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Who wouldn't want to have a drug that is activated only in the target cell? Prodrugs that are metabolically triggered inside the pathogen but not in the host are an attractive concept in antimicrobial chemotherapy. Of particular interest are bioreductive prodrugs such as nitro compounds or quinones that can initiate cytotoxic redox cascades and release active metabolites. The critical points for the selectivity of such molecules are, what is the source of the electrons that activate the prodrug, and which are the enzymes that catalyze the reduction? Meredith et al. conceive an elegant approach to answer these questions, making use of reverse genetics in Trypanosoma brucei. By overexpression of key reductase genes, they engineer trypanosomal indicator lines that are hypersensitive to particular bioreductive prodrugs and allow to discriminate between one‐electron and two‐electron transfer activation mechanisms. Indicator lines that are also defective in DNA repair further indicate whether the resultant metabolites interfere with the parasite's genome. This set of T. brucei indicator lines provides a tool for the deconvolution of the mechanisms of prodrug activation and drug action that will facilitate the rational development of bioreductive prodrugs for parasite chemotherapy.     

Modulating paclitaxel bioavailability for targeting prostate cancer

Four novel water-soluble peptide-paclitaxel conjugates were designed and synthesized as prostate-specific antigen (PSA)-activated prodrugs for prostate cancer therapy. These prodrugs were composed of a peptide, HSSKLQ or SSKYQ, each of which is selectively cleavable by PSA; a self-immolative linker, either para-aminobenzyl alcohol (PABS) or ethylene diamine (EDA); and the parent drug, paclitaxel. Introduction of a PABA or EDA linker between the peptide and paclitaxel in prodrugs 2-5 resulted in products with an increased rate of hydrolysis by PSA. The stability of prodrugs 2 and 3, with the PABA linker, was poor in the serum-containing medium because of the weak carbonate bond between the PABA and paclitaxel; however, this disadvantage was overcome by introducing a carbamate bond using an EDA linker in prodrugs 4 and 5. Thus, the incorporation of an EDA linker increased both the stability and PSA-mediated activation of these prodrugs. The cytotoxicity of each prodrug, as compared to paclitaxel, was determined against a variety of cell lines, including the PSA-secreting CWR22Rv1 prostate cancer cell line. The EDA-derived prodrug of paclitaxel 5 was stable and capable of being efficiently converted to an active drug that killed cells specifically in the presence of PSA, suggesting that this prodrug and similarly designed PSA-cleavable prodrugs may have potential as prostate cancer-specific therapeutic agents.     

Optimization of the auxiliary ligand shell of Cobalt(III)(8-hydroxyquinoline) complexes as model hypoxia-selective radiation-activated prodrugs

A potential approach for activating prodrugs in hypoxic regions of tumors is to use ionizing radiation, rather than bioreductive enzymes, to effect reduction. This study investigates radiolytic release of 8-hydroxyquinoline (8-HQ), as a model for hydroxyaza-chloromethylbenzindoline DNA minor groove alkylators, from Co(III) complexes under hypoxia. 8-HQ release, measured by HPLC, showed higher efficiency (one-electron stoichiometry) when the auxiliary ligand was a tetraazamacrocycle [e.g. 1,4,7,10-tetraazacyclododecane (cyclen)] rather than a triazamacrocycle [1,4,7-triazacyclononane (TACN)]. These complexes differ from the bioreductive cobalt complex SN 24771 in that their reduction provides stable cobalt-containing products rather than free (aquated) Co(2+). Radiolytic release of 8-HQ from Co(cyclen)(8-HQ) and Co(TACN)(CN)(8-HQ) was also demonstrated in deoxygenated human plasma, selectively in the absence of oxygen, again with higher efficiency for the cyclen system. The cobalt complexes were >1000-fold less potent than free 8-HQ as inhibitors of cell proliferation and were metabolically stable in aerobic and hypoxic cell cultures. Investigation of cell uptake of total cobalt, by inductively coupled plasma mass spectrometry, showed that these complexes enter cells but do not accumulate to the high concentrations seen with SN 24771. The results demonstrate the feasibility of masking the cytotoxicity of hydroxyquinoline-based cytotoxins as Co(III) complexes and demonstrate the utility of cyclen-based auxiliary ligands for optimizing radiolytic activation of these novel prodrugs under hypoxia.     

Prototyping kinase inhibitor-cytotoxin anticancer mutual prodrugs activated by tumour hypoxia: A chemical proof of concept study

Amide- and ester-linked kinase inhibitor-cytotoxin conjugates were rationally designed and synthesised as prototype hypoxia-activated anticancer mutual prodrugs. Chemical reduction of an aryl nitro trigger moiety was shown to initiate a spontaneous cyclisation/fragmentation reaction that simultaneously released the kinase inhibitor semaxanib (SU5416) and the amine- or alcohol-linked cytotoxin from the prodrugs. Preliminary cell testing and reduction potential measurements support optimisation of the compounds towards tumour-selective mutual prodrugs.     

New Taxol (paclitaxel) prodrugs designed for ADEPT and PMT strategies in cancer chemotherapy

Two new glucuronide paclitaxel prodrugs have been synthesized. Linked to the 2'-OH of the drug by a carbonate function, they include a self-immolative spacer bearing an arylnitro or arylamino group between the drug and the glucuronic acid residue. Both prodrugs were well detoxified and easily cleaved in the presence of beta-D-glucuronidase with fast removal of the spacer, releasing paclitaxel. The arylamino spacer-containing prodrug, more stable than the corresponding nitro analogue, was selected for further studies.     

Reductively activated disulfide prodrugs of paclitaxel

A series of unsymmetrical polar disulfide prodrugs 2–5 of paclitaxel were designed and synthesized as reductively activated prodrugs. These compounds behaved as prodrugs in vitro on L2987 lung carcinoma cells. In vivo evaluation in mice demonstrated that the mutual prodrug 5 with captopril exhibited significant regressions and cures.     

Synthesis and evaluation of water-soluble paclitaxel prodrugs

A series of water-soluble 2'-paclitaxel prodrugs were synthesized by attaching paclitaxel to polyethylene glycol (PEG) through amino acid spacers. The prodrugs showed highly improved water solubility, enhanced in vitro cytotoxicity and in vivo antitumor activity compared with the native drug, paclitaxel.     

Synthesis and cytotoxicity of pyranonaphthoquinone natural product analogues under bioreductive conditions

We have synthesised a focused library of derivatives of natural products containing the pyranonaphthoquinone moiety including the first report of such a scaffold with an appended tetrazole functionality. Examples include kalafungin derivatives as well as analogues of nanaomycin and eleutherin. These compounds were assessed for cytotoxic activation by breast cancer cell lines engineered to express the prototypic human one- and two-electron quinone bioreductive enzymes, NADPH: cytochrome P450 oxidoreductase (POR) and NAD(P)H: quinoneoxidoreductase 1 (NQO1; DT-diaphorase), respectively. Several compounds were observed to be cytotoxic at sub-micromolar level and a pattern of increased aerobic potency was observed in cells over expressing POR. A subset of analogues was assessed under anoxic conditions, where cytotoxicity was reduced, implicating redox cycling as a major mechanism of toxicity. The substrate specificity for reductive enzymes is relevant to the future design of bioreductive prodrugs to treat cancer.     

Novel 20-carbonate linked prodrugs of camptothecin and 9-aminocamptothecin designed for activation by tumour-associated plasmin

The first prodrugs of camptothecin and 9-aminocamptothecin that are activated by the tumour-associated protease plasmin are reported. The tripartate prodrugs consist of a tripeptide sequence recognised by plasmin, which is linked to the 20-hydroxyl group of the camptothecins via a 1,6-elimination spacer. After selective N-protection of 9-aminocamptothecin with an Aloc group, the promoiety (tripeptide-spacer conjugate) was linked to camptothecin or 9-Aloc-9-aminocamptothecin via a 20-carbonate linkage by reacting parent drugs with the p-nitrophenyl carbonate activated promoiety in the presence of DMAP. Both prodrugs showed to be stable in buffer solution and both parent drugs were released upon incubation in the presence of plasmin. Furthermore, the prodrugs showed an average 10-fold decreased cytotoxicity with respect to their parent drugs upon incubation in seven human tumour cell lines.     

First enzymatically activated Taxotere prodrugs designed for ADEPT and PMT

Described here are the syntheses and preliminary biological evaluations of the first two enzymatically activated prodrugs of docetaxel (Taxotere) reported to date. These prodrugs were designed as potential candidates for selective chemotherapy in ADEPT or PMT. They are constituted of a glucuronic acid moiety, a double spacer and the cytotoxic drug, differing only by the spacer substitution. The prodrugs were stable in a buffer, and the in vitro studies showed good detoxification and hydrolysis kinetics. As docetaxel was efficiently released in both cases, these compounds are very valuable candidates for further biological evaluations.     

Failla Memorial Lecture. Redox, radiation, and reductive bioactivation.

A brief review is presented of the background to, and the principles involved in, the development of redox-sensitive drugs for use in cancer therapy. The role of redox processes in the action of various types of radiosensitizers and in the activation of bioreductive drugs is described. The mechanisms by which many simple hypoxic cell radiosensitizers act are believed to involve fast electron transfer processes involving DNA. Some of these agents can also function as hypoxic cell cytotoxins, although the mechanisms involved are different. These "bioreductive drugs" are activated by intracellular metabolic reduction mediated through various cellular reductases. Usually, though not always, bioreduction is favored under hypoxic conditions, and this is why many of these compounds display differential cytotoxicity to hypoxic cells. This is one of the rationales for selectivity in solid tumors. The potencies of both hypoxic cell radiosensitizers and bioreductive drugs are strongly correlated with their electron affinities. Classes of bioreductive agents of current interest are described briefly. These include simple and dual-function nitroheterocycles including the highly potent compound RB-6145, quinone-based drugs including analogues of mitomycin C, and heterocyclic compounds containing N-oxide functions. The study of bioreductive agents for potential use as adjuncts for various approaches to cancer treatment is described.     

5'-(2-Nitrophenylalkanoyl)-2'-deoxy-5-fluorouridines as potential prodrugs of FUDR for reductive activation

Four 5'-(2-nitrophenylalkanoyl)-2'-deoxy-5-fluorouridines (1a-d) were designed and synthesized as potential prodrugs of FUDR for reductive activation. Two methyl groups were introduced alpha to the ester carbonyl to increase both the rate of cyclization activation and the stability of the conjugates towards serum esterases. Chemical reduction of the nitro group into an amino leads to cyclization and release of the active FUDR. Kinetic analysis of the cyclization activation process indicates that the two methyl groups alpha to the ester carbonyl restrict the rotational freedom of ground state molecule and promote the cyclization reaction. However, the two methyl groups also were found to render the conjugates as poor substrates of E. coli B nitroreductase. Conjugate 1c, without the two methyl groups, was reduced by E. coli B nitroreductase (t(1/2)=8 h) to give two products, a N-hydroxyl lactam and the drug FUDR, suggesting that the enzymatic reduction and subsequent cyclization activation proceeded through the hydroxylamine intermediate. These results indicate that cyclization activation will occur once the nitro group is reduced either to an amino or to a hydroxylamino group. The fact that the amino intermediates cyclized easily to release the incorporated drug FUDR suggests the feasibility of using peptide-linked acyl 2-aminophenylalkanoic acid esters as potential prodrugs for proteolytic activation.     

Pathways of reductive fragmentation of heterocyclic nitroarylmethyl quaternary ammonium prodrugs of mechlorethamine

Nitroarylmethyl quaternary (NMQ) ammonium salts have potential as prodrugs for enzymatic or radiolytic reduction to release amine effectors under hypoxia. Earlier studies demonstrated one-electron release of the cytotoxic amine mechlorethamine (HN2) from 4-nitroimidazolyl and 2-nitropyrrolyl NMQ prodrugs (but not from nitrobenzyl analogs) through intramolecular electron transfer. In this study we determined whether this is a general feature of heterocyclic NMQ prodrugs of HN2 and examined the reductive pathways in detail using pulse and steady-state radiolysis. The kinetics of radical fragmentation varied by more than four orders of magnitude, independently of the one-electron reduction potential, within the series of eight nitroheterocycles examined. In addition to the compounds identified previously, new 2-nitropyrrole and 3-nitrothiophene NMQ prodrugs were found to provide efficient HN2 release (G > 0.5 micromol/J in anoxic formate buffer). However, the nitrothiophene was sensitive to nucleophilic displacement of HN2, making it less promising. Product analysis by HPLC/mass spectrometry identified symmetrical dimers arising from benzyl-type radical intermediates but also demonstrated that these dimers are not reliable markers for the intramolecular fragmentation of the initial nitro radical anion. This study elucidated multiple competing pathways for reductive fragmentation of NMQ prodrugs and identified the preferred electron acceptors for use in the development of analogs that release more potent cytotoxins.     

Nitrobenzocyclophosphamides as potential prodrugs for bioreductive activation: synthesis,stability, enzymatic reduction,and antiproliferative activity in cell culture

In efforts to obtain potential anticancer prodrugs for gene-directed enzyme prodrug therapy using Eschericia coli nitroreductase, a series of four benzocyclophosphamide analogues were designed and synthesized incorporating a strategically placed nitro group in a position para to the benzylic carbon for reductive activation. All four analogues were found to be stable in phosphate buffer at pH 7.4 and 37 degrees C and were good substrates of E. coli nitroreductase with half lives between 7 and 24 min at pH 7.0 and 37 degrees C. However, only two analogues 6a and 6c, both with a benzylic oxygen in the phosphorinane ring para to the nitro group, showed a modest 33-36-fold enhanced cytotoxicity in E. coli nitroreductase-expressing cells. These results suggest that good substrate activity and the para benzylic oxygen are required for activation by E. coli nitroreductase. Compounds 6a and 6c represent a new structure type for reductive activation and a lead for further modification in the development of better analogues with improved selective toxicity to be used in gene-directed enzyme prodrug therapy.     

A differential scanning calorimetry study of phosphocholines mixed with paclitaxel and its bromoacylated taxanes

High sensitivity differential scanning calorimetry (DSC) was used to investigate the thermotropic phase properties of binary mixtures of disaturated phosphocholines (PCs) and alpha-bromoacyl taxane derivatives. The alpha-bromoacyl taxanes were synthesized as hydrolyzable hydrophobic prodrugs of paclitaxel. The PCs used were 1, 2-dimyristoyl-sn-glycero-3-phosphatidyl-choline (DMPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). The bromoacyl chain lengths of the taxane prodrugs were varied from 6 to 12 or 16 carbons. For comparison, paclitaxel and PC mixtures were also examined. DSC data from DPPC and bromoacyl taxane mixtures showed a complete abolition of the pretransition and significant broadening of the main phase transition with increasing amounts of bromoacyl taxane prodrugs. The effects were more pronounced with the long-chain compared to the short-chain prodrugs. Under equivalent DSC conditions, the short-chain DMPC showed greater changes in thermotropic phase behavior than with DPPC on taxane addition, suggesting an enhanced degree of association with the fluid-type bilayers. Under similar conditions, the long-chain DSPC bilayers showed a far less significant change in phase behavior on taxane addition than DPPC. These changes were also chain length-dependent for both the PCs and the taxane prodrugs. In contrast, PC and paclitaxel (lacking the acyl chain) mixtures under similar conditions showed insignificant changes in the endotherms, suggesting only slight insertion of the molecule into the PC bilayers. From the DSC data it is apparent that taxane prodrugs solvated in DMPC bilayers more than in DPPC and DSPC bilayers, and taxane prodrugs with longer acyl chains were able to associate with PCs better than those with shorter chain prodrugs. DSC data also suggest that paclitaxel was poorly associated with any of the PCs. In general, the amount of taxane association with bilayers decreased in order: DMPC > DPPC > DSPC. In contrast, the transition enthalpy (DeltaH) of DMPC, DPPC, and DSPC mixtures with paclitaxel showed significantly lower enthalpies than with taxane prodrugs. Taken together, the DSC data suggest that the acyl chains of paclitaxel prodrugs have some access into the bilayers via alignment with the acyl chain of the PC component.     

Paclitaxel esters of malic acid as prodrugs with improved water solubility

The synthesis of paclitaxel esters of malic acid is described. These compounds were found to have improved water solubility and are stable in solution at neutral pH. The C2' modified compounds behave as prodrugs, that is, paclitaxel is generated upon exposure to human plasma, whereas the C7 modified derivatives do not. 2'-Malyl paclitaxel sodium salt demonstrated enhanced antitumour activity and less toxicity in a P388 murine leukaemia in vivo model when compared to paclitaxel.     

Pyrrolo[2,1-c][1,4]benzodiazepine-beta-glucuronide prodrugs with a potential for selective therapy of solid tumors by PMT and ADEPT strategies

Pyrrolo[2,1-c][1,4]benzodiazepine-beta-glucuronide prodrugs 15a-b, with a potential for selective therapy of solid tumors by PMT and ADEPT have been designed, synthesized and evaluated for selective cytotoxicity in the presence of the enzyme beta-glucuronidase. The prodrugs have been found to possess reduced cytotoxicity compared to the parent moieties, and are excellent substrates for the enzyme, exhibiting cytotoxicity selectively in the presence of the enzyme. Enhanced water solubility and improved stability are the other important outcomes upon modifying these molecules as their prodrugs.     

Design,synthesis and biological evaluation of enzymatically cleavable NSAIDs prodrugs derived from self-immolative dendritic scaffolds for the treatment of inflammatory diseases

It has been reported that delivery systems based on dendritic prodrugs of Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) improved the properties of drug molecules and reduced the side effects and irritation on the gastric mucosa. To find a more effective way in NSAIDs dendritic prodrugs, in this paper, three different dendritic scaffolds of enzymatically cleavable naproxen conjugates have been synthesized in a convergent approach and well characterized by NMR and MS techniques. These self-immolative dendritic NISADs prodrugs programmed to release multiple molecules of the potent naproxen after a single enzymatic activation step, and in 50% human plasma, the drug released from the compound T3 reaching 47.3% after 24 h in vitro assay. Moreover, all prodrugs were also found to maintain more significant anti-inflammatory activity, no significant cytotoxicity against HEK293 cells and less degree of ulcerogenic potential in vivo than their monomeric counterpart naproxen. These results provided an effective entry to the development of new dendritic NSAIDs prodrugs.     

Design,synthesis and ex vivo evaluation of colon-specific azo based prodrugs of anticancer agents

Colon-specific azo based prodrugs of anticancer agents like methotrexate (6), gemcitabine (7) and analogue of oxaliplatin (RTB-4) (8) were synthesized and characterized by modern analytical techniques. The prepared prodrugs were stable in acidic (pH 1.2) and basic (pH 7.4) buffers which showed their stability in upper GIT environment. Further, an assay was performed which demonstrated the presence of azoreductase enzyme in the rat fecal material, rat cecum content and other parts of intestinal content which reduce specifically the azo bond and release the drug. The in vitro cytotoxicity assay was also performed which clearly indicated that these azo based prodrugs are active against human colorectal cancer cell lines (COLO 205, COLO 320 DM and HT-29). The release behavior of prodrugs (10, 11 and 15) was 60–70% after 24 h incubation at 37 °C. Therefore, the synthesized azo linked prodrugs of methotrexate, gemcitabine and RTB-4 are the potential candidates for colon targeted drug delivery system with minimal undesirable side effects.     

Antimicrotubular and cytotoxic activity of geiparvarin analogues, alone and in combination with paclitaxel

Geiparvarin is an antiproliferative compound isolated from the leaves of Geijera parviflora, and may represent a new drug which targets tubulin. To better explore the potential use of this agent, we investigated the antimicrotubular and cytotoxic effects of new synthetic aromatic derivatives of geiparvarin. These drugs inhibited polymerization of microtubular protein, particularly when the assembly was induced by paclitaxel. The microtubular network organization of fibroblasts was altered more effectively by some drugs. Normal microtubule architecture completely disappeared when the cells were treated simultaneously with drugs and paclitaxel: microtubules depolymerized or were reorganized into bundles, in a similar but more disarrayed fashion than that observed after treatment with paclitaxel alone. Cytotoxicity studies showed a dose-dependent effect, whereas combined administration of drugs and paclitaxel increased cytotoxicity, more effectively in paclitaxel versus derivatives administration alone.