Reactivity of 1,1’-(Prop-1-ene-1,3-diyl)-Bridge Functionalised bis(NHC)

A series of reaction on explorative NMR scale towards deprotonation of the allyl bridge of Au2(L7^Mes)2(PF6)2 using Pd(II) bases (Pd(OAc)2 or Pd(acac)2) and well external base/PdCl2

systems in acetonitrile have been conducted.^[203] The mild Pd bases were not strong enough to induce the deprotonation even at 90 °C. A Cs2CO3/PdCl2 system suggested a transmetallation of the NHC ligand to Pd under formation of mono(imidazolium) compounds at 45 °C-90 °C; at RT no reaction was observed. The use of a system with stronger basicity, KNʺ/PdCl2, indicated a reaction already at 45 °C. However, the characterization of the obtained product was impeded by the insolubility of the obtained crude product in acetonitrile or THF.

Therefore, further experiments with other transition metal systems are necessary.

4.2 Conclusion and Outlook

Formation of different conformers of dinuclear silver(I) and gold(I) 1,1’-(2-hydroxyethane-1,1-diyl)-bridge-functionalised bis(NHC) complexes with various wingtip substituents (R = methyl, isopropyl and mesityl) has been investigated by using multinuclear NMR spectroscopy, SC-XRD and DFT calculations. The ratio of anti/syn isomers strongly depends both on wingtip substituents and the metal centre. Moreover, the reaction temperature plays a significant role during the transmetallation process for the ratio of gold(I) conformers, which are further affected by purification procedures. The 1,1’-(2-hydroxyethane-1,1-diyl)-bridge-functionalised bis(NHC) complexes of Au(I) counterbalanced by PF6 anions have been applied in a standard MTT assay performed for screening the antiproliferative activity against human lung and liver cancer cells. An application of sterically hindered mesityl wingtip substituents shows hereby the best results. Likely, the fine-tuning of lipophilicity and conformational isomerism are crucial for designing gold bis(NHC) based anti-cancer drugs. Since isomer formation could be further significantly influenced by purification procedures, an investigation towards the selective application of the different isomers should be conducted.

The experiments towards further functionalization of the OH group in the bridge are an interesting approach. It might be possible to couple a biological marker and other metals, which are known to inhibit the tumour cell growth to that functionality. Also the possibility of inducing stronger aurophilic interactions by further modifying the bridge and wingtip substituents is worth of the investigation. Such modification could enhance the natural luminescent properties of Au(I)-NHC complexes for visualizing its intracellular distribution.^[10a]

First preliminary results in the experiments with Au2(HL3^Me)2(PF6)2 and bis(trimethylsilylamide)bases of alkali metals and iron(II) aimed on modification of the bridge show a high preference for the transfer of the TMS group to the hydroxyl-group of the ligand.

Since higher reaction temperatures were necessary in these cases due to low solubility of Au2(HL3^Me)2(PF6)2 in THF, further experiments with more soluble analogues Au2(HL3^R)2(X)2

(R = i-Pr, Mes) are considered to be more promising. Also the application of other bases is conceivable.

Although it was not possible to obtain Pd(II) complexes supported by 1,1’-(prop-1-ene-1,3-diyl)-bridge functionalised bis(NHC) ligands, novel dinuclear bis(NHC) complexes of Ag(I) and Au(II) were readily isolated and fully characterised by multinuclear NMR spectroscopy, ESI-MA, EA and SC-XRD. Due to rigidity of the bridge these compounds exhibit interesting folded structure in the solid state. A post-synthetic modification of the bridge towards deprotonation and the formation of large delocalized NHC-allyl-NHC system supporting heterometallic Au(I)/Pd(II) complexes was attempted using various Pd(II) internal bases and systems

comprising of Pd(II) salts and various external bases. It is clear that mild bases such as Pd(OA)2, Pd(acac)2 and Cs2CO3 are not sufficiently strong enough to induce the deprotonation.

Further experiments with stronger bases are therefore necessary.

Experimental Section

Chapter 5

5.1 General Procedures

5.1.1 Schlenk Technique

Unless otherwise stated, all syntheses were carried out under argon atmosphere using standard Schlenk and glove box techniques. Anhydrous benzene was obtained from commercial suppliers. All other solvents were dried via a MBraun MB SPS purification system;

where necessary degassed by three freeze-pump-thaw cycles and stored over 4 Å molecular sieves and argon atmosphere. Deuterated solvents were purchased from Eurisotop and if necessary dried over elemental potassium and distilled prior to use.

5.1.2 NMR Spectroscopy

1H NMR spectra were recorded on a Bruker AV400US with broad band probe and a gradient coil (¹H NMR, 400.13 MHz), a Bruker DRX-400 spectrometer with broad band probe (¹H NMR, 400.13 MHz) and a Bruker Avance III 500 with DHC dual cryoprobe (¹H NMR, 500.12 MHz) and Bruker Avance III 500 with prodigy cryoprobe (¹H NMR, 500.12 MHz). ¹³C NMR spectra were run on a Bruker AV400US (¹³C NMR, 100.53 MHz), a Bruker DRX-400 spectrometer (¹³C NMR, 100.61 MHz), a Bruker Avance III 500 with DCH dual cryoprobe (¹³C NMR, 125.77 MHz). ⁷Li NMR were recorded on a Bruker DRX-400 spectrometer with broad band probe (⁷Li NMR, 155.52 MHz) and on a Bruker Avance III 500 with prodigy cryoprobe (⁷Li NMR, 194.40 MHz) ²⁹Si INEPT NMR spectra were run on Bruker AV500C QNP Cryo probe (²⁹Si INEPT-NMR, 99.41 MHz), on a Bruker AV400US (²⁹Si INEPT-NMR, 79.49 MHz) and Bruker Avance III 500 with prodigy cryoprobe (²⁹Si INEPT-NMR, 99.37 MHz). DOSY spectra were recorded on Bruker AVHD400 (¹H NMR, 400.13 MHz). If necessary, 2D-experimetns were used for a correct assignment of the signals. HH-COSY NMR, HSQC NMR, NOESY and DOSY were run either on Bruker AV400US or on Bruker DRX-400 spectrometers (¹H NMR, 400.13 MHz; ¹³C NMR, 100.62 MHz). Chemical shifts (δ) are reported relative to the residual signal of the deuterated solvent. The following abbreviation were used for the characterization of NMR spectra: s – singlet, d –doublet, t – triplet, q – quartet, p – pentet, hept – heptet, br – broad, virt – virtual, dd – doublet of doublets, dt – doublet of triplets, m – multiplet. NMR spectra were analysed by using MestReNova© (Version 8.0.0.-10524, Mestrelab Research S.L.).

5.1.3 Elemental Analysis

Elemental analyses were carried out by the microanalytical laboratory at the Technical University of Munich. All values are given in per cent.

5.1.4 Mass Spectrometry

FAB+ mass spectra were collected at Finnigan MAT 90. ESI mass spectrometry was performed at Thermo Scientific LCQ Fleet Spectrometer. A time-of-flight analyser was used for mass detection. As eluent a mixture of acetonitrile and formic acid (0.1 Vol.%) was used.

5.1.5 UV/VIS Spectroscopy

UV-VIS spectra were recorded at Agilent Cary 60 Spectrometer. The molar extinction coefficient is given L∙mol^-1∙cm^-1.

5.1.6 Fluorescence Spectroscopy

For luminescence experiments, the samples in solution were placed in fluorimetric 1 cm path quartz cuvettes and the solid state samples (powder) were placed in a covered quartz laboratory dish. Uncorrected emission spectra were obtained with a Hamamatsu C11347 Absolute PL Quantum Yield Spectrometer.

5.1.7 DFT-Calculations

DFT calculations were performed by B.Sc. David Mayer and Dr. Marcus Drees from the group of Prof. Roland A. Fisher at Technische Universität München.

All calculations were carried out using the Gaussian 09 package.^[218] To ensure the comparability to the already published results^[173b], the density functional ωB97x-D^[219] and the method PM6^[220] as well as the basis sets 6-31+g(d), 6-311++g(d,p)^[221] and LANL3DZ[221a, 222]

(incl. ECP for metals) were employed as implemented.

Suitable input geometries were based on molecular structures obtained by SC-XRD, as available. To achieve atomic coordinates for the remaining isomers of silver and gold bis(NHC) complexes, the crystallographic data was slightly modified using GaussView 5. The coordinates were pre-optimized on the PM6 theory of level and then refined employing a double zeta basis set (6-31+g(d) for non-metals and LANL3DZ for metals). All optimized geometries were checked by frequency determination for negative eigenfrequencies, corresponding to the local minima on potential energy hypersurface. The optimized parameters using DFT approach have been compared with the mean experimental bonding lengths and are in excellent agreement with them (see Table 4.1.3, Chapter 4, Section 4.1.1.1).

NMR chemical shifts were calculated at the ωB97x-D/6-311++g(d,p) level using a GIAO (gauge independent atomic orbital) approach.^[223] Absolute isotropic magnetic shielding constants were transformed into chemical shifts by referring to TMS (tetramethylsilane, 30.59 ppm for CH3CN and 30.52 ppm for DMSO).

To display the solvent behaviour, the optimization, frequency and NMR-shielding calculations were all performed using an SCRF ansatz with an implicit solvent model (SMD, as recommended by Truhlar and Cramer, c.p.).^[224]

5.1.8 SC-XRD Crystallography

X-ray structural analyses were carried out by Dr. Alexander Pöthig and M.Sc. Christian Jandl at the Sc-XRD laboratory of TUM Catalysis Research Centre.

Data were collected on single crystal X-ray diffractometers equipped with one of the following setups using the APEXII or APEXIII software package: A CCD detector (APEX II, κ-CCD), a fine-focus sealed tube and a graphite monochromator (A, B, C); a CCD detector (APEX II, κ-CCD), a fine-focus sealed tube and a Triumph monochromator (A, B, C); a CCD detector (APEX II, κ-CCD), a FR591 rotating anode and a Montel mirror optic (A, B, C); a CMOS detector (APEX III, κ-CMOS), an IMS microsource and a Helios optic (A, B, C); a CMOS detector (APEX III, κ-CMOS), a TXS rotating anode and a Helios optic (A, B, C).^[225] For all measurements MoKα radiation (λ = 0.71073 Å) was used. The crystals were fixed on the top of a glass fibre or kapton micro sampler with perfluorinated ether, transferred to the diffractometer and frozen under a stream of cold nitrogen. A matrix scan was used to determine the initial lattice parameters. Reflections were merged and corrected for Lorentz and polarization effects, scan speed, and background using SAINT.^[226] Absorption corrections, including odd and even ordered spherical harmonics were performed using SADABS.^[226]

Space group assignments were based upon systematic absences, E statistics, and successful refinement of the structures. Structures were solved using direct methods (SHELXL-97) or intrinsic phasing (SHELXT) with the aid of successive difference Fourier maps, and were refined against all data using the APEXII or APEX III software package in conjunction SHELXL-2014 and SHELXLE. [225, 227] Hydrogen atoms were calculated in ideal positions as follows:

Methyl hydrogen atoms were refined as part of rigid rotating groups, with a C–H distance of 0.98 Å and Uiso(H) = 1.5·Ueq(C). Other H atoms were placed in calculated positions and refined using a riding model, with methylene and aromatic C–H distances of 0.99 Å and 0.95 Å, respectively, other C–H distances of 1.00 Å and Uiso(H) = 1.2·Ueq(C). Non-hydrogen atoms were refined with anisotropic displacement parameters. Full-matrix least-squares refinements were

atom scattering factors for all atoms and anomalous dispersion corrections for the non-hydrogen atoms were taken from International Tables for Crystallography.^[228] A split layer refinement was used to treat with disordered anion/solvent molecules and additional SIMU, DELU and SAME restraints were employed to stabilize the refinement of the layers. In case of unrefinably disordered solvent molecules (e.g. on special positions) these were treated as a diffuse contribution to the overall scattering without specific atom positions using the PLATON/SQUEEZE procedure.^[229] Images were created with Mercury 3.8.^[230]

5.1.9 MTT-Assay

The cell toxicity studies were performed by M.Sc. Wolfgang Heydenreuter and M.Sc. Jonas Drechsel from the group of Prof. Dr. Stephan A. Sieber at Technical University of Munich.

The assay was performed in 96 well plates. A549/HepG2 cells were grown to 30-40 % confluence. The medium was removed and 100 µL medium/well containing 1 µL DMSO compound stock were added to the cells and incubated for 48 h. All concentrations as well as a DMSO control were done in triplicates. 20 µL Thiazolyl Blue Tetrazolium bromide (5 mg/mL in PBS, Sigma Aldrich) were added to the cells and incubated for 2-4 h until complete consumption was observed. After removal of the medium, the resulting formazan was dissolved in 200 µL of DMSO. Optical density was measured at 570 nm (562 nm) and background subtracted at 630 nm (620 nm) by a TECAN Infinite® M200 Pro.

For calculation of IC50 values, residual viabilities for the respective compound concentration were fitted to

𝑉 = 100

1 + 10^{(log⁡(𝐼𝐶50})−log⁡(𝑐))∙𝑁

𝑉: viability [%]; 𝑐: Inhibitor concentration [M]; 𝑁: Hill slope using Graphpad Prism 6.0.