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Enzyme-Mediated Hydrolytic Activation of Prodrugs

Enzyme-Mediated Hydrolytic Activation of Prodrugs

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provided by Elsevier - Publisher Connector Acta Pharmaceutica Sinica B 2011;1(3):143–159

Institute of Materia Medica, Chinese Academy of Medical Sciences Chinese Pharmaceutical Association

Acta Pharmaceutica Sinica B

www.elsevier.com/locate/apsb www.sciencedirect.com

REVIEW -mediated hydrolytic activation of prodrugs

Yan-hui Yang, Herve Aloysius, Daigo Inoyama, Yu Chen, Long-qin Hun

Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA

Received 15 July 2011; revised 4 August 2011; accepted 10 August 2011

KEY WORDS Abstract Prodrug design is an important part of drug discovery. Prodrugs can offer many advantages over parent drugs such as increased , enhanced stability, improved bioavail- Prodrugs; Hydrolytic activation; ability, reduced side effects, and better selectivity. Many prodrugs have been used successfully in ; the clinic; examples include oseltamivir in anti-influenza therapy, enalapril in anti-hypertension ; therapy, in cancer therapy, and omeprazole in the treatment of peptic ulcer. A key step in prodrug design is the incorporation of an activation mechanism that can convert the prodrug into the active species in an efficient and/or controlled manner to meet the needs of a given medical application. Prodrug activation can be achieved through enzyme-mediated hydrolytic or oxido- reductive processes while activation of some prodrugs may proceed through pure chemical nonenzymatic processes. This review focuses on the hydrolytic that have been used in prodrug activation, including , , and .

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nCorresponding author. Tel.: þ1 732 445 5291; fax: þ1 732 445 6312. E-mail address: [email protected] (Long-qin Hu).

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Peer review under responsibility of Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association. doi:10.1016/j.apsb.2011.08.001 144 Yan-hui Yang et al.

1. Introduction specificity of CEs is, in part, determined by con- straints enforced by the dimensions of, and access to, the active With the advance of new technologies such as combinatorial site10. and computational chemistry, more and more compounds are The carboxylesterases are involved in the activation of being identified with extremely potent in vitro activity but are various antiviral, anticancer, and prodrugs. Several found to be inactive in vivo. They may have the optimal representative esterase-activated prodrugs are shown in Fig. 1. configuration and conformation needed to interact with their (1, CPT-11) is converted to its active metabolite, target or enzyme, but they do not necessarily possess 7-ethyl-10-hydroxycamptothecin, which is an inhibitor of the best molecular form and physicochemical properties topoisomerase I8,11; temocapril (2), a long-acting ACE inhibi- needed for their delivery to the site of action. Some of the tor, is quickly metabolized to the active dicarboxylic acid12; problems often encountered include (i) limited solubility and dipivefrin (3), available as an ophthalmic solution for the poor chemical stability preventing the drug from being ade- treatment of glaucoma, is hydrolyzed to epinephrine on quately formulated, (ii) low or variable bioavailability due to penetrating cornea13; lovastatin (4), and simvastain (5), used incomplete absorption across biological membranes or exten- to lower in patients with hypercholesterolemia, sive first-pass , and (iii) lack of site specificity. were hydrolyzed by hCE1 to form the hydroxy acid forms, Prodrugs have been designed and used clinically to solve these which inhibit the activity of HMG-CoA reductase14. problems. A prodrug is inactive or much less active and has to be activated in vivo to form the active drug . 2.2. Acetylcholinesterase Prodrug design has proven to work for many pharmacologi- cally active compounds or drugs in improving their physicochem- Acetylcholinesterase (AChE, EC 3.1.1.7) can rapidly hydro- ical and biological properties and their target selectivity. Currently, lyze the neurotransmitter acetylcholine at cholinergic synapses 5–7% of the drugs approved worldwide can be classified as and neuromuscular junctions. AChE is an extrinsic mem- prodrugs, and approximately 15% of all new drugs approved each brane-bound enzyme that projects into the synapse. It is year are prodrugs1. Prodrugs have been used in a wide variety of responsible for the elimination of ACh released from the therapeutic areas including anti-influenza, anti-hypertension, anti- presynaptic nerve process in response to an action potential biotics, antiulcer, anticoagulant, anti-inflammatory, antifungal, and is critical for maintaining normal cholinergic function15,16. anesthetic, and anticancer. Many prodrugs have gained huge AChE is widely expressed in many tissues throughout the body. clinical success. Activation of prodrugs can involve many enzymes including skeletal muscle, many types of neurons in the central and including like CYP450 and DT-diaphorase as peripheral nervous systems, and numerous other types that are well as hydrolytic enzymes like carboxylesterase and b-glucuroni- targets of cholinergic nerves such as endocrine and exocrine dase2–6. This review will provide an overview of the enzymes glands, smooth and cardiac muscle cells, and sensory organs involved in the hydrolytic activation of prodrugs. embedded in epithelia17. In , AChE is also expressed in non-attached cells such as erythrocytes and lymphocytes. Several prodrugs activated by AChE have been reported. For example, 2. Enzymes butyryl ACV (6, Fig. 2) is an AChE-activated ester prodrug of 2.1. Carboxylesterase acyclovir (ACV), which is a synthetic purine nucleoside analog of and clinically used in the treatment of herpes simplex virus infections because of its affinity for the viral thymidine kinase18. Carboxylesterases (CEs, EC 3.1.1.1) are members of the hydrolase superfamily and they can efficiently hydrolyze a variety of ester-, -, and carbamate-containing xenobio- 2.3. Butyrylcholinesterase tics to their respective free acids. They act as an effective biological barrier to limit the distribution of substrates that Butyrylcholinesterase (BChE, EC 3.1.1.8) is relatively abun- might be toxic and facilitate their elimination by turning them dant in plasma and is sometimes referred to as the serum into more polar . cholinesterase. However, its physiological role remains Although carboxylesterases show varying activity and sub- unclear. BChE can degrade a large number of ester-containing strate specificity, depending on species and location, they are compounds, including ester prodrugs19. The overall 3-D present virtually throughout the body, including intestines, structure of BChE is very similar to that of AChEs. However, blood, brain, skin, and tumor. There are several different BChE does not form the same kind of dimer as observed in isozymes of carboxylesterases; based on sequence homology structures of Torpedo californica AChE, mouse AChE, and and similarity of characteristics, the isozymes have recently AChE15,19. BChE has a broader substrate specificity been classified into five subfamilies, CES1, CES2, CES3, CES4, than AChE, and this difference in specificity can be explained and CES57. However, other names such as human liver by BChE’s more spacious acyl binding pocket at the carboxylesterase 1 (hCE1), carboxylesterase 2 (hCE2), intest- and differences in residues19. inal carboxylesterase (hiCE), and human brain carboxylester- BChE exists predominantly in plasma as well as in various ase (hBr2) continue to be used in the literature. Biochemical tissues such as brain, muscle, , intestine, retina, and analysis indicated that CEs are efficient at hydrolyzing small placenta. Several BChE-activated prodrugs have been developed. substrates, but show vast differences with increasingly larger Bambuterol (7) is slowly converted by BChE to terbutaline, a 20,21 substrates. For example, the human liver hCE1 was 100- to b2-adrenoceptor agonist . Methylprednisolone acetate (8)can 1000-fold less efficient at metabolizing CPT-11 than rabbit liver be hydrolyzed by BChE to its active form—methylprednisolone, a rCE, even though the two enzymes have over 80% amino acid anti-inflammatory agent22. Isosorbide diaspirinate (9), the identity8,9. Analysis of the crystal structures indicates that the diester of isosorbide, is stable towards aqueous hydrolysis Enzyme-mediated hydrolytic activation of prodrugs 145

O O

O N O N CE O O OH N OH N O OH CH3 CH N 3 O N CPT-11 (1) 7-ethyl-10-hydroxycamptothecin

O O

HO O HO O N N NH CE NH S S S S O O O OH Temocapril (2) Temocapril diacid

O OH OH H H HO N O N CE CH O CH3 3 HCl O HO epinephrine Dipivefrin (3)

O OH OOH OH O O HO O H C O 3 H CE H3C R CH H C O 3 3 H H3C R CH3 H3C H C Lovastatin (4) R = H 3 5 hydroxy acid form Simvastatin ( ) R = CH3

Figure 1 Carboxylesterase-mediated activation of representative ester and carbamate prodrugs.

O O

N NH N NH O AChE N N NH N N NH O 2 O 2 O HO Butyryl ACV (6) Acyclovir (ACV)

Figure 2 Acetylcholine esterase-mediated activation of butyryl ACV.

OAc O OH O HO H O H O BChE O O OAc OH + O O H H O OH salicylic acid isosorbide Isosorbide Diaspirinate (9)

Figure 3 Activation of isosorbide diaspirinate by BChE. and in the presence of a-chymotrypsin23. However, it undergoes esterase that can catalyze the hydrolysis of various arylesterase rapid hydrolysis in the presence of BChE as shown in Fig. 3. and organophosphates. PON2 and PON3 share 60% sequence identity with PON1, but, they lack or have very limited arylesterase activities. On the other hand, PON2 and PON3 2.4. Paraoxonase exhibit high lactonase activity. Human PON1 is synthesized in the liver and secreted into the blood, while PON3 is similarly Paraoxonase (PON, EC 3.1.8.1) is a family of hydrolases that expressed predominantly in the liver and at low levels in the include PON1, PON2, and PON3. PON1 is an important kidney24. In recent years, PON1 and its two known Q and R 146 Yan-hui Yang et al. isoenzymes have been shown to possess both arylesterase and require proteolytic processing to produce their active forms that organophosphatase activities, and play important roles in drug usually consist of three main domains, namely a propeptide metabolism and in the prevention of atherosclerosis25. domain, a catalytic domain, and a hemopexin/vitronectin-like Prulifloxacin (10), a lipophilic prodrug of ulifloxacin, is a domain. Several MMPs, including MMP-1, MMP-2, MMP-8, new oral fluoroquinolone with a broad spectrum of in vitro MMP-13, MMP-14, MMP-18, and MMP-22, catalyze the first activity against various Gram-positive and Gram-negative committed step in extracellular matrix degradation by unwinding microorganisms. Its structure contains the skeletal quinolone and cleaving the triple helix of interstitial collagens (type with a four-membered ring in the 1,2-position, including a I–III)29–34. On the other hand, MMP-3 and MMP-9 only possess sulfur atom to increase antibacterial activity and an oxodiox- the ability to bind to single-strand type I collagen. The collagen olenylmethyl group in the 7-piperazine ring to improve its oral cleavage site is similar for most MMPs, and the general motif absorption; it is immediately and quantitatively transformed preceding the scissile bond (between Gly and Ile/Leu) has been by hPON1 into the active metabolite ulifloxacin (Fig. 4)26,27. characterized as a four-triplet region rich in proline with one Lactone-containing drugs, Lovastatin and Simvastatin, are all always at the P3 position. It is important to recognize the ability substrates of hPON315,24,28. to unwind and cleave interstitial collagen in the triple helix form as the main determinant of substrate specificity, rather than the primary structure33,34. 2.5. Matrix metalloproteinase Because of their significant role in tumor angiogenesis, invasion, and metastasis, MMPs have been targeted in poten- Matrix metalloproteinases (MMPs), also known as matrixins, are tial therapeutic strategies for a number of disease conditions -dependent endopeptidases consisting of more than 25 family including cancer, arthritis, glomerulonephritis, periodontal members classified based on domain structure and substrate disease, and ulcers34. Among them, MMP-2 and MMP-9 have specificity (Table 1). They play important roles in the degrada- been used in the targeted activation of potential prodrugs35–38. tion of extracellular matrix components, such as collagen, Model prodrugs have been reported to release, upon activation laminin, fibronectin, and elastin. MMPs also cleave a variety by MMP-9, anthraquinone-type cytotoxic agents that are often of , such as growth factor receptors and cell adhesion used in combination with conventional therapies for the treatment 37 molecules, which may be important for tumor growth and of multiple myeloma . L-Prolyl-N-(3-aminopropyl)-1-aminoan- survival. Synthesized initially as inactive proenzymes, MMPs thraquinone, an adduct known to possess dual topoisomerase

H C H C O 3 O O 3 H3C N N HN O S S S O N N hPON1 O N N N N CH3 O OH H OH OH F F F O O O O O O (10) Ulifloxacin

Figure 4 Activation of prulifloxacin by hPON1.

Table 1 Matrix metalloproteinases and their substrates.

Enzyme Alternative name EC number Substrates

MMP-1 Interstitial collagenase 3.4.24.7 Collagens (I, II, III, VII, VIII, and X); gelatin; aggrecan; L-selectin; IL-1beta; ; entactin; ovostatin; MMP-2; MMP-9 MMP-2 Gelatinase A 3.4.24.24 Collagens (IV, V, VII, and X); gelatin MMP-3 Stromelysin-1, Proteoglycanase 3.4.24.17 Collagens (III, IV, V, and IX); gelatin; aggrecan; perlecan; decorin; laminin; elastin; caesin; osteonectin; ovostatin; entactin; plasminogen; MBP; IL-1beta; MMP-2/TIMP-2; MMP-7; MMP-8; MMP-9; MMP-13 MMP-8 Neutrophil collagenase 3.4.24.34 Collagens (I, II, III, V, VII, VIII, and X); gelatin; aggrecan; fibronectin MMP-9 Gelatinase B 3.4.24.35 Collagens (IV, V, VII, X, and XIV); gelatin; entactin; aggrecan; elastin; fibronectin; osteonectin; plasminogen; MBP; IL-1beta MMP-13 Collagenase-3 – Collagens (I, II, III, IV, IX, X, and XIV); gelatin; plasminogen; aggrecan; perlecan; fibronectin; osteonectin; MMP-9 MMP-14 MT1-MMP 3.4.24.80 Collagens (I-III); gelatin; casein; fibronectin; laminin; vitronectin; entactin; proteoglycans; MMP-2; MMP-13 MMP-18 Xenopus Collagenase-4 – Not known MMP-22 Chicken MMP (C-MMP) – Not known Enzyme-mediated hydrolytic activation of prodrugs 147 inhibitory activity in vivo, was coupled to an optimized substrate class of compounds, including , proteins, and ofMMP-9asshowninEV1(11)37. Cleavage of EV1 by MMP-9 alkaloids. AP is ubiquitously expressed in many tissues with occurs between glycine and norvaline, resulting in the liberation of the highest expression in the liver. The differential expression an active inhibitor of topoisomerases. of various AP isozymes in liver and bone has been used as a Selective delivery of to tumor tissues was diagnostic marker for various cancers and renal dysfunction39. investigated using its conjugates as prodrugs activated AP is believed to be responsible for the activation of several by the combined proteolytic action of MMP-2, MMP-9, and clinically used phosphate prodrugs including fosfluconazole, MMP-14. Doxorubicin is a potent used for the fosphenytoin, fosprepitant, amifostine, phosphate, treatment of various types of cancer. It causes cytotoxicity , and phosphate. Most of these through topoisomerase II-mediated DNA breaks. Coupling of prodrugs were developed to overcome the solubility problems of doxorudicin to several MMP-cleavable generated a parent drugs and are for parental administration while some are series of prodrugs that were readily cleaved by MMP-2, for the targeted treatment of cancer. Amifostine (WR-2721, S-2 MMP-9, and MMP-14. For example, the prodrug Ac–Glu– [3-aminopropylamino]-ethylphosphorothioic acid, 13) was devel- Pro–Cit–Gly–homo-Phe–Tyr–Leu–Dox (12) was cleaved by oped as a protective agent against radiation- and - MMP-2, MMP-9, and MMP-14 to generate a tripeptide–Dox induced cellular injury40. It is selectively activated in normal cells conjugate, which was subsequently degraded by extracellular by AP via dephosphorylation to afford the free WR-1065, proteases to release Leu–doxorubicin as shown in Fig. 5. Leu– which acts a free-radical scavenger (Fig. 6). In addition to free- doxorubicin was known to release doxorubicin upon further radical scavenging, the protective effect of amifostine has been proteolytic cleaveage. Prodrug testing in the fibrosarcoma cell postulated to originate from DNA repair through hydrogen line HT1080 expressing multiple MMPs and HT1080 xeno- donation from the free thiol group, and inhibition of DNA grafts in mice demonstrated the selective conversion of the damage. The protective effects of amifostine have been demon- doxorubicin prodrug to free doxorubicin36. The prodrug also strated in preclinical species in the presence of platinum exhibited a 10-fold increase in tumor/heart distribution ratio, compounds such as . Following incubation of cisplatin greater efficacy and less toxicity as compared to doxorubicin. in salmon sperm DNA in the presence of 50-fold molar excess of amifostine, WR-1065 and related compounds, a maximal 49% 2.6. Alkaline phosphatase reduction in platinum–DNA adduct formation was observed with WR-106541. Clinically, the protective effects of amifostine Alkaline phosphatase (AP, EC 3.1.3.1) is a metalloenzyme administration 30 min prior to chemotherapy have proven (contains zinc and ) with broad substrate specificity. effective although no proof-of-concept could be obtained in It catalyzes the removal of phosphate groups from a diverse in vivo animal studies.

O O OH O OH O CH2OH CH2OH OH OH

OCH3O OH O OCH3 O OH O HO HO

H3C O H3C O MMPs O O O OH H H H O O OH N N N NH H N N N H2N N NH H H H N H3C N O O O H O O O HN MMPs OOHONH2

peptide-doxorubicin prodrug (12) Protease

O OH O CH OH O 2 O OH OH Protease CH2OH OH OCH3O OH O

OCH3O OH O H3C O

H3C O O OH H2N NH OH NH2 Doxorubicin

Figure 5 Activation of a peptide–doxorubicin prodrug designed to be activated by MMPs. 148 Yan-hui Yang et al.

AP-mediated prodrug activation has been applied to cancer acid-containing , such as , imaging and therapy. Such radio-detection of solid tumors . and dermatan sulfate43. The function of in vivo was demonstrated using the radioactive quinazolinone human b-glucuronidase is essential for the restructuring of the 125 phosphate prodrug IQ2-P, the ammonium salt of extracellular matrix components and in the b- 2-(20-phosphoryloxyphenyl)-6-[125I]iodo-4-(3H)-quinazolinone glucuronidase has been shown to cause a rare lysosomal (14). As shown in Fig. 6, the prodrug is hydrolyzed to a much storage disease called mucopolysaccharidosis type-VII (Sly 125 44 less -soluble, radiolabeled quinazolinone IQ2-OH by syndrome) . b-Glucuronidase is a tetrameric glycoprotein AP, which is overexpressed on the exterior surfaces of tumor with four identical subunits of 77 kDa and is localized in cell plasma membranes42. The quinazolinone molecule preci- lysosomes of the cell. The optimal pH for the activity of pitates and is concentrated and permanently trapped within the human b-glucuronidase is around 4, while the physiological extracellular spaces of targeted solid tumors. The prolonged pH of 7.4 leads to decreased enzyme activity43. Since endo- residence time of the quinazolinone molecule should permit the genous extracellular b-glucuronidase is present at high levels in noninvasive detection and therapy of tumors. necrotic tumors, glucuronide prodrugs could potentially be used as a monotherapy. Moreover, in the case of non-necrotic 2.7. b-Glucuronidase areas of tumors where the enzyme concentration is low, exogenous b-glucuronidases can be delivered to these tumor 45 Human b-glucuronidase (EC 3.2.1.31) is a lysosomal enzyme tissues using ADEPT and GDEPT strategies . that plays an important role in the degradation of glucuronic Theactivesitecavityofhumanb-glucuronidase consists of three amino acid residues, Glu540,Glu451,andTyr504. Among them, Glu540 acts as a , while Glu451 acts as an acid– S 2- AP SH 46,47 504 HN PO4 HN base catalyst or the proton donor .Tyr is also important, NH NH2 2 although its role is unclear. b-Glucuronidase have a broader Amifostine (13, WR-2721) WR-1065 substrate specificity; almost any aglycone in a b-linkage can be hydrolyzed to glucuronic acid43. One possible mechanism pro- O O posed for the hydrolytic action by b-glucuronidase is illustrated 125I NH 125I 48 NH in Fig. 7 . N AP Tumor tissues, including breast, lung and gastrointestinal tract N carcinomas, and melanomas have been shown to have increased NH4 O O expression of b-glucuronidase as compared to normal tissues. P HO NH O O The high expression of the b-glucuronidase enzyme is localized 4 43 125 14 125 mainly in necrotic areas of the malignancies . The liberation of IQ2-P ( ) IQ2-OH lysosomal b-glucuronidase extracellularly leads to the accumula- 125 Figure 6 Activation of amifostine and IQ2-P by AP. tion of the enzyme in the extracellular matrix of the malignant

Glu 451 Glu 451 Glu 451 Enz Enz Enz HO R

O O O O OO HOOC HOOC δ+ HOOC H δ− H HO O HO O δ+ O HO O HO O HOHO R R OH OH OH δ− O O O

O O O Enz Enz Enz Glu 540 Glu 540 Glu 540 Enzyme glucuronylation Glucuronyl-Enzyme intermediate

Glu 451 Glu 451 Enz Enz

O O OO HOOC HOOC O H O H H HOHO OH HOHO O OH OH O O

O O Enz Enz Glu 540 Glu 540

Figure 7 The catalytic cycle of b-glucuronidase. The R group shown in red is released upon hydrolysis of the . Enzyme-mediated hydrolytic activation of prodrugs 149 tissues. Cancer tissues have been targeted by a wide variety of acyclovir (ACV), increases the oral bioavailability of ACV glucuronide prodrugs exploiting the presence of high concentra- 3- to 5-fold53. In addition to activating valacyclovir and tions of b-glucuronidase as the enzyme is not present in the valganciclovir, VACVase has been shown to hydrolyze the general circulation. BHAMG (15), a synthetic glucuronide prodrugs of a broad range of antiviral and anticancer nucleoside prodrug of aniline mustard, can be activated by analogs such as zidovudine, floxuridine, and gemcitabine52. b-glucuronidase49. Pyrrolo[2,1-c][1,4]--b-glucuro- nide (16), a prodrug of DNA minor groove binder, has been shown to be activated by b-glucuronidase50. HMR1826 (17), 2.9. Plasmin where the glucuronide group is linked to doxorubicin as showed in Fig. 8,hasaVmax of 635 mmol/(min mg) and a Km of Plasmin (EC 3.4.21.7) is an important present 1.3 mmol/L as a substrate of b-glucuronidase43. Upon b-glucur- in blood that degrades many blood plasma proteins, most onidase-catalyzed hydrolysis, the 4-hydroxybenzyl carbamate- notablely, fibrin clots. In humans, the plasmin is doxorubicin intermediate undergoes self-immolative 1,6-elimina- encoded by the PLG gene. Plasmin is derived from plasmino- tion to release the active doxorubicin drug. gen, a circulating glycoproteins (91 kDa) present in plasma at a concentration of 1.5 mmol/L54. Plasmin is believed to be involved in migration, invasion, 2.8. Human valacyclovirase and metastasis of tumor cells by catalyzing the breakdown of extracellular matrix proteins55,56. Plasmin is predominantly Human valacyclovirase (VACVase) is a highly specific a- present in its inactive pro-enzyme form plasminogen in the amino acid ester hydrolase. VACVase catalyzes the hydrolytic body. Active plasmin is formed locally at or near the surface activation of two clinically important antiviral nucleoside of tumor cells from cell-bound plasminogen activated by cell- prodrugs, valacyclovir and valganciclovir, to their correspond- bound urokinase-type plasminogen activator (u-PA), pro- ing active drugs, acyclovir and ganciclovir51. duced by cancer or stroma cells57. In the general circulation, VACVase is expressed at a high level in human liver and urokinase and plasmin are inactivated by protease inhibitors kidney and lower levels in intestines, heart, and skeletal such as PAI-1 and a2-antiplasmin present in the blood. Many muscle52. Nucleoside analogs valacyclovir (18, VACV) and tumor cell lines and tumor tissues have a significantly higher valganciclovir (19, VGCV) are two typical prodrugs activated u-PA level than their normal counterparts58, and u-PA is a by VACVase. Valacyclovir, the valyl ester prodrug of strong prognostic factor for reduced survival and increased

O2N HOOC HO O O Cl HO OO HO O H OH N O CH2 O N N H 5 H HOOC Cl O HO O N OMe MeO N HO HO O O BHAMG (15) Pyrrolo[2,1-c][1,4]-benzodiazepine--glucuronide (16)

O OH O O OH O OH OH OH OH -glucuronidase

H3CO O OH O H3CO O OH O O H C O H3C HOOC 3 HOOC HO HN O O HOHN O HO O O HO OH HO HO O OH O OH HMR1826 (17)

O OH O OH OH OH + CO2 H3CO O OH O O H3C O NH HO 2 Doxorubicin

Figure 8 Activation of doxorubicin–glucuronide conjugate (HMR1826) by b-glucuronidase followed by 1,6-elimination of the self- immolative linker. 150 Yan-hui Yang et al. relapse in many types of tumors59,60. Therefore, plasmin is and primary prostate cancer, where 80–90% of PSA is considered a promising enzyme for the tumor-specific activa- enzymatically active72. However, intracellular PSA is enzyma- tion of anticancer prodrugs. Characteristic substrate tripep- tically inactive because of the presence of high concentration of tides, D-Ala–Phe–Lys and D-Val–Leu–Lys, with a N-terminal zinc . Normally, PSA is present in blood at very low levels D-amino acid have been used in the design of potential (o4.0 ng/mL). However, increased serum PSA levels are often prodrugs for activation by plasmin. Examples include N- seen in prostate cancer patients, which are believed to be the nitroso-urea-based prodrug (20) with a substrate tripeptide result of cellular PSA leak caused by the absence of a basement coupled via a cyclization linker61. As shown in Fig. 9,an membrane barrier that normally separates the prostate gland ethylene diamine and a mono-methylated ethylene diamine from the surrounding stoma; this lack of confinement is the serve to link the tripeptide substrates to N-nitroso-urea. In the hallmark of invasive prostate cancer73,74. There is evidence that presence of plasminogen, the tripeptide segment is cleaved off serum PSA levels correlate well with the number of malignant by plasmin and subsequent intramolecular cyclization leads to prostate cells, and high serum PSA levels suggest a high risk of the formation of imidazolidin-2-one with concomitant expul- metastatic prostate cancer75. PSA has been approved by FDA sion of the free parent drug. as a clinically important serological biomarker for the screen- Albumin-binding prodrug was reported by ing of prostate cancer. Most importantly, PSA present in conjugation of albumin with the maleimide-linked peptide bloodstream lacks enzymatic activity because it forms an 62 methotrexate 21 . Plasmin cleavage between D-Ala–Phe–Lys inactive complex with protease inhibitors such as a1-antic- 76–78 tripeptide and the lysine linker releases gMTX–eLys–OH, hymotrpsin and a2-macroglobulin . Serum concentrations which is an active cytotoxic agent. Anthracycline-based pro- of these protease inhibitors are 105–106 fold higher than serum drug (22), formed by insertion of a cleavable linker between the PSA levels and, therefore, are able to trap any active PSA tripeptide substrate and doxorubicin, was shown to be stable in present74,79. The loss of enzymatic activity of serum PSA does Tris buffer and bovine serum, and is readily cleaved by not affect the use of PSA as a serological marker because its plasmin63. prodrug (23) with a carbonate linker immunoreactivity is still maintained. These characteristics of was also designed to be selectively activated by plasmin64. The PSA, including specific production in the prostate tissue, paclitaxel-20-carbonate prodrug is stable in Tris buffer, and the enzymatically active form only in extracellular environment, release of free paclitaxel through 1,6-elimination process was enzymatically inactive form in serum and intracellular environ- shown to be instantaneous upon proteolysis by plasmin. ment, and high concentration of PSA in malignant prostate cancer cells, suggest that PSA can be used as a prodrug- converting enzyme to activate anticancer prodrugs to achieve 2.10. Prostate-specific antigen selective therapeutic effects in prostate-derived cancer tissues. Peptide-based prodrugs linking a substrate peptide sequence Prostate-specific antigen (PSA, EC 3.4.21.77) was first identi- to an anticancer drug have been reported for proteolytic fied in human seminal plasma in 1969 (Fig. 11). PSA is a activation by PSA. Upon proteolytic cleavage by PSA, the glycoprotein composed of a single polypeptide chain with a anticancer agent is then released locally in the microenviron- total mass of 33–34 kDa containing approximately 7% carbo- ment that surrounds prostate cancer cells. The anticancer hydrate65. PSA is primarily produced by prostate ductal and selectivity is, therefore, achieved through the site-specific acinar epithelium and is secreted into the lumen, where it activation of PSA. The peptide should be cleaved specifically cleaves semenogelin I and II in the seminal coagulum66. PSA by PSA, and resist the hydrolytic actions of other enzymes; has been shown to activate urokinase-type plasminogen acti- thus, activation of the prodrugs will be localized in the prostate vator, which is thought to be involved in cancer invasion and and prostate-derived cancer tissues where 80–90% of PSA is metastasis67. PSA could also affect tumor spread by proteolytic enzymatically active. Several short peptide sequences have been modulation of cell adhesion receptors68. Furthermore, PSA is identified as PSA-specific, including Ser–Lys–Leu–Gln–, His– able to cleave insulin-like growth factor binding protein 3 Ser–Ser–Lys–Leu–Gln–, and glutaryl-Hyp–Ala–Ser–Chg–Gln–, 80,81 (IGFBP-3) causing the release of active IGF-I, which could all with a unique residue at P1 site . These enhance tumor growth69. However, other studies indicated that unique PSA substrate sequences were attached to anticancer PSA may inhibit tumor growth by generating antiangiogenic drugs with the dipeptide Ser–Leu or Leu as the linker to angiostatin from plasminogen70,71. improve the rate of PSA cleavage (e.g., glutaryl-Hyp–Ala–Ser– The concentration of PSA in the prostate extracellular fluid Chg–Gln–Ser–Leu–Dox (24), N-morpholinocarbonyl-His–Ser– is around 1.6–2.1 mmol/L (50–68 mg/mL) in normal prostate Ser–Lys–Leu–Gln–Leu–Drug). The peptide–drug conjugates

O O + RHN Cl plasmin Cl NN H2N Cl NN NN OH R' NO R' NO N 20 -nitroso-urea-based prodrugs ( ) O

R = D-Ala-Phe-Lys, D-Val-Leu-Lys HN NR' N2 R' = H, CH3 O Cl Cl Cl H , ,

Figure 9 Plasmin activation of N-nitroso-urea-based prodrugs. Enzyme-mediated hydrolytic activation of prodrugs 151 were shown to have increased therapeutic index in preclinical while high molecular mass homohexamers, mainly found in prostate cancer models, validating the use of PSA as a bacteria, accept as substrates both 6-oxo- and/or 6-aminopur- prodrug-converting enzyme82. ines and their nucleosides95. In mammals it is located especially Various anticancer agents, including doxorubicin81,83, thapsi- in the liver, brain, thyroid, kidney, and spleen. The highest gargin84,85, vinblastine86,87, 5-fluoro-20-deoxyuridine88, 3-hydroxy- concentrations of the enzyme are found in the kidney, periph- lapapchone, diazeniumdiolate89,nitrogenmustard90,protoxin91, eral lymphocytes, and granulocytes39. and paclitaxel92,havebeenusedinprodrugsthatshowedselective The selective cleavage of purine nucleoside analogs by E. coli cytotoxicity against PSA-expressing cells. The peptide– PNP is being evaluated as a possible suicide gene therapy doxorubicin conjugate 24 was evaluated in phase I clinical trials82. strategy (GDEPT) for the treatment of solid tumors. Excellent Another design of PSA substrate peptide conjugates of antitumor activity has been demonstrated with 9-(2-deoxy-b-D- anticancer drugs is to incorporate cleavable linkers between ribofuranosyl)-6-methylpurine (MeP-dR, 27) against tumors PSA substrate peptide and anticancer drugs84,86,93,94.As that express E. coli PNP96. The active metabolite 6-methylpur- shown in Fig. 10, the parent drug FUDR could be released ine (MeP), can readily diffuse across cell membranes and kill through cyclization upon proteolytic cleavage of prodrug 25, adjacent tumor cells that do not express E. coli PNP (Fig. 12)96. thus maintaining the potency and physiological property of 2-Fluoro-20-deoxyadenosine (F-dAdo, 28) is another nucleoside the parent drug. analog that is an excellent substrate for E. coli PNP97.Itis Spontaneous conversion of 4-aminocyclophosphamide to better tolerated by mice than MeP-dR, and the active form is phosphoramide mustard under physiologiocal conditions was F-Ade, which is active against human tumor cells that do not demonstrated and exploited towards the design PSA-activated express E. coli PNP due to its conversion to toxic metabolites prodrugs94. A series of 4-aminocylophosphamide prodrugs and the subsequent inhibition of DNA synthesis97. (26) were synthesized by conjugating 4-aminocylophospha- mide to the tetrapeptide Cbz–Ser–Ser–Phe–Tyr sequence, a PSA substrate. The stereoselectivity of PSA-mediated cleavage CH3 of the prodrugs was found to favor the cis-(2R,4R)-conjugate, CH3 N N which has a t1/2 of 12 min in the presence of PSA (Fig. 11). N E. coli PNP N HO N N O N N H 2.11. Purine-nucleoside phosphorylase OH MeP-dR (27) MeP Purine-nucleoside phosphorylase (PNP, EC 2.4.2.1) catalyzes the cleavage of the glycosidic bond of ribo- and deoxyribonu- NH2 cleosides, in the presence of inorganic orthophosphate (Pi) as a NH N 2 second substrate, to generate the purine base and (deox- N N E. coli PNP N yribose)-1-phosphate. For the natural substrates, the reaction is HO N N F 95 O N N F reversible . PNPs are found in a broad range of organisms, H and divided into two main categories based on size, low OH molecule mass homotrimers (about 80–100 kDa) and high F-dAdo (28) F-Ade molecular mass homohexamers (about 110–160 kDa)95.Low molecular mass homotrimers, mainly isolated from mammals, Figure 12 The bioconversion of MeP-dR and F-dAdo by E. coli are specific for of 6-oxopurines and their nucleosides, PNP in GDEPT.

O O O F O NH2 F F O HN O HN HN H N PSA HN Mu-His-Ser-Ser-Lys-Leu-Gln N O O N O O N HO O N H O O O O OH OH OH Mu-His-Ser-Ser-Lys-Leu-Gln-OH peptide-Fluorodeoxyuridine conjugate (25) 5-Fluoro-2'-deoxyuridine

Figure 10 Example of a peptide–fluorodeoxyuridine conjugate and its release mechanism.

OH O O O O HO H H O PSA O O N N P P P Cbz N N N * N N(CH2CH2Cl)2 H2N * N N(CH CH Cl) H H H H H 2 2 2 H2N N(CH CH Cl) O O 2 2 2 OH 26 Rate of proteolytic activation: OH cis-(2R,4R)-26 > trans-(2S,4R)-26 >> trans-(2S,4S)-26 > cis-(2R,4S)-26

Figure 11 Stereoselectivity of PSA cleavage of peptide 4-aminocyclophosphamide conjugates as potential prodrugs of phosphoramide mustard activated by PSA. 152 Yan-hui Yang et al.

2.12. Carboxypeptidase G2 2.13. Penicillin amidase

Glucarpidase under development as a chemoprotective agent Penicillin amidase (PA), also known as penicillin acylase, is to neutralize the toxic effects of methotrexate is a recombinant widely expressed in various microorganisms including, yeast, bacterial carboxypeptidase G2 (CPG2, EC 3.4.17.11). This fungi, and bacteria. It is a member of the N-terminal- CPG2 from Pseudomonas RS-16 is a 42 kDa exopeptidase in nucleophile hydrolase family, a group of enzymes sharing a the aminoacylase-1/M20 family of enzymes that is not found characteristic fold proximal to their active sites as well as a in mammals. The natural substrate of CPG2 is folic acid, but nucleophilic residue at their N-terminus. The PA family it is capable of hydrolyzing glutamate from amidic98,99, members hydrolyze a series of penicillins, and are subdivided urethanic and ureidic bonds100,101, which allows it to be used into three subtypes based on their substrate specificity, namely to activate synthetic prodrugs in GDEPT and ADEPT as penicillin V amidases (PVA, Type I; substrate: penicillin V, represented by the reaction shown in Fig. 13. phenoxymethylpenicillin), penicillin G amidase (PGA, Type A series of CPG2-activated mustard prodrugs has II; substrate: penicillin G, benzylpenicillin) and ampicillin been developed with various cytotoxic nitrogen mustards linked to amidases (Type III; substrate: ampicillin). The E. coli isozyme glutamate directly98,99,102–104 or through a self-immolative lin- (EC 3.5.1.11) is a well-characterized, inexpensive and com- ker105,106. CPG2 catalyzes the hydrolytic activation of these mercially available Type II amidase commonly used for the prodrugs, releasing and the parent cytotoxins, production of semisynthetic penicillins. including benzoic acid nitrogen mustards (BANM), aniline nitro- PGA is known to have broad substrate specificity; several gen mustards (ANM) and phenol nitrogen mustards (PNM). targeted prodrugs activated by PGA have been reported112,113. 4-[(2-mesyloxylethyl)(2-chloroethyl)amino]benzoyl-L-glutamic acid The potent cytotoxin palytoxin exhibits activity against a wide (CMDA, 29) was the first ADEPT prodrug evaluated clinically107. spectrum of cell types including fibroblasts, lymphocytes, A phase I ADEPT clinical trial of CDMA showed promising erythrocytes and epithelial cells. It is cytotoxic against cells results, with one patient showing a partial response and six expressing the Naþ–Kþ-ATPase associated toxin receptor. with stable disease for 4 months. However, the activated drug, The N-acyl palytoxin derivative, N-(40-hydroxyphenylacetyl)- 4-[(2-mesyloxylethyl)-(2-chloroethyl)amino]benzoic acid, had a palytoxin (NHPAP, 32) was developed as a targeted prodrug long half-life that resulted in dose-limiting myelosuppression due for PGA activation via ADEPT112. PGA was covalently to diffusion of the activated drug back into the circulation. linked to the monoclonal antibody L6 mAb. Compared to The pyrrolo[2,1-c][1,4] (PBDs) are a family palytoxin, the prodrug was 1000 times less toxic to carcinoma of antitumor produced by Streptomyces species108. and lymphoma cells lacking PGA expression. NHPAP was They exert their cytotoxicity by covalently bonding to the efficiently converted to palytoxin in H2981 cells expressing the exocyclic C2–NH2 group of guanine residues in the minor L6 antigen, and exhibited comparable cytotoxic activity to the groove of DNA through their N10–C11 imine functionality. free drug (IC50 values were 0.06 and 0.01 nmol/L for NHPAP The more stable carbamate prodrugs are good substrates for and palytoxin, respectively). CPG2. The prodrug 30 was completely converted into the The L6-PGA conjugate has also been used in the ADEPT- cytotoxic parent PBD within 50 min of incubation with the assisted activation of doxorubicin and melphalan113. The enzyme in vitro. prodrugs, N-(phenylactemido)doxorubicin (33) and N-(pheny- Methotrexate conjugates with amino acids or peptides lacetyl) (34) were found to be 10- and 20-fold less covalently linked to the a-carboxyl group of methotrexate cytotoxic than free drugs in H2981 cancer lines lacking PGA. (MTX) are prodrug forms of the parent antifolate109–111. Co-incubation with PGA increased the cytotoxicity of both When tested against UCLA-P3 human lung carcinoma cells prodrugs due to PGA activation. For example, the IC50 values in vitro, the IC50 values for methotrexate–amino acid con- of prodrug 33 were 2 mmol/L in the absence of PGA and jugates in the absence of CPG2 were in the micromolar range 0.2 mmol/L in the presence of PGA, whereas the IC50 of (about 7.0 mmol/L). This value was much higher than the IC50 doxorubicin was 0.2 mmol/L in the same assay. for MTX, which was in nanomolar range (28 nmol/L). When incubated with UCLA-P3 human lung adenocarcinoma cells treated with KS1/4-CPG2 conjugate (KS1/4 is a carcinoma- 2.14. b-Lactamase specific monoclonal antibody), MTX–Phe (31) produced an IC50 of 63 nmol/L. KS1/4–CPG2 conjugate treatment alone b-Lactamase (EC 3.5.2.6) is a serine protease produced by produced no cytotoxicity and, in the absence of KS1/4-CPG2 various bacteria; it catalyzes the hydrolysis of the b-lactam conjugate, cells were much less susceptible to MTX–Phe moiety in penicillin and other similar b-lactam antibiotics to (IC50 ¼2.2 mmol/L). b-amino acid. Based on primary sequences and metal ion

Figure 13 The mechanism of mustard prodrugs activated by CPG2. Enzyme-mediated hydrolytic activation of prodrugs 153 requirement, b-lactamases have been grouped into four As shown in Fig. 14, opening of the b-lactam ring in classes. Classes A, C, and D utilize a serine residue in the cephalosporin is initiated by the nucleophillic attack from the active site, whereas class B enzymes require an additional hydroxyl group of the Ser70 side chain at the carbonyl catalytic zinc atom for activity. All of these enzymes contain (CQO) of the b-lactam ring. The drug attached to the C30 the active site serine residue as well as a conserved triad of position represented as a leaving group (LG) is released by the KS(T)G between the active site serine and the C terminus. The movement of electrons towards the thiazine ring of the cephem class A b-lactamases are largely plasmid-encoded and are core. It has been noted that the propensity for the drug release widely distributed in both Gram-positive and Gram-negative depends on the leaving group ability of the drug molecule and bacteria. The b-lactamases produced by Gram-positive organ- the stability of the linkage to the cephalosporin. isms are usually secreted enzymes. b-Lactamase may be Vinca alkaloid derivative, LY233425, was also conjugated clinically beneficial when orally administered to preserve the to C30 position of cephalosporin through an alkyl thioether, natural intestinal flora during the parenteral administration of antibiotics. NH2 O The advantages of using b-lactamase in prodrug activation F F N HN in ADEPT are that the enzyme is not present in human cells deaminase and that it is highly selective to the b-lactam containing O N O N H H compounds. Cephalosporin has been widely utilized as the 5-Fluorocytosine (39) 5- core of b-lactamase-activated prodrugs due to its ability of releasing the drug from C30 position following the cleavage of Figure 16 The activation of 5-fluorocytosine by cytosine deami- b-lactam ring by b-lactamase. nase in GEDPT.

O O O R NH R NH S R NH S O S O N LGN LG O N + LG Ser O 3' O 70 Ser70 OOH OOH OOH

Ser70 O + H2O -H

O O Enzyme R NH R NH O S O S OH OH N O N Ser70

OOH OOH

Figure 14 The mechanism of activation of cephalosporin prodrugs by b-lactamases114.

O N O N O N

H Cl H Cl H3C H3C H H NH NH N N NH -galactosidase Winstein-cyclization N O O O HO HO O O OH O HO OH prodrug of duocarmycin analog (37) seco-drug drug

O OH O O OH O OH OH OH OH O HO OCH O OH O -galactosidase H 3 O N OCH3 O OH O HO Dox O H C O O HO O 3 O H3C O OH OH NH OH O NH2 + CO2 O Doxorubicin glycoside prodrug of doxorubicin (38)

Figure 15 Activation of glycoside prodrugs by b-galactosidase. 154 Yan-hui Yang et al.

Figure 17 The structures of other compounds. Enzyme-mediated hydrolytic activation of prodrugs 155

OH HO OH O OH OH OH CH3 OH CH3 O O H3C O N N OH O H H CH OH OH OH OH OH H3C 3

OH OH OH CH OH OH OH 3 O O O OH OH OH OH OH HO OH HO OH OH OH OH

OH HO

OH OH HO O O O OH CH3 OH R O HO OH HN OH OH Palytoxin (PTX) R = H

N-(4'-hydroxyphenylacetyl) palytoxin (NHPAP, 32) R = HO CH2 C O

O OH O

CH2OH Cl Cl OH N

OCH3O OH O

H3C O H N OH NH O HOOC O N-(phenylactemido)doxorubicin (33) N-(phenylactemido)melphalan (34)

Et N OH

H N N OAc S O N Et O OH S O H H H COOMe N O N O MeOOC O O O MeO OH O S NH N H O OH S Ph O HO H Me BzOAcO O NHBz O N S NH O N H O OH LY233425 cephalosporin prodrug (35) Cephalosporin taxol (Protax, 36)

Figure 17 (continued)

which would leave as a thiol upon hydrolysis of the b-lactam taxol did not readily react with the p-nitrophenyl carbonate ring115. This vinca alkaloid prodrug 35 was shown to be 5 fold attached to cephalosporin at C30, a GABA linker was used to less toxic than the free drug; but, it was equally potent as the conjugate the C20-hydroxyl group of taxol with cephalosporin. parent drug in the presence of b-lactamase. Since the C20-hydroxyl group is required for biological A carbamate linker was used in a cephalosporin-taxol activity, the prodrug will only be activated after the C20 linker prodrug, Protax (36)116. Because the C20 hydroxyl group of is removed. 156 Yan-hui Yang et al.

2.15. b-Galactosidase deliver active drug molecules to a specific target tissue or organ, non-endogenous enzymes have been employed for site- E. coli b-galactosidase (EC 3.2.1.23), encoded by lacZ, has specific activation of prodrugs. These novel approaches been used in ADEPT. This enzyme is often used for histolo- include ADEPT, GDEPT, and VDEPT strategies for the gical localization with 5-brom-4-chloro-3-indolyl-beta-D- treatment of cancer. Enzymes used in these strategies include galactopyranoside (X-Gal) as the substrate. A glycoside some of the hydrolytic enzymes discussed above. These prodrug of duocarmycin analog (37) consists of the approaches are very promising but are also very challenging. (1S,10R)-methyl-seco-CBI-skeleton as effector, a dimethyla- There are still many problems to solve. 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