1

Supporting Informationfor

Strong Effect of Copper(II) Coordination on Antiproliferative Activity of Thiosemicarbazone-Piperazine and Thiosemicarbazone-Morpholine Hybrids

Felix Bacher, Orsolya Dömötör,† Anastasia Chugunova, Nóra V. Nagy,§ Lana Filipovic, Sinisa Radulović, Éva A. Enyedy,*,‡ Vladimir B. Arion*,

University of Vienna, Faculty of Chemistry, Institute of Inorganic Chemistry, Währinger Strasse 42, A-1090 Vienna, Austria, †MTA-SZTE Bioinorganic Chemistry Research Group, University of Szeged, Dóm tér 7, H-6720 Szeged, Hungary, §Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar Tudósok körútja 2., H-1117 Budapest, Hungary, Institute for Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia, ‡Department of Inorganic and Analytical Chemistry, University of Szeged, Dóm tér 7. H-6720 Szeged, Hungary

Keywords: Thiosemicarbazone-piperazine, Thiosemicarbazone-morpholine, Thiosemicarbazone-methylpyrrole-2-carboxylate hybrids, Solution equilibrium, Stability constants, Antitumor activity

Electronic Supplementary Material (ESI) for Dalton Transactions.This journal is © The Royal Society of Chemistry 2015

2

NN

NH

NH2

SN

564

1

2

37 12

1

313

4

HL1, E-isomer

HL2, Z-isomer

N

589

101114

6

NN

564

137

124N

589

101116

6

NN

H

SN3

213

1

15

14

NN

NH

NH2

SN

564

1

2

37 12

1

313

4

O

589

1011

HL3, E-isomer

NN

NH

N

SN

564

1

2

37 12

1

313

4

O

589

1011

15

14HL4, E-isomer

NN

NH

N

SN

564

1

2

37 13

1

314

4

16

15H3COOC

5

8

12

17

9

10 11

NN

NH

NH2

SN

564

1

2

37 13

1

144H3COOC

5

8

12

15

9

10 11

3

HL5, E-isomer

HL6, E-isomer

Scheme S1. Atom numbering scheme for 1H and 13C NMR data for HL1 – HL6. HL2 is depicted as Z-isomer for clarity reasons.

NOHHO

(i)

NHO Cl

(ii)

NCl

O

(iii)

NCl

MeO OMe

H

(iv)

A B C D

NR

MeO OMe

H

E: R = methylpiperazinylF: R = morpholinylG: R = pyrrolyl-2-carboxylate

(v)N

R(vi)

ON

RNNH

R'2N

S

H: R = methylpiperazinylI: R = morpholinylJ: R = pyrrolyl-2-carboxylate

HL1: R = methylpiperazinyl, R' = HHL2: R = methylpiperazinyl, R' = CH3HL3: R = morpholinyl, R' = HHL4: R = morpholinyl, R' = CH3HL5: R = pyrrolyl-2-carboxylate, R' = HHL5: R = pyrrolyl-2-carboxylate, R' = CH3

Scheme S2. Synthesis of HL1-HL6.aaReagents and conditions: (i) and (ii) see ref.1; (iii) trimethyl orthoformate, methanesulfonic acid, methanol, 78 °C, 3 h; (iv) E: methylpiperazine, triethylamine, THF/CH2Cl2 1:1, 40 °C, 12 h, purification by column chromatography; F: morpholine, triethylamine, THF/CH2Cl2 1:1, 40 °C, 12 h, purification by column chromatography; G: pyrrole-2-carboxylate, sodium hydride, DMF, 0° C, 1 h, than room temperature, 12 h, purification by column chromatography; (v) H and I: HCl, water, 60 °C, 12 h, sat. aqueous NaHCO3, extraction with CH2Cl2; J: water/acetone 5:1, reflux, 12 h; (vi) HL1: thiosemicarbazide, EtOH, 78 °C, 12 h, purification on prep. HPLC; HL2: 4,4-dimethyl-3-thiosemicarbazide, EtOH, room temperature, 12 h, purification on prep. HPLC; HL3: thiosemicarbazide, EtOH, 78 °C, 12 h, recrystallization from water/methanol 5:1; HL4: 4,4-dimethyl-3-thiosemicarbazide, EtOH, room temperature, 12 h. HL5: thiosemicarbazide, EtOH/MeOH 1:1, 78 °C, 12 h, recrystallization from water/methanol 5:1. HL6: 4,4-dimethyl-3-thiosemicarbazide, EtOH/MeOH 1:1, room temperature, 6 h.

3

NN NH

NS

NH+

H+N

H

NN

HN

NS

NH+

H+N

H

H3L2+ (E)

H3L2+ (Z)

NN NH

NS

N

H+N

H

NN

HN

NS

N

H+N

H

H2L+ (E)

H2L+ (Z)

NN NH

NS

NN

H

NN

HN

NS

NN

H

HL (E)

HL (Z)

NN N

N-S

NN

H

NN

N

N-S

NN

H

L- (E)

L- (Z)

(A)

(B)

NN+

NH

NS

NH+

H+N

H

NH+

NHN

NS

NH+

H+N

H

H4L3+ (E)

H4L3+ (Z)

K1 K2

K1 K2

K3

K3 K4

K4

H

Scheme S3. Deprotonation steps of the H4L3+ form of ligand mPip-dm-FTSC (HL2) for its E (A) and Z (B) isomers

Figure S1. Part of the crystal structure of 1 showing complex pairing via intermolecular hydrogen bonding interactions. Donor∙∙∙acceptor (D∙∙∙A) distances (Å) and donor-hydrogen∙∙∙acceptor (DH∙∙∙A) angles (deg) of the intermolecular hydrogen bonds: N4∙∙∙N3i

2.946(3) Å, N4H∙∙∙N3i 173.7°; atoms marked with i are at symmetry position x + 2, y + 1, z + 1.

4

7.3

7.5

7.7

7.9

8.1

8.3

1 3 5 7 9 11

/ p

pm

pH

C12H

C12H

C5H

C5H

C6H

C6H

C4HC4H

(A)

2.3

2.8

3.3

3.8

4.3

1 3 5 7 9 11

/ p

pm

pH

C7H2

C9,10H2

C7H2

C9,10H2

C8,11H2

C14,15H3

C8,11H2

C14,15H3

(B)

Figure S2. pH-Dependence of the chemical shifts of the various protons of the ligand Morph-dm-FTSC (HL4) in the low- (A) and in the high-field (B) regions. Red and blue symbols denote the protons belonging to the major E and minor Z isomers, respectively. [cL= 1.5 mM; T = 298 K; I = 0.10 M (KCl); 10% D2O]

Figure S3. Low- (A) and high-field (B) regions of the 1H NMR spectra of ligand mPip-dm-FTSC (HL2) at different pH values, red and blue symbols denote the peaks belonging to the protons of the major E and minor Z isomers, respectively. [cL= 3 mM; T = 298 K; I = 0.10 M (KCl); 10% D2O]

5

7.3

7.5

7.7

7.9

8.1

8.3

1 3 5 7 9 11

/ p

pm

pH

C12H

C12H

C5HC5H

C6H

C6HC4H

C4H

(A)

2.1

2.6

3.1

3.6

4.1

4.6

1 3 5 7 9 11

/ p

pm

pH

C7H2

C7H2

C16H3

C14,15H3

C16H3

C14,15H3

(B)

Figure S4. pH-Dependence of the chemical shifts of the various protons of the ligand mPip-dm-FTSC (HL2) in the low- (A) and in the high-field (B) regions. Red and blue symbols denote the protons belonging to the major E and minor Z isomers, respectively. [cL= 3 mM; T = 298 K; I = 0.10 M (KCl); 10% D2O]

0.00

0.25

0.50

0.75

1.00

mol

ar fr

actio

n

E izomer

Z izomer (A)

E isomer

Z isomer

0.00

0.25

0.50

0.75

1.00

2 4 6 8 10

mol

ar fr

actio

n

pH

(B)

H3L2+

H2L+

HL

L-

H2L+ HL

H3L2+

Figure S5. pH-Dependence of the molar fraction of the E (red symbols) and Z (blue symbols) isomers of the ligand mPip-dm-FTSC (HL2) calculated on the basis of the integrated areas of the signals of the various ligand protons (A). Concentration distribution curves for the isomeric ligand species (E: red letters; Z: blue letters) calculated with the aid of the microscopic proton dissociation constants (B). [cL= 3 mM; T = 298 K; I = 0.10 M (KCl); 10% D2O]

6

(A) (B)Intensity/ a.u.

Intensity/ a.u.

Figure S6. The 3-dimensional fluorescence spectrum of mPip-dm-FTSC (HL2) (A) and Morph-dm-FTSC (HL4) (B) at physiological pH in water. [cL= 10 M; T = 298 K; I = 0.10 M (KCl); pH = 7.4 (10 mM HEPES)]

3000 3100 3200 3300 3400 3500 3600

Magnetic field [G]

1.77

2.15

2.82

3.43

4.14

5.74

7.05

8.82

10.40

11.63

11.14

pH (a)

3000 3100 3200 3300 3400 3500 3600

Magnetic field [G]

1.62

1.93

2.42

2.97

4.33

5.34

6.19

6.98

7.87

9.93

11.12

11.68

pH (b)

3000 3100 3200 3300 3400 3500 3600

Magnetic field [G]

[CuLH]

[CuL]

[CuLH-1]

[CuL2H3]

[CuL2]

(A) (B) (C)

Magnetic field / G Magnetic field / G Magnetic field / G

[CuL2]

[CuL2H3]3+

[CuLH-1]

[CuL]+

[CuLH]2+

Figure S7. Experimental (black) and simulated (red) solution EPR spectra recorded for the copper(II) – Morph-dm-FTSC (HL4) system at 1:1 (A) and 1:2 (B) metal-to-ligand ratio. Calculated component EPR spectra obtained for the different copper(II) – Morph-dm-FTSC (HL4) species (C). [cligand = 1.0 mM; cCu= 1.0 mM (A) or cCu= 0.5 mM (B); T = 298 K; I = 0.10 M (KCl)]

7

2600 2800 3000 3200 3400 3600

Magnetic field [G]

[CuLH2]

[CuLH]

[CuL]

[CuLH-1]

[CuL2]

2600 2800 3000 3200 3400 3600

Magnetic field [G]

[CuLH]

[CuL]

[CuLH-1]

(A) (B)

Magnetic field / G Magnetic field / G

[CuL2]

[CuLH-1]

[CuL]+

[CuLH]2+

[CuLH2]3+

[CuLH]2+

[CuL]+

[CuLH-1]

Figure S8. Calculated component EPR spectra obtained for the different copper(II) – mPip-dm-FTSC (HL2) (A) and Morph-dm-FTSC (HL4) (B) complexes in frozen solution. [T = 77 K; I = 0.10 M (KCl)]

0.0

0.3

0.6

0.9

1.2

1.5

290 330 370 410 450

Abs

orba

nce

/ nm

0.0

0.2

0.4

0.6

0 50 100 150

Abs.

at 4

20 n

m

cligand / M

Figure S9. UV−vis spectra of the [Cu(EDTA)]2‒ complex (grey solid line, c = 50 M) in the presence of ligand mPip-dm-FTSC (HL2) at increasing concentrations (solid lines in color, c = 7.5 - 167.6 M). UV−vis spectra of EDTA (grey dashed line, c = 50 M), mPip-dm-FTSC (black dashed line, c = 167.6 M) and complex 2 (black solid line, c = 50 M) are shown for comparison. Inset shows the absorbance values recorded at 420 nm for two parallel sets of measurements on the ternary copper(II) – EDTA − ligand system in dependence of the concentration of ligand mPip-dm-FTSC. [T = 298 K; I = 0.10 M (KCl); l = 1 cm; pH = 7.4 (10 mM HEPES)]

8

0.0

0.2

0.4

0.6

0.8

1.0

1.2

230 280 330 380 430 480

Abs

orba

nce

/ nm

ligand1.13

ligand7.40

ligand11.36

1.04

2.25

1.042.25 3.0-8.2

8.46

11.57

3.0-8.2

(A)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

230 280 330 380 430 480

Abs

orba

nce

/ nm

ligand1.11

ligand7.58

ligand11.36

1.04

2.84

10.51

11.49

(B)11.49

1.04

3.50

4.0-10.0 4.0-10.0

Figure S10. UV−vis spectra of the copper(II) – mPip-dm-FTSC (HL2) (A) and copper(II) – Morph-dm-FTSC (HL4) (B) systems at 1:1 metal-to-ligand ratio. (Grey dashed lines show the spectra of the ligands alone at chosen pH values.) [cL= 153 M (A) and 157 M (B); T = 298 K; I = 0.10 M (KCl); l = 0.5 cm]

References

1. G. Paolucci, A. Zanella, M. Bortoluzzi, S. Sostero, P. Longo and M. Napoli, J. Mol. Catal. A: Chem., 2007, 272, 258-264.