Amino Acids as Chelating Ligands for Platinum: Enhanced Stability in an Aqueous Environment Promoted by Biocompatible Molecules

Platinum-based chemotherapeutics are a cornerstone in the treatment of many malignancies. However, their dose-limiting side effects have rooted efforts to develop new drug candidates with higher selectivity for tumor tissues and less problematic side effects. Here, we developed a cytotoxic platinum(II) complex based on Zeise’s salt, containing the nonsteroidal anti-inflammatory drug acetylsalicylic acid and alanine as ligands (4). The previously developed complex (5) displayed high reactivity against sulfur-containing biomolecules; therefore, we put the focus on the optimization of the structure regarding its stability. Different amino acids were used as biocompatible chelating ligands to achieve this aim. Differences in the coordination sphere caused pronounced changes in the stability of Zeise-type precursors 1–3. Coordination with l-Ala through N in the trans position to ethylene showed the most promising results and was employed to stabilize 5. As a result, complex 4 showed improved stability and cytotoxicity, outperforming both 5 and 1.


H-NMR spectrum of complex 1a
Figure S2 13 C-NMR spectrum of complex 1a Figure S3 195 Pt-NMR spectrum of complex 1a

S5
Figure S6 [ 1 H, 13 C]-HMBC 2D-NMR spectrum of complex 1a  Figure S17 shows the IRMPD spectrum of the cluster of signals at m/z 413, which is compatible with the mass and the isotopic pattern of the synthetized complex 3.A few vibrational modes are worth mentioning.The experimental band at 1775 cm -1 can be interpreted by the calculated CO stretching mode of 3_1 and 3_2 at 1762 and 1763 cm -1 , respectively, while the same vibration is calculated for 3_3 at 1676 cm -1 , thus red-shifted due to the interaction of the carboxylic CO with Pt.In addition, the most intense band of the IRMPD spectrum at 1143 cm -1 is correctly reproduced only by the computed OH bending mode of 3_1 and 3_2 (1142 and 1145 cm -1 , respectively).Further spectroscopic evidence for the binding of the amino nitrogen to platinum is the neat experimental band at 1570 cm -1 , which can be assigned to the NH2 scissoring mode of the amino group when bound to platinum.In contrast, 3_3 presents this vibrational mode at 1617 cm -1 .In summary, spectroscopy agrees with the thermodynamic data in assessing the formation of a N,N-bound chelate of histidine to the platinum complex, as in 3_1 and 3_2, while there is no evidence of the formation of an O-bound species through the carboxylate (3_3).[3][4][5]   DFT optimized structures for 1a, 1b, 2a, and 2b Electropherograms of 1a -4 Crystal data and structure refinements for complex 1a  Table S3 Atomic coordinates ( x 10 4 ) and equivalent isotropic displacement parameters (Å 2 x 10 3 ) for complex 1a.U(eq) is defined as one third of the trace of the orthogonalized U ij tensor.

Figure S7 IR spectrum of complex 1a S6
Figure S7 IR spectrum of complex 1a

Figure S15 IR spectrum of complex 3 S10Figure
Figure S15 IR spectrum of complex 3

Figure
Figure S25 Optimized structures at the B3LYP level of isomers 1a, 1b, 2a and 2b.Relative enthalpies (free energies) at 298 K calculated at the M06-2X level are reported in kJ mol -1 .

Figure S26 Electropherogram of complex 1a Figure S27 Electropherogram of complex 2b S17Figure S28 Electropherogram of complex 3 ,
Figure S26 Electropherogram of complex 1a to generate equivalent atoms: to generate equivalent atoms:

Figure
Figure S30 from above: 4) 1 H-NMR spectrum of L-alanine in D2O; 3) 1 H-NMR spectrum of ZS in D2O; 2) 1 H-NMR of the synthesis of complex 1 without base; 1) 1 H-NMR of the precipitate from the synthesis of complex 1 with base in D2O.

Figure
Figure S31 from above: 4) 13 C-NMR spectrum of L-alanine in D2O; 3) 13 C-NMR spectrum of ZS in D2O; 2) 13 C-NMR of the synthesis of complex 1 without base; 1) 13 C-NMR of the precipitate from the synthesis of complex 1 with base in D2O.

Figure S32 from above: 3 )
Figure S32 from above: 3) 195 Pt-NMR spectrum of ZS in D2O; 2) 195 Pt-NMR of the synthesis of complex 1 without base; 1) 195 Pt-NMR of the precipitate from the synthesis of complex 1 with base in D2O.
1 H-and  195  Pt-NMR spectra were recorded as references.The same sample was divided in 2 portions of 250 µL each.The first portion (A) was diluted with 250 µL of CD3OD.The second (B) was diluted with 250 µL of deuterated water (D2O).1 H-and 195  Pt-NMR spectra were recorded at different time points.The NMR tubes were stored at room temperature (rt) with the solution protected from light between one experiment and the other.

Figure
Figure S37 Comparison of the 195 Pt-NMR spectra of ZS in deuterium oxide and of ZS in a concentrated solution (200 nM) of TMG in deuterium oxide.The latter solution was monitored over-time up to 2 days after dissolution of ZS.

4 , and 5 .
Groups investigated: 3 groups of data were considered: a) the COX-1 inhibitory effect, b) the COX-2 inhibitory effect, c) the residual metabolic activity of MDA-MB-231 cell lines.For each group the mean values were considered for compounds 1, Only the values obtained from the incubation of cells or enzymes with the above-mentioned compounds at a concentration of 25 µM were considered.Since only MDA-MB-231 expresses both COX-1 and COX-2 isoenzymes, data obtained employing the A2780cis cell lines were excluded.
Table S1 IRMPD features and theoretical IR frequencies (cm −1 ), infrared absorption intensities (km mol -1 ) in parenthesis, of 3_1 and 3_2 and vibrational normal modes obtained by DFT calculations at the B3LYP level.

Table S2
Crystal data and structure refinement for 1a.

Table S9
Crystal data and structure refinement for 1b.

Table S17
Significance of values determined for MDA-MB-231 and A2780cis incubated with compounds 1, 4, 5, and 6 at 25 µM concentration.The significance is calculated in relation to the reference (cisplatin).Diff stays for difference; CI stays for Confidence Interval.Sidak's multiple comparisons test Mean Diff 95,00% CI of diff Significance Summary

Table S18
Correlation of the % of residual metabolic activity of MDA-MB-231 cells after incubation with compounds 1, 4, and 5, with the corresponding values of COX-1 and COX-2 inhibition