Synthesis, spectroscopic characterization, biological activity, DFT and molecular docking study of novel 4-hydroxycoumarine derivatives and corresponding palladium(II) complexes

Journal Pre-proofs Research paper Synthesis, spectroscopic characterization, biological activity, DFT and molecular docking study of novel 4-hydroxycoumarine derivatives and corresponding palladium(II) complexes Edina H. Avdović, Žiko B. Milanović, Marko N. Živanović, Dragana S. Šeklić, Ivana D. Radojević, Ljiljana R. Čomić, Srećko R. Trifunović, Ana Amić, Zoran S. Marković PII: DOI: Reference:

S0020-1693(19)31973-5 https://doi.org/10.1016/j.ica.2020.119465 ICA 119465

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Inorganica Chimica Acta

Received Date: Revised Date: Accepted Date:

16 December 2019 20 January 2020 20 January 2020

Please cite this article as: E.H. Avdović, Z.B. Milanović, M.N. Živanović, D.S. Šeklić, I.D. Radojević, L.R. Čomić, S.R. Trifunović, A. Amić, Z.S. Marković, Synthesis, spectroscopic characterization, biological activity, DFT and molecular docking study of novel 4-hydroxycoumarine derivatives and corresponding palladium(II) complexes, Inorganica Chimica Acta (2020), doi: https://doi.org/10.1016/j.ica.2020.119465

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1

Synthesis, spectroscopic characterization, biological activity, DFT and

2

molecular docking study of novel 4-hydroxycoumarine derivatives and

3

corresponding palladium(II) complexes

4 5

Edina H. Avdovića,b*, Žiko B. Milanovićb,c, Marko N. Živanovića,c, Dragana S. Šeklića,

6

Ivana D. Radojevića,d, Ljiljana R. Čomićd, Srećko R. Trifunovićb, Ana Amiće, Zoran S.

7

Markovića

8 9

aUniversity

of Kragujevac, Institute for Information Technologies, Department of Science, Jovana Cvijica bb, 34000 Kragujevac, Serbia

10 11

bUniversity

of Kragujevac, Faculty of Science, Department of Chemistry, Radoja Domanovića 12, 34000 Kragujevac, Serbia

12 13 14

cBioengineering dUniversity

of Kragujevac, Faculty of Science, Department of Biology and Ecology, Radoja Domanovića 12, 34000 Kragujevac, Serbia

15 16

Research and Development Center, 34000 Kragujevac, Serbia

eJuraj

Strossmayer University of Osijek, Department of Chemistry, Ulica cara Hadrijana 8/A, Osijek , Croatia

17 18 19

*Corresponding author’s e-mail address: [email protected]

20 21

Abstract

22

In the present manuscript, palladium(II) complexes (C1, C2) with newly synthesized

23

coumarine ligands 3-(1-((3-chlorophenyl)amino)ethylidene)-chroman-2,4-dione (L1) and 3-

24

(1-((4-chlorophenyl)amino)ethylidene)-chroman-2,4-dione

25

structurally characterized by spectroscopic techniques (FT-IR, 1H NMR,

(L2)

were

prepared 13C

and

NMR) in

26

combination with elemental analysis and theoretical methods (DFT). The structures of all

27

compounds were fully optimized using the B3LYP-D3BJ theoretical method. Cytotoxic

28

activity of investigated compounds was tested against two cells lines (human colorectal

29

carcinoma, HCT-116, and human fibroblast lung MRC-5), while their antimicrobial activity

30

was screened against nine strains of pathogenic bacteria, five mould species and two yeast

31

species. Unfortunately, their cytotoxic and antibacterial activities were weak. Docking studies

32

for all compounds with epidermal growth factor receptor (EGFR) were performed. It was found

33

that hydrophobic interactions that include chlorine atom have somewhat lower values of the

34

pairwise interaction energies compared to the purely hydrophobic interactions. In addition, it

35

was found the chlorine atom in the para position contributes to the slightly higher binding free

36

energy and lower values of constant of inhibition.

37 38

Keywords: coumarin-derived ligands; palladium(II) complexes; cytotoxicity; antimicrobial

39

activity; DFT; molecular docking.

40 41

1. Intoduction

42 43

Infectious

diseases

are

a

major

cause

of

morbidity

and

mortality

in

44

immunocompromised and including patients with cancer [1]. The rate of mortality from

45

infectious diseases has increased significantly due to the spread of HIV infections and resistant

46

bacterial pathogens such as methicillin-resistant strains of Staphylococcus aureus (MRSA),

47

vancomycin-resistant enterococci (VRE), multiresistant gram-negative bacteria, and

48

multiresistant strains of Mycobacterium tuberculosis [2].

49

According to data from the World Health Organization (WHO), cancer is the second

50

leading cause of death in the world. It should be noted 9.6 million people died from the

51

consequences of this disease during 2018 [3]. There are more than 100 different types of cancer

52

known in the human population. The most commonly diagnosed cancer types in the world are:

53

breast, prostate, colon, lung, and stomach cancer [4]. Infectious diseases could also be the cause

54

for cancer development induction and progression [5]. For example, some viruses can cause

55

cancer, such as: human papilloma virus (cervical cancer) [6], hepatitis B and C (hepatocellular

56

carcinoma)

57

nasopharyngeal cancer) [9,10]. In addition, bacterial infections may increase the risk of cancer,

58

such as Helicobacter pylori, which causes gastric cancer [11,12]. Epidermal growth factor

59

receptor (EGFR) is upregulated in many types of cancers thus it could be interesting to analyse

60

interaction of EGFR and here investigated substances [13].

[7,8],

Epstein-Barr

virus

(EBV)

(lymphoproliferative

disorders

and

61

Coumarins are simple phenolic compounds that are widespread in the plants world [14].

62

Coumarins can be found in vegetables [15,16], trees [17] seeds [18], fruits [19], coffee, and

63

vine [20]. As secondary metabolites, at higher concentration, these compounds protect the plant

64

against pathogen [21]. In addition, these compounds play a role in the biochemistry and

65

physiology of plants. They can be involved in growth regulation, photosynthesis, and control

66

of respiration [22].

67

Due versatile pharmacological and biological activities, including antitumor [23,24],

68

anticoagulant [25,26], antibacterial [27], antioxidant [16,28], and antifungal [29], coumarins

69

attract significant attention from many chemists from the very beginning of their discovery.

70

Keeping this in mind, chemists have developed different methods for the synthesis of new

71

coumarin derivatives.

72

Complexes of coumarins with transition metals show significant biological activity. A

73

large number of palladium(II) complexes has been synthesized and their antimicrobial and

74

cytotoxic activity has been examined. For example, palladium(II) complexes with coumarin

75

ligands 3-(1-aminoethylidene)-2H-chromene-2,4(3H)-dione and 3-(1-(2-hydroxyethylamino)-

76

-ethylidene)chroman-2,4–dione showed important cytotoxic activity on different cell lines [30-

77

32]. It should be noted that the antitumor activity of these complexes is significantly higher

78

than the well-known therapeutic agents of cisplatin and carboplatin. In addition, numerous

79

Pd(II) complexes have shown antibacterial, antiviral, and antifungal activities [33,34]. Keeping

80

this in mind, our research group synthesized new coumarin derivatives as well as the

81

corresponding Pd(II) complexes, which also showed antimicrobial and cytotoxic activity [35-

82

38]. In this paper, we continue earlier studies of the coumarin derivatives and their Pd(II)

83

complexes [35-38]. The synthesis and characterization of two new coumarin derivatives and

84

the corresponding Pd(II) complexes, is described. Structural characterization is supported by

85

the DFT calculations. The results of antimicrobial and cytotoxic activity of investigated

86

compounds are reported. In addition, molecular docking simulation of synthesised compounds

87

with epidermal growth factor receptor (EGFR) was performed.

88 89 90

2. Experimental 2.1. Materials and methods

91

The chemicals and reagents of high purity: 3-chloroaniline, 4-chloroaniline, 3-acetyl-

92

4-hydroxycoumarin, methanol, 96% ethanol and toluene, were purchased from Sigma-Aldrich

93

Chemical Company (St. Louis, MO), Difco and Merck Laboratory Supplies (Darmstadt,

94

Germany). The chemicals and reagents were used without further purification. IR spectra were

95

recorded by a Perkin–Elmer Spectrum One FT-IR spectrometer using the KBr pellet technique

96

(4000–400 cm-1). 1H and 13C NMR spectra were recorded by a Varian Gemini-2000 (200 MHz)

97

spectrometer in CDCl3 using tetramethylsilane as internal standard. Elemental microanalysis

98

for C, H and N was performed on the Vario EL III C, H, and N Elemental Analyzer.

99 100

2.2. General procedure for the synthesis of ligands

101

General scheme for synthesis of new coumarin derivatives 3 is presented in Scheme 1.

102

The reaction mixture of 3-acetyl-4-hydroxycoumarin 1 (0.41 g, 0.002 mol) and corresponding

103

chloroaniline 2 (0.26 g, 0.002 mol) in methanol (50 mL) was refluxed for 3 h. The progress of

104

reactions was monitored by TLC (toluene:acetone=7:3). When the reaction was completed, the

105

mixture was cooled to the room temperature. The obtained crystals were filtered, air-dried and

106

recrystallized from ethanol. R1 3"

4"

R 2

2" 1" OH

NH2

O

5 MeOH +

O

 R1

O R2

107 108

1

2

3

7

9 8

6"

4

10

6

5"

HN

O

O 1

2

3

1'

2'

O R1 R2 L1: Cl H L2: H Cl

Scheme 1. Synthesis of ligands L1 and L2

109 110

3-(1-((3-chlorophenyl)amino)ethylidene)-chroman-2,4-dione (L1) Yield, 0.487 g

111

(77.42%). Anal. Calcd. for C17H12O3NCl (Mr = 314.78) %: C, 64.86; H, 4.17; N, 4.45. Found:

112

C, 64.81; H, 3.79; N, 4.38. 1H NMR (CDCl3, 200 MHz) δ ppm: 2.70 (3H, s, C2'H3), 7.14 (2H,

113

m, C8H, C6″H), 7.27 (2H, m, C6H, C2″H), 7.42 (1H, m, C4″H), 7.59 (2H, m, C7H, C5″H),

114

8.07 (1H, m, C5H), 15.98 (bs, 1H, NH). 13C NMR (CDCl3, 50 MHz) δ ppm: 20.8 (C2′), 98.2

115

(C3), 116.6 (C8), 119.9 (C10), 123.7 (C6), 123.9 (C6″), 125.9 (C2″), 126.1 (C5), 128.4 (C5),

116

130.6 (C4''), 134.3 (C5″), 135.4 (C7), 137.4 (C1''), 148.1 (C3''), 153.9 (C9), 162.1 (C2), 176.1

117

(C1′), 182.0 (C4). IR (KBr)  cm-1: 3501 (NH), 3053 (=CH), 2923, 2854, 2360 (CH), 1707

118

(C=O), 1609, 1589, 1556, 1487, 1467, 1419 (C=C), 1196 (C–O), 767(C–Cl).

119

3-(1-((4-chlorophenyl)amino)ethylidene)-chroman-2,4-dione (L2) Yield, 0.423 g

120

(67.25%), Anal. Calcd. for C17H12O3NCl (Mr = 314.78) %: C, 64.86; H, 4.17; N, 4.45. Found:

121

C, 64.67; H, 3.89; N, 4.41. 1H NMR (CDCl3, 200 MHz) δ ppm: 2.59 (3H, s, C2'H3), 7.33, (2H,

122

m, C8H, C6H), 7.49 (2H, m, C2''H, C6''H), 7.60 (2H, m, C3''H, C5''H), 7.69 (1H, m, C7H),

123

7.99 (1H, m, C5H), 15.48 (bs, 1H, NH). 13C NMR (CDCl3, 50 MHz) δ ppm: 20.7 (C2'), 97.5

124

(C3), 116.5 (C8), 119.8 (C10), 124.0 (C6), 125.9 (C5), 127.1 (C2″, C6″), 129.7 (C3″, C5″),

125

132.8 (C7), 133.6 (C1″), 134.7 (C4″), 153.3 (C9), 161.5 (C2), 176.1 (C1'), 180.5 (C4). IR (KBr)

126

 cm-1: 3419(NH), 3051 (=CH), 2922, 2924, 2680 (CH), 1718 (C=O), 1609, 1562, 1481, 1467,

127

(C=C), 1199 (C–O), 762(C–Cl).

128 129

2.3. General procedure for the synthesis of complexes

130

Complexes C1 and C2 (Scheme 2) were obtained in reaction between K2[PdCl4] (0.050

131

g, 0.15 mmol) in 10 mL of water and equimolar amount of the ligands L1 and L2 (0.047g, 0.15

132

mmol) in 15 mL of methanol with continuous stirring. After mixing for 5 h, yellow precipitates

133

were obtained. The resulting precipitates were filtered and air-dried.

134 O

O

N

O

R1 R1

R2

O

N

O

O

3

7

9

O 1

8

135 136

3

1" 2"

10

6

4"

4

5

K2 PdCl4

O

R2

Pd

R2

HN

5"

6"

2

4

Scheme 2. Synthesis of complexes C1 and C2

1'

O

3"

R1

2' R1 R2 C1: Cl H C2: H Cl

Bis(3-(1-((3-chlorophenyl)amino)ethylidene)-chroman-2,4-dione)

137

palladium(II)

138

complex (C1) Yield, 0.028 g (25.68%). Anal. Calcd. for C34H22O6N2Cl2Pd (Mr = 731.96) %:

139

C, 55.79; H, 3.03; N,3.48. Found: C, 55.30; H, 3.13; N, 3.53. 1H NMR (CDCl3, 200 MHz) δ

140

ppm: 2.28 (s, 3H, C2'H3), 6.76 (1H, dd, C5), 7.17 (1H, m, C6H), 7.30 (1H, m, C8H), 7.43 (2H,

141

m, C5''H, C6''H), 7.53 (2H, m, C2''H, C4''H), 7.66 (1H, m, C7H). 13C NMR (CDCl3, 50 MHz)

142

δ ppm: 24.0 (C2'), 105.8 (C3), 116.0 (C8), 117.7 (C10), 123.1 (C6), 125.5 (C4''), 126.0 (C5),

143

127.0 (C2''), 130.5 (C5''), 133.5 (C6''), 133.5 (C7), 148.0 (C3''), 152.7 (C1''), 152.8 (C9), 162.5

144

(C2), 170.4 (C4), 173.0 (C1'). IR (KBr, cm-1): 2924, 2853 (CH), 1698 (C=O), 1602 (C=N),

145

1586, 1554 (C=C), 1193 (C–O), 763(C–Cl), 682 (Pd–O), 502 (Pd–N). Bis(3-(1-((4-chlorophenyl)amino)ethylidene)-chroman-2,4-dione)

146

palladium(II)

147

complex (C2) Yield, 0.036 g (33.03%). Anal. Calcd. for C34H22O6N2Cl2Pd (Mr = 731.96) %:

148

C, 55.79; H, 3.03; N, 3.82. Found: C, 55.40; H, 3.00; N, 3.63. 1H NMR (CDCl3, 200 MHz) δ

149

ppm: 2.3 (3H, s, C2'H3), 6.5 (1H, dd, C5H), 7.3 (1H, m, C8H), 7.41 (1H, m, C6H), 7.59 (2H,

150

m, C2''H, C6''H), 7.63 (1H, m, C7H), 8.14 (2H, m, C3''H, C5''H). 13C NMR (CDCl3, 50 MHz)

151

δ ppm: 25.0 (C2'), 105.6 (C3), 116.3 (C10), 117.7 (C8), 123.3 (C2″, C6″), 123.9 (C6), 125.9

152

(C5), 129.6 (C3″, C5″), 133.5 (C7), 134.9 (C4''), 145.5 (C1″), 152.6 (C9), 162.5 (C2), 170.4

153

(C4), 172.6 (C1′). IR (KBr, cm-1): 3067 (=CH), 2924, 2395, 1935 (CH), 1708 (C=O), 1603

154

(C=N), 1551, 1484, 1477, 1425, 1412 (C=C), 1194 (C-O), 757 (C–Cl), 686 (Pd-O), 557 (Pd-

155

N).

156 157

2.4. In vitro cytotoxic assay

158

2.4.1. Cell culturing

159

The human epithelial cell line derived from the colorectal carcinoma, HCT-116 and

160

human fibroblast lung MRC-5 cell lines were obtained from American Tissue Culture

161

Collection (ATCC). The cells were cultured in DMEM supplemented with 10% FBS, 100

162

IU/mL penicillin, 100 μg/mL streptomycin, 1% 100x ZellShield at 37 °C in a humidified

163

atmosphere with 5% CO2.

164 165

2.4.2. Cell viability assay

166

The cells were seeded in 96-well plates at density of 104 cells/well. The standardized

167

protocol for MTT assay was used and is well described in our previous study [35]. Absorbance

168

was measured on ELISA microplate reader (Rayto-2100C) at 550 nm.

169 170

2.5. In vitro antimicrobial assay

171

2.5.1. Test substances, microorganisms and suspension preparation

172

The tested compounds were dissolved in DMSO and then diluted into nutrient liquid

173

medium to achieve a concentration of 10%. DMSO was purchased from Acros Organics

174

(NewJersey, USA). Resazurin was obtained from Alfa Aesar GmbH & Co. (KG, Karlsruhe,

175

Germany). An antibiotic, doxycycline (Galenika A.D., Belgrade), was dissolved in nutrient

176

liquid medium, a Mueller–Hinton broth (Torlak, Belgrade), while antimycotic, fluconazole

177

(Pfizer Inc., USA) was dissolved in Sabouraud dextrose broth (Torlak, Belgrade).

178

The antimicrobial activity of the ligands and complexes was tested against 16

179

microorganisms. The experiment involved 9 strains of pathogenic bacteria (five standard

180

strains and four clinical isolates), five mould and two yeast species. All clinical isolates were a

181

generous gift from the Institute of Public Health, Kragujevac. The other microorganisms were

182

provided from the collection held by the Microbiology Laboratory Faculty of Science,

183

University of Kragujevac.

184

The bacterial suspensions were prepared by the direct colony method. The turbidity of

185

the initial suspension was adjusted using densitometer (DEN-1, BioSan, Latvia). When

186

adjusted to the turbidity of the 0.5 McFarland's standard [39] the bacteria suspension contains

187

about 108 colony forming units (CFU)/mL and the suspension of yeast contains 106 CFU/mL.

188

Ten-fold dilutions of the initial suspension were additionally prepared into sterile 0.85% saline.

189

Bacterial inoculi were obtained from bacterial cultures incubated for 24 h at 37 °C on Müller-

190

Hinton agar substrate and brought up by dilution according to the 0.5 McFarland standard to

191

approximately 106 CFU/ml. Suspensions of fungal spores were prepared from fresh mature (3-

192

to 7-day-old) cultures that grew at 30 °C on a Sabouraud dextrose agar substrate. Spores were

193

rinsed with sterile distilled water, used to determine turbidity spectrophotometrically at 530

194

nm, and then further diluted to approximately 106 CFU/ml according to the procedure

195

recommended by NCCLS [40].

196 197

2.5.2. Microdilution method

198

Antimicrobial activity was tested by determining the minimum inhibitory

199

concentrations (MIC) and minimum microbicidal concentration (MMC) using the

200

microdilution plate method with resazurin [41]. The 96-well plates were prepared by

201

dispensing 100 μL of nutrient broth, Mueller–Hinton broth for bacteria and Sabouraud dextrose

202

broth for fungi, into each well. A 100 μL aliquot from the stock solution of the tested compound

203

(with a concentration of 2000 μg/mL) was added into the first row of the plate. Then, twofold

204

serial dilutions were performed by using a multichannel pipette. The obtained concentration

205

range was from 1000 to 7.8 μg/mL. The method is described in detail in the reported paper

206

[42].

207

Doxycycline and fluconazole were used as a positive control. 10% DMSO (as solvent

208

control test) was recorded not to inhibit the growth of microorganisms. Each test included

209

growth control and sterility control. All the tests were performed in duplicate and the MICs

210

were constant. Minimum bactericidal and fungicidal concentrations were determined by

211

plating 10 μL of samples from wells where no indicator color change, or no mycelia growth

212

was recorded, on nutrient agar medium. At the end of the incubation period the lowest

213

concentration with no growth (no colony) was defined as the minimum microbicidal

214

concentration.

215 216

2.6. Theoretical Methods

217

2.6.1. DFT calculations

218

The computations were performed using Gaussian 09 program with GaussView 6.0.16

219

graphical interface [43]. The geometry for all structures were optimized at the DFT/B3LYP-

220

D3BJ level of theory with a 6-311+G(d,p) basis set for C, N, O, Cl, and H, and def2-TZVPD,

221

triple-zeta-valence, basis set for Pd [44-46]. The latter one contains diffuse and polarization

222

functions, as well as effective core potential. The most stable structures of investigated

223

compounds were obtained by full optimization without any geometrical constraints and no

224

imaginary frequencies were presented. The mentioned theoretical model was chosen to

225

optimize the geometries of the investigated compounds, as it is recommended for determination

226

of the geometry of similar coumarin derivatives. For the simulation of the 1H and 13C NMR

227

spectra of studied compounds GIAO (Gauge Independent Atomic Orbital) approach was used

228

[47,48]. In order to encounter the possible solvent effects of CHCl3, the CPCM solvation model

229

was used [49]. Frequency calculations were not performed at this stage. This solvation model

230

was used to mimic the conditions of experimental measurements and influence of solvents. For

231

this purpose, the geometry of TMS (internal standard) in same solvents was optimized at the

232

same level of theory. In order to obtain values for the chemical shifts of the hydrogen and

233

carbon atoms, it is necessary to subtract the calculated values for TMS from corresponding

234

values of investigated compounds.

235

The complexes C1 and C2 can exist in two conformations, cis and trans. The mutual

236

relationship of these two conformers can be determined based on their relative energies. The

237

Boltzmann distribution formula is used for this purpose:

238

Ncis

239

Ntrans

= e ―(Ecis ― Etrans)/RT

240 241

were Ncis, Ntrans, Ecis, Etrans, k, T represent the number of particles in each state, energy of both

242

conformation, the Boltzmann constant and temperature, respectively [50].

243 244

2.6.2. Molecular docking

245

Several studies have shown that epidermal growth factor receptor (EGFR) represents

246

potential therapeutic target for the treatment of various tumors such as colorectal and breast

247

tumor. Inhibition of this receptor could interrupt cancer cellular proliferation and facilitate

248

cancer cells apoptosis. For this reason, we examined potential inhibitory effect of novel

249

coumarin derivatives and contribution of the position of the halogen atom to the overall

250

inhibitory activity [51].

251

The AutoDock 4.0 software package with AMBER force field was employed to predict

252

the binding interactions between the EGFR protein and L1, L2, C1 and C2 compounds [52].

253

The crystal structure of EGFR receptor (PDB:3W2S) was retrieved from RCSB Protein Data

254

Bank in PDB format (PDB) [53]. The Discovery Studio 4.0 (BIOVIA Discovery Studio 2016)

255

was used to prepare protein structures and remove all non-receptor atoms, including molecules

256

of water and other miscellaneous compounds [54]. In addition, this program was used to

257

visualize and analysed docking results after simulation. The Hydrogen module in

258

AutoDockTools (ADT) graphical interface was used to add polar hydrogen atoms in proteins.

259

The Kollman united atom partial charges for all amino acids in protein, were assigned. The

260

EGFR as a rigid receptor and coumarin derivatives as flexible ligands were for molecular

261

docking. The bonds in ligand were set to be rotatable and number of active torsions was set to

262

2 for all investigated compounds. Grid map was computed using AutoGrid considered grid box

263

of dimension 60×60×60 with point separated by 0.375 Å (grid-point spacing). The Lamarckian

264

Genetic Algorithm (LGA) was performed for protein-ligand rigid-flexible docking with the

265

following settings: the maximum number of energy evaluation was set to 2500000, the

266

maximum number of generations 27000, the maximum number of a top individual that

267

automatically survived was set to 1, a mutation rate of 0.02, and crossover rate of 0.80. During

268

the docking simulation, a maximum of 10 conformers was considered.

269

For the prediction of the binding affinity, between docked receptor and ligands the

270

molecular mechanics force fields were used [55]. An important thermodynamic parameter is

271

free energy of binding (ΔGbind), which represents the energy released by the interaction

272

between the ligand and the protein. The AutoDock program calculates this value according to

273

the following equation:

274

∆𝐆𝐛𝐢𝐧𝐝𝐢𝐧𝐠 = ∆Gvdw + hbond + desolv + ∆Gelec + ∆Gtotal + ∆Gtor ― ∆Gunb

275 276

where ΔGtotal is the final total internal energy, ΔGtor torsional free energy, ΔGunb unbound

277

system’s energy, ΔGelec electrostatic energy and ∆Gvdw + hbond + desolv represents the sum of the

278

following energies: energy of dispersion and repulsion (ΔGvdw), hydrogen bond energy

279

(ΔGhbond) and desolvation (ΔGdesolv) energy. It should be noted that the sum of

280

∆Gvdw + hbond + desolv and ∆Gelec represent free intermolecular energy, ∆Ginter [56].

281 282

Another important parameter is constant of inhibition (Ki) which can be calculated on the basis of free energy binding, using the following equation: ∆Gbinding

283

𝐊𝐢 = e

RT

284

where R is the gas constant (R=1.99 cal/molK), T is the absolute temperature (298.15K), Ki is

285

the constant of inhibition [57].

286

Ligand efficiency (L.E.) represents the binding energy of ligand to protein per atom.

287

Ligand efficiency (L.E.) has unit of kJ/mol/ heavy atom. AutoDock can calculate this value by

288

using the following equation:

L.E.=

289

290

∆Gbinding N

where N is the number of non-hydrogen atoms [58].

291 292

3. Results and Discussion

293

3.1. Chemistry

294

The structures of investigated compounds are presented in Figs. 1 and 2. The structure

295

of synthesized compounds was determined by means of elemental, spectral (IR, 1H NMR, and

296

13C

297

spectra, with the presence of the bands positioned at 3501 cm-1 (L1) and 3419 cm-1 (L2)

298

assigned to the vibrations of the NH groups. Also, stretching vibrations of lactone carbonyl

299

groups were identified at 1718 cm-1 (L1) and 1707 cm-1 (L2), while stretching vibrations

300

corresponding to the C–O group were identified at 1199 and 1196 cm-1, respectively.

NMR), and DFT analysis. The formation of ligands L1 and L2 was confirmed by the IR

301

The 1H NMR spectra of the ligands L1 and L2 in CDCl3 shows singlets at 2.78 and

302

2.59 ppm confirming the presence of methyl groups at the position C2' (L1 and L2). Signals

303

of protons of enamine NH group were identified as broadened singlets at 15.98, and 15.48 ppm,

304

respectively. The resulting signals of aromatic protons of the 2,4-dioxochroman part were in

305

the range from 7.14 to 8.07 ppm, while aromatic protons belonging to the chlorphenyl group

306

were detected in the range from 7.14 to 7.60 ppm.

307

The

13C

NMR spectra of ligands L1 and L2 shows the presence of aromatic carbon

308

atoms in the range of 97.5-1i62.1 ppm, while the signal corresponding to the C4 atom of the

309

both ligands shows the resonance at 182.0 and 180.5 ppm, respectively. In addition, atom C2'

310

of the methyl group shows the resonances at 20.8 and 20.7 ppm, respectively.

311

Significant differences were noted between the IR, 1H, and 13C NMR spectral data of

312

ligands and the corresponding palladium(II) complexes. The IR spectra of complexes showed

313

absence of the NH vibration, and presence of characteristic C=N band at about 1603 cm-1. Also,

314

in the IR spectra of the complexes, bands that appear in the region 686-682 cm-1 and

315

557-502 cm-1, emanate from the stretching vibrations of Pd–O and Pd–N bonds.

316

In the 1H NMR spectra of the complexes, the broad singlet from the NH group is not

317

present, which is the evidence of coordination of ligands via deprotonated nitrogen atoms. The

318

signals of protons belonging to the C2' show lower resonance than the one observed in ligands

319

(Δδ ppm: 0.42, 0.31).

320

The 13C NMR spectra of the Pd(II) complexes showed a signal corresponding to the

321

C-4 carbon atom at 170.4 ppm for both complexes, which are lower chemical shifts compared

322

to the same atom of ligands. This is a consequence of formation of the new Pd-O bond. The

323

signals of carbon atoms C2' of the ligands are at lower chemical shifts compared to the same

324

atoms of the complexes (Δδ ppm: 3.4, 4.3). On the basis of spectral data (1H, 13C NMR, and

325

IR), it can be concluded that the bidentate ligands were coordinated to palladium(II) ion via

326

nitrogen atoms from the enamine NH groups and oxygen atoms of the carbonyl group at C4.

327 328

3.2. DFT calculations

329

Since the structure of the synthesized compounds could not be determined by X-ray

330

crystallography, for this purpose we used the DFT model described in the methodology section.

331

This theoretical model has proven as reliable in our previous investigations of the structure of

332

coumarin derivatives and their complexes [35]. The optimized structures of L1, L2 and cis,

333

trans isomers of C1 and C2 compounds, are presented in Figs. 1 and 2, respectively.

334

Theoretically it is possible to obtain two isomers, cis and trans. Comparing the energies of

335

these isomers, it was found that in both cases the trans isomers were more stable.

336

The energy differences between the two isomers are 18.75 and 20.77 kJ mol-1 for the

337

C1 and C2 complexes, respectively, while the corresponding values of the Boltzmann

338

distribution are 99.95% and 99.98%. Based on these values, it is clear that the trans isomer is

339

dominant in both cases. This is a consequence of the steric and electronic repulsion between

340

the aromatic rings, Fig. 2. For this reason, in the further theoretical investigations trans isomers

341

are examined. The corresponding values of the structural parameters for the most stable

342

structures of all investigated compounds are given in Tables S1 and S2.

343 344 345

Fig. 1. Optimized geometries of L1 and L2 compounds in the gas-phase obtained with the

346

DFT/B3LYP-D3BJ theoretical method

347

348 349 350

Fig. 2. Optimized geometries of cis and trans isomers of C1 and C2 compounds in the gas-

351

phase obtained with the DFT/B3LYP-D3BJ theoretical method

352 353

In order to confirm that the proposed theoretical structures of the investigated

354

compounds correspond to the structure of the synthesized ones, NMR spectra of the most stable

355

structures were simulated. The experimental and calculated values for chemical shifts are

356

shown in Tables S3 and S4. The quality of the linear correlation between the experimental and

357

calculated chemical shift was evaluated by means of two descriptors: the correlation coefficient

358

(R) and mean absolute error (MAE). The relatively large values of correlation coefficients

359

between 0.996 and 0.999 for 1H NMR and 0.998 to 0.999 for 13C NMR, as well as relatively

360

small values for MAE, indicate that the calculated geometries of investigated compounds are

361

in good agreement with the experimentally obtained structures. Good agreement between the

362

experimental and theoretical 1H and 13C NMR spectra is a consequence of geometric rigidity

363

of the obtained compounds [37,59].

364 365

3.3. Cytotoxicity

366

From the results presented in Table 1, we concluded that on HCT-116 cells after 24 h

367

from treatment L2 exerts greater cytotoxic effect than L1, but after 72 h from treatment the

368

effect of ligands is similar, but not significant. Complexes on same cells line did not show

369

cytotoxic effect after 24 h, while after 72 h C2 exerted weak effect. Analyzing the effect of

370

healthy cell lines, MRC-5, we concluded that L2 exerted greater cytotoxic effect than L1. C1

371

complex didn’t show any effect against MRC-5, while C2 showed time and concentration

372

depended cytotoxic effect. In general, all presented results indicate weak influence on cell

373

viability. Finally, can be said that L2 and corresponding Pd(II) complex possess a greater

374

potential for cytotoxic effects then L1 and C1.

375

Table 1

376

Comparison of the IC50 values of the investigated Pd(II) complexes and their ligands alone

377

derived from the growth inhibition assays. The IC50 values were derived from the smooth curve

378

analysis of CalcuSyn Biosoft software, Cambridge, UK software and were calculated from the

379

three independent experiments.

380

IC50(µM) Compound L1 L2 C1 C2 381 382

HCT-116 24h 72h >500 173.8±7.5 >500 >500

94.5±0.3 102.3±0.6 >500 119.0±2.3

MRC-5 24h

72h

>500 182.7±2.4 >500 306.1±1.9

386.5±11.4 94.1±4.1 >500 82.9±3.6

383 384

3.4. Antimicrobial activity

385

The results of in vitro antimicrobial activity of ligands and complexes (L1, L2 and C1,

386

C2) against 16 strains of bacteria and fungi, with control results are presented in Table S5. It

387

was observed that the growth of microorganisms were not inhibited by 10% DMSO. The

388

antimicrobial activity of ligands and complexes are depending on the species of

389

microorganism.The antibacterial activity of tested ligands and complexes were weak. C1

390

exhibits better activity on Staphylococcus aureus (standard and clinical isolat) than ligands and

391

C2. The same activity is shown by C2 on Candida albicans ATCC 10231. The ligands on

392

Trichoderma viridae ATCC 13233 exhibits the same activity like positive control, fluconazole.

393

The influence on G- bacteria was not observed within the tested concentrations (MICs and

394

MMCs was > 1000 μg/mL). In our previous studies of similar compounds, some ligands

395

showed better antibacterial activity than corresponding complexes, against bacteria Bacillus

396

cereus, Staphylococcus aureus ATCC 25923 and Bacillus subtilis IP 5832. On the other hand,

397

complex 3-(1-(2-hydroxypropylamino)-ethylidene)-chroman-2,4-dione-palladium(II) showed

398

antifungal activity against Aspergillus flavus ATCC 9170 (MIC is in the range of positive

399

control) [38]. Generally, palladium(II) complexes with a coumarin ligands showed mostly

400

lower antimicrobial activity compared to commercial antibiotics [60,61]. Some palladium

401

complexes with coumarin-derived ligands have higher activity against gram positive cocci and

402

Candida albicans, whereas on gram-negative bacteria have no activity [62,63].

403 404

3.5. Molecular docking

405

Based on the thermodynamic parameters it is observed that the EGFR-L1 (-35.61 kJ

406

mol-1, 0.58 µM) and EGFR-L2 (-35.73 kJ mol-1, 0.60 µM) complexes have higher predicted

407

binding free energy and constant of inhibition than the EGFR-C1 (-38.91 kJ mol-1, 0.15 µM)

408

and EGFR-C2 (-39.66 kJ mol-1, 0.11 µM) complexes (Table S6). Based on obtained results, it

409

can be concluded that compounds C1 and C2 show better inhibitory activity to the EGFR

410

protein than L1 and L2. In general, the chlorine atom in the para position contributes to the

411

slightly higher binding free energy and lower values of constant of inhibition.

412

Observing the other values in Table S6, for all presented structures, the greatest

413

contribution to the binding free energy comes from the sum of the dispersion and repulsion

414

(ΔGvdw), hydrogen bond (ΔGhbond) and desolvation (ΔGdesolv) energy. A negligible contribution

415

to the binding free energy comes from the electrostatic energy (ΔGelec). It can be concluded

416

that all coumarin derivatives are attached to a hydrophobic binding site in investigated protein.

417

It is noted that the hydrophobic contacts are most frequent type of interactions between

418

L1 and L2 compounds and EGFR receptor (Fig. 3). On the other hand, hydrogen bonds are the

419

most important protein-ligand intermolecular interactions. In the case of EGFR-L1 complex, –

420

NH2 group of amino acid A:PHE 856 builds two weak conventional hydrogen bonds with

421

oxygen atoms of chroman ring of L1 ligand (2.83 Å, Ei=-0.42 kJ mol-1and 3.02 Å, Ei=-0.21 kJ

422

mol-1). On the other hand in the complex EGFR-L2 stronger conventional hydrogen bond was

423

established between -NH2 group of A:LYS 745 and oxygen atom of L2 (2.27 Å), as presented

424

in Fig. 3. This interaction has a significant value of the pairwise interaction energy (Ei=-2.00

425

kJ mol-1). In both cases, partially positive nitrogen atom of A:LYS 745 builds one π-cation

426

interaction with the negatively charged electron cloud of the aromatic ring of L1 (3.04 Å, Ei=-

427

0.12 kJ mol-1) and L2 (4.94 Å, Ei=-0.04 kJ mol-1) ligand. As previously observed by analysing

428

thermodynamic parameters (ΔGelec), this type of electrostatic interaction do not significantly

429

contribute to the total binding energy (Table S6). It is worth mentioning that the halogen

430

interactions occur between the σ-hole (positive electrostatic potential) of chlorine atom in para

431

position of L2 ligand and oxygen atom of amino acid A:MET 793 (2.97 Å). The pairwise

432

interaction energy is low and has value as well as hydrophobic interactions (Ei=-0.16 kJ mol-

433

1).

On the other side, investigated compounds are stabilized in the active site with several

434

hydrophobic interactions, such as alkyl, π-alkyl, and π-sigma interactions. Characteristic weak

435

hydrophobic π-π-T-shaped interaction involving aromatic ring of amino acid A:PHE 856 and

436

chroman ring of L1(5.68 Å, Ei=-0.04 kJ mol-1). The amino acid B:CYS 844 builds alkyl

437

interaction with the chlorine atom of L1 (5.39 Å, Ei=-0.08 kJ mol-1) and π-alkyl interaction

438

with the aromatic ring of L2 (4.82 Å, Ei=-0.04 kJ mol-1). It can be concluded that hydrophobic

439

interactions that include chlorine atom, despite larger interatomic distances, have somewhat

440

lower values of the pairwise interaction energy compared to the purely hydrophobic

441

interactions.

442 443

Fig. 3. The best docking positions of L1 (EGFR-L1) and L2 (EGFR-L2) compounds to the

444

EGFR protein

445

The molecular docking result for the interactions of complexes C1 and C2 with the

446

EGFR protein are shown in Fig. 4 and Table S7. The obtained results show that the dominant

447

types of interactions are hydrophobic interactions, such as alkyl, π-alkyl, and π-sigma

448

interactions. In addition, -SH group from amino acid A:CYS 797 builds a weak π-donor

449

hydrogen bond with the aromatic ring of C1 (3.31 Å, Ei=-0.04 kJ mol-1) and C2 (3.35 Å, Ei=-

450

0.04 kJ mol-1). Partially negatively charged oxygen atom of amino acid ASP 800 and nitrogen

451

atom of amino acid A:LYS 745 build electrostatic π-anion interactions with aromatic rings of

452

C1 (3.69Å, Ei=-0.08 kJ mol-1 and 4.49 Å, Ei=-0.04 kJ mol-1, respectively) and C2 (3.69Å, Ei=-

453

0.08 kJ mol-1 and 4.39 Å, Ei=-0.04 kJ mol-1), respectively.

454

455 456

Fig. 4. The best docking positions of C1 (EGFR-C1) and C2 (EGFR-C2) compounds to the

457

EGFR protein

458 459

4. Conclusion

460

Novel derivative 4-hydroxycoumarine and corresponding Pd(II) complexes were

461

synthesized, characterized and tested as antitumor and antibacterial agents. The geometries of

462

the investigated compounds were determined using the B3LYP-D3BJ theoretical method. The

463

good agreement between the predicted values of the chemical shifts of 1H and 13C NMR with

464

the experimental ones confirms that theoretically proposed geometries well describes the

465

structure of these compounds. Investigation of cytotoxicity and antimicrobial activity showed

466

that these compounds had no strong cytotoxic neither antimicrobial activity. Moderate

467

cytotoxic activity was demonstrated by compounds with the chlorine atom at the para position

468

(L2 and C2).

469

Molecular docking analysis showed that the corresponding C1 and C2 complexes exhibit better

470

inhibitory activity according the EGFR receptor than the L1 and L2 compounds. In addition,

471

compounds with a chlorine atom in the para position have a better inhibitory effect on the

472

EGFR receptor. Observed inhibition of EGFR by these substances could be the main cause of

473

the observed cytotoxic effects. It could be of significant interest to focus our further

474

investigations in the direction of determination of the possible pathway of EGFR inhibition by

475

compounds with a chlorine atom.

476

Graphical Abstract

477

478 479 480

Conflicts of interest

481

There are no conflicts to declare.

482 483

Acknowledgements

484

Authors would like to thank the Ministry of Education, Science and Technological Development

485

of the Republic of Serbia for the support through Grants No. 172016, 172015, 174028, 172040,

486

41007 and 41010.

487 488

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Declaration of interests

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☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Highlights

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

New coumarine derivatives and palladium(II) complexes were synthesized.

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Characterized by microanalysis, infrared, 1H and 13C NMR spectroscopy and DFT methods.

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In vitro antitumor activity for ligands and complexes is investigated.

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In vitro antimicrobial activity for ligands and complexes is investigated.

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Molecular docking studies with epidermal growth factor receptor (EGFR)

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