Highly Cytotoxic Palladium(II) Pincer Complexes Based on

Article pubs.acs.org/IC

Highly Cytotoxic Palladium(II) Pincer Complexes Based on Picolinylamides Functionalized with Amino Acids Bearing Ancillary S‑Donor Groups Svetlana G. Churusova,† Diana V. Aleksanyan,*,† Ekaterina Yu. Rybalkina,‡ Olga Yu. Susova,‡ Valentina V. Brunova,† Rinat R. Aysin,† Yulia V. Nelyubina,† Alexander S. Peregudov,† Evgenii I. Gutsul,† Zinaida S. Klemenkova,† and Vladimir A. Kozlov† †

A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Ulitsa Vavilova 28, Moscow 119991, Russia ‡ Institute of Carcinogenesis, N. N. Blokhin Russian Cancer Research Center, Kashirskoe Shosse 24, Moscow 115478, Russia S Supporting Information *

ABSTRACT: The reactions of picolinyl and 4-chloropicolinyl chlorides with methyl esters of S-methyl-L-cysteine, L- and Dmethionine, and L-histidine afforded a series of functionalized carboxamides, which readily formed pincer-type complexes upon interaction with PdCl2(NCPh)2 in solution under mild conditions. The direct cyclopalladation of the ligands derived was also accomplished in the solid phase, in particular, mechanochemically, although it was complicated by the partial deactivation of the starting amides. The resulting complexes with 5,5- and 5,6membered fused metallocycles were fully characterized by IR and NMR spectroscopy, including variable-temperature and 2D-NMR studies. In the case of some cysteine- and methionine-based derivatives, the realization of κ3-N,N,S-coordination was supported by X-ray diffraction. The cytotoxic effects of these complexes were examined on HCT116, MCF7, and PC3 human cancer cell lines as well as HEK293 as a representative of normal cells. The comparative studies allowed us to determine that the presence of the sulfide ancillary donor group is crucial for cytotoxic activity of this type of Pd(II) complexes. The main structure−activity relationships and the most promising palladocycles were outlined. The additional studies by gel electrophoresis revealed that 4-chloropicolinyl derivatives, despite the nature of an amino acid, can bind with DNA and inhibit topoisomerase I activity.

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INTRODUCTION The discovery of anticancer activity of cis-[PtCl2(NH3)2] provoked an enormous interest of researchers in development of platinum(II)-based chemotherapeutic agents.1 Nowadays, a range of Pt(II) complexes are successfully used in clinical practice for treatment of various cancers (above-mentioned cisplatin and its analogs−carboplatin, oxaliplatin, nedaplatin, lobaplatin, and heptaplatin).2 However, because of the intrinsic and acquired resistance as well as high toxicity of platinumbased drugs, leading to severe side effects,2c there is a constant need for creation of more effective and selective anticancer agents. Many investigations have been focused on nonplatinum transition metal complexes, including gold, ruthenium, and titanium derivatives.3−5 At the same time, despite obvious similarities in structure and coordination behavior between Pt(II) and Pd(II) complexes, the potential of palladium(II)based compounds as anticancer agents seems to be underappreciated.2c,5c,6 The main obstacle consists in the essentially higher reactivity of Pd(II) complexes compared to their Pt(II) analogs, which often leads to deactivation of palladium(II) drug candidates in biological environment (due to hydrolysis and ligand-exchange processes). Nevertheless, a series of © 2017 American Chemical Society

palladium(II) complexes with sterically hindered N-donor ligands were found to possess pronounced cytotoxic properties.7 Even more promising results were demonstrated by application of chelating bidentate ligands, especially those of hemilabile nature.8 The introduction of an additional ancillary donor group into the ligand framework, i.e., transition from bito multidentate ligand systems, offering ample opportunities for directed structural modifications, can provide the resulting palladium complexes with even higher thermodynamic and controlled kinetic stability. Although these advantages of palladocycles based on multidentate ligands has been successfully used in catalysis and organic synthesis for several decades,9 in the field of palladium(II)-based anticancer drug design this is a relatively new and unexplored strategy. However, already known cyclopalladated complexes featuring functionalized thiosemicarbazone,10 diimine,11 terpyridine,12 and bis(2-pyridylmethyl)amine12b,13 ligands and single examples of related derivatives with other skeletons,14−17 including 6-phenyl-2,2′-bipyridine complexes with auxiliary NHC ligands Received: May 26, 2017 Published: August 3, 2017 9834

DOI: 10.1021/acs.inorgchem.7b01348 Inorg. Chem. 2017, 56, 9834−9850

Article

Inorganic Chemistry

Figure 1. Cytotoxic palladium(II) complexes based on multidentate ligands (sac−saccharinate).

Scheme 1. Synthesis of Multidentate Ligands Based on Picolinylamides Functionalized with Amino Acid Esters

possessing high in vivo anticancer activity,18 show a lot of promise (Figure 1). Within the project devoted to pincer complexes having a specific tridentate monoanionic framework, the importance of which expands from catalytic applications to materials science,9,19,20 recently we have shown that functionalized carboxamides with ancillary donor groups both in the amine and acid parts can act as pincer-type ligands upon coordination with the Pd(II) ions.21 The resulting N-metalated palladocycles proved to be efficient and readily tunable catalysts for the Suzuki cross-coupling. Following our interest in such nonclassical pincer complexes based on readily available and metalated ligands with a secondary amide central unit, it seemed interesting to develop related systems potentially attractive from the viewpoint of biological activity of their palladium(II) complexes. Taking into account the high affinity of the soft Pd(II) ions to sulfur and biological relevance of Sdonor amino acids, enantiomerically pure S-methyl-L-cysteine and L-methionine were chosen as the main amino components. Interactions of Pt(II) anticancer agents and their Pd(II) analogs with sulfur-containing amino acids have been the focus of many

researchers, because they have been shown to play an important role in biological activity of these complexes.22 Furthermore, the combination of transition metals with naturally occurring compounds is often exploited in medicinal chemistry, for example, to achieve the desired binding modes with different biological targets.23 Note that the strong binding of potential anticancer Pd(II) complexes with sulfur-containing biomolecules, e.g., glutathione can reduce their bioavailability.18 In this respect, the introduction of a sulfur ancillary donor group into the ligand framework can have positive effect on the cytotoxic activity of the resulting complexes. For comparison, Lhistidine featuring N-donor imidazole moiety was also used as an amino component. In turn, pyridine-2-carboxylic (picolinic) acids were preferred as acid components in order to acquire hemilabile ligation of the metal ions in cysteine- and methionine-based derivatives, especially in view of the strong trans-labilizing effect of sulfur donors. Herein, we report on the synthesis and characterization of novel pincer-type palladium(II) complexes derived from Npicolinylamides functionalized with S-methyl-cysteine, methionine and histidine in enantiomerically pure forms. The results 9835


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Inorganic Chemistry

All the sulfide ligands obtained readily underwent direct cyclometalation upon interaction with PdCl2(NCPh)2 in dichloromethane solution at room temperature in the presence of triethylamine, leading to 5,5- and 5,6-membered pincer complexes 7−12 in high yields (Scheme 3). Note that the same palladocycles were formed in the absence of Et3N, but the yields of the isolated products in these cases were somewhat lower, which was associated with an adverse effect of HCl liberated upon metalation. Therefore, triethylamine was used to trap HCl. Unfortunately, we failed to isolate palladium(II) complex (R)-8o based on L-cysteine bearing the sulfoxide ancillary donor group. This compound appeared to be unstable and gradually decomposed upon storage and isolation. Presumably, this is caused by the highly constrained character of the complex structure, arising from the presence of two fused metallocycles leading to severe distortion of the square-planar geometry around the metal ion. However, its counterpart (S)10o with the elongated S-coordination arm was readily isolated and characterized.

on cytotoxicity studies against several human cancer cell lines and the main structure−activity relationships are discussed.

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RESULTS AND DISCUSSION Synthesis in Solution and Spectroscopic Characterization of Cyclopalladated Complexes. The target functionalized carboxamides were obtained by reactions of methyl esters of S-methyl-L-cysteine, L-methionine and Lhistidine, in situ generated from the corresponding hydrochlorides, with picolinyl chloride in dichloromethane at −5 °C in the presence of Et3N (Scheme 1). Note that the latter actually represented a mixture of picolinyl chloride and 4chloropicolinyl chloride, since, unlike the literature data,21c,24 in our hands the reaction of picolinic acid with an excess of SOCl2 under reflux was accompanied by the chlorination of the pyridine ring. The ratio of 4-chloropicolinyl and picolinyl amides in crude products was ∼2:1. Nevertheless, in each case, the resulting pair of amides of picolinic and 4-chloropicolinic acids were readily separated by column chromatography on silica gel, so that all the ligands were isolated in the individual forms as crystalline solids. In general 4-chloropicolinylamides ((R)-2, (S)-4, (S)-6) featured lower adsorption capacity than the unsubstituted analogs ((R)-1, (S)-3, (S)-5). To compare the cytotoxic properties of palladocycles derived from different enantiomers of amino acids, we synthesized the ligands analogous to (S)-3 and (S)-4 starting from Dmethionine methyl ester hydrochloride (compounds (R)-3 and (R)-4, respectively, Scheme 1). Taking into account an important role of such weak donor ligands as sulfoxides in transition metal chemistry and different biochemical processes,25 it seemed interesting to obtain the corresponding sulfoxide analogs of the above-mentioned ligands. Thus, oxidation of (R)-2 and (S)-4 with H2O2 in t BuOH smoothly afforded the desired sulfoxide derivatives in high yields (Scheme 2). It should be emphasized that the

Complexes 7−12, 10o are moisture- and air-resistant brightly colored crystalline solids. Their structures were unambiguously confirmed based on the IR and NMR spectroscopic data. The IR spectra of all the palladocycles obtained lack the absorption bands corresponding to the stretching and bending vibrations associated with the NH moiety of the amide group. Similarly, their 1H NMR spectra did not reveal the downfield signals of the amide NH protons, indicating the occurrence of metalation. A strong low-frequency displacement of the amide CO stretching vibrations compared to the IR spectra of the free ligands (Δν = 24−53 cm−1) is also characteristic of Nmetalated pincer complexes derived from functionalized carboxamides.21 The realization of S-coordination by the palladium(II) ion in palladocycles 7−10 and (S)-10o is supported by the downfield shifts of the carbon and proton signals corresponding to SMe or S(O)Me groups both in the 1 H (ΔδH = 0.43−0.92 ppm) and 13C (ΔδC = 4.79−8.70 ppm) NMR spectra. The coordination of the imidazole moiety in complexes (S)-11 and (S)-12 is reflected in strong downfield shifts of the NH proton signal and the signal of H(C8) proton between two nitrogen atoms (ΔδH ∼ 4.0 and 0.7 ppm, respectively). An upfield shift of the resonance of tertiary carbon nuclei adjacent to the nitrogen atom of the pyridine moiety (ΔδC5 = 0.31−1.10 ppm), observed in the 13C NMR spectra of most of the complexes obtained, serves as indirect evidence for the heterocycle complexation. Interestingly, the 1H and 13C NMR spectra of N,N,Scomplexes 7−10 in CDCl3 at room temperature show a double set of signals, which correspond to two different isomeric forms. The ratio of the isomers composes approximately 3:2. For 5,5membered palladocycles (R)-7 and (R)-8 based on L-cysteine derivatives, the signals of the major and minor isomers can be readily assigned, while their analogs with the elongated Scoordination arm−complexes (S)-9, (S)-10 and (R)-9, (R)-10,

Scheme 2. Synthesis of Sulfoxide Ligands

products of further oxidation, namely, sulfones were not detected in the reaction mixtures. Note that ligands (R)-2o and (S)-4o actually represent mixtures of two diastereomers, which differ in the configuration of the sulfur atom relative to the chiral carbon center. The diastereomeric ratio for ligand (R)-2o composes 68:32. In the case of its methionine-based counterpart, the diastereomeric ratio cannot be accurately defined due to considerable overlapping of the signals in the NMR spectra. The structures and compositions of the resulting functionalized picolinylamides were supported by the IR and NMR spectroscopic data as well as elemental analyses (see Experimental Section). 9836


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Inorganic Chemistry Scheme 3. Cyclopalladation of Picolinylamide Ligands

featuring more conformationally flexible structures, undergo dynamic transformations at room temperature, which result in the broadened and unresolved signals of the aliphatic carbon and hydrogen nuclei. The variable-temperature NMR spectral studies in CDCl3 showed that the resonances of two isomers collapse at 28−30 °C (see, for example, Figure S1). In contrast, cooling to −30 °C resulted in the resolution of all the signals, including those of both methylene (CH2 and CH2S) units. 2DNMR spectral studies in CDCl3 at −30 °C (COSY, HSQC, and HMBC experiments) allowed us to make the unequivocal assignment of the resonances of two isomeric forms in each case of methionine-based derivatives (complexes 9 and 10, see Figures S2−S11). As for the nature of these isomeric forms, one should note that the coordination of the sulfide group by the Pd(II) ion can lead to configurational stability of the sulfur center. The complicated inversion between the coordinated and noncoordinated electron pairs of the sulfur atom along with the presence of the chiral carbon center in one definite absolute configuration can result in the existence of two diastereomers. Investigation of complex (R)-8 by the ROESY technique confirmed that the major and minor isomers differ in the disposition of the methyl substituent at the sulfur atom and the substituents at the chiral carbon center relative to the main molecular plane (Figure S12). In complex (S)-10o, such an inversion of the sulfur atom configuration is impossible due to the presence of the S−O bond, and its diastereomers are significantly more stable. Thus, the ratio of the isomers of (S)10o almost does not depend on the solvent nature (∼3:2 in CDCl3 and CD3CN and 4:3 in C6D6) and remains unchanged upon heating to 60 °C in C6D6. Therefore, they were readily separated by thin-layer chromatography and fully characterized individually. X-ray Structural Description. The structures of complexes (R)-7, (S)-9, (R)-9, (S)-10, (R)-10 and (S)-10o were additionally confirmed by X-ray diffraction (Figures 2a−f). According to the results obtained, all the palladocycles with the sulfide ancillary donor group crystallize in a noncentrosymmetric space group P21 and have two symmetry-independent molecules with opposite location of the methyl group at the sulfur atom relative to Pd(1)N(1)N(2)Cl(1)S(1) mean plane, which answer to two different diastereomeric forms; the relevant torsion angles (CMeS(1)Pd(1)N(1)) range from − 104.2 to −90.6° and from 105.8 to 120.4°, respectively. In the crystal of the major isomer of (S)-10o, four independent complex molecules, as was expected for a single diastereomer, have one and the same arrangement of the substituents at the sulfur donor atom relative to Pd(1)N(1)N(2)Cl(1)S(1) mean plane. The corresponding torsion angle (CMeS(1)Pd(1)N(1))

composes 123.3−126.5°. Unfortunately we failed to grow single crystals of the minor isomer of this palladocycle. The conformation of a five-membered metal-containing ring formed by the pyridine moiety in all the complexes explored represents a flattened envelope with the N(1) atom deviating by 0.08−0.26 Å. The second five-membered ring in (R)-7 also adopts an envelope conformation with the C(10) atom deviating by 0.62 Å (or 0.53 Å in the second independent molecule of this complex). A six-membered metallocycle in (S)9, (R)-9, (R)-10, (S)-10, and (S)-10o is either in a half-chair conformation (all independent molecules in (S)-10o and one in complexes 9 and 10) with the deviation of the C(10) and C(11) atoms by 0.40−0.64 Å and from −0.55 to −0.18 Å, respectively, or in an envelope conformation (the second independent molecule in 9 and 10) with the deviation of the C(10) atom by 0.72−0.81 Å. The environment of the palladium atom varies from square-planar (all independent molecules in (R)-7 and (S)-10o and one in (S)-10 and (R)-10) to flattened tetrahedral with a folding along S(1)···N(2) line (all independent molecules in (S)-9 and (R)-9 and the second one in (S)-10 and (R)-10); the corresponding angles (N(1)Pd(1)Cl(1)), dihedral angles between Pd(1)N(1)S(1)N(2) and Pd(1)Cl(1)S(1)N(2) planes, and deviations of the chlorine atoms are as follows: 169.9(2)−174.73(18)°, 3.4− 12.9°, and 0.14−0.52 Å, respectively. The main bond lengths and angles of the complexes explored are given in Table S1. Solid-Phase Cyclopalladation. Recently, we have shown that solid-phase cyclopalladation can serve as an efficient and green alternative to the conventional synthesis of organometallic pincer complexes in solution.26 Simple heating of homogenized mixtures of a ligand and appropriate Pd(II) precursor, obtained by manual grinding of the reactants in a mortar, in the absence of a solvent readily afforded several series of C-metalated pincer complexes having ancillary S- and N-donor groups. In the case of the most active ligands, the formation of pincer complexes can be accomplished even at room temperature.26b More importantly, the synthesis of an organometallic palladium(II) pincer complex was realized for the first time via C−H bond activation of the bis(thiocarbamate) ligand with PdCl2(NCPh)2 under mechanochemical conditions both by grinding of the reactants in a mortar and in a vibration ball mill at gram scale.26a In continuation of these studies, it seemed interesting to estimate whether the solid-phase approach can be applied to the systems requiring the activation of the N−H bond. Indeed, heating of a homogenized mixture of L-histidine-based ligand (S)-6 and PdCl2(NCPh)2 at 85−90 °C for 10 min gave rise to palladocycle (S)-12. The cyclopalladation of more reactive 9837


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Figure 2. General views of complexes (a) (R)-7, (b) (S)-9, (c) (R)-9, (d) (S)-10, (e) (R)-10, and (f) (S)-10o in representation of non-hydrogen atoms via thermal ellipsoids at 50% probability level. Only one symmetry-independent molecule of the complex is shown in each case.

Figure 3. Monitoring of a reaction of ligand (R)-2 with PdCl2(NCPh)2 in a mortar: a mixture of (R)-2 and Pd(II) precursor (a) before grinding, and after grinding for (b) 0.5, (c) 1, and (d) 2 min.

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Inorganic Chemistry Scheme 4. Solid-Phase Cyclopalladation of Picolinylamide Ligands (LH, ligand)

Table 1. Cytotoxicity of the Pd(II) Complexes Obtained against Human Cell Lines HCT116, MCF7, PC3, and HEK293 (IC50/ μM) cancer cell lines

normal cell line

entry

compound

HCT116

MCF7

PC3

HEK293

1 2 3 4 5 6 7

(R)-7 (R)-8 (S)-9 (S)-10 (R)-9 (R)-10 Cisplatin

1.8 ± 0.2 1.2 ± 0.4 6.2 ± 0.5 0.45 ± 0.05 7.0 ± 0.2 4.4 ± 0.8 400.0

22.0 ± 1.4 10.0 ± 1.5 15.5 ± 2.5 5.5 ± 0.4 15.0 ± 0.5 11.7 ± 0.3 25.0

>30 16.5 ± 2.0 13.5 ± 0.5 4.2 ± 0.6 8.5 ± 2.5 4.5 ± 0.5 120.0

18.0 ± 3.2 8.0 ± 2.0 16.5 ± 3.5 4.0 ± 2.0 16.2 ± 1.6 11.0 ± 0.5 28.0

(in this case, both pyridine and histidine moieties of the free ligand can bind with HCl). The latter slowly converts to complex (S)-12 in solution (Figures S26 and S27). Scheme 4 outlines the results of the solid-phase experiments. Taking into account the ease of formation of the complexes under consideration in solution even in the absence of a base, we tried to enhance the efficiency of mechanochemical synthesis via performing the reaction of (S)-4 with PdCl2(NCPh)2 in a vibration ball mill of simple construction (Narva DDR GM 9458 vibration ball mill, stainless jar, two grinding balls, 30 W, 50 Hz) (Figure S28). Unfortunately, in this case, the formation of palladocycle (S)-10 was again accompanied by the generation of (S)-4·HCl (Figure S29). Nevertheless, the results obtained show a principal possibility of synthesis of N-metalated Pd(II) pincer complexes by the solidphase methodology. Presumably, limitations connected with the partial deactivation of the ligands explored can be overcome by high-energy ball milling. Cytotoxicity Studies. To estimate the potential of palladocycles 7−12 and (S)-10o as anticancer agents, their cytotoxicity against three human cancer cell lines (human colon cancer HCT116, human breast cancer MCF7, and human prostate cancer PC3) was measured by the colorimetric MTT assay. For comparison, cisplatin from a commercial source was tested under the same experimental conditions. The results are summarized in Table 1. The activity is presented as the concentration required for reduction of cell survival by 50% after 48 h of exposure to the compounds under investigation (IC50). As can be seen from Table 1, almost all the κ3-N,N,Spalladocycles with the sulfide ancillary donor group demon-

ligands (R)-2, (S)-4, and (R)-4 based on sulfur-containing amino acids was accomplished already upon grinding of the reactants in a mortar (Figure 3). In all cases, the IR and Raman spectra of the solid residues did not reveal the absorption bands and lines of free ligands and, at the same time, clearly demonstrated the signals characteristic for the target pincer complexes (see representative IR and Raman spectra in Supporting Information for one series of methionine derivatives (Figures S13−S19) and histidine derivatives (Figures S20−S22)). However, careful inspection of the shape and position of some of the bands and lines along with the additional signals (not typical for the complexes) allowed us to suppose the presence of at least one byproduct. Note that unlike other pincer systems,26 for the ligands under consideration, the liberation of hydrogen chloride (as a result of cyclometalation) was not detected with a paper indicator. This allowed us to assume that liberated HCl could be trapped by the unreacted ligand to form hydrochloride salts. Furthermore, the IR and Raman spectra of the samples rinsed with hexane and Et2O still revealed CN stretching vibrations (Figure S16), which may arise from partially unreacted PdCl2(NCPh)2, because residual benzonitrile should be removed after such a purification procedure. The 1H NMR spectra of the solutions of solid residues obtained after reactions with the ligands bearing sulfur-donor ancillary group, registered directly after dissolution, contained mainly the signals of the pincer complexes and PhCN (see, for example, Figure S23). However, for less reactive histidinederivative (S)-6, the analogous 1H NMR spectrum registered immediately after dissolution (Figures S24 and S25), confirmed the presence of a hydrochloride salt of (S)-6·2HCl formulation 9839


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Using complex (S)-10 as a representative example, it was shown that this type of cyclopalladated derivatives is stable not only in neat DMSO, but also in DMSO−water or DMSO−PBS solutions (see UV−vis spectra in Figures S30 and S31). Thus, the UV−vis spectrum of (S)-10 in DMSO at room temperature shows broad overlapping bands, which refer mainly to MLCT transitions involving metal-based orbitals. In DMSO−water and DMSO−PBS solutions, these bands display hypsochromic shift with increasing medium polarity. This implies the retention of a pincer structure in solution. The only possible change is the displacement of the auxiliary chloride ligand by DMSO in the inner coordination sphere of the metal ion, which is likely to be responsible for the observed increase in the intensity in DMSO−water and DMSO−PBS solutions. However, there is no evidence for the decomposition of a pincer structure over a period of 120 h. A wide range of current chemotherapeutic agents, including cisplatin and its analogs, target DNA either through direct interaction or prevention of DNA relaxation by inhibition of topoisomerase activity. To estimate the possibility of interaction of the pincer palladium(II) complexes under investigation with DNA, we have studied their effect on electrophoretic mobility of supercoiled plasmid DNA. The electrophoretic mobility patterns of the supercoiled form of pHOT1 DNA (TopoGEN), incubated with different amounts of complexes (R)-8, (S)-9, (S)-10, and (S)-12, are shown in Figure 4. A decrease in the DNA mobility with increasing

strated high cytotoxic activity against the explored cancer cell lines, with IC50 values falling in the low micromolar range. Notably, all these complexes were found to be much more active than the reference−cisplatin. Unlike its unoxidized analog, sulfoxide-based complex (S)-10o (both isomers were tested individually) did not afford 50% growth inhibition even at the concentration of 30 μM. Furthermore, in the case of κ3N,N,N-palladocycle (S)-11 with the histidine-derived ligand, only 20−30% growth inhibition was achieved at the concentration of 200 μM. Its counterpart with the chlorine substituent in the pyridine ring (complex (S)-12) even facilitated the growth of cancer cells at the concentration of 80 μM. Therefore, the presence of the sulfide ancillary donor group appears to be crucial for the cytotoxic activity of this type of palladium(II) complexes. In general, complexes 7−10 exhibited better cytotoxicity against HCT116 cancer cell line than the others, which clearly indicates that they are more specific on this particular cancer lineage. An extraordinary high cytotoxic effect on HCT116 cancer cells was observed for palladocycle (S)-10 based on Lmethionine-derived carboxamide ligand (with IC50 value as low as 0.45 ± 0.05 μM). Furthermore, this complex essentially outperformed its analog based on D-methionine, although in the other cases there were no significant differences in the activities between L- and D-methionine derivatives (complexes (S)-9 and (S)-10 (entries 3, 4) vs complexes (R)-9 and (R)-10 (entries, 5, 6)). The comparison of cytotoxic properties of 5,5- and 5,6membered sulfide-based palladium complexes shows that a reduction in the size of one of the fused metallocycles (i.e., transition from methionine to cysteine-based derivatives) leads to a drop in the activity against MCF7 and PC3 cancer cell lines. However, toward HCT116 cancer cells, cysteine-based complexes (R)-7 and (R)-8 still demonstrate the high level of activity (entries 1, 2). Interestingly, unlike histidine derivatives, the introduction of the chlorine substituent into the pyridine ring facilitates the improvement of cytotoxic activity of N,N,Scomplexes (entries 2 vs 1, 4 vs 3, and 6 vs 5). Note that the positive effect of nonreactive chlorine substituents in aromatic or heteroaromatic moieties of some molecules on their intrinsic biological activity has been already reported in the literature and is often connected with an increase in the molecule lipophilicity and possibility of nonbonding interaction of the chlorine atom in the binding site.27 Taking into account that most of the drugs used for cancer treatment are not cancer cell specific and are potentially toxic also against normal cells, the cytotoxicity of palladocycles 7−10 was also evaluated against human embryonic kidney cells (HEK293) as a representative of normal cell lines. The results obtained suggest that in some cases the complexes under study are less toxic to normal cell line than to the assayed cancer cell lineages (for example, the selectivity indices toward HCT116 cancer cells reach up to 10.00), although in most cases similar levels of sensitivity were observed (see Table S2). Finally, it seemed reasonable to estimate the cytotoxic activity of the free ligands against the same cell lines. The experiments showed that the starting functionalized carboxamides exhibited static rather than toxic effect, because at the concentration of 30 μM, they resulted in a reduction of cell growth no more than by 20%. Therefore, the observed cytotoxic properties of the palladocycles under investigation are mainly determined by coordination with the palladium ions.

Figure 4. Gel electrophoresis diagrams showing the effect of complexes (R)-8, (S)-9, (S)-10, and (S)-12 on supercoiled pHOT1 DNA.

concentration of the complexes is observed for all the compounds explored, which clearly indicates their binding with DNA. At the same time, methionine-based derivative (S)9 is likely to form stronger complexes, because it uncoils plasmid DNA already at the concentration of 10 μM, whereas the other complexes cause its retardation only at the concentration of 20 μM. An essential role in the processes of DNA replication, transcription, recombination, and repair is played by nuclear enzymes−topoisomerases. They form potentially toxic singleor double-strand DNA breaks (depending on a particular type of topoisomerases) and, therefore, can also serve as targets for anticancer agents and antibiotics. Owing to the capability of changing the topological state of DNA, they readily transform supercoiled plasmid DNA into a population of relaxed products (topoisomers), which can be distinguished by agarose gel electrophoresis. Figure 5 demonstrates the effect of complexes 9840


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Inorganic Chemistry

Figure 5. Gel electrophoresis diagrams showing the effect of complexes (R)-8, (S)-9, (S)-10, and (S)-12 on human DNA topoisomerase I. NMR spectra of complexes (S)-9, (S)-10, (R)-9, and (R)-10 was carried out based on 1H, 13C{1H}, 1H−1H−COSY, HMQC, and HMBC experiments. The results obtained were used for interpretation of the NMR spectra of the other compounds. Palladocycle (R)-8 was additionally studied by the ROESY technique. UV−vis spectra were registered on a Cary50 spectrometer in CaF2 cells with 0.4 mm path length. IR spectra were recorded on a Nicolet Magna-IR750 FTspectrometer, resolution 2 cm−1, 128 scans. The assignment of absorption bands in the IR spectra was made according to ref.32 Column chromatography was carried out using Macherey-Nagel silica gel 60 (MN Kieselgel 60, 70−230 mesh). Melting points were determined with an MPA 120 EZ-Melt automated melting point apparatus (Stanford Research Systems). Syntheses. General Procedure for the Synthesis of Ligands 1−6. A mixture of pyridine-2-carboxylic acid (0.46 g, 3.74 mmol) and SOCl2 (16 mL) was refluxed for 2 h. After cooling to room temperature, all the volatiles were removed under reduced pressure. The resulting residue, representing a mixture of pyridine- and 4-chloropyridine-2carbonyl chlorides in ∼2:1 ratio,24 was suspended in 15 mL of CH2Cl2 and added dropwise to a suspension of the amino acid methyl ester in situ generated from the corresponding hydrochloride (3.74 mmol) and Et3N (9.32 mmol in the case of the cysteine and methionine derivatives or 13.50 mmol in the case of the histidine derivative) in 10 mL of CH2Cl2 at −5 °C. The reaction mixture was stirred overnight and poured into 50 mL of distilled water. The organic layer was separated, washed with saturated aqueous solution of NaHCO3, dried over anhydrous Na2SO4, and evaporated to dryness. The residue obtained was purified by column chromatography on silica gel (gradient elution using neat CH2Cl2 and CH2Cl2/MeOH mixtures (from 100:1 to 75:1)) to give the desired ligands as white (1−5) or light-beige (6) crystalline solids. Methyl (2R)-3-(methylsulfanyl)-2-[(pyridin-2-ylcarbonyl)amino]propanoate, (R)-1. Yield: 0.16 g (17%). Mp: 50−52 °C (hexane).

(R)-8, (S)-9, (S)-10, and (S)-12 on inhibition of activity of human DNA topoisomerase I. All the tested palladocycles inhibited significantly DNA relaxation in a concentrationdependent manner, with complex (S)-10 being slightly less effective than the others. However, it should be taken into account that the DNA relaxation experiment is very sensitive to DNA intercalators, which can counteract the activity of topoisomerase I by unwinding DNA. Furthermore, there are no substantial differences in the DNA binding and topoisomerase I inhibition abilities between N,N,S- and N,N,N-complexes, although their cytotoxic properties strongly differ. In addition, according to the results of preliminary staining assays using fluorescence microscopic analysis, the cytotoxic complexes under consideration (compound (S)-10 as a representative example) do not induce significant levels of apoptosis in HCT116 cancer cells. Therefore, further detailed investigations are required to elucidate the mechanism of cytotoxic action of palladium(II) pincer complexes based on picolinylamides functionalized with amino acids.

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EXPERIMENTAL SECTION

General Remarks. Unless otherwise noted, all manipulations were carried out without taking precautions to exclude air and moisture. Dichloromethane was distilled from P2O5. Methionine methyl ester hydrochlorides (L - and D -isomers) were synthesized by the esterification of the corresponding amino acids under action of dimethylsulfite, in situ generated by the reaction of SOCl2 with an excess of methanol at low temperature.28 L-S-Methylcysteine methyl ester hydrochloride was obtained from L-cysteine methyl ester hydrochloride via sequential treatment with di-tert-butyl dicarbonate,29 methyl iodide,30 and acetyl chloride.31 A solution of hydrogen peroxide in tBuOH was obtained from 30% aq. H2O2 and tBuOH. The concentration of H2O2 was determined by iodine/thiosulfate titration. All other chemicals, including L-histidine methyl ester dihydrochloride, and solvents were used as purchased. NMR spectra were recorded on Bruker AV-300, AV-400 and AV600 spectrometers, and the chemical shifts (δ) were referenced internally by the residual solvent signals relative to tetramethylsilane. In most cases, 13C{1H} NMR spectra were registered using the JMODECHO mode; the signals for the C nuclei bearing odd and even numbers of protons have opposite polarities. The assignment of the

[α]D25 +2.4 (c = 0.7, CHCl3). 1H NMR (400.13 MHz, CDCl3): δ 2.18 (s, 3H, SMe), 3.06−3.16 (m, 2H, CH2), 3.83 (s, 3H, OMe), 5.03−5.08 (m, 1H, CH), 7.48 (ddd, 1H, H(C4), 3JHH = 7.7 Hz, 3JHH = 4.8 Hz, 4 JHH = 1.2 Hz), 7.88 (dt, 1H, H(C3), 3JHH = 7.7 Hz, 4JHH = 1.6 Hz), 9841


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Inorganic Chemistry 8.20 (d, 1H, H(C2), 3JHH = 7.7 Hz), 8.63 (d, 1H, H(C5), 3JHH = 4.8 Hz), 8.77 (br. d, 1H, NH, 3JHH = 7.6 Hz) ppm. 13C{1H} NMR (75.47 MHz, CDCl3): δ 16.07 (s, SMe), 36.33 (s, CH2S), 51.69 (s, OMe), 52.49 (s, CH), 122.13 and 126.31 (both s, C2 and C4), 137.13 (s, C3), 148.20 (s, C5), 148.95 (s, C1), 163.97 (s, C(O)NH), 170.99 (s, C(O)OMe) ppm. IR (KBr, ν/cm−1): 504(w), 558(w), 619(m), 700(w), 759(m), 824(w), 844(w), 859(w), 942(w), 997(m), 1023(w), 1086(w), 1106(w), 1213(m), 1236(w), 1271(w), 1291(w), 1307(w), 1326(w), 1355(m), 1410(w), 1438(m), 1463(m), 1514(s) (C(O)NH), 1570(w), 1589(w), 1682(s) (νCO in C(O)NH), 1743(s) (νCO in C(O)OMe), 2852(vw), 2901(vw), 2933(w), 2955(w), 3033(vw), 3059(vw), 3383(m) (νNH). Anal. Calcd for C11H14N2O3S: C, 51.95; H, 5.55; N, 11.02. Found: C, 52.23; H, 5.68; N, 10.95%. Methyl (2R)-2-{[(4-chloropyridin-2-yl)carbonyl]amino}-3(methylsulfanyl)propanoate, (R)-2. Yield: 0.34 g (31%). Mp: 51−

NH) ppm. 13C{1H} NMR (100.61 MHz, CDCl3): δ 15.38 (s, SMe), 29.93 and 31.83 (both s, CH2S and CH2), 51.50 (s, OMe), 52.53 (s, CH), 122.90 and 126.51 (both s, C2 and C4), 145.78 (s, C3), 149.10 (s, C5), 150.61 (s, C1), 162.95 (s, C(O)NH), 171.89 (s, C(O)OMe) ppm. IR (KBr, ν/cm−1): 519(w), 563(vw), 681(w), 748(w), 787(vw), 847(w), 905(w), 931(vw), 985(w), 1056(vw), 1110(w), 1158(w), 1177(w), 1220(m), 1284(w), 1355(w), 1431(w), 1449(w), 1536(s) (C(O)NH), 1556(m), 1579(w), 1663(s) and 1674(m) (both νCO in C(O)NH), 1748(s) (νCO in C(O)OMe), 2921(w), 2953(w), 3062(vw), 3330(m) (νNH). Anal. Calcd for C12H15ClN2O3S: C, 47.60; H, 4.99; N, 9.25. Found: C, 47.55; H, 5.06; N, 9.21%. Methyl (2R)-4-(methylsulfanyl)-2-[(pyridin-2-ylcarbonyl)amino]butanoate, (R)-3. Yield: 0.24 g (24%). Mp: 49−51 °C (hexane).

53 °C (hexane). [α]D25 +1.3 (c = 1.9, CHCl3). 1H NMR (300.13 MHz, CDCl3): δ 2.18 (s, 3H, SMe), 3.05−3.18 (m, 2H, CH2), 3.84 (s, 3H, OMe), 5.02−5.08 (m, 1H, CH), 7.48−7.50 (m, 1H, H(C4)), 8.21 (d, 1H, H(C2), 4JHH = 1.6 Hz), 8.54 (d, 1H, H(C5), 3JHH = 5.1 Hz), 8.71 (br. d, 1H, NH, 3JHH = 7.5 Hz) ppm. 13C{1H} NMR (75.47 MHz, CDCl3): δ 16.05 (s, SMe), 36.27 (s, CH2S), 51.72 (s, OMe), 52.55 (s, CH), 122.78 and 126.44 (both s, C2 and C4), 145.63 (s, C3), 149.12 (s, C5), 150.42 (s, C1), 162.80 (s, C(O)NH), 170.79 (s, C(O)OMe) ppm. IR (KBr, ν/cm−1): 528(w), 637(w), 690(m), 697(m), 743(m), 780(vw), 803(vw), 833(w), 854(w), 884(w), 909(vw), 962(vw), 991(w), 1014(w), 1044(w), 1097(w), 1112(w), 1172(m), 1190(w), 1228(m), 1242(m), 1269(m), 1312(w), 1330(w), 1398(w), 1432(m), 1465(m), 1526(s) (C(O)NH), 1557(m), 1580(m), 1666(s) (νCO in C(O)NH), 1718(s) and 1730(s) (both νCO in C(O)OMe), 2836(vw), 2919(w), 2950(w), 3058(w), 3102(vw), 3312(m) and 3350(m) (both νNH). Anal. Calcd for C11H13ClN2O3S: C, 45.75; H, 4.54; N, 9.70. Found: C, 45.72; H, 4.73; N, 9.51%. Methyl (2S)-4-(methylsulfanyl)-2[(pyridin-2-ylcarbonyl)amino]butanoate, (S)-3. Yield: 0.10 g (10%). Mp: 46−48 °C (hexane).

[α]D25 −10.3 (c = 0.6, CHCl3). 1H NMR (400.13 MHz, CDCl3): δ 2.10−2.19 (m, 1H, CH2), 2.12 (s, 3H, SMe), 2.27−2.36 (m, 1H, CH2), 2.55−2.66 (m, 2H, CH2S), 3.80 (s, 3H, OMe), 4.92−4.97 (m, 1H, CH), 7.45−7.48 (m, 1H, H(C4)), 7.87 (dt, 1H, H(C3), 3JHH = 7.7 Hz, 4JHH = 1.3 Hz), 8.19 (d, 1H, H(C2), 3JHH = 7.7 Hz), 8.57−8.61 (m, 2H, H(C5) + NH) ppm. 13C{1H} NMR (100.61 MHz, CDCl3): δ 15.34 (s, SMe), 29.95 and 31.92 (both s, CH2 and CH2S), 51.38 (s, OMe), 52.42 (s, CH), 122.22 and 126.33 (both s, C2 and C4), 137.24 (s, C3), 148.12 (s, C5), 149.10 (s, C1), 164.05 (s, C(O)NH), 172.03 (s, C(O)OMe) ppm. IR (KBr, ν/cm−1): 547(vw), 621(w), 675(w), 755(w), 822(w), 842(w), 925(w), 998(w), 1041(w), 1058(w), 1085(w), 1104(w), 1173(m), 1217(m), 1270(w), 1296(w), 1360(w), 1433(m), 1451(w), 1467(w), 1532(s) (C(O)NH), 1570(w), 1590(w), 1661(s) and 1671(s) (both νCO in C(O)NH), 1728(w) and 1746(s) (both νCO in C(O)OMe), 2922(w), 2953(w), 3053(vw), 3065(vw), 3344(m) (νNH). Anal. Calcd for C12H16N2O3S: C, 53.71; H, 6.01; N, 10.44. Found: C, 53.89; H, 6.02; N, 10.19%. Methyl (2R)-2-{[(4-chloropyridin-2-yl)carbonyl]amino}-4(methylsulfanyl)butanoate, (R)-4. Yield: 0.30 g (26%). Mp: 46−48

[α]D25 +11.0 (c = 0.2, CHCl3). 1H NMR (400.13 MHz, CDCl3): δ 2.11−2.20 (m, 1H, CH2), 2.13 (s, 3H, SMe), 2.28−2.37 (m, 1H, CH2), 2.56−2.67 (m, 2H, CH2S), 3.81 (s, 3H, OMe), 4.93−4.98 (m, 1H, CH), 7.46−7.49 (m, 1H, H(C4)), 7.88 (dt, 1H, H(C3), 3JHH = 7.7 Hz, 4JHH = 1.7 Hz), 8.20 (d, 1H, H(C2), 3JHH = 7.7 Hz), 8.58 (br. d, 1H, NH, 3JHH = 8.3 Hz), 8.62 (d, 1H, H(C5), 3JHH = 4.8 Hz) ppm. 13 C{1H} NMR (100.61 MHz, CDCl3): δ 15.36 (s, SMe), 29.96 and 31.94 (both s, CH2 and CH2S), 51.39 (s, OMe), 52.45 (s, CH), 122.22 and 126.34 (both s, C2 and C4), 137.23 (s, C3), 148.16 (s, C5), 149.13 (s, C1), 164.09 (s, C(O)NH), 172.06 (s, C(O)OMe) ppm. IR (KBr, ν/cm−1): 548(vw), 620(w), 685(m), 753(w), 786(vw), 823(w), 859(w), 925(w), 997(w), 1042(w), 1058(w), 1085(w), 1101(w), 1170(m), 1216(m), 1294(w), 1363(m), 1430(m), 1451(w), 1466(m), 1531(s) (C(O)NH), 1570(w), 1590(w), 1658(s) and 1671(s) (both νCO in C(O)NH), 1747(s) (νCO in C(O)OMe), 2853(w), 2919(m), 2952(w), 3331(m) (νNH). Anal. Calcd for C12H16N2O3S: C, 53.71; H, 6.01; N, 10.44. Found: C, 53.85; H, 5.85; N, 10.22%. Methyl (2S)-2-{[(4-chloropyridin-2-yl)carbonyl]amino}-4(methylsulfanyl)butanoate, (S)-4. Yield: 0.28 g (25%). Mp: 50−52 °C (hexane). [α]D25 +23.0 (c = 0.5, CHCl3). 1H NMR (400.13 MHz, CDCl3): δ 2.10−2.19 (m, 1H, CH2), 2.12 (s, 3H, SMe), 2.27−2.36 (m, 1H, CH2), 2.54−2.65 (m, 2H, CH2S), 3.81 (s, 3H, OMe), 4.91− 4.96 (m, 1H, CH), 7.47 (dd, 1H, H(C4), 3JHH = 5.2 Hz, 4JHH = 1.8 Hz), 8.19 (d, 1H, H(C2), 4JHH = 1.8 Hz), 8.50−8.52 (m, 2H, H(C5) +

°C (hexane). [α]D25 −58.0 (c = 0.25, CHCl3). 1H NMR (400.13 MHz, CDCl3): δ 2.09−2.18 (m, 1H, CH2), 2.11 (s, 3H, SMe), 2.27−2.35 (m, 1H, CH2), 2.55−2.64 (m, 2H, CH2S), 3.80 (s, 3H, OMe), 4.90− 4.95 (m, 1H, CH), 7.47 (dd, 1H, H(C4), 3JHH = 5.2 Hz, 4JHH = 1.8 Hz), 8.19 (d, 1H, H(C2), 4JHH = 1.8 Hz), 8.49−8.52 (m, 2H, H(C5) + NH) ppm. 13C{1H} NMR (100.61 MHz, CDCl3): δ 15.35 (s, SMe), 29.90 and 31.79 (both s, CH2S and CH2), 51.47 (s, OMe), 52.49 (s, CH), 122.87 and 126.47 (both s, C2 and C4), 145.75 (s, C3), 149.06 (s, C5), 150.57 (s, C1), 162.90 (s, C(O)NH), 171.85 (s, C(O)OMe) ppm. IR (KBr, ν/cm−1): 519(w), 563(vw), 681(w), 748(w), 787(vw), 847(w), 905(w), 931(vw), 985(w), 1056(vw), 1093(w), 1110(w), 1158(w), 1177(w), 1220(m), 1284(w), 1355(w), 1431(w), 1449(w), 1536(s) (C(O)NH), 1556(m), 1579(w), 1663(s) and 1674(m) (both νCO in C(O)NH), 1748(s) (νCO in C(O)OMe), 2921(w), 2953(w), 3063(vw), 3330(m) (νNH). Anal. Calcd for C12H15ClN2O3S: C, 47.60; H, 4.99; N, 9.25. Found: C, 47.69; H, 5.06; N, 9.26%. Methyl (2S)-3-(1H-imidazol-4-yl)-2-[(pyridin-2-ylcarbonyl)amino]propanoate, (S)-5. Yield: 0.21 g (20%). Mp: 174−176 °C (compare with 174−175 °C in ref 33). 1H NMR spectroscopy was 9842


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Inorganic Chemistry

m) (νCO in C(O)OMe), 2920(vw), 2953(w), 3003(vw), 3060(w), 3263(br, w) (NH). Anal. Calcd for C11H13ClN2O4S: C, 43.35; H, 4.30; N, 9.19. Found: C, 43.15; H, 4.49; N, 8.91%. Methyl (2S)-2-{[(4-chloropyridin-2-yl)carbonyl]amino}-4(methylsulfinyl)butanoate, (S)-4o. 1H NMR spectrum is described used to control the individuality of compounds (S)-5 and (S)-6 upon separation by column chromatography. Methyl (2S)-2-{[(4-chloropyridin-2-yl)carbonyl]amino}-(1H-imidazol-4-yl)propanoate, (S)-6. Yield: 0.55 g (48%). Mp: 134−136

as for a single compound, because most of the signals of the isomers (except for the NH proton signals) coincide, resulting in slightly broadened patterns. Yield: 0.121 g (76%). 1H NMR (400.13 MHz, CDCl3): δ 2.26−2.36 (m, 1H, CH2), 2.51−2.60 (m, 1H, CH2), 2.60 (br. s, 3H, S(O)Me), 2.73−2.90 (m, 2H, CH2S(O)), 3.83 (br. s, 3H, OMe), 4.90−4.98 (m, 1H, CH), 7.48−7.50 (m, 1H, H(C4)), 8.18 (br. s, 1H, H(C2)), 8.52 (d, 1H, H(C5), 3JHH = 5.2 Hz), 8.62 and 8.64 (both br. d, 0.5H + 0.5H, NH, 3JHH = 8.8 Hz) ppm. 13C{1H} NMR (100.61 MHz, CDCl3): δ 25.80 and 26.26 (both s, CH2), 38.58 and 38.66 (both s, S(O)Me), 50.24 and 50.50 (both s, CH2S(O)), 51.22 and 51.51 (both s, OMe), 52.76 and 52.80 (both s, CH), 122.97 (br. s, C2 or C4), 126.67 and 126.69 (both s, C4 or C2), 145.93 and 145.95 (both s, C3), 149.11 (br. s, C5), 150.25 and 150.29 (both s, C1), 163.19 (br. s, C(O)NH), 171.15 and 171.18 (both s, C(O)OMe) ppm. IR (thin layer, ν/cm−1): 526(m), 692(w), 743(w), 784(w), 846(vw), 996(w), 1024(m) and 1043(m) (both νSO), 1098(w), 1175(w), 1222(m), 1261(w), 1308(w), 1350(w), 1446(w), 1462(w), 1520(s) (C(O)NH), 1556(m), 1579(w), 1674(s) (νCO in C(O)NH), 1742(s) (νCO in C(O)OMe), 2852(vw), 2923(w), 2954(w), 3001(vw), 3058(vw), 3371(br, w) (NH). Anal. Calcd for C12H15ClN2O4S: C, 45.21; H, 4.74; N, 8.79. Found: C, 45.06; H, 4.41; N, 8.43%. General Procedure for the Synthesis of [κ3-N,N,X-(L)Pd(II)Cl] (X = S, N) Complexes 7−12, 10o. A solution of PdCl2(NCPh)2 (76 mg, 0.198 mmol) in 4 mL of dichloromethane was added dropwise to a solution of the corresponding ligand (0.198 mmol) and Et3N (28 μL, 0.198 mmol) in 4 mL of CH2Cl2. The resulting mixture was stirred at room temperature for 1 day and then purified by column chromatography (eluent CH2Cl2/MeOH (100:1 (7−10, 10o) or 50:1 (11, 12)) to give the desired palladocycles as light-yellow ((S)11, (S)-12), yellow ((R)-7, (R)-8, (S)-9, (R)-9, (R)-10, and (S)-10o) or red ((S)-10) crystalline solids. Complex (R)-7 [κ3-N,N,S-(L)Pd(II)Cl]. Yield: 56 mg (72%). Mp: 197−199 °C. 1H NMR (400.13 MHz, CDCl3, major isomer (M) 62%,

°C (EtOAc/Et2O). [α]D25 +41.25 (c = 0.24, CHCl3). 1H NMR (400.13 MHz, CDCl3): δ 3.23−3.32 (m, 2H, CH2), 3.75 (s, 3H, OMe), 5.02−5.07 (m, 1H, CH), 6.10 (br. s, 1H, NH), 6.86 (s, 1H, H(C7)), 7.44 (dd, 1H, H(C4), 3JHH = 5.2 Hz, 4JHH = 1.8 Hz), 7.63 (s, 1H, H(C8)), 8.15 (d, 1H, H(C2), 4JHH = 1.8 Hz), 8.50 (d, 1H, H(C5), 3 JHH = 5.2 Hz), 8.95 (br. d, 1H, NHC(O), 3JHH = 7.8 Hz) ppm. 13 C{1H} NMR (100.61 MHz, CDCl3): δ 29.49 (s, CH2), 52.40 and 52.46 (both s, OMe and CH), 116.19 (s, C7), 122.83 and 126.40 (both s, C2 and C4), 133.47 (s, C6), 135.05 (s, C8), 145.64 (s, C3), 149.24 (s, C5), 150.72 (s, C1), 163.15 (s, C(O)NH), 171.57 (s, C(O)OMe) ppm. IR (KBr, ν/cm−1): 534(vw), 664(w), 692(w), 747(w), 782(vw), 816(w), 848(w), 877(vw), 908(vw), 948(w), 1002(w), 1063(w), 1109(w), 1233(w), 1250(w), 1276(m), 1322(w), 1343(w), 1447(w), 1465(w), 1532(s) (C(O)NH), 1555(m), 1579(w), 1665(s) (νCO in C(O)NH), 1736(s) (νCO in C(O)OMe), 2849(w), 2959(w), 3063(w), 3351(m) (νNH). Anal. Calcd for C13H13ClN4O3: C, 50.58; H, 4.24; N, 18.15. Found: C, 50.51; H, 4.29; N, 18.35%. General Procedure for the Synthesis of Ligands 2o, 4o. A mixture of the corresponding sulfide ((R)-2 or (S)-4) (0.50 mmol) and 7% solution of H2O2 in tBuOH (1.34 g, ∼5.5-fold excess) in 10 mL of CHCl3 was stirred at room temperature for 1 day and then poured into 30 mL of distilled water. The organic layer was separated, dried over anhydrous Na2SO4, and evaporated to dryness. The resulting residue was purified by column chromatography on silica gel (eluent CH2Cl2/MeOH (75:1)) to give the desired sulfoxide ligands as a white crystalline solid ((R)-2o) or a pale-yellow oil ((S)-4o). Methyl (2R)-2{[(4-chloropyridin-2-yl)carbonyl]amino}-3(methylsulfinyl)propanoate, (R)-2o. Yield: 0.137 g (90%). Mp: 92−

94 °C (hexane/Et2O). 1H NMR (400.13 MHz, CDCl3, major isomer (M) 68%, minor isomer (m) 32%): δ 2.70 (br. s, 3H, S(O)Me (M) + 3H, S(O)Me (m)), 3.29−3.53 (m, 2H, CH2 (M) + 2H, CH2 (m)), 3.83 (s, 3H, OMe (M)), 3.86 (s, 3H, OMe (m)), 5.07−5.11 (m, 1H, CH (m)), 5.17−5.22 (m, 1H, CH (M)), 7.47−7.50 (m, 1H, H(C4) (M) + 1H, H(C4) (m)), 8.17 (br. s, 1H, H(C2) (m)), 8.19 (br. s, 1H, H(C2) (M)), 8.51−8.53 (m, 1H, H(C5) (M) + 1H, H(C5) (m)), 8.96 (br. d, 1H, NH (m), 3JHH = 6.2 Hz), 9.10 (br. d, 1H, NH (M), 3 JHH = 7.5 Hz) ppm. 13C{1H} NMR (100.61 MHz, CDCl3): δ 39.06 (s, S(O)Me (m)), 39.30 (s, S(O)Me (M)), 48.76 (s, OMe (m)), 49.04 (s, OMe (M)), 52.99 (s, CH (M)), 53.08 (s, CH (m)), 55.00 (s, CH2 (M)), 55.91 (s, CH2 (m)), 122.85 (s, C2 or C4 (m)), 123.03 (s, C2 or C4 (M)), 126.65 (s, C4 or C2 (M)), 126.72 (s, C4 or C2 (m)), 145.93 (s, C3 (M)), 145.97 (s, C3 (m)), 149.11 (s, C5 (M)), 149.23 (s, C5 (m)), 150.09 (br. s, C1 (M+m)), 163.24 (br. s, C(O)NH (M+m)), 169.66 (s, C(O)OMe (m)), 169.80 (s, C(O)OMe (M)) ppm. IR (KBr, ν/cm−1): 524(w), 693(w), 751(w), 783(vw), 856(w), 900(vw), 995(w), 1020(m) and 1048(sh, w) (both νSO), 1105(w), 1239(m), 1262(m), 1293(m), 1398(w), 1430(w), 1462(w), 1520(s) (C(O)NH), 1557(m), 1581(w), 1681(s) (νCO in C(O)NH), 1725(br,

minor isomer (m) 38%): δ 2.61 (s, 3H, SMe (m)), 2.72 (s, 3H, SMe (M)), 3.18 (dd, 1H, CH2 (M), 2JHH = 11.2 Hz, 3JHH = 6.2 Hz), 3.34 (dd, 1H, CH2 (m), 2JHH = 13.8 Hz, 3JHH = 2.6 Hz), 3.47 (dd, 1H, CH2 (m), 2JHH = 13.8 Hz, 3JHH = 7.0 Hz), 3.61 (dd, 1H, CH2 (M), 2JHH = 11.2 Hz, 3JHH = 1.9 Hz), 3.79 (s, 3H, OMe (M)), 3.81 (s, 3H, OMe (m)), 4.79 (dd, 1H, CH (m), 3JHH = 7.0 Hz, 3JHH = 2.6 Hz), 4.84 (dd, 1H, CH (M), 3JHH = 6.2 Hz, 3JHH = 1.9 Hz), 7.52−7.58 (m, 1H, H(C4) (M) + 1H, H(C4) (m)), 7.91 (d, 1H, H(C2) (m), 3JHH = 8.0 Hz), 7.94 (d, 1H, H(C2) (M), 3JHH = 7.3 Hz), 8.03−8.07 (m, 1H, H(C3) (M) + 1H, H(C3) (m)), 8.82 (d, 1H, H(C5) (M), 3JHH = 4.7 Hz), 8.86 (d, 1H, H(C5) (m), 3JHH = 4.9 Hz) ppm. 13C{1H} NMR (100.61 MHz, CDCl3): δ 20.86 (s, SMe (m)), 22.50 (s, SMe (M)), 45.18 (s, CH2 (m)), 47.71 (s, CH2 (M)), 53.03 (s, OMe (M)), 53.12 (s, OMe (m)), 59.57 (s, CH, M), 61.43 (s, CH (m)), 125.86 (s, C2 (m)), 125.94 (s, C2 (M)), 127.17 (s, C4 (M)), 127.30 (s, C4 (m)), 140.35 (s, C3 (M)), 140.41 (s, C3 (m)), 147.78 (s, C5 (m)), 147.89 (s, C5 (M)), 155.62 (s, C1 (M)), 155.72 (s, C1 (m)), 169.38 (s, C(O) N (M)), 169.77 (s, C(O)N (m)), 170.15 (s, C(O)OMe (M)), 171.44 (s, C(O)OMe (m)) ppm. IR (KBr, ν/cm−1): 508(w), 659(vw), 9843


Article

Inorganic Chemistry 682(m), 757(m), 809(vw), 974(w), 1019(m), 1050(w), 1092(w), 1162(m), 1187(w), 1199(w), 1252(w), 1271(w), 1289(w), 1325(w), 1359(w), 1374(m), 1436(w), 1567(w), 1601(s), 1631(s) and 1637(sh, s) (both νCO in C(O)N), 1741(s) (νCO in C(O)OMe), 2852(vw), 2922(w), 2952(w), 3002(w), 3068(vw), 3090(vw). Anal. Calcd for C11H13ClN2O3PdS: C, 33.43; H, 3.32; N, 7.09. Found: C, 33.61; H, 3.39; N, 6.92%. Complex (R)-8 [κ3-N,N,S-(L)Pd(II)Cl]. Yield: 56 mg (66%). Mp: 203−205 °C. 1H NMR (400.13 MHz, CDCl3, major isomer (M) 64%,

C(O)N), 1747(s) (νCO in C(O)OMe), 2920(w), 2952(w), 3078(vw). Anal. Calcd for C12H15ClN2O3PdS: C, 35.22; H, 3.69; N, 6.85. Found (%): C, 35.12; H, 3.90; N, 6.72%. Complex (S)-10 [κ3-N,N,S-(L)Pd(II)Cl]. Yield: 75 mg (85%). Mp: 165−168 °C. 1H NMR (600.22 MHz, CDCl3, 243 K, major isomer

(M) 62%, minor isomer (m) 38%): δ 1.89−1.94 (m, 1H, CH2 (m)), 2.12−2.17 (m, 1H, CH2 (M) + 1H, CH2S (m)), 2.24−2.32 (m, 2H, CH2S (M)), 2.55−2.60 (m, 1H, CH2 (m)), 2.62 (br. s, 3H, SMe (M) + 3H, SMe (m)), 2.72−2.76 (m, 1H, CH2 (M) + 1H, CH2S (m)), 3.73 (s, 3H, OMe (M)), 3.76 (s, 3H, OMe (m)), 4.48−4.50 (m, 1H, CH (m)), 4.64−4.65 (m, 1H, CH (M)), 7.50−7.52 (m, 1H, H(C4) (M) + 1H, H(C4) (m)), 7.90 (d, 1H, H(C2) (m), 4JHH = 2.2 Hz), 7.94 (d, 1H, H(C2) (M), 4JHH = 2.2 Hz), 8.89 (d, 1H, H(C5) (M), 3 JHH = 6.0 Hz), 8.91 (d, 1H, H(C5) (m), 3JHH = 6.0 Hz) ppm. 13 C{1H} NMR (150.93 MHz, CDCl3, 243 K): δ 22.60 (s, SMe (M)), 24.03 (s, SMe (m)), 26.46 (s, CH2S (M)), 28.07 (s, CH2S (m)), 30.54 (s, CH2 (M)), 30.88 (s, CH2 (m)), 52.72 (s, CH (M)), 52.84 (s, OMe (m)), 52.90 (s, OMe (M)), 53.75 (s, CH (m)), 125.98 (s, C2 (m)), 126.04 (s, C2 (M)), 127.09 (s, C4 (M)), 127.12 (s, C4 (m)), 148.35 (s, C5 (m)), 148.39 (s, C5 (M)), 149.02 (s, C3 (m)), 149.12 (s, C3 (M)), 153.85 (s, C1 (m)), 154.03 (s, C1 (M)), 172.05 (s, C(O)N (m)), 172.11 (s, C(O)N (M)), 172.19 (s, C(O)OMe (M)), 172.79 (s, C(O)OMe (m)) ppm. IR (KBr, ν/cm−1): 517(w), 531(w), 658(vw), 730(w), 758(m), 782(w), 843(w), 972(w), 1035(w), 1071(w), 1107(w), 1124(w), 1148(m), 1158(w), 1174(m), 1192(m), 1281(w), 1337(m), 1353(m), 1421(m), 1559(w), 1595(s), 1622(s) (νCO in C(O)N), 1739(m) and 1754(m) (both νCO in C(O)OMe), 2922(w), 3024(vw), 3076(w). Anal. Calcd for C12H14Cl2N2O3PdS: C, 32.49; H, 3.18; N, 6.31. Found: C, 32.25; H, 3.35; N, 6.49%. Complex (R)-9 [κ3-N,N,S-(L)Pd(II)Cl]. Yield: 80 mg (99%). Mp: 157−159 °C. 1H NMR (600.22 MHz, CDCl3, 243 K, major isomer

minor isomer (m) 36%): δ 2.61 (s, 3H, SMe (m)), 2.71 (s, 3H, SMe (M)), 3.14 (dd, 1H, CH2 (M), 2JHH = 9.0 Hz, 3JHH = 5.0 Hz), 3.36 (dd, 1H, CH2 (m), 2JHH = 11.0 Hz, 3JHH = 1.8 Hz), 3.45 (dd, 1H, CH2 (m), 2JHH = 11.0 Hz, 3JHH = 5.6 Hz), 3.63 (d, 1H, CH2 (M), 2JHH = 9.0 Hz), 3.80 (s, 3H, OMe (M)), 3.83 (s, 3H, OMe (m)), 4.81 (dd, 1H, CH (m), 3JHH = 5.6 Hz, 3JHH = 1.8 Hz), 4.85 (d, 1H, CH (M), 3JHH = 5.0 Hz), 7.54−7.57 (m, 1H, H(C4) (M) + 1H, H(C4) (m)), 7.92 (d, 1H, H(C2) (m), 4JHH = 1.3 Hz), 7.94 (d, 1H, H(C2) (M), 4JHH = 1.5 Hz), 8.78 (d, 1H, H(C5) (M), 3JHH = 4.9 Hz), 8.79 (d, 1H, H(C5) (m), 3JHH = 5.2 Hz) ppm. 13C{1H} NMR (100.61 MHz, CDCl3): δ 20.84 (s, SMe (m)), 22.47 (s, SMe (M)), 45.11 (s, CH2 (m)), 47.69 (s, CH2 (M)), 52.97 (s, OMe (M)), 53.05 (s, OMe (m)), 59.58 (s, CH (M)), 61.50 (s, CH (m)), 126.18 (s, C2 (m)), 126.26 (s, C2 (M)), 127.20 (s, C4 (M)), 127.34 (s, C4 (m)), 148.28 (s, C5 (m)), 148.37 (s, C5 (M)), 149.00 (s, C3 (M)), 149.08 (s, C3 (m)), 156.63 (s, C1 (M)), 156.66 (s, C1 (m)), 168.13 (s, C(O)N (M)), 168.50 (s, C(O)N (m)), 169.79 (s, C(O)OMe (M)), 171.09 (s, C(O)OMe (m)) ppm. IR (KBr, ν/cm−1): 534(w), 701(vw), 767(m), 778(w), 836(w), 852(w), 912(w), 975(w), 1026(m), 1108(m), 1168(m), 1197(m), 1253(m), 1282(w), 1368(m), 1422(m), 1555(w), 1594(s), 1633(s) and 1642(s) (both νC = O in C(O)N), 1732(s) (νCO in C(O)OMe), 2850(vw), 2929(w), 2947(w), 2999(w), 3061(vw), 3078(vw). Anal. Calcd for C11H12Cl2N2O3PdS: C, 30.75; H, 2.82; N, 6.52. Found: C, 31.09; H, 2.88; N, 6.54%. Complex (S)-9 [κ3-N,N,S-(L)Pd(II)Cl]. Yield: 72 mg (89%). Mp: 155−157 °C. 1H NMR (600.22 MHz, CDCl3, 243 K, major isomer

(M) 61%, minor isomer (m) 39%): δ 1.87−1.92 (m, 1H, CH2 (m)), 2.14−2.18 (m, 1H, CH2 (M) + 1H, CH2S (m)), 2.29−2.31 (m, 2H, CH2S (M)), 2.59−2.63 (m, 1H, CH2 (m)), 2.65 (s, 3H, SMe (M)), 2.66 (s, 3H, SMe (m)), 2.74−2.77 (m, 1H, CH2 (M) + 1H, CH2S (m)), 3.76 (s, 3H, OMe (M)), 3.79 (s, 3H, OMe (m)), 4.56−4.57 (m, 1H, CH (m)), 4.70−4.71 (m, 1H, CH (M)), 7.53−7.57 (m, 1H, H(C4) (M) + 1H, H(C4) (m)), 7.98 (d, 1H, H(C2) (m), 3JHH = 7.9 Hz), 7.99 (d, 1H, H(C2) (M), 3JHH = 7.9 Hz), 8.04−8.07 (m, 1H, H(C3) (M) + 1H, H(C3) (m)), 9.02 (d, 1H, H(C5) (M), 3JHH = 5.4 Hz), 9.05 (d, 1H, H(C5) (m), 3JHH = 5.3 Hz) ppm. 13C{1H} NMR (150.93 MHz, CDCl3, 243 K): δ 22.57 (s, SMe (M)), 24.05 (s, SMe (m)), 26.52 (s, CH2S (M)), 28.05 (s, CH2S (m)), 30.64 (s, CH2 (M)), 31.00 (s, CH2 (m)), 52.70 (s, CH (M)), 52.80 (s, OMe (m)), 52.86 (s, OMe (M)), 53.67 (s, CH (m)), 125.61 (s, C2 (m)), 125.63 (s, C2 (M)), 127.01 (s, C4 (M)), 127.03 (s, C4 (m)), 140.55 (s, C3 (m)), 140.63 (s, C3 (M)), 147.75 (s, C5 (m)), 147.79 (s, C5 (M)), 152.83 (s, C1 (m)), 152.97 (s, C1 (M)), 172.45 (s, C(O)OMe (M)), 173.02 (s, C(O)OMe (m)), 173.27 (s, C(O)N (m)), 173.30 (s, C(O) N (M)) ppm. IR (KBr, ν/cm−1): 509(w), 660(w), 683(w), 762(w), 813(w), 983(w), 1028(w), 1056(w), 1070(w), 1112(w), 1163(m), 1193(m), 1242(w), 1281(w), 1294(w), 1342(w), 1370(sh, m), 1375(m), 1432(w), 1481(w), 1569(w), 1600(s), 1628(s) (νCO in C(O)N), 1747(s) (νCO in C(O)OMe), 2852(vw), 2883(vw),

(M) 62%, minor isomer (m) 38%): δ 1.88−1.93 (m, 1H, CH2 (m)), 2.12−2.19 (m, 1H, CH2 (M) + 1H, CH2S (m)), 2.24−2.32 (m, 2H, CH2S (M)), 2.56−2.61 (m, 1H, CH2 (m)), 2.63 (s, 3H, SMe (m)), 2.64 (s, 3H, SMe (M)), 2.72−2.76 (m, 1H, CH2 (M) + 1H, CH2S (m)), 3.74 (s, 3H, OMe (M)), 3.77 (s, 3H, OMe (m)), 4.51−4.53 (m, 1H, CH (m)), 4.66−4.68 (m, 1H, CH (M)), 7.52−7.56 (m, 1H, H(C4) (M) + 1H, H(C4) (m)), 7.95 (d, 1H, H(C2) (m), 3JHH = 7.3 Hz), 7.98 (d, 1H, H(C2) (M), 3JHH = 7.4 Hz), 8.03−8.06 (m, 1H, H(C3) (M) + 1H, H(C3) (m)), 8.96 (d, 1H, H(C5) (M), 3JHH = 5.1 Hz), 9.01 (d, 1H, H(C5) (m), 3JHH = 5.2 Hz) ppm. 13C{1H} NMR (150.93 MHz, CDCl3, 243 K): δ 22.53 (s, SMe (M)), 24.03 (s, SMe (m)), 26.42 (s, CH2S (M)), 28.02 (s, CH2S (m)), 30.54 (s, CH2 (M)), 30.94 (s, CH2 (m)), 52.71 (s, CH (M)), 52.88 (s, OMe (m)), 52.95 (s, OMe (M)), 53.74 (s, CH (m)), 125.62 (s, C2 (m)), 125.68 (s, C2 (M)), 127.07 (s, C4 (M)), 127.12 (s, C4 (m)), 140.63 (s, C3 (m)), 140.73 (s, C3 (M)), 147.73 (br. s, C5 (M+m)), 152.69 (s, C1, (m)), 152.82 (s, C1 (M)), 172.51 (s, C(O)OMe (M)), 173.08 (s, C(O)OMe (m)), 173.28 (s, C(O)N (m)), 173.32 (s, C(O)N (M)) ppm. IR (KBr, ν/cm−1): 509(w), 660(w), 682(w), 726(vw), 762(m), 812(w), 983(w), 1027(w), 1056(w), 1071(w), 1111(w), 1163(m), 1193(m), 1242(w), 1281(w), 1294(w), 1342(w), 1368 (sh, m), 1374(m), 1431(w), 1481(w), 1569(w), 1600(s), 1629(s) (νCO in 9844


Article

Inorganic Chemistry 2

JHH = 15.1 Hz, 3JHH = 3.9 Hz, 4JHH = 1.0 Hz), 3.45 (dd, 1H, CH2, 2JHH = 15.1 Hz, 3JHH = 3.9 Hz), 3.60 (s, 3H, OMe), 4.94 (vt, 1H, CH, 3JHH = 3.9 Hz), 6.93 (s, 1H, H(C7)), 7.47 (dd, 1H, H(C4), 3JHH = 6.1 Hz, 4 JHH = 2.4 Hz), 7.99 (d, 1H, H(C2), 4JHH = 2.4 Hz), 8.30 (s, 1H, H(C8)), 9.05 (d, 1H, H(C5), 3JHH = 6.1 Hz), 10.04 (br. s, 1H, NH) ppm. 13C{1H} NMR (100.61 MHz, (CD3)2SO): δ 30.81 (s, CH2), 52.14 and 52.26 (both s, OMe and CH), 115.11 (s, C7), 125.25 and 127.94 (both s, C2 and C4), 131.71 (s, C6), 141.37 (s, C8), 148.02 (s, C3), 150.34 (s, C5), 155.51 (s, C1), 169.70 and 171.54 (both s, C(O) N and C(O)OMe) ppm. IR (KBr, ν/cm−1): 538(vw), 629(w), 700(vw), 767(m), 824(vw), 988(vw), 1020(vw), 1084(w), 1108(w), 1177(m), 1203(m), 1275(w), 1376(m), 1428(m), 1501(w), 1555(w), 1594(s), 1613(s) (νCO in C(O)N), 1740(m) (νCO in C(O)OMe), 2907(w), 3037(w), 3101(w), 3147(w). Anal. Calcd for C13H12Cl2N4O3Pd: C, 34.73; H, 2.69; N, 12.46. Found: C, 34.79; H, 2.72; N, 12.29%. Complex (S)-10o [κ3-N,N,S-(L)Pd(II)Cl]. A mixture of two isomers (major isomer 62%, minor isomer 38%) was separated by thin layer chromatography on silica gel plates (eluent CHCl3/MeOH (15:1)).

2920(w), 2952(w), 3078(w). Anal. Calcd for C12H15ClN2O3PdS: C, 35.22; H, 3.69; N, 6.85. Found: C, 34.93; H, 3.66; N, 6.62%. Complex (R)-10 [κ3-N,N,S-(L)Pd(II)Cl]. Yield: 87 mg (99%). Mp: 165−170 °C. 1H NMR (600.22 MHz, CDCl3, 243 K, major isomer

(M) 61%, minor isomer (m) 39%): δ 1.89−1.93 (m, 1H, CH2 (m)), 2.13−2.17 (m, 1H, CH2 (M) + 1H, CH2S (m)), 2.25−2.31 (m, 2H, CH2S (M)), 2.56−2.61 (m, 1H, CH2 (m)), 2.63 (br. s, 3H, SMe (M) + 3H, SMe (m)), 2.73−2.76 (m, 1H, CH2 (M) + 1H, CH2S (m)), 3.74 (s, 3H, OMe (M)), 3.77 (s, 3H, OMe (m)), 4.50−4.52 (m, 1H, CH (m)), 4.65−4.66 (m, 1H, CH (M)), 7.51−7.53 (m, 1H, H(C4) (M) + 1H, H(C4) (m)), 7.92 (br. s, 1H, H(C2) (m)), 7.95 (br. s, 1H, H(C2) (M)), 8.90−8.93 (m, 1H, H(C5) (M) + 1H, H(C5) (m)) ppm. 13C{1H} NMR (150.93 MHz, CDCl3, 243 K): δ 22.61 (s, SMe (M)), 24.05 (s, SMe (m)), 26.49 (s, CH2S (M)), 28.08 (s, CH2S (m)), 30.56 (s, CH2 (M)), 30.91 (s, CH2 (m)), 52.72 (s, CH (M)), 52.84 (s, OMe (m)), 52.90 (s, OMe (M)), 53.73 (s, CH (m)), 126.00 (s, C2 (m)), 126.04 (s, C2 (M)), 127.09 (s, C4 (M)), 127.11 (s, C4 (m)), 148.36 (s, C5 (m)), 148.41 (s, C5 (M)), 149.03 (s, C3 (m)), 149.13 (s, C3 (M)), 153.89 (s, C1 (m)), 154.06 (s, C1 (M)), 172.07 (s, C(O)N) (m)), 172.13 (s, C(O)N (M)), 172.19 (s, C(O)OMe (M)), 172.78 (s, C(O)OMe (m)) ppm. IR (KBr, ν/cm−1): 517(w), 531(w), 658(vw), 730(w), 758(m), 782(w), 842(w), 904(w), 972(w), 1035(w), 1108(w), 1124(w), 1148(m), 1158(w), 1174(m), 1192(m), 1281(w), 1337(m), 1353(m), 1422(m), 1558(w), 1595(s), 1622(s) (νCO in C(O)N), 1739(m) and 1754(m) (both νCO in C(O)OMe), 2922(w), 3024(vw), 3076(w). Anal. Calcd for C12H14Cl2N2O3PdS: C, 32.49; H, 3.18; N, 6.31. Found: C, 32.33; H, 2.93; N, 6.23%. Complex (S)-11 [κ3-N,N,N-(L)Pd(II)Cl]. Yield: 64 mg (74%). Mp: 175−177 °C (dec.). 1H NMR (400.13 MHz, CDCl3): δ 2.97 (ddd, 1H,

Major Isomer (isomer with the lower adsorption capacity). Yield: 50 mg (55%). Mp: 223−225 °C. 1H NMR (400.13 MHz, CDCl3): δ 2.63−2.67 (m, 2H, CH2), 2.88−2.95 (m, 1H, CH2S(O)), 3.05−3.10 (m, 1H, CH2S(O)), 3.50 (s, 3H, S(O)Me), 3.79 (s, 3H, OMe), 4.79− 4.81 (m, 1H, CH), 7.55 (dd, 1H, H(C4), 3JHH = 6.0 Hz, 4JHH = 2.0 Hz), 8.00 (d, 1H, H(C2), 4JHH = 2.0 Hz), 9.04 (d, 1H, H(C5), 3JHH = 6.0 Hz) ppm. 13C{1H} NMR (100.61 MHz, CDCl3): δ 24.99 (s, CH2), 43.73 (s, S(O)Me), 47.98 (s, CH2S(O)), 52.37 and 53.96 (both s, OMe and CH), 126.19 (s, C2), 127.00 (s, C4), 148.52 (s, C5), 149.86 (s, C3), 153.81 (s, C1), 171.82 and 172.37 (both s, C(O)N and C(O)OMe) ppm. IR (KBr, ν/cm−1): 536(w), 677(w), 698(w), 734(w), 757(w), 784(w), 843(w), 901(vw), 981(w), 1015(w), 1036(w), 1069(w), 1128(m) and 1158(m) (both νSO), 1173(m), 1199(m), 1260(w), 1281(w), 1297(w), 1351(m), 1401(w), 1426(m), 1557(w), 1595(s), 1624(s) (νCO in C(O)N), 1738(s) (νCO in C(O)OMe), 2917(w), 2953(w), 3000(vw), 3077(vw). Anal. Calcd for C12H14Cl2N2O4PdS: C, 31.36; H, 3.07; N, 6.09. Found: C, 31.50; H, 2.85; N, 5.88%. Minor Isomer (isomer with the higher adsorption capacity). Yield: 30 mg (33%). Mp: > 245 °C (dec.). 1H NMR (400.13 MHz, CDCl3): δ 2.02−2.10 (m, 1H, CH2), 2.57−2.66 (m, 1H, CH2), 3.11− 3.14 (m, 2H, CH2S(O)), 3.52 (s, 3H, S(O)Me), 3.74 (s, 3H, OMe), 4.55 (dd, 1H, CH, 3JHH = 7.8 Hz, 3JHH = 3.5 Hz), 7.47 (dd, 1H, H(C4), 3JHH = 6.0 Hz, 4JHH = 2.4 Hz), 7.97 (d, 1H, H(C2), 4JHH = 2.4 Hz), 8.91 (d, 1H, H(C5), 3JHH = 6.0 Hz) ppm. 13C{1H} NMR (100.61 MHz, CDCl3): δ 25.62 (s, CH2), 43.87 (s, S(O)Me), 49.21 (s, CH2S(O)), 52.47 and 55.38 (both s, OMe and CH), 126.31 (s, C2), 126.93 (s, C4), 148.45 (s, C5), 149.86 (s, C3), 153.61 (s, C1), 171.49 and 171.57 (both s, C(O)N and C(O)OMe) ppm. IR (KBr, ν/cm−1): 532(w), 679(w), 698(w), 731(w), 761(w), 784(w), 842(w), 899(w), 980(w), 1036(w), 1111(w), 1133(m) and 1161(m) (both νSO), 1177(m), 1200(m), 1258(w), 1278(w), 1299(vw), 1354(m), 1428(m), 1557(w), 1595(s), 1626(s) (νCO in C(O)N), 1747(m) (νCO in C(O)OMe), 2918(w), 2952(w), 3009(vw), 3076(vw). Anal. Calcd for C12H14Cl2N2O4PdS: C, 31.36; H, 3.07; N, 6.09. Found: C, 31.06; H, 2.80; N, 5.86%. Solid-Phase Syntheses Using a Mortar. Ligand (R)-2, (S)-4, (R)4, or (S)-6 (0.120 mmol) and PdCl2(NCPh)2 (46 mg, 0.120 mmol) were manually ground in a mortar. In the case of compounds (R)-2, (S)-4, and (R)-4 with ancillary S-donor group, grinding for ∼2 min afforded a dark-yellow or brown paste, which slowly solidified and lightened (for 2 h) to give a yellow ((S)-4) or dark yellow ((R)-2, (R)4) free-flowing powder. The resulting sample was additionally rinsed with hexane and Et2O to remove residual benzonitrile. In the case of L-

CH2, 2JHH = 15.1 Hz, 3JHH = 3.9 Hz, 4JHH = 1.1 Hz), 3.43 (dd, 1H, CH2, 2JHH = 15.1 Hz, 3JHH = 3.9 Hz), 3.59 (s, 3H, OMe), 4.93 (vt, 1H, CH, 3JHH = 3.9 Hz), 6.91 (br. s, 1H, H(C7)), 7.48−7.51 (m, 1H, H(C4)), 7.97 (dd, 1H, H(C2), 3JHH = 7.6 Hz, 4JHH = 1.0 Hz), 8.03 (dt, 1H, H(C3), 3JHH = 7.6 Hz, 4JHH = 1.3 Hz), 8.25 (s, 1H, H(C8)), 9.15 (d, 1H, H(C5), 3JHH = 5.4 Hz), 10.65 (br. s, 1H, NH) ppm. 13C{1H} NMR (100.61 MHz, CDCl3): δ 30.71 (s, CH2), 52.03 and 52.11 (both s, OMe and CH), 113.53 (s, C7), 125.17 and 126.38 (both s, C2 and C4), 132.75 (s, C6), 139.66 (c, C3), 149.24(c, C5), 154.85 (c, C1), 171.34 and 171.62 (both s, C(O)N) and C(O)OMe) ppm (the signal of C8 carbon nucleus was not observed). IR (KBr, ν/cm−1): 502(vw), 628(w), 687(w), 760(w), 808(vw), 987(vw), 1019(vw), 1054(w), 1085(w), 1175(m), 1202(m), 1275(w), 1292(w), 1356(w), 1400(m), 1437(w), 1501(w), 1569(m), 1588(s), 1618(m) (νCO in C(O)N), 1740(m) (νCO in C(O)OMe), 2917(m), 3033(w), 3114(w). Anal. Calcd for C13H13ClN4O3Pd·0.25CH2Cl2: C, 36.47; H, 3.12; N, 12.84. Found: C, 36.34; H, 3.47; N, 12.72%. Complex (S)-12 [κ3-N,N,N-(L)Pd(II)Cl]. Yield: 77 mg (87%). Mp: 215−218 °C. 1H NMR (400.13 MHz, CDCl3): δ 2.96 (ddd, 1H, CH2,

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Inorganic Chemistry Table 2. Crystal Data and Structure Refinement Parameters for (R)-7, (S)-9, (S)-10, (R)-9, (R)-10, and (S)-10o empirical formula formwt T (K) cryst syst space group Z a (Å) b (Å) c (Å) β (deg) V (Å3) Dcalcd (g cm−1) linear absorption, μ (cm−1) F(000) 2θmax (deg) no. of reflns measd no. of independent reflns no. of obsd reflns [I > 2σ(I)] params R1 wR2 GOF Δρmax/ Δρmin (e Å−3)

(R)-7

(S)-9

(S)-10

(R)-9

(R)-10

(S)-10o

C22H26Cl2N4O6Pd2S2 790.29 120 monoclinic P21 4 7.880(2) 12.236(3) 14.380(4) 103.094(5) 1350.5(6) 1.943 17.3

C12H15ClN2O3PdS 409.17 296 monoclinic P21 4 11.4307(8) 8.3369(6) 15.8538(11) 92.6810(10) 1509.16(18) 1.801 15.52

C12H14Cl2N2O3PdS 443.61 120 monoclinic P21 4 8.3614(8) 11.6434(11) 15.9392(15) 102.379(2) 1515.7(2) 1.944 17.24

C12H15ClN2O3PdS 409.17 120 monoclinic P21 4 11.303(8) 8.322(6) 16.007(12) 92.767(19) 1503.9(18) 1.807 15.57



784 56 14 506 6501

816 58 30 500 8032

880 58 12 246 7861

816 54 5240 5148

880 56 11 651 6716

1824 60 9930 9930

5666

7372

7631

4942

6427

7436

347 0.0684 0.1710 1.060 5.295/−0.780

365 0.0224 0.0514 1.001 0.350/−0.386

383 0.0255 0.0478 0.999 0.494/−0.565

365 0.0414 0.0996 1.042 0.753/−1.831

383 0.0259 0.0584 1.009 0.676/−0.950

801 0.0546 0.1299 1.027 1.280/−1.228

histidine-based ligand (S)-6, grinding for 2 min resulted in a lightbeige free-flowing powder, which was heated in an open test tube at 85−90 °C for 10 min to yield a reddish powder. Note that the reaction temperature was optimized by preliminary heating of a sample of the homogenized mixture on an MPA 120 EZ-Melt automated melting point apparatus (Stanford Research Systems) and corresponded to the expected changes in the phase state (melting followed by solidification at the higher temperature). Mechanochemical Synthesis in a Vibration Ball Mill. Ligand (S)-4 (315 mg, 1.04 mmol) and PdCl2(NCPh)2 (400 g, 1.04 mmol) were shaken in a stainless steel jar with two grinding balls using a Narva DDR GM 9458 electrically powdered vibration ball mill. Grinding was stopped after several intervals of time to analyze the reaction mixture (Figure S28). The first 10 s of grinding resulted in an essentially compressed nonuniform mixture comprising large areas of dark semisolid and dry yellow powder. Additional grinding for 30 s afforded a brown amorphous mass. In a further 20 s, the mixture converted to a brown paste because of a large amount of benzonitrile released in the reaction course. The resulting residue was thoroughly rinsed with hexane to give a dark-yellow free-flowing powder. X-ray Diffraction. Single crystals suitable for X-ray experiments were obtained by recrystallization from CH2Cl2/CHCl3/hexane ((R)7), CH2Cl2/CHCl3/Et2O ((S)-9, (R)-9, (S)-10o), CH2Cl2/hexane ((S)-10), and CH2Cl2/hexane/Et2O ((R)-10). X-ray diffraction experiments were carried out with a SMART APEX2 DUO CCD diffractometer for compounds (R)-7 and (R)-10 and with a SMART APEX2 CCD diffractometer for the others, using graphite monochromated Mo−Kα radiation (λ = 0.71073 Å, ω-scans) at room temperature ((S)-9) or 120 K (in the other cases). The structures were solved by direct method and refined by the full-matrix least-squares against F2 in anisotropic approximation for non-hydrogen atoms. Positions of the hydrogen atoms were calculated and refined in isotropic approximation in riding model. Crystal data and structure refinement parameters for complexes (R)-7, (S)-9, (S)-10, (R)-9, (R)10, and (S)-10o are given in Table 2. All calculations were performed using the SHELXTL software.34

Crystallographic data for (R)-7, (S)-9, (S)-10, (R)-9, (R)-10, and (S)-10o have been depostied with the Cambridge Crystallographic Data Centre as CCDC 1473760−1473765. Cytotoxicity Assays. The cytotoxicities of the complexes obtained were tested against human colon cancer cell line HCT116, human breast cancer cell line MCF7, human prostate cancer cell line PC3, and normal human embryonic kidney cells HEK293. RPMI-1640 and DMEM media were obtained from Gibco. Fetal bovine serum (FBS) was purchased from HyClone. Cells were cultured in RPMI-1640 (in the case of PC3) or DMEM (in the other cases) media supplemented with 10% FBS, 100 units/L penicillin, and 100 μg/mL streptomycin in a humidified incubator with 5% CO2 atmosphere. The effect of the compounds on cell viability was evaluated by the standard MTT assay (ICN Biomedicals, Germany). Cells were seeded in triplicate at a cell density of 5 × 103/well in 96-well plates in 100 μL complete medium and preincubated for 24 h. The tested compounds were initially dissolved in DMSO. The compounds at various concentrations were added to the media. The well plates were incubated for 48 h followed by addition of MTT solution (Sigma) (20 μL, 5 mg/mL). The cells were incubated at 37 °C for further 3 h; then the culture medium was removed, and formazan crystals were dissolved in DMSO (70 μL). The absorbance of the resulting solutions was measured on a multiwell plate reader (Uniplan, Picon, Russia) at 590 nm to determine the percentage of surviving cells. The reported values of IC50 are the averages of three independent experiments (Table 1). Cisplatin from a commercial source was used as a reference. DNA Binding Experiments. The DNA binding abilities of complexes (R)-8, (S)-9, (S)-10, and (S)-12 were evaluated by agarose gel electrophoresis. In each experiment, a solution of 0.15 μg of supercoiled pHOT1 plasmid DNA (TopoGEN) in TE buffer (1 M Tris-HCl, pH 8.0, 0.1 M EDTA) and the mentioned amount of the palladocycle (used as a stock solution in DMSO; the final concentration of the complexes ranged from 5 to 40 μM) was incubated in the dark at 37 °C for 30 min. The samples of free DNA were used as controls. After incubation, 20 μL aliquots of the resulting solutions were loaded onto a 0.8% agarose gel. The electrophoresis was carried out at 1.5−2.0 V/cm for 8−10 h in TE buffer. The gels 9846


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Inorganic Chemistry were stained with ethidium bromide (0.5 μg/mL) and visualized under UV light. Topoisomerase I Activity Inhibition Assays. The modulation of topoisomerase I activity by complexes (R)-8, (S)-9, (S)-10, and (S)12 was studied using a TopoGEN Topoisomerase I Drug Screening Kit. In the experiments, each of the mentioned palladocycles and one unit of the purified topoisomerase from calf thymus (Fermentas, Lithuania) were incubated with 0.13 μg of supercoiled pHOT1 plasmid DNA (TopoGEN) in a reaction buffer (10 mM Tris-HCl, pH 7.9, 1 mM EDTA, 0.15 M NaCl, 0.1% BSA, 0.1 mM spermidine, 5% glycerol) for 30 min at 37 °C. The reactions were stopped with SDS. After digestion with proteinase K (50 μg/mL, 30−60 min at 37 °C), the samples were mixed with a DNA loading buffer and loaded onto a 0.9% agarose gel. The electrophoresis was performed at the maximal voltage of 2−3 V/cm in a TAE buffer (2 M Tris base, 0.05 M EDTA, 1.56 M acetic acid). The gels were stained with ethidium bromide (0.5 μg/mL) and visualized via UV fluorescence at wavelengths in the range of 240 to 360 nm using a GelDoc-It TS imaging system. In the absence of inhibitors, topoisomerase I relaxed scDNA through the formation of a series of topoisomers. Topoisomerase I inhibition was revealed by the ability of the complexes under investigation to retard scDNA relaxation, which was observed as a decrease in the amount of topoisomers and a retention of scDNA. Investigation of Morphological Changes by Staining Assays. The possibility of involvement of apoptosis in the cytotoxic action of the complexes under investigation was estimated by a conventional staining assay. For this purpose, HCT116 cancer cells were treated with complex (S)-10 for 24 and 48 h. The morphological changes in the cells were observed using a fluorescence microscope after staining with Hoechst 33342 (Sigma, USA) at 37 °C for 5 min and rinsing with cold PBS. The percentage of apoptotic cells was no more than 1.5%.

Temperature dependence of 1H NMR spectrum of complex (S)-9; 1H, 13C{1H}, COSY, HMQC, and HMBC NMR spectra of complex (S)-9; ROESY spectrum of complex (R)-8; IR and Raman spectra of ligand (S)-4 and complex (S)-10; IR, Raman, and 1H NMR spectra of the solid residues obtained after solventfree reactions of ligands (S)-4 and (S)-6 with PdCl2(NCPh)2; photographs of the reaction mixture during grinding of ligand (S)-4 with PdCl2(NCPh)2 in a vibration ball mill; UV−vis spectra of complex (S)-10 in different media; selected bond lengths and angles for complexes (R)-7, (S)-9, (R)-9, (S)-10, (R)-10, and (S)10o; selectivity indices of complexes 7−10 (PDF) Accession Codes

CCDC 1473760−1473765 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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Corresponding Author

*E-mail: [email protected]. ORCID

Diana V. Aleksanyan: 0000-0002-7083-2537 Yulia V. Nelyubina: 0000-0002-9121-0040

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Notes

CONCLUSIONS To summarize the results presented, we have prepared and fully characterized the rare examples of N-metalated Pd(II) pincer complexes based on carboxamide ligands functionalized with amino acids. We have also shown the possibility of solid-phase synthesis of this type of palladocycles. The comparative studies on cytotoxic activity against several human cancer cell lines allowed us to follow the effect of slight changes to the ligand backbone on the properties of the resulting complexes. Thus, the presence of the sulfide ancillary donor group is crucial for manifestation of cytotoxic activity. The methionine-based derivatives with the elongated S-coordination arm in most cases appeared to be more active than their cysteine analogs. The introduction of the chlorine substituent into the pyridine ring facilitated the improvement of cytotoxic properties of N,N,S-derivatives. In general, the cytotoxic activities of the sulfide-based complexes are essentially higher than that of the clinically used drug, cisplatin, and are mainly determined by the presence of the transition metal ions. Therefore, the palladocycles with multidentate ligands based on functionalized carboxamides can be considered as a new potent class of anticancer agents. Taking into account the strong DNA binding and topoisomerase I inhibition activities of all the complexes explored, further detailed investigations are required to elucidate the mechanism of cytotoxic action of this type of Pd(II) complexes.

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AUTHOR INFORMATION

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Russian Foundation for Basic Research, Projects 16-03-00695-a and 16-03-00915-a (solidphase cyclopalladation). The X-ray diffraction and NMR spectroscopic data were obtained using the equipment of Center for Molecule Composition Studies of INEOS RAS.

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REFERENCES

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b01348. 9847


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Inorganic Chemistry

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