**13. Mechanistic study of Bruice's hydrolysis of di-carboxylic semi-esters 43-47 used for the design of bitterless paracetamol prodrugs**

Five decades ago, Bruice and Pandit have investigated the kinetics of for the hydrolysis reaction of di-carboxylic semi-esters **43-47** depicted in Figure 15 [64,65]. Their findings revealed the relative rate (krel) for **47**>**46**>**45**>**44**>**43**. They attributed the discrepancy in rates to differences in the proximity orientation of the nucleophile to electrophile. Using the observation that alkyl substituent on succinic acid influences rotamer distributions, the ratio between the reactive gauche and the unreactive anti-conformers, they proposed that *gem*-dialkyl substitution increased the probability of the resultant rotamer adopting the more reactive conformation. Hence, for ring-closing reaction to precede, the two reacting centers, the nucleophile and electrophile, must be in the gauche conformation. In the unsubstituted reactant, the nucleo‐ phile and electrophile are almost entirely in the anti-conformation in order to minimize steric interactions [81-82]. In order to design paracetamol prodrugs, *via* linking the active drug with a di-carboxylic semi-ester linker (Bruice's enzyme model), lacking the bitterness of their parent drug, paracetamol, and have the capability to chemically and not enzymatically undergo 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 intermediate to furnish a cyclic anhydride and p-bromophenolate anion.

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

**B**

**t 1/2 (h) k obs (h-1) Medium** 2.4 2.41x10 -4 1 N HCl 14 4.17x10 -5 Buffer pH 2.5 --- No reaction Buffer pH 5.5 --- No reaction Buffer pH 7.4

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

**43-47 used for the design of bitterless paracetamol prodrugs**

**13. Mechanistic study of Bruice's hydrolysis of di-carboxylic semi-esters**

Five decades ago, Bruice and Pandit have investigated the kinetics of for the hydrolysis reaction of di-carboxylic semi-esters **43-47** depicted in Figure 15 [64,65]. Their findings revealed the relative rate (krel) for **47**>**46**>**45**>**44**>**43**. They attributed the discrepancy in rates to differences in the proximity orientation of the nucleophile to electrophile. Using the observation that alkyl substituent on succinic acid influences rotamer distributions, the ratio between the reactive gauche and the unreactive anti-conformers, they proposed that *gem*-dialkyl substitution increased the probability of the resultant rotamer adopting the more reactive conformation. Hence, for ring-closing reaction to precede, the two reacting centers, the nucleophile and electrophile, must be in the gauche conformation. In the unsubstituted reactant, the nucleo‐ phile and electrophile are almost entirely in the anti-conformation in order to minimize steric interactions [81-82]. In order to design paracetamol prodrugs, *via* linking the active drug with a di-carboxylic semi-ester linker (Bruice's enzyme model), lacking the bitterness of their parent drug, paracetamol, and have the capability to chemically and not enzymatically undergo

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

428 Application of Nanotechnology in Drug Delivery

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

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 approaches the electrophile [64,65].

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 **B**

paracetamol the group is hydroxyl, in phenacetin it is ethoxy. On the other hand, acetanilide has a chemical structure similar to that of paracetamol and phenacetin but it lacks any group at the *para* position of the benzene ring. Acetanilide lacks the bitter taste characteristic for paracetamol. The comparisons of the three compounds might suggest that the presence of hydroxy group on the *para* position of the benzene ring plays a major role for paracetamol bitterness. Therefore, it is expected that masking the hydroxyl group in paracetamol with a suitable linker could inhibit the binding of paracetamol to its bitter taste receptor/s and hence masking its bitterness. It is likely that paracetamol binds to the active site of its bitter taste receptor via hydrogen bonding interactions by which its phenolic hydroxyl group is engaged. It is worth noting that linking paracetamol with Bruice's enzyme model linker *via* its phenolic

Based on the DFT calculations on the cyclization of Bruice's **43**-**47** (Figure 15), two paracetamol prodrugs were proposed (Figure 17). As shown in Figure 17, the paracetamol prodrugs, **ProD 1-2**,have a carboxylic acid group as a hydrophilic moiety and the rest of the prodrug, acetani‐ lide, as a lipophilic moiety, where the combination of both groups provides a moderate HLB. It should be noted that the HLB value will be determined upon the physiologic environment by which the prodrug is dissolved. For example, in the stomach, the paracetamol prodrugs will primarily exist in the carboxylic acid form whereas in the blood circulation the carboxylate anion form will be predominant. Since Bruice's cyclization reaction occurs in basic medium paracetamol **ProD 1-2** were obtained as carboxylic free acid form, since this form is expected

**HN H3C**

**O**

**O**

**O**

**Paracetamol ProD 1 Succinic anhydride Paracetamol**

**Paracetamol ProD 2 Maleic anhydride Paracetamol**

**O**

**O**

**O**

**HN H3C**

**O**

**OH**

**OH**

**O**

Prodrugs for Masking the Bitter Taste of Drugs

http://dx.doi.org/10.5772/58404

431

hydroxyl group might hinder paracetamol bitter taste.

to be stable in acidic medium such as the stomach.

**O**

**O**

**Figure 17.** Hydrolysis of paracetamol ProD 1 and paracetamol Prod 2.

**O**

**O**

**HO**

**HO**

**O**

**H2O**

**H2O**

**O**

**H**

**N H3C**

**HN H3C**

**O**

**O**

**Figure 16.** Proposed mechanism for the hydrolysis of di-carboxylic semi-esters **43-47**.

the corresponding experimental relative rates (log krel) [64,65]. The results demonstrated good correlation between the two parameters. On the other hand, attempts to correlate the distance between the two reactive centers and log krel failed to provide any correlation between the two parameters. This reveals that the driving force for acceleration in rates of **43-47** is driven by strain effects and not proximity orientation stemming from Bruice's near attack conformation [64,65].In addition, in accordance with Bruice and Pandit's findings [64,65] we have found that the ring-closing reactions proceed by one mechanism, by which the rate-limiting step is the tetrahedral intermediate dissociation and not its formation.

### **14. Paracetamol Prodrugs Based on Bruice's Enzyme Model**

Paracetamol is an odorless, bitter crystalline compound used as an over the counter analgesic and anti-pyretic drug. Paracetamol is used to relief minor aches. it is used as pain killer by decreasing the synthesis of prostaglandin due to inhibiting cyclooxygenases (COX-1 and COX-2). Paracetamol is favored over aspirin as pain killer in patients have excessive gastric secretion or prolonged bleeding. It was approved to be used as fever reducer in all ages. Pharmacokinetic studies have shown that urine of patients who had taken phenacetin contained paracetamol. Later was demonstrated that paracetamol was a urinary metabolite of acetanilide. Phenacetin known historically to be one of the first non-opioid analgesics without anti-inflammatory properties lacks or has a very slight bitter taste [157,158]. Comparison of the structures of paracetamol and phenacetin shows that shows close similarity between both analgesics except of the nature of the group on the *para* position of the benzene ring. While in paracetamol the group is hydroxyl, in phenacetin it is ethoxy. On the other hand, acetanilide has a chemical structure similar to that of paracetamol and phenacetin but it lacks any group at the *para* position of the benzene ring. Acetanilide lacks the bitter taste characteristic for paracetamol. The comparisons of the three compounds might suggest that the presence of hydroxy group on the *para* position of the benzene ring plays a major role for paracetamol bitterness. Therefore, it is expected that masking the hydroxyl group in paracetamol with a suitable linker could inhibit the binding of paracetamol to its bitter taste receptor/s and hence masking its bitterness. It is likely that paracetamol binds to the active site of its bitter taste receptor via hydrogen bonding interactions by which its phenolic hydroxyl group is engaged. It is worth noting that linking paracetamol with Bruice's enzyme model linker *via* its phenolic hydroxyl group might hinder paracetamol bitter taste.

Based on the DFT calculations on the cyclization of Bruice's **43**-**47** (Figure 15), two paracetamol prodrugs were proposed (Figure 17). As shown in Figure 17, the paracetamol prodrugs, **ProD 1-2**,have a carboxylic acid group as a hydrophilic moiety and the rest of the prodrug, acetani‐ lide, as a lipophilic moiety, where the combination of both groups provides a moderate HLB. It should be noted that the HLB value will be determined upon the physiologic environment by which the prodrug is dissolved. For example, in the stomach, the paracetamol prodrugs will primarily exist in the carboxylic acid form whereas in the blood circulation the carboxylate anion form will be predominant. Since Bruice's cyclization reaction occurs in basic medium paracetamol **ProD 1-2** were obtained as carboxylic free acid form, since this form is expected to be stable in acidic medium such as the stomach.

**Figure 17.** Hydrolysis of paracetamol ProD 1 and paracetamol Prod 2.

**H**

the corresponding experimental relative rates (log krel) [64,65]. The results demonstrated good correlation between the two parameters. On the other hand, attempts to correlate the distance between the two reactive centers and log krel failed to provide any correlation between the two parameters. This reveals that the driving force for acceleration in rates of **43-47** is driven by strain effects and not proximity orientation stemming from Bruice's near attack conformation [64,65].In addition, in accordance with Bruice and Pandit's findings [64,65] we have found that the ring-closing reactions proceed by one mechanism, by which the rate-limiting step is the

**O O**

**O**

**O**

**Reactant Formation transition state**

**O**

**O**

**O**

**Br**

**O O**

**O**

**Intermediate**

**O**

**O**

**O**

**O**

**O**

**Dissociation transition state**

**Br**

**Br**

Paracetamol is an odorless, bitter crystalline compound used as an over the counter analgesic and anti-pyretic drug. Paracetamol is used to relief minor aches. it is used as pain killer by decreasing the synthesis of prostaglandin due to inhibiting cyclooxygenases (COX-1 and COX-2). Paracetamol is favored over aspirin as pain killer in patients have excessive gastric secretion or prolonged bleeding. It was approved to be used as fever reducer in all ages. Pharmacokinetic studies have shown that urine of patients who had taken phenacetin contained paracetamol. Later was demonstrated that paracetamol was a urinary metabolite of acetanilide. Phenacetin known historically to be one of the first non-opioid analgesics without anti-inflammatory properties lacks or has a very slight bitter taste [157,158]. Comparison of the structures of paracetamol and phenacetin shows that shows close similarity between both analgesics except of the nature of the group on the *para* position of the benzene ring. While in

tetrahedral intermediate dissociation and not its formation.

**Products**

**Figure 16.** Proposed mechanism for the hydrolysis of di-carboxylic semi-esters **43-47**.

**r**

**B**

**O O**

**Br O**

**O**

430 Application of Nanotechnology in Drug Delivery

**O**

**14. Paracetamol Prodrugs Based on Bruice's Enzyme Model**
