Synthesis, structural characterization and in vitro anticancer activity of two new nickel complexes bearing imine bonds

Journal Pre-proofs Synthesis, structural characterization and in vitro anticancer activity of two new nickel complexes bearing imine bonds Burak Ay, İlyas Gönül, Burcu Saygıdeğer Demir, Yasemin Saygıdeğer, İbrahim Kani PII: DOI: Reference:

S1387-7003(19)31196-7 https://doi.org/10.1016/j.inoche.2020.107824 INOCHE 107824

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Inorganic Chemistry Communications

Received Date: Revised Date: Accepted Date:

18 November 2019 13 January 2020 31 January 2020

Please cite this article as: B. Ay, I. Gönül, B.S. Demir, Y. Saygıdeğer, I. Kani, Synthesis, structural characterization and in vitro anticancer activity of two new nickel complexes bearing imine bonds, Inorganic Chemistry Communications (2020), doi: https://doi.org/10.1016/j.inoche.2020.107824

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1

Synthesis, structural characterization and in vitro anticancer activity of two new nickel

2

complexes bearing imine bonds

3 4 5 6 7 8 9 10 11

Burak Ay a,*, İlyas Gönüla, Burcu Saygıdeğer Demirb, Yasemin Saygıdeğerb,c, İbrahim Kanid a

Department of Chemistry, Arts and Science Faculty, Çukurova University, 01330, Adana, Turkey b Central Research Laboratory of Çukurova University (CUMERLAB), Adana, Turkey c Department of Pulmonary, Cukurova University School of Medicine, Adana, Turkey d Department of Chemistry, Faculty of Science, Anadolu University,26470, Eskişehir, Turkey Phone: +90 322 338 60 84/2442-20, Fax: +90 322 338 60 70 [email protected]

12 13 14

Abstract

15

Herein, we describe the synthesis and characterization of two novel nickel complexes, [Ni(L1)2]

16

(1)

17

(diethylamino)ethyl)imino)methyl)-6-ethoxyphenol

18

(dimethylamino)ethyl)imino)methyl)-6-methoxyphenol (HL2) ligands. Complexes have been

19

synthesized under conventional methods and characterized by elemental analysis, FT-IR, ICP-

20

OES, TGA, and single crystal X-ray diffraction analysis. The geometry of the 2 is also

21

supported by magnetic susceptibility and Q-TOF LC/MS analysis. The nickel ions are in square

22

planar and distorted octahedral coordination environments, respectively. Complex 1 and 2

23

crystallizes in the monoclinic C1 c1 and C1 2/c1 space group. The antitumor activities of

24

complexes 1-2 have been investigated. Both of the complexes showed dose dependent

25

cytotoxicity and killed the newly established lung cancer cells via apoptotic pathway. The novel

26

nickel complexes were determined as potential antitumor agents.

and

[Ni(L2)2(CH3COO)].9H2O(CH3COOH)

(2),

containing

(E)-2-(((2-

(HL1)

and

(E)-2-(((2-

27 28

Keywords: Nickel complexes, schiff base, primary lung cancer cell, apoptosis, cell viability,

29

antitumor

30 31

1. Introduction

32 33

Therapeutic organometallic compounds have become an acceptable area of research in

34

medicine after the discovery of cis-platin [1, 2]. Because of its proven efficacy, most cancer

35

patients are treated with platinum-based drugs. However, these drugs have negative effects such

36

as lack of selectivity and side effect [3]. Organometallic compounds including nickel, copper,

37

cobalt, zinc, ruthenium, iridium, etc., have been reported to possess much better anticancer

38

activity than cis-platin [4-7]. Platinum based drugs have high costs and toxicity so researchers 1

39

have paid more attention to transition metals, such as nickel [8]. It is well accepted that nickel

40

is as essential ultra-trace nutrient in the human body. It is found in the body in highest

41

concentrations in the nucleic acids, particularly RNA, and is thought to be somehow involved

42

in protein structure or function. It has been speculated that nickel may play a role, as a cofactor,

43

in the activation of certain enzymes related to the breakdown or utilization of glucose [9]. More

44

specifically, nickel complexes with different biological activities have been reported in the

45

literature including antibacterial [10-12], antifungal and anti proliferative/anticancer properties

46

[13-15].

47

Currently, multi‐drug resistance and harmfull side effects have been reported for some

48

of the well known anticancer drugs used clinically such as cisplatin [16, 17]. To overcome the

49

limitations of cisplatin and to develop new potential less toxic, more effective and selective

50

anticancer agents, the interaction of other metal complexes with DNA attracted scientists in

51

medicinal Chemistry [18]. The transition metal complexes are of great interest in this regard

52

because of their binding properties to DNA. In addition, these complexes are potent catalytic

53

inhibitors of DNA gyrase [19, 20]. Schiff bases are also popular compounds with amazing

54

biological properties. They are sophisticate metal complexing ligands and have been used to

55

coordinate almost all d-block metals. Schiff bases and their metal complexes have been reported

56

to exhibit anticancer properties including DNA damage [21]. The interaction of Schiff base

57

metal complexes with Zn-fingers result in displacement of zinc ion from the Zn-finger which

58

prevents binding of DNA with transcription factor [22]. Lung cancer is the leading cause of

59

cancer-related deaths worldwide, representing 25% of all cancer related deaths. Among lung

60

cancers, 80% are classified as non-small cell lung cancer (NSCLC) and 20% as small cell lung

61

cancer (SCLC) [23]. Therefore, studies on the treatment of lung cancer have attracted the

62

attention of worldwide scientists.

63

In this study, we report the synthesis and characterization of nickel complexes [Ni(L1)2]

64

(1) and [Ni(L2)2(CH3COO)].9H2O(CH3COOH) (2). The ligands obtained from condensation

65

reactions of N',N’-diethylethane-1,2-diamine and 3-ethoxy-2-hydroxybenzaldehyde (HL1)

66

(Fig. 1), N',N'-dimethylethane-1,2-diamine and 2-hydroxy-3-methoxybenzaldehyde (HL2)

67

(Fig. 2). The synthesized compounds characterized by elemental analysis, TGA, FT-IR,

68

magnetic susceptibility, Q-TOF LC/MS ICP-OES and single crystal X-ray analysis. We

69

established a primary lung squamous cell carcinoma cell line, which is a type of NSCLC, and

70

evaluated the antitumor activity of complexes 1 and 2 on these cells.

71 72 2

73

2. Experimental

74 75

Experimental details regarding the materials used employed, together with some anticancer

76

procedures are given in the Supplementary material.

77 78

2.1. Synthesis of complex 1

79 80

N1,N1-diethylethane-1,2-diamine (1.162 g, 10.0 mmol) was dissolved in 20 mL of ethanol

81

solution. 3-ethoxy-2-hydroxybenzaldehyde (1.662 g, 10.0 mmol) was then added to the solution

82

and refluxed for 1 h. Ni(CH3COO)2.4H2O (1.244 g, 5.0 mmol) was added as solid onto the

83

homogenous solution. The metal salt was dissolved and refluxed for 3 hours. After the reaction

84

was completed, the mixture was cooled to room temperature and the ethanol was completely

85

evaporated in a rotary evaporator. Crystallization was attempted in many common organic

86

solvents, but was obtained as powder in each trial. To overcome this problem, cellulosic thinner

87

was used as a solvent. The resulting powder mixture was dissolved in 20 mL cellulosic thinner

88

and heated for 1 hour. Then the solution was filtered to remove the impurities. After 3 days the

89

green crystals were obtained suitable for X-ray diffraction analysis were obtained in 92% yield.

90

Anal. Calcd. for C30H46N4NiO4: C, 61.55; H, 7.92; N, 9.57. Found: C, 61.68; H, 8,08; N, 9.82%.

91

The ICP-OES analysis (%) showed that 1 contained Ni: 10.21; Calcd.: 10.03. IR (KBr pellet,

92

cm-1): 3423 (m), 3000-2800 (m), 1612 (s), 1474 (s), 1333 (s), 1246 (s), 1121 (m), 766 (w), 737

93

(m), 726 (m), 461 (w), 408 (w). 1H NMR (CDCl3, ppm, 500 MHz) δ: 8.38 (s, CH=N), 7.28 (s,

94

Cl3-CH), 7.27-6.60, (m, Ar-CH), 5.00, (s, OCH2CH3), 4.00, (d, NCH2CH2), 2.60, (s,

95

OCH2CH3), 2.30, (d, NCH2CH2), (Fig. S1).

96 97 98 NH2

99

O

HO OH

100 101

O

N

N

O

N

C H

102 103

CH3OH

104

3

Ni(CH3COO)2.4H2O

N H C

O O

N Ni N

O O

C H

N

105 106

Figure 1. Reaction pathway for the synthesis of 1.

107 108

2.2. Synthesis of complex 2

109 110

N1,N1-dimethylethane-1,2-diamine (0.882 g, 10.0 mmol) was dissolved in 20 mL of ethanol

111

solution. 2-hydroxy-3-methoxybenzaldehyde (1.522 g, 10.0 mmol) was then added to the

112

solution and refluxed for 1 h. Ni(CH3COO)2.4H2O (1.244 g, 5.0 mmol) was added as solid onto

113

the homogenous solution. The metal salt was dissolved and refluxed for 3 hours. After the

114

reaction was completed, the mixture was cooled to room temperature and the ethanol was

115

completely evaporated in a rotary evaporator. After that, complex 2 was obtained by same

116

procedure as complex 1. Green single crystals suitable for X-ray diffraction analysis were

117

obtained in 90% yield. Anal. Calcd. for C28H56N4NiO15.5: C, 44.52; H, 7.47; N, 7.42. Found: C,

118

44.23; H, 7,83; N, 7.24%. The ICP-OES analysis (%) showed that 1 contained Ni: 7.53; Calcd.:

119

7.77. IR (KBr pellet, cm-1): 3412 (m), 3000-2750 (m), 1625 (s), 1602 (s), 1471 (s), 1318 (m),

120

1219 (s), 1075 (s), 969 (m), 855 (s), 784 (m), 736 (s), 468 (w), 424 (w). Q-TOF MS (70eV):

121

m/z 755.6502 [M+] (Exact Mass: 754.31) (Fig. S2).

122

4

NH2

N

O

N

OH N

OH

HC

O

O

CH3OH

Ni(CH3COO)2.4H2O

123 O CH O C

H3 C

O O

N

N Ni

9H2O (CH3COOH)

O O

N HC

N

124 125

Figure 2. Reaction pathway for the synthesis of 2.

126 127 128

2.3.X-ray structure determination

129

The crystallographic data collection for complex 1 and 2 were collected with Bruker AXS

130

APEX CCD diffractometer equipped with a rotation anode at 296(2) K using graphite

131

monocromated Mo Kα radiation (λ = 0.71073 Å). Diffraction data were collected over the full

132

sphere and were corrected for absorption. The data reduction was performed with the Bruker

133

SMART [24] program package. Structure solution was found with the SHELXS-97 [25]

134

package using the direct methods and was refined SHELXL-97 [26] against F2 using first

135

isotropic and later anisotropic thermal parameters for all nonhydrogen atoms. Hydrogen atoms

136

were added to the structure model on calculated positions. Geometric calculations were

137

performed with Platon [27].

138 139

2.4. Cell viabiliy assay

140 141

The novel metal complexes were tested for their cytotoxicity against a new primary lung cancer

142

cell SA7 using (MTT) Thiazolyl Blue Tetrazolium Bromide method according to [28]. Briefly, 5

143

SA7 cells (1.0×104/200 mL/well) were cultured in a 96-well plate for overnight at 37ºC, 5%

144

CO2 and 80% humidity in their respective medium containing 10% FBS and 1% AB. After 24h

145

old medium was removed and the cells were incubated with 0.1-100 µM concentrations of the

146

compounds for 24 and 48h at 37ºC, 5% CO2. Cells with 0.1% DMSO (vehicle control) and

147

cisplatin (positive control) were also incubated at the same conditions. After incubations, 10 μL

148

of MTT solution (5 mg/mL in PBS buffer) was added and the cells were further incubated at

149

37ºC, 5% CO2 for 4h to metabolize MTT by viable cells. After MTT treatment, the supernatants

150

were carefully removed, 50μL DMSO was added to each well and then absorbance was

151

measured at 630nm subtracted from optical density at 570 nm in a multi-well plate reader. The

152

percentage of viability cells was calculated by comparison with control cells (without

153

synthesized compounds) using the equation of: (A) sample/ (A) control × 100.

154 155

2.5. Flow Cytometry assay

156 157

Annexin-V staining was performed according to the protocol of BIOLEGEND apoptosis

158

detection kit [29]. For quantitative analysis, 100 µM concentration of complexes was tested on

159

SA7 cell. Cancer cell (1.0 × 107 cells/mL) suspension in serum-free medium was incubated with

160

the respective compound in 6-well plates in a CO2 incubator. After treating with compound for

161

48 h, the cancer cells were harvested and incubated with APC Annexin V and PI. The

162

fluorescence emission of APC Annexin-V stained cells was measured at 633 nm (Red laser) in

163

a flow cytometer (Beckman Coulter/CytoFLEX, United States). Dots represent cells as follows:

164

lower left quadrant, normal cells (APC−/PI−); lower right quadrant, early apoptotic cells

165

(APC+/PI−); upper left quadrant, necrotic cells (APC−/PI+); upper right quadrant, late

166

apoptotic cells (APC+/PI+).

167 168

3. Results and discussion

169 170

Experimental results such as IR spectra, thermal properties and magnetic susceptibility of the

171

complexes are given in the Supplementary material.

172 173

3.1. Structure analysis of complex 1 and 2

174 175

Figure 4 shows a molecular drawing of the Ni (II) complex 1 and 2 together with the selective

176

atomic labeling and the crystal data and structure refinement for the 1 and 2, the some bond 6

177

distances and angles are shown in Tables S1 and S2. Complex 1 and 2 crystallizes in the

178

monoclinic C1 c1 and C1 2/c1 space group, respectively. The complex 1 is made up of a

179

deprotonated ligands and the nickel center in a N2O2 square planar geometry (Fig. 3). The four-

180

coordinate geometry around the nickel complex 1 is close to square planar, and the 4 value of

181

0.016 accurately reflects this description [30]. In both complexes the ligand acts as a bidentate

182

chelate donor, the coordination mode that leads to the formation of two five-member chelate

183

rings that confers high stability to the compounds. In complex 1, the torsion angles of two 5-

184

membered chelate rings, namely N(4)–Ni–O3–C28 (0.3°) and O(1)–Ni–N1–C9 (O.5°), were

185

the similar and almost planer. The coordinated ligands are in trans-position. The average bite

186

of N-Ni-O is 89.97 °. The Ni–O distances (1.819 (6) and 1.808 (7) is slightly shorter than Ni–

187

N distances (1.895 (5) and 1.903 (7) A˚).

188 189 190 191 192 193 194 195 196 197 198

Figure 3. Molecular structure of complex 1.

199 200

Crystallographic analysis of mononuclear nickel complex 2, indicates that it consists of one

201

Ni(III) ion, two completely deprotonated ligand, one coordinated acetate ion, nine non-

202

coordinated water molecules and one acetic acid molecule (Fig. 4). The Ni(III) atom is located

203

at the N2O6 coordination sphere of ligand moieties, which is hexacoordinated by two

204

deprotonated oxygen (O1 and O4) and nitrogen atoms (N1 and N2). The amine N atom and O

205

atom from both ligands chelate to the same nickel ion with the ligands cis to each other. The

206

two oxygen atoms of acetate ion chelate to nickel complete the six coordination and adapts to

207

distorted octahedral geometry. The N1–Ni–O2 and O3–Ni–N3 planes are nearly perpendicular

208

(88.43) to each other and O5–Ni–O6 plane is also perpendicular to N1–Ni–O2 and O3–Ni–N3

209

planes, respectively, 88.28 ° and 85.59 °. The Ni–O and Ni–N bond lengths [1.819(6), 1.808(6),

210

and 1.895(8), 1.930(7) A˚, respectively] are comparable with the corresponding values in other 7

211

related complexes [31, 32]. The N–C bond lengths [1.271(3) and 1.283(3) A ˚] are indicative

212

of (C–N) single bond and N=C double bond in th range of 1.475 (4) ° to 1.501 (5) °. For

213

molecule the large distortion from regular octahedral geometry reflects the small ‘bites’ of the

214

3-membered chelate ring of acetate ion (O5NiO6) 59.6° and the small distortion of the bite

215

angles of 6-membered rings of coordinated ligands [88.3(3) ° and 89.56(3) °]. In

216

supramolecular chemistry, intra and intermolecular weak interactions such as p–p stacking and

217

hydrogen bonding contribute significantly to the self-assembly and stabilize the complexes in

218

the solid state.

219 220 221 222 223 224 225 226 227 228 229 230 231 232

Figure 4. Molecular structure of complex 2.

233 234

3.2. Cytotoxicity of the complexes

235 236

In cell viability assay we tested different concentrations of 1 and 2 on lung cancer cells (SA7)

237

for 24h and 48h. Both complexes showed dose dependent cytotoxicity on the cells (Fig. 5). In

238

SA7 cells, compounds 1 and 2 displayed anti-proliferative activity for 24h and 48h. Incubation

239

of both compounds with the cells for 48 hours resulted in a lower percentage of viable cells

240

than 24 hours. 100 µM concentration of the complex 1 left approximately 40% of the cells alive

241

in 24 hours, while approximately 20% in 48 hours. 50% of the cells treated with complex 2 (30

242

µM) survived for 24 hours and 40% for 48 hours. While complex 1 showed better cytotoxicity

243

at 100 µM concentration, complex 2 was good at 30 µM. These finding showed that ligand is

244

very important in terms of cytotoxic activity of the complexes. In a study diazole bearing Schiff 8

245

bases showed different antiproliferative effect with various IC50 values depend on substutient

246

groups [33]. Another imine bearing nickel complex showed good cytotoxicity with 9,6 IC50

247

value on A549 (human alveolar adenocarcinoma epithelial cell line) cells [34]. But at the same

248

study, nickel complexes of ligands including different substutient groups did not show cytotoxic

249

activity on the same cells. It is understood that the structure of the ligand is as important as the

250

metal effect in the cytotoxicity of the metal complexes.

251 Cisplatin vs Complex 1

Cisplatin vs Complex 2

24h

48h

252 253 254 255 256 257 258 259

Figure 5. Cell viability assays for complexes 1, 2 and Cisplatin on SA7 cells were performed via MTT protocol with given concentrations for 24 and 48 hours. Both complexes had increased cytotoxic effects on the cells comparing to cisplatin. 3.3. Apoptotic effect of the complexes on primary lung cancer cells

260 261

We further evaluated the induction of apoptosis in 100 µM concentrations for 24 and 48 h and

262

the results revealed that both compounds had their cytotoxic effects in apoptotic way. Neither

263

of the complexes produced significant toxicity in terms of necrosis to SA7 cells (upper left

264

sections) (Fig. 6A). 48 hours of treatment with both complexes resulted in higher apoptosis than

265

24 hours; The percentage of apoptosis for complex 1 was 35,85% and complex 2 was 45,68%

266

at 24h, apoptosis rate of complex 1 was 52,52% and complex 2 was 57,51% at 48h. Complex

267

2 induced apoptosis more cells than complex 1 during both incubation periods (Fig. 6B). This 9

268

finding is consistent with cytotoxicity results. But the difference between the apoptotic effects

269

of the two complexes is not much. Therefore, it can be said to the contribution of the differences

270

in the structure of the ligands to the apoptosis was not significant. It is known in the literature

271

that Ni (II) complexes showed cytotoxicity and induce apoptosis at different rates in various

272

cell lines [35]. 1 and 2 also showed different results to resemble molecules in the literature. This

273

difference, may be explained by the fact that the structure of the ligand and its binding with Ni

274

(II) are different each other. Coupling of metal complexes with DNA can occur in various ways

275

induced cell death. In fact, a metal complex can often use several types of bonds at the same

276

time [36]. Intercalation is a common pathway [37]. The insertion of an intercalator between

277

adjacent base-pairs results in a substantial change in DNA structure, causing lengthening,

278

stiffening and unwinding of the DNA helix [38]. Nickel complexes disrupt the structure of DNA

279

and induce apoptosis by acting as metallo-intercalator with DNA as other transition metals as

280

mentioned in the literature [39-42]. The conformational difference in the structure of the ligand

281

of complexes 1 and 2, and the resulting electronic distribution, will alter the interaction of DNA

282

with metal, either through the intercalator effect or other bindings. DNA binding properties of

283

the Schiff base nickel complexes according to the molecular geometry was investigated in a

284

review by Barone et al. The evaluations display that tetracoordinate complexes generally

285

behave as DNA-intercalators whereas hexacoordinate complexes can be groove binders,

286

intercalators or both depending on the size of the planar moiety of the coordinated ligands [43].

287

The most common geometry is tetracoordinate among Schiff base metal complexes, assume

288

square-planar or distorted tetrahedral geometry. Tetracoordinate nickel(II) complexes of strong

289

field ligands tend to assume stable square-planar geometry. In addition, the square-planar

290

geometry of nickel(II) complexes is forced by cyclic porphyrin-like tetradentate ligands, with

291

O or N donor atoms [37]. The ligand of complex 1 acts as a bidentate chelate donor in this

292

study, the coordination mode that leads to the formation of two five-member chelate rings that

293

confers high stability to the compounds. So, it can behave as a good DNA- intercalator.

294

Hexacoordinated in a distorted octahedral geometry (overall charge 4+), permits strong

295

electrostatic major groove binding by inducing DNA bending and intramolecular coiling and

296

also allows supramolecular rigid structures according to a hypothesis of Hannon et al. [44]. In

297

current study, nickel ion of complex 2 is in distorted octahedral coordination environments. So

298

the complex 2 is likely to have strong electrostatic interactions with DNA.

299

10

A 24 h

48 h

Control

Complex 1

Complex 2

B

300 301 302 303 304 305 306

Figure 6. After exposure of the cells to 100 µM 1 and 2 complexes for 24 and 48h, the cell viability of SA7 cells was measured by flow cytometry. Upper (Late) and lower (early) right panels show the apoptotic cells of the population (A). Apoptotic cell rates in Complex 1 and 2 treated SA7 cells are given in bar graphs (B).

307

is in the body is a critical requirement for a drug discovery. So we tested physiological condition

308

stability of compounds 1 and 2 in phosphate buffer solution at 37 ºC. As shown in Figure S3, it

309

can be found that the UV-Vis spectra of 1and 2 retained without new emerging absorption peaks

310

and no obvious hypochromic effect, suggesting that 1 and 2 were stable in solution over 48 h.

311

So two novel nickel complexes display significant stability under physiological conditions.

Withstanding physiological challenges without decomposition while a therapeutic compound

11

312

4. Conclusion

313 314

In this study, we described here the synthesis of two asymmetric bidentate and tridentate nickel

315

complexes. The characterization of the complexes was achieved through different spectroscopic

316

and analytic methods. The complex 1 was made up of a deprotonated ligands and the nickel

317

center in a N2O2 square planar geometry. Crystal structure of the complex 2 showed that the

318

two oxygen atoms of acetate ion and ligand chelate to nickel complete the six coordination and

319

adapted to distorted octahedral geometry. The newly synthesized two nickel complexes were

320

found to have cytotoxic effect on primary non-small cell lung cancer cells of SA7. It was

321

determined that these complexes performed cell death by inducing apoptotic pathway.

322

Complexes 1 and 2 are molecules that have the potential to be moderately antitumor agents.

323

This study supports the efficacy of nickel complexes of imine bearing ligands in terms of anti-

324

tumoral activity.

325 326

Acknowledgements

327 328

The authors gratefully acknowledge the Medicinal Plants and Medicine Research Centre of

329

Anadolu University, Eskişehir, Turkey, for the use of X-ray Diffractometer. In-Vitro biological

330

experiments were partially supported by Turkish Thoracic Society and Çukurova University

331

Scientific Research Board.

332 333

Appendix A. Supplementary material

334 335

CCDC 1910802 and 1910803 contains the supplementary crystallographic data for complex 1

336

and

337

http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic

338

Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail:

339

[email protected].

2,

respectively.

The

data

can

340 341 342 343

Abbreviations

344

PBS: Phosphate buffer solution

345

FBS: Fetal bovine serum

MTT: Thiazolyl Blue Tetrazolium Bromide

12

be

obtained

free

of

charge

via

346

AB: Antibiotic

347

DMSO: Dimethylsulfoxide

348

APC: Allophycocyanin

349

PI: Propidium iodide

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