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 ß-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 ß-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 ß-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.

Elongated multiple electronic cascade and cyclization spacer systems in activatible anticancer prodrugs for enhanced drug release.

The design and synthesis of several novel elongated self-elimination spacer systems for application in prodrugs is described. These elongated spacer systems can be incorporated between a cleavable specifier and the parent drug. Naphthalene- and biphenyl-containing spacers were synthesized but did not eliminate. Prodrugs of the anticancer agents doxorubicin and paclitaxel are reported that contain two or three electronic cascade spacers. A novel catalytic application of HOBt was found for the synthesis of N-aryl carbamates through reacting a 4-nitrophenyl carbonate with an aniline derivative, to connect the 1,6-elimination spacers via a carbamate linkage. In addition, a double spacer-containing paclitaxel prodrug was synthesized, comprising a 1,6-elimination spacer and a bis-amine linker connected to paclitaxel via a 2'-carbamate linkage. Prodrugs in which the novel spacer systems were incorporated between a specific tripeptide specifier and the parent drug doxorubicin or paclitaxel proved to be significantly faster activated by plasmin in comparison with prodrugs containing conventional spacer systems. It is expected that the generally applicable novel spacer systems reported herein will contribute to future development of improved enzymatically activated prodrugs.

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.

Molecular clips based on propanediurea: exceptionally high binding affinities for resorcinol guests.

A series of new receptor molecules derived from 2,4,6,8-tetraazabicyclo[3.3.1]nonane-3,7-dione (propanediurea) is described. These molecules possess a cavity which is defined by two nearly parallel aromatic side walls positioned on top of a bis-urea framework. The resulting "U-shaped" clip molecules are ideal hosts for the complexation of flat aromatic guest molecules. The affinity of these new propanediurea based molecular clips for dihydroxybenzene derivatives is exceptionally high, with association constants up to K(a) = 2 400 000 L mol(-)(1). Comparison of the binding mechanism of a variety of clip and half clip hosts, in conjunction with NMR, IR, and X-ray studies, has enabled the reason for this high binding to be elucidated. It is shown that subtle sub-angstrom changes in the geometry of the clip molecules have a great impact on their binding properties.

Synthesis and biological evaluation of 2'-carbamate-linked and 2'-carbonate-linked prodrugs of paclitaxel: selective activation by the tumor-associated protease plasmin.

The nontoxic paclitaxel-2'-carbamate prodrugs 2-5 and paclitaxel-2'-carbonate prodrug 6 were synthesized and tested for activation by the tumor-associated enzyme plasmin. A generally applicable method for the synthesis of paclitaxel-2'-carbamates was developed. In buffer solution, prodrug 2, which contained an unsubstituted ethylenediamine spacer, was not stable, whereas prodrugs 3-6 were highly stable. Prodrugs 3-6 showed on average a decrease in cytotoxicity of more than 8000-fold in comparison with the parent drug in seven human tumor cell lines. Prodrugs 5 and 6 are the most nontoxic prodrugs of paclitaxel that yield the free parent drug upon selective activation currently reported. Enzyme hydrolysis and spacer elimination rates were determined by incubation of prodrugs 5 and 6 in the presence of human plasmin. From these results, prodrug 6 was selected as the promising prodrug for further in vivo studies.

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.

Rearrangement of O-cinnamoyltaxicin I to a novel C-13 spiro-taxane.

During the large-scale synthesis of an O-cinnamoyltaxicin I acetonide, an intermediate for the semisynthesis of 7-deoxypaclitaxel derivatives, side-product 3 was formed via a vinylogous retro-aldol reaction and a long-range hydride shift from O-cinnamoyltaxicin I (1) under alkaline reaction conditions. Compound 3 has two hemi-acetal bridges at C-1,C-9 and C-10,C-13. Compound 4 was formed from side-product 3 under acidic reaction conditions and is the first C-13 spiro-taxane described in the literature. This spiro-taxane has two acetal bridges between C-1, C-13 and C-10,C-13.

Synthesis and biological evaluation of novel prodrugs of anthracyclines for selective activation by the tumor-associated protease plasmin.

New prodrugs of daunorubicin and doxorubicin designed for selective activation by the serine protease plasmin are described. The low toxic prodrugs 3, 4, and 5 are converted to the corresponding cytotoxic drugs upon proteolysis by the tumor-associated protease plasmin. Application of a self-eliminating spacer was essential for enzyme activation. A prodrug containing a chloro-substituted spacer was synthesized with the aim of enhancing the rate of conversion by plasmin. All prodrugs were highly stable in buffer solution and in serum and on the average 15-fold less cytotoxic than the parent drugs in seven human tumor cell lines. A marked in vitro selectivity was demonstrated by incubation of the doxorubicin prodrugs with a plasmin generating MCF-7 breast cancer cell line transfected with urokinase-type plasminogen activator (u-PA) in comparison with the nontransfected nonplasmin generating cell line. Prodrugs 4 and 5 showed the same cytotoxic effect as the free parent drug doxorubicin in the u-PA transfected cells, indicating complete conversion of the prodrug by plasmin. Addition of the plasmin inhibitor Trasylol drastically increased the ID(50) values in the u-PA transfected MCF-7 cells for both prodrugs 4 and 5.

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.

Synthesis and structure-activity data of some new epibatidine analogues.

The high-pressure Diels-Alder reaction of N-carbomethyoxypyrroles and phenyl vinyl sulfone affords versatile intermediates for the palladium-catalyzed preparation of new epibatidine analogues. Structure-activity relationships of new epibatidine analogues are presented. High affinities of Ki = 0.81 and 2.6 nM for the [3H]-cytisine rat brain nicotinic acetylcholine binding sites were found for the 5-pyrimidinyl and the 5-(2-amino)-pyrimidinyl epibatidine analogues, respectively.

Antiviral and tumor cell antiproliferative SAR studies on tetracyclic eudistomins. II.

In a search for the minimum pharmacophore of the naturally occurring tetracyclic eudistomins, five structural analogues (4-8) were evaluated for their in vitro antiviral and tumor cell antiproliferative activities. For the synthesis of these derivatives both intra- and intermolecular Pictet-Spengler reactions have been used. Opening of the beta-carboline annulated 7-membered D-ring in 6 and 7 resulted in a complete loss of activity. On the other hand, replacement of either the oxygen atom or the sulfur atom in the 7-membered ring by a methylene group in 5 and 8, respectively, is allowed. These results combined with previous SAR data underline the crucial importance of the D-ring in eudistomins as a scaffold for the correct positioning of both basic nitrogen atoms. Also bioisosteric replacement of the bicyclic indole system with a dimethoxyphenyl group, to give the isoquinoline skeleton, is allowed. The tricyclic isoquinoline derivative 4 is, so far, the most promising antiviral analogue; it combines a high potency (MIC at 100 ng/ mL (340 nM)) with high MCC/MIC ratios (ranging from 1000 to 5000 against HSV-1, HSV-2, vaccinia virus, and vesicular stomatitis virus.

Synthesis and biological activity of beta-glucuronyl carbamate-based prodrugs of paclitaxel as potential candidates for ADEPT.

The syntheses of prodrugs of paclitaxel, which can be used in ADEPT in order to target paclitaxel towards tumor cells, are described. The prodrugs 1 and 2a, b consist of a spacer molecule connected via a carbamate linkage to a beta-glucuronic acid. The spacer molecule is also connected via an ester linkage to the 2'-OH of paclitaxel. Enzyme-catalyzed hydrolysis of the glucuronic acid moiety by human beta-glucuronidase results in the liberation of the parent drug paclitaxel via gamma or delta lactam formation with half-lives of 45 min and 2 h (1 and 2b). The prodrugs 1 and 2b are two orders of magnitude less cytotoxic than paclitaxel.

Antiviral and antitumor structure-activity relationship studies on tetracyclic eudistomines.

The in vitro antiviral and antitumor activities of (-)-debromoeudistomin K (1a) and 10 structural analogues (1b-1j and 11) were evaluated. The synthesis was accomplished with an intramolecular Pictet-Spengler condensation reaction as the key step. This examination revealed some structural and stereochemical features that are important for both the antiviral and antitumor activities. The most striking points for activity are the necessity to have the correct natural stereochemistry at both C(1) and C(13b) and the presence of the C(1)-NH2 substituent. As was revealed before with naturally isolated eudistomins a substituent in the indole ring greatly influences the biological activity. The 5-OMe derivative 1h shows high potency in both antiviral and antitumor models.