J. Chil. Chem. Soc., 48, N 4 (2003) ISSN 0717-9324

CHEMICAL BASIS FOR THE ANTIMICROBIAL ACTIVITY OF ACETANILIDES

Hector R. Bravo*,¹ Boris Weiss-López,¹ Madeleine Lamborot² and Sylvia Copaja¹

¹Departamento de Química, ²Departamento de Ciencias Ecológicas, Facultad de Ciencias,
Universidad de Chile, Casilla 653,Santiago, Chile.
(Received: January 9, 2003 - Accepted: July 4, 2003)

ABSTRACT

In vitro antimicrobial activity of a series of substituted acetanilides against S. aureus, E. coli and C. albicans were measured at two concentrations, 250 mg/ml and 500 mg/ml. Only the structures substituted with halogens in C_a and electron acceptors in the aromatic ring are bioactive. The results are rationalized in terms of the acid properties of the N-H bond, as calculated using AM1-MO theory.

Keywords- Acetanilides, antimicrobial activity, AM1-MO calculations.

INTRODUCTION

Lactams derived from 1,4-benzoxazin-3-ones (Fig. 1; R1=H, MeO, OH; R₂=OH; R₃=H) are present in higher plants species.¹,²,³ These metabolites are considered to be the most direct precursors in the biosynthesis of the respective hydroxamic acids, Hx (figure 1, R₃=OH).³,⁴,⁵ Both, Hx and lactams show a variety of biological activity, however lactams are usually less active. For instance, Hx show greater antifungal (C. albicans) and antialgal activity (C. xanthella) than the lactams derivatives.⁶ The molecular mode of action of these structures has been related to the electrophilic character of the nitrogen atom, which could be involved in nucleophile-electrophile interactions with biomolecules involved in energetic processes.⁷,⁸ The electrophilic characteristics of the nitrogen atom is mainly determined by the substituents in the aromatic ring, and the leaving group characteristics of the substituent at the nitrogen atom.⁹-¹² This should make lactams less active than Hx, since a previous metabolic step for the N-acylation in lactams should be required to produce a more electrophilic nitrogen atom. It has been also observed that N-acylation of these lactams with acetic anhydride depends on the strength of the N-H bond.¹³

Acetanilide derivatives (figure 1) can be considered as non-cyclic analogs of 1,4-benzoxazin-3-ones, and they are appropriate models to perform a structure-activity study and understand the chemical basis of the biological action. In this work we report a study concerning structural effects of a series of acetanilides in their anti-microbial activity.

Fig. 1. Structures of 1,4-benzoxazin-3-ones and acetanilides

EXPERIMENTAL

Chemicals. Acetanilides were prepared using methods previously described.¹⁴ All the structures were purified by preparative TLC to achieve high purity levels, adequate for biological assays. Melting points of the compounds were compared with literature values and the structures were corroborated by ¹H-NMR and IR spectroscopy.

Antimicrobial Test. Test compounds were dissolved in nutrient media with the aid of either ultrasound and/or gentle heating. The activity was measured at two concentrations, 250 and 500 mg/ml. Mueller-Hinton nutrient medium (DIFCO) was used for Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922). Mueller-Hinton plus 5% dextrose mixture was used for the fungi Candida albicans. The microorganisms were cultivated during 24 hrs at a constant temperature of 35ºC and pH=6.7. Samples were incubated at 35ºC for 24 hours in test tubes containing 104 colony-forming units (CFU). The inhibition percentage was determined spectrophotometrically as a functions of turbidity measured at 590 nm. The activity was measured by comparison with a solution identical to the samples but without acetanilide. The associated errors were always less than 10%.

AM1-MO calculations. The full geometry AM1-MO calculations were performed using the program MOPAC, running in a Pentium IV 1.7 GHz microcomputer. The initial structure for the geometry optimization was the same for all the calculated derivatives.

RESULTS AND DISCUSSION

Table 1 shows the antimicrobial activity of a series of acetanilides with different substituents in C_a and in the aromatic ring. For the two measured concentrations derivatives with electron acceptor substituents in the aromatic ring (3a and 4a) and halogens in C_a (7a, 8a and 9a), showed antimicrobial activity. It has been previously proposed that derivatives containing acid protons, such as phenolic groups, induce decoupling of oxidative phosphorylation processes in mitochondria, chloroplast and other energy-transducing membranes.15 Molecules with hydroxyl groups in positions ortho and para (1a and 2a) and a methyl group in C_a did not show measurable activity. Compounds 3a and 4a have electron acceptor substituents in the aromatic ring and this substitution should increase the acidity of the phenolic OH, as compared to structures 1a and 2a. This difference may account for the antimicrobial activities of 3a and 4a, however derivatives with halogens in C_a showed the greatest activity, despite the small effects, if any, of halogen atoms in the acid properties of the phenolic proton. This result suggests that the phenolic proton should not be involved in the molecular mode of action of these structures.

TABLE 1.

Antimicrobial Activity of Substituted Acetanilides

Ionization of the N-H bond and further acetylation, as suggested for the mechanism of action of 1,4-benzoxazin-3-ones,¹³ could also be considered important in the mode of action of these derivatives. According to this assumption electron acceptor groups in the aromatic ring, as well as in C_a, should favor the ionization of the N-H bond by delocalization of the negative charge generated around the nitrogen atom toward the aromatic ring and the carbonyl oxygen (figure 2). The formation of the isoamide intermediate has been suggested in the mechanism of hydrolysis of para substituted acetanilides, in basic media.^{16, 17} Further support for our assumption was obtained from the pKa values of para substituted acetanilides, measured by Pollack and Stauffer.^17,18 , and these numbers are

Fig. 2. Ionization of acetanilide in basic solution.

TABLE 2.

Experimental pKa ^16,17) , calculated DH and estimated pKa of substituted acetanilides.

included in table 2. As observed, the trifluoromethyl derivatives in C_a (1b-6b) are 10⁴-10⁵ times more acid than the methyl derivatives (7b and 8b). Furthermore, the fluorinated derivative with a NO₂ group in position para of the aromatic ring is the most acid molecule (6b). All the evidence support the assumption that electron acceptor substituents in position para of the ring and in C should play a significant role in the ionization of the N-H bond, by stabilization of the isoamide intermediate anion.

The experimental determination of pKa for the ionization of the N-H group in the studied molecules is a difficult task, mainly because of the presence of O-H groups, probably more acid than the N-H bond. With this in mind we have obtained approximated theoretical values of DH employing the AM1-MO approximation.¹⁹ The enthalpy of an acid-base reaction is defined by the following equations:

Neglecting entropy effects, the equilibrium position of any chemical reaction depends on the enthalpy differences. In order to compare the relative acidity among the different molecules, we can simply define DH = DHº_f (A^-) - DHº_f (HA), for each structure , and since DHº_f (H⁺) is approximately the same in all cases, when comparing any two structures in the same series it is cancelled. We have assumed that solvent effects are the same for all the systems. Full geometry optimization of all the studied molecules, as well as those reported by Pollack and Stauffer,^17,18 were calculated in vacuum. DH values for the dissociation reactions were obtained and the results appear in table 2. The experimentally determined pKa of compounds 1b to 6b correlate with the calculated DH (r² = 0.99). The linear correlation described above was used to estimate the pKa values of molecules 7a-9a. Unfortunately, experimental values of pKa for unsubstituted derivatives in C_a are scarce, possibly due to the very low values of the dissociation constant. We were able to find two values in the literature, only for compounds 7b y 8b. We are aware that a correlation with just two data points is not very reliable. However, we still have performed a procedure similar to that used in the fluorinated derivatives and the experimental pKa values of molecules 7b and 8b were employed to estimate the pKa of molecules 1a to 4a. The experimental determinations of pKa (1b-6b), ¹⁸ as well as the estimated values obtained from the linear correlation of the halogenated derivatives in C_a (7a-9a), always showed the lowest values. Experimentally determined pKa (7b and 8b) and estimated ones (1a-4a) for methyl substituted molecules in C_a, always present greater values than the halogenated molecules. Therefore, the calculated DH should represent an adequate parameter to estimate the relative acid character of the N-H bond in acetanilides.

These variations in the values of pKa are in agreement with the measured differences in the antimicrobial activity (table 1) Halogenated derivatives showed the largest bioactivity and methylated derivatives display the smallest one. The results obtained in this work strongly suggest that ionization of the N-H bond, in these structures, might be an important step in the molecular mode of action for the antimicrobial activity, as previously suggested to explain the bioactivity of 1,4-benzoxazin-3-one derivatives.¹³

ACKNOWLEDGEMENTS

The authors are pleased to acknowledge financial assistance from the Departamento de Química, Facultad de Ciencias, Universidad de Chile and FONDECYT, Grant No. 1010211.

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