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

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 (1)H and (13)C 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.

We present basic spectroscopic studies of 5 triazine dendrimers ranging from generations one through nine, G1, G3, G5, G7, and G9, based on the intrinsic fluorescence of these molecules. The extinction spectra of each generation can be separated into two components; the absorption spectra from triazine chromophores and Rayleigh scattering by dendrimer particles. Rayleigh scattering into the UV spectral range is significant and may contribute more than 50% to the measured light attenuation (extinction) for larger dendrimer generations. Deviations from the Rayleigh model at long wavelengths (where the triazine chromophore does not absorb) are clear indications of dendrimer aggregation. These larger particles can be eliminated by dilution and sonication. Importantly, this model system represents a comprehensive case study where the intrinsic fluorescence of the dendrimer when combined with insights from molecular dynamics (MD) simulations can be utilized to probe molecular conformations and dynamics. Experimental results from fluorescence lifetimes, time-resolved anisotropies, and diffusional quenching indicate an increasingly compact core as size increases from G1 to G5. This trend is reversed for G7 and G9 generations, which present more extended, and porous structures, less dense cores, and a denser peripheries. Simulations corroborate this picture and better anchor intuition of the behavior of these molecules.

Abstract:A readily and rapidly accessible triazine dendrimer was manipulated in four steps with 23% overall yield to give a construct displaying four maleimide groups and DOTA. The maleimide groups of the dendrimer are sensitive to hydrolysis under basic conditions. The addition of up to four molecules of water can be observed via mass spectrometry and HPLC. The evolution in the alkene region of the 1 H-NMR-the transformation of the maleimide singlet to the appearance of two doublets-is consistent with imide hydrolysis and not the Michael addition. The hydrolysis events that proceeded over hours are sufficiently slower than the desired thiol addition reactions that occur in minutes. The addition of thiols to maleimides can be accomplished in a variety of solvents. The thiols examined derived from cysteine and include the protected amino acid, a protected dipeptide, and native oligopeptides containing either 9 or 18 amino acids. The addition reactions were monitored with HPLC and mass spectrometry in most cases. Complete substitution was observed for small molecule reactants. The model peptides containing nine or eighteen amino acids provided a mixture of products averaging between 3 and 4 substitutions/dendrimer. The functionalization of the chelate group with gadolinium was also accomplished easily.