Two square-planar palladium(II) and platinum(II) azido complexes [M(N 3 )(L)] with L = N-phenyl-2-[1-(2-pyridinyl)ethylidene]hydrazine carbothioamide reacted with four different electron-poor alkynes R−CC−R′ with R = R′ = COOCH 3 , COOEt, COOCH 2 CH 2 OCH 3 or R = CF 3 , R′ = COOEt in a [3 + 2] cycloaddition "iClick" reaction. The resulting triazolate complexes [M-(triazolate R,R' )(L)] were isolated by simple precipitation and/or washing in high purity and good yield. Six out of the eight new compounds feature the triazolate ligand coordinated to the metal center via the N2 nitrogen atom, but fortuitous solubility properties allowed isolation of the N1 isomer in two cases from acetone. When the solvent was changed to DMSO, the N1 → N2 isomerization could be studied by NMR spectroscopy and took several days to complete. 19 F NMR studies of the iClick reaction with F 3 C−CC−COOEt led to identification of a putative early linear intermediate in addition to the N1 and N2 isomers, however with the latter as the final product. Rate constants determined by 1 H or 19 F NMR spectroscopy increased in the order Pd > Pt and CF 3 /COOEt > COOR/COOR with R = CH 3 , Et, CH 2 CH 2 OCH 3 . The second-order rate constant k 2 > 3.7 M −1 s −1 determined for the reaction of [Pd(N 3 )(L)] with F 3 C− CC−COOEt is the fastest observed for an iClick reaction so far and compares favorably with that of the most evolved strained alkynes reported for the SPAAC (strain-promoted azide−alkyne cycloaddition) to date. Selected title compounds were evaluated for their anticancer activity on the GaMG human glioblastoma brain cancer cell line and gave EC 50 values in the low micromolar range (2−16 μM). The potency of the Pd(II) complexes increased with the chain length of the substituents in the 4-and 5-positions of the triazolate ligand.

Ten thiosemicarbazone ligands obtained by condensation of pyridine-2-carbaldehyde, quinoline-2-carbaldehyde, 2-acetylpyridine, 2-acetylquinoline, or corresponding 2-pyridyl ketones with thiosemicarbazides RNHC(S)NHNH 2 and R=CH 3 , C 6 H 5 were prepared in good yield. The reaction of [PdCl 2 (cod)] with cod = 1,5-cyclooctadiene or K 2 [PtCl 4 ] resulted in a total of 17 Pd(II) and Pt(II) complexes isolated in excellent purity, as demonstrated by 1 H, 13 C, and, where applicable, 195 Pt NMR spectroscopy combined with CHNS analysis. The cytotoxicity of the title compounds was studied on four human glioblastoma cell lines (GaMG, U87, U138, and U343). The most active compound, with a Pd(II) metal centre, a 2-quinolinyl ring, and methyl groups on both the proximal C and distal N atoms exhibited an EC 50 value of 2.1 μM on the GaMG cell lines, thus being slightly more active than cisplatin (EC 50 3.4 μM) and significantly more potent than temozolomide (EC 50 67.1 μM). Surprisingly, the EC 50 values were inversely correlated with the lipophilicity, as determined with the "shake-flask method", and decreased with the length of the alkyl substituents (C 1 > C 8 > C 10 ). Correlation with the different structural motifs showed that for the most promising anticancer activity, a maximum of two aromatic rings (either quinolinyl or pyridyl plus phenyl) combined with one methyl group are favoured and the Pd(II) complexes are slightly more potent than their Pt(II) analogues.

BACKGROUND Major obstacles for an effective chemotherapy of glioblastomas (GBM) are the blood-brain-barrier (BBB) and serious systemic side effects of the cytotoxic drugs. A new promising strategy could be the delivery of microbubbles, encapsulating the chemotherapeutics, across the BBB to the tumor site. This will shield the drug from detrimental systemic effects. Low intensity focused ultrasound (LIFU) is able to open the BBB and triggers targeted release of the drugs within the tumor. First data on the synthesis of microbubbles, specifically designed new drugs and the targeted rupture of microbubbles by LIFU are presented. MATERIAL AND METHODS Thin-film hydration of lipids was utilized to prepare microbubbles, which were tested for toxicity on the GBM cell lines GaMG, U87, U138 and U343. In addition these cells were treated with 6 platinum(II) and palladium(II) complexes conjugated to lipophilic side chains of different length (C1, C8, C10) for 72h. To evaluate cell viability and calculate EC50 values MTT assays and a real-time proliferation assay using the impedance-based xCELLigence DP-System were executed. RESULTS Microbubbles ≤ 2µm in diameter were synthesized and could be disintegrated by applying LIFU. Neither the intact bubbles nor the lipids alone had any toxic effects on the GBM cells. In contrast, all six drugs were highly effective with EC50 values far below those of Temozolomide (67µM) and in the range of the reference drug cisplatin (3µM). Especially the palladium(II) compound with the C1-chain displayed a very low EC50 value (<10µM), while the longer chains and the platinum(II) compounds were less effective (EC50 10–40µM). An early and concentration-dependent onset of the cytotoxic effect of drugs with C1 and C8 side chains was revealed in the real time proliferation assay. CONCLUSION All components for a new microbubble-based therapeutic strategy are in place. Microbubbles were synthesized without having toxic effects in cell culture. New highly potent palladium(II) and platinum(II) compounds with low EC50 values were developed. The next step will be their encapsulation into the microbubbles via their lipophilic side chains to develop an effective drug-delivery system for the treatment of GBM in combination with LIFU. This will allow increasing the local concentration of chemotherapeutic agents at the tumor site, irrespectively of their molecular size and BBB penetration capacity.

The major obstacles for an effective chemotherapy of glioblastomas (GBM) are the blood-brain barrier (BBB) and serious systemic side effects of the cytotoxic drugs. A new promising strategy could be the delivery of the chemotherapeutics across the BBB to the tumor site encapsulated in microbubbles. The microbubbles will shield the drug from detrimental systemic effects. Low intensity focused ultrasound (LIFU) allows opening of the BBB and a targeted release of the drugs within the brain tumor. We synthesized microbubbles ≤ 2 µm in diameter by thin-film hydration of lipids, which could be disintegrated applying LIFU. The toxicity was tested on GBM cell lines and neither the intact bubbles nor the lipids alone showed any toxic effects. Additionally, these cells were treated with 6 platinum(II) and palladium(II) complexes conjugated to lipophilic side chains of different length (C1, C8, C10) for 72 h. Cell viability was evaluated with MTT assay and in real-time utilizing the impedance-based xCELLigence DP-System. EC50 values were calculated from both assays and all six drugs were highly effective. Especially the palladium(II) compound with the C1-chain had a very low EC50 value (< 10 µM), while the longer chains and the platinum(II) compounds were less effective (EC50 10 - 30 µM). The real time proliferation assay of the drugs revealed an early and concentration-dependent onset of the cytotoxic effect, about 30 h after application. The lipophilic side chains of the drugs should allow encapsulating them into the microbubbles to develop an effective drug-delivery system for the treatment of GBM.