Triazine dendrimers and smaller dendritic scaffolds that present 1,4-diazabicyclo[2.2.2]octane (DABCO) on the periphery were prepared and assessed for antimicrobial activity and human cell toxicity. Hydrophilic linkers on the periphery of these multivalent scaffolds were derivatized with 2 to 6 DABCO groups that presented either methyl, benzyl, or dodecyl substituents. All of these derivatives were highly soluble in water. Antimicrobial assays against Staphylococcus aureus (Newman), methicillin-resistant S. aureus (MRSA; Sanger 252) and Escherichia coli (K-12) revealed that antimicrobial activity is influenced by two factors; the alkyl substituent on the DABCO group and the valency of the construct. Antimicrobial activity decreased from dodecyl > benzyl > methyl. Divalent and trivalent compounds showed greater activity than tetravalent and hexavalent compounds.

The expedited synthesis of odd generation triazine dendrimers up to generation 9 can be executed in high yields using microwave irradiation. The efforts commence from commercially-available and inexpensive materials. Execution is facilitated by automated chromatography.

Condensation of a hydrazine-substituted s-triazine with an aldehyde or ketone yields an equivalent to the widely used, acid-labile acyl hydrazone. Hydrolysis of these hydrazones using a formaldehyde trap as monitored using HPLC reveals that triazine-substituted hydrazones are more labile than acetyl hydrazones at pH >5. The reactivity trends mirror that of the corresponding acetyl hydrazones, with hydrolysis rates increasing along the series (aromatic aldehyde < aromatic ketone < aliphatic ketone). Computational and experimental studies indicate a reversal in stability around the triazine pKa (pH ∼5). Protonation of the triazine moiety retards acid-catalyzed hydrolysis of triazinyl hydrazones in comparison to acetyl hydrazone analogues. This behavior supports mechanistic interpretations suggesting that resistance to protonation of the hydrazone N1 is the critical factor in affecting the reaction rate.

Eight triazine dendrimers were prepared to probe the impact of linker choice on water solubility. Three different linkers were assessed including two hydrophobic diamines that show high reactivity, piperazine and trismethylene bispiperidine, as well as a hydrophilic diamine, 4,7,10-trioxotridecane-1,14-diamine, which is less reactive. Dendrimers 1–8 share a common, generation two, hydrophobic core, 1. Dendrimer 1 is insoluble in water. Of the three generation four dendrimers, 2–4, that were prepared, 2 is also insoluble in water, but substitution of one or two of the hydrophobic linkers with 4,7,10-trioxotridecane-1,14-diamine yields sparingly soluble 3 and more soluble 4, respectively. Molecular dynamics simulations of dendrimers 2–4 in water provide additional insight into their shape, hydration and hydrophobicity. Generation six targets, 5–8, are also sensitive to choice of interior and surface groups. Dendrimer 5 is insoluble in water, but replacing one or two hydrophobic linkers with 4,7,10-trioxotridecane-1,14-diamine yields dendrimers 6 and 7 with modest affect unless the double substitution occurs in tandem at the periphery to yield 8 which shows high solubility in water. The solubility trends suggest that the choice of cationic surface group is critical, and that piperazine groups on the periphery and interior do little to promote solubility of triazine dendrimers in water compared with the hydrophilic amine 4,7,10-trioxotridecane-1,14-diamine.

Microwave assisted synthesis allows for the rapid access of low generation triazine dendrimers in high yields. Products include a macromonomer and alkyne functionalized dendrimers from generation one to three. Using a microwave assisted, convergent synthetic approach, two nucleophilic aromatic substitution reactions are executed on cyanuric chloride in 10 min at 60 °C using primary amines. Substitution of the resulting monochlorotriazine with a diamine requires 95 °C for 30 min. Purifications are accomplished using an automated chromatography system such that the generation three dendrimer can be prepared from the starting materials in less than one day.

The dendrimer chemistry reported offers a route to synthetic target molecules with spherical shape, well-defined surface chemistries, and dimensions that match the size of virus particles. The largest target, a generation-13 dendrimer comprising triazines linked by diamines, is stable across ranges of concentration, pH, temperature, solvent polarity and in the presence of additives. This dendrimer theoretically presents 16 384 surface groups and has a molecular weight exceeding 8.4 MDa. Transmission electron and atomic force microscopies, dynamic light scattering, and computations reveal a diameter of ∼30 nm. The target was synthesized through an iterative divergent approach using a monochlorotriazine macromonomer providing two generations of growth per synthetic cycle. Fidelity in the synthesis is supported by evidence from NMR spectroscopy, mass spectrometry, and high-pressure liquid chromatography.

Triazine dendrimers terminated with either four or eight dichlorotriazines can be prepared in high yields by reacting an amine-terminated dendrimer with cyanuric chloride. These materials exist as white powders and are stable to storage at room temperature. Sequential nucleophilic aromatic substitution with two different amine nucleophiles yields compounds that display the desired compositional diversity. Reaction conditions for the substitution were developed using a model dichlorotriazine with amine nucleophiles at −20, 0, and 25 °C. Selective substitution is favored at lower temperatures and with more nucleophilic amine groups.

The synthesis and characterization of a generation three triazine dendrimer that displays a phenolic group at the core for labeling, up to eight 5 kDa PEG chains for solubility, and 16 paclitaxel groups is described. Three different diamine linkers—dipiperidine trismethylene, piperazine, and aminomethylpiperidine—were used within the dendrimer. To generate the desired stoichiometric ratio of 8 PEG chains to 16 paclitaxel groups, a monochlorotriazine was prepared with two paclitaxel groups attached through their 2′-hydroxyls using a linker containing a labile disulfide. This monochlorotriazine was linked to a dichlorotriazine with aminomethylpiperidine. The resulting dichlorotriazine bearing two paclitaxel groups could be reacted with the eight amines of the dendrimer. NMR and MALDI-TOF confirm successful reaction. The eight monochlorotriazines of the resulting material are used as the site for PEGylation affording the desired 2:1 stoichiometry. The target and intermediates were amenable to characterization by 1H and 13C NMR, and mass spectrometry. Analysis revealed that 16 paclitaxel groups were installed along with 5–8 PEG chains. The final construct is 63% PEG, 22% paclitaxel, and 15% triazine dendrimer. Consistent with previous efforts and computational models, 5 kDa PEG groups were essential for making the target water-soluble. Molecular dynamics simulations showed a high degree of hydration of the core, and a radius of gyration of 2.8 ± 0.2 nm. The hydrodynamic radius of the target was found to be 15.8 nm by dynamic light scattering, an observation indicative of aggregation. Drug release studies performed in plasma showed slow and identical release in mouse and rat plasma (8%, respectively). SPECT/CT imaging was used to follow biodistribution and tumor uptake. Using a two component model, the elimination and distribution half-lives were 2.65 h and 38.2 h, respectively. Compared with previous constructs, this dendrimer persists in the vasculature longer (17.33 ± 0.88% ID/g at 48 h postinjection), and showed higher tumor uptake. Low levels of dendrimer were observed in lung, liver, and spleen (∼6% ID/g). Tumor saturation studies of small prostate cancer tumors (PC3) suggest that saturation occurs at a dose between 23.2 mg/kg and 70.9 mg/kg.Keywords: biodistribution; dendrimer; disulfide linkage; drug delivery; molecular dynamics; paclitaxel; simulation; triazine;

The synthesis, characterization, and host–guest chemistry of high-generation triazine dendrimers are described. With pyrene and camptothecin as guests, experiments revealed that the guest capacity of odd-generation triazine dendrimers increased until generation 7 but decreased at generation 9. Molecular dynamics simulations conducted in explicit solvent showed a useful fingerprint for this behavior in radial distribution functions of water molecules penetrating the interior of the dendrimers. A linear relationship between the guest capacity of dendrimers measured experimentally and the number of water molecules within the interior determined computationally was observed.

The antitumor activities of triazine dendrimers bearing paclitaxel, a well-known mitotic inhibitor, are evaluated in SCID mice bearing human prostate cancer xenografts. To increase the activity of a first generation prodrug 1 that contained twelve paclitaxel molecules tethered via an ester linkage, the new construct described here, prodrug 2, tethers paclitaxel with linkers containing both an ester and disulfide. While PEGylation is necessary for solubility, and may improve biocompatibility and increase plasma half-life, it increases the heterogeneity of the sample with an average of eight to nine PEG chains (2 kDa each) incorporated. The heterogeneous population of PEGylated materials was used without fractionation based on models obtained from molecular dynamics simulations. Three models were examined; hexaPEGylated, nonaPEGylated, and dodecaPEGylated constructs. Intravenous delivery of prodrug 2 was performed by single, double or triple dosing regimes with doses spaced by one week. The doses varied from 50 mg of paclitaxel/kg to 200 mg of paclitaxel/kg. Tumor growth arrest and regression was observed over the 10-week treatment period without mortality for mice treated with the 50 mg of paclitaxel/kg treated three times.Keywords: dendrimer; drug delivery; molecular dynamics; paclitaxel; prostate cancer; simulation; triazine; xenografts;

The use of triazine dendrimers as drug delivery systems benefits from their synthetic versatility and well-defined structure. Triazine dendrimers can be designed and readily synthesized to display orthogonally functional surfaces that facilitate post-synthetic manipulation such as attachment of drug, PEGylation, and/or the installation of ligands or reporting groups. The synthesis is scalable, and large generations can be accessed. To date, triazine dendrimers have been probed for a variety of medicinal applications including drug delivery with an emphasis on cancer, nonviral DNA and RNA delivery systems, in sensing applications, and as bioactive materials. Specifically, triazine adducts with paclitaxel, camptothecin, brefeldin A, and desferrioxamine have been prepared and assessed. Paclitaxel constructs show promising activity in vivo. The use of these materials in fluorescence-based glucose sensors is being pursued. Glycosylated triazine dendrimers interfere with signal transduction in the Toll-4 receptor pathway.Download high-res image (175KB)Download full-size image