**7. Mechanistic study of the acid-catalyzed hydrolysis of maleamic acids 34-42 used for the design of atenolol, amoxicillin and cephalexin prodrugs**

The acid-catalyzed hydrolysis of **34**-**42** (Figure 5) was kinetically investigated by Kirby et al. [84]. The study demonstrated that the amide bond cleavage is due to intramolecular nucleo‐ philic catalysis by the adjacent carboxylic acid group and the rate-limiting step is the tetrahe‐ dral intermediate breakdown (Figure 6) [84]. In 1996, the reaction was computationally investigated by Katagi using AM1 semiempirical calculations. In contrast to what was suggested by Kirby, Katagi's study demonstrated that the rate-limiting step is the formation of the tetrahedral intermediate and not its dissociation [121]. Later on Kluger and Chin have experimentally researched the mechanism of the intramolecular hydrolysis process utilizing several N-alkylmaleamic acids derived from aliphatic amines with a wide range of basicity [122]. The study findings demonstrated that the identity of the rate-limiting step is a function of both the basicity of the leaving group and the solution acidity.

In order to utilize Kirby's enzyme model [84] for the design of prodrugs of the following drugs: atenolol, amoxicillin and cephalexin, a mechanistic study using DFT calculation methods at B3LYP/6-31G (d,p), B3LYP/311+G (d,p) levels and hybrid GGA (MPW1k) on an intramolecular acid catalyzed hydrolysis of maleamic (4-amino-4-oxo-2-butenoic) acids (Kirby's N-alkylma‐ leamic acids) **34**-**42** was conducted. The calculations confirmed that the reaction involves three steps:(1) proton transfer from the carboxylic group to the adjacent amide carbonyl oxygen, (2) nucleophilic attack of the carboxylate anion onto the protonated carbonyl carbon; and (3) dissociation of the tetrahedral intermediate to provide products (Figure 6). Moreover, the calculations demonstrate that the rate-limiting step is dependent on the reaction medium. When the calculations were run in the gas phase the rate-limiting step was the tetrahedral intermediate formation, whereas when the calculations were conducted in the presence of a cluster of water the dissociation of the tetrahedral intermediate was the rate-limiting step. When the leaving group (methylamine) in **34**-**42** was replaced with a group having a low pKa value the rate-limiting step of the hydrolysis in water was the formation of the tetrahedral intermediate. In addition, the calculations revealed that the efficiency of the intramolecular acid-catalyzed hydrolysis by the carboxyl group is remarkably sensitive to the pattern of substitution on the carbon–carbon double bond. The rate of hydrolysis was found to be linearly correlated with the strain energy of the tetrahedral intermediate or the product. Systems having strained tetrahedral intermediates or products experience low rates and vice versa [51,52,54,91].

**Figure 5.** Acid-catalyzed hydrolysis of maleamic acids **34**-**42**.

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a specific class of molecules, for instance proteins, would be expected to only have any relevance when describing other molecules of the same class. These methods can be applied to proteins and other large biological molecules, and allow studies of the approach and docking of potential drug molecules Since the size of the system which *ab initio* calculations can handle is relatively small despite the large sizes of biomacromolecules surrounding solvent water molecules such as in the cases of enzymes and receptors, isolated models of areas of proteins such as active sites have been investigated using *ab initio* calculations. However, the disre‐ garded proteins and solvent surrounding the catalytic centers have also been shown to contribute to the regulation of electronic structures and geometries of the regions of interest. To overcome these discrepancies, quantum mechanics/molecular mechanics (QM/MM) calculations are used, in which the system is divided into QM and MM regions where QM regions correspond to active sites to be studied and are described quantum mechanically. MM regions correspond to the remainder of the system and are treated molecular mechanically. The pioneer work of the QM/MM method was accomplished by Warshel and Levitt [118], and since then, there has been a significant progress on the development of a QM/MM algorithm

**7. Mechanistic study of the acid-catalyzed hydrolysis of maleamic acids 34-42 used for the design of atenolol, amoxicillin and cephalexin prodrugs**

The acid-catalyzed hydrolysis of **34**-**42** (Figure 5) was kinetically investigated by Kirby et al. [84]. The study demonstrated that the amide bond cleavage is due to intramolecular nucleo‐ philic catalysis by the adjacent carboxylic acid group and the rate-limiting step is the tetrahe‐ dral intermediate breakdown (Figure 6) [84]. In 1996, the reaction was computationally investigated by Katagi using AM1 semiempirical calculations. In contrast to what was suggested by Kirby, Katagi's study demonstrated that the rate-limiting step is the formation of the tetrahedral intermediate and not its dissociation [121]. Later on Kluger and Chin have experimentally researched the mechanism of the intramolecular hydrolysis process utilizing several N-alkylmaleamic acids derived from aliphatic amines with a wide range of basicity [122]. The study findings demonstrated that the identity of the rate-limiting step is a function

In order to utilize Kirby's enzyme model [84] for the design of prodrugs of the following drugs: atenolol, amoxicillin and cephalexin, a mechanistic study using DFT calculation methods at B3LYP/6-31G (d,p), B3LYP/311+G (d,p) levels and hybrid GGA (MPW1k) on an intramolecular acid catalyzed hydrolysis of maleamic (4-amino-4-oxo-2-butenoic) acids (Kirby's N-alkylma‐ leamic acids) **34**-**42** was conducted. The calculations confirmed that the reaction involves three steps:(1) proton transfer from the carboxylic group to the adjacent amide carbonyl oxygen, (2) nucleophilic attack of the carboxylate anion onto the protonated carbonyl carbon; and (3) dissociation of the tetrahedral intermediate to provide products (Figure 6). Moreover, the calculations demonstrate that the rate-limiting step is dependent on the reaction medium. When the calculations were run in the gas phase the rate-limiting step was the tetrahedral intermediate formation, whereas when the calculations were conducted in the presence of a

and applications to biological systems [119,120].

414 Application of Nanotechnology in Drug Delivery

of both the basicity of the leaving group and the solution acidity.
