Ibuprofen esters

CASE STUDY ENZYME KINETIC MODELS FOR RESOLUTION OF RACEMIC IBUPROFEN ESTERS IN A MEMBRANE REACTOR... 

In this case study, an enzymatic hydrolysis reaction, the racemic ibuprofen ester, i.e. (R)-and (S)-ibuprofen esters in equimolar mixture, undergoes a kinetic resolution in a biphasic enzymatic membrane reactor (EMR). In kinetic resolution, the two enantiomers react at different rates lipase originated from Candida rugosa shows a greater stereopreference towards the (S)-enantiomer. The membrane module consisted of multiple bundles of polymeric hydrophilic hollow fibre. The membrane separated the two immiscible phases, i.e. organic in the shell side and aqueous in the lumen. Racemic substrate in the organic phase reacted with immobilised enzyme on the membrane where the hydrolysis reaction took place, and the product (S)-ibuprofen acid was extracted into the aqueous phase. 

The reaction under investigation is the enzymatic hydrolysis of racemic ethoxyethyl-ibuprofen ester. The (R)-ester is not active in the above reaction,1-3, thus simplifying the reaction mechanism, as shown in Figure 5.13. Because both enantiomers are converted according to fust-order kinetics, the conversion of one enantiomer is independent of the conversion of the other.4... 

S)-2-ethoxyethyl-ibuprofen ester (S)-ibuprofen acid 2-ethoxyethanol... 

Fig. 5.13. Lipase-catalysed hydrolysis of racemic ibuprofen ester. CRL Candida rugosa lipase.

Substrate and product inhibitions analyses involved considerations of competitive, uncompetitive, non-competitive and mixed inhibition models. The kinetic studies of the enantiomeric hydrolysis reaction in the membrane reactor included inhibition effects by substrate (ibuprofen ester) and product (2-ethoxyethanol) while varying substrate concentration (5-50 mmol-I ). The initial reaction rate obtained from experimental data was used in the primary (Hanes-Woolf plot) and secondary plots (1/Vmax versus inhibitor concentration), which gave estimates of substrate inhibition (K[s) and product inhibition constants (A jp). The inhibitor constant (K[s or K[v) is a measure of enzyme-inhibitor affinity. It is the dissociation constant of the enzyme-inhibitor complex. 

The inhibition analyses were examined differently for free lipase in a batch and immobilised lipase in membrane reactor system. Figure 5.14 shows the kinetics plot for substrate inhibition of the free lipase in the batch system, where [5] is the concentration of (S)-ibuprofen ester in isooctane, and v0 is the initial reaction rate for (S)-ester conversion. The data for immobilised lipase are shown in Figure 5.15 that is, the kinetics plot for substrate inhibition for immobilised lipase in the EMR system. The Hanes-Woolf plots in both systems show similar trends for substrate inhibition. The graphical presentation of rate curves for immobilised lipase shows higher values compared with free enzymes. The value for the... 

The 2-ethoxyethanol was a by-product, as shown in Figure 5.13. The formation rate of 2-ethoxyethanol was the same as the conversion rate of the (S)- or (R)-ibuprofen ester one mole of 2-ethoxyethanol was formed when one mole of ester was catalysed. A known concentration of 2-ethoxyethanol was added in the organic phase before the start of the reaction for product inhibition. The plots of the kinetics for the free lipase system are presented in Figure 5.17 and immobilised enzyme (EMR) in Figure 5.18, respectively. The Kw value was 337.94 mmoFl 1 for the free lipase batch system and 354.20 mmoll 1 for immobilised... 

Enzyme reaction kinetics were modelled on the basis of rapid equilibrium assumption. Rapid equilibrium condition (also known as quasi-equilibrium) assumes that only the early components of the reaction are at equilibrium.8-10 In rapid equilibrium conditions, the enzyme (E), substrate (S) and enzyme-substrate (ES), the central complex equilibrate rapidly compared with the dissociation rate of ES into E and product (P ). The combined inhibition effects by 2-ethoxyethanol as a non-competitive inhibitor and (S)-ibuprofen ester as an uncompetitive inhibition resulted in an overall mechanism, shown in Figure 5.20. 

The plotting of Dixon plot and its slope re-plot (see 5.9.5.9) is a commonly used graphical method for verification of kinetics mechanisms in a particular enzymatic reaction.9 The proposed kinetic mechanism for the system is valid if the experimental data fit the rate equation given by (5.9.4.4). In this attempt, different sets of experimental data for kinetic resolution of racemic ibuprofen ester by immobilised lipase in EMR were fitted into the rate equation of (5.7.5.6). The Dixon plot is presented in Figure 5.22. 

Scheme 7.12 Resolution of an ibuprofen ester derivative by enzymatic ammonolysis.

Long, W.S., Kamaruddin, A.H. and Bhatia, S. (2005) Enzyme kinetics of kinetic resolution of racemic ibuprofen ester using enzymatic membrane reactor. Chemical Engineering Science, 60 (18), 4957—1970. 

The drying step was repeated several times in order to achieve a translucent organic layer. The organic solvent (isooctane) was removed from the (R,5)-ibuprofen ester... 

The racemic (7 ,5)-ibuprofen ester obtained from chemical synthesis was characterized by FTIR and NMR. 

Procedure 3 Enzymatic Kinetic Resolution of (/ ,S)-2-Ethoxyethyl Ibuprofen Ester with Immobilized Lipase... 

The immobilized lipase (0.1 g) in pH 7 phosphate buffer (25 mL) was added to 25 mL (20 mM) of ester stock solution in a 250 mL Erlenmeyer flask (reaction flask). The reaction flask was incubated in an incubator shaker at 40 °C with the agitation speed set to 200 rpm. Samples from the organic phase and aqueous phase were withdrawn at 24 h intervals over a 5-day reaction period. The samples collected were filtered using 0.45 pm nylon filter and injected into the HPLC system to determine the rate of resolution by monitoring both substrate ((/ ,5)-2-ethoxyethyl ibuprofen ester) and product (5-ibuprofen acid concentration). 

To isooctane (25 mL) was added (7 ,5)-ibuprofen ester (0.27 g, 40 mM), 0.5 m sodium hydroxide (25 mL) and immobilized lipase (0.2 g). The reaction medium consisted of two layers of solution, namely ibuprofen ester in isooctane and aqueous NaOH, where the reaction only occurred at the interface between these two layers. The mixture was agitated in an orbital shaker at constant temperature of 45 °C and at 200 rpm. 

The procedure shows that it is feasible to combine racemization with the kinetic resolution process (hence the DKR) of R,S)- ethoxyethyl ibuprofen ester. The chemical synthesis of the ester can be applied to any esters, as it is a common procedure. The immobilized lipase preparation procedure can also be used with any enzymes or support of choice. However, the enzyme loading will need to be optimized first. The procedures for the enzymatic kinetic resolution and DKR will need to be adjusted accordingly with different esters. Through this method, the enantiopurity of (5)-ibuprofen was found to be 99.4 % and the conversion was 85 %. It was demonstrated through our work that the synthesis of (5)-ibuprofen via DKR is highly dependent on the suitability of the reaction medium between enzymatic kinetic resolution and the racemization process. This is because the compatibility between both processes is crucial for the success of the DKR. The choice of base catalyst will vary from one reaction to another, but the basic procedures used in this work can be applied. DKRs of other profens have been reported by Lin and Tsai and Chen et al. ... 

Long, W.S. Kamaruddin, A.H. andBhatia, S., Chiral Resolution of Racemic Ibuprofen Ester in an Enzymatic Membrane Reactor. Journal of Membrane Science., 2005, 247, 185-200. 

In a comparable system, (I ,S)-ibuprofen can be separated by a membrane reactor [83], see Fig. 13.10. The technique comprises a stereo-specific hydrolysis by an enzyme. Subsequently, the enantiomeric ester is extracted into the organic phase on the other side of the membrane. In the system developed by Sepracor Inc., (i )-ibuprofen is selectively hydrolyzed by proteases in a hollow-fiber unit and the (S)-ibuprofen ester can be isolated at 100% yield. This configuration also applies for enantioseparation of other acids such as naproxen and 2-chloropropionic acid. 

Figure 10.10 Enantioselective hydrolysis of naproxen and ibuprofen esters. ...

Recent studies in the pharmaceutical field using MBR technology are related to optical resolution of racemic mixtures or esters synthesis. The kinetic resolution of (R,S)-naproxen methyl esters to produce (S)-naproxen in emulsion enzyme membrane reactors (E-EMRs) where emulsion is produced by crossflow membrane emulsification [38, 39], and of racemic ibuprofen ester [40] were developed. The esters synthesis, like for example butyl laurate, by a covalent attachment of Candida antarctica lipase B (CALB) onto a ceramic support previously coated by polymers was recently described [41]. An enzymatic membrane reactor based on the immobilization of lipase on a ceramic support was used to perform interesterification between castor oil triglycerides and methyl oleate, reducing the viscosity of the substrate by injecting supercritical CO2 [42],... 

On the basis of these three cross coupling reactions, it is probably fair to say that using the OSSOS concept is highly compatible with palladium catalysis but probably not limited to it. For example, a lipase can be used for the kinetic resolution of a racemic ibuprofen ester supported on an imidazolium salt. In a DMSO/phosphate buffer mixture and in the presence of the lipase isolated from Candida antartica, the, V-(+(-supported ibuprofen ester is hydrolyzed selectively (87% ee) in 87% yield. Noticeably, during workup, the support can easily be recovered and reused for another cycle while the other enantiomer can be obtained by hydrolysis using K2CQ3 [137] (Fig. 48). 

Preparation of (S)-ibuprofen by enzyme-catalyzed enantioselective hydrolysis of racemic ibuprofen esters has been investigated by several companies such as Sepracor [50,51], Rhone-Poulenc [52,53], Gist-Brocades [54,55], and the Wisconsin Alumni Research Foundation [56]. These processes are usually performed under mild reaction conditions, yield highly optically pure product, and can be readily scaled up for industrial production. The disadvantage of these processes for (S)-ibuprofen production is the extra step needed to produce the corresponding ester of racemic ibuprofen, as well as the cost of producing the enzyme and microorganism catalysts. 

Nakagawa et al. studied the chiral discrimination in the transport of ketoprofen and ibuprofen esters through an aqueous phase mediated by various serum albumins [41]. Serum albumins that act as carriers discriminated between enantiomers of alkyl esters of ketoprofen and ibuprofen in transport in the O/W/O (oil/water/oil) system using a U-shaped cell. The transport rate and the preferred enantiomer of the esters were substantially affected by pH, temperature, and species of albumin. Among five serum albumins studied, bovine serum albumin (BSA) showed the largest rate constant, and rat serum albumin (RSA) manifested the highest enantio-selectivity. Regarding enantiomer selectivity in overall transport, it is anticipated that the ester uptake step plays an important role for BSA, whereas the ester release is the key step for RSA. 

Nakagawa, H. Shimizu, K. Yamada, K. Chiral discrimination in the transport of ketoprofen and ibuprofen esters through an aqueous phase mediated by various serum albumins. Chirality 1999, 11 (5-6), 516-519. 

In this framework, the class of lipases is made up of enzymes largely employed in pharmaceutical productions, whose product is specific enantiomeric forms of organic compounds (alcohols, adds, esters, amines). The enantioselective hydrolysis of racemic esters and simultaneous separation of the corresponding optically pure (5)-add as pure isomer is of considerable interest to the pharmaceutical industry as a route to non-steroidal anti-inflammatory drugs. In this field, studies have been devoted to the feasibility analysis of MBRs to produce (5)-ibuprofen esters and acids. Studies related to the modeling of the lipase-catalyzed hydrolysis of (S)-ibuprofen acid in MBRs show the feasibility of EMRs for the stereo-selective production of (S )-ibuprofen add indeed, the model indicates a high effidency of the EMR in the kinetic resolution of racemic solutions. ... 

Bhatia S, Long W S, Kamaruddin A H (2004), Enzymatic membrane reactor for the kinetic resolution of racemic ibuprofen ester modeling and experimental studies , Chem. Eng. Sci., 59,5061-5068. 

Long W S, Kamaruddin A, Bhatia S (2005), Chiral resolution of racemic ibuprofen ester in an enzymatic membrane reactor , / Memb. Sci., 247,185-200. 

In 2006, another lipase-catalysed hydrolysis process under in situ racemisation of the remaining (i )-ibuprofen ester substrate with sodium hydroxide as... 

Long, W. S., S. Bhatia, and A. Kamaruddin. 2003. Modeling and Simulation of Enzymatic Membrane Reactor for Kinetic Resolution of Ibuprofen Ester. Journal of Membrane Science 219 (l-2) 69-88. 

Chen, J.C. and Tsai, S.W. (2000) Enantioselective synthesis of (S)-ibuprofen ester prodrug in cyclohexane by Candida rugosa lipase immobilized on... 

---