The synthesis, lipophilicity and cytotoxic effects of new ruthenium(II) arene complexes with chromone derivatives

    The synthesis, lipophilicity and cytotoxic effects of new ruthenium(II) arene complexes with chromone derivatives Adam Pastuszko, Kinga Majchrzak, Malgorzata Czyz, Bogumiła Kupcewicz, Elzbieta Budzisz PII: DOI: Reference:

S0162-0134(16)30045-9 doi: 10.1016/j.jinorgbio.2016.02.020 JIB 9934

To appear in:

Journal of Inorganic Biochemistry

Received date: Revised date: Accepted date:

12 August 2015 27 January 2016 23 February 2016

Please cite this article as: Adam Pastuszko, Kinga Majchrzak, Malgorzata Czyz, Bogumila Kupcewicz, Elzbieta Budzisz, The synthesis, lipophilicity and cytotoxic effects of new ruthenium(II) arene complexes with chromone derivatives, Journal of Inorganic Biochemistry (2016), doi: 10.1016/j.jinorgbio.2016.02.020

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT The synthesis, lipophilicity and cytotoxic effects of new ruthenium(II) arene complexes with chromone derivatives

T

Adam Pastuszkoa, Kinga Majchrzakb, Malgorzata Czyzb, Bogumiła Kupcewiczc, Elzbieta

Department of Cosmetic Raw Materials Chemistry, Faculty of Pharmacy, Medical

SC R

a

IP

Budzisza*

University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland

Department of Molecular Biology of Cancer, Medical University of Lodz, Mazowiecka

NU

b

6/8, 92-215 Lodz, Poland

Department of Inorganic and Analytical Chemistry, Collegium Medicum in Bydgoszcz,

MA

c

Nicolaus Copernicus University in Torun, Jurasza 2, 85-089 Bydgoszcz, Poland

D

*

CE P

Prof. Elzbieta Budzisz

TE

Corresponding author:

Department of Cosmetic Raw Materials Chemistry

AC

Faculty of Pharmacy

Medical University of Lodz, 90-151 Lodz Muszynskiego 1 Poland E-mail: [email protected] tel: + 48 42 272 55 95 fax: + 48 42 678 83 98 Keywords: synthesis, arene ruthenium(II) complexes, lipophilicity, cytotoxic effect

1

ACCEPTED MANUSCRIPT Abstract A series of arene ruthenium(II) complexes with the general formula [(η6-arene)Ru(L)X2]

T

(where arene = p-cymene, benzene, hexamethylbenzene or mesitylene, L = aminoflavone or

IP

aminochromone derivatives and X = Cl, I) were synthesized and characterized by elemental

SC R

analysis, MS, IR and 1H NMR spectroscopy. The stability of the selected complexes was assessed by UV-Vis spectroscopy in 24-hour period. The lipophilicity of the synthesized complexes was determined by the shake-flask method, and their cytotoxicity evaluated in

NU

vitro on patient-derived melanoma populations. The most active complexes against melanoma

MA

cells contain 7-aminoflavone and 6-aminoflavone as a ligand. The relationship between the cytotoxicity of all the obtained compounds and their logP values was determined and briefly analyzed with two different patterns observed.

TE

D

1. Introduction

Our previous paper describes the synthesis, x-ray structure, electrochemical properties

CE P

and cytotoxic effect of (p-cymene) ruthenium(II) complexes with amino derivatives of chromone.1 However, the question of the potential cytotoxic effect of the π-donor substituent

AC

remains to be answered.

The biological applications of organometallic ruthenium(II) complexes have been widely explored because of their lower toxicity and high selectivity, the various properties provided by the combination of a hydrophobic arene ligands and hydrophilic metal center, and the synthetic diversity associated with the arene ligand. It also provides an excellent scaffold for the coupling of organic segments for targeted chemotherapy.2,3,4,5,6,7,8 The resulting complexes exhibit high anticancer activity towards both primary and secondary metastasis tumors.9,10 Furthermore, these complexes also display antiviral, antiparasitic or antimalarial properties. Arene ruthenium complexes with various ligands have been recognized as chemotherapeutic alternatives to cisplatin.11,12 These complexes might work by

2

ACCEPTED MANUSCRIPT interacting with DNA or enzyme/protein active sites. The half-sandwich ruthenium(II) arene complexes form stable complexes with monodentate N-donor or O-donor ligands, as well as

T

with bidentate ligands. The arene substituent is relatively inert towards displacement and is

IP

known to stabilize the ruthenium ion in its +2 oxidation state under physiological

SC R

conditions.13,14 Both the size and hydrophobicity of the coordinated arene substituent, as well as the structure of the mono- or bidentate ligands, influence cytotoxic activity.15,16 All the factors given above enhance the pharmacological properties of half-sandwich ruthenium(II)

NU

arene complexes, making them ideal for preparing multifunctional drugs.17,18

MA

Flavones and chromones are flavonoid derivatives which are structurally related, and whose anti-inflammatory, anticancer, antiviral and anti-allergic properties have been described in depth.19,20 Kurzwernhart and coworkers describe the synthesis and biological

TE

D

activity of these derivatives, including the inhibition of cyclin-dependent kinases by ruthenium(II) flavonoid complexes (3-hydroxyflavone derivatives). These compounds form

CE P

bidentate complexes and were found to exhibit good cytotoxic activity.21 It is generally accepted that these compounds can easily chelate many various metal ions and form

AC

organometallic complexes.22,23,24 Lipophilicity is one of the most important factors in pharmaceutical research and can be considered a key determinant of the pharmacokinetic properties of a drug and its interaction with macromolecular targets. It represents the affinity of a molecule for a lipophilic environment and is commonly measured by the partition coefficient between water and n-octanol, being expressed as logP.25,26,27 Knowledge of the partition coefficient is valuable, and it is frequently used in structure-activity relationship (SAR) and quantitative structure-activity relationship (QSAR) studies.28 Although TLC and traditional shake-flask procedures are widely used and offer clear advantages for analysis, chromatography is not always available, especially for coordination compounds.29

3

ACCEPTED MANUSCRIPT The paper describes the synthesis and characterization of a series of ruthenium(II) arene complexes with derivatives of aminochromone and its analogues. The lipophilic

T

properties and cytotoxic effects of the complexes are assayed in order to characterize the

IP

relationship between structure and activity.

SC R

2. Experimental 2.1. Material and methods

NU

All substances were used without further purification. The ruthenium(II) dimers, 3nitrophtalic anhydride, 7-amino-2-methylchromone, 7-aminoflavone and 6-aminoflavone

MA

were purchased from Sigma-Aldrich. Solvents for synthesis (dichoromethane, isopropanol, acetone, diethyl ether) were reagent grade or better. The melting points were determined using

D

a Büchi Melting Point B-540 apparatus and are uncorrected. C, H and N elemental analysis

TE

was performed on a Perkin Elmer PE 2400 CHNS analyser. The IR spectra were recorded on

CE P

a FTIR–8400S Shimadzu spectrophotometer in KBr. The 1H NMR data was registered using a Varian Gemini 2000 BB 200 MHz spectrometer. The ESI-MS spectroscopy was measured on a Varian 500-MS LC Ion Trap mass spectrometer. The ligands 1-3 were commercially

AC

available. Ligands 4 and 5 were prepared according to published methods.30,31 The ruthenium(II) arene complexes with the formula [(η6-arene)Ru(L)X2] (where arene = p-cymene, benzene, hexamethylbenzene or mesitylene, L = aminoflavone or aminochromone derivatives and X = Cl, I) were synthesized as described in previous report for analogous p-cymene complexes 1a-5a.1 2.2. General procedure to synthesis of ruthenium(II) complexes To a stirred solution of ruthenium(II) dimer (0.1 mmol) in dichloromethane (15 ml) a solution of amino-compounds (0.2 mmol) in dichloromethane (5 ml) was added dropwise. The

4

ACCEPTED MANUSCRIPT reaction mixture was stirred at room temperature for 24 h. The resulting precipitate was separated and dried under reduced pressure.

T

2.2.1. Synthesis of [(η6-p-cymene)Ru(6-amino-2-phenyl-4H-1-benzopyran-4-one-κ1-N)I2] (1b)

IP

The resulting red precipitate was separated and dried under reduced pressure. Yield:

SC R

98.8 mg (68%). M.p.: 238-240°C. Anal. Calc. for C25H25NO2RuI2 (726.350 g/mol): C, 41.34; H, 3.47; N, 1.93. Found: C, 41.56; H, 3.39, N, 1.86. IR (KBr, cm-1) (selected bands): ν(NH2)

NU

3355, 3280; ν(C=O) 1632; νaromat 1581, 1556, 1470. 1H NMR (300 MHz, CDCl3) δ (ppm): 1.23 (d, 6H, 3JHH=7.0 Hz), 2.07 (s, 3H), 3.26 (m, 1H), 5.84(s, 2H, NH2), 6.36 (d, 2H, 3JHH=5.8

MA

Hz), 6.56 (d, 2H, 3JHH=5.8 Hz), 6.79 (s, 1H), 7.07 (d, 1H, 3JHH=8.6 Hz), 7.11 (d, 1H, 3JHH=9.9 Hz), 7.52 (dd, 1H, 3JHH=8.6 Hz, 3JHH=9.9 Hz), 7.58 (d, 1H, 3JHH=5.5 Hz), 7.62 (dd, 1H, JHH=5.8 Hz, 3JHH=4.3 Hz ), 7.66 (d, 1H, 3JHH=5.8 Hz), 7.98 (dd, 1H, 3JHH=5.8 Hz, 3JHH=4.3

D

3

TE

Hz), 8.01 (s, 1H). ESI-MS (m/z): 600 [Ru(C10H14)(C15H11NO2)I2-I]+, 238 [C15H11NO2+H]+.

CE P

2.2.2. Synthesis of [(η6-p-cymene)Ru(7-amino-2-phenyl-4H-1-benzopyran-4-one-κ1-N)I2] (2b) The resulting red precipitate was separated and dried under reduced pressure. Yield: 110.4 mg (76%). M.p.: 172-173°C. Anal. Calc. for C25H25NO2RuI2 (726.350 g/mol): C,

AC

41.34; H, 3.47; N, 1.93. Found: C, 41.40; H, 3.42, N, 1.99. IR (KBr, cm-1)(selected bands): ν(NH2) 3340, 3294; ν(C=O) 1624; νaromat 1604, 1567, 1493. 1H NMR (300 MHz, CDCl3) δ (ppm): 1.24 (d, 6H, 3JHH=7.0 Hz), 2.40 (s, 3H), 3.15 (m, 1H), 5.78 (s, 2H, NH2), 6.12 (d, 2H, 3

JHH=5.8 Hz), 6.30 (d, 2H, 3JHH=5.8 Hz), 6.78 (s, 1H), 7.20 (d, 1H, 3JHH=8.7 Hz), 7.22 (dd,

1H, 3JHH=8.6, 3JHH=Hz, 8.7 Hz), 7.31 (dd, 1H, 3JHH=5.8 Hz, 3JHH=4.1 Hz), 7.32 (d, 1H, 3

JHH=5.8 Hz), 7.39 (d, 1H, 3JHH=4.1 Hz), 7.72 (s, 1H), 7.75 (d, 1H, 3JHH=5.8 Hz), 8.12 (dd,

1H, 3JHH=8.6 Hz, 3JHH=5.8 Hz). ESI-MS (m/z): 600 [Ru(C10H14)(C15H11NO2)I2-I]+, 238 [C15H11NO2+H]+. 2.2.3. Synthesis of [(η6-p-cymene)Ru(7-amino-2-methyl-4H-1-benzopyran-4-one-κ1-N)I2] (3b) 5

ACCEPTED MANUSCRIPT The resulting red precipitate was separated and dried under reduced pressure. Yield: 95.7 mg (72%). M.p.: 176-178°C. Anal. Calc. for C20H23NO2RuI2 (664.281 g/mol): C, 36.16;

T

H, 3.49; N, 2.11. Found: C, 36.50; H, 3.16; N, 2.20. IR (KBr, cm-1)(selected bands): ν(NH2)

IP

3327, 3223; ν(C=O) 1644; νaromat 1584, 1512, 1498. 1H NMR (300 MHz, CDCl3) δ (ppm):

SC R

1.24 (d, 6H, 3JHH=7.0 Hz), 2.22 (s, 3H), 2.41 (s, 3H), 3.30 (m, 1H), 5.25 (s, 2H, NH2), 6.24 (d, 2H, 3JHH=5.9 Hz), 6.63 (d, 2H, 3JHH=5.9 Hz), 6.79 (s, 1H), 7.25 (d, 1H, 3JHH=8.6 Hz), 7.53 (d, 1H, 3JHH=8.6 Hz), 8.10 (s, 1H). ESI-MS (m/z): 538 [Ru(C10H14)(C10H9NO2)I2-I]+, 176

NU

[C10H9NO2+H]+.

MA

2.2.4. Synthesis of [(η6-p-cymene)Ru(5-amino-8-methyl-4H-1-benzopyran-4-one-κ1-N)I2] (4b) The resulting red precipitate was separated and dried under reduced pressure. Yield:

D

73.1 mg (55%). M.p.: 155-156°C. Anal. Calc. for C20H23NO2RuI2 (664.281 g/mol): C, 36.16;

TE

H, 3.49; N, 2.11. Found: C, 36.10; H, 3.27; N, 2.19. IR (KBr, cm-1)(selected bands): ν(NH2) 3318, 3241; ν(C=O) 1639; νaromat 1604, 1567, 1493. 1H NMR (300 MHz, CDCl3) δ (ppm):

CE P

1.25 (d, 6H, 3JHH=6.9 Hz), 2.17 (s, 3H), 2.36 (s, 3H), 3.24 (m, 1H), 5.48 (s, 2H, NH2), 5.98 (d, 2H, 3JHH=5.9 Hz), 6.25 (d, 2H, 3JHH=5.9 Hz), 6.57 (d, 1H, 3JHH=5.3 Hz), 7.20 (d, 1H, 3JHH=8.6

AC

Hz), 7.29 (d, 1H,

3

JHH=8.6 Hz), 8.18 (d, 1H,

3

JHH=5.3 Hz). ESI-MS (m/z): 538

[Ru(C10H14)(C10H9NO2)I2-I]+, 177 [C10H9NO2+H]+. 2.2.5. Synthesis of [(η6-p-cymene)Ru(7-amino-2-benzofuran-1(3H)-one- κ1-N)I2] (5b) The resulting brown precipitate was separated and dried under reduced pressure. Yield: 76,6 mg (60%). M.p.: 149-150°C. Anal. Calc. for C18H21NO2RuI2 (638.244 g/mol): C, 33.87; H, 3.32; N, 2.19. Found: C, 33.94; H, 2.97, N, 2.19. IR (KBr, cm-1)(selected bands): ν(NH2) 3391, 3362; ν(C=O) 1689; νaromat 1628, 1598, 1484. 1H NMR (300 MHz, CDCl3) δ (ppm): 1.26 (d, 6H, 3JHH=6.9 Hz), 2.13 (s, 3H), 3.00 (m, 1H), 5.73 (s, 2H, NH2), 6.30 (d, 2H, 3

JHH=5.9 Hz), 6.52 (d, 2H, 3JHH=5.9 Hz), 7.21 (d, 1H, 3JHH=8.4 Hz), 7.30, 7.33 (dd, 1H,

6

ACCEPTED MANUSCRIPT 3

JHH=8.4 Hz, 3JHH=5.4 Hz), 7.62 (d, 1H, 3JHH=5.4 Hz), 7.99 (s, 2H). ESI-MS (m/z): 512

[Ru(C10H14)(C8H7NO2)I2-I]+, 150 [C8H7NO2+H]+.

T

2.2.6. Synthesis of [(η6-benzene)Ru(6-amino-2-phenyl-4H-1-benzopyran-4-one-κ1-N)Cl2] (1c)

IP

The resulting yellow precipitate was separated and dried under reduced pressure.

SC R

Yield: 60.4 mg (62%). M.p.: 297-298°C. Anal. Calc. for C21H17NO2RuCl2 (487.341 g/mol): C, 51.76; H, 3.52; N, 2.87. Found: C, 51.61; H, 3.41, N, 2.81. IR (KBr, cm-1) (selected

NU

bands): ν(NH2) 3180, 3103; ν(C=O) 1638; νaromat 1612, 1571, 1467. 1H NMR (300 MHz, CDCl3) δ (ppm): 5.74 (s, 2H, NH2), 5.98 (s, 6H), 6.93 (s, 1H), 7.03 (d, 1H, 3JHH=5.8 Hz), 7.23

3

MA

(d, 1H, 3JHH=8.4 Hz), 7.37 (dd, 1H, 3JHH=8.4 Hz, 3JHH=5.8 Hz), 7.54 (dd, 1H, 3JHH=5.3 Hz, JHH=5.8 Hz ), 7.60 (d, 1H, 3JHH=5.8 Hz), 7.76 (d, 1H, 3JHH=5.3 Hz), 7.90 (d, 1H, 3JHH=5.8

D

Hz), 8.15 (s, 1H). ESI-MS (m/z): 452 [Ru(C6H6)(C15H11NO2)Cl2-Cl]+, 238 [C15H11NO2+H]+.

TE

2.2.7. Synthesis of [(η6-benzene)Ru(7-amino-2-phenyl-4H-1-benzopyran-4-one-κ1-N)Cl2] (2c)

CE P

The resulting yellow precipitate was separated and dried under reduced pressure. Yield: 47.8 mg (49%). M.p.: 290-291°C. Anal. Calc. for C21H17NO2RuCl2 (487.341 g/mol): C, 51.76; H, 3.52; N, 2.87. Found: C, 51.59; H, 3.66, N, 2.84. IR (KBr, cm-1)(selected bands):

AC

ν(NH2) 3192, 3096; ν(C=O) 1622; νaromat 1580, 1499, 1453. 1H NMR (300 MHz, CDCl3) δ (ppm): 5.70 (s, 2H, NH2), 5.97 (s, 6H), 6.83 (s, 1H), 7.09 (d, 1H, 3JHH=5.8 Hz), 7.14 (dd, 1H, 3

JHH=8.4 Hz, 3JHH=5.8 Hz), 7.46 (dd, 1H, 3JHH=8.4 Hz, 3JHH=6.1 Hz), 7.52 (d, 1H, 3JHH=5.7

Hz), 7.60 (d, 1H, 3JHH=6.1 Hz), 7.88 (s, 1H), 7.92 (dd, 1H, 3JHH=4.5 Hz, 3JHH=5.7 Hz), 8.05 (d,

1H,

3

JHH=4.5

Hz).

ESI-MS

(m/z):

452

[Ru(C6H6)(C15H11NO2)Cl2-Cl]+,

238

[C15H11NO2+H]+. 2.2.8. Synthesis of [(η6-benzene)Ru(7-amino-2-methyl-4H-1-benzopyran-4-one-κ1-N)Cl2] (3c) The resulting yellow precipitate was separated and dried under reduced pressure. Yield: 47.6 mg (56%). M.p.: 239-240°C. Anal. Calc. for C16H15NO2RuCl2 (425.271 g/mol): 7

ACCEPTED MANUSCRIPT C, 45.19; H, 3.56; N, 3.29. Found: C, 44.93; H, 3.82; N, 3.00. IR (KBr, cm-1)(selected bands): ν(NH2) 3198, 3102; ν(C=O) 1632; νaromat 1604, 1500, 1455. 1H NMR (300 MHz, CDCl3)

T

δ (ppm): 2.34 (s, 3H), 5.55 (s, 2H, NH2), 5.98 (s, 6H), 6.93 (s, 1H), 7.30 (d, 1H, 3JHH=8.6 Hz),

IP

7.65 (d, 1H, 3JHH=8.6 Hz), 8.20 (s, 1H). ESI-MS (m/z): 390 [Ru(C6H6)C10H9NO2)Cl2-Cl]+,

SC R

176 [C10H9NO2+H]+.

2.2.9. Synthesis of [(η6-benzene)Ru(5-amino-8-methyl-4H-1-benzopyran-4-one-κ1-N)Cl2] (4c)

NU

The resulting yellow precipitate was separated and dried under reduced pressure. Yield: 50.2 mg (59%). M.p.: 205-206°C. Anal. Calc. for C16H15NO2RuCl2 (425.271 g/mol):

MA

C, 45.19; H, 3.56; N, 3.29. Found: C, 45.08; H, 3.41; N, 3.41. IR (KBr, cm-1)(selected bands): ν(NH2) 3260, 3115; ν(C=O) 1655; νaromat 1590, 1583, 1475. 1H NMR (300 MHz, CDCl3)

JHH=8.6 Hz), 7.44 (d, 1H, 3JHH=8.6 Hz), 8.09 (d, 1H, 3JHH=5.4 Hz). ESI-MS (m/z): 390

TE

3

D

δ (ppm): 2.28 (s, 3H), 5.66 (s, 2H, NH2), 5.98 (s, 6H), 6.85 (d, 1H, 3JHH=5.4 Hz), 7.15 (d, 1H,

CE P

[Ru(C6H6)(C10H9NO2)Cl2-Cl]+, 176 [C10H9NO2+H]+. 2.2.10. Synthesis of [(η6-benzene)Ru(7-amino-2-benzofuran-1(3H)-one- κ1-N)Cl2] (5c)

AC

The resulting yellow precipitate was separated and dried under reduced pressure. Yield: 35.1 mg (44%). M.p.: 198-199°C. Anal. Calc. for C14H13NO2RuCl2 (399.235 g/mol): C, 42.12; H, 3.28; N, 3.51. Found: C, 42.15; H, 3.33, N, 3.50. IR (KBr, cm-1)(selected bands): ν(NH2) 3232, 3111; ν(C=O) 1633; νaromat 1598, 1561, 1483. 1H NMR (300 MHz, CDCl3) δ (ppm): 5.69 (s, 2H, NH2), 5.99 (s, 6H), 6.90 (d, 1H, 3JHH=8.3 Hz), 7.46 (dd, 1H, 3JHH=8.3 Hz, 3

JHH=5.5 Hz), 7.69 (d, 1H,

3

JHH=5.5 Hz), 8.19 (s, 2H). ESI-MS (m/z): 364

[Ru(C6H6)(C8H7NO2)Cl2-Cl]+, 150 [C8H7NO2+H]+. 2.2.11. Synthesis of [(η6-hexamethylbenzene)Ru(6-amino-2-phenyl-4H-1-benzopyran-4-oneκ1-N)Cl2] (1d)

8

ACCEPTED MANUSCRIPT The resulting brown precipitate was separated and dried under reduced pressure. Yield: 93.7 mg (82%). M.p.: 304-305°C. Anal. Calc. for C27H29NO2RuCl2 (571.501 g/mol):

T

C, 56.74; H, 5.11; N, 2.45. Found: C, 56.75; H, 5.08, N, 2.31. IR (KBr, cm-1) (selected

IP

bands): ν(NH2) 3276, 3189; ν(C=O) 1621; νaromat 1595, 1567, 1479. 1H NMR (300 MHz,

SC R

CDCl3) δ (ppm): 2.07 (s, 18H), 5.33 (s, 2H, NH2), 6.82 (s, 1H), 7.12 (d, 1H, 3JHH=5.8 Hz), 7.16 (d, 1H, 3JHH=8.2 Hz), 7.48 (dd, 1H, 3JHH=5.8 Hz, 3JHH=8.2 Hz), 7.63 (d, 1H, 3JHH=5.7 Hz), 7.71 (dd, 1H, 3JHH=5.7 Hz, 3JHH=5.1 Hz), 7.79 (d, 1H, 3JHH=5.8 Hz), 7.85 (dd, 1H, JHH=5.1 Hz, 3JHH=5.8 Hz), 7.99 (s, 1H). ESI-MS (m/z): 536 [Ru(C12H18)(C15H11NO2)Cl2-

NU

3

MA

Cl]+, 500 [Ru(C12H18)(C15H11NO2)Cl-HCl]+, 238 [C15H11NO2+H]+. 2.2.12. Synthesis of [(η6-hexamethylbenzene)Ru(7-amino-2-phenyl-4H-1-benzopyran-4-one-

D

κ1-N)Cl2] (2d)

TE

The resulting brown precipitate was separated and dried under reduced pressure. Yield: 91.4 mg (80%). M.p.: 259-260°C. Anal. Calc. for C27H29NO2RuCl2 (571.501 g/mol):

CE P

C, 56.74; H, 5.11; N, 2.45. Found: C, 56.68; H, 5.06, N, 2.28. IR (KBr, cm-1)(selected bands): ν(NH2) 3308, 3201; ν(C=O) 1629; νaromat 1592, 1577, 1449. 1H NMR (300 MHz, CDCl3) δ

3

AC

(ppm): 2.04 (s,18H), 5.35 (s, 2H, NH2), 6.71 (s, 1H), 7.00 (d, 1H, 3JHH=5.7 Hz), 7.23 (dd, 1H, JHH=5.7, 3JHH=Hz, 8.6 Hz), 7.29 (dd, 1H, 3JHH=5.8 Hz, 3JHH=4.3 Hz), 7.42 (d, 1H, 3JHH=5.7

Hz), 7.45 (d, 1H, 3JHH=4.3 Hz), 7.63 (s, 1H), 7.85 (d, 1H, 3JHH=5.8 Hz), 7.99 (dd, 1H, 3

JHH=8.6 Hz, 3JHH=5.7 Hz). ESI-MS (m/z): 536 [Ru(C12H18)(C15H11NO2)Cl2-Cl]+, 500

[Ru(C12H18)(C15H11NO2)Cl-HCl]+, 238 [C15H11NO2+H]+. 2.2.13. Synthesis of [(η6-hexamethylbenzene)Ru(7-amino-2-methyl-4H-1-benzopyran-4-oneκ1-N)Cl2] (3d) The resulting brown precipitate was separated and dried under reduced pressure. Yield: 78.5 mg (77%). M.p.: 285-286°C. Anal. Calc. for C22H27NO2RuCl2 (509.431 g/mol): C, 51.87; H, 5.34; N, 2.75. Found: C, 51.68; H, 5.36; N, 2.51. IR (KBr, cm-1)(selected bands): 9

ACCEPTED MANUSCRIPT ν(NH2) 3315, 3196; ν(C=O) 1640. 1H NMR (300 MHz, CDCl3) δ (ppm): 2.07 (s, 18H), 2.51 (s, 3H), 5.33 (s, 2H, NH2), 7.09 (s, 1H), 7.33 (d, 1H, 3JHH=8.4 Hz), 7.40 (d, 1H, 3JHH=8.4 Hz), (s,

1H).

ESI-MS

(m/z):

474

[Ru(C12H18)(C10H9NO2)Cl2-Cl]+,

438

T

7.80

IP

[Ru(C12H18)(C10H9NO2)Cl-HCl]+, 176 [C10H9NO2+H]+.

SC R

2.2.14. Synthesis of [(η6-hexamethylbenzene)Ru(5-amino-8-methyl-4H-1-benzopyran-4-oneκ1-N)Cl2] (4d)

NU

The resulting brown precipitate was separated and dried under reduced pressure. Yield: 76.4 mg (75%). M.p.: 204-205°C. Anal. Calc. for C22H27NO2RuCl2 (509.431 g/mol):

MA

C, 51.87; H, 5.34; N, 2.75. Found: C, 51.74; H, 5.35; N, 2.52. IR (KBr, cm-1)(selected bands): ν(NH2) 3293, 3188; ν(C=O) 1641. 1H NMR (300 MHz, CDCl3) δ (ppm): 2.04 (s, 18H), 2. 40

JHH=5.9 Hz), 6.77 (d, 1H, 3JHH=5.4 Hz), 7.29 (d, 1H, 3JHH=8.5 Hz), 7.39 (d, 1H, 3JHH=8.5

TE

3

D

(s, 3H), 5.25 (s, 2H, NH2), 5.05 (d, 2H, 3JHH=5.9 Hz), 5.36 (d, 2H, 3JHH=5.9 Hz), 5.49 (d, 2H,

Hz), 7.98 (d, 1H, 3JHH=5.4 Hz). ESI-MS (m/z): 474 [Ru(C12H18)(C10H9NO2)Cl2-Cl]+, 438

CE P

[Ru(C12H18)(C10H9NO2)Cl-HCl]+, 176 [C10H9NO2+H]+.

(5d)

AC

2.2.15. Synthesis of [(η6-hexamethylbenzene)Ru(7-amino-2-benzofuran-1(3H)-one-κ1-N)Cl2]

The resulting brown precipitate was separated and dried under reduced pressure. Yield: 66.7 mg (69%). M.p.: 216-217°C. Anal. Calc. for C20H25NO2RuCl2 (483.394 g/mol): C, 49.69; H, 5.21; N, 2.90. Found: C, 49.59; H, 5.22, N, 2.86. IR (KBr, cm-1)(selected bands): ν(NH2) 3316, 3253; ν(C=O) 1657. 1H NMR (300 MHz, CDCl3) δ (ppm): 2.06 (s, 18H), 5.26 (s, 2H, NH2), 7.21 (d, 1H, 3JHH=8.0 Hz), 7.29 (dd, 1H, 3JHH=8.0 Hz, 3JHH=5.4 Hz), 7.55 (d, 1H, 3JHH=5.4 Hz), 7.82 (s, 2H). ESI-MS (m/z): 448 [Ru(C12H18)(C8H7NO2)Cl2-Cl]+, 150 [C8H7NO2+H]+.

10

ACCEPTED MANUSCRIPT 2.2.16. Synthesis of [(η6-mesitylene)Ru(6-amino-2-phenyl-4H-1-benzopyran-4-one-κ1-N)Cl2] (1e)

T

The resulting yellow precipitate was separated and dried under reduced pressure.

IP

Yield: 70.9 mg (67%). M.p.: 274-276°C. Anal. Calc. for C24H23NO2RuCl2 (529.421 g/mol):

SC R

C, 54.45; H, 4.38; N, 2.65. Found: C, 54.38; H, 4.07, N, 2.83. IR (KBr, cm-1) (selected bands): ν(NH2) 3196, 3175; ν(C=O) 1638. 1H NMR (300 MHz, CDCl3) δ (ppm): 2.12 (s, 9H), 5.48 (s, 2H, NH2), 6.07 (s, 3H), 6.85 (s, 1H), 6.99 (d, 1H, 3JHH=8.4 Hz), 7.18 (d, 1H, 3JHH=5.8

JHH=5.7 Hz, 3JHH=5.3 Hz ), 7.85 (d, 1H, 3JHH=5.7 Hz), 7.95 (dd, 1H, 3JHH=4.5 Hz, 3JHH=5.3

Hz),

8.01

(s,

1H).

ESI-MS

MA

3

NU

Hz), 7.37 (dd, 1H, 3JHH=8.4 Hz, 3JHH=5.8 Hz), 7.66 (d, 1H, 3JHH=4.5 Hz), 7.82 (dd, 1H,

(m/z):

494

[Ru(C9H12)(C15H11NO2)Cl2-Cl]+,

459

D

[Ru(C9H12)(C15H11NO2)Cl-HCl]+, 238 [C15H11NO2+H]+.

TE

2.2.17. Synthesis of [(η6-mesitylene)Ru(7-amino-2-phenyl-4H-1-benzopyran-4-one-κ1-N)Cl2]

CE P

(2e)

The resulting yellow precipitate was separated and dried under reduced pressure. Yield: 62.5 mg (59%). M.p.: 178-180°C. Anal. Calc. for C24H23NO2RuCl2 (529.421 g/mol):

AC

C, 54.45; H, 4.38; N, 2.65. Found: C, 54.22; H, 4.14, N, 2.69. IR (KBr, cm-1)(selected bands): ν(NH2) 3342, 3216; ν(C=O) 1626. 1H NMR (300 MHz, CDCl3) δ (ppm): 2.11 (s, 9H), 5.40 (s, 2H, NH2), 6.10 (s, 3H), 6.92 (s, 1H), 7.10 (d, 1H, 3JHH=8.4 Hz), 7.27 (d, 1H, 3JHH=8.4), 7.35, 7.36 (dd, 1H, 3JHH=5.7 Hz, 3JHH=4.5 Hz), 7.44 (d, 1H, 3JHH=5.7 Hz), 7.56 (d, 1H, 3JHH=4.5 Hz), 7.59 (s, 1H), 7.83 (d, 1H, 3JHH=5.7 Hz), 8.10 (d, 1H, 3JHH=5.7 Hz). ESI-MS (m/z): 494[Ru(C9H12)(C15H11NO2)Cl2-Cl]+, 238 [C15H11NO2+H]+. 2.2.18. Synthesis of [(η6-mesitylene)Ru(7-amino-2-methyl-4H-1-benzopyran-4-one-κ1-N)Cl2] (3e)

11

ACCEPTED MANUSCRIPT The resulting yellow precipitate was separated and dried under reduced pressure. Yield: 50.5 mg (54%). M.p.: 247-249°C. Anal. Calc. for C19H21NO2RuCl2 (467.352 g/mol):

T

C, 48.83; H, 4.53; N, 3.00. Found: C, 48.87; H, 4.44; N, 2.81. IR (KBr, cm-1)(selected bands):

IP

ν(NH2) 3327, 3222; ν(C=O) 1643. 1H NMR (300 MHz, CDCl3) δ (ppm): 2.12 (s, 9H), 2.42 (s,

3

SC R

3H), 5.37 (s, 2H, NH2), 6.11 (s, 3H) 6.81 (s, 1H), 7.30 (d, 1H, 3JHH=8.7 Hz), 7. 35 (d, 1H, JHH=8.7 Hz), 8.19 (s, 1H). ESI-MS (m/z): 432 [Ru(C9H12)(C10H9NO2)Cl2-Cl]+, 176

[C10H9NO2+H]+.

NU

2.3. The stability of compounds in aqueous solution

MA

The stability of the ruthenium(II) complexes at concentration of 10μM in PBS/DMSO solution, containing 1% DMSO was assessed using UV-Vis spectroscopy. The spectra were recorded in the range of 200-800 nm at t = 0 hours, t = 6 hours and t = 24 hours, and

TE

D

compared to each other (Table S1). Only the most active complexes, 1a, 1b and 2a-e, were studied.

CE P

The stability were investigated by 1H NMR spectroscopy. Because of the lipophilic character of the compounds all experiments were performed in 10% DMSO-d6/D2O solution.

AC

2.4. Lipophilicity

Partition coefficients (P) between n-octanol and water phases were determined for all synthesized ruthenium(II) complexes by the shake-flask method. The phases of distilled water and n-octanol were saturated with each other by stirring for 24 hours prior to use. Solutions of the test substances were prepared at concentration of 0.5 mg/ml in the water phase. After application of the shake-flask method, the concentration of the complex in both phases was measured by UV-Vis spectrophotometry. The spectra were recorded in the region 200-600 nm using a Shimadzu UV-1800 with a 10 mm quartz cell. For each compound, six measurements were performed.32 2.5. Cytotoxic effects

12

ACCEPTED MANUSCRIPT 2.5.1. Cell cultures Two melanoma cell lines, DMBC11 and DMBC12 cell lines, derived from patients

T

were established in our laboratory as described before.33 DMBC11 represents superficial

IP

spreading melanoma (SSM),34 whereas DMBC12 was obtained from surgical specimens of

SC R

nodular melanoma (NM).33 These cells were maintained in stem cell medium (SCM) consisting of DMEM/F12 (Lonza, Basel, Switzerland), B-27 supplement (Gibco, Paisley, UK), growth factors (10 ng/ml bFGF and 20 ng/ml EGF; BD Biosciences, San Jose, CA),

NU

insulin (10 µg/ml), heparin (1 ng/ml) and antibiotics, and maintained in ultra-low attachment

MA

flask (NUNC). Twice a week medium was exchanged. 2.5.2. Drug treatment

The ruthenium(II) complexes were dissolved in dimethyl sulfoxide (DMSO) at the

TE

D

concentration of 20 mM. They were diluted in SCM for experiments. DMBC11 and DMBC12 cells were were counted after staining with Trypan blue (Sigma-Aldrich) and plated at a

CE P

density of 1.2 x 103 viable cells per well in 96-well plates in 100 μl SCM with compounds at indicated concentrations. An equivalent concentration of DMSO was used in the control cell

AC

cultures.

2.5.3. Flow cytometric analysis of cell viability Drug-induced changes in viability were assessed by propidium iodide (PI) staining or by using an automated cell viability analyzer according to standard procedures. In both assays, the cells were stained with PI for 15 min at room temperature in the dark and analyzed by a FACSVerse flow cytometer (Becton Dickinson). The results were processed by using FACSuite software (Becton Dickinson). The difference in viability between ruthenium(II) complex-treated populations and vehicle-treated populations was calculated using the formula: viable cells in complex-treated population (% of all cells)/viable cells in vehicletreated population (% of all cells), and was expressed as % of control. The results are

13

ACCEPTED MANUSCRIPT represented as means ± SD from at least three independent experiments performed in triplicate.

T

3. Results and Discussion

IP

3.1. Chemistry

SC R

In response to the interesting biological properties previously observed for the series of arene ruthenium(II) complexes 1a – 5a, it was decided to examine the impact of different

NU

π-substituents on these properties. Hence, the present paper describes the synthesis of a series of new ruthenium compounds (Scheme 1) by a reaction with various ruthenium(II) dimers (b-

MA

e) with different amino-derivatives (1-5).

All complexes were prepared at room temperature by mixing equimolar amounts of

D

ligands 1 – 5 and corresponding ruthenium dimers b-e in dichloromethane for 24 hours. As

TE

described before, the ligands behave as strong monodentate chelating agents, forming ruthenium(II) arene complexes through coordination of the amino nitrogen atom. The

CE P

complex has an essentially octahedral coordination geometry comprising the arene ring carbons occupying one face of the octahedron, and another three sites to be coordinated by 1,35

The formation of bidentate compounds

AC

two halogen atoms and a monodentate ligand.

through the coordination of two oxygen or nitrogen atoms may also occur.36 The synthesis of complexes 4e and 5e was not carried out due to the lower cytotoxic activity of the dichloro(mesitylene)ruthenium(II) complexes.

14

ACCEPTED MANUSCRIPT

2

R1

T

R1

IP

1-5

a-e

b=

2: R1 =

c=

3: R1 =

NU

1: R1 =

X = Cl

X=I

X = Cl

TE

D

MA

a=

SC R

R

1a-e - 3a-e; 4a-d, 5a-d

4: R1 =

X = Cl

5: R1 =

X = Cl

CE P

d=

AC

e=

Scheme 1. Synthesis of arene ruthenium(II) complexes. 3.2. IR spectra The most important IR spectral data for all compounds is listed in Table 1. Significant differences were observed in the IR spectra of the complexes in comparison with free ligands. The IR spectra of complexes showed stretching vibrations at 3362-3180 cm-1, which were assigned to νas (NH2), and at 3291-3103 cm-1, which were assigned to νs (NH2). These bands shift negatively in comparison to free ligands, showing that coordination of the amino group

15

ACCEPTED MANUSCRIPT has occurred. The asymmetric and symmetric ν(NH2) vibrations of complexes 1b, 1c, 1d and 1e are shifted to lower energy by 63 and 63 cm-1 for 1b, 238 and 240 cm-1 for 1c, 142 and 154

T

cm-1 for 1d, 222 and 168 cm-1 for 1e in comparison to ligand 1. Similar shifts have been

IP

reported for remaining groups of complexes (2a-e, 3a-e, 4a-d and 5a-d) and corresponding

SC R

free ligands. The ν(C=O) bands observed for each complex, occurring at 1689-1621 cm-1 are shifted insignificantly compared from their positions in the free ligands 1, 2, 3, 4 and 5 respectively at 1611, 1630, 1655, 1648, 1668 cm-1.

NU

Table 1

ν(NH2)

ν(C=O)

Compound

ν(NH2)

ν(C=O)

1

3418, 3343

1611

3c

3198, 3102

1632

1b

3355, 3280

1632

3d

3315, 3196

1640

1c

3180, 3103

1638

3e

3327, 3222

1643

1d

3276, 3189

1621

4

3432, 3324

1648

1e

3196, 3175

1638

4b

3318, 3241

1639

2

3460, 3293

1630

4c

3260, 3115

1655

2b

3340, 3294

1624

4d

3293, 3188

1641

3192, 3096

1622

5

3480, 3376

1668

3308, 3201

1629

5b

3362, 3291

1689

2e

3342, 3216

1626

5c

3232, 3111

1633

3

3431, 3322

1655

5d

3316, 3253

1657

3b

3327, 3223

1644

2d

TE

CE P

AC

2c

D

Compound

MA

IR data (ν, cm-1) for the ruthenium(II) complexes.

3.3. 1H NMR spectra The 1H NMR spectra data of the complexes was recorded in CDCl3 and is presented in the experimental section. The position and intensity of the signals correspond to reagents used

16

ACCEPTED MANUSCRIPT in synthesis. The resonance of the amino group protons gave signals ranging from 5.25 to 5.84 ppm. These signals are slightly shifted compared to the free ligands, probably due to the

T

interaction between the nitrogen of the amino group and the metal centre. In p-cymene

IP

derivatives (1b-5b) signals from methyl protons of the isopropyl group were found at 1.23-

SC R

1.26 ppm as doublets and methylene proton around 3.00-3.30 ppm as multiplets, respectively. The signals from the methyl protons of the hexamethylbenzene complexes (1d-5d) were observed at 2.04 to 2.07 ppm as singlets, and of the mesitylene complexes (1e-3e) at 2.11 to

MA

singlets, doublets and doublets of doublets.

NU

2.12 ppm, also as singlets. The aromatic protons were observed around 6.57-8.19 ppm as

3.4. ESI-MS

ESI-MS spectrometry was carried out for all synthesized complexes. Scanning was

TE

D

performed from m/z = 100 to 1000. The experiments were performed in positive and negative-ion mode. Parent peaks were found at (m/z) 600 for complexes 1b, 2b, at (m/z) 538

CE P

for complexes 3b, 4b, at (m/z) 512 for complex 5b, at (m/z) 452 for complexes 1c, 2c, at (m/z) 391 for complex 4c, 5c, at (m/z) 536 for complexes 1d, 2d, at (m/z) 474 for complexes

AC

3d, 4d, at (m/z) 474 for complex 5d, at (m/z) 600 for complexes 1e, 2e and at (m/z) 538 for complex 3e. Ion peaks corresponding to the ligands have been observed at (m/z) 238 for ligand 1 and 2, at (m/z) 176 for 3 and 4 and at (m/z) 150 for 5. For more mass spectra data, see the experimental section. 3.5. Stability of compounds (time-dependent spectra) As all potential drugs should be able to reach their target under the conditions created by living organisms, an important property of any promising anticancer agents is its thermodynamic stability in aqueous solution. The stability of the most cytotoxic compounds, 1a, 1b and 2a-e, were studied by UV-Vis spectroscopy in a solution of 1% DMSO in PBS. Figure 1 presents the representative time-dependent UV-Vis spectra of complex 1a. All 17

ACCEPTED MANUSCRIPT ruthenium(II) complexes showed characteristic peaks in the region of 220–450 nm and did not exhibit any significant changes during a 24-hour period. The absence of significant changes in

T

the peak absorptions and spectral characteristics for tested complexes over time may suggest

IP

that no structural alternations occurred in aqueous solution.37,38 The data for all studied

AC

CE P

TE

D

MA

NU

SC R

complexes is presented in Table S1.

Fig. 1. Representative time-dependent UV-Vis spectra for complex 1a recorded after t = 0 hours, t = 6 hours and t = 24 hours. The aqueous stability of the representative complex 2b was studied by 1H NMR spectroscopy (Figure 2). Dissolution of arene ruthenium compounds often results in rapid exchange of the chlorido ligand by an aqua moiety.39 The hydrolyzed compounds are stable in aqueous solution for 120 h.

18

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Fig. 2. The aqueous stability of the representative complex 2b studied by 1H NMR

AC

spectroscopy after dissolution in 10% DMSO-d6/D2O after 0h, 24h, 48h and 120h. No additional signals could be seen for 120 h. 3.6. Lipophilicity

The experimental logP for studied compounds was determined by the direct shakeflask method and the results are presented in Table 2. All the compounds exhibit moderate lipophilicity with logP values, within range of 0.81-1.45. It means that their solubility in the n-octanol (lipophilic phase) is 6 to 30-fold higher than in the water (hydrophilic phase). Table 2 Lipophilicity of the ruthenium(II) complexes assessed by direct shake-flask method (mean±SD). 19

ACCEPTED MANUSCRIPT logP

Compound

logP

1a

1.30±0.07

3c

1.02±0.05

1b

1.33±0.04

3d

1.18±0.09

1c

1,14±0.04

3e

0.87±0.04

1d

1.23±0.03

4a

1.10±0.03

1e

1.06±0.04

4b

2a

1.32±0.08

4c

0.89±0.02

2b

1.45±0.08

4d

0.95±0.03

2c

1.08±0.04

5a

1,01±0.08

2d

1.20±0.04

2e

1.11±0.03

3a

0.98±0.05

3b

1.04±0.06

IP

T

Compound

5b

0,99±0.03

5c

0,81±0.04

5d

0,83±0.04

D

MA

NU

SC R

1.15±0.06

TE

Fig. 3 shows the comparison of logP values on the five groups of complexes in

CE P

relation to the arene substituent. Two different patterns are observed, one in the set of complexes a, b and e (with p-cymene and mesitylene), and another in the series c and d (with

AC

benzene and hexamethylbenzene). The relationships between the logP values of complexes in series a and b, as well as c and d, are approximately linear (Pearson correlation coefficient is rab = 0.969 and rcd = 0.0.97). The logP values of series b are higher (with the exception of 5b) than those of series a due to the presence of iodine atoms in the molecule. The most lipophilic complexes in each series are those with flavanone derivatives as ligand 1 and 2, whereas the lowest lipophilicity is exhibited by complexes with 5.

20

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Fig. 3. LogP values for complexes in relation to the arene substituent.

D

3.7. Cytotoxic effects

TE

All compounds were tested in vitro in patient-derived melanoma cultures. Two lines of advanced melanoma, namely nodular melanoma (NM), the most aggressive form of this

CE P

cancer, and superficial spreading melanoma (SSM), the most common one, were used in the study. Both types of melanoma (DMBC11 as SSM; DMBC12 as NM) were cultured in an

AC

anchorage-independent manner as previously described.32 The cytotoxicity of the tested compounds was assessed by flow cytometry as recently described.40 The percentages of viable cells were assessed after PI staining (Fig.4 left panel) or after viable cells were counted (Fig. 4 right panel).

21

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Fig. 4. The effect of arene ruthenium(II) complexes on melanoma cell viability. Two types of

D

melanoma were used: DMBC11 (SSM) and DMB12 (NM). Changes in viability were

TE

assessed after treatment with tested compound at concentrations as indicated. The relative percentages of viable cells were assessed by flow cytometric analysis either after PI staining

CE P

(left panel) or using an automated cell viability analyzer (volumetric assay) (right panel) and were expressed as % of vehicle control. The results are represented as the mean ± SD of three

AC

independent experiments performed in triplicate.

Among all ruthenium(II) complexes, compounds containing 7-aminoflavone 2b-e and 6-aminoflavone 1b as a ligand were the most effective in reducing melanoma cell viability as marked with the grey background in the Table 3. Next, dose-response curves showing changes in cell viability were prepared for the most active complexes (Fig. 5). Based on the dose-response curves, IC50 values were estimated for the most potent ruthenium complexes (Table 4). IC50 values were similar for NM and SSM melanoma populations; however, DMBC12 population (NM) seems to be more sensitive to the obtained complexes than DMBC11 population (SSM). Table 3 22

ACCEPTED MANUSCRIPT The structures of both previously obtained 1a–5a and new series (b-e) of arene ruthenium(II) complexes. The grey background indicate complexes exerting the highest biological activity in patient-derived melanoma cultures.

d

e

1c

1d

1e

2c

2d

2e

D

Ligands (1-5)

3b

3c

3d

3e

4a

4b

4c

4d

-

5a

5b

5c

5d

-

b

1a

1b

2a

2b

3a

c

SC R

a

MA TE

2

AC

CE P

3

5

NU

1

4

IP

T

Ruthenium(II) dimers (a-e)

Fig. 5. Dose-response curves of melanoma cells treated with selected arene ruthenium(II) complexes. Melanoma cells were exposed to ruthenium(II) complexes for 2 days and changes

23

ACCEPTED MANUSCRIPT in viability were assessed by flow cytometry after PI staining. The data are mean of three independent experiments done in triplicates.

T

Table 4

IP

IC50 values of arene ruthenium(II) complexes in patient-derived melanoma cultures.

19.15

0.96

2b

13.18

1.13

2c

4.14

2.53

2d

13.04

2.31

2e

10.16

1.44

NU

1b

MA

[μM]

D

[μM]

SC R

Compound IC50 DMBC11 IC50 DMBC12

TE

3.8. Structure activity relationship

CE P

As lipophilicity is often one of the most influential factors involved in biological activity, the study attempts to determine how the cytotoxicity of ruthenium(II) complexes is related to their logP values (Fig. 6). The LogP values of all compounds are in a relatively

AC

narrow range: 0.81-1.45. The presence of methyl substituents in the ruthenium arene moiety, or a phenyl ring in the ligand, can increase the lipophilicity of the complex. In the whole group, a general trend was observed of cell viability falling with increasing complex lipophilicity, but several compounds characterized by different logP values exhibit comparable results. Therefore, the cytotoxic efficiency of tested complexes appears to be dependent upon the type of lipophilic moiety rather than the lipophilicity itself. As can be seen in Fig. 6 (right panel), four complexes 2c, 2d, 1b and 2b exhibit about 30% of cell viability. These complexes contain similar ligands, flavone derivatives, but have different lipophilicity, which is attributable to differences in the structure of the arene moiety.

24

SC R

IP

T

ACCEPTED MANUSCRIPT

lopP of complexes (concentration 10 µM).

MA

4. Conclusions

NU

Fig. 6. Relationships between melanoma cell viability DMBC11 (a) and DMBC12 (b) and

In summary, eighteen new arene ruthenium(II) complexes with flavone or chromone

D

derivatives as ligands were synthesized and investigated. The complexes coordinate in

TE

a monodentate manner, characteristically to previously obtained series of p-cymene

CE P

ruthenium(II) compounds. The structure of ruthenium(II) complexes is composed of the arene ring carbons occupying one face of the octahedral, with the other three sites to be coordinated by two halogen atoms and a monodentate ligand. The complexes were stable under these

AC

conditions and observed changes in the spectra were negligible (difference in absorbance intensity after 24 h <10%). The logP values measured by the direct shake-flask method range from 0.81 to 1.45. Studies on the lipophilicity of complexes found complexes 1b-e and 2b-e to have the best solubility in the n-octanol phase, partially due to additional arene substituent. The highest cytotoxic activity against melanoma cells derived from surgical specimens was obtained for ruthenium(II) complexes containing 7-aminoflavone as a ligand (2b-e) and complex 1b, which contains 6-aminoflavone as a ligand. Further studies are necessary to determine the potential mechanisms of their action. Both the cytotoxic activity and logP values of the obtained compounds and the previously published 1a – 5a were compared to determine the relationship between 25

ACCEPTED MANUSCRIPT cytotoxicity and lipophilicity. Generally speaking, cytotoxicity against the melanoma cell lines seems to be more dependent on structure and less on lipophilicity.

T

Acknowledgements

IP

Financial support from Medical University of Lodz (grant No 503/3-066-02/503-31-

066-02/502-34-025

and

SC R

001 to E. Budzisz and grant No 503/1-156-01/503-11-001 to M. Czyz), grants No 502-03/3503/3-066-02/503-06-300

AC

CE P

TE

D

MA

NU

acknowledged.

to

26

A.

Pastuszko

are

gratefully

ACCEPTED MANUSCRIPT Graphical abstract

IP

T

2

NU

SC R

R = p-cymene, benzene, hexamethylbenzene or mesitylene; L = 6-aminoflavone, 7-aminoflavone, 7-amino-2-methylchromone, 5-amino-8-methyl-4H-1-benzopyran-4-one and 7-amino-2-benzofuran-1(3H)-one; X = Cl, I.

The ruthenium(II) arene complexes with the formula [(η6-arene)Ru(L)X2] (where

MA

arene = p-cymene, benzene, hexamethylbenzene or mesitylene, L = aminoflavone or aminochromone derivatives and X = Cl, I) have been synthesized. The lipophilic properties and cytotoxic effects of novel complexes has been assayed in order to find structure activity

AC

CE P

TE

D

relationship.

27

ACCEPTED MANUSCRIPT Highlights

 A novel arene ruthenium(II) complexes have been synthesized.  The lipophilicity of synthesized complexes have been determined by shake-flask

T

method.

IP

 The cytotoxicity has been evaluated in vitro in patient derived melanoma cultures.

AC

CE P

TE

D

MA

NU

SC R

 The relationship between cytotoxicity and logP values has been determined.

28

ACCEPTED MANUSCRIPT References A. Pastuszko, K. Niewinna, M. Czyz, A. Jóźwiak, M. Małecka, E. Budzisz, J. Organomet.

Chem. 745-746 (2013) 64-70.

T

1

C.G. Hartinger, P.J. Dyson, Chem. Soc. Rev. 38 (2009) 391–401.

3

Y.K. Yan, M. Melchart, A. Habtemariam, P.J. Sadler, Chem. Commun. 38 (2005) 4764–

SC R

IP

2

4776.

G.S. Smith, B. Therrien, Dalton Trans. 40 (2011) 10793–10800.

5

G. Süss-Fink, Dalton Trans. 39 (2010) 1673–1688.

6

P. Heffeter, U. Jungwirth, M.A. Jakupec, C.G. Hartinger, M. Galanski, L. Elbing, M.

MA

NU

4

Micksche, B.K. Keppler, W. Berger, Drug. Res. Upd. 11 (2008) 1-16.

(2011) 3257-3264.

D

S. Betanzos-Lara, A. Habtemariam, G.J. Clarkson, P J. Sadler, Eur. J. Inorg. Chem. 21

TE

7

I. Kostova, Curr. Med. Chem. 13 (2006) 1085–1107.

9

P.J. Dyson, G. Sava, Dalton Trans. 16 (2006) 1929-1933.

CE P

8

S.J. Dougan, P.J. Sadler, Chimia Int. J. Chem. 61 (2007) 704–715.

11

A.K. Singh, D.S. Pandey, Q. Xu, P. Braunstein, Coord. Chem. Rev. 270-271 (2014) 31-56.

12

S.B. Fricker, Dalton Trans, 43 (2007) 4903-4917.

13

R.E. Morris, R.E. Aird, P. del Socorro Murdoch, H. Chen, J. Cummings, N.D. Hughes, S.

AC

10

Parsons, A. Parkin, G. Boyd, D.J. Jordell, P.J. Sadler, J. Med. Chem. 44 (2001) 3616-3621. 14

F. Wang, A. Habtemariam, E.P. van der Geer, R. Fernández, M. Melchart, J. R. Deeth, R.

Aird, S. Guichard, F. P. Fabbiani, P. Lozano-Casal, I. D. Oswald, D. I. Jodrell, S. Parsons, P.J. Sadler, Proc Natl. Acad. Sci. USA 102 (2005) 18269–18274. 15

M. Melchart, P. J. Sadler, Ruthenium Arene Anticancer Complexes in Bioorganometallics:

Biomolecules, Labeling, Medicine, VCH, Weiheim, 2006. 16

L. Ronconi, P.J. Sadler, Coord. Chem. Rev. 251 (2007) 1633-1648. 29

ACCEPTED MANUSCRIPT W. H. Ang, P. Dyson, Eur. J. Inorg. Chem. 20 (2006) 4003-4018.

18

L.D. Dale, J.H. Tocher, T.M. Dyson, D.I. Edwards, D.A. Tocher, Anticancer Drug Des. 7

(1992) 3-14.

T

17

S. Martens, A. Mithöfer, Phytochemistry 66 (2005) 2399-2407.

20

E. Middleton Jr., C. Kandaswami, T.C. Theoharides, Pharmacol. Rev. 52 (2000) 673-751.

SC R

21

IP

19

A. Kurzwernhart, W. Kandioller, S. Baechler, C. Bartel, S. Martic, M. Buczkowska, G.

NU

Muehlgassner, M.A. Jakupec, H.-B. Kraatz, P.J. Bednarski, V.B. Arion, D. Marko, B.K. Keppler, C.G. Hartinger, J. Med. Chem. 55 (2012) 10512-10522. M. Symonowicz, M. Kolanek, Biotechnol. Food Sci. 76 (2012) 35-41.

23

K. Kowalski, J. Skiba, L. Oehninger, I. Ott, J. Solecka, A. Rajnisz, B. Therrien, J.

MA

22

D

Organomet. Chem. 741-742 (2013) 153-161.

M. Grazul, E. Budzisz, Coord. Chem. Rev. 253 (2009) 2588-2598.

25

M.T. Muller, J.B. Zehnder, B.I. Escher, Environ. Toxicol. Chem. 18 (1999) 2191-2198.

26

J. Paszkowska, B. Kania, I. Wandzik, J. Liq. Chromatogr. Relat. Technol. 35 (2012) 1202-

1212.

V. Pliska, B. Testa, H. Van de Waterbeemd, Lipophilicity in Drug Action and Toxicology in

AC

27

CE P

TE

24

Methods and Principles in Medicinal Chemistry, VCH, Weinheim, 1996. 28

C. Hansch, A. Leo, D.H. Hoekman, Exploring QSAR. Fundamentals and Application in

Chemistry and Biology, American Chemical Society, Washington, 1995. 29

S. Gocan, G. Cimpan, J. Comer, Adv. Chromatogr. 44 (2006) 79-176.

30

M. Grazul, A. Kufelnicki, M. Wozniczka, I.-P. Lorenz, P. Mayer, A. Jóźwiak, M. Czyz, E.

Budzisz, Polyhedron 31 (2012) 150–158. 31

P. Stanetty, I. Rodler, B. Krumpak, J. Prakt. Chem. 335 (1993) 17-22.

32

P. Ruelle, U.W. Kesselring, J. Pharm. Sci. 87 (1998) 1015-1024.

30

ACCEPTED MANUSCRIPT 33

M. Sztiller-Sikorska, K. Koprowska, J. Jakubowska, I. Zalesna, M. Stasiak, M. Duechler, M.

Czyz, Melanoma Res. 22 (2012) 215-224. K. Koprowska, M.L. Hartman, M. Sztiller-Sikorska, M. Czyz, Anticancer Drugs, 24 (2013)

T

34

B. Kupcewicz, M. Grazul, I-P. Lorenz, P. Mayer, E. Budzisz, Polyhedron, 30 (2011) 1177-

SC R

35

IP

835-845.

1184.

A. Kurzwernhart, W. Kandioller, C. Bartel, S. Baechler, R. Trondl, G. Muehlgassner, M.A.

NU

36

Jakupec, V.B. Arion, D. Marko, B.K. Keppler, C.G. Hartinger, Chem. Commun. 48 (2012)

37

M.Grazul, E. Besic-Gyenge, C. Maake, M. Ciolkowski, M. Czyz, R.K.O. Sigel, E. Budzisz,

TE

C. Tan, S. Hu, J. Liu, L. Ji, Eur. J. Med. Chem. 46 (2011) 1555-1563.

W. Kandioller, C.G. Hartinger, A.A. Nazarov, C. Bartel, M. Skocic, M.A. Jakupec, V.B.

CE P

39

D

J. Inorg. Biochem. 135 (2014) 68–76. 38

MA

4839-4841.

Arion, B.K. Keppler, J. Chem. Eur. 15 (2009) 12283-12291. M. Sztiller-Sikorska, K. Koprowska, K. Majchrzak, M.L. Hartman, M. Czyz, PLoS ONE

AC

40

9(3) (2014) e90783.

31