[PDF] Prodrugs Design Based on Inter- and Intramolecular Chemical

8009_2pdf2.pdf Prodrugs Design Based on Inter- and Intramolecular

Chemical Processes

Rafik Karaman

1,2,*

1Bioorganic Chemistry Department, Faculty of Pharmacy,

Al-Quds University, P.O. Box 20002, Jerusalem, Palestine

2Department of Science, University of Basilicata, Via

dell"Ateneo Lucano 10, 85100, Potenza, Italy *Corresponding author: Rafik Karaman, dr_karaman@yahoo.com This review provides the reader a concise overview of the majority of prodrug approaches with the emphasis on the modern approaches to prodrug design. The chemical approach catalyzed by metabolic enzymes which is considered as widely used among all other approaches to minimize the undesirable drug physico- chemical properties is discussed. Part of this review will shed light on the use of molecular orbital methods such as DFT, semiempirical andab initiofor the design of novel prodrugs. This novel prodrug approach implies prodrug design based on enzyme models that were utilized for mimicking enzyme catalysis. The com- putational approach exploited for the prodrug design involves molecular orbital and molecular mechanics (DFT,ab initio,and MM2) calculations and correlations between experimental and calculated values of intra- molecular processes that were experimentally studied to assign the factors determining the reaction rates in certain processes for better understanding on how enzymes might exert their extraordinary catalysis. Key words:Ab initiocalculations, design of prodrugs, DFT calculations, enzyme models, molecular mechanics calcula- tions, prodrugs Received 4 July 2013, revised 13 August 2013 and accepted for publication 16 August 2013 A drug is defined as a substance, which is used in the diagnosis, cure, relief, treatment, or prevention of disease, or intended to affect the structure or function of the body. The development of any potential drug starts with the study of the biochemistry behind a disease for which phar- maceutical intervention is seen. a Drug discovery is a lengthy interdisciplinary endeavor. It is a consecutive process that starts with target and lead discovery, followed by lead optimization and preclinical

in vitroandin vivostudies to evaluate whether acompound satisfies a number of preset criteria to startclinical development. The number of years it takes to intro-

duce a drug to the pharmaceutical market is over 10 years with a cost of more than $1 billion dollars (1,2). Modifying the absorption, distribution, metabolism, and elimination (ADME) properties of an active drug requires a complete understanding of the physicochemical and bio- logical behavior of the drug candidate (3-6). This includes comprehensive evaluation of drug-likeness involving prediction of ADME properties. These predictions can be attempted at several levels:in vitro-in vivousing data obtained from tissue or recombinant material from human and preclinical species, andin silicoor computational pre- dictions projectingin vitroorin vivodata, involving the evaluation of various ADME properties, using computa- tional approaches such as quantitative structure activity relationship (QSAR) or molecular modeling (7-11). Studies have indicated that poor pharmacokinetics and tox- icity are the most important causes of high attrition rates in the drug development process, and it has been widely accepted that these areas should be considered as early as possible in drug discovery to improve the efficiency and cost-effectiveness of the industry. Resolving the pharmaco- kinetic and toxicological properties of drug candidates remains a key challenge for drug developers (12). Thus, the aim is to design drugs that have an efficient permeability to be absorbed into the blood circulation (absorption), to reach their target efficiently (distribution), to be quite stable to survive the physiological journey (metab- olism), and to be eliminated in a satisfactory time (elimina- tion). In other words, designing a drug with optimum pharmacokinetics properties can be achieved by imple- menting one or more of the following strategies:

Improving Absorption

Drug absorption is determined by the drug hydrophilic hydrophobic balance (HLB) value, which depends upon polarity and ionization. Very polar or strongly ionized drugs, having a relatively high HLB values, cannot efficiently cross the cell membranes of the gastrointestinal (GI) barrier. Hence, they are given by the intravenous (I.V.) route, but their disadvantage is being rapidly eliminated. Non-polar ª2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12224643

Chem Biol Drug Des 2013;82:643-668

Review

drugs, on the other hand, having a relatively low HLB val- ues, are poorly soluble in aqueous media and hence are poorly absorbed through membranes. If they are given by injection, most probably, they will be retained in fat tissues (13-21). Generally, the polarity and/or ionization of drug can be altered by changing its substituents, and these changes are classified under the so-called quantitative structure- activity relationships (QSAR). The following are examples for such changes: (1) variation of alkyl or acyl substituents and polar functional groups to vary polarity, (1) variation of N-alkyl substituents to vary pKa; acidic drugs with low pKa and basic drugs with high pKa values tend to be ion- ized and are poorly absorbed through membrane tissues, (2) variation of aromatic substituents to vary pKa: The pKa of aromatic amine or carboxylic acid can be varied by adding electron donating or electron withdrawing groups to the ring. The position of the substituent is important too if the substituent interacts with the ring through resonance and (3) bioisosteres for polar groups; carboxylic acid is a highly polar group which can be ionized and hence decreases the absorption of any drug containing it. To overcome this problem, blocking the free carboxyl group by making the corresponding ester prodrug or replacing it with a bioisostere group, which has similar physiochemical properties and has advantage over carboxylic acid in regard to its pKa, such as 5-substituted tetrazoles, is essential; 5-substituted tetrazole ring contains acidic pro- ton such as carboxylic acid and is ionized at pH 7.4. On the other hand, most of the alkyl and aryl carboxylic groups have a pKa in the range of 2-5 (13-28).

Improving Metabolism

There are different strategies that can be utilized to improve drug metabolism: (i) steric shields: Some func- tional groups are more susceptible to chemical and enzy- matic degradation than others. For example, esters and amides are much more affordable to hydrolysis than others such as carbamates and oximes. Adding steric shields to these drugs increases their stability. Steric shields were designed to hinder the approach of a nucleophile or a nucleophilic center on an enzyme to the susceptible group. These usually involve the addition of a bulky alkyl group such as t-butyl close to the functional group. (ii) Electronic effects of bioisosteres: This approach is used to protect a labile functional group by electronic stabiliza- tion. For example, replacing the methyl group of an ester with an amine group gives a urethane functional group, which is more stable than the parent ester. The amine group has the same size and valance as the methyl group; however, it has no steric effect, but it has totally different electronic properties, because it can donate electronsvia its inductive effect into the carbonyl group resulting in reducing the electrophilicity of the carbonyl carbon and

hence stabilizing it from hydrolysis.Carbachol (1 in Figure 1), a cholinergic agonist, and cefoxi-tin (2 in Figure 1), a cephalosporin, are stabilized in this

way. (iii) Stereoelectronic modification: Steric hindrance and electronic stabilization have been used together to stabilize labile groups. For example, procaine, an ester drug, is quickly hydrolyzed, but changing the ester to the less reac- tive amide group reduces hydrolysis such as in the cases of procainamide (3 in Figure 1) and lidocaine (4 in Figure 1). (iv) Metabolic Blockers: Some drugs are metabolized by introducing polar functional groups at particular positions in their skeleton. For example, megestrol acetate (5 in Figure

1), an oral contraceptive, is oxidized at position 6 to give

hydroxyl group at this position; however, replacing the hydrogen at position 6 with a methyl group blocks its metabolism, and consequently it results in prolonging its duration of action. (v) Removal of susceptible metabolic groups: Certain chemical moieties are particularly suscepti- ble to metabolic enzymes. For example, a methyl group on aromatic rings is often oxidized to carboxylic acid, which then results in a rapid elimination of the drug from the body. Other common metabolic reactions include aliphatic and aromatic C-hydroxylation, O and S-dealkylations, N- and S-oxidations, and deamination. (vi) Group Shifts: Removing or replacing a metabolically vulnerable group is feasible if the group concerned is not involved in important binding interactions within the active site of the receptor or enzyme. If the group is important, then different strategy either masking the vulnerable group using a prodrug or shifting the vulnerable group within the molecule skeleton is undertaken. Salbutamol was developed in 1969 from its analog neurotransmitter, norepinephrine, using this tactic. Norepinephrine is metabolized by methylation of one of its phenolic groups by catechol O-methyl transferase. The other phenolic group is important for receptor-binding inter- action. Removing the hydroxyl or replacing it with a methyl group prevents metabolism but also prevents hydrogen bonding interaction with the binding site. While moving the vulnerable hydroxyl group out from the ring by one carbon unit as in salbutamol makes, this compound unrecogniz- able by the metabolic enzyme, but not to the receptor- binding site (prolonged action) and (vii) ring variation; some ring systems are often found to be susceptible to metabo- lism, and so varying the ring can often improve metabolic stability. For example, replacement of the imidazole ring, which is susceptible to metabolism in tioconazole (6 in Fig- ure 1) with 1,2,4-triazole ring, gives fluconazole (7 in Figure

1) with improved stability.

Making drug less resistance to drug metabolism: drug that is extremely stable to metabolism and is very slowly elimi- nated can cause problems in a similar manner to that sus- ceptible to metabolism, thus resulting in an increase in toxicity and adverse effects. Therefore, designing drugs with decreased chemical and metabolic stability can sometimes be beneficial. Methods for applying such strategy are (i) introducing groups that are susceptible to metabolism is a good way of shorting the lifetime of a drug. For example, methyl group was introduced to some drugs to shorten their

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lifetime because methyl can metabolically undergo oxidation to polar alcohol as well as to a carboxylic acid. (ii) A self- destruct drug is one that is chemically stable under one set of conditions but becomes unstable and spontaneously cleaves under another set of conditions.The advantage of a self-destruct drug is that inactivation does not depend on the activity of metabolic enzymes, which could vary from patient to patient. For example, atracurium, a neuromuscu- lar blocking agent, is stable at acidic pH but self-destructs when it is exposed to the slightly alkaline conditions of the blood (pH 7.4). Thus, the drug has a short duration of action, allowing anesthetists to control its blood concentra- tion levels during surgery by providing it as a continuous intravenous drip (22-28).

Reducing Toxicity

It is often found that a drug fails clinical trials because of its toxic adverse effects. This may be due to toxic metabolites, in which case the drug should be made more resistant to metabolism. It is

known that functional groups such as aromatic nitrogroups, aromatic amines, bromoarenes, hydrazines,hydroxylamines, or polyhalogenated groups are generally

metabolized to toxic metabolites. Side-effects might be reduced or eliminated by varying harmless substituents. For example, addition of fluorine group to UK 47265, antifungal agent, gives the less toxic fluconazole (29-34).

Prodrugs Catalyzed by Metabolic Enzymes

The principle of targeting drugs can be traced back to Paul Ehrlich who developed antimicrobial drugs that were selectively toxic for microbial cells over human cells. Today, targeting tumor cells is considered one of the most important issues that under concern among the health community. A major goal in cancer chemotherapy is to tar- get drugs efficiently against tumor cells rather than against normal cells. One method for achieving this is to design drugs which make use of specific molecular transport sys- tems. The idea is to attach the active drug to an important building block molecule that is needed in large amounts by the rapidly divided tumor cells. This could be an amino

Figure 1:Chemical structures for1-7.

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Inter- and Intramolecular Chemical Processes

acid or a nucleic acid base such as uracil mustard. In the cases where the drug is intended to target against infec- tion of gastrointestinal tract (GIT), it must be prevented from being absorbed into the blood supply. This can easily be done using a fully ionized drug which is incapable of crossing cell membrane barriers. For example, highly ion- ized sulfonamides are used against GIT infections because they are incapable of crossing the gut wall. It is often possible to target drugs such that they act peripherally and not in the central nervous system (CNS). By increasing the polarity of drugs, they are less likely to cross the blood-brain barrier, and thus, they are less likely to have

CNS adverse effects (35-46).

The most efficient approach for overcoming the negative pharmacokinetics characteristics of a drug is the prodrug approach. This approach may be utilized in the cases where the use of the parent drug faces problems associated with solubility, absorption and distribution, site specificity, insta- bility, toxicity, poor patient compliance, or formulation prob- lems (47-52). A metabolic enzyme is usually involved in converting the prodrugs to their active forms. Not all prodrugs are activated by metabolic enzymes. For example, photodynamic therapy involves the use of an external light source to activate prodrugs. When designing a prodrug, it is important to ensure that the prodrug is effectively converted to the active drug once it has been absorbed in blood sup- ply. It is also important to ensure that any groups that are cleaved from the prodrug molecule are non-toxic (22). The prodrug approach is a very versatile strategy to increase the utility of biologically active compounds, because one can optimize any of the ADME properties of potential drug candidates. In most cases, prodrugs contain a promoiety (linker) that is removed by an enzymatic or chemical reac- tion, while other prodrugs release their active drugs after molecular modification such as an oxidation or reduction reaction. The prodrug candidate can also be prepared as a double prodrug, where the second linker is attached to the first promoiety linked to the parent drug molecule. These linkers are usually different and are cleaved by different mechanisms. In some cases, two biologically active drugs can be linked together in a single molecule called a codrug. In a codrug, each drug acts as a linker for the other (8,9). The prodrug approach has been used to overcome various undesirable drug properties and to optimize clinical drug application. Recent advances in molecular biology provide direct availability of enzymes and carrier proteins, including their molecular and functional characteristics. There are two major prodrug design approaches that are considered as widely used among all other approaches to minimize or eliminate the undesirable drug physicochemi- cal properties while maintaining the desirable pharmaco- logical activity. The first approach is the targeted drug design approach by which prodrugs can be designed to target specific enzymes or carriers by considering

enzyme-substrate specificity or carrier-substrate specificityto overcome various undesirable drug properties. This typeof 'targeted-prodrug" design requires considerable knowl-

edge of particular enzymes or carriers, including their molecular and functional characteristics (35-46). The second approach to be discussed in this review is sub- divided to two major approaches: (i) Chemical approach by which the drug is linked to promoiety which upon exposure to physiological environment undergoes enzymatic cata- lyzed degradation to the parent drug and inactive linker. In this approach, the interconversion rate is dependent on the enzyme catalysis. This approach involves carrier-linked prodrugs and contains a group that can be easily removed enzymatically, such as an ester or labile amide, to provide the parent drug. Ideally, the group removed is pharmaco- logically inactive and non-toxic, while the linkage between the drug and promoiety must be labile forin vivoefficient activation. Carrier-linked prodrugs can be further subdivided into (i) bipartite, which is composed of one carrier group attached to the drug, (ii) tripartite, which is a carrier group that is attachedvialinker to drug, and (iii) mutual prodrugs consisting of two drugs linked together, and (1) bioprecur- sors are chemical entities that are metabolized into new compounds that may be active or further are metabolized to active metabolites, such as amine to aldehyde to carboxylic acid (48-51); and (ii) intramolecular chemical approach designed based on calculations using molecular orbital (MO) and molecular mechanics (MM) methods and correla- tions between experimental and calculated values. In this prodrug approach, no enzyme is involved in the intraconver- sion chemical reaction of a prodrug to its parent drug. The interconversion of the prodrug is solely controlled by the rate-limiting step of the intramolecular reaction. The prodrug design can be utilized in the followings cases: (i) enhancing active drug solubility in a physiological envi- ronment/s and consequently its bioavailability since disso- lution of the drug molecule from the dosage form may be the rate-limiting step to absorption (48). It has been docu- mented that more than 30% of drug discovery compounds have poor aqueous solubility (53). Prodrugs are an alterna- tive way to increase the aqueous solubility of the parent drug molecule by increasing dissolution rate through attachment to ionizable or polar groups, such as phos- phates, sugar, or amino acids moieties (51,54). These prodrugs can be used for increasing oral bioavailability and in parenteral or injectable drug delivery. (ii) Upon increasing permeability and hence absorption, membrane permeability has a significant effect on drug effectiveness (7). In oral drug delivery, the most common absorption routes are un-facilitated and largely non-specific, passive transport mechanisms. The lipophilicity of poorly permeable drugs can be enhanced by linking to lipophilic groups. In such cases, the prodrug strategy can be an extremely valuable option and crucially needed. Improvements in lipophilicity have been the most widely researched and successful field of prodrug research. It has been achieved by masking polar ionized or non-ionized functional groups to increase

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either oral or topical absorption (22). (iii) Modification of the distribution profile: Before the drug reaches its physiological target and exert the desired effect, it has to bypass several pharmaceutical and pharmacokinetic barriers. Today, one of the most promising site-selective drug delivery strategies is the prodrug approach, which utilizes target cell- or tis- sue-specific endogenous enzymes and transporters. The suitability of a number of functional groups such as carboxylic, hydroxyl, amine, phosphate, phosphonate, and carbonyl groups for undergoing different chemical modifications facilitates their utilization in prodrug design (1,9) In the past few decades, a variety of prodrugs based on the chemical approach have been designed, synthe- sized, and tested. Among those are the following.

Ester Prodrugs

carboxylic acid, hydroxyl, phosphate, and thiol groups can easily undergo hydrolysisviathe enzymatic catalysis of esterases and phosphatases that are present in many places in the body including liver, blood, and other tissues, orviaoxidative cleavage catalyzed by cytochrome P450 enzymes (CYP) (51,55,56). Carboxyl esterases, acetylcholinesterases, butyrylcholines- terases, paraoxonases, arylesterases and biphenyl hydro- lase-like protein (BPHL) are examples of enzymes that are responsible for the hydrolytic bioactivation of ester prodrugs (56). For example, biphenyl hydrolase-like protein (BPHL) is known to catalyze the hydrolysis of prodrugs such as vala- cyclovir (8in Figure 2) and valganciclovir (9in Figure 2), as well as a number of other amino acid esters of nucleo- side analogs including valyl-AZT, prodrugs of floxuridine (5-fluoro-20-deoxyuridine or FUdR) (10in Figure 2) and gemcitabine (11in Figure 2) (57). Ester prodrugs are com- monly used to enhance lipophilicity, thus increasing mem- brane permeation through masking the charge of polar functional groups and by handling the alkyl chain length and configuration (51). For example, acyclovir aliphatic ester prodrugs were prepared by an esterification of the hydroxyl group with lipophilic acid anhydride or acyl chloride (58); hence, an enhanced lipophilicity can be achieved. Utilizing the lipophilic ester approach, some acyclovir prodrugs were synthesized and have shown an enhanced nasal and skin absorption (51). It has been shown that an increase in the length of an alkyl chain results in a relatively ease cleavage of the ester bond. Therefore, it might be concluded that improved binding to the hydrophobic pocket of carboxyles- terase can be accomplished by increasing the length of the ester alkyl chain, while branching the alkyl chain might result in reduced hydrolysis due to a steric hindrance (51). Further, eighteen amino acid esters of acyclovir were synthesized as potential prodrugs intended for oral administration, and their hydrolytic reaction was shown to be catalyzed by biphenyl hydrolase (59). Acyclic nucleosides such as adefovir and

tenofovir are monophosphorylated and do not rely on viralnucleoside kinases for initial activation; hence, they have

low oral bioavailability due to the ionized phosphonate group (60,61). To overcome this problem, a bis(pivaloyl- oxymethyl) ester prodrug of adefovir (adefovir dipivoxil) and ether lipid ester prodrugs of cidofovir were explored for improving intestinal permeability (51). Other examples for ester prodrugs that were designed and synthesized for dif- ferent purposes are thioester of erythromycin, palmitate ester of clindamycin, a number of angiotensin-converting enzyme (ACE) inhibitors which are (55) presently marketed as ester prodrugs, including enalapril (12in Figure 2), ramipril, benazepril, and fosinopril, and all of them are intended for the treatment for hypertension (47) and ibupro- fen guaiacol ester that was reported to have fewer GI side- effects with similar anti-inflammatory/antipyretic action to its parent drug when is given in equimolar doses (62). As men- tioned before, two active drugs can be joined together such that each one behaves as a carrier moiety for the other, a strategy known as mutual prodrugs (8). The followings are some examples of mutual prodrugs, based on ester linkage, that were produced to overcome several shortcomings associated with therapeutic drugs used in clinical practice: Benorylate (13in Figure 2) is a mutual prodrug of aspirin and paracetamol, coupled through an ester linkage, which is postulated to have reduced gastric irritancy with synergis- tic analgesic effect (63). Moreover, mutual prodrugs of ibu- profen with paracetamol and salicylamide have been reported to have better lipophilicity and diminished gastric toxicity than the parent drug. Another example is naproxen- propyphenazone which was synthesized to prevent GI irrita- tion and bleeding (64). An alternative strategy to avoid GI side-effects is by conjugation of a nitric oxide (NO) releasing moiety to the parent NSAID drug. It has been reported that NO plays a gastro protective role along with prostaglandins (65). Some NO-releasing organic nitrate esters of aspirin, diclofenac, naproxen, ketoprofen, flurbiprofen, and ibupro- fen have been reported to give the corresponding active parent drugs with lower gastro toxicity (66,67). Reduce gas- tro toxicity could be achieved also by linking NSAID drug with histamine H

2antagonist such as in the case of flurbi-

profen-histamine H

2antagonist conjugates (64). The mutual

prodrug approach was also applied to other therapeutic groups. For instance, sultamicillin (14in Figure 2) in which the irreversibleb-lactamase inhibitor sulbactam has been linkedviaan ester linkage with ampicillin has shown a syn- ergistic effect (64), and upon oral administration, sultamicil- lin is completely hydrolyzed to equimolar proportions of sulbactam and ampicillin, thereby acting as an efficient mutual prodrug (68).

Amides Prodrugs

This approach can be exploited to enhance the stability of drugs, provide targeted drug delivery, and change lipophi- licity of drugs such as acids and acid chlorides (69). Drugs that have carboxylic acid or amine group can be con- verted into amide prodrugs.Generally, they are used to a

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limited extent due to highin vivostability. However, pro- drugs using facile intramolecular cyclization reactions have been exploited to overcome this obstacle (70). Similar to mutual ester prodrugs, there are some mutual prodrugs where the two active drugs are linked together by an amide linkage, such as atorvastatin and amlodipine which uponin vivoamide hydrolysis provide the corre- sponding active parent drugs. Amide prodrugs can be con- verted back to the parent drugs either by nonspecific amidases or by specific enzymatic activation such as renalc-glutamyl transpeptidase. Dopamine double prodrug c-glutamyl-l-dopa (gludopa) (15in Figure 2) undergoes specific activation by renalc-glutamyl transpeptidase where it achieves relatively fivefold increase in dopamine level compared with L-dopa prodrug. However, as gludopa has loworalbioavailability,dopamine[N-(N-acetyl-L-methionyl)-O, O-bis(ethoxycarbonyl)dopamine),apseudopeptideprodrugof dopamine, was developed and has shown improved oral absorption; hence, it is given orally and is used in the treat-

ment for renal and cardiovascular diseases. Basically,dopamine prodrugs are developed due to dopamine inac-tivation by COMT and MAO when administered by the oral

route (71,72). A respected number of amine conjugates with amino acids through amide linkage have been considered for providing active drugs with remarkable enhancement in solubility such as dapsone (16in Figure 2) (73). Other examples of amide based prodrugs are allopurinol N-acyl derivatives which were found to be more lipophilic than allopurinol itself (74).

Carbonates and Carbamates Prodrugs

Generally, carbonates and carbamates are more stable than esters but less stable than amides (75). Carbamates and carbonates have no specific enzymes for their hydro- lysis reactions; however, they are degraded by esterases to give the corresponding active parent drugs (75,76). Co-

Figure 2:Chemical structures for8-17.

648ChemBiol Drug Des2013; 82: 643-668Karaman

carboxymethylphenyl ester of amphetamine is an example of carbamates prodrug that can be hydrolyzed by esterase to yield amphetamine (76). Carbamates prodrugs are regarded as double prodrugs (pro-prodrug) because they are enzymatically activated at first which is followed by spontaneous cleavage of the resulting carbamic acid (51). An example for such prodrugs is fluorenylmethoxycarbon- yl]-3 derivatives of insulin and exenatide (77) that undergo slow interconversionviacarbamate bond breakdown, thus providing glucose controlling agents in an adequate rate which consequently results in lowering the risk of hypoglycemia (74).

Another example of carbamates prodrug is the one

obtained by linking phosphorylated steroid, an estradiol, to normustard, an alkylating agent, through a carbamate link- age, which yields estramustine prodrug. The latter is used in the treatment for prostate cancer. The steroid portion has an antiandrogenic action and acts to concentrate the prodrug in the prostate gland where prodrug hydrolysis takes place and normustard action can then be exerted (64). Carbamates prodrugs can also be used to increase the solubility of active drugs such as cephalosporins (78). In addition, carbamates prodrugs have been exploited in targeted therapy such as ADEPT. In this case, the carba- mate group is susceptible to the action of tyrosinase enzyme present in melanomas. This approach is usually utilized in cancer targeted therapy (79). The list of carba- mates prodrugs is long; among other examples is the non- sedating antihistamine loratadine (17in Figure 2), an eth- ylcarbamate, that undergoesin vivointerconversion to its active form, desloratidine, through the action of CYP450 enzymes (80), and capecitabine, an anticancer agent, that undergoes a multistep activation, to finally yield 5-fluoroura- cil in the liver. Capecitabine is less toxic than 5-fluorouracil, more selective, and widely used in clinical practice (81-83).

Oximes Prodrugs

These prodrugs serve to increase the permeability of the corresponding active drugs, and they are converted back to their parent drugs by microsomal cytochrome

P450 enzymes (CYP450) (51). Dopaminergic prodrug

6-(N,N-Di-n-propylamino)-3,4,5,6,7,8-hexahydro-2H-naph-

thalen-1-one is an example for such class (84).

N-Mannich Bases, Enaminones, and Schiff

Bases (Imines) N-Mannich base formation is another approach, which can be utilized to enhance drug"s solubility. N-Mannich bases are prepared by Mannich reaction that involves reacting of NH-acidic compound, an aldehyde and an amine in etha- nol (74). Rolitetracycline (18in Figure 3) is the Mannich derivative of tetracycline, and it is the only one available for

intravenous administration (85). N-Mannich bases of dipy-rone, metamizole (19in Figure 3), the methane sulfonic

acid of the analgesic 4-(methylamino) antipyrine is water soluble and suitable for parenteral route, and when given orally, it is hydrolyzed in the stomach to give the parent active drug (86). Despite the success of N-Mannich base prodrugs to improve bioavailability of active drugs, there still some stability formulation problems arise from poorin vitro stability of some of the prodrugs (85). In addition, thein vivoformation of formaldehyde upon enzymatic breakdown (87) of these prodrugs is considered a limitation of this pro- drug approach. Enamines (88) (a,b-unsaturated amines) are unstable at low pH, which results in their limitation for use in oral administration (74). Nonetheless, an ampicillin prodrug based on enamines was prepared for rectal use, and it exhibits an increased absorption compared with its active parent drug (89). Enaminones are enamines ofb-dic- arbonyl compounds that undergo ketoenolimine-enamine tautomeric equilibrium, which may offer stability to these compounds (86). Enaminones are generally more lipophilic than their parent drugs; hence, they have an improved oral absorption. Typically, enaminones have a relatively high chemical stability; therefore, their use as potential prodrugs is being somewhat limited. It is expected that enaminones derived from ketoesters and lactone may be subjected to enzymatic degradation; hence, a better conversion rate to the active drug can be obtained (90).

Phosphate and Phosphonate Prodrugs

Phosphorylation offers increased aqueous solubility to the parent drugs. A traditional example of phosphate prodrugs is prednisolone sodium phosphate, a water-soluble pro- drug of prednisolone, its water solubility exceeds that of its active form, prednisolone, by 30 times (8), it is often used as an immunosuppressant, and it is formulated as a liquid dosage form (8). Another common phosphate pro- drug is fosamprenavir (20in Figure 3). Similar to predniso- lone, the phosphate promoiety in fosamprenavir is linked to a free hydroxyl group, and the prodrug is 10-fold more water soluble than amprenavir. An enhanced patient com- pliance is achieved when using this antiviral prodrug; instead of administering the drug 8 times daily, dosage regimen is reduced into two times per day (91). In the gut andviathe action of alkaline phosphatases, phosphate prodrugs are cleaved back to their corresponding active drugs and then absorbed into the systemic circulation (8). Another application of this approach is fosphenytoin (21in Figure 3), a prodrug of the anticonvulsant agent phenytoin. Fosphenytoin has an enhanced solubility over its corre- sponding drug (92).

Azo Compounds

Colonic bacteria can be exploited in prodrug approach as a means of prodrug activation through the action of azo- reductases; this approach is applied specially in targeted

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drug strategy (85). Sulfasalazine (22in Figure 3), used in the treatment for ulcerative colitis (93), is a prodrug of

5-aminosalicylic acid and sulfapyridine. Upon reaching the

colon, sulfasalazine undergoes azo bond cleavage to release the active parent drug (64). Osalazine (23in Figure 3), a dimer of 5-aminosalicylic acid, balsalazide, and ipsalazide in which 5-aminosalicylic acid moiety is conjugated to 4-aminobenzoyl-b-alanine and 4-amin- obenzoylglycine, respectively (94), are other examples of prodrugs that are activated by azo-reductases. A prodrug by which 5-aminosalicylic acid is linked to L-aspartic acid is another example for such class that has shown a desir- able colon-specific delivery and a 50% release of 5-amino- salicylic acid from an administered dose (95). Usually this approach is limited to aromatic amines, because azo com- pounds of aliphatic amines exhibit significant instability (74).

Poly Ethylene Glycol (PEG) Conjugates

PEG can be linked to drugs either to increase drug solu- bility

or to prolong drug plasma half-life (74); an ester,carbamate, carbonates, or amide spacer can be used tolink the drug to PEG. Upon enzymatic breakdown of the

spacer, the resultant ester or carbamate drug can be lib- erated by 1,4- or 1,6-benzyl elimination (96). Daunorubi- cin conjugated to PEG is an example of this kind of prodrugs. In this prodrug system, PEG is conjugated to the phenol group of the open lactoneviaa spacer. Con- trolling the rate of the free drug release can be accom- plished by manipulation of the substituents on the aromatic ring (97). The prodrug chemical approach involving enzyme catalysis is perhaps the most unpredicted approach, because there are many intrinsic and extrinsic factors that can affect the bioconversion mechanisms. For example, the activity of many prodrug-activating enzymes may be changed due to genetic polymorphisms, age-related physiological changes, or drug interactions, leading to adverse pharmacokinetic, pharmacodynamics, and clinical effects. In addition, there are wide interspecies variations in both the expression and function of most of the enzyme systems activating pro- drugs which could lead to serious challenges in the pre- clinical optimization phase (3-6).

Figure 3:Chemical structures for18-23.

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Prodrugs Based on Intramolecular

Processes (Enzyme Models)

The novel prodrug approach to be discussed in this sec- tion implies prodrug design based on enzyme models (mimicking enzyme catalysis) that have been advocated to understand how enzymes work. The tool used in the design is a computational approach consisting of calcula- tions using a variety of different molecular orbital and molecular mechanics methods and correlations between experimental and calculated rate values (activation ener- gies) for some intramolecular processes that were utilized to understand the mechanism by which enzymes might exert their high catalysis. In this approach, no enzyme is needed for the catalysis of the intraconversion of a pro- drug to its active parent drug. The release rate of the pro- drug to the active drug is solely determined by the factors affecting the rate-limiting step of the intraconversion pro- cess. Knowledge gained from the mechanisms of the pre- viously studied enzyme models was used in the design. It is worth noting that the use of this approach might elimi- nate all disadvantages that are concerned with prodrug interconversion by enzymes approach. As mentioned in the introduction, the bioconversion of prodrugs has many disadvantages related to many intrinsic and extrinsic fac- tors that can affect the process. For instance, the activity of many prodrug-activating enzymes may be varied due to genetic polymorphisms, age-related physiological changes, or drug interactions, leading to variation in clinical effects. In addition, there are wide interspecies variations in both the expression and function of the major enzyme systems activating prodrugs, and these can pose some obstacles in the preclinical optimization phase.

Intramolecular Processes (Enzyme Models)

Used for the Design of Potential Prodrugs Studies of enzyme mechanisms by Bruice and Benkovic,

Jencks,

Menger, Kirby, Walsh, and Bender, over the past

five decades, have had a tremendous contribution to bet- ter understanding the mode and scope by which enzymes catalyze biochemical transformations (98-101). Nowadays, the scientific community has reached a con- sensus that the catalysis by enzymes is based on the combined effects of the catalysis by functional groups and the ability to reroute intermolecular reactions through alternative pathways by which substrates can bind to pre- organized active sites. The rates for most of enzymatic reactions exceed 10 10- 10

18-fold the nonenzymatic bimolecular counterparts. For

example, reactions catalyzed by the enzyme cyclophilin are accelerated by 10

5, and those by orotidine monophos-

phate decarboxylase are enhanced by 10

17(102).In the last 50 years, as mentioned earlier, scholarly studies

have been carried out by Bruice (103), Cohen (104), Menger (105), Kirby (106), and others (107) to design chemical models that have the capability to reach rates comparable to that with enzyme-catalyzed reactions. Fre- quently cited examples of such models are those based on rate acceleration driven by covalently enforced proxim- ity. The most quoted example is Bruiceet al."s. intramo- lecular ring-closing reaction of dicarboxylic semi-esters to anhydrides (103). Studying this model, Bruiceet al.has shown that a relative rate of anhydride formation can reach 5910

7upon the intramolecular ring-closing reaction of a

dicarboxylic semi-ester when compared to a similar coun- terpart"s intermolecular reaction. Other examples of rate acceleration based on proximity orientation include (1) systems that obey the principles of Koshland"s 'orbital steering" theory (107) that signifies the importance of the ground state angle of attack value of the hydroxyl in hydroxycarboxylic acids on the intramolec- ular lactonization reaction rate; (1) the 'spatiotemporal hypothesis" advocated by Menger, which implies that a type of a reaction, in proton transfer processes, whether intermolecular or intramolecular, is significantly determined by the distance between the two reactive centers involved in the hydroxycarboxylic acids lactonization reaction (105); (2) the stereopopulation control proposed by Cohen to explain the relatively high enhancement rates in the acid- catalyzed lactonization reactions of hydroxyhydrocinnamic acids containing two methyl groups on thebposition of their carboxylic groups (104) and Kirby"s proton transfer models on the acid-catalyzed hydrolysis of acetals and maleamic acid amides which demonstrate the importance of hydrogen bonding formation in the products and transi- tion states leading to them. In the past 15 years, some prodrugs based on hydroxyhy- drocinnamic acids have been introduced. For example, Borchardtet al.reported the use of the 3-(2′-acetoxy-4′,

6′-dimethyl dimethyl)-phenyl-3, 3-dimethylpropionamide

derivative (pro-prodrug) that is capable of releasing the biologically active amine (drug) upon acetate hydrolysis by enzyme triggering. Another successful example of the pharmaceutical applications for a stereopopulation control model is the prodrug Taxol which enhances the drug water solubility and hence affords it to be administered to the human bodyviaintravenous (I.V.) injection. Taxol is the brand name for paclitaxel, a natural diterpene, approved in the USA for use as anticancer agent (108).

Calculation Methods Used in the Prodrugs

Design

In the past six decades, the use of computational chemis- try for calculating molecular properties of ground and tran- sition states has been a progressive task of organic, bioorganic, and medicinal chemists alike. Computational

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Inter- and Intramolecular Chemical Processes

chemistry uses principles of computer science to assist in solving chemical problems. It uses the theoretical chemis- try results, incorporated into efficient computer programs, to calculate the structures and physical and chemical properties of molecules. Reaction rates and equilibrium energy-based calculations for biological systems that have pharmaceutical and bio- medicinal interests are a very important challenge to the health community. Nowadays, quantum mechanics (QM), such asab initio, semi-empirical, and density functional theory (DFT), and molecular mechanics (MM) are increasingly being used and broadly accepted as reliable tools for providing structure-energy calculations for an accurate prediction of potential drugs and prodrugs alike (109). These methods cover both static and dynamic situations. In all cases, the computer time and other resources (such as memory and disk space) increase rapidly with the size of the system being studied.Ab initiomethods typically are feasible only for small systems.Ab initiomethods are based entirely on theory from first principles. The termab initiowas first used in quantum chemistry by Robert Parr and coworkers, including David Craig in a semiempirical study on the excited states of benzene. Theab initio molecular orbital methods (quantum mechanics) such as HF, G1, G2, G2MP2, MP2, and MP3 are based on rigor- ous use of the Schrodinger equation with a number of approximations.Ab initioelectronic structure methods have the advantage that they can be made to converge to the exact solution, when all approximations are sufficiently small in magnitude and when the finite set of basic func- tions tends toward the limit of a complete set. The conver- gence, however, is usually not monotonic, and sometimes the smallest calculation gives the best result for some properties. The disadvantage ofab initiomethods is their computational cost. They often take enormous amounts of computer time, memory, and disk space (110-112). Other less accurate methods are called empirical or semi- empirical because they employ experimental results, often from acceptable models of atoms or related molecules, to approximate some elements of the underlying theory.

Among these methods, the semi-empirical quantum

chemistry methods are based on the Hartree-Fock formal- ism, but make many approximations and obtain some parameters from empirical data. They are very important in computational chemistry for treating large molecules where the full Hartree-Fock method without the approximations is too expensive. Semi-empirical calculations are much faster than theirab initiocounterparts. Their results, how- ever, can be very wrong if the molecule being computed is not similar enough to the molecules in the database used to parameterize the method. Among the most used semi- empirical methods are MINDO, MNDO, MINDO/3, AM1, PM3, and SAM1. The semi-empirical methods have affor-

ded vast information for practical application (113-116).Calculations of molecules exceeding 60 atoms can bemade using such methods.

Another commonly used quantum mechanical modeling

method in physics and chemistry to investigate the elec- tronic structure (principally the ground state) of many-body systems, in particular atoms, molecules, and the con- densed phases, is the density functional theory (DFT). With this theory, the properties of many-electron systems can be determined using functionals, that is, functions of another function, which in this case is the spatially depen- dent electron density. Hence, the name density functional theory comes from the use of functionals of the electron density. DFT is among the most popular and versatile methods available in condensed-matter physics, computa- tional physics, and computational chemistry. The DFT method is used to calculate structures and energies for medium-sized systems (30-60 atoms) of biological and pharmaceutical interest and is not restricted to the second row of the periodic table (117). Despite recent improvements, there are still difficulties in using density functional theory to properly describe inter- molecular interactions, especially van der Waals forces (dispersion), charge transfer excitations, transition states, global potential energy surfaces, and some other strongly correlated systems. Its incomplete treatment of dispersion can adversely affect the accuracy of DFT in the treatment of systems which are dominated by dispersion. The devel- opment of new DFT methods designed to overcome this problem, by alterations to the functional or by the inclusion of additive terms, is a current research topic. On the other hand, molecular mechanics is a mathemati- cal approach used for the computation of structures, energy, dipole moment, and other physical properties. It is widely used in calculating many diverse biological and chemical systems such as proteins, large crystal struc- tures, and relatively large solvated systems. However, this method is limited by the determination of parameters such as the large number of unique torsion angles present in structurally diverse molecules (118). Ab initiois an important tool to investigate functional mechanisms of biological macromolecules based on their

3D and electronic structures. The system size, which

ab initiocalculations can handle, is relatively small despite the large sizes of biomacromolecules surrounding solvent water molecules. Accordingly, isolated models of areas of proteins such as active sites have been studied inab initio calculations. However, the disregarded proteins and solvent surrounding the catalytic centers have also been shown to contribute to the regulation of electronic struc- tures and geometries of the regions of interest. To overcome these discrepancies, quantum mechanics/ molecular mechanics (QM/MM) calculations are utilized, in which the system is divided into QM and MM regions

652Chem Biol Drug Des2013; 82: 643-668Karaman

where QM regions correspond to active sites to be investi- gated and are described quantum mechanically. MM regions correspond to the remainder of the system and are described molecular mechanically. The pioneer work of the QM/MM method was accomplished by Warshel and Levitt (119,127), and since then, there has been much progress on the development of a QM/MM algorithm and applications to biological systems (120,121). Similarly to that utilized for drug discovery, modern com- putational methods based on QM and MM methods could be exploited for the design of innovative prodrugs for drugs containing different functional groups such as hydroxyl, phenol, or amine. For example, mechanisms of intramolecular processes for a respected number of enzyme models that have been previously studied by oth- ers to understand enzyme catalysis have been recently computed by us and used for the design of some novel prodrug linkers (122-140). Using DFT, molecular mechan- ics, andab initiomethods, numerous enzyme models were explored for assigning the factors governed the reac- tion rate in such models. Among the enzyme models that have been studied are (i) proton transfer between two oxy- gens and proton transfer between nitrogen and oxygen in Kirby"s acetals (141-148), (ii) intramolecular acid-catalyzed hydrolysis in N-alkylmaleamic acid derivatives (141-148), (iii) proton transfer between two oxygens in rigid systems as investigated by Menger (149-152), (iv) acid-catalyzed lactonization of hydroxy-acids as researched by Cohen (104,153,154) and Menger (149-152), and (v) SN2-based cyclization as studied by Brown (155), Bruice (156,157), and Mandolini (158). Our recent studies on intramoleculari- ty have demonstrated that there is a necessity to further explore the reaction mechanisms for the above-mentioned processes for determining the factors affecting the reaction rate. Unraveling the reaction mechanism would allow for better design of an efficient chemical device to be utilized as a prodrug linker that can be covalently linked to a drug which can chemically, but not enzymatically, be cleaved to release the active drug in a programmable manner. For example, studying the mechanism for a proton transfer in Kirby"s acetals has led to a design and synthesis of novel prodrugs of aza-nucleosides for the treatment for myelo- dysplastic syndromes (159), atovaquone prodrugs for the treatment for malaria (160), less bitter paracetamol pro- drugs to be administered to children and elderly as antipy- retic and pain killer (161), and prodrugs of phenylephrine as decongestant (162). In these examples, the prodrug moiety was linked to the hydroxyl group of the active drug such that the drug-linker moiety (prodrug) has the potential to interconvert when exposed into physiological environ- ments such as stomach, intestine, and/or blood circula- tion, with rates that are solely dependent on the structural features of the pharmacologically inactive promoiety (Kir- by"s enzyme model). Other different linkers such as Kirby"s maleamic acid amide enzyme model was also explored for the design of a number of prodrugs such as tranexamic

acid for bleeding conditions, acyclovir as antiviral drug forthe treatment for herpes simplex (163), atenolol for treatinghypertension with enhanced stability and bioavailability

(164) and statins for lowering cholesterol levels in the blood (165). In addition, prodrugs for masking the bitter taste of antibacterial drugs such as cefuroxime were also designed and synthesized (166-171). The role of the link- ers in the antibacterial prodrugs such as cefuroxime pro- drugs was to block the free amine, which is responsible for the drug bitterness, and to enable the release of the drug in a controlled manner. Menger"s Kemp acid enzyme model was utilized for the design of dopamine prodrugs for the treatment for Parkinson"s disease as well (172). Prodrugs for dimethyl fumarate for the treatment psoriasis was also designed, synthesized and studied (173).

Computationally Designed Prodrugs Based

on

Intramolecular Amide Hydrolysis of

Kirby"s N-Alkylmaleamic Acids

Kirbyet

al. studied the efficiency of intramolecular catalysis of amide hydrolysis by the carboxyl group of a number of substituted N-methylmaleamic acids24-30(Figure 4) and found that the reaction is remarkably sensitive to the pat- tern of substitution on the carbon-carbon double bond. In addition, the study revealed that the hydrolysis rates for the dialkyl-N-methylmaleamic acids range over more than ten powers of ten, and the 'effective concentration" of the carboxyl group of the most reactive amide, dimethyl-N-n- propylmaleamic acid, is>10

10M. This acid amide was

found to be converted into the more stable dimethyl maleic anhydride with a half-life of<1-second at 39°C below pH 3 (142). Furthermore, Kirby"s study demon- strated that the amide bond cleavage is due to intramolec- ular nucleophilic catalysis by the adjacent carboxylic acid group, and the dissociation of the tetrahedral intermediate is the rate-limiting step (142). Later on, Kluger and Chin researched the intramolecular hydrolysis mechanism of a series of N-alkylmaleamic acids derived from aliphatic amines, having a wide range of basicity (161). Their study revealed that the identity of the rate-limiting step is a func- tion of both the basicity of the leaving group and the acid- ity of the solution. Figure 4:Acid-catalyzed hydrolysis in Kirby"s N-methylmaleamic acids24-30.

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To utilize N-alkylmaleamic acids,24-30, as prodrug linkers for tranexamic acid, atenolol, acyclovir, cefuroxime, and other drugs, having poor bioavailability or/and undesirable (bitter) taste, we have unraveled the mechanism for their acid-catalyzed hydrolysis using DFT and molecular mechanics methods. Our DFT calculation results were found to be in accordance with the reports by Kirbyet al. (142) and Kluger and Chin (174).

Tranexamic Acid Prodrugs Based on

Kirby"s

N-Alkylmaleamic Acids

It is not often that a simple old generic product makes medical news. Yet this is just the case for tranexamic acid. This small molecule that is a synthetic lysine amino acid derivative has been originally developed to prevent and reduce excessive hemorrhage in hemophilia patients and reduce the need for replacement therapy during and fol- lowing tooth extraction. Yet the use of tranexamic acid has been expanding beyond the small number of hemo- philia patients. Perhaps the most exciting new develop- ment about tranexamic acid has been the recent publication of the results of CRASH-2, a randomized con- trolled trial undertaken in 274 hospitals in 40 countries with

20211 adult trauma patients. Tranexamic acid was dem-

onstrated to safely reduce the risk of death in bleeding trauma patients. Tranexamic acid might also have a role in bleeding condi- tions apart from traumatic injury. Postpartum hemorrhage is a leading cause of maternal mortality, accounting for about 100 000 maternal deaths every year. Although preli- minary evidence suggests that this drug reduces postpar- tum bleeding, a large trial is being undertaken to assess the effect of tranexamic acid on the risk of death and hys- terectomy in women with postpartum hemorrhage. Fur- thermore, the similarities of tissue injury after trauma and surgery create a novel model for antifibrinolytic therapy with tranexamic acid. Recently, a new oral formulation of tranexamic acid was shown to be safe and effective for treatment for heavy menstrual bleeding b(175-180). One of the main disadvantages of tranexamic acid is its pharma- cokinetic profile. After an intravenous dose of 1 g, the plasma concentration-time curve shows a terminal elimi- nation half-life of about 2 h. The initial volume of distribu- tion is about 9-12 L. More than 95% of the dose is excreted in the urine as the unchanged drug via glomeru- lar filtration. The plasma protein binding of tranexamic acid is about 3% at therapeutic plasma levels and seems to be fully accounted for by its binding to plasminogen. Tranexa- mic acid does not bind to serum albumin. As a result of this pharmacokinetic profile, tranexamic acid in CRASH-2 study needed to be administered using a loading dose of

1 g by intravenous infusion over 10 min followed by 1 g

infused over 8 h. Although an 8-hr IV infusion may be an easy option in a hospital setting, such option may not

be available in under-developed countries or at sites ofaccidents and battlefields. Similarly, the oral administrationof tranexamic acid results in a 45% oral bioavailability. The

total oral dose recommended in women with heavy men- strual bleeding was two 650-mg tablets three times daily for 5 days. Accumulation following multiple dosing was minimalb (175-180). Improvement in tranexamic acid pharmacokinetic proper- ties may reduce the administration frequencyviaa variety of administration routes. This can be achieved by exploit- ing a carrier-linked prodrug strategyb (175-180). Continuing our study on how to utilize enzyme models as potential linkers for drugs containing amine, hydroxyl, or phenol group (122-140), we have investigated the proton transfer reactions in the acid-catalyzed hydrolysis of N-alkyl maleamic acids24-30(122,142) (Kirby"s enzyme model, Figure 4) reported by Kirbyet al.and based on the calculation results of this system, we have proposed four tranexamic acid prodrugs, tranexamic prodrugsProD

1- ProD 4(Figure 5).

As shown in Figure 5, tranexamic acid prodrugs,ProD

1-Prod 4,consist of a carboxylic group (hydrophilic moi-

ety) and a lipophilic moiety (the rest of the prodrug), where the combination of both moieties secures a relatively mod- erate (adequate) HLB. It is worth noting that our proposal is to exploit tranexamic acid prodrugsProD 1-ProD 4for oral useviaenteric coated tablets. At this physiological environment, the tranexamic acid prodrugs will exist as a mixture of the acidic and ionic forms where the equilibrium constant for the exchange between the two forms is dependent on the pK aof a given prodrug.

Mechanistic Investigation

The DFT kinetic and thermodynamic properties for tra- nexamic acid prodrugsProD 1- ProD 4(Figure 5) were calculated by DFT methods. Using the calculated DFT enthalpy and entropy values for the entities involved in the acid-catalyzed hydrolysis of tranexamic acid prodrugs

ProD 1- ProD 4,the barriers (ΔG

‡) for all steps described in Figure 6 were calculated. The calculated values for ΔG f‡, activation energy for the tetrahedral intermediate for- mation, andΔG d‡, activation energy of the tetrahedral intermediate dissociation, demonstrate that while the rate- limiting step for all prodrugs as calculated in the gas phase is the tetrahedral intermediate formation, the scenario is the opposite when the calculations were made in water. The water DFT calculations indicate that the rate-limiting step in the acid-catalyzed hydrolysis of tranexamic acid ProD 4is the tetrahedral intermediate formation, while that forProD 1-ProD 3is the tetrahedral intermediate col- lapse. To evaluate the factors determining the acid-cata- lyzed hydrolysis rate in tranexamic acid prodrug comparison of their calculated DFT properties with previ- ously calculated properties for the acid-catalyzed hydroly-

654Chem Biol Drug Des2013; 82: 643-668Karaman

sis of24-7(Figure 4), atenolol prodrugsProD 1- ProD 2 (Figure 7), acyclovir prodrugsProD 1- ProD 4(Figure 7), and cefuroximeProD 1- ProD 4(Figure 7) were made. The calculation results reveal that the rate-limiting step (higher barrier) in the gas phase for all systems studied is the tetrahedral intermediate formation. On the other hand, the picture is quite different when the calculations were carried out in dielectric constant of 78.39 (water medium).

While for systems24-30and atenolol prodrugsProD

1-ProD 2,the rate-limiting step was the dissociation of

the tetrahedral intermediate in the reactions of cefuroxime prodrugsProD 1- ProD 4and acyclovir prodrugsProD

1- ProD 4,the rate-limiting step was the formation of the

tetrahedral intermediate. For assigning the factor determining the rate-limiting step, MM2 strain energy values for the reactants (GM) and inter-

mediates (INT2) in24-30and tranexamic acid prodrugsProD 1-ProD 4were calculated and correlated with the

calculated DFT activation energy values, (ΔG d‡). Good correlation was obtained between the Es (INT) (strain energy of intermediate) in24-30and tranexamic acid ProD 1-ProD 3and the activation energies for the tetra- hedral intermediate breakdown (ΔG d‡). In addition, strong correlation was found between log k rel(relative rate) and Es (INT) in24-30. The calculations demonstrate that the reaction rate for systems24-30and tranexamic acidProD

1-ProD 3is dependent on the tetrahedral intermediate

breakdown and its value is largely affected by the strain energy of the tetrahedral intermediate formed. Systems with less-strained intermediates such as25and tranexa- mic acidProD 3undergo hydrolysis with higher rates than those having more strained intermediates such as27and tranexamic acidProD 1. This might be attributed to the fact that the transition state structures in these systems resemble that of the corresponding intermediates.

Figure 5:Acid-catalyzed hydrolysis in

tranexamic acid prodrugs,ProD 1-ProD 4.

Figure 6:Mechanistic pathway for the

acid-catalyzed hydrolysis of24-30and tranexamic acidProD1-ProD 4.

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Inter- and Intramolecular Chemical Processes

Calculation of thet1/2Values for the

Cleavage Reactions of Tranexamic Acid

Prodrugs ProD 1-ProD 4

The effective molarity parameter is considered an excel- lent tool to define the efficiency of an intramolecular process. Generally accepted that the measure for intra- molecular efficiency is the effective molarity (EM), which is defined as a ratio of the intramolecular rate (k intra) and its corresponding intermolecular (k inter) where both pro- cesses are driven by identical mechanisms. The major factors affecting the EM are ring size, solvent, and reac- tion type. Values in the order of 10

9-1013M have been

measured for the EM in intramolecular processes occur- ring through nucleophilic addition. Whereas for proton transfer processes, EM values of less than 10 M were reported (181) until recently where values of 10 10 were reported by Kirby on the hydrolysis of some enzyme models (141-149). Using equation 1 obtained from the correlation of log EM calc(calculated effectivemolarity) vs. log EM exp(experimental effective molarity) and thet

1/2value for process 24 (t1/2=1 second) (141),

thet

1/2values for tranexamic acidProD 1-ProD 4

were calculated. The predictedt

1/2at pH 2 forProD

1-ProD 4is 556, 253 h, 70 seconds, and 1.7 h,

respectively. log EM calc¼0:809 log EMexpþ4:75 (1)

In VitroKineticsStudies

The kinetics for the acid-catalyzed hydrolysis was carried out in aqueous buffer in the same manner as that done by Kirby on his enzyme models24-30. This is in order to explore whether the prodrug hydrolyzes in aqueous med- ium and to what extent or not, suggesting the fate of the prodrug in the system. Acid-catalyzed hydrolysis kinetics of the synthesized tranexamic acidProD 1was studied in four different aqueous media: 1 N HCl, buffer pH 2, buffer Figure 7:Chemical structures for atenololProD 1-ProD 2, acyclovirProD 1-ProD 4.

656ChemBiol Drug Des2013; 82: 643-668Karaman

pH 5 and buffer pH 7.4. Under the experimental condi- tions, the target compounds hydrolyzed to release the par- ent drug as evident by HPLC analysis. At constant pH and temperature, the reaction displayed strict first-order kinet- ics as thek obswas fairly constant and a straight plot was obtained on plotting log concentration of residual prodrug verves time. Half-lives (t

1/2) for tranexamic acid prodrug

ProD 1in 1N HCl, pH 2 and pH 5 were calculated from the linear regression equation correlating the log concen- tration of the residual prodrug versus time, and their values were 0.9, 23.9, and 270 h, respectively. The kinetic data in 1N HCl, pH 2 and pH 5 were selected to examine the interconversion of the tranexamic acid prodrug in pH as of stomach, because the mean fasting stomach pH of adult is approximately 1-2 and increases up to 5 following ingestion of food. In addition, buffer pH 5 mimics the beginning small intestine pathway. Finally, pH 7.4 was selected to examine the interconversion of the tested pro- drug in blood circulation system. Acid-catalyzed hydrolysis of the tranexamic acidProD 1was found to be higher in

1N HCl than at both pH 2 and 5. At 1N HCl, the prodrug

was hydrolyzed to release the parent drug in less than

1 h. On the other hand, at pH 7.4, the prodrug was

entirely stable and no release of the parent drug was observed. As the pK aof tranexamic acidProD1is 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 the a