Maria G. Meirim

Antineoplastic activity of polyaspartamide–ferrocene conjugates †

The ferrocene/ferricenium redox system plays a significant role in biological oxidation, reduction and free-radical reactions. Of particular interest are the findings of earlier investigations which showed certain water-soluble ferricenium salts to possess appreciable antiproliferative activity against various murine tumor lines and a xenografted human colorectal adenocarcinoma. Solubility in water, a prerequisite for efficacious transport and dissipation in central circulation, was then proposed as a principal requirement for the ferrocene complex system to exert antineoplastic activity irrespective of the oxidation state in which it is administered. In order to shed more light on this question, we decided to investigate the antiproliferative properties of polymer–ferrocene conjugates containing the metal complex in the non-oxidized (ferrocene) form while fulfilling the critical requirement of water solubility. To this end, five selected, water-soluble conjugates, synthesized by reversible coupling of 4-ferrocenylbutanoic acid to variously structured polyaspartamides featuring pendant primary amino groups as coupling sites, were tested in vitro against cultured HeLa cells at concentrations up to 50 µg Fe ml−1. Optimal antiproliferative activities, with IC50 in the range of 2–7 µg Fe ml−1, were determined for three compounds possessing tertiary-amine functions susceptible to protonation at physiological pH. Lower activities (IC50 = 45–60 µg Fe ml−1) were demonstrated for two poly(ethylene oxide)-containing conjugates. However, no reasonable structure–performance relationships can be derived at this stage from the small number of compounds tested. Copyright

Antiproliferative Activity of Polymer-Bound, Monoamine-Coordinated Platinum Complexes Against LNCaP Human Metastatic Prostate Adenocarcinoma Cells

The chemotherapeutic treatment of secondary, i.e., disseminated cancers, has until now remained an unsatisfactory modality. The LNCaP human metastatic prostate adenocarcinoma cell line lends itself as a useful tool to probe the efficaciousness of novel antineoplastic agents for the control of metastatic cell growth. In this paper we report on a series of cell culture tests assessing the antiproliferative activity of several water-soluble polymer–platinum conjugates in which the metal is tied (anchored) to various polymeric carriers through coordination by a single carrier-attached primary amine ligand. The conjugates are based on polyaspartamide carriers composed, within the backbone, of a majority fraction of subunits bearing water-solubilizing tertiary amine side-group functions and a minority fraction of subunits featuring side chain-terminating primary amino groups for metal binding. Two similarly structured conjugates in which the platinum center is coordinated by two amine ligands in cisoid orientation mimicking the structural skeleton of cisplatin are included in the study for comparison. In all structures cleavage of a side-chain segment is required for release of the monomeric bioactive platinum complex. The growth of LNCaP human metastatic prostate adenocarcinoma cells in RPMI 1640 medium in the presence and absence of the conjugates in a range of concentrations is assessed by a protein assay, and the IC50 values, representing the drug concentrations required for 50% cell growth inhibition relative to untreated control, are determined. The results show both classes of conjugates, those comprising monoamine-coordinated platinum and those featuring cis-diamine-coordinated metal, to be well comparable in antiproliferative activity. A major program of synthesis and evaluation of polymer-bound monoamineplatinum complexes, prompted by these findings, is forthcoming in this laboratory.

Preliminary toxicological studies of selected water‐soluble polymer–platinum conjugates

The objective of this preliminary investigation of a number of water-soluble carrier-bound platinum(II) complexes for potential use in cancer chemotherapy was to assess the toxicological behavior of representative platinum coordination compounds anchored to, or incorporated into, polymeric carriers via polymer-attached amine ligands. The conjugates included linear polyaspartamides (1-4, 6, 7), each composed of a major fraction of subunits featuring side-chain-attached tertiary amino groups as water-solubilizing entities, and a minor fraction of subunits comprising the anchored platinum complexes, again as side-chain components. Whereas in 1-4 the platinum atom was polymer-bound through a single amino group, both 6 and 7 contained polymer-attached cis-diamine-chelating ligands coordinating to the metal center. Also included in this study was a linear polyamidoamine (5), which contained a poly(ethylene oxide) segment in the backbone in addition to intrachain ethylenediamine segments acting as cis-diamine chelating ligands for coordination to the platinum center. The compounds were injected as aqueous (phosphate-buffered saline) solutions into the tail veins of CD-1 mice (four to eight mice per conjugate), and the maximally tolerated dose was determined for each compound. For polyaspartamides 1-4 the dose levels ranged from about 25 mg Pt (kg body weight -1 ) (in conjugate 4) to 500 mg Pt kg -1 (in compound 1), the latter conjugate proving some 100-fold less toxic than cisplatin (3-4 mg Pt kg -1 ), which was included in this study for comparison. Low toxicity (tolerated dose 160 mg Pt kg -1 ) was also observed for the intrachain cis-diamineplatinum complex polymer (5). The polyaspartamide conjugates 6 and 7, on the other hand, both characterized by a cis-diamineplatinum complex system in the side chain, were toxic even below the dose level of 20-25 mg Pt kg -1 . The preliminary findings of this study, while providing a basis for more extensive and broad-based toxicological studies, will serve to direct and optimize structural conjugate designs in forthcoming synthetic programs.

Synthesis and spectroscopic features of bis(tetramethylammonium) and bis(benzyltriethylammonium) salts possessing a μ-oxo-bis(trichloroferrate) anion structure

SummaryThe two μ-oxo-bis(trichloroferrate) salts (1) and (3) with diamagnetic ammonium cations are prepared as prototype compounds possessing an oxo-bridged dimer anion structure in which each iron(III) centre coordinates tetrahedrally to the three Cl ligands in addition to the bridging ligand. Antiferromagnetic coupling between the two iron centres in the anion complex is indicated by the low room-temperature effective magnetic moments (1.71–1.74 μB). The i.r. and electronic absorption spectra are in accord with the proposed anionic oxygen bridge structure.

Poly(ethylene Oxide)-Modified Polyaspartamide–Ferrocene Conjugates

In continuation of earlier investigations of polymer–ferrocene conjugates for biomedical applications, this article deals with conjugates prepared by N-acylation of linear, amine-functionalized polyaspartamide carriers with 4-ferrocenylbutanoic acid. Acylation is brought about both by mediation of HBTU coupling agent and by the N-hydroxysuccinimide active ester method. The polymeric carriers contain oligo- or poly(ethylene oxide) side chains introduced here for enhancement of water solubility. The longer side chains, in addition, are to impart such biomedically important properties as increased resistance to uptake by the reticuloendothelial system and to protein binding, extended circulation life time, and lowered immunogenicity. The conjugates comprise from 10 to 25 mol% ferrocenylated subunits, corresponding to ca. 2–5% Fe by mass. Freshly prepared and isolated in the solid state, they dissolve smoothly in aqueous media, with upper concentration limits (>0.2g/ml) dictated solely by their viscosity behavior. The conjugates are of interest in biomedical applications.

A Chlorotitanocene Tetrachloroferrate Complex Stabilized by Acetonitrile Coordination

SummaryTitanocene derivatives of the dichlorodicyclopentadienyltitanium type(1) are known to possess antineoplastic activity against Ehrlich ascites and other tumor systems in mice. Good solubility in water is generally a prerequisite for biomedical applications of this kind. A titanocene derivative possessing excellent cold water solubility (being converted into an aquated species in the process of dissolution) is the complex(2), acetonitrilechlorodicyclopentadienyltitanium tetrachloroferrate, readily obtained from(1) and iron(III) chloride in acetonitrile. Some spectroscopic properties of(2) are presented.

Antiproliferative Activity of Polyaspartamide–Ferrocene Conjugates Containing the Biofissionable Carboxamide Function in the Anchoring Links

In continuation of earlier studies probing the cytotoxic properties of polymer-bound ferrocene against the HeLa cell line, the present communication presents the results of cell culture tests performed on a number of polymer–ferrocene conjugates against the LNCaP human metastatic prostate adenocarcinoma line. The water-soluble substrates are polyaspartamide carriers containing primary amine functions as side-group terminals to which the ferrocenylation agent, 4-ferrocenylbutanoic acid, is covalently bound (anchored) through N-acylation, the carboxamide groups so generated in the tether acting as the biofissionable links for release of the monomeric ferrocene compound from the conjugate in the lysosomal compartment. The carrier backbone structures are of the α, β-DL-peptide type preferred in our ongoing work, as this structural configuration allows ultimate chain degradation for efficacious excretion yet prevents fast and premature, α-peptidase-mediated “unzipping” of the peptidic chain. The screens are performed by exposing the selected conjugates 1–7 at various concentrations to cultured LNCaP cells in RPMI medium over a period of 7 days and assessing cell viability by a protein assay. From cell growth data relative to control, plotted against conjugate concentration, the IC50 values, expressed as metal concentrations required for 50% cell growth inhibition, are found to be in the narrow range of 1–10 μg Fe/ml. This compares well with earlier results obtained on selected ferrocene conjugates against the HeLa cell line and, more strikingly still, on analogous, previously tested conjugates containing the square-planar structural skeleton of the cisplatin-type anticancer drug system as the bioactive agent. The present findings, hence, should provide a healthy motivation for more extended studies of polymer-anchored ferrocenes in the biomedical realm.

Aqueous-phase synthesis of di-(η5-cyclopentadienyl)salicylato- anddi-(η5-cyclopentadienyl)phthalatotitanium(IV) complexes

Di-(η5-cyclopentadienyl)dichlorotitanium(IV) reacts with salicylic acid or some of its ring-substituted derivatives in aqueous medium in the presence of alkali carbonate, giving (substituted) di-(η5-cyclopentadienyl)-salicylatotitanium(IV) complexes(3). Analogously, although less efficaciously, the dichlorotitanium compound reacts with phthalic acid to give the phthalato complex(5), and with dipicolinic acid to yield the pyridinedicarboxylato compound(7). Meticulous control of the experimental conditions is necessary to minimize hydrolytic side reactions. The product complexes(3) and(5) can be recrystallized from chloroform, in which they dissolve completely when freshly prepared; prolonged storage at ambient temperature causes reductions in solubility. I.r. and n.m.r. spectroscopic features of the product complexes are presented.

Aliphatic polyamides prepared by ester-amine polycondensation as potential drug carrier polymers

Aliphatic polyamides of interest as macromolecular drug carriers are synthesized by base-catalyzed polycondensation of aliphatic diesters with diamines. The reactions are conducted in the presence of anhydrous sodium carbonate at temperatures ranging from ambient to 65°C, initially in the undilute state. The addition of aprotic solvent at a later stage serves to maintain sufficiently low viscosity for proper homogenization. The comonomers, diethyl 3,6,9-trioxaundecanedioate and 4,7,10-trioxa-1,13-tridecanediamine, copolymerize to form polymer 1, a straight-chain polyamide devoid of specific functionality. Use of diethyl tartrate in lieu of the aforementioned diester leads to polyamide 2 possessing hydroxyl side groups. Other experiments in which diamines incorporating additional (secondary) amino groups are employed afford polyamides 3–8 containing such secondary amine functions as main-chain constituents. The water-soluble target polymers are crudely fractionated by aqueous dialysis (12000–14000 molecular mass cut-off) and collected by freeze-drying in yields of 20 to 40%. The low-yield range has been accepted in this investigation as the price to be paid for the realization of linear polyamide structures in accordance with compositional expectations, a requirement vital for the proper functioning of the polymers as drug carriers. The practicability of drug binding (conjugating) is exemplified by the coupling of ferrocene as a drug model to polyamide 5 via amide linkage. The water-soluble conjugate 5-Fc features an iron content corresponding to one ferrocene group in the repeat unit.

Water-Soluble Polymer–Ferrocene Conjugates Based on Polyamide Carriers Containing Intrachain-Type Secondary Amine Functions as Binding Sites

It is the objective of this project to synthesize polymer–ferrocene conjugates from macromolecular carriers in which the “anchoring” sites are main-chain constituents, thus contrasting with previously described conjugates featuring side-chain terminals as anchoring sites. To this end, earlier-developed polyamide carriers containing secondary amino groups in the main chain are treated with the active N-succinimidyl ester of 4-ferrocenylbutanoic acid in DMF solution. Molar feed ratios are chosen so as to favor the incorporation of a single ferrocenyl group per recurring unit. The water-soluble, microanalytically and spectroscopically characterized conjugates are of interest as antiproliferative agents in cancer research.