**12. In vitro intraconversion of amoxicillin and cephalexin prodrugs to their parent drugs**

Based on our previously reported DFT calculations and on experimental data for the acidcatalyzed hydrolysis of amide acids **34**-**42** (Figure 5) [84,91], two amoxicillin and cephalexin prodrugs were proposed (Figures11 and 12, respectively). As shown in Figures11 and 12, the antibacterial prodrugs, amoxicillin **ProD 1** and cephalexin **ProD 1** molecules are composed of an amide acid promoiety, containing a carboxylic acid group (hydrophilic moiety) and the rest of the antibacterial prodrug molecule (a lipophilic moiety). **<sup>N</sup>**

**Figure 11.** Acid-catalyzed hydrolysis of amoxicillin ProD 1.

administration. Up to 50% of cefuroxime in the circulation is bound to plasma proteins. The plasma half-life is about 70 minutes and is prolonged in patients with renal impairments and in neonates. Cefuroxime axetil is widely distributed in the body including plural fluid, sputum bone synovial fluid, and aqueous humor, but only achieves therapeutic concentration in the CSF when the meninges are inflamed. It crosses the placenta and has been detected in breast milk. Cefuroxime is excreted unchanged, by glomerular filtration and renal tubular secretion,

Amoxicillin, cephalexin and cefuroxime axetil as mentioned before suffer low stability and bitter taste sensation. Several attempts were made in order to enhance their aqueous solubility and bioavailability. Among several research approaches, the prodrug approach has been widely used for an improvement of drugs delivery to their site of action by physicochemical modulation properties that affect absorption or by targeting to specific enzymes or membrane transporters [147,148]. Generally, enzymatic catalysis is required for most of prodrugs that are in clinical use in order to be converted into the parent drug. This is mostly particular for those prodrugs designed to liberate the parent drug in the blood stream following gastro-intestinal absorption. These prodrugs are typically ester derivatives of drugs containing carboxyl or hydroxyl groups which are converted into the parent drug by esterase catalyzed hydrolysis. However, a high chemical reactivity that precludes either liquid or solid formulation of the prodrug (e.g. some phenol esters) or low chemical reactivity, resulting in reduced regeneration of the parent drug due to enzymatic activation for other functional groups. Thus, nonenzymatic pathways for some prodrugs that can regenerate the parent drug, have emerged as an alternative approach by which prodrug activation is not influenced by inter-and intraindividual variability that affects the enzymatic activity. In particular, since the middle-1980s, cyclization-activated prodrugs have been capturing the attention of medicinal chemists, and reached maturity in prodrug design in the late 1990s. Activation of prodrugs *via* a cyclization pathway allows a fine tuning of the rate of drug release through the appropriate choice of the functional groups involved in ring closure and stereoelectronic constraints in the course of the cyclization step. As noticed from the history of prodrugs mostly in preclinical and clinical consideration of prodrug bioconversion, the most common that several hydrolyses-activated prodrugs of penicillins, cephalosporins, and angiotensin-converting enzyme inhibitors have less than complete absorption which was observed and highlights yet another challenge with prodrugs susceptible to esterase hydrolysis. The oral bioavailability of these mentioned types of prodrugs is typically around 50% since these prodrugs undergo premature hydrolysis during the absorption process in the enterocytes of the gastrointestinal tract [149]. Another approach which has been utilized to enhance bioavailability of antibacterial drugs is by making the corresponding prodrugs with optimum lipophilicity. Some drugs remain poorly absorbed from most of the administration routes due to their poor lipophilicity. Two approaches were utilized to enhance the bioavailability of antibacterial drugs by increasing their lipophilicity: (a) membrane/water partition coefficient of the lipophilic form of a drug has been enhanced as compared to the hydrophilic form, thus favoring passive diffusion such as in the cases of pivampicillin, bacampicillin and talamipicillin (prodrugs of ampicillin)which are more lipophilic and better absorbed than amoxicillin and are rapidly interconverted and (b) the

and high concentration is achieved in urine [146].

424 Application of Nanotechnology in Drug Delivery

The combination of both, the hydrophilic and lipophilic groups provides a prodrug entity with a potential to be with a high permeability (a moderate HLB). It should be emphasized, that the HLB value of the prodrug entity will be determined upon the pH of the target physiological environment. In the stomach where the pH is in the range 1-2, it is expected that prodrugs, amoxicillin **ProD1** and cephalexin **ProD1** will be in a free carboxylic acid form (a relatively high hydrophobicity) whereas in the blood stream circulation where the is pH 7.4 a carboxylate anion (a relatively low hydrophobicity) is expected to be predominant form. Our strategy was to prepare amoxicillin **ProD 1** and cephalexin **ProD 1** as sodium or potassium carboxylates due to their high stability in neutral aqueous medium. It should be indicated that compounds

**34**-**42** undergo a relatively fast hydrolysis in acidic aqueous medium whereas they are quite stable at neutral pH. **<sup>S</sup>**

cephalexin **ProD 1** is in the range of 3-4, it is expected that at pH 5 the anionic form of the prodrug will be dominant and the percentage of the free acidic form that undergoes an acid-catalyzed hydrolysis will be relatively low. At 1N HCl and pH 2.5 most of the prodrug will exist as the free acid form and at pH 7.4 most of the prodrug will be in the anionic

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**Figure 13.** First order hydrolysis plot of amoxicillin **ProD 1** in (a) 1N HCl, (b) buffer pH 2.5 and (c) buffer pH 5.

**t 1/2 (h) k obs (h-1) Medium** 2.5 2.33x10 -4 1 N HCl 7 9.60 x10 -5 Buffer pH 2.5 81 7.55x10-6 Buffer pH 5 ---- No reaction Buffer pH 7.4

**Table 2.** The observed *k* value and *t*1/2 of amoxicillin **ProD 1** in 1N HCl and at pH 2, 5 and 7.4

form. Thus, the discrepancy in rates at the different pH buffers.

**Figure 12.** Acid-catalyzed hydrolysis of cephalexin ProD 1.

The hydrolysis kinetic studies for amoxicillin **ProD 1** and cephalexin **ProD 1** were carried out in aqueous buffers in the same manner to that executed by Kirby *et al.* on maleamic acids **34**-**40**. This is to investigate whether the antibacterial prodrugs undergo hydrolysis in aqueous medium and to what extent or not, suggesting the fate of the prodrugs in the system. The kinetics for the acid-catalyzed hydrolysis of the synthesized amoxicillin **ProD 1** and cephalexin **ProD 1** were carried out in four different aqueous media: 1 N HCl, buffer pH 2.5, buffer pH 5 and buffer pH 7.4. Under the experimental conditions the two antibacterial prodrugs intraconverted to release the parent drugs (Figures 13 and 14) as was determined by HPLC analysis. For both amoxicillin and cephalexin prodrugs, at constant temperature and pH the hydrolysis reaction displayed strict first order kinetics as the *k*obs was quite constant and a straight line was obtained on plotting log concentration of residual prodrug verves time. The rate constant (*k*obs) and the corresponding half-lives (t1/2) for amoxicillin **ProD 1**and cephalexin **ProD 1** in the different media were calculated from the linear regression equation obtained from the correlation of log concentration of the residual prodrug verses time. The kinetic data for amoxicillin **ProD 1**and cephalexin **ProD 1** are listed in Tables 2 and 3, respectively. It is worth noting that 1N HCl, pH 2.5 and pH 5 were selected to examine the intraconversion of amoxicillin **ProD 1**and cephalexin **ProD 1** in the pH as of stomach, since the mean fasting stomach pH of adult is approximately 1-2.5. Furthermore, environment of buffer pH 5 mimics that of beginning small intestine route, whereas pH 7.4 was selected to determine the intraconversion of the tested prodrugs in blood circulation system. Acid-catalyzed hydrolysis of both, amoxicillin **ProD 1**and cephalexin **ProD 1** was found to be much higher in 1N HCl than at pH 2.5 and 5 (Figures13 and 14). At 1N HCl the t ½ values for the intraconversion of amoxicillin **ProD 1**and cephalexin **ProD 1** were about 2.5 hours. On the other hand, at pH 7.4, both prodrugs amoxicillin **ProD 1**and cephalexin **ProD 1** were quite stable and no release of the parent drugs was observed. At pH 5 the hydrolysis of both prodrugs amoxicillin **ProD 1**and cephalexin **ProD 1** was too slow. This is because the pKa of amoxicillin **ProD 1**and

cephalexin **ProD 1** is in the range of 3-4, it is expected that at pH 5 the anionic form of the prodrug will be dominant and the percentage of the free acidic form that undergoes an acid-catalyzed hydrolysis will be relatively low. At 1N HCl and pH 2.5 most of the prodrug will exist as the free acid form and at pH 7.4 most of the prodrug will be in the anionic form. Thus, the discrepancy in rates at the different pH buffers.

**34**-**42** undergo a relatively fast hydrolysis in acidic aqueous medium whereas they are quite

**H2O**

**O**

**S**

**O OH**

**N**

**<sup>H</sup> <sup>H</sup> <sup>N</sup>**

**O**

**O**

**NH2**

**O**

**O**

**Maleic anhydride**

**Cephalexin ProD 1 Cephalexin**

The hydrolysis kinetic studies for amoxicillin **ProD 1** and cephalexin **ProD 1** were carried out in aqueous buffers in the same manner to that executed by Kirby *et al.* on maleamic acids **34**-**40**. This is to investigate whether the antibacterial prodrugs undergo hydrolysis in aqueous medium and to what extent or not, suggesting the fate of the prodrugs in the system. The kinetics for the acid-catalyzed hydrolysis of the synthesized amoxicillin **ProD 1** and cephalexin **ProD 1** were carried out in four different aqueous media: 1 N HCl, buffer pH 2.5, buffer pH 5 and buffer pH 7.4. Under the experimental conditions the two antibacterial prodrugs intraconverted to release the parent drugs (Figures 13 and 14) as was determined by HPLC analysis. For both amoxicillin and cephalexin prodrugs, at constant temperature and pH the hydrolysis reaction displayed strict first order kinetics as the *k*obs was quite constant and a straight line was obtained on plotting log concentration of residual prodrug verves time. The rate constant (*k*obs) and the corresponding half-lives (t1/2) for amoxicillin **ProD 1**and cephalexin **ProD 1** in the different media were calculated from the linear regression equation obtained from the correlation of log concentration of the residual prodrug verses time. The kinetic data for amoxicillin **ProD 1**and cephalexin **ProD 1** are listed in Tables 2 and 3, respectively. It is worth noting that 1N HCl, pH 2.5 and pH 5 were selected to examine the intraconversion of amoxicillin **ProD 1**and cephalexin **ProD 1** in the pH as of stomach, since the mean fasting stomach pH of adult is approximately 1-2.5. Furthermore, environment of buffer pH 5 mimics that of beginning small intestine route, whereas pH 7.4 was selected to determine the intraconversion of the tested prodrugs in blood circulation system. Acid-catalyzed hydrolysis of both, amoxicillin **ProD 1**and cephalexin **ProD 1** was found to be much higher in 1N HCl than at pH 2.5 and 5 (Figures13 and 14). At 1N HCl the t ½ values for the intraconversion of amoxicillin **ProD 1**and cephalexin **ProD 1** were about 2.5 hours. On the other hand, at pH 7.4, both prodrugs amoxicillin **ProD 1**and cephalexin **ProD 1** were quite stable and no release of the parent drugs was observed. At pH 5 the hydrolysis of both prodrugs amoxicillin **ProD 1**and cephalexin **ProD 1** was too slow. This is because the pKa of amoxicillin **ProD 1**and

stable at neutral pH.

426 Application of Nanotechnology in Drug Delivery

**O**

**HN**

**OH <sup>O</sup>**

**N**

**Figure 12.** Acid-catalyzed hydrolysis of cephalexin ProD 1.

**<sup>H</sup> <sup>H</sup> <sup>N</sup>**

**O OH**

**O**

**O**

**S**

**Figure 13.** First order hydrolysis plot of amoxicillin **ProD 1** in (a) 1N HCl, (b) buffer pH 2.5 and (c) buffer pH 5.


**Table 2.** The observed *k* value and *t*1/2 of amoxicillin **ProD 1** in 1N HCl and at pH 2, 5 and 7.4


hydrolysis in physiological environment we have unraveled the mechanism for the ringclosing reaction of **43-47** using DFT and molecular mechanics calculation methods [93].

Quantum molecular mechanics using DFT methods at B3LYP 6-31G (d,p) and B3LYP/311+G (d,p) levels were exploited to calculate the thermodynamic and kinetic parameters for all reactants, transition states, intermediates and products involved in the proposed mechanism for process **43-47** (Figure 16). As shown in Figure 16 the mechanism for these processes consists of two steps; (1) formation of a tetrahedral intermediate and (2) collapse of a tetrahedral

**Br**

**Br**

429

**O O**

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**O**

**C2H5 C2H5**

**Br**

intermediate to furnish a cyclic anhydride and p-bromophenolate anion.

**O O**

**<sup>O</sup> <sup>O</sup> <sup>O</sup>**

**O**

**O**

**H H**

**43 44 45**

**O**

**O O**

The phenomenon of rate enhancements in several intramolecular processes was ascribed by Bruice and Menger to the importance of the proximity of the nucleophile to the electrophile of the ground state molecules [64,65,155]. Menger in his "spatiotemporal" hypothesis advocated a mathematical equation correlating activation energy to distance and based on this that, he came to the conclusion that enormous rate accelerations in reactions catalyzed by enzymes are feasible when imposing short distances between the reactive centers of the substrate and enzyme [155]. Differently from Menger, Bruice attributed the catalysis by enzymes to favorable 'near attack conformations'; systems that have a high quota of near attack conformations will have a higher intramolecular reaction rate and *vice versa*. Bruice's idea invokes a combination of distance between the two reacting centers and the angle of attack by which the nucleophile

In contrast to the proximity orientation proposal, others proposed the high rate enhancements in intramolecular processes to steric effects (relief of the strain energy of the reactant) [156].

To test whether the acceleration in rates for processes **43-47** (Figure 15) is a result of proximity orientation or due to steric effects (difference in strain energies of the reactants), the strain energy values for the reactants and the intermediates in systems **43-47** were calculated using Allinger's MM2 method. The calculated strain energy values for **43-47** were correlated with

**O**

**H3C H3C**

**46 47**

**r**

**Br**

**Figure 15.** Hydrolysis of di-carboxylic semi-esters **43-47**.

approaches the electrophile [64,65].

**B**

**O O**

> **O O**

**O**

**O**

**O**

in 1N HCl and at pH 2, 5 and 7.4

**Table 3.** The observed *k* value and *t*1/2 of cephalexin **ProD 1**

**Figure 14.** First order hydrolysis plot of cephalexin ProD 1 in (a) 1N HCl, (b) buffer pH 2.5 and (c) buffer pH 5.
