Kinetic investigation of reactions of a 3-arylidene-2-thiohydantoin derivative with palladium(II) salts

: 1 H NMR spectroscopy was used to monitor the reactions of an arylidene 2-thiohydantoin derivative, 3-((phenylmethylene)amino)-2-thioxo-4- imidazolidinone ( 3 ), with PdCl 2 , cis -[PdCl 2 (dmso-S ) 2 ] and K 2 [PdCl 4 ] in DMSO-d 6 in order to elucidate the reaction kinetics and mechanism. The 2-thiohydantoin derivative 3 formed cis -[Pd( 3 - N,S )(dmso-S ) 2 ] + complex ( 5 ) in reactions with PdCl 2 and cis -[PdCl 2 (dmso-S ) 2 ], while no reaction with K 2 [PdCl 4 ] was observed. A two-step mechanism for the reactions of 3 with PdCl 2 and cis -[PdCl 2 (dmso-S ) 2 ] is proposed, in which fast coordination to the side chain nitrogen occurs in the first step, while chelation and coordination to the sulfur atom in the 2-thiohydantoin ring is the second, slower, rate-determining step. The reaction rate constants were calculated and reactivities of the 2-thiohydantoin derivative 3 towards the palladium(II) salts were compared and discussed. Reaction of 3 with cis -[PdCl 2 (dmso-S ) 2 ] was faster than with PdCl 2 . The investigated palladium(II) salts also react with the solvent, DMSO-d 6 , and the influence of these side reactions on the outcome and kinetics of the 2-thiohydantoin derivative complexation reaction is discussed in detail. The obtained results of this study can have an impact in explanation of the coordination behavior of antitumor active palladium(II) and platinum(II) complexes.


INTRODUCTION
Thiohydantoins are sulphur derivatives of hydantoin, in which one or both of the carbonyl groups in the cyclic ureide structure are replaced with a thiocarbonyl group. 1 Out of this class of compounds, 2-thiohydantoins are certainly the most prominent and extensively researched. 2-Thiohydantoins represent a valuable molecular scaffold, exhibiting various biological and pharmacological activities and they have found applications in both medicine and industry. 2,3 They display a wide range of biological activities, such as antibacterial and antifungal, 4 anti-HIV, 5 anticarcinogenic, 6,7 anti-ulcer and anti-inflammatory, 8 anticonvulsive, 9 antimutagenic 10 and antimelanogenic. 11 2-Thiohydantoins found various applications in industry, such as C-terminal protein sequencing standards, 12 textile printing reagents 13 and polymerization and complexation catalysts. 14 Coordination of active compounds with biologically relevant transition metal ions can, at times, increase their activities, especially in regards to anticarcinogenic activity. 15 A newer, hybrid approach, in discovering new potential antitumor agents is coordination of active compounds with metal ions in order to improve their activity and selectivity. 16,17 2-Thiohydantoins have a great affinity for coordination with transition metal ions. 18,19 Even though it is a small molecule, 2thiohydantoin has four derivatization points, making its derivatives very versatile ligands. In addition to the heteroatoms in the ring, 2-thiohydantoin derivatives most often contain heteroatoms in the side chains of its substituents. Many kinds of various 2-thiohydantoin complexes have been synthesized and reported so far. [20][21][22][23][24][25][26] In particular, transition metal complexes of arylidene 2-thiohydantoin derivatives have been researched extensively, largely due to the biological activities they exhibit, primarily antimicrobial and anticancer. 21,27,28 The aim of this study was to investigate the kinetics and mechanism of the coordination reactions of 3-((phenylmethylene)amino)-2-thioxo-4imidazolidinone, an arylidene 2-thiohydantoin derivative, with some palladium(II) salts. As arylidene 2-thiohydantoin metal complexes, palladium(II) in particular, 28 show some promise as prospective antitumor agents, a better understanding of the mechanisms of their formation, coordination modes and kinetics might prove beneficial for the design and conceptualization of novel, more potent compounds.

Materials and methods
All chemicals and reagents used in this investigation were commercially obtained (from either Sigma-Aldrich or Acros) and were high in purity. They were used as received, without additional purification. NMR spectra were recorded on a Varian Gemini-2000 spectrometer at 50 MHz and 200 MHz. DMSO-d6 was used as the solvent and all chemical shifts were referenced accordingly. Downfield shifts were recorded as positive numbers. Tetramethylsilane was used as the internal reference and all chemical shits were rounded to the nearest 0.01 ppm.
A c c e p t e d m a n u s c r i p t

Synthesis and characterization of 3-((phenylmethylene)amino)-2-thioxo-4-imidazolidinone (3)
3-((Phenylmethylene)amino)-2-thioxo-4-imidazolidinone (3) was synthesized using a slight modification of a formerly published procedure. 28 Benzaldehyde (0.01 mol) and thiosemicarbazide (0.01 mol) in methanol (30 mL) were heated for 3 h under reflux and cooled thereafter. Ethyl chloroacetate (0.01 mol) and anhydrous sodium acetate (0.03 mol) were added in situ and the mixture was heated for another 6 h under reflux. Upon the completion of the reaction, the mixture was cooled at room temperature and then added to cold water, for the resulting product to precipitate. The product was filtered off, rinsed with hot water, dried and re-crystallized from hot methanol. Structure and purity of the compound was confirmed by NMR ( 1 H and 13 C) and IR spectroscopy. The corresponding IR and NMR ( 1 H and 13 C) spectra are provided in the ESI ( in DMSO-d6 (0.6 mL) were performed in standard 5 mm NMR tubes at room temperature in an overnight experiment. DMSO-d6 solutions of the reactants (0.3 mL each) were freshly prepared right before the start of the experiment. After the mixing of the reactants, 29 spectra in total were recorded overnight for each one of the experiments. The first six spectra were recorded with no delay, then the next three with a 5 min delay, then sets of three every 10, 15 and 30 min, and finally the last 11 spectra were recorded with an hour delay between them. The concentrations of the products at given experiment intervals were determined by integrating suitable proton signals in the 1 H NMR spectra. The first-order rate constants were determined according to Equation 1. ln(c) = -kt + ln(c0) (1) where c is concentration, c0 is starting concentration, k is the first-order rate constant and t is experiment time.

3-((Phenylmethylene)amino)-2-thioxo-4-imidazolidinone
(3) was synthesized from benzaldehyde in a reaction with thiosemicarbazide (Scheme 1). Nucleophilic addition of thiosemicarbazide to benzaldehyde (1) yield thiosemicarbazone (2). Thiosemicarbazone (2) then undergoes a cyclocondenzation reaction with ethyl chloroacetate in the presence of anhydrous sodium acetate, forming the arylidene 2-thiohydantoin derivative, 3-    One more thing that implies coordination is the absence of the broad singlet of the 2-thiohydantoion ring NH proton. 2-Thiohydantoins are known to exist in two tautomeric forms in equilibrium (Scheme 2). 29 It is proposed that the thio-enol tautomeric form (4) is responsible for coordination and furthermore, that this 'ketoenol' equilibrium shifts to the thio-enol form during the reaction. 27 The SH protons of the thio-enol tautomer can be seen in the spectra as a singlet at 1.85 ppm ( during the course of the experiment (see Fig. S-4). The reaction as a whole is proposed to proceed in two steps. In the first step (Equation 2), initial coordination takes place through the nitrogen in the side chain. This is the faster reaction step, which is supported by the presence of the signals of the complex (a, b and c) in the first spectrum of the experiment, that then do not change in intensity during the experiment. The second, slower reaction step is deprotonation of the 2-thiohydantoin ring and coordination through the sulfur atom in the ring (Equation 3). The resulting complex (5)  A c c e p t e d m a n u s c r i p t

Fig. 2. Changes in product concentration, cis-[Pd(3-N,S)(dmso-S)2] + , during the substitution reaction of cis-[PdCl2(dmso-S)2] with 2-thiohydantoin derivative 3 in DMSO-d6.
It can be seen in the plot that after about an hour into the experiment, complex formation slows down drastically. If we take into consideration the stoichiometry of the system, at the end of the experiment, more than half of the initial amount of the 2-thiohydantoin derivative 3 has not undergone any sort of reaction, implying that the reaction system is a bit more complex and that cis-[PdCl2(dmso-S)2] undergoes multiple competing reactions.
In order to gain a deeper insight into the details of the mechanism of the reaction, a plot of the logarithm of the product concentration vs experiment time was analyzed (Fig. 3). In the plot, it is clearly visible that this is not a linear firstorder reaction, but instead, two linear slopes can be observed. This goes along with the conclusion that multiple processes are occurring, not just the reaction of cis-   There is a slight deviation from the ideal square-planar structure in cis-[Pd(dmso-O)2(dmso-S)2] 2+ , the biggest of which being the angle between the two sulfur bonded DMSO molecules, due to steric repulsions between the methyl groups of one DMSO and the sulfoxy group of the other. These steric repulsions prohibit the S-bonding of the other DMSO molecules, which is believed to be the main reason for coordination through oxygen. 30 Two All the same signals with identical chemical shifts can be observed in the spectra of the reaction (Fig. 4). Pairs of signals of the coordinated and uncoordinated 3, among which are singlets of the 2-thiohydantoin ring CH2 group protons (a), multiplets of the aromatic benzene ring protons (b) and singlets of the double bond CH proton (c), can be seen at the same chemical shift in the spectra. Thio-enol tautomer -SH proton is at 1.85 ppm, the broad singlet of the 2thiohydantoin ring -NH proton is missing and the HCl singlet at 10.15 ppm increases throughout the experiment.  ) from the spectral data, an obvious difference in reaction rates was observed and it was noticed that the reaction with PdCl2 is slower than with cis-[PdCl2(dmso-S)2]. Changes in complex 5 concentrations over the course of the experiment are shown in Fig. 5. The difference in the kinetics of the systems are somewhat perplexing, as spectral data confirms that the same reaction product is formed in both cases. In order to get to the bottom of this, a plot of the logarithm of the product concentration vs experiment time was analyzed ( Figure 6). As with cis-[PdCl2(dmso-S)2], in this case there are also two phases, with two linear slopes that intercept after little over an hour. The first phase can be described with the equation y = (1.80±0.22)•10 -4 x -6.59, while the second phase can be described with the equation y = (1.25±0.09)•10 -5 x -5.77. The first phase, where most of the complex is formed, has a coefficient k1 = 1.80•10 -4 s -1 , which is very close to the slope coefficient of the first phase of the reaction of cis-[PdCl2(dmso-S)2] (k1 = 1.63•10 -4 s -1 ). It is known that PdCl2 has great affinity towards DMSO and reacts with it to form cis-[PdCl2(dmso-S)2], 32    In the case of the third examined palladium(II) salt, K2[PdCl4], there was no reaction with 3-((phenylmethylene)amino)-2-thioxo-4-imidazolidinone (3) during the course of the experiment. No signals of a newly formed 2-thiohydantoin complex species of any kind could be observed ( Figure S-4). The four chlorido ligands in K2[PdCl4] are kinetically equivalent and a strong possibility is that all of them were substituted with DMSO, as tetrachloroplatinate(II) and also tetrachrloropalladate(II) can react with DMSO in this manner. 33 This would prohibit the reaction with the 2-thiohydantoin derivative 3.

Reactions
of an arylidene 2-thiohydantoin derivative, 3-((phenylmethylene)amino)-2-thioxo-4-imidazolidinone (3) with PdCl2, cis-[PdCl2(dmso-S)2] and K2[PdCl4] in DMSO-d6 were monitored in a time dependent kinetic 1 H NMR experiment. In the cases of PdCl2 and cis-[PdCl2(dmso-S)2], the complex cis-[Pd(3-N,S)(dmso-S)2] + (5) was formed, with palladium(II) coordinated through the nitrogen in the side chain and the 2-thiohydantoin ring sulfur atom. The mechanism of complex 5 formation consists of two steps. The first step is fast monodentate coordination of 3 via its nitrogen atom in the side chain. This step is too fast for the NMR time-scale, but it is confirmed with the corresponding signals of the complex 5 that are unchanged during the course of the experiment. The second, rate determining step of the reaction is chelation of