Hans W. Scheeren

Anticancer Prodrugs for Application in Monotherapy Targeting Hypoxia, Tumor-Associated Enzymes, and Receptors

In order to improve current chemotherapeutic treatment and diminish severe side effects, several prodrug strategies have evolved to achieve site-specific delivery of cytotoxic anticancer agents. This review concentrates on recent developments of antitumor prodrug monotherapy with prodrugs that are designed for direct recognition of tumor-associated factors, such as hypoxia, tumor-associated enzymes and receptors. Firstly, oxygen deficiency in the core of solid tumors leads to enhanced activity of reducing enzymes, like for example nitroreductases, which can be used for site- specific conversion of prodrug to drug. Secondly, some enzymes are present in elevated levels in tumor tissue: beta-glucuronidase leaks from necrotic areas within tumors, while tumor cells for invasive and metastatic activities need several tumor-associated proteases, like plasmin. These enzymes form an attractive target for designing selective prodrugs. Finally, tumor-selective expression of receptors can be exploited for the delivery of antitumor agents. Low molecular weight binding motifs for these receptors can be coupled to cytotoxic drugs in order to obtain tumor-homing conjugates. At present, receptor-binding motifs for a number of receptors that are required for angiogenesis are used for prodrug monotherapy. There exists an increasing body of literature, which describes the complex interplay not only between tumor-associated enzymes, but also between these enzymes and tumor-associated receptors in the process of tumor invasion and metastasis, indicating the feasibility of targeting cytotoxic drugs to these key players in tumor growth. This paper reviews the development and evaluation of anticancer prodrugs, and their application in the various prodrug monotherapy approaches.

Beta-glucuronidase-mediated drug release

The selective activation of a relatively non-toxic prodrug by an enzyme present only in the tumour should enhance the drug concentration at the tumour site and result in a better anti-tumour effect and a reduction in systemic toxicity as compared to conventional chemotherapy. beta-Glucuronidase is such an enzyme. It is normally expressed in the lysosomes of cells. In larger tumours, however, high levels of the enzyme are present in necrotic areas. Several glucuronide prodrugs have been synthesised that can be activated by beta-glucuronidase. They are relatively non-toxic due to their hydrophilic nature, which prevents them from entering cells and thus from contact with lysosomal beta-glucuronidase. The main problem of glucuronide prodrugs for clinical use is their fast renal clearance. Special attention should be paid to the development of new less hydrophilic prodrugs with slower clearance, as this would result in a prolonged exposure to beta-glucuronidase at the site of the tumour and a reduction of the amount of prodrug needed. A number of interesting anthracyclin-based glucuronide prodrugs have been synthesised and have shown favourable therapeutic effects compared to treatment with the parent drug. The tumoural levels of beta-glucuronidase can even be enhanced by two-step approaches, in which exogenous enzyme is targeted to the tumour by an antibody (ADEPT) or by the gene encoding the enzyme in transduced tumour cells (GDEPT). The ADEPT and GDEPT approaches in combination with glucuronide prodrugs have shown enhanced efficacy in experimental tumour models. Further improvement of ADEPT and GDEPT is warranted to optimise the tumour uptake and retention of antibody-enzyme fusion proteins and the efficiency and safety of current gene delivery methods. In conclusion, it is clear that glucuronide prodrugs hold promise for future use in the treatment of cancer in patients as monotherapy. Enhancement of the therapeutic effects of glucuronide prodrugs, also in patients with small tumour lesions, may possibly be achieved by techniques that target beta-glucuronidase specifically to the site of the tumour.

Asymmetric 13-dipolar cycloaddition of nitrones with ketene acetals catalyzed by chiral oxazaborolidines

Abstract Asymmetric 1,3-dipolar cycloaddition of nitrones with ketene acetals is strongly catalyzed by chiral oxazaborolidines derived from N-tosyl-L-α-amino acids. The 5,5-dialkoxyisoxazolidines are obtained regioselectivity in high yield with high stereoselectivity and moderate enantioselectivity up to 62% ee. Mild hydrogenolysis of the NO bond yields quantitatively the corresponding β-amino-ester.

1,3-DIPOLAR CYCLOADDITION REACTIONS OF NITRONES WITH ALKYL VINYL ETHERS CATALYZED BY CHIRAL OXAZABOROLIDINES

Abstract The 1,3-dipolar cycloaddition reactions of various Z - and E -nitrones with ethyl vinyl ether and 2,3-dihydrofuran are catalyzed by 20 mol% of chiral oxazaborolidines at room temperature. The effect of high pressure on these reactions is discussed.

Dramatic solvents effects on the enantioselectivity of chiral oxazaborolidine catalyzed asymmetric 1,3-dipolar cycloadditions of nitrones with ketene acetals☆

Abstract The enantioselectivity of the chiral oxazaborolidine catalyzed asymmetric 1,3-dipolar cycloaddition of nitrones with 1,1-dialkoxypropenes can be controlled by the α-side-chain substituent in the catalyst and the solvent. Remarkable reversal of enantioselectivity is achieved for catalysts having aryl substituents in the α-side-chain by addition of ligand-like solvents. Both enantiomers of a chiral β-amino ester have been prepared in two catalytic steps.

Synthesis of novel paclitaxel prodrugs designed for bioreductive activation in hypoxic tumour tissue.

The syntheses and preliminary evaluation of the first potential bioreductive paclitaxel prodrugs are described. These prodrugs were designed as potential candidates in more selective chemotherapy by targeting hypoxic tumour tissue. Aromatic nitro and azide groups were used as the bioreductive trigger. Generation of paclitaxel occurs after reduction and subsequent 1,6-elimination or 1,8-elimination. All prodrugs are stable in buffer and indeed give paclitaxel after chemical reduction of the aromatic nitro or azide functionality. In aerobic cytotoxicity assays several prodrugs exhibit diminished cytotoxicity. These compounds are interesting candidates for further biological evaluation.

Novel anthracycline-spacer-beta-glucuronide, -beta-glucoside, and -beta-galactoside prodrugs for application in selective chemotherapy

A series of anthracycline prodrugs containing an immolative spacer was synthesized for application in selective chemotherapy. The prodrugs having the general structure anthracycline-spacer-beta-glycoside were designed to be activated by beta-glucuronidase or beta-galactosidase. Prodrugs with -chloro, -bromo or -n-hexyl substituents on the spacer were synthesized as well as prodrugs containing a -beta-glucuronyl, -beta-glucosyl or -beta-galactosyl carbamate specifier. The key step in the synthesis of all prodrugs is the highly beta-diastereoselective addition reaction of the anomeric hydroxyl of a glycosyl donor to a spacer isocyanate resulting in the respective beta-glycosyl carbamate pro-moieties. The resulting protected pro-moieties were coupled to an anthracycline. Prodrugs were evaluated with respect to activation rate by the appropriate enzyme and additionally, their IC50 values were determined. Optimal prodrugs in this study were at least 100- to 200-fold less toxic than their corresponding drug in vitro and were activated to the parent drug in a half-life time of approximately 2 h.

BETA-GLUCURONYL CARBAMATE BASED PRO-MOIETIES DESIGNED FOR PRODRUGS IN ADEPT

A number of pro-moieties 8a - • designed for prodrug preparation have been synthesized (chart 2). The pro-moieties, containing a glucuronyl carbamate group linked to a spacer possessing a terminal carboxylic acid group, have been synthesized from isocyanates 6 and anomerically unprotected glucuronic acids I0 (chart 2). The requisite isocyanates had to be prepared using the Curtius rearrangement. Glucuronyl carbamates proved to be excellent substrates for human ~-glucuronidase. The pro-moieties 8a - e can be coupled to hydroxy- or amino group containing dmgs, The resulting prodrugs are designed to be activated by ~-glucuronidase (chart 1) and to be used in ADEPT. Application is demonstrated with the synthesis of daunomycin prodrugs 12a - e (chart 3). The lack of selectivity of cytostatic agents for tumor cells is a serious drawback in conventional cancer chemotherapy. This major problem dictates research in the field of selective chemotherapy. In this context the selectivity of monoclonal antibodies (mAb) for their target tumor cells can be used to selectively deliver cytotoxicity to tumor tissue. In the ADEPT 1 (Antibody Directed Enzyme Prodrug Therapy) approach, specific enzymes are delivered to tumor sites by using mAbs. These enzymes are selected to convert a prodrug, which is administered in a second step after localization of the mAb-enzyme conjugate, to the parent cytotoxic drug 2. We focused our attention on the (3-glucuronidase (GUS) / (3-glucuronide prodrug 3 couple. This system possesses the advantage that GUS is a human lysosomal enzyme present only at minor concentrations in blood 4 and that (3-glucuronides are highly polar compounds which have a poor cell-membrane permeability. In our approach towards glucuronide based prodrugs, a (3-glucuronide is attached to a spacer molecule via a carbamate linkage. A spacer was used in order to couple the 13-glucuronide to a hydroxy- or amino group containing drug as well as to facilitate GUS hydrolysis 5.

The preparation of resin-bound nitroalkenes and some applications in high pressure promoted cycloadditions

Abstract The preparation of resin-bound nitroalkenes via a microwave assisted Knoevenagel reaction of resin-bound nitroacetic acid with aryl and alkyl substituted aldehydes is described. The potential of these resin-bound nitroalkenes for application in combinatorial chemistry is demonstrated by a Diels–Alder reaction with 2,3-dimethylbutadiene and one-pot three component tandem [4+2]/[3+2] reactions with ethyl vinyl ether and styrene. The cycloadditions were promoted by high pressure.

Synthesis and Characterization of Biodegradable and Thermosensitive Polymeric Micelles with Covalently Bound Doxorubicin-Glucuronide Prodrug via Click Chemistry

Doxorubicin is an anthracycline anticancer agent that is commonly used in the treatment of a variety of cancers, but its application is associated with severe side effects. Biodegradable and thermosensitive polymeric micelles based on poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-p(HPMAmLac(n))) have been studied as drug delivery systems for therapeutic and imaging agents and have shown promising in vitro and in vivo results. The purpose of this study was to investigate the covalent coupling of a doxorubicin-glucuronide prodrug (DOX-propGA3) to the core of mPEG-b-p(HPMAmLac(2)) micelles. This prodrug is specifically activated by human β-glucuronidase, an enzyme that is overexpressed in necrotic tumor areas. To this end, an azide modified block copolymer (mPEG(5000)-b-p(HPMAmLac(2)-r-AzEMA)) was synthesized and characterized, and DOX-propGA3 was coupled to the polymer via click chemistry with a high (95%) coupling efficiency. Micelles formed by this DOX containing polymer were small (50 nm) and monodisperse and released 40% of the drug payload after 5 days incubation at 37 °C in the presence of β-glucuronidase, but less than 5% in the absence of the enzyme. In vitro cytotoxicity experiments demonstrated that DOX micelles incubated with 14C cells showed the same cytotoxicity as free DOX only in the presence of β-glucuronidase, indicating full conversion of the polymer-bound DOX into the parent drug. Overall, this novel system is very promising for enzymatically responsive anticancer therapy.