz: H11 0.8[s, 2H], H12 1.27[s, 2H], H10 2.08[s, 2H, CH2], 3.46[C-HN], N-H1 9.78 [s, 1H], 6.48[HC=N], Ar-H (6.92-7.75) [m,3H], 5.34[s, OH alcohol], 10.60[s, OH phenol]. C15H15N5O7: C, 54.69, H, 4.58; N, 21.26.

(E)-2-((2-hydroxy-4-methoxybenzylidene) amino)-9-((2-hydroxyethoxy) methyl)-1, 9-dihydro-6H-purin-6-one

FTIR (KBr) cm-1:3479(OH alcohol) 3549 (OH phenol), 3244 (NH), 1589(C=C), 2854 (CH aliphatic), 3043(CH aromatic), 1725 (C=O), 1647 (C=N). NMR [CDCl3] 500MHz: H121.27 [t,2H,CH2], H111.35 [t,2H,CH2], OCH3 2.49[s,3H], H10 4.10[s, 2H, CH2], 4.11 [s, OH alcohol] [HC=N] 6.88, H8 6.91[s,1H], Ar-H (7.23-7.24)[m, 3H], N-H1 10.14[s,1H] 10.24[s, OH phenol]. C16H17N5O8: C, 53.49, H, 4.77; N, 19.47.

Antibacterial activities

The results of antibacterial effects of the synthesized compounds against five microorganisms including Gram-positive and Gram-negative bacteria using Hahn method revealed that the Schiff base 2 showed higher activity on Gram-positive bacteria, Bacillus with inhibition zone 30 mm and on Gram-negative bacteria, Proteus with inhibition zone 28mm compare with 1 shows little inhibition against these bacteria. In addition, Schiff base 2 showed higher activity than Schiff base 1 on other Gram-negative bacteria such as E- Coli, Pseudomonas, and Klebsiella, as illustrated in Table 2.

Conclusions

The novel series of acyclovir schiff bases synthesized by the chemical reaction of different aldehydes with acyclovir. The synthesized compounds tested for antibacterial activity using five microorganisms including Gram-positive Bacillus and Gram-negative Escherichia coli (E- Coli), Proteus, Pseudomonas aeruginosa and Klebsiella, by the use of nutrient agar medium and disc diffusion method with incubation at 37◦C for 24 h. DMSO was used for the preparation of the test solutions. In general, the synthesized compounds have showed more antibacterial activity than acyclovir that they derived from it. Schiff base 2 showed higher activity on Gram-positive bacteria, Bacillus with inhibition zone 30 mm, and on Gram-negative bacteria, Proteus with inhibition zone 28mm compare with Schiff base 1. In addition, Schiff base 2 showed higher activity than Schiff base 1 on other Gram-negative bacteria such as E- Coli, Pseudomonas, and Klebsiella. This may be related to the structure of Schiff base 2, which contains an additional methoxy group compared with Schiff base 1.

Author’s contribution

All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

Fundings

The authors declare that they did not receive any specific funding from agencies in the commercial, public, profit sectors.