The Prodrug Approach: a Successful Tool for Improving Drug Solubility

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Review The Prodrug Approach: A Successful Tool for Improving Drug Solubility

Daniela Hartmann Jornada, Guilherme Felipe dos Santos Fernandes, Diego Eidy Chiba, Thais Regina Ferreira de Melo, Jean Leandro dos Santos and Man Chin Chung *

Received: 14 October 2015 ; Accepted: 15 December 2015 ; Published: 29 December 2015 Academic Editors: Thomas Rades, Holger Grohganz and Korbinian Löbmann

Faculdade de Ciências Farmacêuticas, UNESP—University Estadual Paulista, Rodovia Araraquara Jaú Km 01, 14801-902 Araraquara, São Paulo, Brasil; [email protected] (D.H.J.); [email protected] (G.F.S.F.); [email protected] (D.E.C.); [email protected] (T.R.F.M.); [email protected] (J.L.S.) * Correspondence: [email protected]; Tel.: +55-16-3301-6962; Fax: +55-16-3301-6960

Abstract: Prodrug design is a widely known molecular modiﬁcation strategy that aims to optimize the physicochemical and pharmacological properties of drugs to improve their solubility and pharmacokinetic features and decrease their toxicity. A lack of solubility is one of the main obstacles to drug development. This review aims to describe recent advances in the improvement of solubility via the prodrug approach. The main chemical carriers and examples of successful strategies will be discussed, highlighting the advances of this ﬁeld in the last ten years.

Keywords: prodrug; solubility; water-soluble prodrugs; solubility of prodrugs; molecular modification

1. Introduction Poor solubility is one of the main problems faced by researchers during drug development. Commonly, even with the use of current computational “filters” to minimize this problem, compounds that are active in vitro may lack adequate pharmacokinetic properties and/or may be difficult to formulate [1]. A study conducted with the top 200 oral drug products in Japan, Great Britain, United States and Spain revealed that approximately 37% of drugs had solubilities of less than 0.1 mg/mL. One explanation may include the need for drugs that are highly potent in low doses, however, this issue represents a challenge in drug discovery [2]. Although the prodrug approach is often considered only when the prototype presents unexpected problems, this strategy offers a versatile approach to drug development and should not be considered a last resort. The prodrug approach is a promising molecular modification by which drug developers and designers can modulate drug pharmacokinetics, pharmacodynamics and toxicology [3]. A prodrug is a poorly active or inactive compound containing the parental drug that undergoes some in vivo biotransformation through chemical or enzymatic cleavage, enabling the delivery of the active molecule at efficacious levels [1,3,4]. Prodrugs are conventionally classified in two major classes: carrier-linked prodrugs and bioprecursors. Carrier-linked prodrugs can be classified as bipartite prodrugs, in which the carrier is linked directly to the parent drug, and tripartite prodrugs, in which a spacer links the carrier to the parent drug [5]. Carriers are commonly attached by chemical groups such as ester, amide, carbamate, carbonate, ether, imine, phosphate, among others [1,6,7] (Figure1). Mutual prodrugs are a type of carrier-linked prodrug in which two active compounds are linked each acting as the carrier to the other. These prodrugs have increased effectiveness through synergistic action [5,8]. Another type of carrier-linked prodrug is the macromolecular prodrug; these prodrugs use polymeric backbones as carriers. Macromolecular prodrugs are commonly used to design prodrugs that will be cleaved inside of a cell and in targeted drug-delivery systems. This approach offers improved drug

Molecules 2016, 21, 42; doi:10.3390/molecules21010042 www.mdpi.com/journal/molecules Molecules 2016, 21, 42 2 of 31 Molecules 2016, 21, 42 2 of 30 solubility,offers improved stability, drug drug solubility, release and stability, pharmacokinetics. drug release Additionally, and pharmacokinetics. it can facilitate Additionally, the accumulation it can offacilitate a drug at the the accumulation site of action of and a drug improve at the site safety of action [9,10]. and Bioprecursors improve safety are [9,10]. inactive Bioprecursors compounds are that doinactive not have compounds a carrier and that are do rapidly not have converted a carrier to and an activeare rapidly drug converted after metabolic to an reactionsactive drug (normally after redoxmetabolic reactions) reactions [5,8]. (normally redox reactions) [5,8].

FigureFigure 1. 1.In In vivo vivobioactivation bioactivation of of prodrugsprodrugs byby enzymaticenzymatic and/or and/or chemical chemical transformations. transformations.

Undesirable properties, including poor aqueous solubility, chemical instability, insufficient oral or Undesirablelocal absorption, properties, fast pre-syst includingemic poor metabolism, aqueous solubility,low half-life, chemical toxicity instability, and local insufficient irritation oralare or localcommonly absorption, resolved fast pre-systemic using the prodrug metabolism, approach. low In half-life, addition, toxicity problems and related local irritation to drug formulation are commonly resolvedand delivery using thecan prodrugalso be overcome approach. by In using addition, this st problemsrategy [1,11–13]. related toOccasionally, drug formulation strategies and such delivery as canparticle-size also be overcome reduction, by solubilizing using this strategy excipients, [1, 11complexation–13]. Occasionally, agents and strategies use of suchsurfactants as particle-size fail to reduction,improve solubilizingthe solubility excipients,in water profile complexation and reduce agents toxicity and to usedesirable of surfactants levels [13,14]. fail toFor improve example, the solubilitysome surfactants in water profileused in and parenteral reduce toxicityformulations to desirable frequently levels have [13 ,toxic14]. Foreffects example, such as some anaphylactic surfactants usedreactions in parenteral [14]. formulations frequently have toxic effects such as anaphylactic reactions [14]. However,However, in in some some cases, cases, low-solubility low-solubility compoundscompounds yield yield false-positive false-positive results results on on in invitro vitro assaysassays duedue to to non-specific non-specific binding binding [15[15].]. Moreover,Moreover, in clinical trials, trials, low-solubility low-solubility drugs drugs can can lead lead to to precipitationprecipitation and and crystalluria, crystalluria, raising raising additionaladditional safety concerns. concerns. Poorly Poorly soluble soluble drugs drugs have have recently recently beenbeen discontinued discontinued in in clinical clinical assays assays forfor thisthis reasonreason [[15,16].15,16]. ForFor these these reasons, reasons, the the prodrug prodrug approach approach presents presents a safe a andsafe effectiveand effective strategy strategy by which by which to improve to theimprove solubility the ofsolubility drugs. Thisof drugs. review This aims review to presentaims to thepresent latest the strategies latest strategies in prodrug in prodrug design design used to obtainused water-solubleto obtain water-soluble compounds comp forounds oral andfor oral parenteral and parenteral uses. We uses. selected We selected research research from thefrom last the ten last ten years (2005 through August of 2015) showing increased solubility through the prodrug years (2005 through August of 2015) showing increased solubility through the prodrug approach. approach. In the literature search, we used the following terms: “water-soluble prodrugs”, “increased In the literature search, we used the following terms: “water-soluble prodrugs”, “increased solubility solubility prodrugs” and “enhanced solubility prodrugs” in published databases including PubMed, prodrugs” and “enhanced solubility prodrugs” in published databases including PubMed, LILACS, LILACS, Scielo, Cochrane, Web of Science and Scopus. Scielo, Cochrane, Web of Science and Scopus. 2. Ester Prodrugs 2. Ester Prodrugs The features of an ideal prodrug include the following: (a) hydrolysis resistance during The features of an ideal prodrug include the following: (a) hydrolysis resistance during absorption; absorption; (b) weak or no activity; (c) aqueous solubility; (d) good permeability through the cells; (b) weak or no activity; (c) aqueous solubility; (d) good permeability through the cells; (e) chemical

Molecules 2016, 21, 42 3 of 31 stabilityMolecules at different 2016, 21, 42 pHs; (f) kinetics that allow release of the parental drug [17]. Among the3 chemicalof 30 bonds(e) used chemical to link stability the parentalat different drug pHs; and (f) kinetics carrier, that esters allow have release proven of the toparental be promising drug [17]. dueAmong to their amenabilitythe chemical to hydrolysis bonds used both to linkin vivothe parentaland in vitrodrug. and Some carrier, examples esters have of the proven use of to estersbe promising in prodrug designdue are to discussedtheir amenability below. to hydrolysis both in vivo and in vitro. Some examples of the use of esters in Theprodrug enzyme design thioredoxin–thioredoxin are discussed below. reductase plays an important role in thioredoxin system by catalyzingThe the enzyme reduction thioredoxin–thioredoxin of thioredoxin. Speciﬁcally, reductase play the thioredoxins an important system role in participatesthioredoxin system in protecting by DNAcatalyzing against oxidativethe reduction damage of thioredoxin. and has Specifically been implicated, the thioredoxin in several system diseases, participates including in protecting cancer and rheumatoidDNA against arthritis oxidative [18]. Among damage theand described has been inhibitors,implicated in the several naphthoquinone diseases, including spiroketal cancer compound and palmarumycinrheumatoid ( 1arthritis) has shown [18]. Amongin vitro theanticancer described activity; inhibitors, however, the naphthoquinone it failed in in spiroketal vivo assays. compound The authors palmarumycin (1) has shown in vitro anticancer activity; however, it failed in in vivo assays. The hypothesized that the lack of activity could be due to its high lipophilicity; therefore, they designed authors hypothesized that the lack of activity could be due to its high lipophilicity; therefore, they prodrugsdesigned containing prodrugs containing amino-esters amino-esters and two and morpholine two morpholine analogues. analogues. The The glycyl ester ester derivative derivative 2 2 (Figure(Figure2) was 2) morewas more than than seven seven times times more more soluble soluble inin waterwater than than its its parent parent drug. drug. Despite Despite the higher the higher solubilitysolubility compared compared to theto the parent parent drug, drug, the the compound compound did did not not exhibi exhibitt superior superior activity activity compared compared to to palmarumycinpalmarumycin CP1 CP[191 ].[19].

FigureFigure 2. 2.Glycyl Glycyl ester ester and and amino-acid-esteramino-acid-ester prodrugs prodrugs [19,20]. [19,20 ].

The adenosine A2A receptor is a G-protein-coupled receptor expressed at high levels in blood Theplatelets, adenosine GABAergic A2A receptorneurons, isand a G-protein-coupledcells of the thymus, receptorthe olfactory expressed bulb and at highthe spleen. levels This in blood platelets,receptor GABAergic has been widely neurons, known and as cellsa target of since the thymus,the approval the of olfactory the drug regadenoson bulb and the by spleen.the U.S. This receptorFood has and beenDrug widelyAdministration known (FDA). as a targetAmong since the ac thetivities approval described of for the adenosine drug regadenoson antagonists are by the U.S. Foodneuroprotective, and Drug antidepressant, Administration and (FDA). analgesic Among effects. the In activitiesaddition, this described receptorfor has adenosine been explored antagonists as a are neuroprotective,target of new therapeutic antidepressant, agents to and treat analgesic Parkinson’s effects. disease In addition, [21]. Compound this receptor 3, known has beenas MSX-2 explored as a target(Figure of 2), new is a therapeutic potent and agentsselective to antagonist treat Parkinson’s of the adenosine disease [21 A].2A Compoundreceptor [22,23].3, known Despite as its MSX-2 in vitro activity, this molecule shows low solubility in water, hindering in vivo studies. Two different (Figure2), is a potent and selective antagonist of the adenosine A receptor [22,23]. Despite its approaches were undertaken to resolve this issue. The first involved the2A introduction of a phosphate in vitro in vivo subunitactivity, to increase this molecule solubility. shows Phosphate low solubility prodrugs have in water, shown hindering high solubility instudies. water; however, Two different it approachesis not readily were undertakenabsorbed by the to resolve oral route. this The issue. second The approach ﬁrst involved involved the the introduction introduction of of a amino phosphate subunitacids, to increasespecifically solubility. L-valine. PhosphateThe solubility prodrugs in water have of compound shown high 4 (Figure solubility 2), as in evaluated water; however, by a it is notspectrophotometric readily absorbed method by the oralis 7.3 route.mg/mL, The less second than that approach of the phosphate involved prodrug the introduction (9.0 mg/mL) of but amino acids, speciﬁcally L-valine. The solubility in water of compound 4 (Figure2), as evaluated by a spectrophotometric method is 7.3 mg/mL, less than that of the phosphate prodrug (9.0 mg/mL) but Molecules 2016, 21, 42 4 of 31

superiorMolecules to that 2016, of21, the42 parent drug. According to the authors, the valine prodrug was stable in4 of aqueous 30 solution at room temperature for up to four days; however, in the presence of pig liver esterase, the compoundsuperior undergoes to that of the bioconversion, parent drug. According releasing to thethe parentalauthors, the drug, valine with prodrug a half-life was stable of 6.9 in min aqueous [20]. solution at room temperature for up to four days; however, in the presence of pig liver esterase, the New water-soluble antiviral compounds also use ester amino-acid prodrugs to increase solubility compound undergoes bioconversion, releasing the parental drug, with a half-life of 6.9 min [20]. with a bicyclicNew water-soluble nucleoside subunit.antiviral compounds This scaffold also was use initially ester amino-acid described prodrugs as a by-product to increase of the condensationsolubility ofwith 5-iodo a bicyclic nucleosides nucleoside within subunit. terminal This scaffold alkynes was catalyzed initially described by palladium; as a by-product however, ofin vitro studiesthe revealed condensation activity of 5-iodo against nucleosides several viruses, within includingterminal alkynes herpes catalyzed simplex (typeby palladium; 1 and type however, 2), varicella zosterin andvitro cytomegalovirus.studies revealed activity Oneof against the most several active viruses, compounds including was herpes the psimplex-penthylphenylbicyclic (type 1 and nucleosidetype 2), analogue varicella Cf1743;zoster and however, cytomegalovirus. its low solubility One of inthe water most limitsactive itscompounds use [24]. Thewas prodrugthe approachp-penthylphenylbicyclic was used to resolve nucleoside this limitation, analogue Cf1743; with dipeptides however, its as low carriers solubility of Cf1743 in water (Val, limits Asn, its Lys, Asp)use (Figure [24]. 3The). The prodrug amino-acid approach pro-residue was used to is resolve recognized this limitation, by the enzyme with dipeptides dipeptidyl-peptidase as carriers of IV Cf1743 (Val, Asn, Lys, Asp) (Figure 3). The amino-acid pro-residue is recognized by the enzyme (DPPIV/CD26), which is expressed in leukocytes as well as epithelial, endothelial and ﬁbroblast cells. dipeptidyl-peptidase IV (DPPIV/CD26), which is expressed in leukocytes as well as epithelial, It was hypothesized that leukocyte enzymes would activate the prodrug. Interestingly, the authors endothelial and fibroblast cells. It was hypothesized that leukocyte enzymes would activate the foundprodrug. an analog Interestingly,5 with 4000-fold the authors greater found an solubility analog 5 inwith water 4000-fold and 7–15-foldgreater solubility greater in bioavailabilitywater and compared7–15-fold to thegreater parent bioavailability drug [25]. compared to the parent drug [25].

O N Cf1743 DPPIV/CD26 HO O enzyme N O O HN spacer Xaa-Pro O ester hydrolysis O (5)

Xaa- Val, Asn, Lys, Asp Spacer- Val, Ala, Leu, Phe

FigureFigure 3. 3.Bicyclic Bicyclic furanopyrimidine furanopyrimidine nucleoside nucleoside analogue analogue [25]. [25 ].

Amino acids were also used to improve the physicochemical properties of oleanolic acid (6). AminoThis triterpenoid acids were molecule also used is a tonatural improve product the found physicochemical in Asian herbs properties whose pharmacological of oleanolic acid effects (6). This triterpenoidinclude anti-inflammatory, molecule is a natural analgesic product and found antitumoral in Asian activities. herbs whoseDespite pharmacological these effects, oleanolic effects acid include anti-inflammatory,has low bioavailability analgesic in andrats antitumoral(7%), probably activities. due to its Despite low solubility these effects, in water oleanolic (0.0012 acidμg/mL). has low bioavailabilityAttempts to in improve rats (7%), the probablysolubility dueusing to differen its lowt formulations solubility in failed, water probably (0.0012 µdueg/mL). to its Attemptslow to improvepermeability. the solubility To explore using the transpor differentters formulationsinvolved in the failed,absorption probably of oleanolic due acid, to its the low researchers permeability. designed a series of prodrugs intended to be recognized by the PepT1 transporter. This transporter To explore the transporters involved in the absorption of oleanolic acid, the researchers designed a is important in drug absorption because it improves bioavailability. One commercially successful series of prodrugs intended to be recognized by the PepT1 transporter. This transporter is important prodrug that exploits this transporter is valaciclovir, which has improved bioavailability (55%) in drugcompared absorption to its parental because drug, it improves acyclovir bioavailability.(15%–20%) [26–28]. One This commercially transporter could successful also improve prodrug the that exploitsabsorption this transporter of oleanolic-acid is valaciclovir, derivatives. which A series has of improvednovel ethylene-glycol-linked bioavailability (55%)amino-acid-diester compared to its parentalprodrugs drug, of acyclovir oleanolic (15%–20%)acid was synthesized [26–28]. Thisand tested transporter for solubility could and also pharmacokinetic improve the absorption profile. of oleanolic-acidSix analogs derivatives. showed improved A series solubility of novel in water. ethylene-glycol-linked The compound 7 containing amino-acid-diester an L-valine prodrugssubunit of oleanolic(Figure acid 4) was was the synthesized most promising and analog, tested exhibiting for solubility solubility and greater pharmacokinetic than 25 μg/mL profile. and increased Six analogs showedintestinal improved perfusion solubility and oral in bioavailability water. The in compound rats [29]. In7 a containingdifferent paper, an Lthe-valine same research subunit group (Figure 4) was thedescribed most promisinga series of seven analog, propylene exhibiting glycol-linked solubility amino-acid/dipeptide-diester greater than 25 µg/mL prodrugs and increased of oleanolic intestinal acid. In this study, propylene glycol (PEG) was chosen as a spacer due to its very low toxicity in vivo. perfusion and oral bioavailability in rats [29]. In a different paper, the same research group described a The authors synthesized seven diester prodrugs and evaluated the influence of the linker on series of seven propylene glycol-linked amino-acid/dipeptide-diester prodrugs of oleanolic acid. In this pharmacokinetic properties. Compound 8 was the most water soluble (up to 1.0 mg/mL; Figure 4). study, propylene glycol (PEG) was chosen as a spacer due to its very low toxicity in vivo. The authors synthesized seven diester prodrugs and evaluated the influence of the linker on pharmacokinetic Molecules 2016, 21, 42 5 of 31

Molecules 2016, 21, 42 5 of 30 properties. Compound 8 was the most water soluble (up to 1.0 mg/mL; Figure4). Compared to ethylene-glycolMoleculesCompared 2016 ,to 21 derivatives, ,ethylene-glycol 42 the propylene-glycolderivatives, the propyl seriesene-glycol was the mostseries soluble,was the stablemost soluble, and bioavailable stable5 of 30 andand had bioavailable the greatest and afﬁnity had the for greatest the PepT1 affinity enzyme for the [30 PepT1]. enzyme [30]. Compared to ethylene-glycol derivatives, the propylene-glycol series was the most soluble, stable

and bioavailable and had the greatest affinity for the PepT1 enzyme [30]. L-Valine

O L-Valine O NH H O 2 O O H O H C NHCH2 H O 3 H 3 HO H O H (7) H C CH Solubility3 H= 25 µg3 /mL HO H H (7) OH Solubility = 25 µg /mL H O H OH L-Alanine, L-Valine HO (6) H O O L-Alanine,O L-Valine H H H N NH2 HO (6) O O ParentalH drug O O H Oleanoic acid O H H CH3N H C CHNH2 Solubility = 0.012 µg/mL O O3 H 3 Parental drug O Oleanoic acid O H CH3 H3C CH3 Solubility = 0.012 µg/mL HO H H O(8) Solubility > 1 mg/mL HO FigureFigure 4. 4.Ethylene Ethylene glycol glycol and and propylenepropyleneH glycol prodrugs prodrugs(8) of of oleanoic oleanoicSolubility acid acid [29,30]. > [ 129 mg/mL,30 ]. Another Figureterpenoid 4. Ethylene with low glycol solubility and propylene in water glycol whose prodrugs properties of oleanoic were acid enhanced [29,30]. by the use of theAnother prodrug terpenoid approach withis oridonin low solubility (9). This natural in water product whose is properties found in Rabdosia were enhanced rubescens, bya Chinese the use of Another terpenoid with low solubility in water whose properties were enhanced by the use of themedicinal prodrug approachplant, and is it oridonin exhibited (9 ).antitumoral This natural acti productvity against is found several in Rabdosiatypes of rubescenscancer, including, a Chinese the prodrug approach is oridonin (9). This natural product is found in Rabdosia rubescens, a Chinese medicinalleukemia plant, [31,32]. and Exploring it exhibited polyethylene antitumoral glycol activityas carrier, against the authors several linked types oridonin of cancer, to PEG, including using medicinal plant, and it exhibited antitumoral activity against several types of cancer, including leukemiasuccinic [31 acid,32 ].as Exploring a spacer. Four polyethylene different molecular glycol as weights carrier, of the PEGs authors were linked tested oridoninto increase to solubility PEG, using leukemia [31,32]. Exploring polyethylene glycol as carrier, the authors linked oridonin to PEG, using succinicin water. acid The asa greatest spacer. increase Four different in solubility molecular was observed weights for of PEGsa low werePEG-molecular-weight tested to increase (5 solubility kDa) succinic acid as a spacer. Four different molecular weights of PEGs were tested to increase solubility in water.conjugate The 10 greatest (Figure 5). increase This prodrug in solubility had 99.2 was times observed the solubility for a lowof oridonin. PEG-molecular-weight A chemical hydrolysis (5 kDa) in water. The greatest increase in solubility was observed for a low PEG-molecular-weight (5 kDa) study showed a sustained-release effect for the prodrugs. In vivo studies using the two intermediate conjugateconjugate10 10(Figure (Figure5). 5). This This prodrug prodrug had had 99.2 99.2 times times thethe solubilitysolubility of of oridonin. oridonin. A A chemical chemical hydrolysis hydrolysis conjugates (10 and 20 kDa) demonstrated a successful use of this prodrug approach, as these studystudy showed showed a sustained-release a sustained-release effect effectfor for thethe prodrugs.prodrugs. InIn vivo vivo studiesstudies using using the the two two intermediate intermediate derivatives have better pharmacokinetic profiles than oridonin [33]. conjugatesconjugates (10 and(10 and 20 kDa) 20 kDa) demonstrated demonstrated a successful a successful use of use this of prodrug this prodrug approach, approach, as these as derivatives these havederivatives better pharmacokinetic have better pharmacokinetic proﬁles than profiles oridonin than [33 oridonin]. [33]. OH CH2 OH OH CH2 O O OH Oridonin OH O O (9) OH Oridonin OH (9) OH 99.2-fold increase in solubility O 99.2-fold increase CH3 in solubility n H O N O O CH3 OH CH2 n H PEG (MW = 5KDa) O N O O O OH CH2 O PEG (MW(10) = 5KDa) O O Prodrug conjugateO O (10) OH O Prodrug conjugate OH OH Figure 5. PEG prodrugs of oridonin [33]. OH

FigureFigure 5. 5.PEG PEG prodrugsprodrugs of of oridonin oridonin [33]. [33 ].

Molecules 2016, 21, 42 6 of 31

MoleculesA similar 2016 approach, 21, 42 exploring the use of PEG to increase solubility was carried out using6 anotherof 30 anticancerMolecules compound,2016, 21, 42 gambogic acid (11). This natural product inhibits tumor growth and6 inducesof 30 A similar approach exploring the use of PEG to increase solubility was carried out using another apoptosis in various cancer cells [34]. The authors synthesized a series of PEGylate prodrugs 12 anticancerA similar compound, approach gambogic exploring acid the use(11). of This PEG natural to increase product solubility inhibits was tumor carried growth out using and anotherinduces with molecular weights of 2 kDa, 4 kDa, 10 kDa and 20 kDa using L-leucine as a spacer (Figure6). anticancerapoptosis incompound, various cancer gambogic cells [34].acid The(11). auth Thisors natural synthesized product a seriesinhibits of tumorPEGylate growth prodrugs and induces12 with The solubility in water ranged from 645 mg/mL to 1750 mg/mL. The most soluble prodrug contained apoptosismolecular inweights various of cancer 2 kDa, cells 4 kDa, [34]. 10The kDa auth andors 20synthesized kDa using a Lseries-leucine of PEGylateas a spacer prodrugs (Figure 126). withThe PEG-2 kDa-L-leucine-GA. For all prodrugs, the half-life measured in plasma ranged from 1.26 to molecularsolubility inweights water rangedof 2 kDa, from 4 645kDa, mg/mL 10 kDa to and1750 20mg kDa/mL. using The most L-leucine soluble as prodruga spacer contained (Figure 6). PEG-2 The 6.12solubilitykDa- h [35L-leucine-GA.]. in water rangedFor all prodrugs,from 645 mg/mLthe half-life to 1750 measur mg/mL.ed in The plasma most ranged soluble from prodrug 1.26 tocontained 6.12 h [35]. PEG-2 kDa-L-leucine-GA. For all prodrugs, the half-life measured in plasma ranged from 1.26 to 6.12 h [35]. OH H O O O O O OH H O O O O OH OH O (11) H GambogicOH O acid (11) < 0.5 mg/mL Gambogic acid H O < 0.5 mg/mL O O R O O NH O H O O O O (CH CH O) CH CH O O O H 2 2 n 2 2 R OH O HN O OH O O O NH R O (CH CH O) CH CH O O H O O 2 2 n 2 2 (12)OH O HN O H O O HO R O HO O (12) Solubility: 645 to 1750 mg/mL HO CH O H 3 Solubility: 645 to 1750 mg/mL R= H2CCHCH3 CH3 R= H CCHCH FigureFigure 6. 6.General General structure structure ofof gambogic-acid PEG PEG prodrugs prodrugs2 [35]. [35 3 ]. Figure 6. General structure of gambogic-acid PEG prodrugs [35]. The poor solubility in water of taxoids is well established in the literature [36]. Using the The poor solubility in water of taxoids is well established in the literature [36]. Using the prodrug prodrugThe approach,poor solubility Skwarczynski in water et of al. taxoids, synthesized is well prodrugs established whose in soluthe bilitiesliterature ranged [36]. fromUsing 0.8 the to approach, Skwarczynski et al., synthesized prodrugs whose solubilities ranged from 0.8 to 1.1 mg/mL. prodrug1.1 mg/mL. approach, Interestingly, Skwarczynski the authors et al., synthesizedidentified a prodrugsnovel mechanism whose solu ofbilities parental-drug ranged from release: 0.8 toa Interestingly, the authors identiﬁed a novel mechanism of parental-drug release: a pH-dependent 1.1pH-dependent mg/mL. Interestingly, O–N intramolecular the authors acyl identified migration a reactionnovel mechanism (Figure 7). ofThis parental-drug strategy increased release: the a O–NpH-dependentstability intramolecular of taxoids, O–N acyl released intramolecular migration the parental reaction acyl drugmigration (Figure at pH7 ).reaction7.4 This and strategyimproved (Figure increased7). drug This solubility. strategy the stability Someincreased of of these taxoids, the releasedstabilityprodrugs the of parental containing taxoids, drug released the at 2′ pH-O the-isoform 7.4 parental and of improved drugtaxoids, at pH such drug 7.4 asand solubility. compound improved Some 14 drug, exhibited of solubility. these prodrugs4000-fold Some ofgreater containing these 1 the 2prodrugssolubility-O-isoform containingthan ofthe taxoids, parent the 2 drug′- suchO-isoform 13 as [37]. compound of taxoids, 14such, exhibited as compound 4000-fold 14, exhibited greater 4000-fold solubility greater than the parentsolubility drug 13 than[37 the]. parent drug 13 [37]. O O HO O O O HO O O (14) O O O O HO O O O HO O O O N O (14) PBS O O NH2 H O H O O O OH H O O O O OH O N O pH = 7.4 O O PBS O OH O NH2 H O H O OH (13) OH O O O-N intramolecular O O O OH pH = 7.4 O O acyl migration reaction Parent drug OH O O (13) O O O O-N intramolecular acyl migration reaction Parent drug PBS = phosphate-buffered saline Increase solubility: 4000-fold

PBS = phosphate-buffered saline Increase solubility: 4000-fold

Water-soluble moiety

Water-solubleHO OHmoiety HO O OH O OH O O O O HO OH O OH HO O O O OH OH OH O O N O O O H O H O OH O OH O O O OH OH O O O O N O O H H Increase solubility: H O O OH 52-fold O O N O O OH (15) O O H O Parent drug O OH OH O O Increase solubility: H O O O N O (16) (15) 52-fold O H Parent drug OH OH O O (16) Figure 7. Taxoid prodrugs [37,38]. Figure 7. Taxoid prodrugs [37,38]. Figure 7. Taxoid prodrugs [37,38].

Molecules 2016, 21, 42 7 of 31 Molecules 2016, 21, 42 7 of 30 Molecules 2016, 21, 42 7 of 30 One approach to improve the solubility of taxoids used docetaxel-glycopyranoside ester-linked One approach to improve the solubility of taxoids used docetaxel-glycopyranoside ester-linked prodrugs. The compounds were prepared by chemo-enzymatic reactions using β-xylosidase, lactase prodrugs. The compounds were prepared by chemo-enzymatic reactions using β-xylosidase, lactase and β-galactosidase. All All of of them them demonstrated demonstrated improved improved solubility in water compared to the parental and β-galactosidase. All of them demonstrated improved solubility in water compared to the parental drug, docetaxel, l and the best compound 16 showedshowed aa 52-fold52-fold increaseincrease (Figure(Figure7 7).). InIn addition,addition, thethe drug, docetaxel, l and the best compound 16 showed a 52-fold increase (Figure 7). In addition, the compounds were able to release the parental drug inin vitrovitro and exhibited cytotoxic effects against KB compounds were able to release the parental drug in vitro and exhibited cytotoxic effects against KB and MCF-7 human cancer cell lines [[38].38]. and MCF-7 human cancer cell lines [38]. A paclitaxel prodrug containing a disulfidedisulfide moiety was described by Gund etet al.,, asas anan anticanceranticancer A paclitaxel prodrug containing a disulfide moiety was described by Gund et al., as an anticancer compound. The rationale behind the design of these molecules is based on the recognition of disulfidedisulfide compound. The rationale behind the design of these molecules is based on the recognition of disulfide subunit by the enzymeenzyme glutathione,glutathione, which releases thethe paclitaxelpaclitaxel ((17).). High levels of this enzymeenzyme subunit by the enzyme glutathione, which releases the paclitaxel (17). High levels of this enzyme have been described in cancer cells, and one hypothesishypothesis speculates that this enzyme is involved in have been described in cancer cells, and one hypothesis speculates that this enzyme is involved in resistance to many anticancer drugs [[39,40].39,40]. Th Thee prodrug compounds were 6–100 times more soluble resistance to many anticancer drugs [39,40]. The prodrug compounds were 6–100 times more soluble than paclitaxel. Moreover, Moreover, the most active compound 18 (Figure8 8)) waswas 6565 timestimes moremore waterwater solublesoluble than paclitaxel. Moreover, the most active compound 18 (Figure 8) was 65 times more water soluble than paclitaxel. For For this this molecule, superior superior antitumoral antitumoral activity activity was was identified identified in in all types of cells than paclitaxel. For this molecule, superior antitumoral activity was identified in all types of cells evaluated, except for MCF10A. After oral administration, the bioavailability in mice was five-foldfive-fold evaluated, except for MCF10A. After oral administration, the bioavailability in mice was five-fold greater than that of the parental drugdrug [[41].41]. greater than that of the parental drug [41].

Figure 8. Paclitaxel-disulfidePaclitaxel-disulﬁde prodrug withwith anticancer activity [[41].41]. Figure 8. Paclitaxel-disulfide prodrug with anticancer activity [41]. Etoposide (19) is a topoisomerase inhibitor with variable pharmacokinetics and low solubility in Etoposide ( 19)) is a topoisomerase inhibitor with variab variablele pharmacokinetics and low solubility in water. Using the prodrug approach, a series of water-soluble etoposide ester prodrugs were produced. water. Using the prodrug approach, a series of of wate water-solubler-soluble etoposide etoposide ester ester prodrugs prodrugs were were produced. produced. These prodrugs with an attached malic acid demonstrated solubility in water 23 to 120-fold greater These prodrugs with an attached malic acid demonstrateddemonstrated solubility in water 23 to 120-fold greater than that of the parental drug. In addition, the most active compound 20 (Figure 9), demonstrated than that of the parental drug.drug. In addition, the most active compound 20 (Figure9 9),), demonstrateddemonstrated in vitro cytotoxic effects comparable to those of etoposide [42]. in vitro cytotoxic effects comparable to those of etoposide [[42].42]. OH OH O O O O O O O O O O O O HO OH O O O O HO OH O O O O O O O O O O O O O O O O O O OH O O (19) OH O O (19) O OH (20) OH O OH OH (20) Parental drug - Etoposide Prodrug Prodrug ParentalWater-solubility drug - 0.1Etoposide mg/mL Water-solubility~ 9.0 mg/mL Water-solubility 0.1 mg/mL Water-solubility~ 9.0 mg/mL Figure 9. Etoposide ester prodrugs containing malic acid [42]. Figure 9. Etoposide ester prodrugs containing malic acid [42]. [42].

Molecules 2016, 21, 42 8 of 31 Molecules 2016, 21, 42 8 of 30

Non-steroidal anti-inflammatory anti-inflammatory drugs (NSAIDs) are widely prescribed worldwide. The use of NSAID-ester prodrugs is extensively reported in th thee literature. Controversially, Controversially, some authors report that the esterification esterification of NSAIDs can reduce the adverse gastric effects by masking the carboxylic acid function; however, the changes in the pharmacokineticpharmacokinetic profile profile that provide sustained release can explain in part the reduced adverse effects seen in some cases [[43,44].43,44]. In many cases, the lack of solubility limitslimits the the use use and and worsens worsens the adversethe adverse effects effects of NSAIDs. of NSAIDs. Therefore, Therefore, to improve to improve the solubility the solubilityin water ofin thewater NSAID of the 6-methoxy-2-naphthylacetic NSAID 6-methoxy-2-naphthylacetic acid (6-MNA, acid 21(6-MNA,), a series 21), of a esterseries prodrugs of ester prodrugsintended forintended percutaneous for percutaneous drug delivery drug was delive designedry was anddesigned synthesized. and synthesized. The authors The designed authors designedpiperazine piperazine derivatives derivatives attached by attached an ester linkage by an toester enhance linkage the solubilityto enhance of thesethe solubility compounds. of these They compounds.identified a compound They identified22 that a proved compound to be 22 water that soluble proved and to 11.2-foldbe water moresoluble permeable and 11.2-fold through more the permeableskin than 6-MNA through at the pH skin 7.4 (Figurethan 6-MNA 10)[45 at]. pH 7.4 (Figure 10) [45].

Figure 10. NSAID ester prodrugs [45,46]. [45,46].

Exploring transdermal delivery, Lobo et al., have synthesized and evaluated series of diclofenac Exploring transdermal delivery, Lobo et al., have synthesized and evaluated series of diclofenac (23)-ester prodrugs. The most promising compound, glycerol diclofenac ester (24), demonstrated (23)-ester prodrugs. The most promising compound, glycerol diclofenac ester (24), demonstrated better better solubility, a lower partition coefficient and higher flux across the skin than its parental drug solubility, a lower partition coefficient and higher flux across the skin than its parental drug (Figure 10). (Figure 10). In vitro studies using rat plasma showed rapid conversion of prodrugs to diclofenac due In vitro studies using rat plasma showed rapid conversion of prodrugs to diclofenac due to the activity to the activity of esterases [46]. of esterases [46]. The natural product quercetin (25) exhibited promising anti-inflammatory properties; however, The natural product quercetin (25) exhibited promising anti-inflammatory properties; however, its low skin permeation limits its use [47]. Therefore, to improve the passage of quercetin through the its low skin permeation limits its use [47]. Therefore, to improve the passage of quercetin through the skin, a series of prodrugs was synthesized and evaluated. The researchers observed that prodrugs skin, a series of prodrugs was synthesized and evaluated. The researchers observed that prodrugs with with long acyl chains were less soluble than the parent drug. However, compounds with shorter acyl long acyl chains were less soluble than the parent drug. However, compounds with shorter acyl chains chains were more water soluble, with an increase of 64-fold for compound 26. Thus, the prodrug were more water soluble, with an increase of 64-fold for compound 26. Thus, the prodrug exhibited exhibited greater solubility and skin permeation than quercetin (Figure 11) [48]. greater solubility and skin permeation than quercetin (Figure 11)[48]. 2R-γ-Tocotrienol (γ-T3), which is present in vitamin E, and has antioxidant activity against 2R-γ-Tocotrienol (γ-T3), which is present in vitamin E, and has antioxidant activity against brain microsomes and nitric oxide and inhibits cholesterol increases and cancer proliferation [49–58]; brain microsomes and nitric oxide and inhibits cholesterol increases and cancer proliferation [49–58]; however, γ-T3 is poorly soluble in aqueous solutions, impairing its pharmacological use. Three however, γ-T3 is poorly soluble in aqueous solutions, impairing its pharmacological use. Three aminoalkylcarboxylic acid esters generated as water-soluble prodrugs of γ-T3 showed better solubility aminoalkylcarboxylic acid esters generated as water-soluble prodrugs of γ-T3 showed better solubility in water compared to the parental drug. Compound 27 (Figure 11) was the most promising for in water compared to the parental drug. Compound 27 (Figure 11) was the most promising for parenteral use, with high solubility in water, stability and rapid hydrolysis in rat and human plasma, parenteral use, with high solubility in water, stability and rapid hydrolysis in rat and human plasma, making it a good potential alternative for cancer therapy [59]. making it a good potential alternative for cancer therapy [59].

Molecules 2016, 21, 42 9 of 31 Molecules 2016, 21, 42 9 of 30 Molecules 2016, 21, 42 9 of 30

Figure 11. Prodrugs of quercetin and γ-T3 [48,59]. Figure 11. Prodrugs of quercetin and γ-T3 [48,59]. [48,59]. Screening of an in-house library was used to identify compound 28 as a promising candidate Screening of an in-house library was used to identify compound 28 as a promising candidate targeting Human Immunodeficiency Virus (HIV) replication; however, its poor solubility in water targeting Human Immunodeficiency Immunodeﬁciency Virus Virus (HIV) replication; however, its poor solubility in water limits additional preclinical pharmacokinetic studies. Therefore, researchers have designed novel limits additional preclinical pharmacokineticpharmacokinetic studies. Therefore, Therefore, researchers researchers have designed novel indazole derivatives. The most active molecule 29 (Figure 12) contains an N-acyloxymethyl group as indazole derivatives.derivatives. TheThe mostmost activeactive moleculemolecule29 29(Figure (Figure 12 12)) contains contains an anN N-acyloxymethyl-acyloxymethyl group group as as a a pro-moiety and was 300-fold more water soluble than its parent compound. Additionally, this pro-moietya pro-moiety and and was was 300-fold 300-fold more more water water soluble soluble than itsthan parent its parent compound. compound. Additionally, Additionally, this prodrug this prodrug was more active against HIV, exhibiting an EC50 of 2 μM, while the parental drug showed wasprodrug more was active more against active HIV, against exhibiting HIV, exhibiting an EC50 of an 2 µ ECM,50 while of 2 μ theM, parentalwhile the drug parental showed drug an showed EC50 of an EC50 of 3 μM [60]. 3anµ ECM[5060 of]. 3 μM [60]. O H O NH N H N N (28) H (29) N (28) H (29) N H N O N N N O N N N H O N N H O N ~300-fold more O O O ~300-fold more O O O water-soluble water-soluble O O

Parent drug Parent drug Cl Solubility: 0.2 µM Cl Solubility: 0.2 µM (phosphate buffer at pH 7.4) Prodrug (phosphate buffer at pH 7.4) Prodrug Solubility: 60 µM Solubility: 60 µM (phosphate buffer at pH 7.4) (phosphate buffer at pH 7.4) Figure 12. N-acyloxymethyl ester prodrug [60]. Figure 12. N-acyloxymethyl ester prodrug [[60].60]. 3. Amide Prodrugs 3. Amide Amide Prodrugs Prodrugs Acyclovir (30) is an antiviral drug that has poor solubility in water and also has low Acyclovir (30) is an antiviral drug that has poor solubility in water and also has low bioavailabilityAcyclovir ((between30) is an antiviral10% and drug 20%) that [61]. has To poor impr solubilityove these in characteristics, water and also two has peptide low bioavailability prodrugs, bioavailability (between 10% and 20%) [61]. To improve these characteristics, two peptide prodrugs, (betweenan amide 10%31 and and an 20%) ester [61 prodrug]. To improve 32 were these developed characteristics, (Figure two 13). peptide The first prodrugs, was designed an amide to release31 and an amide 31 and an ester prodrug 32 were developed (Figure 13). The first was designed to release acycloviran ester prodrug directly32 throughwere developed the action (Figureof enzyme 13). dipeptidyl The ﬁrst was peptidase designed IV to (DPPIV release or acyclovir CD26), and directly the acyclovir directly through the action of enzyme dipeptidyl peptidase IV (DPPIV or CD26), and the secondthrough requires the action two of steps enzyme to release dipeptidyl the parental peptidase drug. IV (DPPIVBoth showed or CD26), a signific andant the increase second requiresin solubility two second requires two steps to release the parental drug. Both showed a significant increase in solubility (17-foldsteps to releasefor amide the and parental 9-fold drug. for ester Both derivatives) showed a signiﬁcant compared increase to the inparent solubility drug. (17-fold Furthermore, for amide the (17-fold for amide and 9-fold for ester derivatives) compared to the parent drug. Furthermore, the compoundsand 9-fold for were ester stable derivatives) in phosphate-buffered compared to the saline parent (PBS) drug. and Furthermore,were rapidly thecleaved compounds by plasmatic were compounds were stable in phosphate-buffered saline (PBS) and were rapidly cleaved by plasmatic enzymes.stable in phosphate-buffered Although the antiviral saline activity (PBS) andwas werenot superior rapidly cleavedto that of by free plasmatic acyclovir, enzymes. both presented Although enzymes. Although the antiviral activity was not superior to that of free acyclovir, both presented good antiviral activity against herpes simplex virus type 1 and type 2 [62]. good antiviral activity against herpes simplex virus type 1 and type 2 [62].

Molecules 2016, 21, 42 10 of 31 the antiviral activity was not superior to that of free acyclovir, both presented good antiviral activity against herpes simplex virus type 1 and type 2 [62]. Molecules 2016, 21, 42 10 of 30 SB-3CT (33) is a highly selective inhibitor of the endopeptidases matrix metalloproteinases 2 and 9 (MMP-2SB-3CT and (33 MMP-9),) is a highly also selective known inhibitor as gelatinases, of the endopeptidases which have attracted matrix metalloproteinases interest in research 2 due to theirand participation 9 (MMP-2 and in MMP-9), a signiﬁcant also known number as gelatinases, of human which pathologies have attracted such asinterest cancer, in research neuronal due death, atherosclerosisto their participation and aneurysms in a significant [63]. Due number to the of limited human solubility pathologies in watersuch as of cancer, SB-3CT neuronal (2.3 µg/mL), death, two seriesatherosclerosis of prodrugs wereand aneurysms developed [63]. using Due amino to the acids limited (Gly, solubilityL-Lys, Lin-Glu water and ofL SB-3CT-Arg) and (2.3 the μg/mL), dipeptide L-Arg-twoL-Arg series as of pro-moieties, prodrugs were linked developed by an using ester amino or amide acids group.(Gly, L-Lys, All prodrugsL-Glu and showedL-Arg) and improved the solubilitydipeptide in water L-Arg- (aL more-Arg as than pro-moieties, 5000-fold linked increase). by an The ester ester or compoundsamide group.were All prodrugs unstable showed in aqueous improved solubility in water (a more than 5000-fold increase). The ester compounds were unstable in solution, human plasma and blood, while amides demonstrated resistance to fast hydrolysis. The aqueous solution, human plasma and blood, while amides demonstrated resistance to fast hydrolysis. amide prodrugs released the active drug into the blood in 30 min. The arginyl-amide prodrug 34 The amide prodrugs released the active drug into the blood in 30 min. The arginyl-amide prodrug 34 (Figure(Figure 13) was 13) metabolicallywas metabolically stable stable in mouse, in mouse, rat and rat humanand human liver liver microsomes. microsomes. Moreover, Moreover, the prodrugthe 34 wasprodrug non-mutagenic 34 was non-mutagenic in an Ames in assay an Ames [64]. assay [64].

HN N

HN N N Amide prodrug (Val - Pro)2 O (31) O

OH O 17-fold increase in solubility HN N N O H2N N OH O N acyclovir HN (30) N Ester prodrug N H2N O (32) O O Val - Pro - Val

9-fold increase in solubility

O O S S S S O O O H N 2 N O (34) O H R

SB-3CT Amino acids Gly, L-Lys, L-Glu, L-Arg (33) dipeptide L-Arg-L-Arg

5000-fold increase in solubility Figure 13. Peptide prodrugs of acyclovir and SB-3CT [62,64]. Figure 13. Peptide prodrugs of acyclovir and SB-3CT [62,64]. DW2282 (35) is a potent antiproliferative compound with a broad spectrum of action against DW2282various cancer (35) iscell a lines, potent but antiproliferative in preclinical assays, compound this analog with showed a broad gastrointestinal spectrum toxicity of action [65]. against A variousseries cancer of novel cell lines,amino-acid butin conjugates preclinical were assays, designed this to analog improve showed the aqueous gastrointestinal solubility toxicityof these [65]. A seriesanalogs of novel and prevent amino-acid toxic effects. conjugates All synthesized were designed compounds to improvewere more the soluble aqueous and three solubility were more of these analogsactive and than prevent the parental toxic effects.drug. The All most synthesized promising compounds analog 36 (Figure were 14) more had soluble a good reconversion and three were ratemore activein than human the plasma parental and drug. good The bioavailability most promising by the analog oral route36 (Figure in mice. 14 Moreover,) had a good this molecule reconversion was rate more active and approximately 36-fold more soluble than DW2282 [66]. in human plasma and good bioavailability by the oral route in mice. Moreover, this molecule was Bone tissues are difficult to treat because of limited drug delivery and retention and low moresanguineous active and approximatelyflow [67]. Hydroxyapatite 36-fold more (HA), soluble a constituent than DW2282 of bones, [ 66can]. be a target for these drugs Bonethrough tissues the interaction are difﬁcult between to treatnegative because groups of and limited the calcium drug ions delivery in HA and[68–71]. retention Researchers and low sanguineousdesigned ﬂowand evaluated [67]. Hydroxyapatite the solubility and (HA), pharmacoki a constituentnetic profile of bones, of novel can dendritic be a target naproxen-peptide for these drugs throughprodrugs, the interaction specifically between L-aspartate negative and L-glutamate. groups and the calcium ions in HA [68–71]. Researchers

Molecules 2016, 21, 42 11 of 31 designed and evaluated the solubility and pharmacokinetic proﬁle of novel dendritic naproxen-peptide prodrugs,Molecules 2016 speciﬁcally, 21, 42 L-aspartate and L-glutamate. 11 of 30 Molecules 2016, 21, 42 11 of 30

Figure 14. Prodrug derivative of DW2282 [[66].66]. Figure 14. Prodrug derivative of DW2282 [66]. Their solubility in water was evaluated at three different pH values, and the prodrugs were Their solubility in water was evaluated at three different pH values, and the prodrugs were more moreTheir soluble solubility at increased in water pH. Thewas authors evaluated proposed at thre the atdifferent the solubility pH values, of the analogsand the can prodrugs be related were to soluble at increased pH. The authors proposed that the solubility of the analogs can be related to moresurface soluble area and at increased carboxyl pH.groups The inauthors dendritic proposed peptides; that in the this solubility way, the of second-generation the analogs can be moleculesrelated to surface area and carboxyl groups in dendritic peptides; in this way, the second-generation molecules surfacehave more area interactions and carboxyl with groups water in anddendritic thus arepept moides;re waterin this soluble way, the than second-generation the first-generation molecules drugs have moremore interactionsinteractions withwith waterwater and and thus thus are are more more water water soluble soluble than than the the ﬁrst-generation first-generation drugs drugs39 39 (Figure 15). The most promising analog NAP-G2-Asp (38) attached to L-aspartate subunits showed (Figure 15). The most promising analog NAP-G2-Asp (38) attached to L-aspartate subunits showed an 39an (Figureapproximately 15). The 90-fold most promising increased analogsolubility NAP-G2-Asp in water at pH(38 )7. attached In the HA-binding to L-aspartate assay, subunits NAP-G2-Asp showed approximately 90-fold increased solubility in water at pH 7. In the HA-binding assay, NAP-G2-Asp anshowed approximately 80% binding 90-fold activity increased in 25 solubility mg/mL inin watePBS.r atIn pH50% 7. humanIn the HA-binding plasma it actedassay, as NAP-G2-Asp a prodrug, showed 80% binding activity in 25 mg/mL in PBS. In 50% human plasma it acted as a prodrug, rapidly showedrapidly and80% effectively binding activity releasing in the25 parentmg/mL drug in PBS. [72]. In 50% human plasma it acted as a prodrug, andrapidly effectively and effectively releasing releasing the parent the drug parent [72 drug]. [72].

L- aspartate HO O L- aspartate OHHO O OH O H O HN OH HO O H OH O Naproxen moiety HNH OH HO O Naproxen moiety OH O H OH CH3 HN N CH3 H H H OH CH3 N HN N CH3 OH H O H H N OH H O O H C O H3C H 3 O O O (37) H3C O H3C O O HN O O (37) O HNH O Parental drug naproxen HN O ParentalWater-solubility: drug naproxen 0.19 mg/mL (pH 7) HNH O OH H HNH Water-solubility: 0.19 mg/mL (pH 7) HN O OHO O H H O O OH OH OHO OH OH (38) O OH (38) Prodrug NAP-G2-Asp HO O Water-solubility: 17.22 mg/mL (pH 7) Naproxen moiety O Prodrug NAP-G2-Asp HO O Water-solubility: 17.22 mg/mL (pH 7) Naproxen moiety H O CH HNH OH 3 H CH3 N HN OH H O N H O H3C O H O O H3C O O HN O HNH O Prodrug NAP-G1-Asp OH Water-solubility: 6.12 mg/mL (pH 7) H Prodrug NAP-G1-Asp HO O OH Water-solubility: 6.12 mg/mL (pH 7) (39) HO O (39) L- aspartate L- aspartate Figure 15. Dendritic naproxen-peptides prodrugs [72]. Figure 15. Dendritic naproxen-pep naproxen-peptidestides prodrugs [[72].72]. A benzamide derivative, PC190723 (40, Figure 16), was proposed as a potent antimicrobial agentA targeting benzamide the derivative,FtsZ protein. PC190723 This enzyme (40, Figureplays a 16),key wasrole inproposed bacterial as cell a potentdivision, antimicrobial and studies agentsuggest targeting that it is thea promising FtsZ protein. new Thistarget enzyme for antibiot playics development.a key role in bacterialTo overcome cell division, its poor formulation,and studies suggesttwo new that prodrugs it is a promising were developed. new target The for first, antibiot the compoundic development. TXY436 To overcome(41, Figure its 16) poor is a formulation, N-Mannich twobase newderivative prodrugs that were was developed.2.8-fold more The soluble first, the th ancompound the parent TXY436 drug in(41 PBS, Figure (pH 16)= 7.4) is aand N-Mannich 100-fold base derivative that was 2.8-fold more soluble than the parent drug in PBS (pH = 7.4) and 100-fold

Molecules 2016, 21, 42 12 of 31

A benzamide derivative, PC190723 (40, Figure 16), was proposed as a potent antimicrobial agent targeting the FtsZ protein. This enzyme plays a key role in bacterial cell division, and studies suggest that it is a promising new target for antibiotic development. To overcome its poor formulation, two new prodrugs were developed. The ﬁrst, the compound TXY436 (41, Figure 16) is a N-Mannich Moleculesbase derivative 2016, 21, 42 that was 2.8-fold more soluble than the parent drug in PBS (pH = 7.4) and 100-fold12 of 30 more soluble in 10 mM citrate solution (pH = 2.6). The latter is suitable for in vivo drug administration. moreMoreover, soluble this in compound10 mM citrate is stablesolution in (pH citrate = 2.6). solution The latter and is is suitable rapidly for converted in vivo drug to the administration. parent drug Moreover,under physiological this compound conditions is stable(pH in = citrate 7.4). In solu addition,tion and it maintainedis rapidly converted anti-staphylococcal to the parent activity drug underdue to physiologicalin vitro FtsZ inhibitionconditions and(pH showed= 7.4). In 73%additi oralon, bioavailability.it maintained anti-staphylococcalIn vivo studies demonstrated activity due tothat in thevitro prodrug FtsZ inhibition was effective and showed in a murine73% oral model bioavailability. of peritonitis In vivo with studies systematic demonstrated infection that with the prodrugmethicillin-susceptible was effective inStaphylococcus a murine model aureus of peritonitis(MSSA, survival with systematic of 100%) infection and methicillin-resistant with methicillin- Staphylococcussusceptible Staphylococcus aureus (MRSA, aureus survival(MSSA, survival of 67%), of while100%) and the methicillin-resistant parent drug was not Staphylococcus effective in aureus this (MRSA,model [73 survival]. of 67%), while the parent drug was not effective in this model [73]. The second prodrug TXY541 (42, Figure 1616)) is a carboxamide deri derivativevative with increased solubility in water water (143-fold (143-fold in in citrate citrate solution). solution). Interestingly, Interestingly, the the solubility solubility in inPBS PBS decreased, decreased, to almost to almost half half the solubilitythe solubility of the of theparental parental compound. compound. The The stability stability study study revealed revealed that that TXY541 TXY541 is is stable stable in in citrate solution and is converted under physiological cond conditionsitions to PC190723, but at slower rates than the parental compoundcompound is is41 .41 In. mouseIn mouse serum, serum, the conversion the conversion occurred occurred faster, which faster, suggest which an suggest enzymatic an enzymaticconversion. conversion. The oral bioavailability The oral bioavailability of the prodrug of the42 wasprodrug 29.6%, 42 andwasin 29.6%, vivo evaluation and in vivo showed evaluation that showedthis analog that was this effective analog was against effective systematic against infection systematic with infection MSSA (83% with survival) MSSA (83% and survival) MRSA (100% and MRSAsurvival) (100% [74]. survival) [74].

N S O F

Cl N F NH2 O

(40) Parent Drug (PC190723)

No survival (in vivo model of systematic infection of MSSA or MRSA)

N S O F N S O F

Cl N F NH Cl N F NH +H O N+ H O N O

Prodrug TXY436 (41) Prodrug TXY541 (42)

100-fold increase of solubility in citrate solution (pH=2.6) 143-fold increase of solubility in citrate solution (pH=2.6)

100% survival 83% survival (in vivo model of systematic infection of MSSA) (in vivo model of systematic infection of MSSA) 67% survival 100% survival (in vivo model of systematic infection of MRSA) (in vivo model of systematic infection of MRSA)

Figure 16. Benzamide and carboxamide prodrugs of PC190723 [[73,74].73,74].

4. Carbamate Prodrugs 4. Carbamate Prodrugs The benzamide compound CI-994 (43) is a potent inhibitor of histone deacetylases (HDACs). The benzamide compound CI-994 (43) is a potent inhibitor of histone deacetylases (HDACs). HDACs have an essential role in the regulation of gene expression, and several types of inhibitors HDACs have an essential role in the regulation of gene expression, and several types of inhibitors exhibit antitumor activity [75,76], although adverse effects and poor solubility in water limit its use. exhibit antitumor activity [75,76], although adverse effects and poor solubility in water limit its use. Two glucuronide prodrugs, one (compound 44) linked to the glucuronide moiety by a spacer and the Two glucuronide prodrugs, one (compound 44) linked to the glucuronide moiety by a spacer and the other linked directly by a carbamate group (compound 45), were used to resolve these limitations other linked directly by a carbamate group (compound 45), were used to resolve these limitations (Figure 17). While the solubility of CI-994 is 0.08 mg/mL, those of the prodrugs were greater than 1 mg/mL. Both compounds were stable at different pH values (2.1 and 7.0). In addition, the prodrugs were evaluated in vitro using an antiproliferative assay with NCI-H661 non-small cell lung cancer cells. The results demonstrated that this compound, when incubated with β-glucuronidase, showed the same antiproliferative activity (IC50 = 20 μM) shown by CI-994. Without the enzyme, the prodrugs had decreased cytotoxicity, which may decrease the toxic effects of the parent drug [77].

Molecules 2016, 21, 42 13 of 31

(Figure 17). While the solubility of CI-994 is 0.08 mg/mL, those of the prodrugs were greater than 1 mg/mL. Both compounds were stable at different pH values (2.1 and 7.0). In addition, the prodrugs were evaluated in vitro using an antiproliferative assay with NCI-H661 non-small cell lung cancer cells. The results demonstrated that this compound, when incubated with β-glucuronidase, showed the same antiproliferative activity (IC50 = 20 µM) shown by CI-994. Without the enzyme, the prodrugs Moleculeshad 2016 decreased, 21, 42 cytotoxicity, which may decrease the toxic effects of the parent drug [77]. 13 of 30

spacer O OH O NH2 O HO O O NH HO OH NH O N 2 O H

O NH (44) NH N 2 H Water solubility > 1mg/mL (43) glucoronide

Parent drug CI-994

Water solubility = 0.08 mg/mL O O OH OH NH HO O HO NH2 OH O N H (45)

water-soluble moiety Br Br

N O N HN O N

N N N N N S N S N N

(47) (46)

Water solubility = 0.01 mg/mL Water solubility = 6 mg/mL Membrane permeability = 2.11 × 10-6 cm s-1 -6 -1 Membrane permeability = 0.01 × 10 cm s

FigureFigure 17.17. GlucuronideGlucuronide and and pyrazolo[3,4- pyrazolo[3,4-dd]pyrimides]pyrimides prodrugs prodrugs [77 [77,78].,78].

Pyrazolo[3,4-Pyrazolo[3,4-d]pyrimidesd]pyrimides compounds compounds are are able able toto inhibitinhibit oncogenic oncogenic tyrosine tyrosine kinases kinases and and may may be be usedused to treat to treat cancer. cancer. In Ingeneral, general, pyrazolo[3,4- pyrazolo[3,4-dd]pyrimides]pyrimides compoundscompounds have have low low solubility solubility in water in water [79]. [79]. ProdrugsProdrugs of pyrazolo[3,4- of pyrazolo[3,4-d]pyrimidined]pyrimidine compounds compounds were were designeddesigned to to enhance enhance its pharmacokineticits pharmacokinetic propertiesproperties using using a awater-soluble water-soluble NN-methylpiperazino-methylpiperazino promoiety promoiety linked linked by a O -alkylby a carbamateO-alkyl carbamate linker. linker.The The prodrug prodrug47 showed 47 showed 600-fold 600-fold improvement improvement in solubility in comparedsolubility to compared the parent drugto the46 parent(Figure drug17). 46 ´6 ´1 (FigureIn vitro 17).assay In vitro showed assay good showed increase good in increase passive membrane in passive permeability, membrane frompermeability, 0.01 ˆ 10 fromcm¨ s0.01to × 10−6 ´6 ´1 cm·s−2.111 to ˆ2.1110 × 10cm−6¨ scm·s. In−1. addition,In addition, this compoundthis compound was more was cytotoxic more cytotoxic compared compared to the parental to the drug; parental drug;however, however, the the mechanism mechanism of action of action is still is not still well not characterized well characterized [78]. [78]. Photodynamic therapy is a non-invasive treatment for several types of cancer and infectious Photodynamic therapy is a non-invasive treatment for several types of cancer and infectious diseases [80]. This strategy consists in administration of a sensitizer and non-mutagenic substance diseases [80]. This strategy consists in administration of a sensitizer and non-mutagenic substance that will be irradiated only in the affected area, where it will be activated by a specific wavelength of light [81]. This tool can be considered a prodrug approach, in that it uses the light to convert the photosensitizer (prodrug) to an active compound and in that both the selectivity and the solubility of the photosensitizer are important in design [80]. Three photopaclitaxel prodrugs were generated by linking paclitaxel (17) with a coumarinic derivative. The most soluble compound 48 showed at least 400,000-fold higher solubility in water compared to paclitaxel (Figure 18). This same compound was stable under physiological and storage conditions. In a photoconversion assay, the carbamate prodrug 48 rapidly generated paclitaxel with minimal tissue damage under 365 nm UV-A light irradiation at low power [82].

Molecules 2016, 21, 42 14 of 31

that will be irradiated only in the affected area, where it will be activated by a speciﬁc wavelength of light [81]. This tool can be considered a prodrug approach, in that it uses the light to convert the photosensitizer (prodrug) to an active compound and in that both the selectivity and the solubility of the photosensitizer are important in design [80]. Three photopaclitaxel prodrugs were generated by linking paclitaxel (17) with a coumarinic derivative. The most soluble compound 48 showed at least 400,000-fold higher solubility in water compared to paclitaxel (Figure 18). This same compound was stable under physiological and storage Molecules 2016, 21, 42 14 of 30 conditions. In a photoconversion assay, the carbamate prodrug 48 rapidly generated paclitaxel with Moleculesminimal 2016 tissue, 21, 42 damage under 365 nm UV-A light irradiation at low power [82]. 14 of 30

Figure 18. Photopaclitaxel prodrug [82]. Figure 18. Photopaclitaxel prodrug [[82].82]. 5. Carbonate Prodrugs 5. Carbonate Carbonate Prodrugs Prodrugs In an attempt to improve the solubility of an anticancer compound, CHS8281 (49), a series of In an attempt to improve the solubility of an anticanceranticancer compound, CHS8281 ( 49), a series of prodrugsprodrugs was waswas synthesized synthesized synthesized using using aa carbonate acarbonate carbonate group groupgroup to link toto a link tetraethylene-glycollink a atetraethylene-glycol tetraethylene-glycol moiety moiety to themoiety parental to the to the parentalparentaldrug. drug. The drug. prodrug The The prodrug EB1627prodrug EB1627 (50 EB1627) was ( 600-fold50(50) )was was more 600-fold600-fold soluble moremore than soluble soluble the parental than than the drugthe parental parental at pH drug 5.5 drug (Figure at pH at 19 5.5pH). 5.5 (FigureThe(Figure 19). selection 19).The The ofselection the selection carbonate of ofthe groupthe carbonate carbonate provided groupgroup the required provided hydrolytic the the required required labiality hydrolytic andhydrolytic rapid labiality release labiality of and the and rapidrapidactive release release drug ofin the of vivo the active[ 83active]. drug drug in in vivo vivo [83]. [83].

H H H H N HN H H NH N O Cl (CH ) O (CH2)6 N N 2 6 N N O Cl (CH ) O N N(CH2)6 O O O O N N 2 6 CN Cl O O O O O O N CNN Cl N N O CN Cl O O CN Cl (49) O CHS8281 (49) EB1627 (50) (50) Solubility:CHS8281 0.5 µg/mL at pH 7.4 Solubility: 120 µg /mLEB1627 at pH 7.4 >1200 µg /mL at pH 5.5 Solubility: 0.5 µg/mL2.0 µg/mL at pHat pH 7.4 5.5 Solubility: 120 µg /mL at pH 7.4 2.0 µg/mL at pH 5.5 >1200 µg /mL at pH 5.5 Figure 19. CHS8281 prodrug [83]. FigureFigure 19. 19. CHS8281CHS8281 prodrug [ 83[83].]. To overcome the poor solubility of paclitaxel (17) and release the drug selectively into malignant tissue, an N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer-drug conjugate 51 with an AB3 To overcomeTo overcome the the poor poor solubility solubility of of paclitaxel paclitaxel (17)) andand release release the the drug drug selectively selectively into into malignant malignant self-immolative dendritic linker was designed. The HPMA was used as a water-soluble group, and tissue,tissue, an N an-(2-hydroxypropyl)-methacrylamideN-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer-drug copolymer-drug conjugate conjugate51 with 51 with an AB an AB3 the dendritic peptide linked the HPMA to the parent drug (Figure 20). This enzyme-cleavable linker3 self-immolativeself-immolative dendritic dendritic linker linker was was designed. The The HPMA HPMA was was used used as a water-solubleas a water-soluble group, andgroup, the and served as a platform with a triple payload of paclitaxel attached by a carbonate group [84]. Interestingly, the dendriticdendritic peptidepeptide linked linked the the HPMA HPMA to the toparent the parent drug (Figuredrug (Figure 20). This 20). enzyme-cleavable This enzyme-cleavable linker served linker HPMA not only showed improved solubility but also selectively accumulated in tumors due to its as a platform with a triple payload of paclitaxel attached by a carbonate group [84]. Interestingly, servedenhanced as a platform permeability with a and triple retention payload [85]. of paclitaxel attached by a carbonate group [84]. Interestingly, HPMAHPMA not notonly only showed showed improved improved solubility solubility but but alsoalso selectivelyselectively accumulated accumulated in tumorsin tumors due due to its to its enhancedenhanced permeability permeability and and retention retention [85]. [85].

Figure 20. Copolymer-paclitaxel conjugate [84].

Molecules 2016, 21, 42 14 of 30

Figure 18. Photopaclitaxel prodrug [82].

5. Carbonate Prodrugs In an attempt to improve the solubility of an anticancer compound, CHS8281 (49), a series of prodrugs was synthesized using a carbonate group to link a tetraethylene-glycol moiety to the parental drug. The prodrug EB1627 (50) was 600-fold more soluble than the parental drug at pH 5.5 (Figure 19). The selection of the carbonate group provided the required hydrolytic labiality and rapid release of the active drug in vivo [83].

H H H H N N N N O Cl (CH ) O (CH2)6 2 6 N N O O O O N N CN Cl O O CN Cl O (49) CHS8281 EB1627 (50) Solubility: 0.5 µg/mL at pH 7.4 Solubility: 120 µg /mL at pH 7.4 2.0 µg/mL at pH 5.5 >1200 µg /mL at pH 5.5 Figure 19. CHS8281 prodrug [83].

To overcome the poor solubility of paclitaxel (17) and release the drug selectively into malignant tissue, an N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer-drug conjugate 51 with an AB3 self-immolative dendritic linker was designed. The HPMA was used as a water-soluble group, and the dendritic peptide linked the HPMA to the parent drug (Figure 20). This enzyme-cleavable linker served as a platform with a triple payload of paclitaxel attached by a carbonate group [84]. Interestingly, MoleculesHPMA2016, 21not, 42 only showed improved solubility but also selectively accumulated in tumors due to its15 of 31 enhanced permeability and retention [85].

FigureFigure 20. 20.Copolymer-paclitaxel Copolymer-paclitaxel conjugate conjugate [84]. [84 ].

Molecules 2016, 21, 42 15 of 30 6. Ether Prodrugs 6. Ether Prodrugs A glucuronide prodrug of the anticancer compound 10-hydroxycamptothecin (52) was reported by Leu etA al. glucuronideThe authors prodrug used of the the prodrug anticancer approach compound to 10-hydroxycamptothecin improve the solubility ( of52) the was parental reporteddrug, whichby showed Leu et al. anticancer The authors activity used the against prodrug various approach cancer to improve cell lines the [86 solubility,87]. To improveof the parental the solubility drug, of which showed anticancer activity against various cancer cell lines [86,87]. To improve the solubility of 10-hydroxycamptothecin, a prodrug was designed using glucuronic acid as a water-soluble moiety 10-hydroxycamptothecin, a prodrug was designed using glucuronic acid as a water-soluble moiety linked by a 3-nitrobenzyl spacer. The compound 53 was 80-fold more soluble than the parental drug. linked by a 3-nitrobenzyl spacer. The compound 53 was 80-fold more soluble than the parental drug. The prodrugThe prodrug was was activated activated by enzymaticby enzymatic cleavage cleavage followed followed by by a a 1,6-elimination 1,6-elimination reaction reaction to to release release the parentalthe parental drug (Figure drug (Figure 21)[88 21)]. [88].

O N HOOC 2 O O Prodrug HO O O N HO Solubility: 1.10 µM at pH 4.0 OH N 2.60 µM at pH 7.0 O (53) ß-glucuronidase HO O 80-fold more water-soluble

O2N O O O N N O

1,6-elimination HO O

HO O Parent drug N Solubility: 0.0137 µM at pH 4.0 N 0.137 µM at pH 7.0 O (52) HO O 10-hydroxycamptothecin Figure 21. Prodrug release based on an enzymatic cleavage followed by a 1,6-elimination reaction [88]. Figure 21. Prodrug release based on an enzymatic cleavage followed by a 1,6-elimination reaction [88]. Cadalene (54) is a flavonoid isolated from Zelkova serrata Makino. This molecule has a wide Cadalenerange of biological (54) is aactivities, ﬂavonoid including isolated anticancer from Zelkova activities; serrata however, Makino its low. This solubility molecule in water has is a a wide rangedrawback of biological [89]. Glycosylated activities, including cadalene derivatives anticancer linked activities; by ether however, orethoxy its spacers low solubility were synthesized in water is a drawbackas prodrugs. [89]. Glycosylated Prodrug 55 cadalene(Figure 22) derivatives exhibited linkedthe best by solubility ether orethoxy profile. spacersIn vivo, weretreatment synthesized with as cadalene did not affect tumor volume, but treatment with the glycosylated prodrug reduced tumor volume by 50% compared to the control group. The authors hypothesized that the better activity of the prodrug could be due to its better solubility compared to cadalene [90].

Water-soluble moiety

HO O HO O HO OH O O (54) (55)

Cadalene Increase solubility Figure 22. Cadalene prodrug with improved solubility in water [90].

Molecules 2016, 21, 42 15 of 30

6. Ether Prodrugs A glucuronide prodrug of the anticancer compound 10-hydroxycamptothecin (52) was reported by Leu et al. The authors used the prodrug approach to improve the solubility of the parental drug, which showed anticancer activity against various cancer cell lines [86,87]. To improve the solubility of 10-hydroxycamptothecin, a prodrug was designed using glucuronic acid as a water-soluble moiety linked by a 3-nitrobenzyl spacer. The compound 53 was 80-fold more soluble than the parental drug. The prodrug was activated by enzymatic cleavage followed by a 1,6-elimination reaction to release the parental drug (Figure 21) [88].

O N HOOC 2 O O Prodrug HO O O N HO Solubility: 1.10 µM at pH 4.0 OH N 2.60 µM at pH 7.0 O (53) ß-glucuronidase HO O 80-fold more water-soluble

O2N O O O N N O

1,6-elimination HO O

Molecules 2016Cadalene, 21, 42 (54) is a flavonoid isolated from Zelkova serrata Makino. This molecule has a wide16 of 31 range of biological activities, including anticancer activities; however, its low solubility in water is a drawback [89]. Glycosylated cadalene derivatives linked by ether orethoxy spacers were synthesized prodrugs. Prodrug 55 (Figure 22) exhibited the best solubility proﬁle. In vivo, treatment with cadalene as prodrugs. Prodrug 55 (Figure 22) exhibited the best solubility profile. In vivo, treatment with didcadalene not affect did tumor not affect volume, tumor but volume, treatment but withtreatm theent glycosylated with the glycosylated prodrug prodrug reduced reduced tumor volumetumor by 50%volume compared by 50% to the compared control to group. the control The authors group. The hypothesized authors hypothesized that the better that the activity better of activity the prodrug of couldthe be prodrug due to could its better be due solubility to its better compared solubility to compared cadalene [to90 cadalene]. [90].

Water-soluble moiety

HO O HO O HO OH O O (54) (55)

Cadalene Increase solubility Figure 22. Cadalene prodrug with improved solubility in water [90]. Molecules 2016, 21, 42 Figure 22. Cadalene prodrug with improved solubility in water [90]. 16 of 30

7. Imine Imine Prodrugs Prodrugs Amphotericin B and nystatin prodrugs containingcontaining the pyridoxal phosphatephosphate as a water-soluble moiety were synthesized and evaluated. An An imine imine group group was was used used to allow the attachment of both subunits, and high water-soluble prodrugs 56 andand 5757 withwith solubilitiessolubilities upup toto 100100 mg/mLmg/mL were characterized (Figure 2323))[ [91].91].

OH OH a) O OH

HO O OH OH OH OH O O

X OH X O O X = CH (Amphotericin B prodrug) (56) X = CH (Nystatin prodrug) (57) 2 HO OH OH N O P O O Solubility >100 mg/mL N OH

Pyridoxal phosphate

OH O OH O H NH4 N N N S NH4 O O OH O O S P O O O OH O hydrazone NH H3C P O 4 bond O NH4 O O HCl (58) H2N CH3 OH Bisphosphonate Doxorubicin moiety promoiety

Increase solubility and stability in human plasma

FigureFigure 23. 23. (a()a )Amphotericin Amphotericin B B and and nystatin nystatin prodrugs; prodrugs; ( b) Bisphosphonate doxorubicindoxorubicin prodrug prodrug [ 91[91,92].,92].

Another paper described the synthesis of a novel bisphosphonate doxorubicin prodrug to treat bone metastases. To improve the solubility of doxorubicin, the water-soluble group bisphosphonate was attached to the parental drug by a hydrazone acid-sensitive bond. Moreover, the bisphosphonate moiety acts as a bone-targeting ligand, improving the drug’s selectivity. The prodrug 58 showed considerably higher stability at pH 7.4, with approximately 12% of the doxorubicin being released after 18 h of incubation (Figure 23) [92]. Synthesis of a soluble paclitaxel prodrug using an acid-sensitive hydrazone bond was reported by Moktan et al. The authors used a biopolymer elastin-like polypeptide (ELP) as a water-soluble group to improve the solubility of paclitaxel. ELP also enables thermal targeted delivery of paclitaxel, as hyperthermia above a specific transition temperature at the site of a tumor causes ELP to aggregate and accumulate, thus increasing the local concentration of the drug. The ELP paclitaxel prodrug 59 was able to increase solubility and cytotoxicity against a paclitaxel-resistant breast cancer cell line (Figure 24) [93].

Molecules 2016, 21, 42 17 of 31

Another paper described the synthesis of a novel bisphosphonate doxorubicin prodrug to treat bone metastases. To improve the solubility of doxorubicin, the water-soluble group bisphosphonate was attached to the parental drug by a hydrazone acid-sensitive bond. Moreover, the bisphosphonate moiety acts as a bone-targeting ligand, improving the drug’s selectivity. The prodrug 58 showed considerably higher stability at pH 7.4, with approximately 12% of the doxorubicin being released after 18 h of incubation (Figure 23)[92]. Synthesis of a soluble paclitaxel prodrug using an acid-sensitive hydrazone bond was reported by Moktan et al. The authors used a biopolymer elastin-like polypeptide (ELP) as a water-soluble group to improve the solubility of paclitaxel. ELP also enables thermal targeted delivery of paclitaxel, as hyperthermia above a speciﬁc transition temperature at the site of a tumor causes ELP to aggregate and accumulate, thus increasing the local concentration of the drug. The ELP paclitaxel prodrug 59 was able to increase solubility and cytotoxicity against a paclitaxel-resistant breast cancer cell line Molecules(Figure 2016 ,24 21)[, 4293 ]. 17 of 30

O O HO O O O (59)

O O N H H O O O O OH O O

O H N N N O O Elastin-like S Acid-sensitive polypeptide (ELP) hydrazone bond FigureFigure 24. 24. Paclitaxel-biopolymer-conjugatedPaclitaxel-biopolymer-conjugated prodrug prodrug [93 [93].].

8. Phosphate8. Phosphate Prodrugs Prodrugs PhosphatePhosphate ester ester compounds compounds are are relatively relatively stable whenwhen free free in in metabolic metabolic surroundings surroundings because because they theyhave have a low a low pKa, pK betweena, between 1 and 1 and 2. 2. At At a a physiological physiological pHpH of of 7.0–7.4, 7.0–7.4, the the compounds compounds are permanentlyare permanently deprotonated and therefore negatively charged but are readily activated upon complexation to various deprotonated and therefore negatively charged but are readily activated upon complexation to counterions in an enzyme’s active site [94,95]. Intestinal alkaline phosphatases play an important role various counterions in an enzyme’s active site [94,95]. Intestinal alkaline phosphatases play an important in drug metabolism by cleaving phosphate prodrugs and releasing the parental drugs [96]. Within role inthe drug cell, metabolism the regeneration by cleaving of the parental phosphate drug prod fromrugs the phosphate and releasing prodrug the parental is difﬁcult drugs to determine. [96]. Within the cell,In most the cases,regeneration this process of the is assumedparental todrug involve from a speciﬁcthe phosphate enzyme orprodrug enzymes is suchdifficult as an to esterase, determine. In mostan amidase,cases, this a phosphatase,process is assumed or even to a redoxinvolve process a specific [95,97 enzyme,98]. Therefore, or enzymes the phosphate-prodrugsuch as an esterase, an amidase,approach a phosphatase, has been successfully or even a usedredox to process enhance [95,97,98]. the solubility Therefore, and bioavailability the phosphate-prodrug of the parental drug.approach has been Degoeysuccessfullyet al., used synthesized to enhance a series the of solubility prodrugs and of lopinavir bioavailability (LPV, 60 of) andthe parental ritonavir drug. (RTV, 61), HIVDegoey protease et al. inhibitors., synthesized The a oxymethylphosphate series of prodrugs andof lopinavir oxyethylphosphate (LPV, 60) prodrugsand ritonavir (62/63 (RTV,and 64/65 61),, HIV proteaserespectively) inhibitors. had moreThe thanoxymethylphosp 700 and 1600-foldhate enhanced and oxyethylphosphate solubility in water comparedprodrugs to ( LPV62/63 and and RTV, 64/65, respectively)respectively had (Figure more 25 than). Pharmacokinetic 700 and 1600-fold studies enhanced in rats and solubility dogs showed in water high plasmacompared levels to ofLPV the and RTV,parent respectively drugs after (Figure administration 25). Pharmacokinetic via the oral route stud [99ies]. in rats and dogs showed high plasma levels of the parent drugs after administration via the oral route [99]

O O- P Na+ O - O OH O Na+ H N O O R N O H H N O N O N O H (60) O N O HN HN Lopinavir R= CH3 (62) R= H (64) >700-fold more water soluble

S N

O O S O HO HN O NH O O NH P + O - Na HN -O O HN O + S N R Na O N (61) S O O NH R= CH (63) Ritonavir 3 N N R= H (65) >1600-fold more water soluble Figure 25. Structures of the prodrugs of lopinavir and ritonavir [99].

Molecules 2016, 21, 42 17 of 30

O O HO O O O (59)

O O N H H O O O O OH O O

O H N N N O O Elastin-like S Acid-sensitive polypeptide (ELP) hydrazone bond Figure 24. Paclitaxel-biopolymer-conjugated prodrug [93].

8. Phosphate Prodrugs Phosphate ester compounds are relatively stable when free in metabolic surroundings because they have a low pKa, between 1 and 2. At a physiological pH of 7.0–7.4, the compounds are permanently deprotonated and therefore negatively charged but are readily activated upon complexation to various counterions in an enzyme’s active site [94,95]. Intestinal alkaline phosphatases play an important role in drug metabolism by cleaving phosphate prodrugs and releasing the parental drugs [96]. Within the cell, the regeneration of the parental drug from the phosphate prodrug is difficult to determine. In most cases, this process is assumed to involve a specific enzyme or enzymes such as an esterase, an amidase, a phosphatase, or even a redox process [95,97,98]. Therefore, the phosphate-prodrug approach has been successfully used to enhance the solubility and bioavailability of the parental drug. Degoey et al., synthesized a series of prodrugs of lopinavir (LPV, 60) and ritonavir (RTV, 61), HIV protease inhibitors. The oxymethylphosphate and oxyethylphosphate prodrugs (62/63 and 64/65, respectively) had more than 700 and 1600-fold enhanced solubility in water compared to LPV and RTV, respectively (Figure 25). Pharmacokinetic studies in rats and dogs showed high plasma levels Molecules 2016, 21, 42 18 of 31 of the parent drugs after administration via the oral route [99]

O O- P Na+ O - O OH O Na+ H N O O R N O H H N O N O N O H (60) O N O HN HN Lopinavir R= CH3 (62) R= H (64) >700-fold more water soluble

S N

O O S O HO HN O NH O O NH P + O - Na HN -O O HN O + S N R Na O N (61) S O O NH R= CH (63) Ritonavir 3 N N R= H (65) >1600-fold more water soluble FigureFigure 25.25. StructuresStructures of of the the prodrugs prodrugs ofof lopinavirlopinavir and and ritonavir ritonavir [99 [99].]. Molecules 2016, 21, 42 18 of 30

Another example of water soluble prodrug with phosphate group was synthesized by Flores-ramos etet al. al., ,using using as as prototype prototype the the compound compound α-6-chloro-2-(methylthio)-5-(naphthalen-1-α-6-chloro-2-(methylthio)-5-(naphthalen- 1-yloxy)-1yloxy)-1H-benzo[H-benzo[d]imidazoled]imidazole (66 (),66 a), benzimidazole a benzimidazole derivative. derivative. The Thesolubility solubility of the of prodrug the prodrug 67 was67 wasincreased increased 50,000-fold 50,000-fold when whencompared compared to its precursor to its precursor by linking by a linking disodium a disodium phosphate phosphate to the structure to the (Figurestructure 26). (Figure In vitro 26 ).andIn vitroin vivoand assaysin vivo demonstratedassays demonstrated that the prodrug that the prodrughave faciolicidal have faciolicidal activity; however,activity; however, administered administered dose in vivo dose wasin vivo lowerwas than lower the thanparent the drug parent [100]. drug [100].

(66) (67)

O O N N SCH SCH3 3 N Cl N Cl H O Increase >50,000 fold O P O Na water solubility O Na

Figure 26. Benzimidazole phosphate prodrug [100]. [100].

The chalcone family is known to prevent CXCL12 chemokine from binding to its CXCR4 or The chalcone family is known to prevent CXCL12 chemokine from binding to its CXCR4 or CXCR7 receptors, preventing inflammatory reactions, but their poor solubility in water limits their use CXCR7 receptors, preventing inﬂammatory reactions, but their poor solubility in water limits their use (9 μM/mL) [101]. To overcome this problem, chalcone prodrugs 68 were synthesized with different (9 µM/mL) [101]. To overcome this problem, chalcone prodrugs 68 were synthesized with different functional groups, such as phosphate, an L-seryl, and a sulfate. The phosphate prodrug 69 (Figure 27) functional groups, such as phosphate, an L-seryl, and a sulfate. The phosphate prodrug 69 (Figure 27) showed a greater increase in solubility, to at least 3000 times greater solubility than the parent chalcone. showed a greater increase in solubility, to at least 3000 times greater solubility than the parent chalcone. These results allowed low-dose intranasal administration, with ≥50% inhibited eosinophil recruitment These results allowed low-dose intranasal administration, with ě50% inhibited eosinophil recruitment in the airways at a dose as low as 30 nmol/kg without any signs of toxicity [102]. in the airways at a dose as low as 30 nmol/kg without any signs of toxicity [102]. O O O O Na+ Cl O O- Cl OH P Na+ O O- Chalcone Increse solubility: >3000-fold (68) (69) Figure 27. Chalcone-phosphate prodrugs [102].

Propofol (70) is another drug with high lipophilicity and is administered in an oil-in-water emulsion. To overcome this drawback, phosphate prodrugs of propofol have been generated using a phosphate group attached directly to the hydroxyl group of propofol with oxymethyl (compound 71) and ethylenedioxy moieties (compound 72) as spacers linked to a phosphate group (Figure 28). All these prodrugs demonstrated stability and enhanced solubility in water. Solubility of the ethyl dioxy phosphate derivative 72 was increased more than 70-fold compared to propofol. Furthermore, neither prodrug produced formaldehyde, a toxic subproduct, and thus both represent useful water-soluble prodrugs suitable for i.v. administration [103]. SB-3CT (73) is a selective inhibitor of matrix metalloproteinases 2 and 9 that is active in the treatment of traumatic brain injury (TBI) [63]. Its phosphate prodrug 74 (Figure 29) showed more than 2000-fold enhanced solubility in water. The prodrug was readily hydrolyzed to the active p-hydroxy SB-3CT (75), which crossed the blood-brain barrier and reached a therapeutic concentration in the brain. In an in vivo assay in TBI mice, the prodrug significantly decreased brain-lesion volume and improved neurological outcomes [104].

Molecules 2016, 21, 42 18 of 30

Another example of water soluble prodrug with phosphate group was synthesized by Flores-ramos et al., using as prototype the compound α-6-chloro-2-(methylthio)-5-(naphthalen-1- yloxy)-1H-benzo[d]imidazole (66), a benzimidazole derivative. The solubility of the prodrug 67 was increased 50,000-fold when compared to its precursor by linking a disodium phosphate to the structure (Figure 26). In vitro and in vivo assays demonstrated that the prodrug have faciolicidal activity; however, administered dose in vivo was lower than the parent drug [100].

(66) (67)

O O N N SCH SCH3 3 N Cl N Cl H O Increase >50,000 fold O P O Na water solubility O Na

Figure 26. Benzimidazole phosphate prodrug [100].

The chalcone family is known to prevent CXCL12 chemokine from binding to its CXCR4 or CXCR7 receptors, preventing inflammatory reactions, but their poor solubility in water limits their use (9 μM/mL) [101]. To overcome this problem, chalcone prodrugs 68 were synthesized with different functional groups, such as phosphate, an L-seryl, and a sulfate. The phosphate prodrug 69 (Figure 27) showed a greater increase in solubility, to at least 3000 times greater solubility than the parent chalcone. MoleculesThese2016 results, 21, 42allowed low-dose intranasal administration, with ≥50% inhibited eosinophil recruitment19 of 31 in the airways at a dose as low as 30 nmol/kg without any signs of toxicity [102].

O O O O Na+ Cl O O- Cl OH P Na+ O O- Chalcone Increse solubility: >3000-fold (68) (69)

FigureFigure 27.27. Chalcone-phosphateChalcone-phosphate prodrugs prodrugs [102]. [102].

Propofol (70) is another drug with high lipophilicity and is administered in an oil-in-water Propofol (70) is another drug with high lipophilicity and is administered in an oil-in-water emulsion. To overcome this drawback, phosphate prodrugs of propofol have been generated using emulsion.a phosphate To overcome group attached this drawback, directly to phosphate the hydrox prodrugsyl group of of propofol propofol with have oxymethyl been generated (compound using a phosphate71) and ethylenedioxy group attached moieties directly (compound to the hydroxyl 72) as groupspacers of linked propofol to a withphosphate oxymethyl group (compound (Figure 28).71 ) andAll ethylenedioxy these prodrugs moieties demonstrated (compound stability72 )and as spacersenhanced linked solubility to a phosphatein water. Solubility group (Figure of the 28ethyl). All thesedioxy prodrugs phosphate demonstrated derivative 72 stability was increased and enhanced more than solubility 70-fold incompared water. Solubility to propofol. of Furthermore, the ethyl dioxy phosphateneither prodrug derivative produced72 was increasedformaldehyde, more thana toxic 70-fold subproduct, compared and to propofol.thus both Furthermore, represent useful neither prodrugwater-soluble produced prodrugs formaldehyde, suitable for a toxici.v. administration subproduct, and[103]. thus both represent useful water-soluble prodrugsSB-3CT suitable (73) for is i.v.a selective administration inhibitor [ 103of matrix]. metalloproteinases 2 and 9 that is active in the treatmentSB-3CT of ( 73traumatic) is a selective brain injury inhibitor (TBI) [63]. of matrix Its phosphate metalloproteinases prodrug 74 (Figure 2 and 29) 9 showed that is activemore than in the treatment2000-fold of enhanced traumatic solubility brain injury in water. (TBI) The [63]. prodrug Its phosphate was readily prodrug hydrolyzed74 (Figure to the29) active showed p-hydroxy more than 2000-foldSB-3CT enhanced(75), which solubility crossed the in water.blood-brain The prodrug barrier and was reached readily hydrolyzeda therapeutic to concentration the active p-hydroxy in the SB-3CTbrain. (In75 ),an which in vivo crossedassay in the TBI blood-brain mice, the prodrug barrier signific and reachedantly decreased a therapeutic brain-lesion concentration volume and in the brain.improved In an neurologicalin vivo assay outcomes in TBI mice, [104]. the prodrug signiﬁcantly decreased brain-lesion volume and improvedMolecules 2016 neurological, 21, 42 outcomes [104]. 19 of 30 Molecules 2016, 21, 42 19 of 30

O O P Na+ O O P O- Na+ O O O- O- O- Na+ (71) Na+ (71)

OH Solubility not determined OH Solubility not determined

(70) (70) Propofol Propofol O O P Na+ O O P O- Na+ O O O- O- O- Na+ (72) Na+ (72) Increase in water-solubility: >70 fold Increase in water-solubility: >70 fold Figure 28. Propofol prodrug [103]. Figure 28. Propofol prodrug [[103].103].

O O

S Prodrug S S Prodrug O O S approach O O approach SB-3CT (73) SB-3CT (73)

O O O OH O Biotransformation HO OH Biotransformation HO P O P O S HO S O O S S HO S S O O S O O S O O O O (74) (75) (74) (75) Increase in water solubility:Increase >2000 in water fold p-hydroxy SB-3CT solubility: >2000 fold p-hydroxy SB-3CT Active metabolite MMP-9 inhibitor Active metabolite MMP-9 inhibitor Figure 29. SB-3CT prodrug and its active metabolite [104]. Figure 29. SB-3CT prodrug and its active metabolite [[104].104].

Compound 76 is a benzimidazole derivative with antibacterial activity that inhibits DNA gyrase and topoisomerase IV [105,106]. Due to its poor solubility in water, Dowd et al., synthesized a benzimidazole phosphate-ester prodrug. The compound 77 was up to 30,000-fold more water soluble than the parent drug at physiological pH (Figure 30). Interestingly, the phosphate-ester prodrug was much less potent in vitro against all bacteria strains tested. Nevertheless, in the target-level assay, the prodrug exhibited similar activity to the parent drug against S. aureus gyrase and topoisomerase IV [107].

F O F O F O F O N N N N NH O HO NH O HO N NH O HO O N NH O HO N P O N N N N O P N N N N HN HN O OH N HN HN OH H H H H (77) (76) (77) (76) Increase >30,000-fold Increasewater solubility>30,000-fold Prodrug water solubility Prodrug

OH O OH O P P OH O OH O N N N N NH N HN S NH S N S NH HN NH HN NH HN S S O O NH N O S N O S N S Cl N Cl N Cl N Cl N N (78) (79) (78) (79) Increase >335-fold Prodrug SNS-314 Increasewater solubilit >335-foldy Prodrug SNS-314 water solubility Figure 30. Benzimidazole and SNS-314 phosphate prodrugs [107,108]. Figure 30. Benzimidazole and SNS-314 phosphate prodrugs [107,108].

Molecules 2016, 21, 42 19 of 30

O P Na+ O O O- O- Na+ (71)

OH Solubility not determined

(70) Propofol O P Na+ O O O- O- Na+ (72)

Increase in water-solubility: >70 fold Figure 28. Propofol prodrug [103].

S Prodrug O S approach O SB-3CT (73)

O O OH HO Biotransformation P O O S HO S S S O O O O (74) (75) Increase in water solubility: >2000 fold p-hydroxy SB-3CT Active metabolite MMP-9 inhibitor Figure 29. SB-3CT prodrug and its active metabolite [104]. Molecules 2016, 21, 42 20 of 31 Compound 76 is a benzimidazole derivative with antibacterial activity that inhibits DNA gyrase and topoisomeraseCompound IV76 is[105,106]. a benzimidazole Due to derivative its poor with solubility antibacterial in activitywater, thatDowd inhibits et al. DNA, synthesized gyrase a benzimidazoleand topoisomerase phosphate-ester IV [105, 106prodrug.]. Due toThe its compound poor solubility 77 was in water, up to Dowd30,000-foldet al., synthesizedmore water a soluble than thebenzimidazole parent drug phosphate-ester at physiological prodrug. pH (Figure The compound 30). Interestingly,77 was up to 30,000-foldthe phosphate-ester more water prodrug soluble was than the parent drug at physiological pH (Figure 30). Interestingly, the phosphate-ester prodrug was much less potent in vitro against all bacteria strains tested. Nevertheless, in the target-level assay, the much less potent in vitro against all bacteria strains tested. Nevertheless, in the target-level assay, prodrugthe exhibited prodrug exhibited similar similar activity activity to the to theparent parent drug drug against against S. S. aureus aureusgyrase gyrase and topoisomeraseand topoisomerase IV [107].IV [107].

F O F O N N NH O HO NH O HO N O N P N N N O N N N H H OH H H (77) (76) Increase >30,000-fold water solubility Prodrug

OH O P OH O N N NH N HN S S NH HN NH S O O N S N Cl Cl N N (78) (79) Increase >335-fold Prodrug SNS-314 water solubility

FigureFigure 30. Benzimidazole 30. Benzimidazole and and SNS-314 SNS-314 phosphatephosphate prodrugs prodrugs [107 [107,108].,108].

Aurora serine/threonine kinases have been described as a promising anti-cancer target [109]. To overcome the poor solubility in water of SNS-314 (78), an aurora kinase inhibitor, a prodrug was designed with several moieties attached to the parent drug, such as acyl-oxymethylene, amine-containing acyl-oxymethylene and phosphate group. Prodrug 79, a phosphate-ester derivative, was 335-fold more water soluble than the parent drug (Figure 30). Moreover, although the acyl-oxymethylene derivatives showed increased solubility (8–20-fold), they were much less effective than the phosphate-ester prodrug [108].

9. Other Chemical Prodrugs Carbamazepine (CBZ) (80) is an effective anticonvulsant drug that causes less sedation and cognitive impairment than other anticonvulsants; however, its aqueous solubility is 120 µg/mL, hindering intravenous administration [110]. To overcome this problem, CBZ prodrugs 81 and 82 with improved solubility were synthesized (Figure 31). The aqueous chemical stability of 81 was superior to that of 82. Compound 81 was dissolved in water, with apparent solubility in excess of 100 mg/mL (ﬁnal pH value of 2.6). An in vivo pharmacokinetic assay with i.v. administration demonstrated that compound 81 was rapidly converted to CBZ [111]. Molecules 2016, 21, 42 20 of 30

9. Other Chemical Prodrugs Carbamazepine (CBZ) (80) is an effective anticonvulsant drug that causes less sedation and cognitive impairment than other anticonvulsants; however, its aqueous solubility is 120 μg/mL, hindering intravenous administration [110]. To overcome this problem, CBZ prodrugs 81 and 82 with improved solubility were synthesized (Figure 31). The aqueous chemical stability of 81 was superior to that of 82. Compound 81 was dissolved in water, with apparent solubility in excess of Molecules1002016 mg/mL, 21, 42 (final pH value of 2.6). An in vivo pharmacokinetic assay with i.v. administration21 of 31 demonstrated that compound 81 was rapidly converted to CBZ [111].

(81) (80) (82)

N NH2 .TFA N N S NHS NH .TFA ON ONH2 O 2 H Carbamazepine O Solubility: 100,000 µg/mL 100 µg/mL O Solubility: not determined

O O S NH2 SO2N(Na)COCH2CH2CO2Na

(83) (84)

N N N N

CF 3 CF3 PC407 Prodrug Solubilty: 1.6 µg/mL Solubilty: 20,300 µg/mL

FigureFigure 31. 31.Carbamazepine Carbamazepine and PC407 PC407 prodrugs prodrugs [111,112]. [111,112 ].

The sulfonamide group was also utilized to develop a cyclooxygenase 2 (COX-2)-inhibitor prodrug Theof PC407 sulfonamide (83) for group parenteral was alsoadministration utilized to (Figure develop 31 a). cyclooxygenase The prodrug showed 2 (COX-2)-inhibitor highly improved prodrug of PC407solubility, (83) forfrom parenteral 1.6 μg/mL to administration 20.3 mg/mL. Prodrug (Figure 84 showed31). The analgesic prodrug activity showed in vivo highlyand aqueous improved solubility,stability from and 1.6 is µag/mL promising to 20.3 candidate mg/mL. COX-2 Prodrug inhibitor84 forshowed injectable analgesic formulation activity [112].in vivo and aqueous stability andTetrahydrocurcumin is a promising candidate (THCur) COX-2(85) is inhibitora metabolite for injectableof curcumin formulation that shows [112 antioxidant]. and anticancer activities [113–115]. A series of THCur prodrugs was synthesized using Tetrahydrocurcumin (THCur) (85) is a metabolite of curcumin that shows antioxidant carboxymethylcellulose (CMC) as a water-soluble group linked by an azo bond. 4-Amino-THCur and anticancer(86, Figure 32) activities has shown [increased113–115]. solubility A series in water of up THCur to 5 mg/mL prodrugs and was was released synthesized in the colon using carboxymethylcellulosevia azoreductase enzymes (CMC) of colonic as a water-soluble bacteria (up to group 62% within linked 24 h). by In an human azo bond. colon 4-Amino-THCuradenocarcinoma (86, Figurecell 32 )lines has (HT-29), shown increasedthe prodrug solubility was cytotoxic, in water with a up IC50 to of 5 28.67 mg/mL ± 1.01 and μg/mL, was and released selective in theto these colon via azoreductasecells, unlike enzymes curcumin, of colonicwhich was bacteria non-selective (up to [116]. 62% within 24 h). In human colon adenocarcinoma cell lines (HT-29),Famotidine the ( prodrug87) is a histamine was cytotoxic, H2-receptor with a blocker IC50 of us 28.67ed to˘ treat1.01 µandg/mL, prevent and ulcers selective [117]. to these cells, unlikeVijayaraj curcumin, et al., synthesized which a was water-soluble non-selective prodrug [116 of]. famotidine by introducing a sulphoxide group. This modification (compound 88) increased solubility in water 6.7-fold compared to famotidine Famotidine (87) is a histamine H2-receptor blocker used to treat and prevent ulcers [117]. (Figure 32). The prodrug was stable at pH 1.2 and 7.4 and might be effective in ulcer therapy [118]. Vijayaraj et al., synthesized a water-soluble prodrug of famotidine by introducing a sulphoxide group. This modiﬁcation (compound 88) increased solubility in water 6.7-fold compared to famotidine

(FigureMolecules 32). The 2016, prodrug 21, 42 was stable at pH 1.2 and 7.4 and might be effective in ulcer therapy21 of 30 [118 ].

OO OO O O O O

N HO OH HO N OH (85) Tetrahydrocurcumin PABA/PAH (86) Solubility: 0.1mg/mL hexadeamine

Na-CMC

Solubility: 5 mg/mL

NH2 NH2 S S O H2N (87) H2N (88) O N O N S N S N N S N S NH NH O 2 O 2 NH2 NH2 Famotidine 6.7-fold more water-soluble Figure 32. Tetrahydrocurcumin and famotidine prodrugs [116,118]. Figure 32. Tetrahydrocurcumin and famotidine prodrugs [116,118]. 10. Marketed Prodrugs From 2005 to 2015, several prodrugs were discovered and launched in the market (Table 1). Some of these drugs are summarized in Table 1 and will be discussed below.

Table 1. Prodrugs launched in the USA, 2005–2015 a.

Year Prodrug Name Chemical Structure Indication

Nelarabine 2005 Arranon® Lymphoblastic leukemia GlaxoSmithKline plc

2006 - - - Lisdexamfetamine dimesylate Attention–deficit/ Vyvanse® hyperactivity disorder New River, Inc.

2007 Temsirolimus Advanced renal cell Torisel® carcinoma Wyeth Pharm., Inc.

Fesoterodinefumarate Overactive bladder 2008 Toviaz® disorder Pfizer, Inc.

Molecules 2016, 21, 42 21 of 30 Molecules 2016, 21, 42 21 of 30 Molecules 2016, 21, 42 21 of 30 Molecules 2016, 21, 42 21 of 30 OO OO OO O OO O OO OO O OO O O O O OO O O O O O O O O O HO OH N HO OH HO N N OH HO OH HO N N OH HO (85)OH HO N N OH Tetrahydrocurcumin (85) HO N OH Tetrahydrocurcumin (85) PABA/PAH (86) Tetrahydrocurcumin (85) PABA/PAH (86) Solubility:Tetrahydrocurcumin 0.1mg/mL PABA/PAH (86) Solubility: 0.1mg/mL PABA/PAH (86) Solubility: 0.1mg/mL Solubility: 0.1mg/mL hexadeamine hexadeamine hexadeamine hexadeamine Na-CMC Na-CMC Na-CMC Na-CMC Solubility: 5 mg/mL Solubility: 5 mg/mL Solubility: 5 mg/mL Solubility: 5 mg/mL

NH2 NH2 NH NH2 NH2 S H N NH2 S O (88) H2N 2 S (87) 2 S H N NH2 S O (87) H2N NHN 2 S O O (88) H2N N (87) H2N OS N O (88) 2 N S S N O N SN O O H2N N N S N SO (87) H2N N S N S (88) N S N SONH N N S N SONH2 N N S NOS 2 N S NHNOS NH2 NH2 N 2 NH2 N NH OS NH2 NH2 OS NH2 NH2O NH NH2 O NH Molecules 2016, 21Famotidine, 42 2O 2 6.7-fold more water-soluble NH O 2 22 of 31 Famotidine NH2 6.7-fold more water-soluble 2 Famotidine 6.7-fold more water-soluble FamotidineFigure 32. Tetrahydrocurcumin and6.7 -famofold mtidineore water-soluble prodrugs [116,118]. Figure 32. Tetrahydrocurcumin and famotidine prodrugs [116,118]. 10. Marketed ProdrugsFigure 32. Tetrahydrocurcumin and famotidine prodrugs [116,118]. 10. Marketed Prodrugs 10. Marketed Prodrugs 10. MarketedFrom 2005 Prodrugs to 2015, several prodrugs were discovered and launched in the market (Table3). Some 10. MarketedFrom 2005 Prodrugs to 2015, several prodrugs were discovered and launched in the market (Table 1). of theseFrom drugs2005 to are 2015, summarized several prodrugs in Table3 andwere will discov be discussedered and below.launched in the market (Table 1). SomeFrom of these 2005 drugs to 2015, are summarizedseveral prodrugs in Table were 1 and discov willered be discussed and launched below. in the market (Table 1). Some of these drugs are summarized in Table 1 and will be discussed below. Some of these drugs are summarizedTable 1. Prodrugs in Table launched 1 and will in thebe USA,discussed 2005–2015 below.a. a Table 1. Prodrugs launched in the USA, 2005–2015 a. Table 1. Prodrugs launched in the USA, 2005–2015 a. Table 1. Prodrugs launched in the USA, 2005–2015 a. YeaYearr Prodrug Prodrug Name NameTable 1. Prodrugs launched Chemical in the Structure USA, 2005–2015 . Indication Indication Year Prodrug Name Chemical Structure Indication Year Prodrug Name Chemical Structure Indication

Nelarabine Nelarabine ® NelarabineNelarabine Arranon® 20052005 NelarabineArranon® LymphoblasticLymphoblastic leukemia leukemia 2005 GlaxoSmithKlineArranon® plc Lymphoblastic leukemia 2005 GlaxoSmithKlineArranon® plc Lymphoblastic leukemia GlaxoSmithKline plc GlaxoSmithKline plc

2006 - - - 20062006 - - - - - 2006 Lisdexamfetamine- - - 2006 Lisdexamfetamine- - - Lisdexamfetaminedimesylate Attention–deficit/ LisdexamfetamineLisdexamfetaminedimesylate Attention–deficit/ dimesylate® ® Attention–deﬁcit/hyperactivityAttention–deficit/ dimesylatedimesylateVyvanse Vyvanse® hyperactivityAttention–deficit/ disorder Vyvanse® hyperactivitydisorder disorder NewNewVyvanse River, River, ®Inc. Inc. hyperactivity disorder New River, Inc. New River, Inc. 2007 20072007 2007 Temsirolimus 2007 Temsirolimus Advanced renal cell TemsirolimusTorisel® Advanced renal cell TemsirolimusTemsirolimus® Torisel ® AdvancedAdvanced renal renal cell cell Torisel® Advancedcarcinoma renal cell WyethWyethTorisel Pharm., Pharm.,® Inc. Inc. carcinomacarcinoma Wyeth Pharm., Inc. carcinoma Wyeth Pharm., Inc.

Fesoterodinefumarate Fesoterodinefumarate Overactive bladder Fesoterodinefumarate® Overactive bladder 2008 FesoterodinefumarateToviaz® 2008 Toviaz® Overactivedisorder bladder 2008 FesoterodinefumaratePfizer,Toviaz Inc.® Overactivedisorder bladder 2008 Pfizer,Toviaz® Inc. Overactivedisorder bladder disorder Molecules 2016, 21, Toviaz42 Pfizer,Pﬁzer, Inc. Inc. disorder 22 of 30 Molecules 2016, 21, 42 Pfizer, Inc. 22 of 30 Molecules 2016, 21, 42 22 of 30 Table 1. Cont. Table 1. Cont. 2008 Table 1. Cont.

FospropofoldisodiumFospropofoldisodium Monitored anesthesia Fospropofoldisodium Monitored anesthesia care FospropofoldisodiumLusedraLusedra®® Monitoredcare sedation. anesthesia Lusedra® sedation.care sedation. # discontinued ElanPharm.ElanPharm.Lusedra® plc plc #care discontinued sedation. ElanPharm. plc # discontinued ElanPharm. plc # discontinued

Prasugrel Prevention of thrombotic Prasugrel PreventionPrevention of ofthrombotic thrombotic PrasugrelPrasugrelEffient Efﬁent® ® Preventioncardiovascular of thrombotic Effient® cardiovascularcardiovascular EliEli Lilly Lilly (developedEffient (developed® with with complicationscardiovascular in acute Eli Lilly (developed with complicationscomplications in inacute acute Eli LillyDaiichiDaiichi (developed Sankyo) Sankyo) with complicationscoronary syndromes in acute Daiichi Sankyo) coronarycoronary syndromes syndromes Daiichi Sankyo) coronary syndromes 2009 20092009 2009 Romidepsin Romidepsin Cutaneous T cell RomidepsinIstodax® Cutaneous T cell RomidepsinIstodax Istodax® ® Cutaneouslymphoma T cell GloucesterIstodax Pharm.® Cutaneouslymphoma T celllymphoma GloucesterGloucester Pharm. Pharm. lymphoma Gloucester Pharm.

Dabigatranetexilate Dabigatranetexilate Dabigatranetexilatemesylate mesylate Thromboembolism Pradaxamesylate® Thromboembolism Pradaxa® acuteThromboembolism coronary syndrome BoehringerPradaxa Ingelheim® acute coronary syndrome Boehringer Ingelheim acute coronary syndrome BoehringerGmbH Ingelheim GmbH GmbH Multiple sclerosis Fingolimod Multiple sclerosis 2010 Fingolimod [sphingosin-1-phosphateMultiple sclerosis 2010 FingolimodGilenya® [sphingosin-1-phosphate 2010 Gilenya® [sphingosin-1-phosphate(S1P) agonist with Novartis GilenyaInternational® AG (S1P) agonist with Novartis International AG cannabinoid(S1P) agonist antagonist] with Novartis International AG cannabinoid antagonist] H3C O cannabinoid antagonist] H3C O H C O H H Ceftarolinefosamil 3 N HN H S Ceftarolinefosamil S S ® N NH H S CeftarolinefosamilTeflaro S N N S S N CH3 Bacterial skin infection Teflaro® O S N O N S N CH Bacterial skin infection ® NH N O O N S N 3 Forest Laboratories,Teflaro Inc. O P N O N S N N CH3 Bacterial skin infection O NH O COO N Forest Laboratories, Inc. HOP OHNH O S P OH COO Forest Laboratories, Inc. HO COO HO OH

Hormone-refractory Abiraterone acetate Hormone-refractory Abiraterone acetate Hormone-refractoryprostate cancer AbirateroneZytiga ®acetate prostate cancer Zytiga® (17-prostateα-hydrolase/ cancer JanssenZytiga Biotech,® Inc. (17-α-hydrolase/ Janssen Biotech, Inc. (17-C17,20lyase)α-hydrolase/ Janssen Biotech, Inc. C17,20lyase) C17,20lyase)

2011 2011 Azilsartanmedoxomil Hypertension 2011 Azilsartanmedoxomil Hypertension AzilsartanmedoxomilEdarbi® (angiotensinHypertension II Edarbi® (angiotensin II TakedaEdarbi Pharm.® (angiotensinantagonist) II Takeda Pharm. antagonist) Takeda Pharm. antagonist)

Restless leg syndrome Gabapentin encarbil Restless leg syndrome Gabapentin encarbil (GABARestless and leg Casyndrome channel GabapentinHorizant encarbil® (GABA and Ca channel Horizant® (GABAmodulator) and Ca newchannel GlaxoSmithKlineHorizant® plc modulator) new GlaxoSmithKline plc modulator)indication new GlaxoSmithKline plc indication indication

MoleculesMolecules 2016 2016,, , 21 21,, , 42 4242 2222 of of 30 30 Molecules 2016, 21, 42 22 of 30 Molecules 2016, 21, 42 22 of 30 Molecules 2016, 21, 42 22 of 30 TableTable 1. 1. Cont Cont.. . Table 1. Cont. Table 1. Cont. Table 1. Cont.

FospropofoldisodiumFospropofoldisodium MonitoredMonitored anesthesia anesthesia Fospropofoldisodium Monitored anesthesia FospropofoldisodiumLusedraLusedra®® Monitoredcarecare sedation. sedation. anesthesia FospropofoldisodiumLusedra® Monitoredcare sedation. anesthesia ElanPharm.ElanPharm.Lusedra ®plc plc ## carediscontinued discontinued sedation. ElanPharm.Lusedra® plc #care discontinued sedation. ElanPharm. plc # discontinued ElanPharm. plc # discontinued

PrasugrelPrasugrel PreventionPrevention of of thrombotic thrombotic Prasugrel Prevention of thrombotic PrasugrelEffientEffient®® Preventioncardiovascularcardiovascular of thrombotic PrasugrelEffient® Preventioncardiovascular of thrombotic EliEli Lilly Lilly (developed (developedEffient® with with complicationscomplicationscardiovascular in in acute acute Eli Lilly (developedEffient® with complicationscardiovascular in acute Eli LillyDaiichiDaiichi (developed Sankyo) Sankyo) with complicationscoronarycoronary syndromes syndromes in acute Eli LillyDaiichi (developed Sankyo) with complicationscoronary syndromes in acute Daiichi Sankyo) coronary syndromes Daiichi Sankyo) coronary syndromes 20092009 2009 Molecules2009 2016, 21, 42 23 of 31 2009 RomidepsinRomidepsin Romidepsin CutaneousCutaneous T T cell cell RomidepsinIstodaxIstodaxIstodax®® Cutaneous T cell RomidepsinIstodax® Cutaneouslymphomalymphomalymphoma T cell GloucesterGloucesterIstodax Pharm. Pharm.® Table 2. Cont. Cutaneouslymphoma T cell GloucesterIstodax Pharm.® lymphoma Gloucester Pharm. lymphoma Gloucester Pharm. Year Prodrug Name Chemical Structure Indication DabigatranetexilateDabigatranetexilate DabigatranetexilateDabigatranetexilate Dabigatranetexilatemesylatemesylate Dabigatranetexilatemesylatemesylate Pradaxa ® ThromboembolismThromboembolism acute PradaxaPradaxamesylate®® Thromboembolism BoehringerPradaxamesylate® acuteacuteThromboembolism coronary coronary syndromesyndromesyndrome BoehringerBoehringerPradaxa Ingelheim Ingelheim® acuteThromboembolism coronary syndrome BoehringerIngelheimPradaxa Ingelheim GmbH® acute coronary syndrome BoehringerGmbHGmbH Ingelheim acute coronary syndrome BoehringerGmbH Ingelheim GmbH MultipleMultiple sclerosissclerosis FingolimodFingolimodGmbH ® MultipleMultiple sclerosis sclerosis FingolimodFingolimod Gilenya [sphingosin-1-phosphateMultiple sclerosis 201020102010 Fingolimod®® [sphingosin-1-phosphate[sphingosin-1-phosphate[sphingosin-1-phosphateMultiple sclerosis 2010 FingolimodGilenyaGilenyaNovartis® [sphingosin-1-phosphateMultiple sclerosis 2010 FingolimodGilenya® [sphingosin-1-phosphate(S1P)(S1P)(S1P)(S1P) agonist agonistagonist agonist with withwith with 2010 NovartisNovartisInternational GilenyaInternationalInternational® AG AGAG [sphingosin-1-phosphate(S1P) agonist with Novartis InternationalGilenya® AG cannabinoidcannabinoidcannabinoid(S1P) agonist antagonist]antagonist] antagonist] with Novartis International AG cannabinoid(S1P) agonist antagonist] with Novartis International AG HH3CC OO cannabinoid antagonist] Novartis International AG H33C O cannabinoid antagonist] H3C O HH HH CeftarolinefosamilCeftarolinefosamil N H H cannabinoid antagonist] Ceftarolinefosamil H3CN O NN SS Ceftarolinefosamil SS NH HS SS Ceftarolinefosamil®® H3SC N O NH S S TeflaroTeflaro® ® H NN CHCH3 BacterialBacterial skinskin infectioninfection CeftarolinefosamilTeﬂaroTeflaroForest S NNN OO HN NNH S S N CH33 BacterialBacterial skin skin infection infection Ceftarolinefosamil® OO S NN O N SS SNN N CH Teflaro® O NHNH N O ONO N S S N 3 Bacterial skin infection ForestForest Laboratories,Laboratories,Teflaro Inc.Inc. OPP NHS O S N N CH Bacterial skin infection ForestLaboratories, Laboratories,® Inc. Inc. OHNH N O O N COOCOO S 3 Forest Laboratories,Teflaro Inc. HHOOOPOH COO S N N CH3 Bacterial skin infection HOO NH N O O N COO Forest Laboratories, Inc. HOP OHNH O S N P OH COO Forest Laboratories, Inc. HO COO HO OH Hormone-refractoryHormone-refractory AbirateroneAbiraterone acetateacetate Hormone-refractory Abiraterone acetate prostateprostate cancercancer AbirateroneAbiraterone® ®acetate acetate Hormone-refractoryHormone-refractoryprostate cancer prostate AbirateroneZytigaZytiga® acetate Hormone-refractoryprostate cancer AbirateroneZytigaZytiga ®acetate® (17-(17-(17-prostateαα-hydrolase/-hydrolase/-hydrolase/cancer cancer JanssenJanssenJanssenZytiga Biotech, Biotech,Biotech,® Inc. Inc.Inc. (17-prostateα-hydrolase/ cancer JanssenJanssenZytiga Biotech, Biotech,® Inc. Inc. (17-α(17-C17,20lyase)-hydrolase/C17,20lyase)C17,20lyase)α-hydrolase/ Janssen Biotech, Inc. (17-C17,20lyase)α-hydrolase/ Janssen Biotech, Inc. C17,20lyase) C17,20lyase)

20112011 Azilsartanmedoxomil Hypertension 20112011 Azilsartanmedoxomil Hypertension 2011 Azilsartanmedoxomil®® Hypertension 2011 AzilsartanmedoxomilEdarbiEdarbi® (angiotensin(angiotensin(angiotensinHypertension II IIII AzilsartanmedoxomilAzilsartanmedoxomilEdarbi® (angiotensinHypertensionHypertension II TakedaTakedaEdarbi Pharm. Pharm.® (angiotensinantagonist)antagonist) II EdarbiTakeda®EdarbiTakeda Pharm.® Pharm. (angiotensin(angiotensinantagonist) II antagonist) II Takeda Pharm. antagonist) Takeda Pharm. antagonist)

RestlessRestless leg leg syndrome syndrome GabapentinGabapentin encarbil encarbil Restless leg syndrome Gabapentin encarbil (GABA(GABA(GABARestless and andand leg Ca Ca Casyndrome channel channelchannel GabapentinHorizantHorizant encarbil®® (GABARestless and leg Casyndrome channel GabapentinGabapentinHorizant encarbil encarbil® (GABARestlessmodulator)modulator) and leg Ca new syndrome newchannel Molecules 2016, 21GlaxoSmithKlineGlaxoSmithKline, 42 Horizant® plc plc (GABAmodulator) and Ca newchannel 23 of 30 GlaxoSmithKlineHorizantHorizant®® plc (GABAmodulator)indicationindicationindication and Ca new channel GlaxoSmithKline plc modulator)indication new GlaxoSmithKlineGlaxoSmithKline plc plc modulator)indication new indication Table 1. Cont. indication

Tafluprost ElevatedElevated intraocular intraocular Taﬂuprost Zioptan® 20122012 Zioptan® pressure (prostaglandinpressure Merck & Co., Inc. Merck & Co., Inc. (prostaglandinanalog) analog)

Dimethyl fumarate Relapsing multiple Tecfidera® sclerosis Biogen Idec, Inc.

Eslicarbazepineacetate Anticonvulsant Aptiom® (partial-onset seizures) Bial-Portela & Ca. S.A. 2013

Sofosbuvir Treatment of hepatitis C Sovaldi® virus (HCV) infection Gilead Sciences, Inc.

Droxidopa Neurogenic orthostatic Northera® hypotension Lundbeck A/S

2014 Tedizolidphosphate Acute bacterial skin and Sivextro® skin structure infections Cubist Pharma., Inc.

Isavuconazonium sulfate Invasive aspergillosis and 2015 Cresemba® invasive mucormycosis Astellas Pharma, Inc.

a The table does not include the launch of reformulations or new indications of already-marketed prodrugs. Drugs approved till the first half 2015.

Eslicarbazepine (ESL) is an antiepileptic prodrug developed from oxcarbazepine and approved by the European Medicines Agency (EMA), the Food and Drug Administration and Health Canada as an adjunctive therapy in adults with partial-onset seizures. The development of the ESL acetate prodrug was based on the observation that the active S-enantiomer of ESL is produced after biotransformation at a rate 20-fold higher than the inactive R-enantiomer [119,120].

Molecules 2016, 21, 42 23 of 30 Molecules 2016, 21, 42 23 of 30 Molecules 2016, 21, 42 23 of 30 Molecules 2016, 21, 42 23 of 30 Molecules 2016, 21, 42 23 of 30 Molecules 2016, 21, 42 23 of 30 Table 1. Cont. Table 1. Cont. Table 1. Cont. Table 1. Cont.

Molecules 2016, 21, 42Tafluprost Elevated intraocular 24 of 31 Tafluprost® Elevated intraocular 2012 TafluprostZioptan® pressureElevated (prostaglandin intraocular 2012 TafluprostZioptan® pressureElevated (prostaglandin intraocular 2012 MerckTafluprostZioptan & Co.,® Inc. pressureElevatedanalog) (prostaglandin intraocular 2012 MerckZioptan & Co.,® Inc. pressureanalog) (prostaglandin 2012 MerckZioptan & Co.,® Inc. Table 3. Cont. pressureanalog) (prostaglandin Merck & Co., Inc. analog) Merck & Co., Inc. analog) Year Prodrug Name Chemical Structure Indication

Dimethyl fumarate DimethylDimethyl fumarate fumarate Relapsing multiple Dimethyl fumarate®® Relapsing multiple DimethylTecfideraTecﬁdera fumarate® RelapsingRelapsing multiple multiple sclerosis DimethylTecfidera fumarate® Relapsingsclerosis multiple DimethylBiogenBiogenTecfidera Idec, Idec,fumarate ®Inc.Inc. Relapsingsclerosis multiple BiogenTecfidera Idec,® Inc. Relapsingsclerosis multiple BiogenTecfidera Idec, ®Inc. sclerosis Biogen Idec, Inc. sclerosis Biogen Idec, Inc.

Eslicarbazepineacetate EslicarbazepineacetateEslicarbazepineacetate Anticonvulsant Eslicarbazepineacetate® Anticonvulsant EslicarbazepineacetateAptiom®® AnticonvulsantAnticonvulsant EslicarbazepineacetateAptiomAptiom® (partial-onsetAnticonvulsant seizures) Bial-PortelaEslicarbazepineacetateAptiom & Ca.® S.A. (partial-onset(partial-onsetAnticonvulsant seizures) seizures) Bial-PortelaBial-PortelaAptiom & & ®Ca. Ca. S.A. S.A. (partial-onsetAnticonvulsant seizures) 2013 Bial-PortelaAptiom & Ca.® S.A. (partial-onset seizures) 20132013 Bial-Portela & Ca. S.A. (partial-onset seizures) 2013 Bial-Portela & Ca. S.A. 2013 2013

Sofosbuvir Sofosbuvir Treatment of hepatitis C Sofosbuvir® Treatment of hepatitis C SofosbuvirSovaldi® ® Treatment of hepatitis C SofosbuvirSofosbuvirSovaldi Sovaldi® TreatmentvirusTreatment (HCV) of hepatitis ofinfection hepatitis C GileadSofosbuvirSovaldi Sciences,® Inc. Treatmentvirus (HCV) of hepatitis infection C GileadGileadSovaldi Sciences, Sciences,® Inc. Inc. TreatmentvirusC virus (HCV) (HCV)of hepatitis infection infection C GileadSovaldi Sciences,® Inc. virus (HCV) infection Gilead Sciences, Inc. virus (HCV) infection Gilead Sciences, Inc.

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a a The table does not include the launch of reformulations or new indications of already-marketed a The table does not include the launch of reformulations or new indications of already-marketed prodrugs.a The table Drugs does approvednot include till the the launch first half of 2015.reform ulations or new indications of already-marketed aprodrugs. The table Drugs does notapproved include till the the launch first half of 2015.reform ulations or new indications of already-marketed prodrugs.a Drugs approved till the first half 2015. prodrugs. Thea Thetable table Drugs does does approvednot not include include till the the launchlaunch first half of of reformulations 2015.reform ulations or new or new indications indications of already-marketed of already-marketed prodrugs. prodrugs. Drugs approved till the first half 2015. Eslicarbazepineprodrugs.Drugs approvedDrugs approved (ESL) till the is ﬁrst antill half antiepilepticthe 2015. first half 2015. prod rug developed from oxcarbazepine and approved Eslicarbazepine (ESL) is an antiepileptic prodrug developed from oxcarbazepine and approved by theEslicarbazepineEslicarbazepine European Medicines (ESL)(ESL) isis Agency anan antiepilepticantiepileptic (EMA), the prodprod Forugrugod anddevelopeddeveloped Drug Administration fromfrom oxcarbazepineoxcarbazepine and Health andand approvedapproved Canada by theEslicarbazepine EslicarbazepineEuropean Medicines (ESL) (ESL) is Agency isan an antiepileptic antiepileptic (EMA), the prod prodrug Forugod anddeveloped developed Drug Administration from oxcarbazepine and Health andand approvedapproved Canada by byasby anthethe adjunctive EuropeanEuropean MedicinestherapyMedicines in AgencyAgencyadults with (EMA),(EMA), partial-onset thethe FoFoodod andseizures.and DrugDrug The AdministrationAdministration development andand of theHealthHealth ESL Canada Canadaacetate asbythe anthe Europeanadjunctive European Medicines therapyMedicines in Agency Agencyadults (EMA),with (EMA), partial-onset the the Food Food and seizures.and Drug Drug Administration The Administration development and and Healthof theHealth ESL Canada Canadaacetate as an asprodrugas anan adjunctiveadjunctive was based therapytherapy on theinin adults adultsobservation withwith partial-onsetpartial-onset that the active seizures.seizures. S-enantiomer TheThe developmentdevelopment of ESL ofisof theproducedthe ESLESL acetateacetate after prodrugasadjunctive an adjunctive was therapy based therapy inon adults thein adults observation with with partial-onset partial-onset that the seizures. active seizures. TheS-enantiomer development The development of ofESL the isof ESL producedthe acetate ESL acetate prodrug after prodrugbiotransformationprodrug waswas basedbased at aonon rate thethe 20-fold observationobservation higher thanthatthat thethethe inactive activeactive SSR-enantiomer-enantiomer-enantiomer of[119,120].of ESLESL isis producedproduced afterafter biotransformationprodrugwas based was on based the at observation aon rate the 20-fold observation that higher the active thanthatS the-enantiomerthe inactive active SR of-enantiomer-enantiomer ESL is produced [119,120].of ESL after is biotransformationproduced after biotransformationbiotransformation atat aa raterate 20-fold20-fold higherhigher thanthan thethe inactiveinactive RR-enantiomer-enantiomer [119,120].[119,120]. biotransformationat a rate 20-fold higherat a rate than 20-fold the inactive higher thanR-enantiomer the inactive [119 R-enantiomer,120]. [119,120].

Fospropofol disodium, a phosphate ester prodrug of propofol, was approved by the FDA in 2008

as a sedative–hypnotic agent for monitored anesthesia-care sedation in patients who are undergoing diagnostic or therapeutic procedures. The aqueous solubility of fospropofol provides an advantage over propofol, which is available only as a lipid-containing oil-water emulsion. These advantages Molecules 2016, 21, 42 25 of 31 include lack of pain on injection, different pharmacokinetic proﬁle due to in vivo conversion of fospropofol to propofol and rapid recovery from sedation [121,122]. Ceftaroline fosamil, a phosphoramidate prodrug launched in 2011 for the treatment of bacterial skin infections and bacterial pneumonia, showed 50-fold enhancement of solubility relative to the parental drug, thus enabling formulation of a marketable injectable solution [123]. Tedizolid phosphate, which has been marketed for the treatment of acute bacterial skin and skin-structure infections since 2014, is a phosphate-ester prodrug of the active compound tedizolid. The additional phosphate group provides improved aqueous solubility and thus bioavailability [124,125].

11. Conclusions The prodrug approach has been a successful tool for improving solubility in water, as can be observed from several publications that showed up to 400,000-fold increased solubility compared to the parent drug, as described herein. This approach can make it possible to avoid discarding promising active prototypes or drugs with therapeutic uses limited by poor solubility. The rational selection of the adequate pro-moiety and the type of linkage, (e.g., ester, amide, carbamate and phosphate), may determine the prodrug selectivity, toxicity, and ideal bioconversion proﬁle. Furthermore, the prodrug approach could be viewed as an alternative in the early phases of drug discovery. This technique may be used to modulate pharmacokinetic properties (absorption, distribution, metabolism and excretion), as well as poor water solubility, a critical step in pre-clinical phases. The majority of prodrugs presented herein were esters and amides because esterases and amidases could activate them, releasing the parent drug. Amino acids were the most-used water-soluble pro-moieties and, as demonstrated by several reviewed authors, efﬁciently increase solubility in water. Nevertheless, several other chemical groups are represented herein to a lesser degree, such as glycol groups (e.g., polyethylene glycol and ethylene glycol) and glycosides. Several prodrugs were approved in the last 10 years by the FDA, although not all prodrugs were designed to increase solubility. Nevertheless, many prodrugs were designed for this purpose, such as tedizolid phosphate, ceftaroline fosamil and fospropofol disodium. Therefore, the prodrug approach is an important tool in rational drug design to improve drug solubility in water.

Acknowledgments: The authors thank the Programa de Apoio ao Desenvolvimento Científico da Faculdade de Ciências Farmacêuticas da UNESP (PADC-FCF UNESP) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2014/02240-1; 2014/24811-0; 2014/06755-6 and 2014/14980-0) for financial support. Author Contributions: Authors D.H.J., G.F.S.F., D.E.C., T.R.F.M. and C.M.C. designed the study, analyzed and organized the literature papers. J.L.S. and C.M.C. helped rewrite the revised manuscript; J.L.S. and C.M.C. revised the manuscript and approved it in its final form. All authors edited and contributed to drafts of the manuscript. All authors approved the final form of the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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