Prodrugs Design Based on Inter- and Intramolecular Chemical Processes

Home , Molecular modification, Salbutamol

Chem Biol Drug Des 2013; 82: 643–668 Review

Rafik Karaman1,2,* compound satisfies a number of preset criteria to start clinical development. The number of years it takes to intro- 1Bioorganic Chemistry Department, Faculty of Pharmacy, duce a drug to the pharmaceutical market is over 10 years Al-Quds University, P.O. Box 20002, Jerusalem, Palestine with a cost of more than $1 billion dollars (1,2). 2Department of Science, University of Basilicata, Via dell’Ateneo Lucano 10, 85100, Potenza, Italy Modifying the absorption, distribution, metabolism, and *Corresponding author: Rafik Karaman, elimination (ADME) properties of an active drug requires a [email protected] complete understanding of the physicochemical and biological behavior of the drug candidate (3 6). This includes This review provides the reader a concise overview of – the majority of prodrug approaches with the emphasis comprehensive evaluation of drug-likeness involving on the modern approaches to prodrug design. The prediction of ADME properties. These predictions can be chemical approach catalyzed by metabolic enzymes attempted at several levels: in vitro–in vivo using data which is considered as widely used among all other obtained from tissue or recombinant material from human approaches to minimize the undesirable drug physico- and preclinical species, and in silico or computational pre- chemical properties is discussed. Part of this review dictions projecting in vitro or in vivo data, involving the will shed light on the use of molecular orbital methods evaluation of various ADME properties, using computa- such as DFT, semiempirical and ab initio for the design tional approaches such as quantitative structure activity of novel prodrugs. This novel prodrug approach relationship (QSAR) or molecular modeling (7–11). implies prodrug design based on enzyme models that were utilized for mimicking enzyme catalysis. The com- Studies have indicated that poor pharmacokinetics and tox- putational approach exploited for the prodrug design icity are the most important causes of high attrition rates in involves molecular orbital and molecular mechanics the drug development process, and it has been widely (DFT, ab initio, and MM2) calculations and correlations between experimental and calculated values of intra- accepted that these areas should be considered as early as molecular processes that were experimentally studied possible in drug discovery to improve the efficiency and to assign the factors determining the reaction rates in cost-effectiveness of the industry. Resolving the pharmaco- certain processes for better understanding on how kinetic and toxicological properties of drug candidates enzymes might exert their extraordinary catalysis. remains a key challenge for drug developers (12).

Key words: Ab initio calculations, design of prodrugs, DFT Thus, the aim is to design drugs that have an efficient calculations, enzyme models, molecular mechanics calcula- permeability to be absorbed into the blood circulation tions, prodrugs (absorption), to reach their target efficiently (distribution), to be quite stable to survive the physiological journey (metab- Received 4 July 2013, revised 13 August 2013 and accepted olism), and to be eliminated in a satisfactory time (elimina- for publication 16 August 2013 tion). In other words, designing a drug with optimum pharmacokinetics properties can be achieved by imple- menting one or more of the following strategies: A drug is defined as a substance, which is used in the diagnosis, cure, relief, treatment, or prevention of disease, Improving Absorption or intended to affect the structure or function of the body. The development of any potential drug starts with the Drug absorption is determined by the drug hydrophilic study of the biochemistry behind a disease for which phar- hydrophobic balance (HLB) value, which depends upon maceutical intervention is seen.a polarity and ionization. Very polar or strongly ionized drugs, having a relatively high HLB values, cannot efficiently cross Drug discovery is a lengthy interdisciplinary endeavor. It is the cell membranes of the gastrointestinal (GI) barrier. a consecutive process that starts with target and lead Hence, they are given by the intravenous (I.V.) route, but discovery, followed by lead optimization and preclinical their disadvantage is being rapidly eliminated. Non-polar in vitro and in vivo studies to evaluate whether a

ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12224 643 Karaman drugs, on the other hand, having a relatively low HLB val- Carbachol (1 in Figure 1), a cholinergic agonist, and cefoxi- ues, are poorly soluble in aqueous media and hence are tin (2 in Figure 1), a cephalosporin, are stabilized in this poorly absorbed through membranes. If they are given by way. (iii) Stereoelectronic modification: Steric hindrance and injection, most probably, they will be retained in fat tissues electronic stabilization have been used together to stabilize (13–21). labile groups. For example, procaine, an ester drug, is quickly hydrolyzed, but changing the ester to the less reac- Generally, the polarity and/or ionization of drug can be tive amide group reduces hydrolysis such as in the cases altered by changing its substituents, and these changes of procainamide (3 in Figure 1) and lidocaine (4 in Figure 1). are classified under the so-called quantitative structure– (iv) Metabolic Blockers: Some drugs are metabolized by activity relationships (QSAR). The following are examples introducing polar functional groups at particular positions in for such changes: (1) variation of alkyl or acyl substituents their skeleton. For example, megestrol acetate (5 in Figure and polar functional groups to vary polarity, (1) variation of 1), an oral contraceptive, is oxidized at position 6 to give N-alkyl substituents to vary pKa; acidic drugs with low hydroxyl group at this position; however, replacing the pKa and basic drugs with high pKa values tend to be ion- hydrogen at position 6 with a methyl group blocks its ized and are poorly absorbed through membrane tissues, metabolism, and consequently it results in prolonging its (2) variation of aromatic substituents to vary pKa: The pKa duration of action. (v) Removal of susceptible metabolic of aromatic amine or carboxylic acid can be varied by groups: Certain chemical moieties are particularly suscepti- adding electron donating or electron withdrawing groups ble to metabolic enzymes. For example, a methyl group on to the ring. The position of the substituent is important too aromatic rings is often oxidized to carboxylic acid, which if the substituent interacts with the ring through resonance then results in a rapid elimination of the drug from the and (3) bioisosteres for polar groups; carboxylic acid is a body. Other common metabolic reactions include aliphatic highly polar group which can be ionized and hence and aromatic C-hydroxylation, O and S-dealkylations, N- decreases the absorption of any drug containing it. To and S-oxidations, and deamination. (vi) Group Shifts: overcome this problem, blocking the free carboxyl group Removing or replacing a metabolically vulnerable group is by making the corresponding ester prodrug or replacing it feasible if the group concerned is not involved in important with a bioisostere group, which has similar physiochemical binding interactions within the active site of the receptor or properties and has advantage over carboxylic acid in enzyme. If the group is important, then different strategy regard to its pKa, such as 5-substituted tetrazoles, is either masking the vulnerable group using a prodrug or essential; 5-substituted tetrazole ring contains acidic pro- shifting the vulnerable group within the molecule skeleton is ton such as carboxylic acid and is ionized at pH 7.4. On undertaken. Salbutamol was developed in 1969 from its the other hand, most of the alkyl and aryl carboxylic analog neurotransmitter, norepinephrine, using this tactic. groups have a pKa in the range of 2–5 (13–28). Norepinephrine is metabolized by methylation of one of its phenolic groups by catechol O-methyl transferase. The other phenolic group is important for receptor-binding inter- Improving Metabolism action. Removing the hydroxyl or replacing it with a methyl group prevents metabolism but also prevents hydrogen There are different strategies that can be utilized to bonding interaction with the binding site. While moving the improve drug metabolism: (i) steric shields: Some func- vulnerable hydroxyl group out from the ring by one carbon tional groups are more susceptible to chemical and enzy- unit as in salbutamol makes, this compound unrecogniz- matic degradation than others. For example, esters and able by the metabolic enzyme, but not to the receptor- amides are much more affordable to hydrolysis than others binding site (prolonged action) and (vii) ring variation; some such as carbamates and oximes. Adding steric shields to ring systems are often found to be susceptible to metabo- these drugs increases their stability. Steric shields were lism, and so varying the ring can often improve metabolic designed to hinder the approach of a nucleophile or a stability. For example, replacement of the imidazole ring, nucleophilic center on an enzyme to the susceptible which is susceptible to metabolism in tioconazole (6 in Fig- group. These usually involve the addition of a bulky alkyl ure 1) with 1,2,4-triazole ring, gives fluconazole (7 in Figure group such as t-butyl close to the functional group. 1) with improved stability. (ii) Electronic effects of bioisosteres: This approach is used to protect a labile functional group by electronic stabiliza- Making drug less resistance to drug metabolism: drug that tion. For example, replacing the methyl group of an ester is extremely stable to metabolism and is very slowly elimi- with an amine group gives a urethane functional group, nated can cause problems in a similar manner to that sus- which is more stable than the parent ester. The amine ceptible to metabolism, thus resulting in an increase in group has the same size and valance as the methyl group; toxicity and adverse effects. Therefore, designing drugs with however, it has no steric effect, but it has totally different decreased chemical and metabolic stability can sometimes electronic properties, because it can donate electrons via be beneficial. Methods for applying such strategy are (i) its inductive effect into the carbonyl group resulting in introducing groups that are susceptible to metabolism is a reducing the electrophilicity of the carbonyl carbon and good way of shorting the lifetime of a drug. For example, hence stabilizing it from hydrolysis. methyl group was introduced to some drugs to shorten their

644 Chem Biol Drug Des 2013; 82: 643–668 Inter- and Intramolecular Chemical Processes

Figure 1: Chemical structures for 1–7. lifetime because methyl can metabolically undergo oxidation groups, aromatic amines, bromoarenes, hydrazines, to polar alcohol as well as to a carboxylic acid. (ii) A self- hydroxylamines, or polyhalogenated groups are generally destruct drug is one that is chemically stable under one set metabolized to toxic metabolites. of conditions but becomes unstable and spontaneously cleaves under another set of conditions. The advantage of a Side-effects might be reduced or eliminated by varying self-destruct drug is that inactivation does not depend on harmless substituents. For example, addition of fluorine the activity of metabolic enzymes, which could vary from group to UK 47265, antifungal agent, gives the less toxic patient to patient. For example, atracurium, a neuromuscu- fluconazole (29–34). lar blocking agent, is stable at acidic pH but self-destructs when it is exposed to the slightly alkaline conditions of the blood (pH 7.4). Thus, the drug has a short duration of Prodrugs Catalyzed by Metabolic Enzymes action, allowing anesthetists to control its blood concentration levels during surgery by providing it as a continuous The principle of targeting drugs can be traced back to intravenous drip (22–28). Paul Ehrlich who developed antimicrobial drugs that were selectively toxic for microbial cells over human cells. Today, targeting tumor cells is considered one of the most Reducing Toxicity important issues that under concern among the health community. A major goal in cancer chemotherapy is to tar- It is often found that a drug fails clinical trials because of get drugs efficiently against tumor cells rather than against its toxic adverse effects. normal cells. One method for achieving this is to design drugs which make use of specific molecular transport sys- This may be due to toxic metabolites, in which case the tems. The idea is to attach the active drug to an important drug should be made more resistant to metabolism. It is building block molecule that is needed in large amounts known that functional groups such as aromatic nitro by the rapidly divided tumor cells. This could be an amino

Chem Biol Drug Des 2013; 82: 643–668 645 Karaman acid or a nucleic acid base such as uracil mustard. In the to overcome various undesirable drug properties. This type cases where the drug is intended to target against infec- of ‘targeted-prodrug’ design requires considerable knowl- tion of gastrointestinal tract (GIT), it must be prevented edge of particular enzymes or carriers, including their from being absorbed into the blood supply. This can easily molecular and functional characteristics (35–46). be done using a fully ionized drug which is incapable of crossing cell membrane barriers. For example, highly ion- The second approach to be discussed in this review is sub- ized sulfonamides are used against GIT infections because divided to two major approaches: (i) Chemical approach by they are incapable of crossing the gut wall. It is often which the drug is linked to promoiety which upon exposure possible to target drugs such that they act peripherally to physiological environment undergoes enzymatic cata- and not in the central nervous system (CNS). By increasing lyzed degradation to the parent drug and inactive linker. In the polarity of drugs, they are less likely to cross the this approach, the interconversion rate is dependent on the blood–brain barrier, and thus, they are less likely to have enzyme catalysis. This approach involves carrier-linked CNS adverse effects (35–46). prodrugs and contains a group that can be easily removed enzymatically, such as an ester or labile amide, to provide The most efficient approach for overcoming the negative the parent drug. Ideally, the group removed is pharmaco- pharmacokinetics characteristics of a drug is the prodrug logically inactive and non-toxic, while the linkage between approach. This approach may be utilized in the cases where the drug and promoiety must be labile for in vivo efficient the use of the parent drug faces problems associated with activation. Carrier-linked prodrugs can be further subdivided solubility, absorption and distribution, site specificity, insta- into (i) bipartite, which is composed of one carrier group bility, toxicity, poor patient compliance, or formulation prob- attached to the drug, (ii) tripartite, which is a carrier group lems (47–52). A metabolic enzyme is usually involved in that is attached via linker to drug, and (iii) mutual prodrugs converting the prodrugs to their active forms. Not all consisting of two drugs linked together, and (1) bioprecur- prodrugs are activated by metabolic enzymes. For example, sors are chemical entities that are metabolized into new photodynamic therapy involves the use of an external light compounds that may be active or further are metabolized to source to activate prodrugs. When designing a prodrug, it is active metabolites, such as amine to aldehyde to carboxylic important to ensure that the prodrug is effectively converted acid (48–51); and (ii) intramolecular chemical approach to the active drug once it has been absorbed in blood sup- designed based on calculations using molecular orbital ply. It is also important to ensure that any groups that are (MO) and molecular mechanics (MM) methods and correla- cleaved from the prodrug molecule are non-toxic (22). tions between experimental and calculated values. In this prodrug approach, no enzyme is involved in the intraconver- The prodrug approach is a very versatile strategy to increase sion chemical reaction of a prodrug to its parent drug. The the utility of biologically active compounds, because one interconversion of the prodrug is solely controlled by the can optimize any of the ADME properties of potential drug rate-limiting step of the intramolecular reaction. candidates. In most cases, prodrugs contain a promoiety (linker) that is removed by an enzymatic or chemical reac- The prodrug design can be utilized in the followings cases: tion, while other prodrugs release their active drugs after (i) enhancing active drug solubility in a physiological envi- molecular modification such as an oxidation or reduction ronment/s and consequently its bioavailability since disso- reaction. The prodrug candidate can also be prepared as a lution of the drug molecule from the dosage form may be double prodrug, where the second linker is attached to the the rate-limiting step to absorption (48). It has been docu- first promoiety linked to the parent drug molecule. These mented that more than 30% of drug discovery compounds linkers are usually different and are cleaved by different have poor aqueous solubility (53). Prodrugs are an alterna- mechanisms. In some cases, two biologically active drugs tive way to increase the aqueous solubility of the parent can be linked together in a single molecule called a codrug. drug molecule by increasing dissolution rate through In a codrug, each drug acts as a linker for the other (8,9). attachment to ionizable or polar groups, such as phos- The prodrug approach has been used to overcome various phates, sugar, or amino acids moieties (51,54). These undesirable drug properties and to optimize clinical drug prodrugs can be used for increasing oral bioavailability and application. Recent advances in molecular biology provide in parenteral or injectable drug delivery. (ii) Upon increasing direct availability of enzymes and carrier proteins, including permeability and hence absorption, membrane permeability their molecular and functional characteristics. has a significant effect on drug effectiveness (7). In oral drug delivery, the most common absorption routes are There are two major prodrug design approaches that are un-facilitated and largely non-specific, passive transport considered as widely used among all other approaches to mechanisms. The lipophilicity of poorly permeable drugs minimize or eliminate the undesirable drug physicochemi- can be enhanced by linking to lipophilic groups. In such cal properties while maintaining the desirable pharmaco- cases, the prodrug strategy can be an extremely valuable logical activity. The first approach is the targeted drug option and crucially needed. Improvements in lipophilicity design approach by which prodrugs can be designed to have been the most widely researched and successful field target specific enzymes or carriers by considering of prodrug research. It has been achieved by masking enzyme–substrate specificity or carrier–substrate specificity polar ionized or non-ionized functional groups to increase

646 Chem Biol Drug Des 2013; 82: 643–668 Inter- and Intramolecular Chemical Processes either oral or topical absorption (22). (iii) Modification of the nucleoside kinases for initial activation; hence, they have distribution profile: Before the drug reaches its physiological low oral bioavailability due to the ionized phosphonate target and exert the desired effect, it has to bypass several group (60,61). To overcome this problem, a bis(pivaloyl- pharmaceutical and pharmacokinetic barriers. Today, one oxymethyl) ester prodrug of adefovir (adefovir dipivoxil) and of the most promising site-selective drug delivery strategies ether lipid ester prodrugs of cidofovir were explored for is the prodrug approach, which utilizes target cell- or tis- improving intestinal permeability (51). Other examples for sue-specific endogenous enzymes and transporters. ester prodrugs that were designed and synthesized for different purposes are thioester of erythromycin, palmitate The suitability of a number of functional groups such as ester of clindamycin, a number of angiotensin-converting carboxylic, hydroxyl, amine, phosphate, phosphonate, and enzyme (ACE) inhibitors which are (55) presently marketed carbonyl groups for undergoing different chemical as ester prodrugs, including enalapril (12 in Figure 2), modifications facilitates their utilization in prodrug design ramipril, benazepril, and fosinopril, and all of them are (1,9) In the past few decades, a variety of prodrugs based intended for the treatment for hypertension (47) and ibupro- on the chemical approach have been designed, synthe- fen guaiacol ester that was reported to have fewer GI side- sized, and tested. Among those are the following. effects with similar anti-inflammatory/antipyretic action to its parent drug when is given in equimolar doses (62). As mentioned before, two active drugs can be joined together such Ester Prodrugs that each one behaves as a carrier moiety for the other, a strategy known as mutual prodrugs (8). The followings are carboxylic acid, hydroxyl, phosphate, and thiol groups can some examples of mutual prodrugs, based on ester linkage, easily undergo hydrolysis via the enzymatic catalysis of that were produced to overcome several shortcomings esterases and phosphatases that are present in many associated with therapeutic drugs used in clinical practice: places in the body including liver, blood, and other tissues, Benorylate (13 in Figure 2) is a mutual prodrug of aspirin or via oxidative cleavage catalyzed by cytochrome P450 and paracetamol, coupled through an ester linkage, which enzymes (CYP) (51,55,56). is postulated to have reduced gastric irritancy with synergis- tic analgesic effect (63). Moreover, mutual prodrugs of ibu- Carboxyl esterases, acetylcholinesterases, butyrylcholines- profen with paracetamol and salicylamide have been terases, paraoxonases, arylesterases and biphenyl hydro- reported to have better lipophilicity and diminished gastric lase-like protein (BPHL) are examples of enzymes that are toxicity than the parent drug. Another example is naproxen- responsible for the hydrolytic bioactivation of ester prodrugs propyphenazone which was synthesized to prevent GI irrita- (56). For example, biphenyl hydrolase-like protein (BPHL) is tion and bleeding (64). An alternative strategy to avoid GI known to catalyze the hydrolysis of prodrugs such as vala- side-effects is by conjugation of a nitric oxide (NO) releasing cyclovir (8 in Figure 2) and valganciclovir (9 in Figure 2), as moiety to the parent NSAID drug. It has been reported that well as a number of other amino acid esters of nucleo- NO plays a gastro protective role along with prostaglandins side analogs including valyl-AZT, prodrugs of floxuridine (65). Some NO-releasing organic nitrate esters of aspirin, (5-fluoro-20-deoxyuridine or FUdR) (10 in Figure 2) and diclofenac, naproxen, ketoprofen, flurbiprofen, and ibupro- gemcitabine (11 in Figure 2) (57). Ester prodrugs are com- fen have been reported to give the corresponding active monly used to enhance lipophilicity, thus increasing mem- parent drugs with lower gastro toxicity (66,67). Reduce gas- brane permeation through masking the charge of polar tro toxicity could be achieved also by linking NSAID drug functional groups and by handling the alkyl chain length with histamine H2 antagonist such as in the case of flurbi- and configuration (51). For example, acyclovir aliphatic ester profen-histamine H2 antagonist conjugates (64). The mutual prodrugs were prepared by an esterification of the hydroxyl prodrug approach was also applied to other therapeutic group with lipophilic acid anhydride or acyl chloride (58); groups. For instance, sultamicillin (14 in Figure 2) in which hence, an enhanced lipophilicity can be achieved. Utilizing the irreversible b-lactamase inhibitor sulbactam has been the lipophilic ester approach, some acyclovir prodrugs were linked via an ester linkage with ampicillin has shown a syn- synthesized and have shown an enhanced nasal and skin ergistic effect (64), and upon oral administration, sultamicil- absorption (51). It has been shown that an increase in the lin is completely hydrolyzed to equimolar proportions of length of an alkyl chain results in a relatively ease cleavage sulbactam and ampicillin, thereby acting as an efficient of the ester bond. Therefore, it might be concluded that mutual prodrug (68). improved binding to the hydrophobic pocket of carboxylesterase can be accomplished by increasing the length of the ester alkyl chain, while branching the alkyl chain might result Amides Prodrugs in reduced hydrolysis due to a steric hindrance (51). Further, eighteen amino acid esters of acyclovir were synthesized as This approach can be exploited to enhance the stability of potential prodrugs intended for oral administration, and their drugs, provide targeted drug delivery, and change lipophi- hydrolytic reaction was shown to be catalyzed by biphenyl licity of drugs such as acids and acid chlorides (69). Drugs hydrolase (59). Acyclic nucleosides such as adefovir and that have carboxylic acid or amine group can be con- tenofovir are monophosphorylated and do not rely on viral verted into amide prodrugs.Generally, they are used to a

Chem Biol Drug Des 2013; 82: 643–668 647 Karaman

Figure 2: Chemical structures for 8–17. limited extent due to high in vivo stability. However, pro- dopamine prodrugs are developed due to dopamine inac- drugs using facile intramolecular cyclization reactions have tivation by COMT and MAO when administered by the oral been exploited to overcome this obstacle (70). route (71,72).

Similar to mutual ester prodrugs, there are some mutual A respected number of amine conjugates with amino acids prodrugs where the two active drugs are linked together by through amide linkage have been considered for providing an amide linkage, such as atorvastatin and amlodipine active drugs with remarkable enhancement in solubility which upon in vivo amide hydrolysis provide the corre- such as dapsone (16 in Figure 2) (73). sponding active parent drugs. Amide prodrugs can be converted back to the parent drugs either by nonspecific Other examples of amide based prodrugs are allopurinol amidases or by specific enzymatic activation such as N-acyl derivatives which were found to be more lipophilic renal c-glutamyl transpeptidase. Dopamine double prodrug than allopurinol itself (74). c-glutamyl-l-dopa (gludopa) (15 in Figure 2) undergoes specific activation by renal c-glutamyl transpeptidase where it achieves relatively fivefold increase in dopamine level Carbonates and Carbamates Prodrugs compared with L-dopa prodrug. However, as gludopa has low oral bioavailability, dopamine [N-(N-acetyl-L-methionyl)-O, Generally, carbonates and carbamates are more stable O-bis(ethoxycarbonyl)dopamine), a pseudopeptide prodrug of than esters but less stable than amides (75). Carbamates dopamine, was developed and has shown improved oral and carbonates have no specific enzymes for their hydro- absorption; hence, it is given orally and is used in the treat- lysis reactions; however, they are degraded by esterases ment for renal and cardiovascular diseases. Basically, to give the corresponding active parent drugs (75,76). Co-

648 Chem Biol Drug Des 2013; 82: 643–668 Inter- and Intramolecular Chemical Processes carboxymethylphenyl ester of amphetamine is an example rone, metamizole (19 in Figure 3), the methane sulfonic of carbamates prodrug that can be hydrolyzed by esterase acid of the analgesic 4-(methylamino) antipyrine is water to yield amphetamine (76). Carbamates prodrugs are soluble and suitable for parenteral route, and when given regarded as double prodrugs (pro-prodrug) because they orally, it is hydrolyzed in the stomach to give the parent are enzymatically activated at first which is followed by active drug (86). Despite the success of N-Mannich base spontaneous cleavage of the resulting carbamic acid (51). prodrugs to improve bioavailability of active drugs, there still An example for such prodrugs is fluorenylmethoxycarbon- some stability formulation problems arise from poor in vitro yl]-3 derivatives of insulin and exenatide (77) that undergo stability of some of the prodrugs (85). In addition, the in slow interconversion via carbamate bond breakdown, thus vivo formation of formaldehyde upon enzymatic breakdown providing glucose controlling agents in an adequate rate (87) of these prodrugs is considered a limitation of this pro- which consequently results in lowering the risk of drug approach. Enamines (88) (a,b-unsaturated amines) hypoglycemia (74). are unstable at low pH, which results in their limitation for use in oral administration (74). Nonetheless, an ampicillin Another example of carbamates prodrug is the one prodrug based on enamines was prepared for rectal use, obtained by linking phosphorylated steroid, an estradiol, to and it exhibits an increased absorption compared with its normustard, an alkylating agent, through a carbamate link- active parent drug (89). Enaminones are enamines of b-dic- age, which yields estramustine prodrug. The latter is used arbonyl compounds that undergo ketoenolimine-enamine in the treatment for prostate cancer. The steroid portion tautomeric equilibrium, which may offer stability to these has an antiandrogenic action and acts to concentrate the compounds (86). Enaminones are generally more lipophilic prodrug in the prostate gland where prodrug hydrolysis than their parent drugs; hence, they have an improved oral takes place and normustard action can then be exerted absorption. Typically, enaminones have a relatively high (64). Carbamates prodrugs can also be used to increase chemical stability; therefore, their use as potential prodrugs the solubility of active drugs such as cephalosporins (78). is being somewhat limited. It is expected that enaminones In addition, carbamates prodrugs have been exploited in derived from ketoesters and lactone may be subjected to targeted therapy such as ADEPT. In this case, the carba- enzymatic degradation; hence, a better conversion rate to mate group is susceptible to the action of tyrosinase the active drug can be obtained (90). enzyme present in melanomas. This approach is usually utilized in cancer targeted therapy (79). The list of carbamates prodrugs is long; among other examples is the non- Phosphate and Phosphonate Prodrugs sedating antihistamine loratadine (17 in Figure 2), an eth- ylcarbamate, that undergoes in vivo interconversion to its Phosphorylation offers increased aqueous solubility to the active form, desloratidine, through the action of CYP450 parent drugs. A traditional example of phosphate prodrugs enzymes (80), and capecitabine, an anticancer agent, that is prednisolone sodium phosphate, a water-soluble pro- undergoes a multistep activation, to finally yield 5-fluoroura- drug of prednisolone, its water solubility exceeds that of cil in the liver. Capecitabine is less toxic than 5-fluorouracil, its active form, prednisolone, by 30 times (8), it is often more selective, and widely used in clinical practice (81–83). used as an immunosuppressant, and it is formulated as a liquid dosage form (8). Another common phosphate prodrug is fosamprenavir (20 in Figure 3). Similar to predniso- Oximes Prodrugs lone, the phosphate promoiety in fosamprenavir is linked to a free hydroxyl group, and the prodrug is 10-fold more These prodrugs serve to increase the permeability of the water soluble than amprenavir. An enhanced patient com- corresponding active drugs, and they are converted pliance is achieved when using this antiviral prodrug; back to their parent drugs by microsomal cytochrome instead of administering the drug 8 times daily, dosage P450 enzymes (CYP450) (51). Dopaminergic prodrug regimen is reduced into two times per day (91). In the gut 6-(N,N-Di-n-propylamino)-3,4,5,6,7,8-hexahydro-2H-naph- and via the action of alkaline phosphatases, phosphate thalen-1-one is an example for such class (84). prodrugs are cleaved back to their corresponding active drugs and then absorbed into the systemic circulation (8). Another application of this approach is fosphenytoin (21 in N-Mannich Bases, Enaminones, and Schiff Figure 3), a prodrug of the anticonvulsant agent phenytoin. Bases (Imines) Fosphenytoin has an enhanced solubility over its corresponding drug (92). N-Mannich base formation is another approach, which can be utilized to enhance drug’s solubility. N-Mannich bases are prepared by Mannich reaction that involves reacting of Azo Compounds NH-acidic compound, an aldehyde and an amine in etha- nol (74). Rolitetracycline (18 in Figure 3) is the Mannich Colonic bacteria can be exploited in prodrug approach as derivative of tetracycline, and it is the only one available for a means of prodrug activation through the action of azo- intravenous administration (85). N-Mannich bases of dipy- reductases; this approach is applied specially in targeted

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Figure 3: Chemical structures for 18–23. drug strategy (85). Sulfasalazine (22 in Figure 3), used in carbamate, carbonates, or amide spacer can be used to the treatment for ulcerative colitis (93), is a prodrug of link the drug to PEG. Upon enzymatic breakdown of the 5-aminosalicylic acid and sulfapyridine. Upon reaching the spacer, the resultant ester or carbamate drug can be lib- colon, sulfasalazine undergoes azo bond cleavage to erated by 1,4- or 1,6-benzyl elimination (96). Daunorubi- release the active parent drug (64). Osalazine (23 in cin conjugated to PEG is an example of this kind of Figure 3), a dimer of 5-aminosalicylic acid, balsalazide, prodrugs. In this prodrug system, PEG is conjugated to and ipsalazide in which 5-aminosalicylic acid moiety is the phenol group of the open lactone via a spacer. Con- conjugated to 4-aminobenzoyl-b-alanine and 4-amin- trolling the rate of the free drug release can be accom- obenzoylglycine, respectively (94), are other examples of plished by manipulation of the substituents on the prodrugs that are activated by azo-reductases. A prodrug aromatic ring (97). by which 5-aminosalicylic acid is linked to L-aspartic acid is another example for such class that has shown a desir- The prodrug chemical approach involving enzyme catalysis able colon-speciﬁc delivery and a 50% release of 5-amino- is perhaps the most unpredicted approach, because there salicylic acid from an administered dose (95). Usually this are many intrinsic and extrinsic factors that can affect the approach is limited to aromatic amines, because azo com- bioconversion mechanisms. For example, the activity of pounds of aliphatic amines exhibit signiﬁcant instability many prodrug-activating enzymes may be changed due to (74). genetic polymorphisms, age-related physiological changes, or drug interactions, leading to adverse pharmacokinetic, pharmacodynamics, and clinical effects. In addition, there Poly Ethylene Glycol (PEG) Conjugates are wide interspecies variations in both the expression and function of most of the enzyme systems activating pro- PEG can be linked to drugs either to increase drug solu- drugs which could lead to serious challenges in the pre- bility or to prolong drug plasma half-life (74); an ester, clinical optimization phase (3–6).

650 Chem Biol Drug Des 2013; 82: 643–668 Inter- and Intramolecular Chemical Processes

Prodrugs Based on Intramolecular In the last 50 years, as mentioned earlier, scholarly studies Processes (Enzyme Models) have been carried out by Bruice (103), Cohen (104), Menger (105), Kirby (106), and others (107) to design The novel prodrug approach to be discussed in this sec- chemical models that have the capability to reach rates tion implies prodrug design based on enzyme models comparable to that with enzyme-catalyzed reactions. Fre- (mimicking enzyme catalysis) that have been advocated to quently cited examples of such models are those based understand how enzymes work. The tool used in the on rate acceleration driven by covalently enforced proxim- design is a computational approach consisting of calcula- ity. The most quoted example is Bruice et al.’s. intramo- tions using a variety of different molecular orbital and lecular ring-closing reaction of dicarboxylic semi-esters to molecular mechanics methods and correlations between anhydrides (103). Studying this model, Bruice et al. has experimental and calculated rate values (activation ener- shown that a relative rate of anhydride formation can reach gies) for some intramolecular processes that were utilized 5 9 107 upon the intramolecular ring-closing reaction of a to understand the mechanism by which enzymes might dicarboxylic semi-ester when compared to a similar coun- exert their high catalysis. In this approach, no enzyme is terpart’s intermolecular reaction. needed for the catalysis of the intraconversion of a prodrug to its active parent drug. The release rate of the pro- Other examples of rate acceleration based on proximity drug to the active drug is solely determined by the factors orientation include (1) systems that obey the principles of affecting the rate-limiting step of the intraconversion pro- Koshland’s ‘orbital steering’ theory (107) that signiﬁes the cess. Knowledge gained from the mechanisms of the pre- importance of the ground state angle of attack value of viously studied enzyme models was used in the design. the hydroxyl in hydroxycarboxylic acids on the intramolecular lactonization reaction rate; (1) the ‘spatiotemporal It is worth noting that the use of this approach might elimi- hypothesis’ advocated by Menger, which implies that a nate all disadvantages that are concerned with prodrug type of a reaction, in proton transfer processes, whether interconversion by enzymes approach. As mentioned in intermolecular or intramolecular, is signiﬁcantly determined the introduction, the bioconversion of prodrugs has many by the distance between the two reactive centers involved disadvantages related to many intrinsic and extrinsic fac- in the hydroxycarboxylic acids lactonization reaction (105); tors that can affect the process. For instance, the activity (2) the stereopopulation control proposed by Cohen to of many prodrug-activating enzymes may be varied due to explain the relatively high enhancement rates in the acid- genetic polymorphisms, age-related physiological changes, catalyzed lactonization reactions of hydroxyhydrocinnamic or drug interactions, leading to variation in clinical effects. acids containing two methyl groups on the b position of In addition, there are wide interspecies variations in both their carboxylic groups (104) and Kirby’s proton transfer the expression and function of the major enzyme systems models on the acid-catalyzed hydrolysis of acetals and activating prodrugs, and these can pose some obstacles maleamic acid amides which demonstrate the importance in the preclinical optimization phase. of hydrogen bonding formation in the products and transition states leading to them.

Intramolecular Processes (Enzyme Models) In the past 15 years, some prodrugs based on hydroxyhy- Used for the Design of Potential Prodrugs drocinnamic acids have been introduced. For example, Borchardt et al. reported the use of the 3-(2′-acetoxy-4′, Studies of enzyme mechanisms by Bruice and Benkovic, 6′-dimethyl dimethyl)-phenyl-3, 3-dimethylpropionamide Jencks, Menger, Kirby, Walsh, and Bender, over the past derivative (pro–prodrug) that is capable of releasing the ﬁve decades, have had a tremendous contribution to bet- biologically active amine (drug) upon acetate hydrolysis by ter understanding the mode and scope by which enzymes enzyme triggering. Another successful example of the catalyze biochemical transformations (98–101). pharmaceutical applications for a stereopopulation control model is the prodrug Taxol which enhances the drug Nowadays, the scientiﬁc community has reached a con- water solubility and hence affords it to be administered to sensus that the catalysis by enzymes is based on the the human body via intravenous (I.V.) injection. Taxol is the combined effects of the catalysis by functional groups and brand name for paclitaxel, a natural diterpene, approved in the ability to reroute intermolecular reactions through the USA for use as anticancer agent (108). alternative pathways by which substrates can bind to pre- organized active sites. Calculation Methods Used in the Prodrugs Design The rates for most of enzymatic reactions exceed 1010– 1018 -fold the nonenzymatic bimolecular counterparts. For In the past six decades, the use of computational chemis- example, reactions catalyzed by the enzyme cyclophilin try for calculating molecular properties of ground and tran- are accelerated by 105, and those by orotidine monophos- sition states has been a progressive task of organic, phate decarboxylase are enhanced by 1017 (102). bioorganic, and medicinal chemists alike. Computational

Chem Biol Drug Des 2013; 82: 643–668 651 Karaman chemistry uses principles of computer science to assist in Calculations of molecules exceeding 60 atoms can be solving chemical problems. It uses the theoretical chemis- made using such methods. try results, incorporated into efficient computer programs, to calculate the structures and physical and chemical Another commonly used quantum mechanical modeling properties of molecules. method in physics and chemistry to investigate the electronic structure (principally the ground state) of many-body Reaction rates and equilibrium energy-based calculations systems, in particular atoms, molecules, and the con- for biological systems that have pharmaceutical and bio- densed phases, is the density functional theory (DFT). With medicinal interests are a very important challenge to the this theory, the properties of many-electron systems can health community. Nowadays, quantum mechanics (QM), be determined using functionals, that is, functions of such as ab initio, semi-empirical, and density functional another function, which in this case is the spatially depen- theory (DFT), and molecular mechanics (MM) are dent electron density. Hence, the name density functional increasingly being used and broadly accepted as reliable theory comes from the use of functionals of the electron tools for providing structure-energy calculations for an density. DFT is among the most popular and versatile accurate prediction of potential drugs and prodrugs methods available in condensed-matter physics, computa- alike (109). tional physics, and computational chemistry. The DFT method is used to calculate structures and energies for These methods cover both static and dynamic situations. medium-sized systems (30–60 atoms) of biological and In all cases, the computer time and other resources (such pharmaceutical interest and is not restricted to the second as memory and disk space) increase rapidly with the size row of the periodic table (117). of the system being studied. Ab initio methods typically are feasible only for small systems. Ab initio methods are Despite recent improvements, there are still difficulties in based entirely on theory from first principles. The term ab using density functional theory to properly describe inter- initio was first used in quantum chemistry by Robert Parr molecular interactions, especially van der Waals forces and coworkers, including David Craig in a semiempirical (dispersion), charge transfer excitations, transition states, study on the excited states of benzene. The ab initio global potential energy surfaces, and some other strongly molecular orbital methods (quantum mechanics) such as correlated systems. Its incomplete treatment of dispersion HF, G1, G2, G2MP2, MP2, and MP3 are based on rigor- can adversely affect the accuracy of DFT in the treatment ous use of the Schrodinger equation with a number of of systems which are dominated by dispersion. The devel- approximations. Ab initio electronic structure methods opment of new DFT methods designed to overcome this have the advantage that they can be made to converge to problem, by alterations to the functional or by the inclusion the exact solution, when all approximations are sufficiently of additive terms, is a current research topic. small in magnitude and when the finite set of basic functions tends toward the limit of a complete set. The conver- On the other hand, molecular mechanics is a mathemati- gence, however, is usually not monotonic, and sometimes cal approach used for the computation of structures, the smallest calculation gives the best result for some energy, dipole moment, and other physical properties. It is properties. The disadvantage of ab initio methods is their widely used in calculating many diverse biological and computational cost. They often take enormous amounts of chemical systems such as proteins, large crystal struc- computer time, memory, and disk space (110–112). tures, and relatively large solvated systems. However, this method is limited by the determination of parameters such Other less accurate methods are called empirical or semi- as the large number of unique torsion angles present in empirical because they employ experimental results, often structurally diverse molecules (118). from acceptable models of atoms or related molecules, to approximate some elements of the underlying theory. Ab initio is an important tool to investigate functional Among these methods, the semi-empirical quantum mechanisms of biological macromolecules based on their chemistry methods are based on the Hartree–Fock formal- 3D and electronic structures. The system size, which ism, but make many approximations and obtain some ab initio calculations can handle, is relatively small despite parameters from empirical data. They are very important in the large sizes of biomacromolecules surrounding solvent computational chemistry for treating large molecules where water molecules. Accordingly, isolated models of areas of the full Hartree–Fock method without the approximations proteins such as active sites have been studied in ab initio is too expensive. Semi-empirical calculations are much calculations. However, the disregarded proteins and faster than their ab initio counterparts. Their results, how- solvent surrounding the catalytic centers have also been ever, can be very wrong if the molecule being computed is shown to contribute to the regulation of electronic struc- not similar enough to the molecules in the database used tures and geometries of the regions of interest. to parameterize the method. Among the most used semiempirical methods are MINDO, MNDO, MINDO/3, AM1, To overcome these discrepancies, quantum mechanics/ PM3, and SAM1. The semi-empirical methods have affor- molecular mechanics (QM/MM) calculations are utilized, in ded vast information for practical application (113–116). which the system is divided into QM and MM regions

652 Chem Biol Drug Des 2013; 82: 643–668 Inter- and Intramolecular Chemical Processes where QM regions correspond to active sites to be investi- the treatment for herpes simplex (163), atenolol for treating gated and are described quantum mechanically. MM hypertension with enhanced stability and bioavailability regions correspond to the remainder of the system and (164) and statins for lowering cholesterol levels in the are described molecular mechanically. The pioneer work blood (165). In addition, prodrugs for masking the bitter of the QM/MM method was accomplished by Warshel and taste of antibacterial drugs such as cefuroxime were also Levitt (119,127), and since then, there has been much designed and synthesized (166–171). The role of the link- progress on the development of a QM/MM algorithm and ers in the antibacterial prodrugs such as cefuroxime pro- applications to biological systems (120,121). drugs was to block the free amine, which is responsible for the drug bitterness, and to enable the release of the Similarly to that utilized for drug discovery, modern com- drug in a controlled manner. Menger’s Kemp acid enzyme putational methods based on QM and MM methods could model was utilized for the design of dopamine prodrugs be exploited for the design of innovative prodrugs for for the treatment for Parkinson’s disease as well (172). drugs containing different functional groups such as Prodrugs for dimethyl fumarate for the treatment psoriasis hydroxyl, phenol, or amine. For example, mechanisms of was also designed, synthesized and studied (173). intramolecular processes for a respected number of enzyme models that have been previously studied by others to understand enzyme catalysis have been recently Computationally Designed Prodrugs Based computed by us and used for the design of some novel on Intramolecular Amide Hydrolysis of prodrug linkers (122–140). Using DFT, molecular mechan- Kirby’s N-Alkylmaleamic Acids ics, and ab initio methods, numerous enzyme models were explored for assigning the factors governed the reac- Kirby et al. studied the efﬁciency of intramolecular catalysis tion rate in such models. Among the enzyme models that of amide hydrolysis by the carboxyl group of a number of have been studied are (i) proton transfer between two oxy- substituted N-methylmaleamic acids 24–30 (Figure 4) and gens and proton transfer between nitrogen and oxygen in found that the reaction is remarkably sensitive to the pat- Kirby’s acetals (141–148), (ii) intramolecular acid-catalyzed tern of substitution on the carbon–carbon double bond. In hydrolysis in N-alkylmaleamic acid derivatives (141–148), addition, the study revealed that the hydrolysis rates for (iii) proton transfer between two oxygens in rigid systems the dialkyl-N-methylmaleamic acids range over more than as investigated by Menger (149–152), (iv) acid-catalyzed ten powers of ten, and the ‘effective concentration’ of the lactonization of hydroxy-acids as researched by Cohen carboxyl group of the most reactive amide, dimethyl-N-n- 10 (104,153,154) and Menger (149–152), and (v) SN2-based propylmaleamic acid, is > 10 M. This acid amide was cyclization as studied by Brown (155), Bruice (156,157), found to be converted into the more stable dimethyl and Mandolini (158). Our recent studies on intramoleculari- maleic anhydride with a half-life of <1-second at 39 °C ty have demonstrated that there is a necessity to further below pH 3 (142). Furthermore, Kirby’s study demon- explore the reaction mechanisms for the above-mentioned strated that the amide bond cleavage is due to intramolec- processes for determining the factors affecting the reaction ular nucleophilic catalysis by the adjacent carboxylic acid rate. Unraveling the reaction mechanism would allow for group, and the dissociation of the tetrahedral intermediate better design of an efﬁcient chemical device to be utilized is the rate-limiting step (142). Later on, Kluger and Chin as a prodrug linker that can be covalently linked to a drug researched the intramolecular hydrolysis mechanism of a which can chemically, but not enzymatically, be cleaved to series of N-alkylmaleamic acids derived from aliphatic release the active drug in a programmable manner. For amines, having a wide range of basicity (161). Their study example, studying the mechanism for a proton transfer in revealed that the identity of the rate-limiting step is a func- Kirby’s acetals has led to a design and synthesis of novel tion of both the basicity of the leaving group and the acid- prodrugs of aza-nucleosides for the treatment for myelo- ity of the solution. dysplastic syndromes (159), atovaquone prodrugs for the treatment for malaria (160), less bitter paracetamol prodrugs to be administered to children and elderly as antipyretic and pain killer (161), and prodrugs of phenylephrine as decongestant (162). In these examples, the prodrug moiety was linked to the hydroxyl group of the active drug such that the drug-linker moiety (prodrug) has the potential to interconvert when exposed into physiological environ- ments such as stomach, intestine, and/or blood circulation, with rates that are solely dependent on the structural features of the pharmacologically inactive promoiety (Kir- by’s enzyme model). Other different linkers such as Kirby’s maleamic acid amide enzyme model was also explored for the design of a number of prodrugs such as tranexamic Figure 4: Acid-catalyzed hydrolysis in Kirby’s N-methylmaleamic acid for bleeding conditions, acyclovir as antiviral drug for acids 24–30.

Chem Biol Drug Des 2013; 82: 643–668 653 Karaman

To utilize N-alkylmaleamic acids, 24–30, as prodrug linkers accidents and battlefields. Similarly, the oral administration for tranexamic acid, atenolol, acyclovir, cefuroxime, and of tranexamic acid results in a 45% oral bioavailability. The other drugs, having poor bioavailability or/and undesirable total oral dose recommended in women with heavy men- (bitter) taste, we have unraveled the mechanism for their strual bleeding was two 650-mg tablets three times daily acid-catalyzed hydrolysis using DFT and molecular for 5 days. Accumulation following multiple dosing was mechanics methods. Our DFT calculation results were minimalb (175–180). found to be in accordance with the reports by Kirby et al. (142) and Kluger and Chin (174). Improvement in tranexamic acid pharmacokinetic properties may reduce the administration frequency via a variety of administration routes. This can be achieved by exploit- Tranexamic Acid Prodrugs Based on ing a carrier-linked prodrug strategyb (175–180). Kirby’s N-Alkylmaleamic Acids Continuing our study on how to utilize enzyme models as It is not often that a simple old generic product makes potential linkers for drugs containing amine, hydroxyl, or medical news. Yet this is just the case for tranexamic acid. phenol group (122–140), we have investigated the proton This small molecule that is a synthetic lysine amino acid transfer reactions in the acid-catalyzed hydrolysis of derivative has been originally developed to prevent and N-alkyl maleamic acids 24–30 (122,142) (Kirby’s enzyme reduce excessive hemorrhage in hemophilia patients and model, Figure 4) reported by Kirby et al. and based on the reduce the need for replacement therapy during and fol- calculation results of this system, we have proposed four lowing tooth extraction. Yet the use of tranexamic acid tranexamic acid prodrugs, tranexamic prodrugs ProD has been expanding beyond the small number of hemo- 1- ProD 4 (Figure 5). philia patients. Perhaps the most exciting new development about tranexamic acid has been the recent As shown in Figure 5, tranexamic acid prodrugs, ProD publication of the results of CRASH-2, a randomized con- 1-Prod 4, consist of a carboxylic group (hydrophilic moi- trolled trial undertaken in 274 hospitals in 40 countries with ety) and a lipophilic moiety (the rest of the prodrug), where 20211 adult trauma patients. Tranexamic acid was dem- the combination of both moieties secures a relatively mod- onstrated to safely reduce the risk of death in bleeding erate (adequate) HLB. It is worth noting that our proposal trauma patients. is to exploit tranexamic acid prodrugs ProD 1-ProD 4 for oral use via enteric coated tablets. At this physiological Tranexamic acid might also have a role in bleeding condi- environment, the tranexamic acid prodrugs will exist as a tions apart from traumatic injury. Postpartum hemorrhage mixture of the acidic and ionic forms where the equilibrium is a leading cause of maternal mortality, accounting for constant for the exchange between the two forms is about 100 000 maternal deaths every year. Although preli- dependent on the pKa of a given prodrug. minary evidence suggests that this drug reduces postpartum bleeding, a large trial is being undertaken to assess the effect of tranexamic acid on the risk of death and hys- Mechanistic Investigation terectomy in women with postpartum hemorrhage. Fur- thermore, the similarities of tissue injury after trauma and The DFT kinetic and thermodynamic properties for tra- surgery create a novel model for antifibrinolytic therapy nexamic acid prodrugs ProD 1- ProD 4 (Figure 5) were with tranexamic acid. Recently, a new oral formulation of calculated by DFT methods. Using the calculated DFT tranexamic acid was shown to be safe and effective for enthalpy and entropy values for the entities involved in the treatment for heavy menstrual bleedingb (175–180). One of acid-catalyzed hydrolysis of tranexamic acid prodrugs the main disadvantages of tranexamic acid is its pharma- ProD 1- ProD 4, the barriers (∆G‡) for all steps described cokinetic profile. After an intravenous dose of 1 g, the in Figure 6 were calculated. The calculated values for ‡ plasma concentration–time curve shows a terminal elimi- ∆Gf , activation energy for the tetrahedral intermediate for- ‡ nation half-life of about 2 h. The initial volume of distribu- mation, and ∆Gd , activation energy of the tetrahedral tion is about 9–12 L. More than 95% of the dose is intermediate dissociation, demonstrate that while the rate- excreted in the urine as the unchanged drug via glomeru- limiting step for all prodrugs as calculated in the gas phase lar filtration. The plasma protein binding of tranexamic acid is the tetrahedral intermediate formation, the scenario is is about 3% at therapeutic plasma levels and seems to be the opposite when the calculations were made in water. fully accounted for by its binding to plasminogen. Tranexa- The water DFT calculations indicate that the rate-limiting mic acid does not bind to serum albumin. As a result of step in the acid-catalyzed hydrolysis of tranexamic acid this pharmacokinetic profile, tranexamic acid in CRASH-2 ProD 4 is the tetrahedral intermediate formation, while that study needed to be administered using a loading dose of for ProD 1- ProD 3 is the tetrahedral intermediate col- 1 g by intravenous infusion over 10 min followed by 1 g lapse. To evaluate the factors determining the acid-cata- infused over 8 h. Although an 8-hr IV infusion may be an lyzed hydrolysis rate in tranexamic acid prodrug easy option in a hospital setting, such option may not comparison of their calculated DFT properties with previ- be available in under-developed countries or at sites of ously calculated properties for the acid-catalyzed hydroly-

654 Chem Biol Drug Des 2013; 82: 643–668 Inter- and Intramolecular Chemical Processes

Figure 5: Acid-catalyzed hydrolysis in tranexamic acid prodrugs, ProD 1- ProD 4.

Figure 6: Mechanistic pathway for the acid-catalyzed hydrolysis of 24–30 and tranexamic acid ProD1-ProD 4. sis of 24–7 (Figure 4), atenolol prodrugs ProD 1- ProD 2 ProD 1-ProD 4 were calculated and correlated with the ‡ (Figure 7), acyclovir prodrugs ProD 1- ProD 4 (Figure 7), calculated DFT activation energy values, (∆Gd ). Good and cefuroxime ProD 1- ProD 4 (Figure 7) were made. correlation was obtained between the Es (INT) (strain The calculation results reveal that the rate-limiting step energy of intermediate) in 24–30 and tranexamic acid (higher barrier) in the gas phase for all systems studied is ProD 1- ProD 3 and the activation energies for the tetra- ‡ the tetrahedral intermediate formation. On the other hand, hedral intermediate breakdown (∆Gd ). In addition, strong the picture is quite different when the calculations were correlation was found between log krel (relative rate) and carried out in dielectric constant of 78.39 (water medium). Es (INT) in 24–30. The calculations demonstrate that the While for systems 24–30 and atenolol prodrugs ProD reaction rate for systems 24–30 and tranexamic acid ProD 1-ProD 2, the rate-limiting step was the dissociation of 1-ProD 3 is dependent on the tetrahedral intermediate the tetrahedral intermediate in the reactions of cefuroxime breakdown and its value is largely affected by the strain prodrugs ProD 1- ProD 4 and acyclovir prodrugs ProD energy of the tetrahedral intermediate formed. Systems 1- ProD 4, the rate-limiting step was the formation of the with less-strained intermediates such as 25 and tranexa- tetrahedral intermediate. mic acid ProD 3 undergo hydrolysis with higher rates than those having more strained intermediates such as 27 and For assigning the factor determining the rate-limiting step, tranexamic acid ProD 1. This might be attributed to the MM2 strain energy values for the reactants (GM) and inter- fact that the transition state structures in these systems mediates (INT2) in 24–30 and tranexamic acid prodrugs resemble that of the corresponding intermediates.

Chem Biol Drug Des 2013; 82: 643–668 655 Karaman

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

Calculation of the t1/2 Values for the molarity) vs. log EMexp (experimental effective molarity) Cleavage Reactions of Tranexamic Acid and the t1/2 value for process 24 (t1/2 = 1 second) (141), Prodrugs ProD 1-ProD 4 the t1/2 values for tranexamic acid ProD 1 – ProD 4 were calculated. The predicted t1/2 at pH 2 for ProD The effective molarity parameter is considered an excel- 1 - ProD 4 is 556, 253 h, 70 seconds, and 1.7 h, lent tool to define the efficiency of an intramolecular respectively. process. Generally accepted that the measure for intramolecular efficiency is the effective molarity (EM), which log EMcalc ¼ 0:809 log EMexp þ 4:75 (1) is defined as a ratio of the intramolecular rate (kintra) and its corresponding intermolecular (kinter) where both processes are driven by identical mechanisms. The major In Vitro Kinetics Studies factors affecting the EM are ring size, solvent, and reaction type. Values in the order of 109-1013 M have been The kinetics for the acid-catalyzed hydrolysis was carried measured for the EM in intramolecular processes occur- out in aqueous buffer in the same manner as that done by ring through nucleophilic addition. Whereas for proton Kirby on his enzyme models 24–30. This is in order to transfer processes, EM values of less than 10 M explore whether the prodrug hydrolyzes in aqueous med- were reported (181) until recently where values of 1010 ium and to what extent or not, suggesting the fate of the were reported by Kirby on the hydrolysis of some prodrug in the system. Acid-catalyzed hydrolysis kinetics enzyme models (141–149). Using equation 1 obtained of the synthesized tranexamic acid ProD 1 was studied in from the correlation of log EMcalc (calculated effective four different aqueous media: 1 N HCl, buffer pH 2, buffer

656 Chem Biol Drug Des 2013; 82: 643–668 Inter- and Intramolecular Chemical Processes pH 5 and buffer pH 7.4. Under the experimental condi- administration is necessary when high doses are needed tions, the target compounds hydrolyzed to release the par- (183). Orally acyclovir is mostly used as 200-mg tablets, ent drug as evident by HPLC analysis. At constant pH and five times daily. In addition, 6 months to a year administra- temperature, the reaction displayed strict first-order kinet- tion of acyclovir is required in immune-competent patient ics as the kobs was fairly constant and a straight plot was with relapsing herpes simplex infection (183). obtained on plotting log concentration of residual prodrug verves time. Half-lives (t1/2) for tranexamic acid prodrug The oral administration therapy that currently available is ProD 1 in 1N HCl, pH 2 and pH 5 were calculated from associated with a number of drawbacks such as highly the linear regression equation correlating the log concen- variable absorption and low bioavailability (10–20%). The tration of the residual prodrug versus time, and their values main problem with the therapeutic effectiveness of acyclo- were 0.9, 23.9, and 270 h, respectively. The kinetic data vir is its absorption that is highly variable and dose depen- in 1N HCl, pH 2 and pH 5 were selected to examine the dent, thus reducing the bioavailability to 10–20%. In interconversion of the tranexamic acid prodrug in pH as of commercially available dosage forms of acyclovir, the stomach, because the mean fasting stomach pH of adult amount of drug absorbed is very low due to short resi- is approximately 1–2 and increases up to 5 following dence time of the dosage forms at the absorption site. In ingestion of food. In addition, buffer pH 5 mimics the humans, acyclovir showed poor and variable oral bioavail- beginning small intestine pathway. Finally, pH 7.4 was ability (10–20%), probably due to the relatively low lipophi- selected to examine the interconversion of the tested pro- licity of the drug. Thus, the rate-limiting factor in acyclovir drug in blood circulation system. Acid-catalyzed hydrolysis absorption is its membrane penetration (184). of the tranexamic acid ProD 1 was found to be higher in 1N HCl than at both pH 2 and 5. At 1N HCl, the prodrug Several approaches have been investigated to improve the was hydrolyzed to release the parent drug in less than oral bioavailability of acyclovir: (i) Luengo and coworkers 1 h. On the other hand, at pH 7.4, the prodrug was have used different preparations of acyclovir with b-cyclo- entirely stable and no release of the parent drug was dextrin to increase its solubility and hence its bioavailability; observed. As the pKa of tranexamic acid ProD1 is in the however, no significant effect of b-cyclodextrin on the oral range of 3–4, it is expected that at pH 5, the anionic form drug bioavailability was observed (185). (ii) Encapsulation of the prodrug will be dominant and the percentage of the of acyclovir in lipophilic vesicular structure to enhance the free acidic form that undergoes the acid-catalyzed hydroly- oral absorption and prolong the existence of the drug in sis will be relatively low. At 1N HCl and pH 2, most of the the systemic circulation (186). (iii) Yadav and coworkers prodrug will exist as the free acid form, and at pH 7.4, have used acyclovir-loaded mucoadhesive microspheres most of the prodrug will be in the anionic form, thus the for increasing the retention time and hence the bioavail- difference in rates at the different pH buffers. ability of acyclovir (187), and (iv) the search for an effective prodrug that would provide acyclovir with higher bioavail-

The t1/2 experimental value at pH 5 was 270 h, and at pH ability led to the synthesis of a number of aliphatic and 7.4, no interconversion was observed. The lack of the amino acid esters of acyclovir (188,189). reaction at the latter pH might be due to the fact that at this pH, tranexamic acid ProD 1 exists solely in the ion- Improvement in acyclovir pharmacokinetic properties may ized form (pKa about 4). As mentioned before, the free increase the absorption of acyclovir via a variety of dosing acid form is a mandatory requirement for the reaction to routes. This can be achieved by utilizing a carrier-linked proceed. prodrug strategy which could be implemented by covalently linking acyclovir to a linker to provide a drug-host On the other hand, tranexamic acid ProD 4 has a higher system which upon exposure to physiological environment, pKa than tranexamic acid ProD 1 (about 6 versus 4). There- such as stomach or intestine, can penetrate the mem- fore, it is expected that the interconversion rate of tranexa- brane tissues and release the active drug, acyclovir, in a mic acid ProD 4 to its parent drug, tranexamic acid, at all programmable manner. pHs studied will be higher (log EM for tranexamic acid ProD 4 is 14.33 versus 9.53 for tranexamic acid ProD 1). To expand our approach for utilizing intramolecularity to design potential linkers for amine drugs, we have studied the mechanism and driving forces determining the rate of Acyclovir Prodrugs Based on Kirby’s the acid-catalyzed hydrolysis in some of Kirby’s acid N-Alkylmaleamic Acid Enzyme Model amides (prodrugs linkers) (122,142). This work was carried out with the hope that such linkers might have a potential Acyclovir is a synthetic acyclic purine nucleoside analog to be good carriers to the antiviral agent, acyclovir. that is the ﬁrst agent to be registered for the treatment for and prevention of viral infections caused by herpes Based on the DFT calculation results on the acid-catalyzed simplex (HSV), varicella zoster (chicken pox), and herpes hydrolysis of maleamic acid amides 24–30 (Figure 4), four zoster (shingles) (170,182). Acyclovir water solubility is very acyclovir prodrugs were proposed (Figure 7). The acyclovir poor and has an oral bioavailability of < 20%; hence, prodrugs, ProD 1 – ProD 4 are composed of the amide

Chem Biol Drug Des 2013; 82: 643–668 657 Karaman acid linker having a carboxylic acid group (hydrophilic moi- amer adopting the more reactive conformation. Therefore, ety) and the rest of the prodrug molecule (a lipophilic moi- for cyclization to occur, the two reacting centers must be ety); the combination of both groups secures a prodrug in the gauche conformation. In the unsubstituted reactant, moiety, having a potential to be with a high permeability the reactive centers are almost completely in the anticon- (adequate HLB). The plan was to prepare acyclovir ProD formation to minimize steric interactions (156,157). 1- ProD 4 as sodium or potassium carboxylate salts due to their stability in neutral aqueous medium. It is worth not- For exploiting systems 31–35 (Figure 8) as prodrug linkers ing that N-alkylmaleamic acids such as 24–30 undergo for drugs containing hydroxyl or phenol group, we have hydrolysis in acidic aqueous medium, whereas they are recently unraveled the mechanism for their cyclization relatively stable at pH 7.4. using DFT and molecular mechanics calculation methods (130). In accordance with the results by Bruice and Pandit Based on a linear correlation between the calculated and (156,157), we have found that the cyclization reaction pro- experimental effective molarities (EM), the study on the ceeds by one mechanism, by which the rate-limiting step systems reported herein could provide a good basis for is the tetrahedral intermediate collapse and not its forma- designing prodrug systems that are less hydrophilic than tion. However, contrarily to the conclusion by Bruice’s their parent drugs and can be used, in different dosage et al., we have found that the acceleration in rate is due to forms, to release the parent drug in a controlled manner. steric effects rather than to proximity orientation stemming For example, based on the calculated log EM values, the from the ‘rotamer effect’ (190). predicted t1/2 (a time needed for 50% of the reactant to be hydrolyzed to products) for acyclovir prodrugs, ProD 1–4, were 29.2 h, 6097 days, 4.6 min, and 8.34 h, Atovaquone (ATQ) Prodrugs Based on respectively. Hence, the rate by which acyclovir prodrug Bruice’s Enzyme Model releases acyclovir can be determined according to the structural features of the linker (Kirby’s acid amide moiety). Malaria is a global public health problem, affecting 300 million clinical cases annually, and causes about 2 million deaths per year (191–193). This protozoan dis- Computationally Designed Prodrugs Based ease is caused by 5 parasites species of the genus Plas- on Bruice’s Enzyme Model modium that affect humans (P. falciparum, P. vivax, P. ovale, P. malariae, and P knowlesi) (194). The only one In this section, we summarize the results of our study on among these parasites that can cause life-threatening design and synthesis of novel prodrugs (170) via linking complications is P. falciparum (192), which is dominated the active drug with a di-carboxylic semi-ester linker in Africa and to which most drug-resistant cases are (Bruice’s enzyme model) to produce a system that having attributed. Malaria can exist in a mild form that most better physicochemical properties (adequate HLB value) commonly associated with ﬂulike symptoms: fever, vomit- than its active parent drug and is able to release the latter ing, and general malaise. While in the severe form caused in a chemically driven controlled manner. Bruice et al. by P. falciparum, nervous, respiratory, and renal compli- studied the hydrolysis of di-carboxylic semi-esters 31–35 cations frequently coexist due to serious organ failurec shown in Figure 8 and found that the relative rate (krel) for (195). 5 > 4 > 3 > 2 > 1. They attributed the acceleration in rate to proximity orientation. Using the observation that alkyl Several medications, alone or in combination such as substitution on succinic acid inﬂuences rotamer distribu- chloroquine, antifolates, artemisinins and others, show tions, the ratio between the reactive gauche and the unre- effectiveness and were considered as being the corner active anti-conformations, they proposed that gem-dialkyl stone in malaria treatment. However, drug or multidrug substitution increased the probability of the resultant rot- resistance to these agents has been escalated and consti-

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

658 Chem Biol Drug Des 2013; 82: 643–668 Inter- and Intramolecular Chemical Processes tutes a major challenge in malaria treatment (192). Accord- hydrophilic than its parent drug and is able to release ATQ ingly, the need for new anti-malarial drugs is now widely in a chemically driven controlled manner. This will result in recognized, particularly those that are structurally different introducing novel ATQ prodrugs with improved bioavailabil- from existing antimalarial drugs and possess a novel ity and better clinical profile. mechanism of action (195). Based on our calculation results that enabled us to unravel Atovaquone (ATQ) (for the structure see Figure 9), a the cyclization reaction mechanism and to assign the fac- hydroxynaphthoquinone, is relatively new treatment option, tors determining the reaction rate, we have designed five active against Plasmodium spp (196). It has a novel mech- ATQ prodrugs with a potential to have better water solubil- anism of action, acts by inhibition of the electron transport ity than ATQ and to release the active parent drug in a system at the level of cytochrome bc1 complex (197). In sustained controlled manner (Figure 9). The relative rate, malaria parasites, the mitochondria act as a sink for the log krel (effective molarity), for processes ATQ ProD electrons generated from dihydrofolate dehydrogenase, an 1- ProD 5 The experimental relative rates for the intramo- essential enzyme for pyrimidine biosynthesis; inhibition of lecular cyclization of 31–35 (Figure 8) were obtained from electron transport by ATQ leads to dihydrofolate dehydro- the division of the intramolecular rate and the correspond- genase inhibition resulting in reduced pyrimidine biosynthe- ing intermolecular reaction (156,157). For obtaining the rel- sis and thus parasite replication inhibition (198). ative rates (effective molarity, EM) for processes ATQ ProD 1– ProD 5, we assume that their corresponding in- It is well established that ATQ has an excellent safety pro- termolecular process is similar to that for systems 1–5. file and long half-life; besides, ATQ can be administered via oral route. However, ATQ has poor oral bioavailability As an excellent correlation was obtained between the acti- (less than 10% under fasted condition) and variable oral vation free energy values (∆G‡) for 31–35 and ATQ ProD absorption (199) due to its poor solubility that results from 1– ProD 5 and the difference in the strain energy values its lipophilic structure. Consequently, low and variable of the reactants and intermediates, DEs (INT-GM), the calcu- plasma and intracellular levels of the drug which is an lated values of DEs(INT-GM) for ATQ ProD 1–ProD 5 were important determinant of therapeutic outcome are used to calculate their corresponding relative rates (log krel) expected (200). Moreover, ATQ is an expensive medica- which were 6.96, 6.47, 3.78, 6.50, and -12.82, respec- tion (201). Therefore, to achieve therapeutic success and tively. These values demonstrate that ATQ ProD 1 and to meet the medical needs in malaria treatment, ATQ solu- ATQ ProD 4 are the most efficient processes among all bility improvement strategies should be addressed. The systems studied, and the least efficient are ATQ ProD 3 prodrug approach has the potential to be the most and ATQ ProD 5. Using the experimental t1/2 (the time successful among other approaches to overcome this needed for the conversion of 50% of the reactants to shortcoming. products) value for the cyclization reaction of di-carboxylic

semi-ester 31 and the calculated log krel values for pro- The study herein was conducted to design ATQ prodrugs drugs ATQ ProD 1-ProD 5, we have calculated the t1/2 through linking ATQ to a di-carboxylic semi-ester linker values for the conversion of ATQ ProD 1- ProD 5 to their

(Bruice’s enzyme model) to produce a system that is more parent drug. The calculated t1/2 values were found to be

Figure 9: Chemical structures for atovaquone prodrugs ATQ ProD 1- ProD 5.

Chem Biol Drug Des 2013; 82: 643–668 659 Karaman

Figure 10: Chemical structures of paracetamol, phenacetin and acetanilide.

ATQ ProD 3, 22.44 h, ATQ ProD1, ATQ ProD2, and ATQ humans, bitter taste perception is mediated by 25 G- ProD 4, few seconds and ATQ ProD 5 few years. There- protein-coupled receptors of the hTAS2R gene family fore, the interconversion rates of atovaquone prodrugs to (211). atovaquone can be programmed according to the nature of the prodrug linker. Drugs such as macrolide antibiotics, non-steroidal anti- inflammatory, and penicillin derivatives have a pronounced bitter taste (212). Masking the taste of water-soluble bitter Paracetamol Prodrugs Based on Bruice’s drugs, especially those given in high doses, is difficult to Enzyme Model achieve using sweeteners alone. As a consequence, several approaches have been studied and have resulted in The palatability of active drugs possesses significant the development of more efficient techniques for masking obstacle in developing a patient convenient dosage form. the bitter taste of molecules. All of the developed Organoleptic properties, such as taste, are an important techniques are based on the physical modification of the factor when selecting a drug from the generic products formulation containing the bitter tastant. available in the market that have the same active ingredi- ent. It is a key issue for doctors and pharmacists adminis- Although these approaches have helped to improve the tering the drugs and particularly for the pediatric and taste of some drugs formulations, the problem of the bitter geriatric populations (202). taste of drugs in pediatric and geriatric formulations still creates a serious challenge to the health community. Organic and inorganic molecules dissolve in saliva and Thus, different strategies should be developed to over- bind to taste receptors on the tongue giving a bitter, come this serious problem. sweet, salty, sour, or umami sensation. Bitter taste is sensed by the receptors on the posterior part of the Bitter tastant molecules interact with taste receptors on tongue. The sensation is a result of signal transduction the tongue to give bitter sensation. Altering the ability of from taste receptors located in areas known as taste the drug to interact with its bitter taste receptors could buds. The taste buds contain very sensitive nerve end- reduce or eliminate its bitterness. This could be achieved ings, which are responsible for the production and trans- by an appropriate modification of the structure and the mission of electrical impulses via cranial nerves VII, IX, size of a bitter compound. and X to certain areas in the brain that are devoted to the perception of taste (203). Bitter taste receptors are Paracetamol is an odorless, bitter crystalline compound believed to have evolved for organism protection against widely used as pain killer and antipyretic. Paracetamol was the ingestion of poisonous food products. Molecules with found in the urine of patients who had taken phenacetin, bitter taste (204–208) are very diverse in their chemical and later on it was demonstrated that paracetamol was a structure and physicochemical properties (209,210). In urinary metabolite of acetanilide (Figure 10) (161).

Figure 11: Acid-catalyzed hydrolysis of paracetamol ProD 1- ProD 2 to paracetamol and inactive linker.

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Phenacetin, on the other hand, lacks or has a very slight Prodrug design based on a computational approach con- bitter taste (161). Careful inspection of the structures of sisting of calculations using molecular orbital (MO) and paracetamol and phenacetin indicates that the only differ- molecular mechanics (MM) methods and correlations ence in the structural features in both is the nature of the between experimental and calculated values of intramolec- group on the para position of the benzene ring. While in ular processes has the potential to be a very effective tool the case of paracetamol, the group is hydroxy, in phenac- to be utilized for obtaining effective prodrugs that are capa- etin it is ethoxy. Another related example is acetanilide that ble of releasing the parent drug in a programmable fashion. has a chemical structure similar to that of paracetamol In this prodrug approach, no enzyme is needed for the and phenacetin, but it lacks the group at the para position catalysis of the intraconversion of a prodrug to its parent of the benzene ring. Acetanilide has a burning taste and drug. The interconversion of the prodrug is solely depen- lacks the bitter taste characteristic for paracetamol (161). dent on the rate-limiting step for the intramolecular reaction. The combined facts described above suggest that the presence of hydroxy group on the para position is the The future of prodrug design is forthcoming yet extremely major contributor for the bitter taste of paracetamol. challenging. Progresses must be made in better under- Hence, it is expected that blocking the hydroxy group in standing the chemistry of many organic mechanisms that paracetamol with a suitable linker could inhibit the interac- can be effectively exploited to push forward the develop- tion of paracetamol with its bitter taste receptor/s and, ment and advances of even more types of prodrugs. The hence masking its bitterness. understanding of the organic reactions mechanisms of intramolecular processes will be the next major milestone in It seems reasonable to assume that the phenolic hydroxyl this ﬁeld. It is envisioned that the future of prodrug design group in paracetamol is crucial for obtaining the bitter holds the ability to produce safe and efﬁcacious delivery of taste characteristic for paracetamol. This might be due to a wide range of active small molecule and biotherapeutics. the ability of paracetamol to be engaged in a hydrogen bonding net with the active site of its bitter taste receptor via its phenolic hydroxyl group. Acknowledgments

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