Synthesis, X-ray structure and cytotoxic effect of nickel(II) complexes with pyrazole ligands

European Journal of Medicinal Chemistry 46 (2011) 5917e5926

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Original article

Synthesis, X-ray structure and cytotoxic effect of nickel(II) complexes with pyrazole ligands Marta Sobiesiak a, Ingo-Peter Lorenz b, Peter Mayer b, Magdalena Wo
zniczka d, Aleksander Kufelnicki d, Urszula Krajewska c, Marek Rozalski c, Elzbieta Budzisz a, e, * a

Collegium Medicum in Bydgoszcz, Nicholaus Copernicus University in Torun, Faculty of Pharmacy, Department of Inorganic and Analytical Chemistry, 85-094 Bydgoszcz, Poland Department of Chemistry, Ludwig-Maximilians-University, Butenandtstr. 5-13 (D), D-81377 Munich, Germany Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland d Department of Physical and Biocoordination Chemistry, Faculty of Pharmacy, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland e Department of Cosmetic Raw Materials Chemistry, Faculty of Pharmacy, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 February 2011 Received in revised form 7 September 2011 Accepted 1 October 2011 Available online 15 October 2011

Here we present the synthesis of the new Ni(II) complexes with chelating ligands 1-benzothiazol-2-yl3,5-dimethyl-1H-pyrazole (a), 5-(2-hydroxyphenyl)-3-methyl-1-(2-pyridylo)-1H-pyrazole-4-carboxylic acid methyl ester (b) and 1-benzothiazol-2-yl-5-(2-hydroxyphenyl)-3-methyl-1H-pyrazole-4carboxylic acid methyl ester (c). These ligands aec create solid complexes with Ni(II). The crystal and molecular structures of two complexes were determined by X-ray diffraction method. Thermal stability of two complexes with ligand c by TG/DTG and DSC methods were also shown. Cytotoxic activity of all the complexes against three tumour cell lines and to normal endothelial cells (HUVEC) was also estimated. Complexes with ligand c exhibited relatively high cytotoxic activity towards HL-60 and NALM-6 leukaemia cells and WM-115 melanoma cells. Cytotoxic effectiveness of one of these complexes against melanoma WM-115 cells was two times higher than that of cisplatin. The protonation constant log K ¼ 9.63 of ligand b corresponding to the phenol 2-hydroxy group has been determined in 10% (v/v) DMSO/water solution (25  C). The coordination modes (formation of two monomeric species: NiL and NiL2) in the complexes with Ni(II) are discussed for b on the basis of the potentiometric and UV/Vis data. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: Synthesis X-ray structure Cytotoxic effect Pyrazole ligands

1. Introduction In recent years, considerable attention has been paid to pyrazoles and related N-containing heterocyclic derivatives. In search for better antitumour treatment, a large series of pyrazole derivatives were synthesized. In the last decade several pyrazole derivatives proved to have potent anticancer action [1]. The pyrazolecontaining derivatives have been used as ligands in the formation of transition-metal complexes. This class of complexes has been reported to possess antitumour activity comparable to that of cisplatin [2]. In our previous papers we described the synthesis of 5-(2hydroxybenzoyl)-3-methyl-1-(2-pyridinyl)pyrazol-4-carboxylic acid methyl ester and its complexes with platinum(II), palladium(II) and

* Corresponding author. Department of Cosmetic Raw Materials Chemistry, Faculty of Pharmacy, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland. E-mail address: [email protected] (E. Budzisz). 0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2011.10.001

copper(II). These complexes showed lower cytotoxicity than cisplatin and the platinum(II) and copper(II) complexes were found to be more efficient in the induction of leukaemia cell death than the palladium(II) complex [3,4]. Structure and cytotoxic activities of cobalt(II) complexes with 5-(2-hydroxybenzoyl)-3-methyl-1-(2-pyridinyl) pyrazol-4-carboxylic acid methyl ester and 1-benzothiazol-2-yl-3,5dimethyl-1H-pyrazole we have been also investigated [5]. Nickel compounds are human carcinogens. However, nickel(II) complexes have potential application in medicine. A large number of these compounds have been extensively studied because of their antibacterial, fungicidal and anticancer activities [6e11]. The complexes of Ni(II) with 40 -methoxy-5,7-dihydroxy-isoflavone ligand showed high activity and selectivity against human cancer cell lines (SW620 colon and A549 lung carcinoma) [12]. Nickel(II) complexes with thiosemicarbazones-derived ligands were tested for their anticancer activity. Biological studies, performed with these complexes, including inhibition of cell proliferation and apoptosis test in vitro on the human leukaemia cell line U937, indicated that some of them could induce apoptosis [13]. The nickel(II) complexes of ortho-naphthaquinone thiosemicarbazones

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a

b HC

OH COOCH

COOCH

N

S

c

OH

N

N

N

CH

CH

N

N

N

N

S

N

CH

Fig. 1. Selected ligands aec.

were evaluated for their antiproliferative activities against the human breast cancer cell line MCF7. It was found that nickel(II) complexes were able to stabilize the cleavable complex formed by DNA and Topoisomerase II [14e16]. It has been also reported that Ni(II)esalphen complex induced quadruplex DNA stabilization and telomerase inhibition [17]. The results of these studies implicate the potential of nickel complexes as telomere-targeted chemotherapeutics [18]. Here we present the synthesis, structural characterization and cytotoxic effect of new Ni(II) complexes with pyrazole-containing 1-benzothiazol-2-yl-3,5-dimethyl-1H-pyrazole (a), 5-(2-hydroxyphenyl)-3-methyl-1-(2-pyridylo)-1H-pyrazole-4-carboxylic acid methyl ester (b) and (1-benzothiazol-2-yl-5-(2-hydroxyphenyl)-3methyl-1H-pyrazole-4-carboxylic acid methyl ester (c)) ligands. Physical and protolytic properties of the ligand b as well as its interaction with Ni(II) in solution are also shown.

2. Results and discussion 2.1. Preparation of the ligands and their complexes The ligands aec (Fig. 1) were synthesized according to the procedure described in our previous paper [3e5]. All ligands in the reactions with nickel(II) salts created the solid complexes 1aec, 2aec, 3c and 4c(4c0 þ 4c00 ) (Schemes 1e4). The ligands a and c with nickel(II) perchlorate hexahydrate in ethyl acetate solution formed the complexes 1a [19] and 1c in molar ratio of 2:1 (Scheme 1). Surprisingly, complex 1b is formed by ligand b and nickel(II) perchlorate hexahydrate in a molar ratio of 3:1, irrespective of the L:M molar ratios (2:1 or 3:1) (Scheme 2). The ligands a and b react with anhydrous NiCl2 in 1:1 molar ratio in ethyl acetate/methanol with formation of complexes 2a and 2b of the type [MLCl2(solvent)2] (Scheme 3). Single crystal X-ray diffraction studies revealed the presence of methanol and water molecules in cis-position in the structure of complex 2a. In the case of complex 2b, satisfactory elemental analyses of C, H and N were obtained, while the observed nickel content (9.5%) deviates significantly from the calculated one

(12.36%) for unknown reasons. Complexes 2c and 3c with ligand c and nickel(II) chloride formed in a molar ratio of 2:1, irrespective of the L:M molar ratio (1:1 or 2:1) in which the reaction proceeded (Scheme 4). The obtained products had the same elemental composition, regardless of whether the reaction proceeded with ligand c and nickel(II) chloride hexahydrate in ethyl acetate (compound 2c) or anhydrous nickel(II) chloride in acetonitrile (compound 3c). The differences in thermal stability and in the farinfrared spectra of both complexes suggest that we have obtained two isomeric compounds. In the reaction of nickel chloride hexahydrate trans-isomer (2c) was obtained. The formation of the cisisomer in the reaction with anhydrous nickel chloride has been confirmed by X-ray analysis. Single crystals of complexes have been obtained by slow diffusion of diethyl ether into mixture of ethyl acetate/methanol. Recrystallization of complex 3c by dissolving in DMF and the diffusion of diethyl ether into the solution gave product 4c(4c0 þ 4c00 ). 2.2. Structural studies. Spectroscopic characterization of ligands and their complexes 2.2.1. Thermal analysis of complexes 2c and 3c Thermal stability of the complexes was determined by DSC and TG studies from 40 to 1000  C. The TG/DTG and DSC curves of the complexes are shown in Fig. 2a and b, respectively. The compounds were heated to 950e1000  C, all relevant weight loss was completed by 850  C. From the thermal investigation (TG/DTG) it is concluded that the decomposition occurs in different ways for the 2c and 3c complexes. The thermal degradation of both complexes is presented in Table 1. The complex 2c decomposed in four steps. The first two endothermic processes are due to the loss of 3 mol of water and 2 mol of OH with mass losses of 10.43% (calcd. ¼ 9.63%). The successive decomposition occurs within a temperature range of 240e460  C and is attributed to the loss of organic moieties 4C6H4, 4CH3 and O2 (found weight loss 42.19%, calcd. ¼ 43.37%). The last step involves the loss of 2 mol of the organic moiety C5N3SO, at 460e770  C with mass losses of 33.57% (calcd. ¼ 32.83%). The remaining residue is NiCl2. The decomposition of the complex 3c occurred in six phases. The first step corresponds to the loss of one water molecule with mass losses of 2.21% (calcd. ¼ 2.05%). The second phase is due to the loss of 0.5 mol of O2 (of the ester group) between 90 and 175  C with mass losses of 1.93%, (calcd. ¼ 1.82%). The next step ranges from 180 to 220  C and is assigned to the loss of 2 mol of OH with mass losses of 3.93% (calcd. ¼ 3.87%). The successive decomposition occurs within a temperature range of 220e260  C and is attributed to the loss of the fragment 4CH3 6.93% (calcd. ¼ 6.85%). The next phase involves the loss of 2 mol of Cl2 and 4C6H4 organic moiety from 260 to 430  C with mass losses of

2+ R1 2:1 2 x a, c + Ni(ClO4)2 x 6H2O

CH3 H2O

R N N

ethyl acetate/methanol

H2O Ni

S

N

N N

H 3C 1a R = - CH3

2 x ClO4

N

R1 = -H R1

R

1c R = 2-OH-Ph R1 = COOCH3 1a, 1c Scheme 1. Synthesis of the Ni(II) complexes 1a, 1c.

S

-

M. Sobiesiak et al. / European Journal of Medicinal Chemistry 46 (2011) 5917e5926

5919

2+

OH CO2CH3 CH3

N N N

3:1 3xb

+ Ni(ClO4)2 x 6 H2O

H3CO2C HO

CH3 N N

N

Ni

2 x ClO4

N N H3C N

-

OH CO2CH3

1b Scheme 2. Synthesis of the Ni(II) complex 1b.

42.33% (calcd. ¼ 42.74%). The final step corresponds to the loss of the organic moiety 2C5N3SO with mass losses of 31.92% (calcd. ¼ 34.17%). The observed residue corresponds to NiO. 2.2.2. FAB-MS For valuable structural information all obtained Ni(II) complexes were investigated by mass spectrometric measurement. For compounds in series 1aec, 2aec and 3c parent peak of complexes have not been observed in FAB-MS spectra. The FAB-MS analysis of all compounds exhibited molecular peaks corresponding to compounds with nickel bounded to one [NiL]2þand two ligand molecules [NiL2]2þ. For the compounds 1aec two characteristic signals for this structure observed: [NiLClO4]þ (Mþ  522, 466, 386) and [NiL2ClO4]þ (Mþ  887, 775, 615), respectively. Mass peaks of [NiLCl]þ at (m/z) 402, 322, 458, 458 and [NiL2Cl]þ at (m/z) 711, 551, 823, 823 ions observed in the mass spectra of coordination 2a, 2b and 2c, 3c. FAB-MS spectra of 1b,c, 2a,b, and 3c complexes presented ion peak corresponded to the ligand. The data presented above are in good agreement with the results obtained by elemental analysis, and therefore confirm the proposed structure by X-ray diffractometry of investigated complexes. 2.2.3. FTIR analysis The IR spectrum of the ligands b, c and complexes 1b, 1c, 2b, 3c shows bands at 3447e3062 cm1 assigned to the hydroxy group of the phenyl ring. The characteristic bands at 3118e2924 cm1 of the methyl group of the ligand a and its complexes 1a, 2a are assigned to CeH vibration. The band arising from v(C]O) was observed at

H3C

H3C

S

N

S

N

CH3 H2O

N N

Cl

Ni OHCH3

N

CH3

Cl

N anh. NiCl2

a

2a

1 : 1/ethyl acetate/MeOH

OH

OH

COOCH3 COOCH3

N N N b

CH3

N N N Ni Cl Cl 2b

Scheme 3. Synthesis of complexes 2a and 2b.

CH3

1705e1723 cm1 in the spectra of ligands b, c and their complexes. The band in the 1514e1568 cm1 region of complexes with ligands a, c has been assigned to C]N of the benzothiazole moiety. The most characteristic bands are those at 1639e1600 cm1, attributable to the pyrazole C]N group. These bands observed for ligands are shifted towards lower frequencies for complexes. This observation can be explained by participation of the nitrogen atom in the coordination with metal ions in complexes. The infrared spectra showed that ligandemetal bonds in obtained complexes with ligands a and c were being created by nitrogen atoms and not by sulphur atoms. No shift of absorption bands characteristic to the vibrations of C]S group in region 580e700 cm1 is in its obtained spectra, so the sulphur atom is not involved in coordination to nickel(II). The complexes 1aec show an intense band due to the perchlorate ion at 1120e1106 cm1 and a moderate and sharp band at 625 cm1 characteristic for uncoordinated perchlorate ions [20]. The important differences are shown in the far-infrared spectra of the compounds 2c and 3c. These complexes show stretching bands in the 418e434 cm1 range, which may correspond to the NieN vibrations involving the N-atoms of the pyrazole ring. However, the cis-isomer (3c) shows a symmetric NieN stretching band. The absorption observed in the 343e351 cm1 range is assigned to NieCl stretching vibration. The trans-isomer 2c shows a single band due to stretching near 347 cm1, the cis-isomer 3c shows two stretching bands at 345 and 351 cm1 [21]. 2.2.4. Potentiometric and spectrophotometric studies in solution The titrations in the presence of ligand b were carried out within the range of ligand neutralization coefficient (base equivalent) a ¼ mmol of base/mmol of ligand ¼ 0.5 to 2.0. The experiments were started with neutralization of the excessive mineral acid related to the negative values of a. Representative titrations obtained under conditions ensuring attainment of equilibrium are shown in Fig. 3. Observed deflections of the curves for the ligand alone and for ligand-to-metal ratio 2:1 and 3:1 were corresponding to precipitation and by that indicating the last points used in calculations. For ligand-to-metal ratio 1:1 the precipitation was visible already at a w1 (pH w8.5) and then the curves flattened decidedly due to predomination of aqua-ion hydrolysis. Consequently, those curves were not included in further calculations. Ligand b exhibited only one measurable protonation constant (reciprocal dissociation constant), corresponding to the benzyl ring OH (Table 2). The species distribution of protonation is shown in Fig. 4a. Calculations based on the potentiometric data suggest the formation of two monomeric species NiL and NiL2 (Table 2 and Fig. 4b). Complexation starts above pH w7 from the NiL species, in

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H3CO2C OH

+ NiCl2x6H2O ethyl acetate/methanol

N

CO2CH3

HC CH3 3 Cl N N

N

Ni S

N

HO S

N Cl

2xc 2c H3COOC H 3C N + anh. NiCl2 OH

acetonitrile

S

Cl

N

N N

HO

N

Ni

S

N

CH3 Cl

H3COOC

3c rec. DMF

-Ligand c 4c OCHN(CH3)2

OCHN(CH3)2

OH

S

N N H3COOC

Cl

N

+

OH

S

OCHN(CH3)2

N Ni

N N

Ni H3COOC 4c'

Cl

-

OCHN(CH 3)2

OCHN(CH3)2

CH3 Cl

+

CH3 Cl 4c"

Scheme 4. Synthesis of the Ni(II) complexes 2c, 3c and 4c(4c0 þ 4c00 ) with ligand c.

which the ligand acts most likely as a bidentate ligand through its pyridine and pyrazole nitrogens. It may be observed that complexation precedes deprotonation of the ligand hydroxyl group e then a negative charge of the ligand is promoting the donation of both the nitrogen lone electron pairs and by that alleviating the formation of dative bonds with the metal. Moreover, the formation of NiL is followed by formation of the NiL2 complex (Fig. 4b). As can also be seen, the first product of metal hydrolysis (NiOH or in an alternative notation NiH1) is competing with complexation. Unfortunately, because of predominating hydrolysis of the metal aqua-ion at higher pH leading to enhanced light scattering, the UV/

Vis spectroscopy could confirm only the formation of the first species (NiL) e Table 2. 2.2.5. X-ray measurements Single crystals of 2a and 4c(4c0 þ 4c00 ) suitable for X-ray diffraction studies were obtained by slow diffusion of diethyl ether into a solution of 2a in ethyl acetate and analogously for 4c(4c0 þ 4c00 ), when 3c was dissolved in DMF. Details of the relevant data collection and refinement are summarized in Table 3. The molecular structures of 2a, 4c0 and 4c00 are illustrated in Figs. 5 and 6, their corresponding selected bond lengths and angles

Fig. 2. Thermal analysis of complexes 2c and 3c. a) TG/DTG curves of complexes: 2c, 3c; b) DSC curves of complexes: 2c, 3c.

M. Sobiesiak et al. / European Journal of Medicinal Chemistry 46 (2011) 5917e5926 Table 1 Thermal data for complexes 2c and 3c. Comp Step TG range [ C] Mass loss [%] Assignment

DSC

Final product

Found Calcd. 2c

I þ II 70e240 III

3c

240e460

Endo Endo 42.19 43.37 4C6H4, 4CH3, O2 Endo Endo Endo Exo Exo 33.57 32.83 2C5N3SO 86.19 85.83 NiCl2 (14.18%) 10.43

9.63 3H2O, 2OH

IV S

460e770

I II III IV

30e60 90e175 180e220 220e260

V

260e430

42.33 42.74 4C6H4, 2Cl

VI S

430e850

31.92 34.17 2C5N3SO 89.25 91.50

2.21 1.93 3.93 6.93

2.05 1.82 3.87 6.85

1H2O 0.5O2 2OH 4CH3

Endo Endo Exo Endo Exo Endo Endo Exo

Table 2 Potentiometric and spectrophotometric data for the Ni(II)eb system based on a comprehensive file of titrations. Temperature 25  C. Standard deviations in parentheses. Solvent in potentiometry: 10/90% (v/v) DMSO/water. Solvent in UV/Vis: DMSO. UV/vis

l (nm) Protonation of L LH 9.63 (1)

3

(M1 cm1)

9.63

eb

3.89 3.31

601 ec

2þ

Ni complexes NiL 3.89 (2) 7.20 (3) NiL2

c

Fig. 3. Titration of the Ni(II)eb system at various ligand-to-metal ratio (1:1; 2:1; 3:1) and of ligand L in absence of the metal. Total concentration of the ligand CL ¼ 2.0  103 M. The value of base equivalent a ¼ 0.5 corresponds to HNO3 in excess as related to ligand.

log K

a

a

are given in Table 4. All three complexes exhibit a distorted octahedral environment for the Ni(II) centres each bearing one bidentate benzothiazole containing pyrazole-N,N0 ligand, but different further monodentate ligands. In complex 2a there are two chlorido ligands in cis-position and one H2O and CH3OH molecule as additional ligands. The chelating ligand a binds a little bit different with its thiazole-N (Ni1eN1 2.127(2)  A) and pyrazole-N atoms (Ni1eN3 2.081(3)  A). This is due to the better p-acceptor character of the pyrazole part which also causes a shorter bond lengths of its transoidal bound Cl1 (Ni1eCl1 2.3588(9)  A) compared to Cl2 (Ni1eCl2 2.4537(9)  A) in trans-position to O1 of CH3OH ligand. For the same reason, O2 of the aqua ligand in trans-position to N1 is rather shorter bound to Ni1 than O1 A). to CH3OH (Ni1eO2 2.063(2), Ni1eO1 2.147(2)  All the bond lengths CeN of the central metallacycle and its condensed heterocycles lie within the range 1.290(4) (N1-C1) and 1.403(4)  A (N3-C2). The distortion of the octahedron is demonstrated by the trans-axial angles Cl1eNi1eN3 177.06(7) , Cl2eNi1eO1 172.35(6) and O2eNi1eN1 167.16(10) as well as by some of the cisoidal angles as Cl1eNi1eN1 99.33(8) , Cl2eNi1eN1 97.22(8) , O1eNi1eO2 84.73(8) and O1eNi1eN1 84.70(10) . The bite angle of planar chelate system a in 1a is of course the smallest one (N1eNi1eN3 77.74(10) ). As the torsion angles indicate, the

log b

Species

b

NiO (8.5%)

5921

48

L ¼ L (with deprotonated hydroxyl group in b). Lack of spectrophotometric features, absorption only in UV (<400 nm). Undetectable in UV/Vis due to light disturbances at pH > 8.10.

whole ligand a is rather planar with the greatest deviation for Ni1eN3eN2eC9 172.5(2) . The asymmetric unit of compound 4c contains two different complexes 4c0 and 4c00 whose structures are discussed separately as follows. In the neutral cis-configurated complex 4c0 there are two chlorido and two DMF ligands bound to the Ni(II) centre besides the planar N,N0 -chelating ligand c as in 2a, both NieCl lengths are different (Ni1eCl1 2.3805(8), Ni1eCl2 2.4341(8)  A) due to their different trans-axial ligands. For the same reason the bond Ni1eO5 2.051(2)  A is shorter than Ni1eO4 2.120(2)  A. Ligand c with its thiazole and pyrazole parts shows nearly no difference of its NieN bond lengths (Ni1eN1 2.124(2), Ni1eN3 2.132(2)  A). The bond lengths CeN and N2eN3 within the metallacycle and to the adjacent atoms are similar to those found in complex 2a. The distortion of the octahedral configuration of 3c is more obvious than in 2a as the trans-axial angles show Cl1eNi1eO4 174.20(6), Cl2eNi1eN3 172.07(7), O5eNi1eN1 161.46(8) . Some of the cisoidal angles also show a great deviation of the ideal 90 (Cl2eNi1eN1 99.27(6), O4eNi1eN3 81.37(8) ). The bite angle of ligand c (N1eNi1eN3 77.40(8) ) in 4c0 is again the smallest one and nearly the same as that of a in 2a. In spite of the three different substituents of the pyrazole part one can say that the basic framework of ligand c in 4c0 is planar. In the ionic complex 4c00 , again with one N,N0 -bidentate ligand c, there is only one chlorido ligand bound to the Ni(II) centre, a second chlorido anion is the counterion. The distorted octahedral environment of 4c00 is completed by three DMF ligands in a facial manner. The bond length Ni2eCl3 2.3795(8)  A is similar to those in 2a and 4c0 . The three DMF ligands also show similar bond lengths to the Ni(II) centre, they vary only between 2.06 (Ni2eO11) and 2.10  A (Ni2eO11) in spite of their different trans-axial bonding partners. As the bonds Ni2eN6 2.141(2) and Ni2eN8 2.139(2)  A indicate there is no more any difference in the bonding behaviour between the pyrazole and thiazole part of ligand c (and a) like it was found in 4c0 (and in 2a). Only two of the trans-axial angles in 4c00 differ significantly from 180 (O11eNi2eN6 173.43(8) and O10eNi2eN8 171.07(8) ) and only two of the cisoidal angles differ more than 5 from the octahedral one (O10eNi2eN6 95.51(8) and O11eNi2eN8 96.61(8) ). Thus, the octahedral distortion of 4c00 is less than in 2a and 4c0 , the bite angle of c in 4c00 is again equal to those in 2a and 4c0 . The planar structure of ligand c, however, is in 4c00 more distorted than in 4c0 as especially the torsion angles show Ni2eN6eC26eS2 164.45(3) and Ni2eN6eC27eC28 159.2(2) . Hydrogen bonds exist in all three complexes, in 2a they are of OeHeCl type as inter- and intramolecular ones. As both complexes 4c0 and 4c00 are found together in crystalline compound 4c, hydrogen bonds of intermolecular OeHeO type as well as of intraand intermolecular OeHeCl type are found. It seems they are

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M. Sobiesiak et al. / European Journal of Medicinal Chemistry 46 (2011) 5917e5926

Fig. 4. Species distribution profile at 25  C for a) protonation of L ¼ b, CL ¼ 1.0  103 M; b) Ni2þ complexes of b. Solvent: 10/90% (v/v) DMSO/water. Total concentration of the metal CNi(II) ¼ 1.0  103 M. Ligand-to-metal ratio 3:1.

responsible for the strong contact and package of 4c0 and 4c00 in crystals of 4c. 3. Biological assay The cytotoxicity of ligands aec and their nickel complexes 1aec, 2aec and 3c was measured against human melanoma WM-115 cells as well as leukaemia promyelocytic HL-60 and lymphoblastic NALM-6 cells. Cisplatin and carboplatin were used as the reference compounds [3e5,22]. Cells were exposed to a broad Table 3 Crystallographic data for complexes 2a and 4c. Compound

Net formula Mr/g mol1 Crystal size/mm Crystal system Space group a/ A b/ A  c/A a/ b/ g/ V/ A3 Z Calcd. density/g cm3 m/mm1 Absorption correction Transmission factor range Refls. measured Rint Mean s(I)/I q Range Observed refls. x, y (weighting scheme) Refls. in refinement Parameters Restraints R(Fobs) Rw(F2) S Shift/errormax Max electron density/e  A3 Min electron density/e  A3

2a

4c(4c0 þ 4c00 )

C13H17Cl2N3NiO2S 408.958 0.14  0.13  0.12 Triclinic P1 bar 6.9132(5) 9.7951(6) 12.6448(9) 108.451(6) 99.843(6) 90.727(5) 798.23(9) 2 1.70152(19) 1.689 ‘Multi-scan’ 0.87638e1.00000 5601 0.0317 0.0550 4.17e26.35 2450 0.0479, 0 3232 211 3 0.0376 0.0885 0.940 0.001 0.882 0.872

C53H67Cl4N11Ni2O12S2 1373.496 0.12  0.10  0.04 Monoclinic P21/n 17.1359(4) 17.7179(3) 20.7631(4) 90 96.2486(11) 90 6266.5(2) 4 1.45585(5) 0.905 None e 37591 0.0547 0.0537 3.15e25.34 8147 0.0365, 2.7105 11430 779 0 0.0382 0.0946 1.019 0.001 0.358 0.383

range of drug concentrations (107 to 103 M) for 48 h and cell viability was analysed by MTT assay. IC50 values of ligands and their complexes are shown in Table 5. Ligands a and b were not toxic to all three tumour cell lines. Ligand c was also inactive in the case of HL-60 leukaemia cells but expressed moderate cytotoxicity against NALM-6 and WM-115 cells. The highest cytotoxic activity against both leukaemia and melanoma WM-115 cell lines has been observed for complexes 1c and 3c with the ligand c (IC50 coefficient in the range from 8.0 to 34.7 mM). It should be noted that compound 1c with an IC50 of 9.5 mM was significantly more effective in the case of skin melanoma WM-115 cells when compared to cisplatin (IC50 18.2 mM). It seems that compounds 1c and 3c could serve as models of Ni(II)-complexes for further cytotoxicity studies. Ni(II) complexes 1b and 2b with the ligand b exhibited the lowest cytotoxicity for all tumour cell lines studied. Complexes of Ni(II) 1a, 2a with the ligand a expressed rather moderate and similar cytotoxity against all cell lines but lower than the complexes with ligand c. We decided to check whether Ni(II) complexes could be more effective against tumour cells than to normal cells. To this aim

Fig. 5. Molecular structure of complex 2a. Displacement ellipsoids are drawn at 30% of probability level.

M. Sobiesiak et al. / European Journal of Medicinal Chemistry 46 (2011) 5917e5926

C5

C6

X-ray spectroscopy enabled us to establish that in the procedure adopted in this study the cis-isomer 4c was produced. The complexing properties of ligand b with Ni(II) in solution were also examined. Data obtained indicate only one measurable protonation constant corresponding to the phenol 2-hydroxy group and formation of two monomeric species: NiL and NiL2. Cytotoxic study demonstrates that Ni(II) complexes 1c and 3c with ligand c exhibits relatively high cytotoxic activity towards HL60 and NALM-6 leukaemia cells and WM-115 melanoma cells as compared to complexes with ligands a and b. Cytotoxic effectiveness of complex 1c against melanoma WM-115 cells was two times higher than that of cisplatin. Therefore, complexes 1c and 3c could serve as nickel models for further cytotoxicity studies. Unfortunately, these complexes showed also high toxic effect to the noncancerogenic HUVECs.

C22

C4 C7 C3

N4 C2 C21

S1

C20

C14

Cl2

O4

C13

O1

C15

N1

C1

Ni1

N2

C12

O5

C16 C17

C23

N3

C11

Cl1 C18

O2

N5

C9

C10

C25 C8

C24

O3

C19

4c’

5. Experimental C46 N9

C31

C30

C47

C29 C32

O6 C39

C28 S2

C27

C38 C40

C37

C26 N7

C36

C42

C43

O8

C35

N10

N6 Ni2

O10 C48

C50

O11

C34 C33

O7

C49

C45

O9

N8

C41

Cl3

C51 C52

N11

C44

5923

C53

4c” Fig. 6. Molecular structures of compound 4c containing two different complexes 4c0 and 4c00 within the asymmetric unit. Displacement ellipsoids are drawn at 30% of probability level, the chloride anion of 4c00 is not shown.

cytotoxic activity of synthesized complexes 1a, 2c, 2a, 1c and 3c against normal (healthy) human umbilical vein endothelial cells (HUVECs) was determined and compared. Analysis of the cytotoxicity data revealed lack of difference in susceptibility of normal and tumour cells to the most of compounds. The only exception was observed for compound 2c which presented higher effectiveness against HL-60 and NALM-6 leukaemia cells as compared to HUVECs (p value less than 0.05 in Student’s t-test).

4. Conclusions In this paper we have shown synthesis of nickel(II) complexes with pyrazole-derived ligands. The structure of complexes was confirmed by spectral and elemental analysis. The molecular structures of the two complexes (2a and 4c(4c0 þ 4c00 )) were confirmed by X-ray analysis. In the reaction of ligand c and nickel(II) chloride hexahydrate or nickel(II) chloride anhydrous we have obtained different complexes 2c and 3c, respectively. The differences in the decomposition profiles and in their thermal stability as well as the presence of the different bands in the far-infrared spectra of both complexes suggest that we have obtained two isomeric (cis and trans) compounds. These complexes also exhibited different biological activity. Complex 1c showed a less cytotoxic effect than the more unstable complex 3c (cis-isomer) against tested cancer cell lines.

Materials and methods: All substances were used without further purification. Nickel(II) chloride and nickel(II) perchlorate hexahydrate were purchased from Aldrich. Solvents for synthesis (acetonitrile, dichloromethane, diethyl ether, dimethylformamide, ethyl acetate, and methanol) were reagent grade or better and were dried according to standard protocols [23]. The melting points were determined using an Electrothermal 1A9100 apparatus and they are uncorrected. The IR and far-IR spectra were recorded on KBr and CsI pellets on FT-IR-8400S Shimadzu and PerkineElmer FT-IR Spectrum 2000 spectrophotometer, respectively. The FAB-MS data were determined on Finnigan Matt 95 mass spectrometer (NBA, Csþ gun operating at 13 keV). For the new compounds elemental analyses (C, H and N) were obtained using a Perkin Elmer PE 2400 CHNS analyser and S content using an Elementar Vario El III analyser. The nickel content was performed on a Thermo Elektron Solar M6. The thermal decompositions of w10 mg of the prepared complexes were measured under the static air atmosphere with Mettler-Toledo Star TG/SDTA 851e thermal analyzer at a heating rate of 5 K/min. For all complexes in the temperature range of 298e1273 K alumina open crucibles were used. DSC analysis was carried out using DSC Mettler-Toledo instrument in standard closed sample pans, static air atmosphere and heating rate of 5 K/min. The thermo analytical curves were obtained using STARe System METTLER-TOLEDO software. Potentiometric equilibria studies were carried out for ligand b (soluble in DMSO and then diluted with water) by using a Molspin automatic titration kit equipped with combined microelectrode Russell CMAWL/4/5/S7 (Auchtermuchty, Scotland). The remaining ligands (a and c) were insoluble in mixed, mainly aqueous, solvents. Therefore glass electrode potentiometry could not be applied in these cases. The samples for b were prepared in 10/90% (v/v) DMSO/water solution. A constant temperature of 25  C and ionic strength I ¼ 0.1 (KNO3) were maintained in all the experiments. The Ni(II) hydrolysis, ligand protonation and complex formation constants were determined by pH-metric titrations of 4.0 cm3 thermo stated and argonated samples. Alkali (0.1 M NaOH carbonate-free, Malinckrodt Baker B.V.) was added from a 0.250 cm3 calibrated micro syringe. The measurement cell was daily calibrated in the log [Hþ] scale by titration of 0.005 M HNO3 (I ¼ 0.1, KNO3) in 10/90% (v/v) DMSO/water with 0.1 M NaOH, temp. 25  C. The calculated ionic product of water for the 10/90% (v/v) DMSO/water solution amounted to 14.06. Overall concentration formation constants: bmlh ¼ [MmLlHh]/ [M]m[L]l[H]h were determined from at least four titration files by SUPERQUAD [24,25] and then HYPERQUAD 2008 [25,26]. Standard deviations computed by both the procedures refer to random errors only. The total concentration of the ligand in each

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M. Sobiesiak et al. / European Journal of Medicinal Chemistry 46 (2011) 5917e5926

Table 4 Selected bond lengths, bond angles and torsion angles of complexes 2a, 4c0 and 4c00 . Compound 4c0

Compound 2a

Compound 4c00

Ni1eCl1 Ni1eCl2 Ni1eO1 Ni1eO2 Ni1eN1 Ni1eN3 N1eC1 C1eN2 N2eN3 N1eC3 C1eS1 N2eC9 N3eC11 O1eC13

2.3588(9) 2.4537(9) 2.147(2) 2.063(2) 2.127(2) 2.081(3) 1.290(4) 1.386(4) 1.382(4) 1.403(4) 1.739(3) 1.370(4) 1.325(4) 1.419(4)

Ni1eCl1 Ni1eCl2 Ni1eO4 Ni1eO5 Ni1eN1 Ni1eN3 N1eC1 C1eN2 N2eN3 N1eC2 C1eS1 N2eC11 N3eC9

2.3805(8) 2.4341(8) 2.120(2) 2.051(2) 2.124(2) 2.132(2) 1.301(3) 1.399(4) 1.385(3) 1.406(4) 1.730(3) 1.366(3) 1.323(4)

Ni2eCl3 Ni2eO9 Ni2eO10 Ni2eO11 Ni2eN6 Ni2eN8 N6eC26 N7eC26 N7eN8 N6eC27 C26eS2 N7eC36 N8eC34 O9eC45 O10eC38 O11eC51

2.3795(8) 2.0644(19) 2.0614(19) 2.103(2) 2.141(2) 2.139(2) 1.297(3) 1.402(3) 1.379(3) 1.400(4) 1.727(3) 1.367(3) 1.328(4) 1.248(3) 1.235(3) 1.235(4)

Cl1eNi1eCl2 Cl1eNi1eO1 Cl1eNi1eO2 Cl1eNi1eN1 Cl1eNi1eN3 Cl2eNi1eO1 Cl2eNi1eO2 Cl2eNi1eN1 Cl2eNi1eN3 O1eNi1eO2 O1eN1eN1 O1eNi1eN3 O2eNi1eN1 O2eNi1eN3 N1eNi1eN3 Ni1eO1eC13

93,75(3) 93.25(6) 88.54(6) 99.33(8) 177.06(7) 172.35(6) 92.34(6) 97.22(8) 86.35(8) 84.73(8) 84.70(10) 86.83(10) 167.16(10) 94.40(9) 77.74(10) 126.5(9)

Cl1eNi1eCl2 Cl1eNi1eO4 Cl1eNi1eO5 Cl1eNi1eN1 Cl1eNi1eN3 Cl2eNi1eO4 Cl2eNi1eO5 Cl2eNi1eN1 Cl2eNi1eN3 O4eNi1eO5 O4eNi1eN1 O4eNi1eN3 O5eNi1eN1 O5eNi1eN3 N1eNi1eN3 Ni1eO4eC20 Ni1eO5eC23

94.28(3) 174.20(6) 91.38(6) 91.93(6) 93.03(6) 91.39(6) 90.45(6) 99.27(6) 172.07(7) 87.35(8) 88.36(8) 81.37(8) 161.46(8) 92.44(8) 77.40(8)

Cl3eNi2eO9 Cl3eNi2eO10 Cl3eNi2eO11 Cl3eNi2eN6 Cl3eNi2eN8 O9eNi2eO10 O9eNi2eO11 O9eNi2eN6 O9eNi2eN8 O10eNi2eO11 O10eNi2eN6 O10eNi2eN8 O11eNi2eN6 O11eNi2eN8 N6eNi2eN8

179.42(6) 93.05(6) 92.43(6) 86.96(6) 91.19(6) 86.50(7) 87.22(8) 93.44(8) 89.31(8) 91.06(8) 95.51(8) 171.07(8) 173.43(8) 96.61(8) 76.86(8)

Ni1eN1eC1eN2 Ni1e1eC1eS1 Ni1eN1eC3eC2 Ni1eN3eN2eC1 Ni1eN3eN2eC9 N1eC1eN2eN3

1.0(4) 176.8(9) 174.9(2) 3.4(3) 172.5(2) 3.0(4)

Ni1eN1eC1eN2 Ni1eN1eC1eS1 Ni1eN1eC2eC3 Ni1eN3eN2eC1 Ni1eN3eN2eC11 N1eC1eN2eN3

1.4(3) 177.99(12) 177.09(19) 2.6(3) 174.22(17) 2.8(4)

Ni2eN6eC26eN7 Ni2eN6eC26eS2 Ni2eN6eC27eC28 Ni2eN8eN7eC26 Ni2eN8eN7eC36 N6eC26eN7eN8

12.2(3) 164.45(13) 159.2(2) 7.6(3) 171.46(16) 3.1(3)

Hydrogen bonds O1eH1eCl2i O2eH21eCl2ii O2eH22eCl1ii

0.74(4) 2.42(4) 3.128(2) 161(4) 0.77(3) 2.50(3) 3.228(2) 157(4) 0.81(4) 2.31(4) 3.095(2) 164(4)

Symmetry i ¼ [1655.00] ¼ 1 þ x, y, z ii ¼ [2555.00] ¼ x, y, z

sample ranged within 2.0 O 3.0  103 M. The metaleligand interaction was studied at ligand-to-metal ratios 1:1, 2:1 and 3:1. Ni(NO3)2 p.a. of POCh Gliwice was used e the standard solution was titrated with disodium salt of EDTA in the presence of murexide. Species distribution profiles were obtained by means of HySS 2006 (Protonic Software) [27]. UV/visible absorption spectra were recorded on a Cary 50 Bio spectrophotometer, slit width 1.5 nm, range 300e800 nm. Owing to relatively low molar absorptivities of the solutions and on the other hand limited solubility of b in DMSO/water solutions the measurements were carried out in pure DMSO. The concentrations of ligand and metal were 3.0  103 M and 1.0  103 M, respectively. Silica cells of path length 1 cm were applied. A Peltier accessory was used to maintain the temperature constant at 25  0.1  C. Acidity of the solutions was changed directly in the cell by small aliquots of base (NaOH) or acid (HNO3). The pH was measured by means of a multifunctional microprocessor digital instrument CX-731 (ELMETRON, Poland) equipped with a combined Inlab Micro (METTLER-TOLEDO) electrode. The values

O1eH1eO12 O6eH6eCl4 O12eH121eCl4i O12eH122eCl2ii

0.840 1.800 2.628(4) 170 0.840 2.160 2.988(2) 167 0.82(6) 2.32(6) 3.115(3) 166(5) 0.83(6) 2.34(6) 3.166(3) 172(5)

i ¼ [3765.00] ¼ 2  x, 1  y, z ii ¼ [2755.00] ¼ 5/2  x, ½ þ y, ½  z

Table 5 Cytotoxicity of the ligands and their nickel complexes expressed as the IC50 values (in mM). Compound

HL-60

NALM-6 IC50

ab bb cb 1a 1b 1c 2a 2b 2c 3c Cisplatinb Carboplatinb

368.8  18.8 649.0  74.1 >1000 60.8  8.9 42.7  1.6 15.6  4.4 69.0  3.1 75.6  5.9 23.1  3.9 8.6  0.9 0.8  0.1 4.3  1.3

WM-115

HUVEC

743.8  76.4 >1000 55.1  8.2 87.9  4.4 230.3  24.2 9.5  0.5 85.8  3.1 283.9  36.9 65.8  4.8 34.7  6.0 18.2  4.3 422.2  50.2

e e e 75.3  7.5 e 8.97  2.0 68.0  3.6 e 85.1  8.7 6.01  1.2 96.0  5.7 e

a

367.4  22.2 >1000 74.0  12.9 59.3  6.5 49.4  1.5 10.7  3.2 75.3  2.1 75.4  3.2 31.0  5.4 8.0  0.4 0.7  0.3 0.7  0.2

a IC50 e concentration of a tested compound required to reduce the fraction of surviving cells to 50% of that observed in the control, non-treated cells. Mean values of IC50 (in mM)  S.D. from 4 experiments are presented. b See literature [3e5,22].

M. Sobiesiak et al. / European Journal of Medicinal Chemistry 46 (2011) 5917e5926

of 3 were calculated at the maximum concentrations of the respective species obtained from the potentiometric data.

5.1. Chemistry 5.1.1. Synthesis of the complexes Caution! Perchlorate salts are potentially explosive and were handled only in small quantities with care. 5.1.1.1. Synthesis of complex 1a. Nickel perchlorate Ni(ClO4)2$6H2O (27.9 mg, 0.08 mmol) was dissolved in 1 ml methanol and was added at room temperature to a stirred solution of ligand a (35.8 mg, 0.16 mmol) in ethyl acetate (7 ml). The reaction solution was stirred and refluxed for 24 hours. The resulting blue-green crystals of 1a were obtained by the diffusion of diethyl ether into the mixture. Yield: 27.0 mg (55.0%), mp 324.3e325.8  C. Anal. Calcd. for 1a C24H22N6S2NiCl2O8$3.5H2O (779.14 g/mol) C, 37.00%; H, 3.75%, N, 10.79%; S, 8.22%; Ni, 7.53%. Found: C, 37.40%; H, 3.40%, N, 10.39%; S, 7.91%; Ni, 7.37%. FTIR (KBr, cm1): n(OeH) 3400; n(CeCH3) 3118; n(C]N) 1580, 1514; n(ClO 4 ) 1109, 625. MS-FAB (m/ z): 287 (75%) [Ni(a)]2þ, 386 (58%) [Ni(a)]2þClO 4 , 516 (40%) [Ni(a)2]2þ, 615 (100%) [Ni(a)2]2þClO 4. 5.1.1.2. Synthesis of complex 1b. Nickel perchlorate Ni(ClO4)2$6H2O (16.5 mg, 0.045 mmol) was dissolved in 0.5 ml methanol and was added dropwise at room temperature to a stirred solution of ligand b (41.7 mg, 0.13 mmol) in ethyl acetate (8 ml). The reaction solution was stirred for 24 h at room temperature. The resulting pink-violet crystals of 1b were obtained by the diffusion of diethyl ether into the mixture. Yield: 34.5 mg (61.7%), mp 264.7e266.2  C. Anal. Calcd. for 1b C51H45N9O9NiCl2O8$3H2O (1239.61 g/mol) C, 49.42%; H, 4.15%; N, 10.17%; Ni, 4.73%. Found: C, 49.50; H, 3.50%; N, 9.74%; Ni, 5.13%. FTIR (KBr, cm1): n(OeH) 3391; n(CeCH3) 2953; n(C¼O) 1723; n(C]N) 1614; n(ClO 4 ) 1106, 625. MS-FAB (m/z): 307 (18%) [(b)], 367 (35%) [Ni(b)]2þ, 466 (8%) [Ni(b)]2þClO 4 , 676 (35%) [Ni(b)2]2þ, 775 (20%) [Ni(b)2]2þClO 4. 5.1.1.3. Synthesis of complex 1c. Nickel perchlorate Ni(ClO4)2$6H2O (21.2 mg, 0.06 mmol) was dissolved in 0.5 ml methanol and was added dropwise at room temperature to a stirred solution of ligand c (42.3 mg, 0.12 mmol) in ethyl acetate (10 ml). The reaction solution was stirred for 24 h at room temperature. The resulting greenblue crystals of 1c were obtained by the diffusion of diethyl ether into the mixture. Yield: 54.5 mg (95%), mp 294.6e295.8  C. Anal. Calcd. for 1c C38H30N6O6S2NiCl2O8$4H2O (1059.35 g/mol) C, 43.08%; H, 3.52%; N, 7.93%; S, 6.04%; Ni, 5.54%. Found: C, 42.85%; H, 3.79%; N, 7.40%; S, 6.11%; Ni, 5.60%. FTIR (KBr, cm1): n(OeH) 3442; n(CeCH3) 3099; n(C]O) 1708; n(C]N) 1573, 1517; n(ClO 4 ) 1120, 624. MS-FAB (m/z): 366 (8%) [(c)], 423 (64%) [Ni(c)]2þ, 522 (10%)  2þ [Ni(c)]2þClO 4 , 887 (10%) [Ni(c)2] ClO4 . 5.1.1.4. Synthesis of complex 2a. Nickel(II) chloride anh. NiCl2 (22.5 mg, 0.17 mmol) was dissolved in 2 ml methanol and was added dropwise at room temperature to a stirred solution of ligand 1benzothiazol-2-yl-3,5-dimethyl-1H-pyrazole a (39.7 mg, 0.17 mmol) in ethyl acetate (10 ml). The reaction solution was stirred for 48 h at room temperature. The resulting green solid 2a was obtained by the diffusion of diethyl ether into the mixture. Yield: 48.9 mg (%), mp 292.5e293.0  C. Anal. Calcd. for 2a C12H11N3SNiCl2$4H2O (431.2 g/ mol) C, 33.45%; H, 4.44%; N, 9.75%; S, 7.43%; Ni, 13.62%. Found: C, 33.48%; H, 4.15%; N, 9.65%; S, 7.29%; Ni, 13.25%. FTIR (KBr, cm1): n(OeH) 3284; n(CeCH3) 3079; n(C]N) 1579, 1518. MS-FAB (m/z): 229 (10%) [(a)], 287 (30%) [Ni(a)]2þ, 322 (85%) [Ni(a)Cl]þ, 516 (8%) [Ni(a)2]2þ, 551 (12%) [Ni(a)2Cl]þ.

5925

5.1.1.5. Synthesis of complex 2b. Nickel(II) chloride anh. NiCl2 (18.3 mg, 0.14 mmol) was dissolved in 2 ml methanol and was added dropwise at room temperature to a stirred solution of ligand 5-(2hydroxyphenyl)-3-methyl-1-(2-pyridylo)-1H-pyrazole-4-carboxylic acid methyl ester b (43.7 mg, 0.14 mmol) in ethyl acetate (10 ml). The reaction solution was stirred for 24 h at room temperature. The resulting green solid 2b was obtained by the diffusion of diethyl ether into the mixture. Yield: 50.3 mg (75%), mp dec. >243.5  C. Anal. Calcd. for 2b C17H15N3O3NiCl2$2H2O (475.0 g/mol) C, 42.99%; H, 4.03%; N, 8.85%; Ni, 12.36%. Found: C, 42.76%; H, 4.42%; N, 8.43%; Ni, 9.5%. FTIR (KBr, cm1): n(CeOH) 3347; n(CeCH3) 2953; n(C]O) 1718, n(C]N) 1613, n(CeOeC) 1117, 1106. MS-FAB (m/z): 307 (28%) [(b)], 367 (28%) [Ni(b)]2þ, 402 (90%) [Ni(b)Cl]þ, 676 (16%) [Ni(b)2]2þ, 711 (28%) [Ni(b)2Cl]þ. 5.1.1.6. Synthesis of complex 2c. Nickel(II) chloride NiCl2$6H2O (23.6 mg, 0.1 mmol) was dissolved in 1 ml methanol and was added dropwise at room temperature to a stirred solution of 1-benzothiazol2-yl-5-(2-hydroxyphenyl)-3-methyl-1H-pyrazole-4-carboxylic acid methyl ester c (72.3 mg, 0.2 mmol) in ethyl acetate (10 ml). The stirring was continued for 1 h at room temperature and the reaction mixture was left standing overnight. A light green micro-crystalline product was obtained, filtered off and dried. Yield: 41.7 mg (46%). Anal. Calcd. for 2c C38H30N6O6S2NiCl2$3H2O (914.35 g/mol) C, 49.92%; H, 3.97%; N, 9.19%; S, 6.04%; Ni, 5.54%. Found: C, 50.18%; H, 3.79%; N, 9.25%; S, 6.87%; Ni, 6.34%. FTIR (KBr, CsI, cm1): n(CeOH) 3305; n(CeCH3) 2925; n(C]O) 1721, n(C]N) 1611, 1515; n(CeOeC)1128, 1081, n(NieN) 431; n(NieN) 347. MS-FAB (m/z): 423 (28%) [Ni(c)]2þ, 458 (15%) [Ni(c)Cl]þ, 788 (9%) [Ni(c)2]2þ, 823 (4%) [Ni(c)2Cl]þ. 5.1.1.7. Synthesis of complex 3c. Ligand c (109.6 mg, 0.3 mmol) was suspended in acetonitrile (10 ml) and anhydrous nickel(II) chloride NiCl2 (19.4 mg, 0.15 mmol) was added. The reaction suspension was stirred for 18 h and light green micro-crystalline product was obtained. The mixture was evaporated, light green micro-crystalline product 3c was washed with dichloromethane and dried in vacuum. The solid product 3c was recrystallized by slow diffusion of diethyl ether into DMF solution of this complex. The resulting green crystals 4c were filtered off and dried. Yield: 97.0 mg (74.0%). Anal. Calcd. for 3c C38H30N6O6S2NiCl2$H2O (878,42 g/mol) C, 51.96%; H, 3.67%; N, 9.57%, S, 7.29%; Ni, 6.68%. Found: C, 51.67%; H, 3.63%; N, 9.60%, S, 7.60%; Ni, 6.21%. FTIR (KBr, CsI, cm1): n(OeH) 3442; n(CeCH3) 3099; n(C]O) 1717; n(C]N) 1616, 1516; n(CeOeC) 1112, 1095; n(NieN) 434; n(NieN) 345, 351. MS-FAB (m/z): 366 (20%) [(c)], 423 (90%) [Ni(c)]2þ, 458 (72%) [Ni(c)Cl]þ, 788 (25%) [Ni(c)2]2þ, 823 (22%) [Ni(c)2Cl]þ. 5.2. Cells and cytotoxicity assay 5.2.1. Cell cultures Human skin melanoma WM-115 cells as well as human leukaemia promyelocytic HL-60 and lymphoblastic NALM-6 cell lines were used. Leukaemia cells were cultured in RPMI 1640 medium supplemented with 10% foetal bovine serum and antibiotic (gentamicin). For melanoma WM-115 cells Dulbecco’s minimal essential medium (DMEM) instead of RPMI 1640 was used. In some cases normal human umbilical vein endothelial cells (HUVECs) were also used. HUVECs and all reagents for cell culture were purchased from Cascade Biologics (Portland, Oregon, USA). The HUVECs were cultured according to the manufacturer’s instructions and the cells underwent 3e8 passages. Cells were grown in 37  C in a humidified atmosphere of 5% CO2 in air. 5.2.2. Cytotoxicity assay by MTT Cytotoxicity of ligands, their complexes, carboplatin and cisplatin was determined by the MTT [3-(4,5-dimethylthiazol-2-

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yl)-2,5-diphenyltetrazolium bromide, Sigma, St. Louis, MO] assay as described [28]. Briefly, after 46 h of incubation with drugs, the cells were treated with the MTT reagent and incubation was continued for 2 h. MTT e formazan crystals were dissolved in 20% SDS and 50% DMF at pH 4.7 and absorbance was read at 562 and 630 nm on an ELISA e plate reader (ELX 800, Bio-Tek, USA). The values of IC50 (the concentration of test compound required to reduce the cells survival fraction to 50% of the control) were calculated from concentrationeresponse curves and used as a measure of cellular sensitivity to a given treatment. Stock solutions of ligands, nickel complexes, carboplatin and cisplatin were freshly prepared in DMSO and diluted with complete culture medium to obtain a concentration range from 107 to 103 M. DMSO concentration never exceeded 0.2% and had no influence on cell growth. As a control, cultured cells were grown in the absence of drugs.

5.3. X-ray measurements X-ray data were collected at 173 K (2a) and 200 K 4c(4c0 þ 4c00 ) A) with an Oxford Diffraction with MoKa radiation (l ¼ 0.71073  Xcalibur diffractometer (2a) and a Nonius Kappa CCD diffractometer equipped with a rotating anode generator (4c). The structures were solved with direct methods with S/R 97 [29] and refined with SHELXL-97 by full matrix least-squares on F2 [30]. All non-hydrogen atoms were refined anisotropically. The crystal data and X-ray details are given in Table 2. Further details are available from the Crystallographic Data Centre under the depository numbers CCDC 810830 (2a) and CCDC 810829 4c(4c0 þ 4c00 ). Copies of the data can be obtained free of charge upon application to CCDC, 12, Union Road, Cambridge CB2 1EZ, UK, e-mail: [email protected].

Acknowledgements Financial supports from Medical University of Lodz (grant No. 503/3-066-02/503-01 to Prof. Budzisz, grant No. 503/3-014-02/ 503-01 to Department of Physical and Biocoordination Chemistry at Medical University of Lodz and grant No. 503/3-015-02/503-01 to Department of Pharmaceutical Biochemistry at Medical University of Lodz) as well as grant No. 411 at Collegium Medicum in Bydgoszcz are gratefully acknowledged.

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