Synthesis, structures and mechanistic pathways of

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Synthesis, structures and mechanistic pathways of anticancer activity of palladium(II) complexes with indole-3-carbaldehyde thiosemicarbazones† Jebiti Haribabu,a Manoharan Muthu Tamizh, b Chandrasekar Balachandran,c Yuvaraj Arun, d Nattamai S. P. Bhuvanesh,e Akira Endof and Ramasamy Karvembu *a New PdII complexes of the types [PdCl(L)(PPh3)] (1–5) and [Pd(L)2] (6 and 7) (HL = indole-3-carbaldehyde thiosemicarbazones, HL1–HL5) have been synthesized in order to ascertain the effect of substitution at the terminal nitrogen of thiosemicarbazones on the biological properties of their PdII complexes. The compounds were characterized by elemental analyses, and UV-visible, FT-IR, 1H NMR, DEPT-135 NMR,

13

C NMR,

31

P NMR, 1H–1H COSY, 1H–13C HSQC, 1H–13C HMBC, 1H–31P HMBC and mass spectro-

scopic techniques. The solid state structures of the ligand (HL3) and complexes (2–5 and 6) were determined by single crystal X-ray diffraction analysis. Spectroscopic and crystallographic studies revealed that thiosemicarbazone is coordinated to the PdII ion as a monobasic bidentate (NS ) ligand by forming a five membered ring. To determine the potential of the PdII complexes towards biomolecular interactions, additional experiments involving interaction with calf thymus DNA (CT DNA) and bovine serum albumin (BSA) were carried out. Further, the complexes cleaved DNA (pUC19 and pBR322) without any co-oxidant at pH 7.2 and temperature 37 1C. The effect of substitution on the DNA and BSA binding ability of the complexes was revealed through molecular docking studies. In addition, in vitro anticancer activity was examined by using MTT assay in three cancer cell lines (HepG-2, A549 and MCF7) and one normal cell line (L929). Complexes 4 and 5 which contain triphenylphosphine showed better activity (HepG-2) with an Received 30th September 2017, Accepted 27th April 2018

IC50 value of 22.8 and 67.1 mM respectively. The anticancer activity of the complexes was compared

DOI: 10.1039/c7nj03743k

comparable activity. All the complexes displayed moderate anticancer activity against A549 and MCF7 cancer cell lines and less toxicity towards the normal cell line. The morphological changes assessed by

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staining methods and DNA fragmentation revealed that the cell death occurred by apoptosis.

with that of the well-known anticancer drug cisplatin and it was inferred that complex 4 exhibited

Introduction a

Department of Chemistry, National Institute of Technology, Tiruchirappalli – 620015, India. E-mail: [email protected]; Fax: +91 431 2500133; Tel: +91 431 2503636 b Department of Chemistry, Siddha Central Research Institute, Central Council for Research in Siddha, Arumbakkam, Chennai – 600106, India c Division of Natural Drug Discovery, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan d Organic Chemistry Division, CSIR-Central Leather Research Institute, Chennai – 600020, India e Department of Chemistry, Texas A & M University, College Station, TX 77842, USA f Department of Materials and Life Sciences, Sophia University, 7-1 Kioicho, Chiyoda-ku, Tokyo 102-8554, Japan † Electronic supplementary information (ESI) available. CCDC 1569816, 1569817, 1569818, 1569819, 1569820 and 1814486 for HL3, 2, 4, 5, 3 and 6 respectively. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/ c7nj03743k

Although cisplatin is a well-known anticancer drug, multifactorial resistance, serious side effects on normal cells, and general toxicity have limited its use in cancer chemotherapy.1–3 Therefore, less toxic and highly efficient metal based anticancer compounds have been sought and developed,4–6 and drug delivery systems have been exploited to improve the selectivity of anticancer drugs and decrease their side effects.7 Palladium is one of the alternatives that has shown considerable promise in the development of metal based anticancer drugs.8–13 Palladium complexes are closely related to their platinum analogues due to their structural similarities and significant overlap of coordination chemistry of the two metals. One of the early studies by Graham and co-workers14 advocated the use of palladium complexes as possible anticancer agents. Since this report, many novel palladium complexes have been synthesized, which exhibited promising

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activity against tumor cell lines (lung, prostate, etc.). In several cases, the palladium complexes have exhibited better antitumor activity than their platinum counterparts (cisplatin, carboplatin, etc.). In particular, palladium(II) and palladium(IV) complexes displayed significant cytotoxic activity and apoptosis.15,16 Thiosemicarbazones are a type of N, S donor ligands that have been extensively used in coordination chemistry because of their structural flexibility and versatility since they exist as thione–thiol tautomers, and consequently bind to metal centers as monodentate, bidentate or bridging ligands.17 Thiosemicarbazone derivatives and their metal complexes have aroused considerable interest in chemistry and biology because of their pharmacological properties, particularly as antiphrastic, antibacterial, antioxidant and antitumor agents.18–21 In particular, heterocyclic thiosemicarbazones exhibit beneficial therapeutic properties in mammalian cells by inhibiting ribonucleotide reductase enzyme which inhibits DNA synthesis.22 The effect of the substituent at the terminal nitrogen atom of thiosemicarbazones has been evaluated, with an emphasis on biological properties such as DNA/protein binding and cytotoxic activity.23–29 The results revealed that the binding affinity of the complexes increases with the increase in the bulkiness of the substituent. Indole and its derivatives have occupied a unique place in chemistry because of their biological applications such as antiinflammatory, antimicrobial, anticancer, antidiabetic, and antihypertensive agents.20,30–34 Palladium(II) complexes featuring heterocyclic thiosemicarbazones have attracted considerable attention and many of them exhibit good biomolecular interactions and anticancer activity.35–37 Inspired by the utility of palladium, thiosemicarbazones and indole derivatives in the pharmaceutical field, we report the synthesis, structure, DNA/protein binding, molecular docking, and in vitro anticancer activity of new palladium(II) complexes of indole-3-carbaldehyde thiosemicarbazones.

Scheme 1

Results and discussion Synthesis The indole-3-carbaldehyde thiosemicarbazone ligands (HL1, HL2, HL4 and HL5) used in this work have been synthesized and reported by us earlier.20 One new ligand [(E)-2-((1H-indol-3yl)methylene)-N-ethylhydrazinecarbothioamide (HL3)] was prepared from 3-indole aldehyde and 4-ethyl thiosemicarbazide. The palladium(II) complexes of indole-3-carbaldehyde thiosemicarbazones (1–5) have been obtained by combining the ligands (HL1–HL5) with [PdCl2(PPh3)2] in a 1 : 1 molar ratio (Scheme 1). Complexes 6 and 7 have been made from ligands HL2 and HL4, respectively, and PdCl2 (2 : 1 molar ratio) (Scheme 1) at room temperature. All the new palladium(II) complexes were characterized by analytical and various spectroscopic studies. Spectroscopic characterization The electronic spectra of the complexes (1–7) recorded in DMF showed three bands. Two bands which appeared around 262–269 and 328–360 nm corresponded to p - p* and n - p* intra-ligand transitions respectively. The absorption band at 370–392 nm was due to the ligand-to-metal charge transfer transition (LMCT).38,39 FT-IR absorption bands of the palladium(II) complexes (1–7) were compared with those of the corresponding ligands. The ligands showed an intense band at 1567–1548 cm 1 for CQN stretching. This band was shifted to lower frequency (1515– 1500 cm 1) after complexation. The sharp (813–797 cm 1) and broad (3238–3169 cm 1) bands due to CQS and N–H stretching, respectively, existed in the FT-IR spectra of the ligands. There was a disappearance of both (CQS and N–H) the bands in the spectra of all the complexes and a new band corresponding to the C–S group was observed at 759–747 cm 1.25 These facts suggest the coordination of the ligands to the palladium ion through

Synthetic route toward the palladium(II) complexes.

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thiolate sulfur and azomethine nitrogen. The FT-IR spectra of the complexes showed two bands around 3448–3409 and 3343–3300 cm 1 corresponding to indole N–H and terminal N–H stretching respectively, which are quite similar to those observed in the spectra of the ligands.40,41 The appearance of three new bands around 1434–1429, 1095–1092 and 745–741 cm 1 in the spectra of complexes 1–5 confirmed the presence of triphenylphosphine in these complexes.42 The 1H NMR spectra of the complexes (1–5) exhibited multiplets in the region 7.03–7.98 ppm for the aromatic protons of the coordinated ligand and triphenylphosphine. Assignment for individual aromatic protons has been made based on 1H–1H COSY, 1H–13C HSQC and 1H–13C HMBC spectra. The NMR (1H, 13C, DEPT-135 and 31P) and 2D NMR (1H–1H COSY, 1 H–13C HSQC, 1H–13C HMBC and 1H–31P HMBC) spectra of the compounds (1–5) are shown in Fig. S1–S34 (ESI†). A doublet (4JP,H = 3.8–4.0 Hz) observed in the 1H NMR spectra of the complexes (1–5) in the region 8.65–9.15 ppm has been assigned to the HCQN resonance. 1H–31P HMBC spectra of the complexes revealed that this doublet was due to the four bond cis coupling of phosphorus in triphenylphosphine with the azomethine proton.43 Our group has previously reported the 4JP,H value of 15.13–15.78 Hz (trans coupling) for palladium(II) Schiff base complexes but in the present case the 4JP,H value was low (3.8–4.0 Hz) because of the cis coupling between azomethine proton and phosphorus.44 The coupling between the phosphorus and the aromatic protons of the phenyl rings attached to it was also identified from the 1H–31P HMBC spectra (Fig. 1, 2 and Fig. S12, ESI†). The signal which appeared at 8.15–8.58 ppm as a doublet corresponded to the indole CH proton. The signals due to NH2 protons in 1 appeared as two broad singlets at 7.33 and 11.86 ppm. Complexes 2 and 6 exhibited a doublet in their NMR spectra at 2.94–2.99 ppm that corresponded to methyl protons. The CH3 and CH2 protons of complex 3 resonated

Fig. 1

at 1.20 and 3.38 ppm as a triplet and a quartet of doublet respectively. In the spectra of 2–5, a signal at 4.52–6.39 ppm corresponding to the NH proton was detected. A signal due to the NH proton of complexes 6 and 7 appeared around 12.13– 12.18 ppm. In the spectra of complexes 4 and 7, the terminal cyclohexyl protons were found in the regions 1.10–1.41 and 1.54–2.17 ppm respectively. In the 13C {1H} NMR spectra of all the complexes, azomethine carbon resonance appeared in the 145.9–148.4 ppm range. In the spectra of complexes 1–5, five doublets were observed due to 31P–13C coupling. Three doublets appearing at 130.1–130.7, 134.5–134.6 and 128.2–128.3 ppm were attributed to ipso, ortho and meta carbons in the phenyl rings of triphenylphosphine respectively. A doublet at 168.0– 176.2 ppm was assigned to thiolate carbon (C–S). A week 31P–13C coupling (4JP,C = 2.7–5.0 Hz) observed for indole C2 carbon attributed to long range coupling with a through bond distance of B7.1 Å. In the spectra of 6 and 7, the C–S carbon was observed at 185.1 and 176.1 ppm respectively. In the spectra of 4 and 7, the cyclohexyl carbons were observed at 25.0–25.3, 25.7–25.8, 32.4–32.5 and 55.5–55.6 ppm. The methyl (22.6–31.1 ppm) and ethyl (41.5 and 14.4 ppm) carbon atoms present in complexes 2, 3 and 6 exhibited signals in the expected regions. Assignment for all the aromatic carbon resonances was made with the help of 1H–13C HSQC and 1H–13C HMBC spectra. 31 P NMR spectra of complexes 1–5 exhibited a singlet around 27.0–27.2 ppm suggesting the presence of one coordinated triphenylphosphine in each of these palladium(II) complexes (Fig. S29–S34 and Table S3, ESI†).44 Crystal structure of the complexes The crystal structures of the ligand (HL3) and complexes (2–5 and 6) are shown in Fig. 3, 4 and Fig. S35–S38 (ESI†). The complexes (2–5) crystallized in a monoclinic system with the space group P21/c (2 and 3), P21/n (4) or Pc (5). In the complexes

1

H–31P HMBC NMR spectrum of complex 4.


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Fig. 2

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1

H–31P HMBC NMR spectrum of complex 5.

Fig. 3 Thermal ellipsoid plot of 2 (some of the atoms are not labelled for clarity). Selected bond distances (Å) and angles (1): Pd(1)–Cl(1) 2.3389(5), Pd(1)–P(1) 2.2679(5), Pd(1)–N(1) 2.0791(14), Pd(1)–S(1) 2.2517(5), P(1)–Pd(1)–Cl(1) 89.27(2), N(1)–Pd(1)–Cl(1) 94.62(4), N(1)–Pd(1)–P(1) 174.73(4), N(1)–Pd(1)–S(1) 82.88(4), S(1)–Pd(1)–Cl(1) 171.032(17), S(1)–Pd(1)–P(1) 93.79(2), N(2)–N(1)–Pd(1) 121.12(10). Selected torsion angles (1): Pd(1)–N(1)– N(2)–C(10) 0.64(19), Pd(1)–S(1)–C(10)–N(2) 2.18(16), Pd(1)–S(1)–C(10)–N(4) 178.15(12).

(2–5), the coordination geometry around palladium(II) can be described as distorted square planar; the palladium ion was bonded to the monobasic bidentate NS donor ligand in such a way that a five membered ring was formed and the remaining sites were occupied by one chlorine and one triphenylphosphine.

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One molecule of DMF and one fourth of H2O molecule were found in complexes 2 and 4 respectively. There was a decrease in the CQN and increase in the C–S bond lengths in the complexes compared to those of the corresponding free ligands.20 The N(1)–Pd(1)–P(1) bond angle (1), 174.73(4) [2], 175.07(12) [3],


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Fig. 4 Thermal ellipsoid plot of 6 (symmetry atoms are not labelled for clarity). Selected bond distances (Å) and angles (1): Pd(1)–S(1) 2.2876(3), Pd(1)–N(1) 2.0229(11), N(1)–Pd(1)–S(1) 82.95(3), C(10)–S(1)–Pd(1) 95.19(4), N(2)–N(1)–Pd(1) 120.82(8), C(1)–N(1)–Pd(1) 123.28(9). Selected torsion angles (1): Pd(1)–S(1)–C(10)–N(2) 12.83(12), Pd(1)–S(1)–C(10)–N(4) 167.59(10), Pd(1)–N(1)–N(2)–C(10) 13.41(14).

175.09(14) [4], 176.39(6) [5], and the S(1)–Pd(1)–Cl(1) bond angle (1), 171.032(17) [2], 176.6(7) [3], 171.11(5) [4], 177.06(3) [5], of the complexes deviated from the expected values, suggesting distortion in the square planar geometry. In complexes 2 and 4, the indole hydrogen (N–H) was involved in intermolecular hydrogen bonding with the oxygen atom of DMF (2) and H2O (4). N(4)–H(4) O(1S) and O(1W) N(4)–H(4) bond lengths and angles are shown in Table S4 (ESI†). The other bond lengths and angles are in agreement with the corresponding values of previously reported palladium(II) complexes.24,45 Complex 6 crystallized in the triclinic system with the P1% space group, and adopted a perfect square planer geometry [S(1)–Pd(1)–S(1)#1 = N(1)#1–Pd(1)–N(1) = 180.01] with two indole appended thiosemicarbazone ligands coordinated to the palladium(II) ion in a trans fashion, to form two five membered rings with a bite angle N(1)–Pd(1)–S(1) of 82.95(3)1. The Pd(1)–S(1) and Pd(1)–N(1) bond lengths were found to be 2.2876(3) and 2.0229(11) Å respectively, which were comparable with previous reports.23b,45 In this complex, the indole hydrogen (N–H) was involved in intermolecular hydrogen bonding with the oxygen atom of DMF solvent [N(4)–H(4) O(1S) = 2.9043(15) Å]. DNA interaction studies Electronic absorption spectral characteristics of DNA binding. Metal based compounds bind to DNA through covalent and/or non-covalent interactions.46 In the covalent interactions, the

labile group of metal complexes is exchanged by a nitrogen donor atom from the nucleotide, while non-covalent interactions occur via intercalative, electrostatic or groove binding. Electronic absorption spectroscopy was used to determine the binding mode and binding constant (Kb) of the complexes with CT DNA.47 The electronic absorption spectra in the absence and presence of CT DNA are shown in Fig. 5 and Fig. S39 (ESI†). A compound that binds to DNA via non-covalent intercalation results in hypochromism with or without red shift, which is due to the stacking interaction between the aromatic chromophore and the base pairs of DNA. Upon titration of the complexes with DNA, hypochromism (10–57%) occurred with 1–6 nm red shift. The observed spectral changes were plotted by using [DNA]/ (ea ef) on the y-axis and [DNA] on the x-axis according to the equation48 [DNA]/(ea ef) = [DNA]/(eb ef) + 1/Kb(eb ef), where [DNA] is the concentration of DNA in base pairs, ea is the apparent extinction coefficient value found by calculating A(observed)/[complex], ef is the extinction coefficient for the free compound and eb is the extinction coefficient for the compound in the fully bound form. Kb was calculated from the ratio of the slope and the intercept (Fig. 6). The calculated Kb values were 1.97 105, 1.52 105, 2.09 105, 2.97 105 and 2.20 105 M 1 for complexes 1–5 respectively (Table 1). The results derived from electronic absorption spectral studies showed the high binding efficacy of complexes 4 and 5, which might be due to the bulky N-terminal substituent.


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Fig. 5 Absorption spectra of complex 4 in Tris-HCl buffer upon addition of CT DNA. [Complex] = 2.0 10 5 M, [DNA] = 0–35 mM. The arrow shows that the absorption intensities decrease upon increasing DNA concentration.

Fig. 7 Fluorescence quenching curves of EB bound to DNA in the presence of 4. [DNA] = 5 mM, [EB] = 5 mM and [complex] = 0–50 mM.

complexes to DNA.49 This competitive method gives indirect evidence for the mode of DNA binding.50 Fig. 7 and Fig. S40 (ESI†) show the emission spectra of the EB-DNA system in the absence and presence of the palladium(II) complexes. On addition of the complexes to EB-DNA, the fluorescence intensity at 605 nm was decreased remarkably. The extent of quenching reveals the extent of displacement of the EB molecules from the DNA by the complexes. The quantitative assessment of the interaction of the complexes with DNA is given by the Stern–Volmer equation,51 Fo/F = 1 + Kq[Q], where Fo and F are the fluorescence intensities in the absence and presence of the complex respectively, Kq is a linear Stern–Volmer quenching constant, and [Q] is the concentration of the complex. The slope of the plot of Fo/F versus [Q] gave Kq (Fig. 8). The apparent DNA binding constant (Kapp) values were calculated by using the equation52 KEB[EB] = Kapp [complex], where [complex] is the concentration of the complex at 50% reduction in the

Fig. 6 Plot of [DNA]/(ea with CT DNA.

ef) versus [DNA] for the titration of the complexes

Table 1 DNA binding constant (Kb), Stern–Volmer constant (Kq) and the apparent binding constant (Kapp) for complexes 1–5

Complex 1 2 3 4 5

Kb (M 1) 1.97 1.52 2.09 2.97 2.20

Kq (M 1) 5

10 105 105 105 105

2.79 2.75 3.06 4.39 3.31

Kapp (M 1) 4

10 104 104 104 104

1.39 1.37 1.53 2.19 1.65

106 106 106 106 106

Ethidium bromide (EB) displacement studies The complexes showed no fluorescence at room temperature in solution or in the presence of DNA, and hence, their binding with DNA could not be directly predicted through the emission spectral studies. The extent of quenching of fluorescence due to EB bound to DNA (EB-DNA) is used to predict the binding of the

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Fig. 8 Stern–Volmer plot of fluorescence titrations of the complexes with CT DNA.


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fluorescence intensity of EB, KEB = 1.0 107 M 1 and [EB] = 5 mM. Kq and Kapp values are listed in Table 1. The interaction of the complexes with DNA followed the order 4 4 5 4 3 4 1 4 2. The results were in agreement with that found from the electronic absorption studies, supporting the better DNA binding affinity of 4 and 5 compared to the other complexes. The enhanced binding of 4 and 5 may be due to the presence of bulky cyclohexyl and phenyl groups respectively in the N-terminal position.53 Further, the intercalative binding mode of the complexes with CT DNA was confirmed by viscometry and circular dichroism spectra. The detailed discussion of the results of these experiments is provided in the ESI.† Unwinding of supercoiled DNA The interaction of the palladium(II) complexes (1–5) with pUC19 DNA and pBR322 DNA was investigated. The supercoiled (SC) pUC19 DNA/pBR322 DNA (40 mM) was incubated at 37 1C with complexes 1–5 (100 mM) in 5% DMF/5 mM Tris-HCl/50 mM NaCl buffer at pH 7.2 for 4 h. The cleaving efficiency of palladium(II) complexes 1–5 was assessed from their ability to convert SC (Form I) DNA into nicked circular (NC) (Form II) DNA. No linear DNA (Form III) was observed by agarose gel electrophoresis (Fig. 9 and 10). The pUC19 DNA and pBR322 DNA cleavage efficiency followed the order 5 (90.6%) 4 4 (79.2%) 4 3 (47.9%) 4 2 (23.2%) and 4 (98.1%) 4 5 (80.5%) 4 1 (18.8%) respectively. Complex 1 did not show appreciable cleavage while 5 cleaved pUC19 DNA more efficiently than the other complexes. Complexes 2 and 3 did not cleave while 4 and 5 showed more percentage of pBR322 DNA cleavage than the other complexes.

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BSA interaction studies Electronic absorption and fluorescence spectroscopic studies. UV-visible spectra of bovine serum albumin (BSA) in the presence and absence of the complexes are shown in Fig. 11. On addition of the complexes, the absorption intensity of BSA at 280 nm was enhanced. The formation of unique absorption spectra on addition of the complexes to BSA showed a static quenching mechanism as reported.54 To understand the mechanism of interaction between the palladium(II) complexes and BSA, fluorescence quenching experiments have been carried out. The fluorescence property of BSA arises from intrinsic characteristics of the proteins, mainly due to the presence of tryptophan and tyrosine residues. The fluorescence spectral changes before and after the addition of the palladium(II) complexes are shown in Fig. 12 and Fig. S43

Fig. 11 Absorption spectra of BSA (10 mM) and BSA with 1–5 (4 mM).

Fig. 9 Cleavage of SC pUC19 DNA (40 mM) by complexes 1–5 in a buffer containing 5% DMF/5 mM Tris-HCl/50 mM NaCl at pH = 7.2 and temperature 37 1C after 4 h. Lane 1, DNA control; Lane 2, DNA + 1 (100 mM); Lane 3, DNA + 2 (100 mM); Lane 4, DNA + 3 (100 mM); Lane 5, DNA + 4 (100 mM); Lane 6, DNA + 5 (100 mM).

Fig. 10 Cleavage of SC pBR322 DNA (40 mM) by complexes 1–5 in a buffer containing 5% DMF/5 mM Tris-HCl/50 mM NaCl at pH = 7.2 and temperature 37 1C after 4 h. Lane 1, DNA control; Lane 2, DNA + 1 (100 mM); Lane 3, DNA + 2 (100 mM); Lane 4, DNA + 3 (100 mM); Lane 5, DNA + 4 (100 mM); Lane 6, DNA + 5 (100 mM).

Fig. 12 Fluorescence quenching curves of BSA in the absence and presence of 4. [BSA] = 1 mM and [complex] = 0–20 mM.


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NJC Table 2 Protein binding constant (Kb), quenching constant (Kq) and number of binding sites (n) for complexes 1–5

Kb (M 1)


Complex 1 2 3 4 5

Fig. 13 Stern–Volmer plot of the fluorescence titrations of the complexes with BSA.

(ESI†). Addition of palladium(II) complexes 1–5 to BSA resulted in the quenching of its fluorescence intensity at 340 nm by 74.9, 71.1, 75.7, 80.3 and 83.2% with hypsochromic shift of 1, 4, 2, 1 and 3 nm respectively. The results obtained confirmed the interaction of the palladium(II) complexes with BSA. To have a quantitative insight into the quenching process, the quenching constant (Kq) was assessed following the Stern–Volmer equation (Fig. 13). Further, the equilibrium binding constant was evaluated using the Scatchard equation log[(Fo F)/F] = log Kb + n log[Q], where F and Fo are the fluorescence intensities in the presence and absence of the complex respectively, Kb is the binding constant and n is the number of binding sites. The Kb values were derived from the graph between log[(Fo F)/F] and log[Q] (Fig. 14). The values of Kb and Kq revealed the enhanced binding ability of complex 4 compared to the other complexes under

1.48 1.31 1.97 2.69 2.09

Kq (M 1) 7

1.14 1.05 1.21 1.91 1.58

10 107 107 107 107

n 5

10 105 105 105 105

1.09 1.04 1.14 1.17 1.03

investigation (Table 2). Synchronous fluorescence spectra confirmed that the interaction of the complexes with BSA affected the microenvironments of both tyrosine and tryptophan residues. The detailed discussion of synchronous fluorescence spectroscopic studies is provided in the ESI.† Effect of substituents on DNA and BSA binding: molecular docking with B-DNA, DNA-T and BSA. The palladium(II) complexes and free PPh3 were subjected to molecular docking studies using the AutoDock Tools (ADT) version 1.5.6 and AutoDock version 4.2.5.1 to gain insights into the possible reasons for the biological activity.55 Docking of the compounds was done with the receptor binding site of B-DNA (PDB ID: 1BNA) dodecamer d(CGCGAATTCGCG)2, human DNA-Topoisomerase I complex (DNA-T) (PDB ID: 1SC7) and bovine serum albumin (BSA) (PDB ID: 3V03).56 To find the binding mode of the potent inhibitors, docked conformation of the complexes with receptors was analyzed in terms of energy, hydrogen bonding and hydrophobic interactions. The detailed analysis of complex–receptor interactions was carried out, and the final coordinates were saved. Then, the output was exported to PyMol software to display the interaction of the complexes with the receptor binding site.57 The free energy of binding (FEB) of the compounds was estimated from the docking scores and the details are shown in Table 3. Inhibition of DNA is vital in the designing of chemotherapeutic drugs, in order to arrest the replication step in the cell cycle. The complexes block the cell functions by destabilizing the DNA structure. In the present study, the palladium(II) complexes and PPh3 are docked with the 1BNA receptor. The results revealed that all the docked palladium complexes bound efficiently with the 1BNA receptor and showed the binding energy value from 8.63 to 9.40 kcal mol 1. Complex 4 showed a binding energy of 9.40 kcal mol 1 with two hydrogen bonding interactions with the docked 1BNA receptor. Indole N–H interacted with the CQO of DC-9 to form a hydrogen bond (1.9 Å). Table 3 The calculated FEB of the complexes with B-DNA, DNATopoisomerase I and BSA

Free energy of binding (kcal mol 1)a Compound

Fig. 14 Scatchard plot of the fluorescence titrations of the complexes with BSA.

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1 2 3 4 5 PPh3 a

B-DNA (PDB ID: 1BNA) 9.28 8.63 9.22 9.40 8.96 6.60

DNA-Topoisomerase-I (PDB ID: 1SC7) 11.16 11.05 11.38 12.30 12.04 7.39

BSA (PDB ID: 3V03) 6.94 7.05 7.03 8.05 7.98 6.71

Calculated by Autodock.


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Imine nitrogen interacted with the N–H hydrogen of DG-16 and formed a hydrogen bond (2.2 Å). Furthermore, terminal N–H exhibited polar interactions with DG-10 and DG-16. In addition, phenyl rings of PPh3 exhibited hydrophobic interactions with the 1BNA receptor. The interactions of the compounds (1–5 and PPh3) with the 1BNA receptor are shown in Fig. S46 (ESI†). Topoisomerase I is bound to the oligonucleotide sequence 50-AAAAAGACTTsX-GAAAATTTTT-30 in the human DNA-Topoisomerase I complex (PDB ID: 1SC7), in which ‘s’ is 50-bridging phosphorothiolate of the cleaved strand and ‘X’ represents any of the four bases A, G, C or T. The SH of G11 on the scissile strand was changed to OH and the phosphoester bond of G12 in 1SC7 was rebuilt.58 Docking of the complexes and PPh3 with DNA-Topoisomerase I revealed that the complexes approached towards the DNA cleavage site to form a stable complex and subsequently leading to an inhibitory effect. The binding energy of the compounds (1–5 and PPh3) with DNA-Topoisomerase I was from 6.94 to 12.30 kcal mol 1. Among all the palladium(II) complexes and PPh3, complex 4 showed the highest binding energy value ( 12.30 kcal mol 1) with the docked 1SC7. N–H attached with the cyclohexyl ring in 4 interacted with the pentose oxygen of TGP-11 and formed a hydrogen bond with a bond length of 2.5 Å. Furthermore, p - p and polar interactions were found between the indole ring and DA-113. Also, phenyl rings of PPh3 exhibited hydrophobic interactions with the docked receptor. Interactions of the compounds (1–5 and PPh3) with DNA-Topoisomerase I receptor are shown in Fig. S47 (ESI†). Docking studies of all the complexes and PPh3 with the BSA (PDB ID: 3V03) receptor showed that all the compounds bound efficiently with the receptor and exhibited free energy of binding from 6.71 to 8.05 kcal mol 1. The possible drug binding site in BSA was taken from the literature.59 The study revealed that all the complexes effectively bound to the 22 active sites of amino acids, namely, PHE-205, ARG-208, ALA-209, LYS-211, TRP-213, VAL-215, PHE-227, THR-231, VAL-234, THR-235, ASP-323, LEU-326, GLY-327, LEU-330, SER-343, LEU-346, ALA-349, LYS-350, GLU-353, SER-479, LEU-480 and VAL-481. Among all the compounds docked with the 3V03 receptor, complex

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4 exhibited the highest binding energy of 8.05 kcal mol 1. N–H attached with the cyclohexyl ring in 4 formed a hydrogen bond (2.1 Å) with the CQO of ARG-208. Furthermore, there was a polar interaction between indole N–H and ARG-208. In addition to the polar interaction, the cyclohexyl ring and phenyl rings of PPh3 exhibited hydrophobic interactions with LYS-211, VAL-215, PHE227, GLY-327, LEU-346 and LYS-350. Interactions of the compounds with the BSA receptor are shown in Fig. S48 (ESI†). In vitro anticancer activity The anticancer activity of the complexes (1–7) has been evaluated against HepG-2, A549, MCF7 and L929 normal cell lines. Though all the tested complexes showed good activity against HepG-2 cells (Table S5 (ESI†) and Fig. 15), complexes 4 and 5 showed promising cell death with an IC50 value of 22.8 and 67.1 mM respectively. The results were compared with the activity of the anticancer drug cisplatin and it was inferred that complex 4 showed similar activity. All the complexes showed moderate activity against A549 and MCF7 cells (Table S5 and Fig. 16 and Fig. S49, ESI†). Samples with all concentrations used in the experiment decreased the cell viability significantly in a concentration dependent manner (Table S5, ESI†). Complexes 6 and 7 which do not contain triphenylphosphine showed poor/ moderate activity in the cancer cell lines. Complexes 1–5 showed better activity than 6 [IC50 4 100 mM (HepG-2 and MCF7)] and 7 [IC50 = 96.3 (HepG-2) and 4100 mM (MCF7)] in HepG-2 and MCF7 cells (Fig. 15 and Fig. S49, ESI†). Complex 7 showed slightly better activity in A549 cells (Table S5 (ESI†) and Fig. 16) with an IC50 value of 80.4 mM but still it was inferior to complexes 1–5. Overall, complex 4 displayed significant activity than the other complexes, which may be due to the presence of a bulky substituent (cyclohexyl) at the nitrogen atom of the thiosemicarbazone and triphenylphosphine ligand.53 The complexes exhibited different activity in various cell lines, which exposed the specificity of the complexes against various cancer targets. Additional studies on the structure–activity relationship and gene profiling, in future, will provide more insight into the specific activity of the complexes against various cancer cell lines. Similarly, the activity

Fig. 15 Comparison of anticancer activity of complexes 1–7 against HepG-2 cancer cells. Data are mean SD of three independent experiments with each experiment conducted in triplicate (24 h incubation).


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Fig. 16 Comparison of anticancer activity of complexes 1–7 against A549 cancer cells. Data are mean SD of three independent experiments with each experiment conducted in triplicate (24 h incubation).

profiles of the complexes against HepG-2, A549 and MCF7 cell lines are comparable to those of previously reported similar palladium(II) complexes.23b,28b,53,60 Fortunately, all the complexes were less toxic towards the normal cell (L929) as evident from the higher IC50 values (552.9–880 mM) (Table S5 and Fig. S50, ESI†). DNA fragmentation and fluorescence staining studies There are many pathways in which cell death occurs in response to a variety of external and physiological stimuli. Apoptosis is an important mechanism, by which the cell death occurs in a controlled and regulated fashion whereas other cell death pathways like necrosis lead to serious health issues.61–63 This makes apoptosis distinct from the other forms of cell death. Therefore, in order to have an insight into the cell death mechanism, DNA fragmentation and fluorescence studies have been carried out.

DNA fragmentation was demonstrated by incubating HepG-2 cells with complexes 1–5 at their IC50 concentrations for 24 h. DNA fragmentation became apparent and dose dependent (Fig. 17). When cells were treated with IC50 concentration of the complexes, DNA ladders were visible after 24 h. Complexes 4 and 5 showed effective DNA fragmentation compared to the other complexes (1–3), which was in agreement with the anticancer results. HepG-2 cells treated with palladium(II) complexes 2 (93.5 mM), 4 (22.8 mM) and 5 (67.1 mM) were subjected to confocal microscopic studies. In this study, DAPI, FITC and PI fluorescence stains were used. DAPI binds strongly to A–T rich regions in DNA and can pass through an intact cell membrane. FITC acts as a phosphatidyl serine tracer and suggests the presence of apoptosis. PI can only penetrate cells where the cell membrane has been compromised. The confocal microscopic images showed that significant morphological changes were found in HepG-2 cells

Fig. 17 Detection of DNA fragmentation by agarose gel electrophoresis (2%). The apoptotic DNA fragmentation was detected by agarose gel electrophoresis in HepG-2 cells treated with complexes 1–5 for 24 h. Lane 1, 1 kb DNA ladder; Lane 2, control; Lane 3, + 1; Lane 4, + 2; Lane 5, + 3; Lane 6, + 4; Lane 7, + 5 and Lane 8, 100 bp DNA ladder. The arrows indicate DNA fragmentation compared to the control.

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Fig. 18 DAPI (blue), FITC (green) and PI (red) fluorescence staining for the detection of apoptosis in HepG-2 cells. Cells were treated with complexes 2, 4 and 5 at their IC50 concentration. The fluorescence signals of DAPI, FITC and PI were examined under a confocal laser scanning microscope. Control (a1 – DAPI, a2 – FITC, a3 – PI and a4 – merged) and treated (2: b1 – DAPI, b2 – FITC, b3 – PI and b4 – merged; 4: c1 – DAPI, c2 – FITC, c3 – PI and c4 – merged; 5: d1 – DAPI, d2 – FITC, d3 – PI and d4 – merged). The arrows indicate apoptotic cancer cells.

after treatment with the complexes. Apoptotic cells are indicated by arrows in Fig. 18.

Conclusion In this paper, we report the synthesis and complete characterization of the palladium(II) complexes (1–7) which were obtained from the reaction between [PdCl2(PPh3)2] or PdCl2 and 4N-un/substituted thiosemicarbazone ligands (HL1–HL5). The molecular structures of the ligand (HL3) and the complexes (2–5 and 6) were confirmed by single crystal X-ray crystallography. In order to know about the potential binding ability of the palladium(II) complexes, CT DNA and BSA protein were taken as models. The binding ability of the complexes with CT DNA was assessed using absorption, emission, viscosity and circular dichroism measurements, which showed that complexes 4 and 5 have higher binding affinity with CT DNA than the rest of the complexes. EB displacement studies revealed the capacity of the complexes to displace EB from the EB-DNA complex. The DNA cleavage experiments showed that complexes 4 and 5 have better cleaving ability in pBR322 DNA and pUC19 DNA respectively. The complexes bound with BSA protein through static quenching. Among the palladium(II) complexes, 4 exhibited better binding affinity with BSA protein. Molecular docking was employed to

understand the binding of the palladium(II) complexes with the molecular targets B-DNA, human DNA-Topoisomerase I and BSA. The palladium(II) complexes showed prominent cytotoxic activity against HepG-2 and moderate activity against A549 and MCF7 cells. The morphological changes in HepG-2 cell lines assessed by DNA fragmentation and staining methods disclosed that the possible way of cell death was through apoptosis. The bulkiness of the substituent at the terminal nitrogen of thiosemicarbazone and the presence of triphenylphosphine had a positive effect on the anticancer activity of the complexes; as a result, 4 and 5 were promising candidates. Animal model studies are to be carried out with the palladium(II) complexes in order to establish their potential as clinical drugs.

Experimental Materials and methods Chemicals and solvents obtained from commercial suppliers (Sigma Aldrich and Alfa Asear) were used as received and were of analytical grade. Protein free calf thymus DNA (CT DNA), plasmid DNA, bovine serum albumin (BSA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) were purchased from Hi Media Ltd. A549, MCF7 and L929 cell lines were purchased


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from NCCS (National Centre for Cell Science), Pune, India, and the HepG-2 cell line was purchased from ATCC (American Type Culture Collection), Manassas, VA, USA. FT-IR spectra were recorded as KBr pellets using a Nicolet-iS5 spectrophotometer. UV-visible spectra were recorded using a Shimadzu-2600 spectrophotometer. Emission spectra were measured on a Jasco V-630 spectrophotometer using 5% DMF in buffer as solvent. 1H, 13C, DEPT-135 and 31P NMR spectra were recorded in DMSO-d6/CDCl3 solvent on a Bruker spectrometer using tetramethylsilane (TMS) as the internal standard. The 1H–1H COSY, 1H–13C HSQC, 1H–13C HMBC and 1H–31P HMBC NMR spectra were obtained by using the standard Bruker pulse program. Melting points were determined in open capillary tubes on a Sigma melting point apparatus and are uncorrected. ESI-MS spectra were recorded using a high resolution Bruker maXis impact mass spectrometer. The DNA/protein binding, molecular docking and anticancer activity studies were carried out for the complexes according to the literature procedures.20,54,64 Synthesis of (E)-2-((1H-indol-3-yl)methylene)-Nethylhydrazinecarbothioamide (HL3) The synthesis and characterization of the ligands (HL1, HL2, HL4 and HL5) were reported by us earlier.20 (E)-2-((1H-indol-3yl)methylene)-N-ethylhydrazinecarbothioamide (HL3) was synthesized from 3-indole aldehyde (0.145 g, 0.001 mol) and 4-ethyl thiosemicarbazide (0.119 g, 0.001 mol) in ethanol (15 mL) in the presence of acetic acid (1–2 drops). The solution was refluxed for 3 h and the white precipitate that appeared was filtered off. It was then washed with cold ethanol and hexane, and dried under vacuum. The single crystals of HL3 grown from an acetonitrile–dichloromethane mixture (1 : 1) were found to be suitable for X-ray diffraction. Yield: 89%. White. M.p.: 161 1C. Anal. calc. for C12H14N4S (%): C, 58.51; H, 5.73; N, 22.74; S, 13.02. Found: C, 58.39; H, 5.82; N, 22.89; S, 13.11. UV-vis (CH3OH): lmax, nm 260, 328. FT-IR (KBr): n, cm 1 3437 (N–H), 3320, 3231 (H–N–CQS), 1550 (CQN), 795 (CQS). 1H NMR (500 MHz, DMSO-d6): d ppm 1.16 (t, J = 7.1 Hz, 3H, H12), 3.58–3.65 (m, 2H, H11), 7.17 (td, J = 10.8, 4.0 Hz, 1H, H6), 7.20 (td, J = 11.0, 4.1 Hz, 1H, H5), 7.45 (d, J = 7.9 Hz, 1H, H7), 7.75 (d, J = 2.6 Hz, 1H, H4), 7.90 (t, J = 5.8 Hz, 1H, NH), 8.17 (d, J = 7.7 Hz, 1H, H9), 8.28 (s, 1H, H1), 10.95 (s, 1H, NH), 11.53 (s, 1H, NH). 13C NMR (126 MHz, DMSO-d6): d ppm 15.2 (C12), 38.8 (C11), 111.4 (C2), 112.4 (C7), 121.3 (C4), 122.1 (C5), 123.3 (C6), 124.3 (C3), 131.2 (C9), 137.4 (C8), 141.3 (C1), 175.8 (C10). TOF-MS-ES+ (m/z): found (calcd) 247.1022 (247.1017) [M + H]+. Synthesis of the [PdCl(L)(PPh3)] type complexes The ligand (1 mmol) dissolved in CH2Cl2 (10 mL) was added to the suspension of [PdCl2(PPh3)2] (1 mmol) in ethanol (10 mL). Then a few drops of Et3N were added. The resultant orange colour solution was refluxed at 80 1C for 5 h, and then reduced to 3 mL and cooled. Hexane (20 mL) was then added whereupon the product complex separated. The orange coloured complex was isolated by filtration, and washed with ethanol and hexane, before drying in vacuum. [PdCl(L1)(PPh3)] (1). [PdCl2(PPh3)2] (701 mg, 1 mmol) and HL1 (218 mg, 1 mmol) were used. Yield: 80%. Orange coloured

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solid. M.p.: 221 1C. Anal. calc. for C28H24ClN4PPdS (%): C, 54.12; H, 3.89; N, 9.02; S, 5.16. Found: C, 54.03; H, 3.81; N, 9.16; S, 5.05. UV-vis (DMF): lmax, nm 268, 360, 377. FT-IR (KBr): n, cm 1 3448, 3343 (N–H), 1500 (CQN), 757 (C–S), 1430, 1097, 745 (bands due to PPh3). 1H NMR (500 MHz, CDCl3): d ppm 7.16–7.23 (m, 2H, H5, H6), 7.33 (bs, 1H, NH(NH2)), 7.38 (dd, J = 6.7, 1.7 Hz, 1H, H7), 7.40–7.46 (m, 6H, Hm), 7.46–7.51 (m, 3H, Hp), 7.73–7.81 (m, 6H, Ho), 7.90–7.94 (m, 1H, H4), 8.58 (d, J = 2.9 Hz, 1H, H9), 9.03 (d, J = 4.0 Hz, 1H, H1), 9.32 (bs, 1H, NH), 11.86 (bs, 1H, NH(NH2)). 13C NMR (126 MHz, CDCl3): d ppm 108.8 (C2, d, J = 2.7 Hz), 111.5 (C7), 118.8 (C4), 121.4 (C5), 122.9 (C6), 127.7 (C3), 128.2 (d, J = 10.9 Hz, Cm), 130.1 (d, J = 53.6 Hz, Cq), 131.0 (Cp), 133.0 (C9), 134.5 (d, J = 10.9 Hz, Co), 135.1 (C8), 147.1 (C1), 171.2 (d, J = 10.9 Hz, C10). 31P NMR (203 MHz, CDCl3): d ppm 27.0 (P1). ESI-MS (m/z): found (calcd) 585.0551 (585.0594) [M Cl]+. [PdCl(L2)(PPh3)] (2). [PdCl2(PPh3)2] (701 mg, 1 mmol) and HL2 (232 mg, 1 mmol) were used. Yield: 77%. Orange coloured solid. M.p.: 258 1C (dec.). Anal. calc. for C29H26ClN4PPdS (%): C, 54.81; H, 4.12; N, 8.82; S, 5.05. Found: C, 54.75; H, 4.04; N, 8.90; S, 5.11. UV-vis (DMF): lmax, nm 267, 357, 373. FT-IR (KBr): n, cm 1 3412, 3340 (N–H), 1500 (CQN), 749 (C–S), 1429, 1095, 745 (bands due to PPh3). 1H NMR (500 MHz, CDCl3): d ppm 2.99 (d, J = 4.9 Hz, 3H, H11), 4.68 (bs, 1H, NH), 7.19–7.25 (m, 2H, H5, H6), 7.39 (dd, J = 7.1, 1.0 Hz, 1H, H7), 7.41–7.45 (m, 6H, Hm), 7.46–7.50 (m, 3H, Hp), 7.74–7.81 (m, 6H, Ho), 7.98 (d, J = 7.3 Hz, 1H, H4), 8.57 (d, J = 2.3 Hz, 1H, H9), 8.61 (bs, 1H, NH), 9.10 (d, J = 3.8 Hz, 1H, H1). 13C NMR (126 MHz, CDCl3): d ppm 31.6 (C11), 109.4 (C2, d, J = 5.0 Hz,), 111.4 (C7), 118.9 (C4), 121.3 (C5), 122.9 (C6), 128.1 (C3), 128.2 (d, J = 10.0 Hz, Cm), 130.7 (d, J = 50.4 Hz, Cq), 130.9 (Cp), 132.6 (C9), 134.6 (d, J = 10.9 Hz, Co), 135.0 (C8), 146.3 (C1), 176.2 (d, J = 12.6 Hz, C10). 31P NMR (203 MHz, CDCl3): d ppm 27.14 (P1). ESI-MS (m/z): found (calcd) 599.0681 (599.0651) [M Cl]+. The crystals of 2 grown from CHCl3/DMF solution were found to be suitable for X-ray diffraction. [PdCl(L3)(PPh3)] (3). [PdCl2(PPh3)2] (701 mg, 1 mmol) and HL3 (246 mg, 1 mmol) were used. Yield: 82%. Orange coloured solid. M.p.: 253 1C. Anal. calc. for C30H28ClN4PPdS (%): C, 55.48; H, 4.35; N, 8.63; S, 4.94. Found: C, 55.59; H, 4.43; N, 8.55; S, 5.03. UV-vis (DMF): lmax, nm 266, 358, 374. FT-IR (KBr): n, cm 1 3434, 3338 (N–H), 1504 (CQN), 748 (C–S), 1434, 1094, 745 (bands due to PPh3). 1H NMR (500 MHz, CDCl3): d ppm 1.20 (t, J = 7.2 Hz, 3H, H12), 3.38 (qd, J = 7.2, 5.2 Hz, 2H, H11), 4.52 (t, J = 5.1 Hz, 1H, NH), 7.21 (dtd, J = 16.2, 7.2, 7.2, 1.2 Hz, 2H, H5, H6), 7.38 (dt, J = 7.9, 0.9 Hz, 1H, H7), 7.39–7.45 (m, 6H, Hm), 7.46–7.50 (m, 3H, Hp), 7.74–7.81 (m, 6H, Ho), 7.94–7.98 (m, 1H, H4), 8.51 (d, J = 2.9 Hz, 1H, H9), 8.64 (bs, 1H, NH), 9.08 (d, J = 4.0 Hz, 1H, H1). 13C NMR (126 MHz, CDCl3): d ppm 14.4 (C12), 41.5 (C11), 109.6 (d, J = 2.7 Hz, C2), 111.3 (C7), 119.04 (C4), 121.4 (C5), 123.0 (C6), 127.8 (C3), 128.2 (d, J = 11.8 Hz, Cm), 130.4 (d, J = 53.6 Hz, Cq), 130.9 (Cp), 132.2 (C9), 134.6 (d, J = 10.9 Hz, Co), 135.0 (C8), 146.2 (C1), 170.3 (d, J = 12.0 Hz, C10). DEPT-135 (126 MHz, CDCl3): d ppm 14.4 (C12), 41.5 (C11), 111.2 (C7), 119.0 (C4), 121.3 (C5), 123.0 (C6), 128.2 (d, J = 11.3 Hz, Cm), 130.9 (Cp), 132.1 (C9), 134.6 (d, J = 11.3 Hz, Co), 146.2 (C1). 31P NMR (203 MHz, CDCl3): d ppm 27.1 (P1). ESI-MS


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(m/z): found (calcd) 613.0647 (613.0607) [M Cl]+. The crystals of 3 grown from CHCl3/DMF solution were found to be suitable for X-ray diffraction. [PdCl(L4)(PPh3)] (4). [PdCl2(PPh3)2] (701 mg, 1 mmol) and HL4 (300 mg, 1 mmol) were used. Yield: 75%. Orange coloured solid. M.p.: 265 1C. Anal. calc. for C34H34ClN4PPdS (%): C, 58.04; H, 4.87; N, 7.96; S, 4.56. Found: C, 57.97; H, 4.81; N, 8.09; S, 4.50. UV-vis (DMF): lmax, nm 266, 359, 377. FT-IR (KBr): n, cm 1 3425, 3300 (N–H), 1505 (CQN), 755 (C–S), 1433, 1094, 745 (bands due to PPh3). 1H NMR (500 MHz, CDCl3): d ppm 1.14–1.29 (m, 3H, H12a, H14a), 1.30–1.41 (m, 2H, H13a), 1.61–1.66 (m, 1H, H14e), 1.75 (dt, J = 13.3, 3.7 Hz, 2H, H13e), 2.08–2.17 (m, 2H, H12e), 3.64 (tdt, J = 10.3, 10.3, 7.0, 3.7, 3.7 Hz, 1H, H11), 4.56 (d, J = 7.2 Hz, 1H, NH), 7.19–7.26 (m, 2H, H5, H6), 7.39 (dd, J = 7.1, 1.1 Hz, 1H, H7), 7.41–7.46 (m, 6H, Hm), 7.47–7.51 (m, 3H, Hp), 7.74–7.83 (m, 6H, Ho), 7.98 (d, J = 7.5 Hz, 1H, H4), 8.51 (d, J = 2.7 Hz, 1H, H9), 8.71 (bs, 1H, NH), 9.08 (d, J = 4.0 Hz, 1H, H1). 13C NMR (126 MHz, CDCl3): d ppm 25.0 (C13), 25.7 (C14), 32.5 (C12), 55.5 (C11), 109.7 (d, J = 5.0 Hz, C2), 111.1 (C7), 119.1 (C4), 121.3 (C5), 123.0 (C6), 127.8 (C3), 128.2 (d, J = 10.9 Hz, Cm), 130.4 (d, J = 54.5 Hz, Cq), 130.9 (Cp), 131.5 (C9), 134.6 (d, J = 10.9 Hz, Co), 135.0 (C8), 145.9 (C1), 169.3 (d, J = 12.0 Hz, C10). DEPT-135 (126 MHz, CDCl3): d ppm 25.0 (C13), 25.7 (C14), 32.4 (C12), 55.5 (C11), 111.1 (C7), 119.1 (C4), 123.0 (C6), 128.2 (d, J = 11.3 Hz, Cm), 130.9 (Cp), 131.5 (C9), 134.6 (d, J = 11.3 Hz, Co), 145.9 (C1). 31P NMR (203 MHz, CDCl3): d ppm 27.2 (P1). ESI-MS (m/z): found (calcd) 667.1317 (667.1377) [M Cl]+. The single crystals of 4 grown from CHCl3/ DMF solution were found to be suitable for X-ray diffraction. [PdCl(L4)(PPh3)] (5). [PdCl2(PPh3)2] (701 mg, 1 mmol) and HL5 (294 mg, 1 mmol) were used. Yield: 81%. Orange coloured crystals. M.p.: 286 1C. Anal. calc. for C34H28ClN4PPdS (%): C, 58.54; H, 4.05; N, 8.03; S, 4.60. Found: C, 58.62; H, 4.00; N, 8.10; S, 4.67. UV-vis (DMF): lmax, nm 269, 360, 370. FT-IR (KBr): n, cm 1 3445, 3343 (N–H), 1500 (CQN), 749 (C–S), 1430, 1095, 741 (bands due to PPh3). 1H NMR (500 MHz, CDCl3): d ppm 6.39 (bs, 1H, NH), 7.03–7.08 (m, 1H, H14), 7.18–7.25 (m, 2H, H5, H6), 7.27–7.31 (m, 2H, H12), 7.33–7.37 (m, 1H, H7), 7.42–7.47 (m, 8H, Hm, H13), 7.48–7.52 (m, 3H, Hp), 7.76–7.83 (m, 6H, Ho), 7.92–7.96 (m, 1H, H4), 8.52 (d, J = 2.8 Hz, 1H, H9), 8.58 (bs, 1H, NH), 9.15 (d, J = 3.8 Hz, 1H, H1). 13C NMR (126 MHz, CDCl3): d ppm 109.1 (d, J = 3.6 Hz, C2), 111.27 (C7), 118.9 (C4), 120.8 (C13), 121.6 (C5), 123.1 (C6), 123.4 (C14), 127.7 (C3), 128.3 (d, J = 11.8 Hz, Cm), 128.8 (C12), 130.3 (d, J = 54.5 Hz Cq), 131.0 (Cp), 132.0 (C9), 134.6 (d, J = 10.9 Hz Co), 134.9 (C11), 140.9 (C8), 148.4 (C1), 168.0 (d, J = 12.0 Hz, C10). DEPT-135 (126 MHz, CDCl3): d ppm 111.2 (C7), 118.9 (C4), 120.8 (C13), 121.6 (C5), 123.1 (C6), 123.4 (C14), 128.3 (d, J = 12.5 Hz, Cm), 128.8 (C12), 131.0 (Cp), 134.6 (d, J = 11.3 Hz, Co), 134.9 (C11), 148.4 (C1). 31P NMR (203 MHz, CDCl3): d ppm 27.1 (P1). ESI-MS (m/z): found (calcd) 661.0844 (661.0807) [M Cl]+. The single crystals of 5 grown from CH3CH2OH/DMF were found to be suitable for X-ray diffraction.

appropriate indole thiosemicarbazone ligand (2 mmol) in acetonitrile (15 mL). The reaction mixture was stirred for 4 h at room temperature, and then the precipitate formed was filtered off, washed with hexane and dried in vacuo. Suitable crystals for X-ray diffraction were grown from the CHCl3–DMF mixture (3 : 1). [Pd(L2)2] (6). PdCl2 (170 mg, 1 mmol) and HL2 (464 mg, 2 mmol) were used. Yield: 81%. Orange colored solid. M.p.: 297 1C. Anal. calc. for C22H22N8PdS2 (%): C, 46.44; H, 3.90; N, 19.69; S, 11.27. Found: C, 46.36; H, 3.99; N, 19.84; S, 11.14. UV-vis (DMF): lmax, nm 262, 331, 387. FT-IR (KBr): n, cm 1 3436, 3321 (N–H), 1513 (CQN), 747 (C–S). 1H NMR (400 MHz, DMSO-d6): d ppm 2.94 (d, J = 4.8 Hz, 3H, CH3), 7.19–7.34 (m, 2H, aromatic-H), 7.57 (d, J = 6.8 Hz, 1H, aromatic-H), 7.73 (d, J = 7.0 Hz, 1H, aromatic-H), 8.35 (s, 1H, CH), 8.51 (q, J = 3.9 Hz, 1H, NH), 8.73 (s, 1H, CHQN), 12.18 (bs, 1H, NH). 13C NMR (100 MHz, DMSO-d6): d ppm 31.1 (methyl carbon), 107.4, 112.9, 121.1, 123.0, 124.0, 128.7, 135.7, 137.6 (aromatic carbons), 145.2 (CQN), 185.4 (C–S). ESI-MS (m/z): found (calcd) 569.0521 (569.0522) [M + H]+. [Pd(L4)2] (7). PdCl2 (170 mg, 1 mmol) and HL4 (600 mg, 2 mmol) were used. Yield: 79%. Orange colored solid. M.p.: 308 1C. Anal. calc. for C32H38N8PdS2 (%): C, 54.50; H, 5.43; N, 15.89; S, 9.09. Found: C, 54.61; H, 5.39; N, 15.74; S, 9.21. UV-vis (DMF): lmax, nm 269, 328, 392. FT-IR (KBr): n, cm 1 3420, 3332 (N–H), 1515 (CQN), 750 (C–S). 1H NMR (400 MHz, DMSO-d6): d ppm 1.10–1.22 (m, 3H, cyclohexyl-H), 1.26–1.35 (m, 2H, cyclohexyl-H), 1.54–1.95 (m, 5H, cyclohexyl-H), 4.21 (m, 1H, cyclohexyl-H), 7.17–7.12 (m, 2H, aromatic-H), 7.44 (d, J = 6.4 Hz, 1H, aromatic-H), 7.50 (d, J = 6.3 Hz 1H, aromatic-H), 8.15 (s, 1H, CH), 8.34 (s, 1H, NH), 8.65 (s, 1H, CHQN), 12.13 (bs, 1H, NH). 13C NMR (100 MHz, DMSO-d6): d ppm 25.3, 25.8, 32.4, 55.6 (cyclohexyl carbons), 109.1, 113.0, 117.0, 121.5, 123.2, 127.0, 135.3, 135.6 (aromatic carbons), 147.2 (CQN), 176.1 (C–S). ESI-MS (m/z): found (calcd) 705.1798 (705.1774) [M + H]+.

Synthesis of the [Pd(L)2] type complexes

Conflicts of interest

The [Pd(L)2] type complexes (6 and 7) were synthesized by the reaction between PdCl2 (1 mmol) in acetonitrile (25 mL) and an

X-ray crystallography Data collection details and a summary of the structure refinement of the compounds (HL3, 2–5 and 6) are given in Tables S1 and S2 (ESI†). A Bruker APEX2 (Mo Ka) X-ray (three-circle) diffractometer was used for crystal screening, unit cell determination and data collection. Integrated intensity information for each reflection was obtained by reduction of the data frames using the program APEX2.65 The integration method employed a three dimensional profiling algorithm and all data were corrected for Lorentz and polarization factors as well as for crystal decay effects. Finally, the data were merged and scaled to produce a suitable data set. SADABS66 was employed to correct the data for absorption effects. A solution was obtained readily using SHELXTL (SHELXS).67 Typically, H atoms were added at the calculated positions in the final refinement cycles. All other H atoms were refined with anisotropic thermal parameters. The structural solution was obtained readily using XT/XS in APEX265,66 and refined by full matrix least squares on F2 using Olex2.68

There are no conflicts to declare.


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Acknowledgements


J. H. thanks the University Grants Commission for the fellowship (F1-17.1/2012-13/RGNF-2012-13-ST-AND-18716).

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