Prodrugs Design Based on Inter-and Intramolecular Chemical Processes

Prodrugs Design Based on Inter- and IntramolecularChemical Processes

Rafik Karaman1,2,*

1Bioorganic Chemistry Department, Faculty of Pharmacy,Al-Quds University, P.O. Box 20002, Jerusalem, Palestine2Department of Science, University of Basilicata, Viadell’Ateneo Lucano 10, 85100, Potenza, Italy*Corresponding author: Rafik Karaman,[email protected]

This review provides the reader a concise overview ofthe majority of prodrug approaches with the emphasison the modern approaches to prodrug design. Thechemical approach catalyzed by metabolic enzymeswhich is considered as widely used among all otherapproaches to minimize the undesirable drug physico-chemical properties is discussed. Part of this reviewwill shed light on the use of molecular orbital methodssuch as DFT, semiempirical and ab initio for the designof novel prodrugs. This novel prodrug approachimplies prodrug design based on enzyme models thatwere utilized for mimicking enzyme catalysis. The com-putational approach exploited for the prodrug designinvolves molecular orbital and molecular mechanics(DFT, ab initio, and MM2) calculations and correlationsbetween experimental and calculated values of intra-molecular processes that were experimentally studiedto assign the factors determining the reaction rates incertain processes for better understanding on howenzymes might exert their extraordinary catalysis.

Key words: Ab initio calculations, design of prodrugs, DFTcalculations, enzyme models, molecular mechanics calcula-tions, prodrugs

Received 4 July 2013, revised 13 August 2013 and acceptedfor publication 16 August 2013

A drug is defined as a substance, which is used in thediagnosis, 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 thestudy of the biochemistry behind a disease for which phar-maceutical intervention is seen.a

Drug discovery is a lengthy interdisciplinary endeavor. It isa consecutive process that starts with target and leaddiscovery, followed by lead optimization and preclinicalin vitro and in vivo studies to evaluate whether a

compound 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 yearswith a cost of more than $1 billion dollars (1,2).

Modifying the absorption, distribution, metabolism, andelimination (ADME) properties of an active drug requires acomplete understanding of the physicochemical and bio-logical behavior of the drug candidate (3–6). This includescomprehensive evaluation of drug-likeness involvingprediction of ADME properties. These predictions can beattempted at several levels: in vitro–in vivo using dataobtained from tissue or recombinant material from humanand preclinical species, and in silico or computational pre-dictions projecting in vitro or in vivo data, involving theevaluation of various ADME properties, using computa-tional approaches such as quantitative structure activityrelationship (QSAR) or molecular modeling (7–11).

Studies have indicated that poor pharmacokinetics and tox-icity are the most important causes of high attrition rates inthe drug development process, and it has been widelyaccepted that these areas should be considered as early aspossible in drug discovery to improve the efficiency andcost-effectiveness of the industry. Resolving the pharmaco-kinetic and toxicological properties of drug candidatesremains a key challenge for drug developers (12).

Thus, the aim is to design drugs that have an efficientpermeability to be absorbed into the blood circulation(absorption), to reach their target efficiently (distribution), tobe 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 optimumpharmacokinetics properties can be achieved by imple-menting one or more of the following strategies:

Improving Absorption

Drug absorption is determined by the drug hydrophilichydrophobic balance (HLB) value, which depends uponpolarity and ionization. Very polar or strongly ionized drugs,having a relatively high HLB values, cannot efficiently crossthe cell membranes of the gastrointestinal (GI) barrier.Hence, they are given by the intravenous (I.V.) route, buttheir disadvantage is being rapidly eliminated. Non-polar

ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12224 1

Chem Biol Drug Des 2013

Review

drugs, on the other hand, having a relatively low HLB val-ues, are poorly soluble in aqueous media and hence arepoorly absorbed through membranes. If they are given byinjection, most probably, they will be retained in fat tissues(13–21).

Generally, the polarity and/or ionization of drug can bealtered by changing its substituents, and these changesare classified under the so-called quantitative structure–activity relationships (QSAR). The following are examplesfor such changes: (1) variation of alkyl or acyl substituentsand polar functional groups to vary polarity, (1) variation ofN-alkyl substituents to vary pKa; acidic drugs with lowpKa 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 pKaof aromatic amine or carboxylic acid can be varied byadding electron donating or electron withdrawing groupsto the ring. The position of the substituent is important tooif the substituent interacts with the ring through resonanceand (3) bioisosteres for polar groups; carboxylic acid is ahighly polar group which can be ionized and hencedecreases the absorption of any drug containing it. Toovercome this problem, blocking the free carboxyl groupby making the corresponding ester prodrug or replacing itwith a bioisostere group, which has similar physiochemicalproperties and has advantage over carboxylic acid inregard to its pKa, such as 5-substituted tetrazoles, isessential; 5-substituted tetrazole ring contains acidic pro-ton such as carboxylic acid and is ionized at pH 7.4. Onthe other hand, most of the alkyl and aryl carboxylicgroups have a pKa in the range of 2–5 (13–28).

Improving Metabolism

There are different strategies that can be utilized toimprove drug metabolism: (i) steric shields: Some func-tional groups are more susceptible to chemical and enzy-matic degradation than others. For example, esters andamides are much more affordable to hydrolysis than otherssuch as carbamates and oximes. Adding steric shields tothese drugs increases their stability. Steric shields weredesigned to hinder the approach of a nucleophile or anucleophilic center on an enzyme to the susceptiblegroup. These usually involve the addition of a bulky alkylgroup such as t-butyl close to the functional group.(ii) Electronic effects of bioisosteres: This approach is usedto protect a labile functional group by electronic stabiliza-tion. For example, replacing the methyl group of an esterwith an amine group gives a urethane functional group,which is more stable than the parent ester. The aminegroup has the same size and valance as the methyl group;however, it has no steric effect, but it has totally differentelectronic properties, because it can donate electrons via

its inductive effect into the carbonyl group resulting inreducing the electrophilicity of the carbonyl carbon andhence stabilizing it from hydrolysis.

Carbachol (1 in Figure 1), a cholinergic agonist, and cefoxi-tin (2 in Figure 1), a cephalosporin, are stabilized in thisway. (iii) Stereoelectronic modification: Steric hindrance andelectronic stabilization have been used together to stabilizelabile groups. For example, procaine, an ester drug, isquickly hydrolyzed, but changing the ester to the less reac-tive amide group reduces hydrolysis such as in the casesof procainamide (3 in Figure 1) and lidocaine (4 in Figure 1).(iv) Metabolic Blockers: Some drugs are metabolized byintroducing polar functional groups at particular positions intheir skeleton. For example, megestrol acetate (5 in Figure1), an oral contraceptive, is oxidized at position 6 to givehydroxyl group at this position; however, replacing thehydrogen at position 6 with a methyl group blocks itsmetabolism, and consequently it results in prolonging itsduration of action. (v) Removal of susceptible metabolicgroups: Certain chemical moieties are particularly suscepti-ble to metabolic enzymes. For example, a methyl group onaromatic rings is often oxidized to carboxylic acid, whichthen results in a rapid elimination of the drug from thebody. Other common metabolic reactions include aliphaticand aromatic C-hydroxylation, O and S-dealkylations, N-and S-oxidations, and deamination. (vi) Group Shifts:Removing or replacing a metabolically vulnerable group isfeasible if the group concerned is not involved in importantbinding interactions within the active site of the receptor orenzyme. If the group is important, then different strategyeither masking the vulnerable group using a prodrug orshifting the vulnerable group within the molecule skeleton isundertaken. Salbutamol was developed in 1969 from itsanalog neurotransmitter, norepinephrine, using this tactic.Norepinephrine is metabolized by methylation of one of itsphenolic groups by catechol O-methyl transferase. Theother phenolic group is important for receptor-binding inter-action. Removing the hydroxyl or replacing it with a methylgroup prevents metabolism but also prevents hydrogenbonding interaction with the binding site. While moving thevulnerable hydroxyl group out from the ring by one carbonunit 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; somering systems are often found to be susceptible to metabo-lism, and so varying the ring can often improve metabolicstability. 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 Figure1) with improved stability.

Making drug less resistance to drug metabolism: drug thatis 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 intoxicity and adverse effects. Therefore, designing drugs withdecreased chemical and metabolic stability can sometimesbe beneficial. Methods for applying such strategy are (i)introducing groups that are susceptible to metabolism is agood way of shorting the lifetime of a drug. For example,methyl group was introduced to some drugs to shorten their

2 Chem Biol Drug Des 2013

Karaman

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

Reducing Toxicity

It is often found that a drug fails clinical trials because ofits toxic adverse effects.

This may be due to toxic metabolites, in which case thedrug should be made more resistant to metabolism. It isknown that functional groups such as aromatic nitro

groups, aromatic amines, bromoarenes, hydrazines,hydroxylamines, or polyhalogenated groups are generallymetabolized to toxic metabolites.

Side-effects might be reduced or eliminated by varyingharmless substituents. For example, addition of fluorinegroup to UK 47265, antifungal agent, gives the less toxicfluconazole (29–34).

Prodrugs Catalyzed by Metabolic Enzymes

The principle of targeting drugs can be traced back toPaul Ehrlich who developed antimicrobial drugs that wereselectively toxic for microbial cells over human cells.Today, targeting tumor cells is considered one of the mostimportant issues that under concern among the healthcommunity. A major goal in cancer chemotherapy is to tar-get drugs efficiently against tumor cells rather than againstnormal cells. One method for achieving this is to designdrugs which make use of specific molecular transport sys-tems. The idea is to attach the active drug to an importantbuilding block molecule that is needed in large amountsby the rapidly divided tumor cells. This could be an amino

Figure 1: Chemical structures for 1–7.

Chem Biol Drug Des 2013 3

Inter- and Intramolecular Chemical Processes

acid or a nucleic acid base such as uracil mustard. In thecases where the drug is intended to target against infec-tion of gastrointestinal tract (GIT), it must be preventedfrom being absorbed into the blood supply. This can easilybe done using a fully ionized drug which is incapable ofcrossing cell membrane barriers. For example, highly ion-ized sulfonamides are used against GIT infections becausethey are incapable of crossing the gut wall. It is oftenpossible to target drugs such that they act peripherallyand not in the central nervous system (CNS). By increasingthe polarity of drugs, they are less likely to cross theblood–brain barrier, and thus, they are less likely to haveCNS adverse effects (35–46).

The most efficient approach for overcoming the negativepharmacokinetics characteristics of a drug is the prodrugapproach. This approach may be utilized in the cases wherethe use of the parent drug faces problems associated withsolubility, absorption and distribution, site specificity, insta-bility, toxicity, poor patient compliance, or formulation prob-lems (47–52). A metabolic enzyme is usually involved inconverting the prodrugs to their active forms. Not allprodrugs are activated by metabolic enzymes. For example,photodynamic therapy involves the use of an external lightsource to activate prodrugs. When designing a prodrug, it isimportant to ensure that the prodrug is effectively convertedto the active drug once it has been absorbed in blood sup-ply. It is also important to ensure that any groups that arecleaved from the prodrug molecule are non-toxic (22).

The prodrug approach is a very versatile strategy to increasethe utility of biologically active compounds, because onecan optimize any of the ADME properties of potential drugcandidates. 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 aftermolecular modification such as an oxidation or reductionreaction. The prodrug candidate can also be prepared as adouble prodrug, where the second linker is attached to thefirst promoiety linked to the parent drug molecule. Theselinkers are usually different and are cleaved by differentmechanisms. In some cases, two biologically active drugscan 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 variousundesirable drug properties and to optimize clinical drugapplication. Recent advances in molecular biology providedirect availability of enzymes and carrier proteins, includingtheir molecular and functional characteristics.

There are two major prodrug design approaches that areconsidered as widely used among all other approaches tominimize or eliminate the undesirable drug physicochemi-cal properties while maintaining the desirable pharmaco-logical activity. The first approach is the targeted drugdesign approach by which prodrugs can be designed totarget specific enzymes or carriers by consideringenzyme–substrate specificity or carrier–substrate specificity

to overcome various undesirable drug properties. This typeof ‘targeted-prodrug’ design requires considerable knowl-edge of particular enzymes or carriers, including theirmolecular and functional characteristics (35–46).

The second approach to be discussed in this review is sub-divided to two major approaches: (i) Chemical approach bywhich the drug is linked to promoiety which upon exposureto physiological environment undergoes enzymatic cata-lyzed degradation to the parent drug and inactive linker. Inthis approach, the interconversion rate is dependent on theenzyme catalysis. This approach involves carrier-linkedprodrugs and contains a group that can be easily removedenzymatically, such as an ester or labile amide, to providethe parent drug. Ideally, the group removed is pharmaco-logically inactive and non-toxic, while the linkage betweenthe drug and promoiety must be labile for in vivo efficientactivation. Carrier-linked prodrugs can be further subdividedinto (i) bipartite, which is composed of one carrier groupattached to the drug, (ii) tripartite, which is a carrier groupthat is attached via linker to drug, and (iii) mutual prodrugsconsisting of two drugs linked together, and (1) bioprecur-sors are chemical entities that are metabolized into newcompounds that may be active or further are metabolized toactive metabolites, such as amine to aldehyde to carboxylicacid (48–51); and (ii) intramolecular chemical approachdesigned based on calculations using molecular orbital(MO) and molecular mechanics (MM) methods and correla-tions between experimental and calculated values. In thisprodrug approach, no enzyme is involved in the intraconver-sion chemical reaction of a prodrug to its parent drug. Theinterconversion of the prodrug is solely controlled by therate-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 bethe rate-limiting step to absorption (48). It has been docu-mented that more than 30% of drug discovery compoundshave poor aqueous solubility (53). Prodrugs are an alterna-tive way to increase the aqueous solubility of the parentdrug molecule by increasing dissolution rate throughattachment to ionizable or polar groups, such as phos-phates, sugar, or amino acids moieties (51,54). Theseprodrugs can be used for increasing oral bioavailability andin parenteral or injectable drug delivery. (ii) Upon increasingpermeability and hence absorption, membrane permeabilityhas a significant effect on drug effectiveness (7). In oraldrug delivery, the most common absorption routes areun-facilitated and largely non-specific, passive transportmechanisms. The lipophilicity of poorly permeable drugscan be enhanced by linking to lipophilic groups. In suchcases, the prodrug strategy can be an extremely valuableoption and crucially needed. Improvements in lipophilicityhave been the most widely researched and successful fieldof prodrug research. It has been achieved by maskingpolar ionized or non-ionized functional groups to increase


Karaman

either oral or topical absorption (22). (iii) Modification of thedistribution profile: Before the drug reaches its physiologicaltarget and exert the desired effect, it has to bypass severalpharmaceutical and pharmacokinetic barriers. Today, oneof the most promising site-selective drug delivery strategiesis the prodrug approach, which utilizes target cell- or tis-sue-specific endogenous enzymes and transporters.

The suitability of a number of functional groups such ascarboxylic, hydroxyl, amine, phosphate, phosphonate, andcarbonyl groups for undergoing different chemicalmodifications facilitates their utilization in prodrug design(1,9) In the past few decades, a variety of prodrugs basedon the chemical approach have been designed, synthe-sized, and tested. Among those are the following.

Ester Prodrugs

carboxylic acid, hydroxyl, phosphate, and thiol groups caneasily undergo hydrolysis via the enzymatic catalysis ofesterases and phosphatases that are present in manyplaces in the body including liver, blood, and other tissues,or via oxidative cleavage catalyzed by cytochrome P450enzymes (CYP) (51,55,56).

Carboxyl esterases, acetylcholinesterases, butyrylcholines-terases, paraoxonases, arylesterases and biphenyl hydro-lase-like protein (BPHL) are examples of enzymes that areresponsible for the hydrolytic bioactivation of ester prodrugs(56). For example, biphenyl hydrolase-like protein (BPHL) isknown to catalyze the hydrolysis of prodrugs such as vala-cyclovir (8 in Figure 2) and valganciclovir (9 in Figure 2), aswell as a number of other amino acid esters of nucleo-side analogs including valyl-AZT, prodrugs of floxuridine(5-fluoro-20-deoxyuridine or FUdR) (10 in Figure 2) andgemcitabine (11 in Figure 2) (57). Ester prodrugs are com-monly used to enhance lipophilicity, thus increasing mem-brane permeation through masking the charge of polarfunctional groups and by handling the alkyl chain lengthand configuration (51). For example, acyclovir aliphatic esterprodrugs were prepared by an esterification of the hydroxylgroup with lipophilic acid anhydride or acyl chloride (58);hence, an enhanced lipophilicity can be achieved. Utilizingthe lipophilic ester approach, some acyclovir prodrugs weresynthesized and have shown an enhanced nasal and skinabsorption (51). It has been shown that an increase in thelength of an alkyl chain results in a relatively ease cleavageof the ester bond. Therefore, it might be concluded thatimproved binding to the hydrophobic pocket of carboxyles-terase can be accomplished by increasing the length of theester alkyl chain, while branching the alkyl chain might resultin reduced hydrolysis due to a steric hindrance (51). Further,eighteen amino acid esters of acyclovir were synthesized aspotential prodrugs intended for oral administration, and theirhydrolytic reaction was shown to be catalyzed by biphenylhydrolase (59). Acyclic nucleosides such as adefovir andtenofovir are monophosphorylated and do not rely on viral

nucleoside kinases for initial activation; hence, they havelow oral bioavailability due to the ionized phosphonategroup (60,61). To overcome this problem, a bis(pivaloyl-oxymethyl) ester prodrug of adefovir (adefovir dipivoxil) andether lipid ester prodrugs of cidofovir were explored forimproving intestinal permeability (51). Other examples forester prodrugs that were designed and synthesized for dif-ferent purposes are thioester of erythromycin, palmitateester of clindamycin, a number of angiotensin-convertingenzyme (ACE) inhibitors which are (55) presently marketedas ester prodrugs, including enalapril (12 in Figure 2),ramipril, benazepril, and fosinopril, and all of them areintended 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 itsparent drug when is given in equimolar doses (62). As men-tioned before, two active drugs can be joined together suchthat each one behaves as a carrier moiety for the other, astrategy known as mutual prodrugs (8). The followings aresome examples of mutual prodrugs, based on ester linkage,that were produced to overcome several shortcomingsassociated with therapeutic drugs used in clinical practice:Benorylate (13 in Figure 2) is a mutual prodrug of aspirinand paracetamol, coupled through an ester linkage, whichis postulated to have reduced gastric irritancy with synergis-tic analgesic effect (63). Moreover, mutual prodrugs of ibu-profen with paracetamol and salicylamide have beenreported to have better lipophilicity and diminished gastrictoxicity 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 GIside-effects is by conjugation of a nitric oxide (NO) releasingmoiety to the parent NSAID drug. It has been reported thatNO 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 activeparent drugs with lower gastro toxicity (66,67). Reduce gas-tro toxicity could be achieved also by linking NSAID drugwith histamine H2 antagonist such as in the case of flurbi-profen-histamine H2 antagonist conjugates (64). The mutualprodrug approach was also applied to other therapeuticgroups. For instance, sultamicillin (14 in Figure 2) in whichthe irreversible b-lactamase inhibitor sulbactam has beenlinked via an ester linkage with ampicillin has shown a syn-ergistic effect (64), and upon oral administration, sultamicil-lin is completely hydrolyzed to equimolar proportions ofsulbactam and ampicillin, thereby acting as an efficientmutual prodrug (68).

Amides Prodrugs

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



limited extent due to high in vivo stability. However, pro-drugs using facile intramolecular cyclization reactions havebeen exploited to overcome this obstacle (70).

Similar to mutual ester prodrugs, there are some mutualprodrugs where the two active drugs are linked together byan amide linkage, such as atorvastatin and amlodipinewhich upon in vivo amide hydrolysis provide the corre-sponding active parent drugs. Amide prodrugs can be con-verted back to the parent drugs either by nonspecificamidases or by specific enzymatic activation such asrenal c-glutamyl transpeptidase. Dopamine double prodrugc-glutamyl-l-dopa (gludopa) (15 in Figure 2) undergoesspecific activation by renal c-glutamyl transpeptidase whereit achieves relatively fivefold increase in dopamine levelcompared with L-dopa prodrug. However, as gludopa haslow oral bioavailability, dopamine [N-(N-acetyl-L-methionyl)-O,O-bis(ethoxycarbonyl)dopamine), a pseudopeptide prodrug ofdopamine, was developed and has shown improved oralabsorption; 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 oralroute (71,72).

A respected number of amine conjugates with amino acidsthrough amide linkage have been considered for providingactive drugs with remarkable enhancement in solubilitysuch as dapsone (16 in Figure 2) (73).

Other examples of amide based prodrugs are allopurinolN-acyl derivatives which were found to be more lipophilicthan allopurinol itself (74).

Carbonates and Carbamates Prodrugs

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



Karaman

carboxymethylphenyl ester of amphetamine is an exampleof carbamates prodrug that can be hydrolyzed by esteraseto yield amphetamine (76). Carbamates prodrugs areregarded as double prodrugs (pro-prodrug) because theyare enzymatically activated at first which is followed byspontaneous cleavage of the resulting carbamic acid (51).An example for such prodrugs is fluorenylmethoxycarbon-yl]-3 derivatives of insulin and exenatide (77) that undergoslow interconversion via carbamate bond breakdown, thusproviding glucose controlling agents in an adequate ratewhich consequently results in lowering the risk ofhypoglycemia (74).

Another example of carbamates prodrug is the oneobtained by linking phosphorylated steroid, an estradiol, tonormustard, an alkylating agent, through a carbamate link-age, which yields estramustine prodrug. The latter is usedin the treatment for prostate cancer. The steroid portionhas an antiandrogenic action and acts to concentrate theprodrug in the prostate gland where prodrug hydrolysistakes place and normustard action can then be exerted(64). Carbamates prodrugs can also be used to increasethe solubility of active drugs such as cephalosporins (78).In addition, carbamates prodrugs have been exploited intargeted therapy such as ADEPT. In this case, the carba-mate group is susceptible to the action of tyrosinaseenzyme present in melanomas. This approach is usuallyutilized in cancer targeted therapy (79). The list of carba-mates prodrugs is long; among other examples is the non-sedating antihistamine loratadine (17 in Figure 2), an eth-ylcarbamate, that undergoes in vivo interconversion to itsactive form, desloratidine, through the action of CYP450enzymes (80), and capecitabine, an anticancer agent, thatundergoes 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 thecorresponding active drugs, and they are convertedback to their parent drugs by microsomal cytochromeP450 enzymes (CYP450) (51). Dopaminergic prodrug6-(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 SchiffBases (Imines)

N-Mannich base formation is another approach, which canbe utilized to enhance drug’s solubility. N-Mannich basesare prepared by Mannich reaction that involves reacting ofNH-acidic compound, an aldehyde and an amine in etha-nol (74). Rolitetracycline (18 in Figure 3) is the Mannichderivative of tetracycline, and it is the only one available forintravenous administration (85). N-Mannich bases of dipy-

rone, metamizole (19 in Figure 3), the methane sulfonicacid of the analgesic 4-(methylamino) antipyrine is watersoluble and suitable for parenteral route, and when givenorally, it is hydrolyzed in the stomach to give the parentactive drug (86). Despite the success of N-Mannich baseprodrugs to improve bioavailability of active drugs, there stillsome stability formulation problems arise from poor in vitro

stability of some of the prodrugs (85). In addition, the in

vivo formation 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 foruse in oral administration (74). Nonetheless, an ampicillinprodrug based on enamines was prepared for rectal use,and it exhibits an increased absorption compared with itsactive parent drug (89). Enaminones are enamines of b-dic-arbonyl compounds that undergo ketoenolimine-enaminetautomeric equilibrium, which may offer stability to thesecompounds (86). Enaminones are generally more lipophilicthan their parent drugs; hence, they have an improved oralabsorption. Typically, enaminones have a relatively highchemical stability; therefore, their use as potential prodrugsis being somewhat limited. It is expected that enaminonesderived from ketoesters and lactone may be subjected toenzymatic degradation; hence, a better conversion rate tothe active drug can be obtained (90).

Phosphate and Phosphonate Prodrugs

Phosphorylation offers increased aqueous solubility to theparent drugs. A traditional example of phosphate prodrugsis prednisolone sodium phosphate, a water-soluble pro-drug of prednisolone, its water solubility exceeds that ofits active form, prednisolone, by 30 times (8), it is oftenused as an immunosuppressant, and it is formulated as aliquid dosage form (8). Another common phosphate pro-drug is fosamprenavir (20 in Figure 3). Similar to predniso-lone, the phosphate promoiety in fosamprenavir is linkedto a free hydroxyl group, and the prodrug is 10-fold morewater soluble than amprenavir. An enhanced patient com-pliance is achieved when using this antiviral prodrug;instead of administering the drug 8 times daily, dosageregimen is reduced into two times per day (91). In the gutand via the action of alkaline phosphatases, phosphateprodrugs are cleaved back to their corresponding activedrugs and then absorbed into the systemic circulation (8).Another application of this approach is fosphenytoin (21 inFigure 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 asa means of prodrug activation through the action of azo-reductases; this approach is applied specially in targeted



drug strategy (85). Sulfasalazine (22 in Figure 3), used inthe treatment for ulcerative colitis (93), is a prodrug of5-aminosalicylic acid and sulfapyridine. Upon reaching thecolon, sulfasalazine undergoes azo bond cleavage torelease the active parent drug (64). Osalazine (23 inFigure 3), a dimer of 5-aminosalicylic acid, balsalazide,and ipsalazide in which 5-aminosalicylic acid moiety isconjugated to 4-aminobenzoyl-b-alanine and 4-amin-obenzoylglycine, respectively (94), are other examples ofprodrugs that are activated by azo-reductases. A prodrugby which 5-aminosalicylic acid is linked to L-aspartic acidis 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 thisapproach 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 thespacer, 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 ofprodrugs. In this prodrug system, PEG is conjugated tothe phenol group of the open lactone via a spacer. Con-trolling the rate of the free drug release can be accom-plished by manipulation of the substituents on thearomatic ring (97).

The prodrug chemical approach involving enzyme catalysisis perhaps the most unpredicted approach, because thereare many intrinsic and extrinsic factors that can affect thebioconversion mechanisms. For example, the activity ofmany prodrug-activating enzymes may be changed due togenetic polymorphisms, age-related physiological changes,or drug interactions, leading to adverse pharmacokinetic,pharmacodynamics, and clinical effects. In addition, thereare wide interspecies variations in both the expression andfunction of most of the enzyme systems activating pro-drugs which could lead to serious challenges in the pre-clinical optimization phase (3–6).



Karaman

Prodrugs Based on IntramolecularProcesses (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 tounderstand how enzymes work. The tool used in thedesign is a computational approach consisting of calcula-tions using a variety of different molecular orbital andmolecular mechanics methods and correlations betweenexperimental and calculated rate values (activation ener-gies) for some intramolecular processes that were utilizedto understand the mechanism by which enzymes mightexert their high catalysis. In this approach, no enzyme isneeded 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 factorsaffecting 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 prodruginterconversion by enzymes approach. As mentioned inthe introduction, the bioconversion of prodrugs has manydisadvantages related to many intrinsic and extrinsic fac-tors that can affect the process. For instance, the activityof many prodrug-activating enzymes may be varied due togenetic polymorphisms, age-related physiological changes,or drug interactions, leading to variation in clinical effects.In addition, there are wide interspecies variations in boththe expression and function of the major enzyme systemsactivating prodrugs, and these can pose some obstaclesin 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 pastfive decades, have had a tremendous contribution to bet-ter understanding the mode and scope by which enzymescatalyze biochemical transformations (98–101).

Nowadays, the scientific community has reached a con-sensus that the catalysis by enzymes is based on thecombined effects of the catalysis by functional groups andthe ability to reroute intermolecular reactions throughalternative pathways by which substrates can bind to pre-organized active sites.

The rates for most of enzymatic reactions exceed 1010–1018 -fold the nonenzymatic bimolecular counterparts. Forexample, reactions catalyzed by the enzyme cyclophilinare accelerated by 105, and those by orotidine monophos-phate decarboxylase are enhanced by 1017 (102).

In the last 50 years, as mentioned earlier, scholarly studieshave been carried out by Bruice (103), Cohen (104),Menger (105), Kirby (106), and others (107) to designchemical models that have the capability to reach ratescomparable to that with enzyme-catalyzed reactions. Fre-quently cited examples of such models are those basedon rate acceleration driven by covalently enforced proxim-ity. The most quoted example is Bruice et al.’s. intramo-lecular ring-closing reaction of dicarboxylic semi-esters toanhydrides (103). Studying this model, Bruice et al. hasshown that a relative rate of anhydride formation can reach5 9 107 upon the intramolecular ring-closing reaction of adicarboxylic semi-ester when compared to a similar coun-terpart’s intermolecular reaction.

Other examples of rate acceleration based on proximityorientation include (1) systems that obey the principles ofKoshland’s ‘orbital steering’ theory (107) that signifies theimportance of the ground state angle of attack value ofthe hydroxyl in hydroxycarboxylic acids on the intramolec-ular lactonization reaction rate; (1) the ‘spatiotemporalhypothesis’ advocated by Menger, which implies that atype of a reaction, in proton transfer processes, whetherintermolecular or intramolecular, is significantly determinedby the distance between the two reactive centers involvedin the hydroxycarboxylic acids lactonization reaction (105);(2) the stereopopulation control proposed by Cohen toexplain the relatively high enhancement rates in the acid-catalyzed lactonization reactions of hydroxyhydrocinnamicacids containing two methyl groups on the b position oftheir carboxylic groups (104) and Kirby’s proton transfermodels on the acid-catalyzed hydrolysis of acetals andmaleamic acid amides which demonstrate the importanceof 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,Borchardt et al. reported the use of the 3-(2′-acetoxy-4′,6′-dimethyl dimethyl)-phenyl-3, 3-dimethylpropionamidederivative (pro–prodrug) that is capable of releasing thebiologically active amine (drug) upon acetate hydrolysis byenzyme triggering. Another successful example of thepharmaceutical applications for a stereopopulation controlmodel is the prodrug Taxol which enhances the drugwater solubility and hence affords it to be administered tothe human body via intravenous (I.V.) injection. Taxol is thebrand name for paclitaxel, a natural diterpene, approved inthe USA for use as anticancer agent (108).

Calculation Methods Used in the ProdrugsDesign

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



chemistry uses principles of computer science to assist insolving chemical problems. It uses the theoretical chemis-try results, incorporated into efficient computer programs,to calculate the structures and physical and chemicalproperties of molecules.

Reaction rates and equilibrium energy-based calculationsfor biological systems that have pharmaceutical and bio-medicinal interests are a very important challenge to thehealth community. Nowadays, quantum mechanics (QM),such as ab initio, semi-empirical, and density functionaltheory (DFT), and molecular mechanics (MM) areincreasingly being used and broadly accepted as reliabletools for providing structure-energy calculations for anaccurate prediction of potential drugs and prodrugsalike (109).

These methods cover both static and dynamic situations.In all cases, the computer time and other resources (suchas memory and disk space) increase rapidly with the sizeof the system being studied. Ab initio methods typicallyare feasible only for small systems. Ab initio methods arebased entirely on theory from first principles. The term ab

initio was first used in quantum chemistry by Robert Parrand coworkers, including David Craig in a semiempiricalstudy on the excited states of benzene. The ab initio

molecular orbital methods (quantum mechanics) such asHF, G1, G2, G2MP2, MP2, and MP3 are based on rigor-ous use of the Schrodinger equation with a number ofapproximations. Ab initio electronic structure methodshave the advantage that they can be made to converge tothe exact solution, when all approximations are sufficientlysmall 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 sometimesthe smallest calculation gives the best result for someproperties. The disadvantage of ab initio methods is theircomputational cost. They often take enormous amounts ofcomputer time, memory, and disk space (110–112).

Other less accurate methods are called empirical or semi-empirical because they employ experimental results, oftenfrom acceptable models of atoms or related molecules, toapproximate some elements of the underlying theory.Among these methods, the semi-empirical quantumchemistry methods are based on the Hartree–Fock formal-ism, but make many approximations and obtain someparameters from empirical data. They are very important incomputational chemistry for treating large molecules wherethe full Hartree–Fock method without the approximationsis too expensive. Semi-empirical calculations are muchfaster than their ab initio counterparts. Their results, how-ever, can be very wrong if the molecule being computed isnot similar enough to the molecules in the database usedto 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 modelingmethod in physics and chemistry to investigate the elec-tronic structure (principally the ground state) of many-bodysystems, in particular atoms, molecules, and the con-densed phases, is the density functional theory (DFT). Withthis theory, the properties of many-electron systems canbe determined using functionals, that is, functions ofanother function, which in this case is the spatially depen-dent electron density. Hence, the name density functionaltheory comes from the use of functionals of the electrondensity. DFT is among the most popular and versatilemethods available in condensed-matter physics, computa-tional physics, and computational chemistry. The DFTmethod is used to calculate structures and energies formedium-sized systems (30–60 atoms) of biological andpharmaceutical interest and is not restricted to the secondrow of the periodic table (117).

Despite recent improvements, there are still difficulties inusing 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 stronglycorrelated systems. Its incomplete treatment of dispersioncan adversely affect the accuracy of DFT in the treatmentof systems which are dominated by dispersion. The devel-opment of new DFT methods designed to overcome thisproblem, by alterations to the functional or by the inclusionof 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 iswidely used in calculating many diverse biological andchemical systems such as proteins, large crystal struc-tures, and relatively large solvated systems. However, thismethod is limited by the determination of parameters suchas the large number of unique torsion angles present instructurally diverse molecules (118).

Ab initio is an important tool to investigate functionalmechanisms of biological macromolecules based on their3D and electronic structures. The system size, whichab initio calculations can handle, is relatively small despitethe large sizes of biomacromolecules surrounding solventwater molecules. Accordingly, isolated models of areas ofproteins such as active sites have been studied in ab initio

calculations. However, the disregarded proteins andsolvent surrounding the catalytic centers have also beenshown 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, inwhich the system is divided into QM and MM regions


Karaman

where QM regions correspond to active sites to be investi-gated and are described quantum mechanically. MMregions correspond to the remainder of the system andare described molecular mechanically. The pioneer workof the QM/MM method was accomplished by Warshel andLevitt (119,127), and since then, there has been muchprogress on the development of a QM/MM algorithm andapplications to biological systems (120,121).

Similarly to that utilized for drug discovery, modern com-putational methods based on QM and MM methods couldbe exploited for the design of innovative prodrugs fordrugs containing different functional groups such ashydroxyl, phenol, or amine. For example, mechanisms ofintramolecular processes for a respected number ofenzyme models that have been previously studied by oth-ers to understand enzyme catalysis have been recentlycomputed by us and used for the design of some novelprodrug linkers (122–140). Using DFT, molecular mechan-ics, and ab initio methods, numerous enzyme modelswere explored for assigning the factors governed the reac-tion rate in such models. Among the enzyme models thathave been studied are (i) proton transfer between two oxy-gens and proton transfer between nitrogen and oxygen inKirby’s acetals (141–148), (ii) intramolecular acid-catalyzedhydrolysis in N-alkylmaleamic acid derivatives (141–148),(iii) proton transfer between two oxygens in rigid systemsas investigated by Menger (149–152), (iv) acid-catalyzedlactonization of hydroxy-acids as researched by Cohen(104,153,154) and Menger (149–152), and (v) SN2-basedcyclization 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 furtherexplore the reaction mechanisms for the above-mentionedprocesses for determining the factors affecting the reactionrate. Unraveling the reaction mechanism would allow forbetter design of an efficient chemical device to be utilizedas a prodrug linker that can be covalently linked to a drugwhich can chemically, but not enzymatically, be cleaved torelease the active drug in a programmable manner. Forexample, studying the mechanism for a proton transfer inKirby’s acetals has led to a design and synthesis of novelprodrugs of aza-nucleosides for the treatment for myelo-dysplastic syndromes (159), atovaquone prodrugs for thetreatment 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 phenylephrineas decongestant (162). In these examples, the prodrugmoiety was linked to the hydroxyl group of the active drugsuch that the drug-linker moiety (prodrug) has the potentialto interconvert when exposed into physiological environ-ments such as stomach, intestine, and/or blood circula-tion, with rates that are solely dependent on the structuralfeatures of the pharmacologically inactive promoiety (Kir-by’s enzyme model). Other different linkers such as Kirby’smaleamic acid amide enzyme model was also explored forthe design of a number of prodrugs such as tranexamicacid for bleeding conditions, acyclovir as antiviral drug for

the treatment for herpes simplex (163), atenolol for treatinghypertension with enhanced stability and bioavailability(164) and statins for lowering cholesterol levels in theblood (165). In addition, prodrugs for masking the bittertaste of antibacterial drugs such as cefuroxime were alsodesigned 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 responsiblefor the drug bitterness, and to enable the release of thedrug in a controlled manner. Menger’s Kemp acid enzymemodel was utilized for the design of dopamine prodrugsfor the treatment for Parkinson’s disease as well (172).Prodrugs for dimethyl fumarate for the treatment psoriasiswas also designed, synthesized and studied (173).

Computationally Designed Prodrugs Basedon Intramolecular Amide Hydrolysis ofKirby’s N-Alkylmaleamic Acids

Kirby et al. studied the efficiency of intramolecular catalysisof amide hydrolysis by the carboxyl group of a number ofsubstituted N-methylmaleamic acids 24–30 (Figure 4) andfound that the reaction is remarkably sensitive to the pat-tern of substitution on the carbon–carbon double bond. Inaddition, the study revealed that the hydrolysis rates forthe dialkyl-N-methylmaleamic acids range over more thanten powers of ten, and the ‘effective concentration’ of thecarboxyl group of the most reactive amide, dimethyl-N-n-propylmaleamic acid, is > 1010 M. This acid amide wasfound to be converted into the more stable dimethylmaleic anhydride with a half-life of <1-second at 39 °Cbelow 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 acidgroup, and the dissociation of the tetrahedral intermediateis the rate-limiting step (142). Later on, Kluger and Chinresearched the intramolecular hydrolysis mechanism of aseries of N-alkylmaleamic acids derived from aliphaticamines, having a wide range of basicity (161). Their studyrevealed 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-methylmaleamicacids 24–30.



To utilize N-alkylmaleamic acids, 24–30, as prodrug linkersfor tranexamic acid, atenolol, acyclovir, cefuroxime, andother drugs, having poor bioavailability or/and undesirable(bitter) taste, we have unraveled the mechanism for theiracid-catalyzed hydrolysis using DFT and molecularmechanics methods. Our DFT calculation results werefound to be in accordance with the reports by Kirby et al.

(142) and Kluger and Chin (174).

Tranexamic Acid Prodrugs Based onKirby’s N-Alkylmaleamic Acids

It is not often that a simple old generic product makesmedical news. Yet this is just the case for tranexamic acid.This small molecule that is a synthetic lysine amino acidderivative has been originally developed to prevent andreduce excessive hemorrhage in hemophilia patients andreduce the need for replacement therapy during and fol-lowing tooth extraction. Yet the use of tranexamic acidhas been expanding beyond the small number of hemo-philia patients. Perhaps the most exciting new develop-ment about tranexamic acid has been the recentpublication of the results of CRASH-2, a randomized con-trolled trial undertaken in 274 hospitals in 40 countries with20211 adult trauma patients. Tranexamic acid was dem-onstrated to safely reduce the risk of death in bleedingtrauma patients.

Tranexamic acid might also have a role in bleeding condi-tions apart from traumatic injury. Postpartum hemorrhageis a leading cause of maternal mortality, accounting forabout 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 assessthe 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 andsurgery create a novel model for antifibrinolytic therapywith tranexamic acid. Recently, a new oral formulation oftranexamic acid was shown to be safe and effective fortreatment for heavy menstrual bleedingb (175–180). One ofthe main disadvantages of tranexamic acid is its pharma-cokinetic profile. After an intravenous dose of 1 g, theplasma 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 isexcreted in the urine as the unchanged drug via glomeru-lar filtration. The plasma protein binding of tranexamic acidis about 3% at therapeutic plasma levels and seems to befully accounted for by its binding to plasminogen. Tranexa-mic acid does not bind to serum albumin. As a result ofthis pharmacokinetic profile, tranexamic acid in CRASH-2study needed to be administered using a loading dose of1 g by intravenous infusion over 10 min followed by 1 ginfused over 8 h. Although an 8-hr IV infusion may be aneasy option in a hospital setting, such option may notbe available in under-developed countries or at sites of

accidents and battlefields. Similarly, the oral administrationof tranexamic acid results in a 45% oral bioavailability. Thetotal oral dose recommended in women with heavy men-strual bleeding was two 650-mg tablets three times dailyfor 5 days. Accumulation following multiple dosing wasminimalb (175–180).

Improvement in tranexamic acid pharmacokinetic proper-ties may reduce the administration frequency via a varietyof 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 aspotential linkers for drugs containing amine, hydroxyl, orphenol group (122–140), we have investigated the protontransfer reactions in the acid-catalyzed hydrolysis ofN-alkyl maleamic acids 24–30 (122,142) (Kirby’s enzymemodel, Figure 4) reported by Kirby et al. and based on thecalculation results of this system, we have proposed fourtranexamic acid prodrugs, tranexamic prodrugs ProD

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), wherethe combination of both moieties secures a relatively mod-erate (adequate) HLB. It is worth noting that our proposalis to exploit tranexamic acid prodrugs ProD 1-ProD 4 fororal use via enteric coated tablets. At this physiologicalenvironment, the tranexamic acid prodrugs will exist as amixture of the acidic and ionic forms where the equilibriumconstant for the exchange between the two forms isdependent on the pKa of a given prodrug.

Mechanistic Investigation

The DFT kinetic and thermodynamic properties for tra-nexamic acid prodrugs ProD 1- ProD 4 (Figure 5) werecalculated by DFT methods. Using the calculated DFTenthalpy and entropy values for the entities involved in theacid-catalyzed hydrolysis of tranexamic acid prodrugsProD 1- ProD 4, the barriers (ΔG‡) for all steps describedin Figure 6 were calculated. The calculated values forΔGf

‡, activation energy for the tetrahedral intermediate for-mation, and ΔGd

‡, activation energy of the tetrahedralintermediate dissociation, demonstrate that while the rate-limiting step for all prodrugs as calculated in the gas phaseis the tetrahedral intermediate formation, the scenario isthe opposite when the calculations were made in water.The water DFT calculations indicate that the rate-limitingstep in the acid-catalyzed hydrolysis of tranexamic acidProD 4 is the tetrahedral intermediate formation, while thatfor ProD 1- ProD 3 is the tetrahedral intermediate col-lapse. To evaluate the factors determining the acid-cata-lyzed hydrolysis rate in tranexamic acid prodrugcomparison of their calculated DFT properties with previ-ously calculated properties for the acid-catalyzed hydroly-


Karaman

sis of 24–7 (Figure 4), atenolol prodrugs ProD 1- ProD 2

(Figure 7), acyclovir prodrugs ProD 1- ProD 4 (Figure 7),and cefuroxime ProD 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 isthe tetrahedral intermediate formation. On the other hand,the picture is quite different when the calculations werecarried out in dielectric constant of 78.39 (water medium).While for systems 24–30 and atenolol prodrugs ProD

1-ProD 2, the rate-limiting step was the dissociation ofthe tetrahedral intermediate in the reactions of cefuroximeprodrugs ProD 1- ProD 4 and acyclovir prodrugs ProD

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

For assigning the factor determining the rate-limiting step,MM2 strain energy values for the reactants (GM) and inter-mediates (INT2) in 24–30 and tranexamic acid prodrugs

ProD 1-ProD 4 were calculated and correlated with thecalculated DFT activation energy values, (ΔGd

‡). Goodcorrelation was obtained between the Es (INT) (strainenergy of intermediate) in 24–30 and tranexamic acidProD 1- ProD 3 and the activation energies for the tetra-hedral intermediate breakdown (ΔGd

‡). In addition, strongcorrelation was found between log krel (relative rate) andEs (INT) in 24–30. The calculations demonstrate that thereaction rate for systems 24–30 and tranexamic acid ProD

1-ProD 3 is dependent on the tetrahedral intermediatebreakdown and its value is largely affected by the strainenergy of the tetrahedral intermediate formed. Systemswith less-strained intermediates such as 25 and tranexa-mic acid ProD 3 undergo hydrolysis with higher rates thanthose having more strained intermediates such as 27 andtranexamic acid ProD 1. This might be attributed to thefact that the transition state structures in these systemsresemble that of the corresponding intermediates.

Figure 5: Acid-catalyzed hydrolysis intranexamic acid prodrugs, ProD 1- ProD4.

Figure 6: Mechanistic pathway for theacid-catalyzed hydrolysis of 24–30 andtranexamic acid ProD1-ProD 4.



Calculation of the t1/2 Values for theCleavage Reactions of Tranexamic AcidProdrugs ProD 1-ProD 4

The effective molarity parameter is considered an excel-lent tool to define the efficiency of an intramolecularprocess. Generally accepted that the measure for intra-molecular efficiency is the effective molarity (EM), whichis defined as a ratio of the intramolecular rate (kintra) andits corresponding intermolecular (kinter) where both pro-cesses are driven by identical mechanisms. The majorfactors affecting the EM are ring size, solvent, and reac-tion type. Values in the order of 109-1013 M have beenmeasured for the EM in intramolecular processes occur-ring through nucleophilic addition. Whereas for protontransfer processes, EM values of less than 10 Mwere reported (181) until recently where values of 1010

were reported by Kirby on the hydrolysis of someenzyme models (141–149). Using equation 1 obtainedfrom the correlation of log EMcalc (calculated effective

molarity) vs. log EMexp (experimental effective molarity)and the t1/2 value for process 24 (t1/2 = 1 second) (141),the t1/2 values for tranexamic acid ProD 1 – ProD 4

were calculated. The predicted t1/2 at pH 2 for ProD

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

log EMcalc ¼ 0:809 log EMexp þ 4:75 (1)

In Vitro Kinetics Studies

The kinetics for the acid-catalyzed hydrolysis was carriedout in aqueous buffer in the same manner as that done byKirby on his enzyme models 24–30. This is in order toexplore whether the prodrug hydrolyzes in aqueous med-ium and to what extent or not, suggesting the fate of theprodrug in the system. Acid-catalyzed hydrolysis kineticsof the synthesized tranexamic acid ProD 1 was studied infour different aqueous media: 1 N HCl, buffer pH 2, buffer

Figure 7: Chemical structures for atenolol ProD 1- ProD 2, acyclovir ProD 1- ProD 4.


Karaman

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 andtemperature, the reaction displayed strict first-order kinet-ics as the kobs was fairly constant and a straight plot wasobtained on plotting log concentration of residual prodrugverves time. Half-lives (t1/2) for tranexamic acid prodrugProD 1 in 1N HCl, pH 2 and pH 5 were calculated fromthe linear regression equation correlating the log concen-tration of the residual prodrug versus time, and their valueswere 0.9, 23.9, and 270 h, respectively. The kinetic datain 1N HCl, pH 2 and pH 5 were selected to examine theinterconversion of the tranexamic acid prodrug in pH as ofstomach, because the mean fasting stomach pH of adultis approximately 1–2 and increases up to 5 followingingestion of food. In addition, buffer pH 5 mimics thebeginning small intestine pathway. Finally, pH 7.4 wasselected to examine the interconversion of the tested pro-drug in blood circulation system. Acid-catalyzed hydrolysisof the tranexamic acid ProD 1 was found to be higher in1N HCl than at both pH 2 and 5. At 1N HCl, the prodrugwas hydrolyzed to release the parent drug in less than1 h. On the other hand, at pH 7.4, the prodrug wasentirely stable and no release of the parent drug wasobserved. As the pKa of tranexamic acid ProD1 is in therange of 3–4, it is expected that at pH 5, the anionic formof the prodrug will be dominant and the percentage of thefree acidic form that undergoes the acid-catalyzed hydroly-sis will be relatively low. At 1N HCl and pH 2, most of theprodrug will exist as the free acid form, and at pH 7.4,most of the prodrug will be in the anionic form, thus thedifference in rates at the different pH buffers.

The t1/2 experimental value at pH 5 was 270 h, and at pH7.4, no interconversion was observed. The lack of thereaction at the latter pH might be due to the fact that atthis pH, tranexamic acid ProD 1 exists solely in the ion-ized form (pKa about 4). As mentioned before, the freeacid form is a mandatory requirement for the reaction toproceed.

On the other hand, tranexamic acid ProD 4 has a higherpKa than tranexamic acid ProD 1 (about 6 versus 4). There-fore, it is expected that the interconversion rate of tranexa-mic acid ProD 4 to its parent drug, tranexamic acid, at allpHs studied will be higher (log EM for tranexamic acid ProD

4 is 14.33 versus 9.53 for tranexamic acid ProD 1).

Acyclovir Prodrugs Based on Kirby’sN-Alkylmaleamic Acid Enzyme Model

Acyclovir is a synthetic acyclic purine nucleoside analogthat is the first agent to be registered for the treatment forand prevention of viral infections caused by herpessimplex (HSV), varicella zoster (chicken pox), and herpeszoster (shingles) (170,182). Acyclovir water solubility is verypoor and has an oral bioavailability of < 20%; hence,

administration is necessary when high doses are needed(183). Orally acyclovir is mostly used as 200-mg tablets,five times daily. In addition, 6 months to a year administra-tion of acyclovir is required in immune-competent patientwith relapsing herpes simplex infection (183).

The oral administration therapy that currently available isassociated with a number of drawbacks such as highlyvariable absorption and low bioavailability (10–20%). Themain problem with the therapeutic effectiveness of acyclo-vir is its absorption that is highly variable and dose depen-dent, thus reducing the bioavailability to 10–20%. Incommercially available dosage forms of acyclovir, theamount of drug absorbed is very low due to short resi-dence time of the dosage forms at the absorption site. Inhumans, acyclovir showed poor and variable oral bioavail-ability (10–20%), probably due to the relatively low lipophi-licity of the drug. Thus, the rate-limiting factor in acyclovirabsorption is its membrane penetration (184).

Several approaches have been investigated to improve theoral bioavailability of acyclovir: (i) Luengo and coworkershave used different preparations of acyclovir with b-cyclo-dextrin to increase its solubility and hence its bioavailability;however, no significant effect of b-cyclodextrin on the oraldrug bioavailability was observed (185). (ii) Encapsulationof acyclovir in lipophilic vesicular structure to enhance theoral absorption and prolong the existence of the drug inthe systemic circulation (186). (iii) Yadav and coworkershave used acyclovir-loaded mucoadhesive microspheresfor increasing the retention time and hence the bioavail-ability of acyclovir (187), and (iv) the search for an effectiveprodrug that would provide acyclovir with higher bioavail-ability led to the synthesis of a number of aliphatic andamino acid esters of acyclovir (188,189).

Improvement in acyclovir pharmacokinetic properties mayincrease the absorption of acyclovir via a variety of dosingroutes. This can be achieved by utilizing a carrier-linkedprodrug strategy which could be implemented by cova-lently linking acyclovir to a linker to provide a drug-hostsystem which upon exposure to physiological environment,such as stomach or intestine, can penetrate the mem-brane tissues and release the active drug, acyclovir, in aprogrammable manner.

To expand our approach for utilizing intramolecularity todesign potential linkers for amine drugs, we have studiedthe mechanism and driving forces determining the rate ofthe acid-catalyzed hydrolysis in some of Kirby’s acidamides (prodrugs linkers) (122,142). This work was carriedout with the hope that such linkers might have a potentialto be good carriers to the antiviral agent, acyclovir.

Based on the DFT calculation results on the acid-catalyzedhydrolysis of maleamic acid amides 24–30 (Figure 4), fouracyclovir prodrugs were proposed (Figure 7). The acyclovirprodrugs, ProD 1 – ProD 4 are composed of the amide



acid linker having a carboxylic acid group (hydrophilic moi-ety) and the rest of the prodrug molecule (a lipophilic moi-ety); the combination of both groups secures a prodrugmoiety, having a potential to be with a high permeability(adequate HLB). The plan was to prepare acyclovir ProD

1- ProD 4 as sodium or potassium carboxylate salts dueto their stability in neutral aqueous medium. It is worth not-ing that N-alkylmaleamic acids such as 24–30 undergohydrolysis in acidic aqueous medium, whereas they arerelatively stable at pH 7.4.

Based on a linear correlation between the calculated andexperimental effective molarities (EM), the study on thesystems reported herein could provide a good basis fordesigning prodrug systems that are less hydrophilic thantheir parent drugs and can be used, in different dosageforms, to release the parent drug in a controlled manner.For example, based on the calculated log EM values, thepredicted t1/2 (a time needed for 50% of the reactant tobe hydrolyzed to products) for acyclovir prodrugs, ProD

1–4, were 29.2 h, 6097 days, 4.6 min, and 8.34 h,respectively. Hence, the rate by which acyclovir prodrugreleases acyclovir can be determined according to thestructural features of the linker (Kirby’s acid amide moiety).

Computationally Designed Prodrugs Basedon Bruice’s Enzyme Model

In this section, we summarize the results of our study ondesign and synthesis of novel prodrugs (170) via linkingthe active drug with a di-carboxylic semi-ester linker(Bruice’s enzyme model) to produce a system that havingbetter physicochemical properties (adequate HLB value)than its active parent drug and is able to release the latterin a chemically driven controlled manner. Bruice et al.

studied the hydrolysis of di-carboxylic semi-esters 31–35shown in Figure 8 and found that the relative rate (krel) for5 > 4 > 3 > 2 > 1. They attributed the acceleration in rateto proximity orientation. Using the observation that alkylsubstitution on succinic acid influences rotamer distribu-tions, the ratio between the reactive gauche and the unre-active anti-conformations, they proposed that gem-dialkylsubstitution increased the probability of the resultant rot-

amer adopting the more reactive conformation. Therefore,for cyclization to occur, the two reacting centers must bein the gauche conformation. In the unsubstituted reactant,the reactive centers are almost completely in the anticon-formation to minimize steric interactions (156,157).

For exploiting systems 31–35 (Figure 8) as prodrug linkersfor drugs containing hydroxyl or phenol group, we haverecently unraveled the mechanism for their cyclizationusing DFT and molecular mechanics calculation methods(130). In accordance with the results by Bruice and Pandit(156,157), we have found that the cyclization reaction pro-ceeds by one mechanism, by which the rate-limiting stepis the tetrahedral intermediate collapse and not its forma-tion. However, contrarily to the conclusion by Bruice’set al., we have found that the acceleration in rate is due tosteric effects rather than to proximity orientation stemmingfrom the ‘rotamer effect’ (190).

Atovaquone (ATQ) Prodrugs Based onBruice’s Enzyme Model

Malaria is a global public health problem, affecting300 million clinical cases annually, and causes about2 million deaths per year (191–193). This protozoan dis-ease is caused by 5 parasites species of the genus Plas-

modium that affect humans (P. falciparum, P. vivax,

P. ovale, P. malariae, and P knowlesi) (194). The only oneamong these parasites that can cause life-threateningcomplications is P. falciparum (192), which is dominatedin Africa and to which most drug-resistant cases areattributed. Malaria can exist in a mild form that mostcommonly associated with flulike symptoms: fever, vomit-ing, and general malaise. While in the severe form causedby P. falciparum, nervous, respiratory, and renal compli-cations frequently coexist due to serious organ failurec

(195).

Several medications, alone or in combination such aschloroquine, antifolates, artemisinins and others, showeffectiveness and were considered as being the cornerstone in malaria treatment. However, drug or multidrugresistance to these agents has been escalated and consti-

Figure 8: Chemical structures for di-carboxylic semi-esters 31-35.


Karaman

tutes a major challenge in malaria treatment (192). Accord-ingly, the need for new anti-malarial drugs is now widelyrecognized, particularly those that are structurally differentfrom existing antimalarial drugs and possess a novelmechanism of action (195).

Atovaquone (ATQ) (for the structure see Figure 9), ahydroxynaphthoquinone, is relatively new treatment option,active against Plasmodium spp (196). It has a novel mech-anism of action, acts by inhibition of the electron transportsystem at the level of cytochrome bc1 complex (197). Inmalaria parasites, the mitochondria act as a sink for theelectrons generated from dihydrofolate dehydrogenase, anessential enzyme for pyrimidine biosynthesis; inhibition ofelectron transport by ATQ leads to dihydrofolate dehydro-genase inhibition resulting in reduced pyrimidine biosynthe-sis and thus parasite replication inhibition (198).

It is well established that ATQ has an excellent safety pro-file and long half-life; besides, ATQ can be administeredvia oral route. However, ATQ has poor oral bioavailability(less than 10% under fasted condition) and variable oralabsorption (199) due to its poor solubility that results fromits lipophilic structure. Consequently, low and variableplasma and intracellular levels of the drug which is animportant determinant of therapeutic outcome areexpected (200). Moreover, ATQ is an expensive medica-tion (201). Therefore, to achieve therapeutic success andto meet the medical needs in malaria treatment, ATQ solu-bility improvement strategies should be addressed. Theprodrug approach has the potential to be the mostsuccessful among other approaches to overcome thisshortcoming.

The study herein was conducted to design ATQ prodrugsthrough linking ATQ to a di-carboxylic semi-ester linker(Bruice’s enzyme model) to produce a system that is more

hydrophilic than its parent drug and is able to release ATQin a chemically driven controlled manner. This will result inintroducing novel ATQ prodrugs with improved bioavailabil-ity and better clinical profile.

Based on our calculation results that enabled us to unravelthe cyclization reaction mechanism and to assign the fac-tors determining the reaction rate, we have designed fiveATQ prodrugs with a potential to have better water solubil-ity than ATQ and to release the active parent drug in asustained controlled manner (Figure 9). The relative rate,log krel (effective molarity), for processes ATQ ProD

1- ProD 5 The experimental relative rates for the intramo-lecular cyclization of 31–35 (Figure 8) were obtained fromthe division of the intramolecular rate and the correspond-ing intermolecular reaction (156,157). For obtaining the rel-ative rates (effective molarity, EM) for processes ATQProD 1– ProD 5, we assume that their corresponding in-termolecular process is similar to that for systems 1–5.

As an excellent correlation was obtained between the acti-vation free energy values (ΔG‡) for 31–35 and ATQ ProD

1– ProD 5 and the difference in the strain energy valuesof the reactants and intermediates, DEs (INT-GM), the calcu-lated values of DEs(INT-GM) for ATQ ProD 1–ProD 5 wereused to calculate their corresponding relative rates (log krel)which were 6.96, 6.47, 3.78, 6.50, and -12.82, respec-tively. These values demonstrate that ATQ ProD 1 andATQ ProD 4 are the most efficient processes among allsystems studied, and the least efficient are ATQ ProD 3

and ATQ ProD 5. Using the experimental t1/2 (the timeneeded for the conversion of 50% of the reactants toproducts) value for the cyclization reaction of di-carboxylicsemi-ester 31 and the calculated log krel values for pro-drugs ATQ ProD 1-ProD 5, we have calculated the t1/2values for the conversion of ATQ ProD 1- ProD 5 to theirparent drug. The calculated t1/2 values were found to be

Figure 9: Chemical structures foratovaquone prodrugs ATQ ProD 1- ProD5.



ATQ ProD 3, 22.44 h, ATQ ProD1, ATQ ProD2, and ATQProD 4, few seconds and ATQ ProD 5 few years. There-fore, the interconversion rates of atovaquone prodrugs toatovaquone can be programmed according to the natureof the prodrug linker.

Paracetamol Prodrugs Based on Bruice’sEnzyme Model

The palatability of active drugs possesses significantobstacle in developing a patient convenient dosage form.Organoleptic properties, such as taste, are an importantfactor when selecting a drug from the generic productsavailable in the market that have the same active ingredi-ent. It is a key issue for doctors and pharmacists adminis-tering the drugs and particularly for the pediatric andgeriatric populations (202).

Organic and inorganic molecules dissolve in saliva andbind to taste receptors on the tongue giving a bitter,sweet, salty, sour, or umami sensation. Bitter taste issensed by the receptors on the posterior part of thetongue. The sensation is a result of signal transductionfrom taste receptors located in areas known as tastebuds. The taste buds contain very sensitive nerve end-ings, which are responsible for the production and trans-mission of electrical impulses via cranial nerves VII, IX,and X to certain areas in the brain that are devoted tothe perception of taste (203). Bitter taste receptors arebelieved to have evolved for organism protection againstthe ingestion of poisonous food products. Molecules withbitter taste (204–208) are very diverse in their chemicalstructure and physicochemical properties (209,210). In

humans, bitter taste perception is mediated by 25 G-protein-coupled receptors of the hTAS2R gene family(211).

Drugs such as macrolide antibiotics, non-steroidal anti-inflammatory, and penicillin derivatives have a pronouncedbitter taste (212). Masking the taste of water-soluble bitterdrugs, especially those given in high doses, is difficult toachieve using sweeteners alone. As a consequence, sev-eral approaches have been studied and have resulted inthe development of more efficient techniques for maskingthe bitter taste of molecules. All of the developedtechniques are based on the physical modification of theformulation containing the bitter tastant.

Although these approaches have helped to improve thetaste of some drugs formulations, the problem of the bittertaste of drugs in pediatric and geriatric formulations stillcreates a serious challenge to the health community.Thus, different strategies should be developed to over-come this serious problem.

Bitter tastant molecules interact with taste receptors onthe tongue to give bitter sensation. Altering the ability ofthe drug to interact with its bitter taste receptors couldreduce or eliminate its bitterness. This could be achievedby an appropriate modification of the structure and thesize of a bitter compound.

Paracetamol is an odorless, bitter crystalline compoundwidely used as pain killer and antipyretic. Paracetamol wasfound in the urine of patients who had taken phenacetin,and later on it was demonstrated that paracetamol was aurinary metabolite of acetanilide (Figure 10) (161).

Figure 10: Chemical structures ofparacetamol, phenacetin and acetanilide.

Figure 11: Acid-catalyzed hydrolysis ofparacetamol ProD 1- ProD 2 toparacetamol and inactive linker.


Karaman

Phenacetin, on the other hand, lacks or has a very slightbitter taste (161). Careful inspection of the structures ofparacetamol and phenacetin indicates that the only differ-ence in the structural features in both is the nature of thegroup on the para position of the benzene ring. While inthe case of paracetamol, the group is hydroxy, in phenac-etin it is ethoxy. Another related example is acetanilide thathas a chemical structure similar to that of paracetamoland phenacetin, but it lacks the group at the para positionof the benzene ring. Acetanilide has a burning taste andlacks the bitter taste characteristic for paracetamol (161).The combined facts described above suggest that thepresence of hydroxy group on the para position is themajor contributor for the bitter taste of paracetamol.Hence, it is expected that blocking the hydroxy group inparacetamol with a suitable linker could inhibit the interac-tion of paracetamol with its bitter taste receptor/s and,hence masking its bitterness.

It seems reasonable to assume that the phenolic hydroxylgroup in paracetamol is crucial for obtaining the bittertaste characteristic for paracetamol. This might be due tothe ability of paracetamol to be engaged in a hydrogenbonding net with the active site of its bitter taste receptorvia its phenolic hydroxyl group.

In a similar manner to the design of ATQ prodrugs, twoparacetamol prodrugs were designed by linking the activedrug paracetamol to Bruice’s enzyme model (linker), whichupon exposure to physiological environment releases theactive parent drug (Figure 11).

It is worth noting that linking the paracetamol with suchlinkers via its phenolic hydroxyl group will hinder its bittertaste. Paracetamol ProD 1 and ProD 2 were synthe-sized, and their kinetics at pH 2 was studied (stomachphysiological environment). The kinetics results revealedthat while paracetamol ProD 1 has a t1/2 value of 1 h inpH 2, paracetamol ProD 2 showed very fast kinetics andit underwent complete interconversion to paracetamolwithin few minutes. The difference in the hydrolysis ratesfor both prodrugs is attributed to strained effects imposedin the case of paracetamol ProD 2, which upon cleavagegives maleic anhydride while in the case of paracetamolProD 1, the byproduct is the less-strained succinicanhydride.

Studies have revealed that poor pharmacokinetics proper-ties and toxicity-related issues are the crucial causes ofhigh attrition rates in the drug development process.Resolving the pharmacokinetic and toxicological propertiesof drug candidates remains a key challenge for drug devel-opers. The most efficient approach for overcoming thesenegative drug characteristics is the prodrug approach. Therationale behind the use of prodrugs is to optimize theADME properties and to increase the selectivity of drugsfor their intended target.

Prodrug design based on a computational approach con-sisting of calculations using molecular orbital (MO) andmolecular mechanics (MM) methods and correlationsbetween experimental and calculated values of intramolec-ular processes has the potential to be a very effective toolto be utilized for obtaining effective prodrugs that are capa-ble of releasing the parent drug in a programmable fashion.In this prodrug approach, no enzyme is needed for thecatalysis of the intraconversion of a prodrug to its parentdrug. The interconversion of the prodrug is solely depen-dent on the rate-limiting step for the intramolecular reaction.

The future of prodrug design is forthcoming yet extremelychallenging. Progresses must be made in better under-standing the chemistry of many organic mechanisms thatcan be effectively exploited to push forward the develop-ment and advances of even more types of prodrugs. Theunderstanding of the organic reactions mechanisms ofintramolecular processes will be the next major milestone inthis field. It is envisioned that the future of prodrug designholds the ability to produce safe and efficacious delivery ofa wide range of active small molecule and biotherapeutics.

Acknowledgments

The author would like to acknowledge funding by theGerman Research Foundation (DFG, ME 1024/8-1) andExo Research Organization, Potenza, Italy.

References

1. DiMasi J.A., Hansen R.W., Grabowski H.G. (2003)The price of innovation: new estimates of drug devel-opment costs. J Health Econ;22:151–185.

2. DiMasi J.A. (2002) The value of improving the produc-tivity of the drug development process: faster timesand better decisions. Pharmacoeconomics;20:1–10.

3. Gonzalez F.J., Tukey R.H. (2006) Drug metabolism. In:Brunton L.L., Lazo J.S., Parker K.L., editors. Good-man and Gilman’s The Pharmacological Basis of Ther-apeutics. New York: The McGraw-Hill Companies,Inc.; p. 71–91.

4. Testa B., Kr€amer S.D. (2007) The biochemistry of drugmetabolism–an introduction: part 2. Redox reactionsand their enzymes. Chem Biodivers;4:257–405.

5. Gangwar S., Pauletti G.M., Wang B., Siahaan T.J.,Stella V.J., Borchardt R.T. (1997) Prodrug strategiesto enhance the intestinal absorption of peptides.DDT;2:148–155.

6. Wang W., Jiang J., Ballard C.E., Wang B. (1999) Pro-drug approaches to the improved delivery of peptidedrugs. Curr Pharm Des;5:265–287.

7. Chan O.H., Stewart B.H. (1996) Physicochemical anddrug-delivery considerations for oral drug bioavailabil-ity. Drug Discov Today;1:461–473.



8. Huttunen K.M., Raunio H., Rautio J. (2011) Prodrugsfrom serendipity to rational design. PharmacolRev;63:750–771.

9. Stella V.J., Nti-Addae K.W. (2007) Prodrug strategiesto overcome poor water solubility. Adv Drug DelivRev;59:677–694.

10. Dahan A., Khamis M., Agbaria R., Karaman R. (2012)Targeted prodrugs in oral delivery: the modern molec-ular biopharmaceutical approach. Expert Opinion onDrug Delivery;9:1001–1013.

11. Karaman R., Fattash B., Qtait A. (2013) The future ofprodrugs – design by quantum mechanics methods.Expert Opinion on Drug Delivery;10:713–729.

12. Ohlstein E.H., Ruffolo R.R. Jr, Elliott J.D. (2000) Drugdiscovery in the next millennium. Annu Rev PharmacolToxicol;40:177–191.

13. Stella V.J. (2010) Prodrugs: some thoughts and cur-rent issues. J Pharm Sci;99(12):4755–4765.

14. Muller C.E. (2009) Prodrug approaches for enhancingthe bioavailability of drugs with low solubility. ChemBiodivers;6:2071–2083.

15. Tunek A., Levin E., Svensson L.A. (1988) Hydrolysisof 3H-bambuterol, a carbamate prodrug of terbutaline,in blood from humans and laboratory animals in vitro.Biochem Pharmacol;37:3867–3876.

16. Browne T.R., Kugler A.R., Eldon M.A. (1996) Pharma-cology and pharmacokinetics of fosphenytoin. Neurol-ogy;46:S3–S7.

17. Wolff M.E. (editor) (1995) Medicinal Chemistry andDrug Discovery, 5th edn. New York: John Wiley &Sons.

18. Williams D.A., Foye W.O., Lemke T.L. editors. (2002)Foye’s Principles of Medicinal Chemistry. Baltimore,MD: Wolters Kluwer Health; p. 26–53.

19. Kenny B.A., Bushfield M., Parry-Smith D.J., FogartyS., Trehene M. (1998) The application of highthroughput screening to novel lead discovery, prog.Drug Res;41:246–269.

20. Blundell T. (1996) Structure-based drug design.Nature;384:23–26.

21. Williams M. (1993) Strategies for drug discovery. NIDARes Monogr;132:1–22.

22. Beaumont K., Webster R., Gardner I., Dack K. (2003)Design of ester prodrugs to enhance oral absorptionof poorly permeable compounds: challenges to thediscovery scientist. Curr Drug Metab;4:461–485.

23. Bellott R., Le Morvan V., Charasson V., Laurand A.,Colotte M., Zanger U.M., Robert J. (2008) Functionalstudy of the 830C >G polymorphism of the humancarboxylesterase 2 gene. Cancer Chemother Pharma-col;61:481–488.

24. Reddy K.R., Matelich M.C., Ugarkar B.G., G�omez-Galeno J.E., DaRe J., Ollis K., Erion M.D. (2008) Pra-defovir: a prodrug that targets adefovir to the liver forthe treatment of hepatitis B. J Med Chem;51:666–676.

25. Kumpulainen H., M€ah€onen N., Laitinen M.L., Ja-urakkaj€arvi M., Raunio H., Juvonen R.O., Veps€al€ainen

J., J€arvinen T., Rautio J. (2006) Evaluation of hydrox-yimine as cytochrome P450 selective prodrug struc-ture. J Med Chem;49:1207–1211.

26. Saunders M.P., Patterson A.V., Chinje E.C., HarrisA.L., Stratford I.J. (2000) NADPH: cytochrome c(P450) reductase activates tirapazamine (SR4233) torestore hypoxicandoxic cytotoxicity in anaerobic resis-tant derivative of the A549 lung cancer cell line. Br JCancer;82:651–656.

27. Dobesh P.P. (2009) Pharmacokinetics and pharmaco-dynamics of prasugrel, a thienopyridine P2Y12 inhibi-tor. Pharmacotherapy;29:1089–1102.

28. Pereillo J.M., Maftouh M., Andrieu A., Uzabiaga M.F.,Fedeli O., Savi P., Picard C. (2002) Structure and ste-reochemistry of the active metabolite of clopidogrel.Drug Metab Dispos;30:1288–1295.

29. Svensson L.A., Tunek A. (1988) The design and bio-activation of presystemically stable prodrugs. DrugMetab Rev;19:165–194.

30. Stella V.J. (2007) A case for prodrugs. In: Stella V.J.,Borchardt R.T., Hageman M.J., Oliyai R., Magg H., Til-ley J.W., editors. Prodrugs: Challenges and Rewards.Part 1. New York, NY: AAPS Press/Springer; p. 3–33.

31. Albert A. (1958) Chemical aspects of selective toxicity.Nature;182:421–422.

32. Harper N.J. (1959) Drug latentiation. J Med PharmChem;1:467–500.

33. Harper N.J. (1962) Drug latentiation. Prog DrugRes;4:221–294.

34. Sinkula A.A., Yalkowsky S.H. (1975) Rationale fordesign of biologically reversible drug derivatives:prodrugs. J Pharm Sci;64:181–210.

35. Stella V.J. (1975) Pro-drugs: an overview and definition.In: Higuchi T., Stella V., editors. Prodrugs as Novel DrugDelivery Systems. ACS Symposium Series. Washington,DC: American Chemical Society; p. 1–115.

36. Amidon G.L., Leesman G.D., Elliott R.L. (1980)Improving intestinal absorption of water-insolublecompounds: a membrane metabolism strategy.J Pharm Sci;69:1363–1368.

37. Fleisher D., Stewart B.H., Amidon G.L. (1985) Designof prodrugs for improved gastrointestinal absorptionby intestinal enzyme targeting. Methods Enzy-mol;112:360–381.

38. Bai J.P., Amidon G.L. (1992) Structural specificity ofmucosal-cell transport and metabolism of peptidedrugs: implication for oral peptide drug delivery.Pharm Res;9:969–978.

39. Stella V.J., Himmelstein K.J. (1980) Prodrugs and site-specific drug delivery. J Med Chem;23:1275–1282.

40. Stella V.J., Himmelstein K.J. (1982) Critique of pro-drugs and site specific delivery. In: Bundgaard H.,editor. Optimization of Drug Delivery. Alfred BenzonSymposium Copenhagen: Munksgaard; p. 134–155.

41. Friend D.R., Chang G.W. (1984) A colon-specificdrug-delivery system based on drug glycosides andthe glycosidases of colonic bacteria. J MedChem;27:261–266.


Karaman

42. Philpott G.W., Shearer W.T., Bower R.J., Parker C.W.(1973) Selective cytotoxicity of hapten-substitutedcells with an antibody-enzyme conjugate. J Immu-nol;111:921–929.

43. Deonarain M.P., Spooner R.A., Epenetos A.A. (1996)Genetic delivery of enzymes for cancer therapy. GeneTher;2:235–244.

44. Singhal S., Kaiser L.R. (1998) Cancer chemotherapyusing suicide genes. Surg Oncol Clin NorthAm;7:505–536.

45. Aghi M., Hochberg F., Breakefield X.O. (2000) Pro-drug activation enzymes in cancer gene therapy. JGene Med;2:148–164.

46. Greco O., Dachs G.U. (2001) Gene directed enzyme/prodrug therapy of cancer: historical appraisal andfuture prospectives. J Cell Physiol;187:22–36.

47. Hu L. (2004) Prodrugs: effective solutions for solubility,permeability and targeting challenges. Drugs;7:736–742.

48. Stella V.J., Borchardt R.T., Hageman M.J., Oliyai R.,Maag H., Tilley J.W. (2007) Prodrugs: Challenges andRewards Part 1 and 2. New York: Springer Science +Business Media.

49. Stella V.J., Charman W.N., Naringrekar V.H. (1985)Prodrugs. Do they have advantages in clinical prac-tice? Drugs;29:455–473.

50. Banerjee P.K., Amidon G.L. (1985) Design of pro-drugs based on enzymes-substrate specificity. In:Bundgaard H., editor. Design of Prodrugs. New York:Elsevier; p. 93–133.

51. Li F., Maag H., Alfredson T. (2008) Prodrugs of nucle-oside analogues for improved oral absorption and tis-sue targeting. J Pharm Sci;97:1109–1134.

52. Roche E.B. (1977) Design of Biopharmaceutical Prop-erties through Prodrugs and Analogs. Washington,DC: American Pharmaceutical Association.

53. Di L., Kerns E.H. (2007) Solubility issues in earlydiscovery and HTS. In: Augustijins P., Brewster M.,editors. Solvent Systems and Their Selection in Phar-maceutics and Biopharmaceutics. New York: SpringerScience + Business Media; p. 111–136.

54. Fleisher D., Bong R., Stewart B.H. (1996) Improvedoral drug delivery: solubility limitations overcome bythe use of prodrugs. Adv Drug Deliv Rev;19:115–130.

55. Tammara V.K., Narurkar M.M., Crider A.M., KhanM.A. (1994) Morpholinoalkyl ester prodrugs of diclofe-nac: synthesis, in vitro and in vivo evaluation. J PharmSci;83:644–648.

56. del Amo E.M., Urtti A., Yliperttula M. (2008) Pharma-cokinetic role of L-type amino acid transporters LAT1and LAT2. Eur J Pharm Sci;35:161–174.

57. Kim I., Song X., Vig B.S., Mittal S., Shin H.C., LorenziP.J., Amidon G.L. (2004) A novel nucleoside prodrug-activating enzyme: substrate specificity of biphenylhydrolase-like protein. Mol Pharm;1:117–127.

58. Bando H., Takagi T., Yamashita F., Takakura Y.,Hashida M. (1996) Theoretical design of prodrug-enhancer combination based on a skin diffusion

model: prediction of permeation of acyclovir prodrugstreated with l-Geranylazacycloheptan-2-one. PharmRes;13:427–432.

59. Kim I., Chu X.Y., Kim S., Provoda C.J., Lee K.D.,Amidon G.L. (2003) Identification of a human valacy-clovirase: biphenyl hydrolase-like protein as valacyclo-vir hydrolase. J Biol Chem;278:25348–25356.

60. Bronson J.J., HO H.T., Boeck H.D., Woods K.,Ghazzouli I., Martin J.C., Hitchcock M.J. (1990)Biochemical pharmacology of acyclic nucleotide ana-logues. Ann N Y Acad Sci;616:398–407.

61. Cundy K.C., Barditch-Crovo P., Walker R.E., CollierA.C., Ebeling D., Toole J., Jaffe H.S. (1995) Clinicalpharmacokinetics of adefovir in human immunodefi-ciency virus type 1-infected patients. AntimicrobAgents Chemother;39:2401–2405.

62. Cioli V., Putzolu S., Rossi V., Corradino C. (1980)A toxicological and pharmacological study of ibupro-fen guaiacol ester (AF 2259) in the rat. Toxicol ApplPharmacol;54:332–339.

63. Croft D.N., Cuddigan J.H., Sweetland C. (1972)Gastric bleeding and benorylate, a new aspirin. BrMed J;3:545–547.

64. Bhosle D., Bharambe S., Gairola N., DhaneshwarS.S. (2006) Mutual prodrug concept: fundamentalsand applications. Indian J Pharm Sci;68:286–294.

65. Muscara M.N., McKnight W., Soldato P.D., WallaceJ.L. (1998) Effect of a nitric oxide-releasing naproxenderivative on hypertension and gastric damageinduced by chronic nitric oxide inhibition in the rat.Life Sci;62:PL235–PL240.

66. Burgaud J.L., Riffaud J.P., Del Soldato P. (2002)Nitric-oxide releasing molecules: a new class of drugswith several major indications. Curr Pharm Des;8:201–213.

67. Shanbhag V.R., Crider A.M., Gokhale R., Harpalani A.,Dick R.M. (1992) Ester and amide prodrugs of ibupro-fen and naproxen: synthesis, anti-inflammatory activity,and gastrointestinal toxicity. J Pharm Sci;81:149–154.

68. English A.R., Girard D., Haskell S.L. (1984) Pharma-cokinetics of sultamicillin in mice, rats, and dogs.Antimicrob Agents Chemother;25:599–602.

69. Kalgutkar A.S., Marnett A.B., Crews B.C., RemmelR.P., Marnett L.J. (2000) Ester and amide derivativesof the nonsteroidal antiinflammatory drug, indometha-cin, as selective cyclooxygenase-2 inhibitors. J MedChem;43:2860–2870.

70. Shan D., Nicolaou M.G., Borchardt R.T., Wang B.(1997) Prodrug strategies based on intramolecularcyclization reactions. J Pharm Sci;86:765–767.

71. Lee M.R. (1990) Five years’ experience with gamma-L-glutamyl-L-dopa: a relatively renally specificdopaminergic prodrug in man. J Auton Pharmacol;10(Suppl 1):s103–s108.

72. Casagrande C., Merlo L., Ferrini R., Miragoli G.,Semeraro C. (1989) Cardiovascular and renal actionof dopaminergic prodrugs. J Cardiovasc Pharma-col;14(Suppl 8):S40–S59.



73. Pochopin N.L., Charman W.N., Stella V.J. (1994)Pharmacokinetics of dapsone and amino acid pro-drugs of dapsone. Drug Metab Dispos;22:770–775.

74. Simplicio A.L., Clancy J.M., Gilmer J.F. (2008) Pro-drugs for amines. Molecules;13:519–547.

75. Bundgaard H. (1985) Design of Prodrug. p. 3–79.76. Safadi M., Oliyai R., Stella V.J. (1993) Phosphoryl-

oxymethyl carbamates and carbonates–novelwater-soluble prodrugs for amines and hindered alco-hols. Pharm Res;10:1350–1355.

77. Shechter Y., Tsubery H., Fridkin M. (2003) [2-Sulfo-9-fluorenylmethoxycarbonyl]3-exendin-4-a long-actingglucose-lowering prodrug. Biochem Biophys ResCommun;305:386–391.

78. Hecker S.J., Calkins T., Price M.E., Huie K., Chen S.,Glinka T.W., Dudley M.N. (2003) Prodrugs of cephalo-sporin RWJ-333441 (MC-04,546) with improvedaqueous solubility. Antimicrob Agents Chemo-ther;47:2043–2046.

79. Jordan A.M., Khan T.H., Malkin H., Osborn H.M.I.(2002) Synthesis and analysis of urea and carbamateprodrugs as candidates for melanocyte-directedenzyme prodrug therapy (MDEPT). Bioorg MedChem;10:2625–2633.

80. Yumibe N., Hule K., Chen K.-J., Snow M., ClementR.P., Cayen M.N. (1996) Identification of human livercytochrome P450 enzymes that metabolize the non-sedating antihistamine loratadine. Formation of des-carboethoxyloratadine by CYP3A4 and CYP2D6.Biochem Pharmacol;51:165–172.

81. Shimma N., Umeda I., Arasaki M., Murasaki C., Ma-subuchi K., Kohchi Y., Miwa M., Ura M., Sawada N.,Tahara H., Kuruma I., Horii I., Ishitsuka H. (2000) Thedesign and synthesis of a new tumor-selective fluoro-pyrimidine carbamate, capecitabine. Bioorg MedChem;8:1697–1706.

82. Ajani J. (2006) Review of capecitabine as oral treat-ment of gastric, gastroesophageal, and esophagealcancers. Cancer;107:221–231.

83. Alexander J., Carqill R., Michelson S.R., Schwam H.(1988) (Acyloxy)alkyl carbamates as novel biorevers-ible prodrugs for amines: increased permeationthrough biological membranes. J Med Chem;31:318–322.

84. Venhuis B.J., Dijkstra D., Wustrow D., Meltzer L.T.,Wise L.D., Johnson S.J., Wikstr€om H.V. (2003) Orallyactive oxime derivatives of the dopaminergic prodrug6-(N,N-di-n-propylamino)-3,4,5,6,7,8-hexahydro-2H-naphthalen-1-one. Synthesis and pharmacologicalactivity. J Med Chem;46:4136–4140.

85. Madsen U., Krogsgaard-Larsen P., Liljefors T. (2002)Textbook of Drug Design and Discovery. Washington,DC: Taylor & Francis; p. 410–458.

86. Testa B., Mayer J.M. (2003) Hydrolysis in Drug andProdrug metabolism, Chemistry, biochemistry andenzymology. 690–695.

87. Bundgaard H., Johansen M. (1980) Prodrugs as drugdelivery systems IV: N-Mannich bases as potential

novel prodrugs for amides, ureides, amines, and otherNH-acidic compounds. J Pharm Sci;69:44–46.

88. Caldwell H.C., Adams H.J., Jones R.G., Mann W.A.,Dittert L.W., Chong C.W., Swintosky J.V. (1971)Enamine prodrugs. J Pharm Sci;60:1810–1812.

89. Murakami T., Tamauchi H., Yamazaki M., Kubo K.,Kamada A., Yata N. (1981) Biopharmaceutical studyon the oral and rectal administrations of enamine pro-drugs of amino acid-like beta-lactam antibiotics inrabbits. Chem Pharm Bull;29:1986–1997.

90. Naringrekar V.H., Stella V.J. (1990) Mechanism ofhydrolysis and structure-stability relationship of enami-nones as potential prodrugs of model primary amines.J Pharm Sci;79:138–146.

91. Chapman T.M., Plosker G.L., Perry C.M. (2004)Fosamprenavir: a review of its use in the managementof antiretroviral therapy-naive patients with HIV infec-tion. Drugs;64:2101–2124.

92. Boucher B.A. (1996) Fosphenytoin: a novel phenytoinprodrug. Pharmacotherapy;16:777–791.

93. Azadkhan A.K., Truelove S.C., Aronson J.K. (1982)The disposition and metabolism of sulphasalazine(salicylazosulphapyridine) in man. Br J Clin Pharma-col;13:523–528.

94. Sandborn W.J. (2002) Rational selection of oral5-aminosalicylate formulations and prodrugs for thetreatment of ulcerative colitis. Am J Gastroenter-ol;97:2939–2941.

95. Jung Y., Lee J., Kim Y. (2001) Colon-specific prodrugof 5-aminosalicylic acid: synthesis and in vitro/in vivo

properties of acidic amino acid derivatives of 5-amino-salicylic acid. J Pharm Sci;90:1767–1775.

96. Greenwald R.B., Pendri A., Conover C.D., Zhao H.,Choe Y.H., Martinez A., Shum K., Guan S. (1999)Drug delivery systems employing 1,4- or 1,6-elimina-tion: poly(ethylene glycol) prodrugs of amine-contain-ing compounds. J Med Chem;42:3657–3667.

97. Greenwald R.B., Choe Y.H., McGuire J., ConoverC.D. (2003) Effective drug delivery by PEGylated drugconjugates. Adv Drug Deliv Rev;55:217–250.

98. Hanson K.R., Havir E.A. (1972) The enzymic elimina-tion of ammonia. In: Boyer P.D., editor. The Enzymes,3rd edn. vol. 7. New York: Academic Press; p 75–166.

99. Czarnik A.W. (1988) Intramolecularity: proximity andstrain. In: Liebman J.F., Greenberg A., editors. Mech-anistic Principles of Enzyme Activity. New York, NY:VCH Publishers; p. 75–117.

100. Sweigers G.F. (2008) Mechanical Catalysis. Hoboken,NJ: John Wiley & Sons.

101. Fersht A. (1979) Structure and Mechanism in ProteinScience: A guide to Enzyme Catalysis and ProteinFolding. New York: Freeman, W. H. and Company.

102. Nelson D.L., Cox M.M. (2003) Lehninger Principles ofBiochemistry. New York: Worth Publishers.

103. Lightstone F.C., Bruice T.C. (1997) Separation ofground state and transition state effects in intramolec-ular and enzymatic reactions. 2. A theoretical study of


Karaman

the formation of transition states in cyclic anhydrideformation. J Am Chem Soc;119:9103–9113.

104. Milstein S., Cohen L.A. (1972) Stereopopulationcontrol I. Rate enhancement in the lactonizations ofo-hydroxyhydrocinnamic acids. J Am Chem Soc;94:9158–9165.

105. Menger F.M. (2005) An alternative view of enzymecatalysis. Pure Appl Chem;77:1873–1876.

106. Kirby A.J., Hollfelder F. (2009) From Enzyme Modelsto Model Enzymes. Cambridge UK: RSC Publishing;p. 1–273.

107. Dafforn A., Koshland D.E. Jr (1973) Proximity, entropyand orbital steering. Biochem Biophys Res Commun;52:779–785.

108. Liederer B.M., Borchardt R.T. (2006) Enzymesinvolved in the bioconversion of ester-based pro-drugs. J Pharm Sci;95:1177–1195.

109. Reddy M.R., Erion M.D., editors. (2001) Free EnergyCalculations in Rational Drug Design. New York, NY:AAPS Press/Springer.

110. Parr R.G., Craig D.P., Ross I.G. (1950) Molecularorbital calculations of the lower excited electronic lev-els of benzene, configuration interaction included. JChem Phys;18:1561–1563.

111. Parr R.G. (1990) On the genesis of a theory. Int JQuantum Chem;37:327–347.

112. Chen T.C. (1955) Expansion of electronic wave func-tions of molecules in terms of ‘united-atom’ wavefunctions. J Chem Phys;23:2200–2201.

113. Dewar M.J.S., Thiel W. (1977) Ground states of mole-cules. The MNDO method. Approximations andparameters. J Am Chem Soc;99:4899–4907.

114. Bingham R.C., Dewar M.J.S., Lo D.H. (1975) Groundstates of molecules. XXV. MINDO/3. An improvedversion of the MNDO semiempirical SCF-MO method.J Am Chem Soc;97:1285–1293.

115. Dewar M.J.S., Zoebisch E.G., Healy E.F., StewartJ.J.P. (1985) AM1: a new general purpose quantummechanical molecular model. J Am Chem Soc;107:3902–3907.

116. Dewar M.J.S., Jie C., Yu J. (1993) The first of newseries of general purpose quantum mechanicalmolecular models. Tetrahedron;49:5003–5038.

117. Parr R.G., Yang W. (1989) Density Functional Theoryof Atoms and Molecules. Oxford: Oxford UniversityPress.

118. Burker U., Allinger N.L. (1982) Molecular Mechanics.Washington, DC, USA: American Chemical Society.

119. Warshel A., Levitt M. (1976) Theoretical studies ofenzymatic reactions: dielectric, electrostatic and stericstabilization of the carbonium ion in the reaction oflysozyme. J Mol Biol;103:227–249.

120. Field M.J. (2002) Simulating enzyme reactions: chal-lenges and perspectives. J Comput Chem;23:48–58.

121. Mulholland A.J. (2005) Modelling enzyme reactionmechanisms, specificity and catalysis. Drug DiscoveryToday;10:1393–1402.

122. Karaman R. (2008) Analysis of Menger’s spatiotem-poral hypothesis. Tetrahedron Lett;49:5998–6002.

123. Karaman R. (2009) Cleavage of Menger’s aliphaticamide: a model for peptidase enzyme solelyexplained by proximity orientation in intramolecularproton transfer. J Mol Struct;910:27–33.

124. Karaman R. (2010) The efficiency of proton transfer inKirby’s enzyme model, a computational approach.Tetrahedron Lett;51:2130–2135.

125. Karaman R., Pascal R.A. (2010) Computational analy-sis of intramolecularity in proton transfer reactions.Org Biol Chem;8:5174–5178.

126. Karaman R. (2010) A general equation correlatingintramolecular rates with attack” parameters: distanceand angle. Tetrahedron Lett;51:5185–5190.

127. Karaman R. (2011) Analyzing the efficiency of pro-ton transfer to carbon in Kirby’s enzyme model- acomputational approach. Tetrahedron Lett;52:699–704.

128. Karaman R. (2011) Analyzing the efficiency in intra-molecular amide hydrolysis of Kirby’s N-alkylmaleamicacids - a computational approach. Comput TheorChem;974:133–142.

129. Karaman R. (2009) A new mathematical equationrelating activation energy to bond angle and distance:a key for understanding the role of acceleration inlactonization of the trimethyl lock system. BioorgChem;37:11–25.

130. Karaman R. (2009) Revaluation of Bruice’s ProximityOrientation. Tetrahedron Lett;50:452–458.

131. Karaman R. (2009) Accelerations in the Lactonizationof Trimethyl Lock Systems are Due to ProximityOrientation and not to Strain Effects. Res Lett OrgChem:1–5.

132. Karaman R. (2009) The gem-disubstituent effect- acomputational study that exposes the relevance ofexisting theoretical models. Tetrahedron Lett;50:6083–6087.

133. Karaman R. (2009) Analyzing Kirby’s amine olefin – amodel for amino-acid ammonia lyases. TetrahedronLett;50:7304–7309.

134. Karaman R. (2009) The Effective Molarity (EM) Puzzlein Proton Transfer Reactions. Bioorg Chem;37:106–110.

135. Karaman R. (2010) Effects of substitution on theeffective molarity (EM) for five membered ring-closurereactions- a computational approach. J MolStruct;939:69–74.

136. Karaman R. (2010) The Effective Molarity (EM) Puzzlein Intramolecular Ring-Closing Reactions. J MolStruct;940:70–75.

137. Menger F.M., Karaman R. (2010) A Singularity Modelfor Chemical Reactivity. Eur J Chem;16:1420–1427.

138. Karaman R. (2010) The Effective Molarity (EM) –a computational approach. Bioorg Chem;38:165–172.

139. Karaman R. (2010) Proximity vs. Strain in Ring-ClosingReactions of Bifunctional Chain Molecules- a Compu-tational Approach. J Mol Phys;108:1723–1730.



140. Karaman R. (2011) The role of proximity orientation inintramolecular proton transfer reactions. J ComputTheor Chem;966:311–321.

141. Barber S.E., Dean K.E., Kirby A.J. (1999) A mecha-nism for efficient proton-transfer catalysis. Intramolec-ular general acid catalysis of the hydrolysis of1-arylethyl ethers of salicylic acid. Can JChem;77:792–801.

142. Kirby A.J., Lancaster P.W. (1972) Structure and effi-ciency in intramolecular and enzymatic catalysis.Catalysis of amide hydrolysis by the carboxy-group ofsubstituted maleamic acids. J Chem Soc PerkinTrans;2:1206–1214.

143. Kirby A.J., de Silva M.F., Lima D., Roussev C.D.,Nome F. (2006) Efficient intramolecular general acidcatalysis of nucleophilic attack on a phosphodiester.J Am Chem Soc;128:16944–16952.

144. Kirby A.J., Williams N.H. (1994) Efficient intramolecu-lar general acid catalysis of enol ether hydrolysis.Hydrogen-bonding stabilization of the transition statefor proton transfer to carbon. J Chem Soc PerkinTrans;2:643–648.

145. Kirby A.J., Williams N.H. (1991) Efficient intramolecu-lar general acid catalysis of vinyl ether hydrolysis bythe neighbouring carboxylic acid group. J Chem SocChem Commun:1643–1644.

146. Kirby A.J. (1996) Enzyme Mechanisms, Models, andMimics. Angew Chem Int Ed Engl;35:706–724.

147. Fife T.H., Przystas T.J. (1979) Intramolecular generalacid catalysis in the hydrolysis of acetals with ali-phatic alcohol leaving groups. J Am ChemSoc;101:1202–1210.

148. Kirby A.J. (1997) Efficiency of proton transfer catalysisin models and enzymes. Acc Chem Res;30:290–296.

149. Menger F.M., Ladika M. (1988) Fast hydrolysis of an ali-phatic amide at neutral pH and ambient temperature. Apeptidase model. J Am Chem Soc;110:6794–6796.

150. Menger F.M. (1985) On the source of intramolecularand enzymatic reactivity. Acc Chem Res;18:128–134.

151. Menger F.M., Chow J.F., Kaiserman H., VasquezP.C. (1983) Directionality of proton transfer in solu-tion. Three systems of known angularity. J Am ChemSoc;105:4996–5002.

152. Menger F.M., Galloway A.L., Musaev D.G. (2003)Relationship between rate and distance. Chem Com-mun:2370–2371.

153. Milstein S., Cohen L.A. (1970) Concurrent general-acid and general-base catalysis of esterification. J AmChem Soc;92:4377–4382.

154. Milstein S., Cohen L.A. (1970) Rate acceleration bystereopopulation control: models for enzyme action.Proc Natl Acad Sci USA;67:1143–1147.

155. Brown R.F., van Gulick N.M. (1956) The geminal alkyleffect on the rates of ring closure of bromobutylam-ines. J Org Chem;21:1046–1049.

156. Bruice T.C., Pandit U.K. (1960) The effect of geminalsubstitution ring size and rotamer distribution on theintra molecular nucleophilic catalysis of the hydrolysis

of monophenyl esters of dibasic acids and the solvol-ysis of the intermediate anhydrides. J Am ChemSoc;82:5858–5865.

157. Bruice T.C., Pandit U.K. (1960) Intramolecular mod-els depicting the kinetic importance of ‘‘Fit’’ inenzymatic catalysis. Proc Natl Acad Sci USA;46:402–404.

158. Galli C., Mandolini L. (2000) The role of ring strain onthe ease of ring closure of bifunctional chain mole-cules. Eur J Org Chem;2000:3117–3125.

159. Karaman R. (2010) Prodrugs of Aza NucleosidesBased on Proton Transfer Reactions. J ComputAided Mol Des;24:961–970.

160. Karaman R., Hallak H. (2010) Computer-assisteddesign of pro-drugs for antimalarial atovaquone.Chem Biol Drug Des;76:350–360.

161. Hejaz H., Karaman R., Khamis M. (2012) Computer-Assisted Design for paracetamol Masking Bitter TasteProdrugs. J Mol Model;18:103–114.

162. Karaman R., Karaman D., Zeiadeh I. (2013) Compu-tationally designed phenylephrine prodrugs - A modelfor enhancing bioavailability. J Mol Phys. doi: 10.1080/00268976.2013.779395.

163. Karaman R., Dajani K.K., Qtait A., Khamis M. (2012)Prodrugs of Acyclovir - a computational approach.Chem Biol Drug Des;79:819–834.

164. Karaman R., Dajani K.K., Hallak H. (2012) Computer-assisted design for atenolol prodrugs for the use inaqueous formulations. J Mol Model;18:1523–1540.

165. Karaman R., Amly W., Scrano L., Mecca G., BufoS.A. (2013) Computationally designed prodrugs ofstatins based on Kirby’s enzyme model. J MolModel;???:1–14.

166. Karaman R. (2013) Prodrugs for Masking Bitter Taste ofAntibacterial Drugs - A Computational Approach. J MolModel;19:2399–2412.

167. Karaman R. (2012) The future of prodrugs designedby computational chemistry. J Drug Design;1:e103.

168. Karaman R. (2012) Computationally designed pro-drugs for masking the bitter taste of drugs. J DrugDesign;1:e106.

169. Karaman R. (2013) prodrugs design by computationmethods- a new era. J Drug Design;1:e113.

170. Karaman R. (2012) Computationally designed enzymemodels to replace natural enzymes in prodrugapproaches. J Drug Design;1:e111.

171. Karaman R. (2013) Prodrug design vs. drug design. JDrug Design;2:e114.

172. Karaman R. (2011) Computational aided design fordopamine prodrugs based on novel chemicalapproach. Chem Biol Drug Des;78:853–863.

173. Karaman R., Dokmak G., Bader M., Hallak H., Kha-mis M., Scrano L., Bufo S.A. (2013) Prodrugs offumarate esters for the treatment of psoriasis andmultiple sclerosis (MS)- A computational approach” J.Mol Model;19:439–452.

174. Kluger R., Chin J. (1982) Carboxylic acid participationin amide hydrolysis. Evidence that separation of a


Karaman

nonbonded complex can be rate determining. J AmChem Soc;104:2891–2897.

175. CRASH-2 Trial Collaborators (2010) Effects of tra-nexamic acid on death, vascular occlusive events,and blood transfusion in trauma patients with signifi-cant hemorrhage (CRASH-2): a randomized, placebo-controlled trial. Lancet;6736:60835–5.

176. Gohel M., Patel P., Gupta A., Desai P. (2007) Efficacyof tranexamic acid in decreasing blood loss duringand after cesarean section: a randomized case con-trolled prospective study. J Obstet GynecolIndia;57:227–230.

177. Giancarlo L., Francesco B., Angela L., Pierluigi P.,Gina R. (2011) Recommendations for the transfusionmanagement of patients in the peri-operative period.II. The intra-operative period. Blood Transfus;9:189–217.

178. Lukes A.S., Kouides P.A., Moore K.A. (2011) Tra-nexamic acid: a novel oral formulation for the treat-ment of heavy menstrual bleeding. Women’sHealth;7:151–158.

179. Lukes A.S., Moore K.A., Muse K.N., Gersten J.K.,Hecht B.R., Edlund M Richter H.E., Eder S.E., AttiaG.R., Patrick D.L., Rubin A., Shangold G.A. (2010)Tranexamic acid treatment for heavy menstrual bleed-ing: a randomized controlled trial. Obstet Gyne-col;116:865–875.

180. Pilbrant A., Schannong M., Vessman J. (1981) Phar-macokinetics and bioavailability of tranexamic acid.Eur J Clin Pharmacol;20:65–72.

181. Kirby A.J. (2005) effective molarities for intramolecularreactions. J Phys Org Chem;18:101–278.

182. de Clercq E., Field H.J. (2006) Antiviral prodrugs —the development of successful prodrug strategies forantiviral chemotherapy. Br J Pharmacol;147:1–11.

183. de Miranda P., Blum M.R. (1983) Pharmacokineticsof acyclovir after intravenous and oral administration.J Antimicrob Chemother;12(Suppl B): 29–37.

184. Blum M.R., Liao S.H.T., de Miranda P. (1982) Over-view of aciclovir pharmacokinetic disposition in adultsand children. Am J Med;73(Suppl. 1A):186–192.

185. Luengo J., Aranguiz T., Sepulveda J. (2002) Prelimin-ary pharmacokinetic study to different preparations ofacyclovir with b-cyclodextrin. J Pharm Sci;91:2593–2598.

186. Attia I.A., El-Gizawy S.A., Fouda M.A., Donia A.M.(2007) Influence of a niosomal formulation on the oralbioavailability of acyclovir in rabbits. AAPS Pharm SciTech;8:Article 106 doi: 10.1208/pt0804106.

187. Yadav S., Jain S., Prajapati S., Motwani M., KumarS. (2011) Formulation and in vitro and in vivo charac-terization of acyclovir loaded mucoadhesive micro-spheres. J Pharm Sci Tech;3:411–447.

188. Soul-Lawton J., Seaber E., On N., Wootton R., RolanP., Posner J. (1995) Absolute bioavailability and met-abolic disposition of valacyclovir, the L-valyl ester ofacyclovir, following oral administration to humans.Antimicrob Agents Chemother;39:2759–2764.

189. Tolle-Sander S., Lentz K.A., Maeda D.Y., Coop A.,Polli J.E. (2004) Increased Acyclovir Oral Bioavailabil-ity via a Bile Acid Conjugate. Mol Pharm;1:40–48.

190. Houk K.N., Tucker J.A., Dorigo A.E. (1990) Quantita-tive modeling of proximity effects on organic reactiv-ity. Acc Chem Res;23:107–113.

191. Reinaldo T. (2003) Antimalarial drug discovery: oldand new approaches. J Exp Biol;206:3735–3744.

192. Chung M.C., Ferreira E.I., Santos J.L., Giarolla J.,Rando D.G., Almeida A.E., Bosquesi P.L., MenegonR.F., Blau L. (2008) Prodrugs for the treatment ofneglected diseases. Molecules;13:616–677.

193. Peterson A.T. (2009) Shifting suitability for malariavectors across Africa with warming climates. BMCInfect Dis;9:59.

194. Hitani A., Nakamura T., Ohtomo H., Nawa Y., KimuraM. (2006) Efficacy and safety of atovaquone-proguanilcompared with mefloquine in the treatment of nonim-mune patients with uncomplicated P. falciparummalaria in Japan. J Infect Chemother;12:277–282.

195. Vial H.J., Wein S., Farenc C., Kocken C., Nicolas O.,Ancelin M.L., Bressolle F., Thomas A., Calas M.(2004) Prodrugs of bisthiazolium salts are orallypotent antimalarials. Proc Natl Acad Sci USA;101:15458–15463.

196. Hudson A.T., Dickins M., Ginger C.D., GutteridgeW.E., Holdich T., Hutchinson D.B., Pudney M.,Randall A.W., Latter V.S. (1991) 566C80: a potentbroad spectrum anti-infective agent with activityagainst malaria and opportunistic infections in AIDSpatients. Drugs Exp Clin Res;17:427–435.

197. Fry M., Pudney M. (1992) Site of action of the anti-malarial hydroxynaphthoquinone, 2-[trans-4-(4′-chlor-ophenyl) cyclohexyl]-3- hydroxy-1,4-naphthoquinone(566C80). Biochem Pharmacol;43:1545–1553.

198. Looareesuwan S., Chulay J.D., Canfield C.J., Hutch-inson D.B. (1999) Malarone (atovaquone and progua-nil hydrochloride): a review of its clinical developmentfor treatment of malaria. Malarone Clinical Trials StudyGroup. Am J Trop Med Hyg;60:533–541.

199. Comley J.C., Yeates C.L., Frend T.J. (1995)Antipneumocystis activity of 17C91, a prodrug ofatovaquone. Antimicrob Agents Chemother;39:2217–2219.

200. Haile L.G., Flaherty J.F. (1993) Atovaquone: a review.Ann Pharmacother;27:1488–1494.

201. Torres R.A., Weinberg W., Stansell J., Leoung G.,Kovacs J., Rogers M., Scott J. (1997) Atovaquone forsalvage treatment and suppression of toxoplasmicencephalitis in patients with AIDS. Atovaquone/Toxo-plasmic Encephalitis Study Group. Clin InfectDis;24:422–429.

202. Sohi H., Sultana Y., Khar R.K. (2004) Taste MaskingTechnologies in oral pharmaceuticals, recent develop-ment and approaches. Drug Develop Ind Pharm;30:429–448.

203. Reilly W.J. (2002) Pharmaceutical Necessities in Rem-ington: The Science and Practice of Pharmacy. Balti-



more, MD: Mack Publishing Company; p. 1018–1020.

204. Drewnowski A., Gomez-Carneros C. (2000) Bittertaste, phytonutrients, and the consumer: a review.Am J Clin Nutr;72:1424–1435.

205. Hofmann T. (2009) Identification of the key bittercompounds in our daily diet is a prerequisite for theunderstanding of the hTAS2R gene polymorphismsaffecting food choice. Ann N Y Acad Sci;1170:116–125.

206. Rodgers S., Busch J., Peters H., Christ-Hazelhof E.(2005) Building a tree of knowledge: analysis of bittermolecules. Chem Senses;30:547–557.

207. Rodgers S., Glen R.C., Bender A. (2006) Characteriz-ing bitterness: identification of key structural featuresand development of a classification model. J ChemInf Model;46:569–576.

208. Maehashi K., Huang L. (2009) Bitter peptides and bit-ter taste receptors. Cell Mol Life Sci;66:1661–1671.

209. Behrens M., Meyerhof W. (2006) Bitter taste recep-tors and human bitter taste perception. Cell Mol LifeSci;63:1501–1509.

210. Meyerhof W., Born S., Brockhoff A., Behrens M.(2011) Molecular biology of mammalian bitter tastereceptors. A review. Flavour Frag J;26:260–268.

211. Behrens M., Meyerhof W. (2009) Mammalian bittertaste perception. Results Probl Cell Differ;47:203–220.

212. Ayenew Z., Puri V., Kumar L., Bansal A.K. (2009)Trends in pharmaceutical taste masking technologies:a patent review. Recent Pat Drug Deliv Formul;3:26–39.

Author Information

Rafik Karaman, Bioorganic Chemistry Department, Collegeof Pharmacy, Al-Quds University, Jerusalem, Palestine.P.O. Box 20002. Tel./Fax: +972-2-2790413. E-mail:[email protected].

Notes

aDrug.” Dictionary.com Unabridged (v 1.1) (2007), RandomHouse, Inc., via dictionary.com. Retrieved on 20 Septem-ber 2007.bCyklokapron� tranexamic acid injection prescribing infor-mation: http://www.pfizer.com/files/products/uspi_cykloka-pron.pdf.cwww.cdc.gov/malaria/about/disease.htm.


Karaman

Prodrugs Design Based on Inter-and Intramolecular Chemical Processes

Documents

Transcript of Prodrugs Design Based on Inter-and Intramolecular Chemical Processes