Synthesis of a novel legumain-cleavable colchicine prodrug with cell-specific toxicity

Conventional chemotherapy has undesirable toxic side-effects to healthy tissues due to low cell selectivity of cytotoxic drugs. One approach to increase the specificity of a cytotoxic drug is to make a less toxic prodrug which becomes activated at the tumour site. The cysteine protease legumain have remarkable restricted substrate specificity and is the only known mammalian asparaginyl (Asn) endopeptidase. Over-expression of legumain is reported in cancers and unstable atherosclerotic plaques, and utilizing legumain is a promising approach to activate prodrugs.In this study we have synthesized the legumain-cleavable peptide sequence N-Boc-Ala-Ala-Asn-Val-OH. The peptide was subsequently conjugated to deacetyl colchicine during three steps to produce Suc-Ala-Ala-Asn-Val-colchicine (prodrug) with >90% chemical purity. Several cell lines with different expressions and activities of legumain were used to evaluate the general toxicity, specificity and efficacy of the microtubule inhibitor colchicine, valyl colchicine and the legumain-cleavable colchicine prodrug. The prodrug was more toxic to the colorectal cancer HCT116 cells (expressing both the 36 kDa active and 56 kDa proform of legumain) than SW620 cells (only expressing the 56 kDa prolegumain) indicating a relationship between toxicity of the prodrug and activity of legumain in the cells. Also, in monoclonal legumain over-expressing HEK293 cells the prodrug toxicity was higher compared to native HEK293 cells. Furthermore, co-administration of the prodrug either with the potent legumain inhibitor cystatin E/M or the endocytosis inhibitor Dyngo-4a inhibited cell death, indicating that the prodrug toxicity was dependent on both asparaginyl endopeptidase activity and endocytosis. This colchicine prodrug adds to a legumain-activated prodrug strategy approach and could possibly be of use both in targeted anticancer and anti-inflammatory therapy.     

Water-soluble prodrugs of an Aurora kinase inhibitor

Compound 1 (SNS-314) is a potent and selective Aurora kinase inhibitor that is currently in clinical trials in patients with advanced solid tumors. This communication describes the synthesis of prodrug derivatives of 1 with improved aqueous solubility profiles. In particular, phosphonooxymethyl-derived prodrug 2g has significantly enhanced solubility and is converted to the biologically active parent (1) following iv as well as po administration to rodents.     

Synthesis and biological activity of novel potent endothelin-converting enzyme-1 inhibitors

Through directed screening of metalloprotease inhibitors, CGS 30084 (1) has been identified as a potent endothelin-converting enzyme-1 (ECE-1) inhibitor in vitro (IC50 = 77 nM). Herein we report the syntheses and biological activities of analogues derived from this lead, based on modifications of the biphenyl moiety. Compound 10, the thioacetate methyl ester prodrug derivative of compound 6m, was found to be an orally active and potent inhibitor of ECE-1 activity in rats.     

Synthesis and evaluation of N-acyl sulfonamides as potential prodrugs of cyclin-dependent kinase inhibitor JNJ-7706621

A novel prodrug strategy for cyclin-dependent kinase inhibitor JNJ-7706621 has been explored. Through N-acylation of a sulfonamide substituent, tails containing different solubilizing groups (amino, carboxyl, alkoxyl, and hydroxyl) were attached to JNJ-7706621. Most of the prodrugs exhibited good aqueous solubility and the N-acyl groups on the sulfonamide were metabolically cleaved to generate active drug in rat PK study.     

Metabolism of GS-7340, a novel phenyl monophosphoramidate intracellular prodrug of PMPA, in blood

PMPA, an acyclic nucleoside phosphonate analog, is a potent inhibitor of HIV. In the cells, PMPA is efficiently phosphorylated by intracellular kinases to produce PMPApp, the pharmacologically active metabolite. Despite its demonstrated antiviral potency, PMPA has limited cell permeability presumably resulting from the presence of two negative charges on the phosphonyl group. To enhance intracellular concentrations of PMPA, we developed a prodrug, selectively metabolized inside cells. GS-7340 (9-[(R)-2-[[[[(S)-1-(isopropoxycarbonyl)ethyl] amino] phenoxy-phosphinyl]-methoxy] propyl] adenine) is a prodrug which is orally bioavailable in dogs as the intact prodrug and has demonstrated anti-HIV activity in cell culture of over 1000-fold greater than that of PMPA. The metabolism of PMPA in peripheral blood mononuclear cells (PBMC), red blood cells (RBC) and plasma was examined following exposure of whole blood to PMPA or GS-7340 at concentrations similar to ones observed systemically following oral administration in dogs. Following 1 hour incubation with whole blood, GS-7340 was stable in plasma, produced high levels of PMPA and its phosphorylated metabolites in PBMC but not in RBC. No intact prodrug was present in PBMC. The only other species present in PBMC was monoalaninyl PMPA. The levels of PMPA and the phosphorylated metabolites were over 20 times greater than those after incubation with PMPA. The dog and human blood data were similar. The intracellular levels of PMPA and PMPApp were roughly proportional to GS-7340 over a 10-fold concentration range indicating a lack of saturability of uptake and phosphorylation. Since PMPApp is the species responsible for antiviral activity of PMPA, the high intracellular levels of PMPApp should be an important indicator of greater clinical efficacy of GS-7340.     

Prodrug Mono Therapy: synthesis and biological evaluation of an etoposide glucuronide-prodrug

A glucuronide-based prodrug of etoposide has been synthesized for a Prodrug Mono Therapy strategy. The aim is to selectively liberate the active compound by beta-D-glucuronidase already present in necrotic tumours. Outside from these sites, this enzyme is known to be localised inside the lysosomes. The three components of this prodrug are the glucuronic acid (substrate of the enzyme), the spacer (for a faster cleavage), and the active etoposide. In vitro, the prodrug was shown to be less cytotoxic and more water-soluble than etoposide itself. Finally, in the presence of the beta-D-glucuronidase, cleavage of the prodrug with complete release of the drug has been observed.     

Synthesis and biological activity of potent heterocyclic thiol-based inhibitors of endothelin-converting enzyme-1

Directed screening of metalloprotease inhibitors identified CGS 30084 (1) as a potent inhibitor of endothelin-converting enzyme-1 (ECE-1) in vitro (IC(50)=77 nM). Herein we report the syntheses and biological activities of analogues containing modified biphenyl moieties, bearing heterocyclic proximal rings. Compound 20, the thioacetate ethyl ester prodrug derivative of compound 19a, was found to be an orally active and potent inhibitor of ECE-1 activity in rats.     

Inhibition of carbonic anhydrase II by thioxolone: a mechanistic and structural study

This paper examines the functional mechanism of thioxolone, a compound recently identified as a weak inhibitor of human carbonic anhydrase II by Iyer et al. (2006) J. Biomol. Screening 11, 782-791 . Thioxolone lacks sulfonamide, sulfamate, or hydroxamate functional groups that are typically found in therapeutic carbonic anhydrase (CA) inhibitors, such as acetazolamide. Analytical chemistry and biochemical methods were used to investigate the fate of thioxolone upon binding to CA II, including Michaelis-Menten kinetics of 4-nitrophenyl acetate esterase cleavage, liquid chromatography-mass spectrometry (LC-MS), oxygen-18 isotope exchange studies, and X-ray crystallography. Thioxolone is proposed to be a prodrug inhibitor that is cleaved via a CA II zinc-hydroxide mechanism known to catalyze the hydrolysis of esters. When thioxolone binds in the active site of CA II, it is cleaved and forms 4-mercaptobenzene-1,3-diol via the intermediate S-(2,4-thiophenyl)hydrogen thiocarbonate. The esterase cleavage product binds to the zinc active site via the thiol group and is therefore the active CA inhibitor, while the intermediate is located at the rim of the active-site cavity. The time-dependence of this inhibition reaction was investigated in detail. Because this type of prodrug inhibitor mechanism depends on cleavage of ester bonds, this class of inhibitors may have advantages over sulfonamides in determining isozyme specificity. A preliminary structure-activity relationship study with a series of structural analogues of thioxolone yielded similar estimates of inhibition constants for most compounds, although two compounds with bromine groups at the C1 carbon of thioxolone were not inhibitory, suggesting a possible steric effect.     

METABOLISM OF GS-7340, A NOVEL PHENYL MONOPHOSPHORAMIDATE INTRACELLULAR PRODRUG OF PMPA,IN BLOOD

PMPA, an acyclic nucleoside phosphonate analog, is a potent inhibitor of HIV. In the cells, PMPA is efficiently phosphorylated by intracellular kinases to produce PMPApp, the pharmacologically active metabolite. Despite its demonstrated antiviral potency, PMPA has limited cell permeability presumably resulting from the presence of two negative charges on the phosphonyl group. To enhance intracellular concentrations of PMPA, we developed a prodrug, selectively metabolized inside cells. GS-7340 (9-[R)-2-[[[[S)-1-(isopropoxycarbonyl)ethyl] amino] phenoxy-phosphinyl]-methoxy] propyl] adenine) is a prodrug which is orally bioavailable in dogs as the intact prodrug and has demonstrated anti-HIV activity in cell culture of over 1000-fold greater than that of PMPA. The metabolism of PMPA in peripheral blood mononuclear cells (PBMC), red blood cells (RBC) and plasma was examined following exposure of whole blood to PMPA or GS-7340 at concentrations similar to ones observed systemically following oral administration in dogs. Following 1 hour incubation with whole blood, GS-7340 was stable in plasma, produced high levels of PMPA and its phosphorylated metabolites in PBMC but not in RBC. No intact prodrug was present in PBMC. The only other species present in PBMC was monoalaninyl PMPA. The levels of PMPA and the phosphorylated metabolites were over 20 times greater than those after incubation with PMPA. The dog and human blood data were similar. The intracellular levels of PMPA and PMPApp were roughly proportional to GS-7340 over a 10-fold concentration range indicating a lack of saturability of uptake and phosphorylation. Since PMPApp is the species responsible for antiviral activity of PMPA, the high intracellular levels of PMPApp should be an important indicator of greater clinical efficacy of GS-7340.     

Development of dual-acting prodrugs for circumventing multidrug resistance

We have developed a novel dual-acting maleimide-bearing prodrug that incorporates the anticancer agent doxorubicin and an inhibitor of the P-glycoprotein efflux pump that is over-expressed in multidrug resistant tumor cells. Additionally, the prodrug contains a 1,6-self-immolative spacer coupled to the dipeptide Phe-Lys that acts as a substrate for cathepsin B. The prodrug, once bound through its maleimide moiety to the cysteine-34 group of human serum albumin, was cleaved by cathepsin B and in tumor homogenates demonstrating a release of the anticancer agent doxorubicin and the inhibitor.     

6-Substituted Derivatives of Carbovir: Anti-HIV Activity

Abstract

A series of 6-alkoxy and 6-alkylamino carbovir derivatives were synthesized in order to evaluate prodrug approaches to increased bioavailability of the anti-HIV agent, carbovir. All of the compounds were active against HIV with the N-alkyl derivatives less active than the corresponding O-alkyl derivatives. The adenosine deaminase inhibitor, EHNA, had no effect on the anti-HIV activity of 6-propoxycarbovir, while the adenylic acid deaminase inhibitor, 2′-deoxycoformycin, significantly decreased antiviral activity. These observations suggest that the 6-alkoxycarbovirs are metabolized directly to the monophosphates and are subsequently converted to carbovir monophosphate via adenylic acid deaminase     

Structural and functional analysis of laninamivir and its octanoate prodrug reveals group specific mechanisms for influenza NA inhibition

The 2009 H1N1 influenza pandemic (pH1N1) led to record sales of neuraminidase (NA) inhibitors, which has contributed significantly to the recent increase in oseltamivir-resistant viruses. Therefore, development and careful evaluation of novel NA inhibitors is of great interest. Recently, a highly potent NA inhibitor, laninamivir, has been approved for use in Japan. Laninamivir is effective using a single inhaled dose via its octanoate prodrug (CS-8958) and has been demonstrated to be effective against oseltamivir-resistant NA in vitro. However, effectiveness of laninamivir octanoate prodrug against oseltamivir-resistant influenza infection in adults has not been demonstrated. NA is classified into 2 groups based upon phylogenetic analysis and it is becoming clear that each group has some distinct structural features. Recently, we found that pH1N1 N1 NA (p09N1) is an atypical group 1 NA with some group 2-like features in its active site (lack of a 150-cavity). Furthermore, it has been reported that certain oseltamivir-resistant substitutions in the NA active site are group 1 specific. In order to comprehensively evaluate the effectiveness of laninamivir, we utilized recombinant N5 (typical group 1), p09N1 (atypical group 1) and N2 from the 1957 pandemic H2N2 (p57N2) (typical group 2) to carry out in vitro inhibition assays. We found that laninamivir and its octanoate prodrug display group specific preferences to different influenza NAs and provide the structural basis of their specific action based upon their novel complex crystal structures. Our results indicate that laninamivir and zanamivir are more effective against group 1 NA with a 150-cavity than group 2 NA with no 150-cavity. Furthermore, we have found that the laninamivir octanoate prodrug has a unique binding mode in p09N1 that is different from that of group 2 p57N2, but with some similarities to NA-oseltamivir binding, which provides additional insight into group specific differences of oseltamivir binding and resistance.     

Crystal structure of yeast cytosine deaminase. Insights into enzyme mechanism and evolution

Yeast cytosine deaminase is an attractive candidate for anticancer gene therapy because it catalyzes the deamination of the prodrug 5-fluorocytosine to form 5-fluorouracil. We report here the crystal structure of the enzyme in complex with the inhibitor 2-hydroxypyrimidine at 1.6-A resolution. The protein forms a tightly packed dimer with an extensive interface of 1450 A2 per monomer. The inhibitor was converted into a hydrated adduct as a transition-state analog. The essential zinc ion is ligated by the 4-hydroxyl group of the inhibitor together with His62, Cys91, and Cys94 from the protein. The enzyme shares similar active-site architecture to cytidine deaminases and an unusually high structural homology to 5-aminoimidazole-4-carboxamide-ribonucleotide transformylase and thereby may define a new superfamily. The unique C-terminal tail is involved in substrate specificity and also functions as a gate controlling access to the active site. The complex structure reveals a closed conformation, suggesting that substrate binding seals the active-site entrance so that the catalytic groups are sequestered from solvent. A comparison of the crystal structures of the bacterial and fungal cytosine deaminases provides an elegant example of convergent evolution, where starting from unrelated ancestral proteins, the same metal-assisted deamination is achieved through opposite chiral intermediates within distinctly different active sites.     

Structural studies of nucleoside analog and feedback inhibitor binding to Drosophila melanogaster multisubstrate deoxyribonucleoside kinase

The Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (dNK; EC 2.7.1.145) has a high turnover rate and a wide substrate range that makes it a very good candidate for gene therapy. This concept is based on introducing a suicide gene into malignant cells in order to activate a prodrug that eventually may kill the cell. To be able to optimize the function of dNK, it is vital to have structural information of dNK complexes. In this study we present crystal structures of dNK complexed with four different nucleoside analogs (floxuridine, brivudine, zidovudine and zalcitabine) and relate them to the binding of substrate and feedback inhibitors. dCTP and dGTP bind with the base in the substrate site, similarly to the binding of the feedback inhibitor dTTP. All nucleoside analogs investigated bound in a manner similar to that of the pyrimidine substrates, with many interactions in common. In contrast, the base of dGTP adopted a syn-conformation to adapt to the available space of the active site.     

Identification of 2-hydroxymethyl-4-[5-(4-methoxyphenyl)-3-trifluoromethyl-pyrazol-1-yl]-N-propionylbenzenesulfonamide sodium as a potential COX-2 inhibitor for oral and parenteral administration

Synthesis of prodrugs of orally active COX-2 inhibitor 3 involving sulfamoyl (SO₂NH₂) and hydroxymethyl (CH₂OH) groups, and their biological evaluation are described. Of these prodrugs, the N-propionyl sulfonamide sodium 3k was found to be much superior to the parent compound 3 and other marketed COX-2 inhibitors in carrageenan induced rat paw edema model of inflammation due to highly elevated drug levels in systemic circulation. This prodrug has a potential both for oral as well as parenteral administration due to impressive analgesic activity, antipyretic potency, and extraordinary water solubility.     

Targeted inhibition of hedgehog signaling by cyclopamine prodrugs for advanced prostate cancer

A promising agent for use in prostate cancer therapy is the Hedgehog (Hh) signaling pathway inhibitor, cyclopamine. This compound, however, has the potential for causing serious side effects in non-tumor tissues. To minimize these bystander toxicities, we have designed and synthesized two novel peptide-cyclopamine conjugates as prostate-specific antigen (PSA)-activated prodrugs for use against prostate cancer. These prodrugs were composed of cyclopamine coupled to one of two peptides (either HSSKLQ or SSKYQ) that can be selectively cleaved by PSA, converting the mature prodrug into an active Hedgehog inhibitor within the malignant cells. Of the two prodrugs, Mu-SSKYQ-Cyclopamine was rapidly hydrolyzed, with a half-life of 3.2h, upon incubation with the PSA enzyme. Thus, modulating cyclopamine at the secondary amine with PSA-cleavable peptides is a promising strategy for developing prodrugs to target prostate cancer.     

Formulating a new basis for the treatment against botulinum neurotoxin intoxication: 3,4-Diaminopyridine prodrug design and characterization

Botulism is a disease characterized by neuromuscular paralysis and is produced from botulinum neurotoxins (BoNTs) found within the Gram positive bacterium Clostridium botulinum. This bacteria produces the most deadliest toxin known, with lethal doses as low as 1 ng/kg. Due to the relative ease of production and transport, the use of these agents as potential bioterrorist weapons has become of utmost concern. No small molecule therapies against BoNT intoxication have been approved to date. However, 3,4-diaminopyridine (3,4-DAP), a potent reversible inhibitor of voltage-gated potassium channels, is an effective cholinergic agonist used in the treatment of neuromuscular degenerative disorders that require cholinergic enhancement. 3,4-DAP has also been shown to facilitate recovery of neuromuscular action potential post botulinum intoxication by blocking K(+) channels. Unfortunately, 3,4-DAP displays toxicity largely due to blood-brain-barrier (BBB) penetration. As a dual-action prodrug approach to cholinergic enhancement we have designed carbamate and amide conjugates of 3,4-DAP. The carbamate prodrug is intended to be a slowly reversible inhibitor of acetylcholinesterase (AChE) along the lines of the stigmines thereby allowing increased persistence of released acetylcholine within the synaptic cleft. As a secondary activity, cleavage of the carbamate prodrug by AChE will afford the localized release of 3,4-DAP, which in turn, will enhance the pre-synaptic release of additional acetylcholine. Being a competitive inhibitor with respect to acetylcholine, the activity of the prodrug will be greatest at the synaptic junctions most depleted of acetylcholine. Here we report upon the synthesis and biochemical characterization of three new classes of prodrugs intended to limit previously reported stability and toxicity issues. Of the prodrugs examined, compound 32, demonstrated the most clinically relevant half-life of 2.76 h, while selectively inhibiting AChE over butyrylcholinesterase--a plasma-based high activity esterase. Future in vivo studies could provide validation of prodrug 32 as a potential treatment against BoNT intoxication as well as other neuromuscular disorders.     

Design and synthesis of factor Xa inhibitors and their prodrugs

In addition to our previously reported fluoro acrylamides Xa inhibitors 2 and 3, a series of potent and novel cyclic diimide amidine compounds has been identified. In efforts to improve their oral bioavailability, replacement of the amidine group with methyl amidrazone gives compounds of moderate potency (14, IC(50)=0.028 microM). In the amidoxime prodrug approach, the amidoxime compounds show good oral bioavailability in rats and dogs. High plasma level of prodrug 26 and significant concentration of active drug 26a were obtained upon oral administration of prodrug 26 in rats.     

Prodrug AST-003 Improves the Therapeutic Index of the Multi-Targeted Tyrosine Kinase Inhibitor Sunitinib

Patients have responded well to the multi-targeted tyrosine kinase inhibitor (TKI) Sunitinib in the clinic. But the severe toxic side effects associated with Sunitinib limit its therapeutic index. To improve the therapeutic index of Sunitinib, a prodrug strategy was employed to modify Sunitinib. The inactive prodrug AST-003 can be converted to Sunitinib in vitro and in vivo. Compared with Sunitinib, AST-003 has unique biochemical, cellular and pharmacokinetic properties with improved tolerability in mice and yield higher efficacy in tumor xenograft models. This prodrug strategy may constitute a novel paradigm to improve the therapeutic index of Sunitinib and other TKI or anti-angiogenesis drugs in general.