Supporting Information

Nanoencapsulation of a ruthenium(II) complex with triazolopyrimidine in liposomes as a tool for improving its anticancer activity against melanoma cell lines

Marzena Fandzloch,*a,b,† Anna Jaromin, *c,† Magdalena Zaremba-Czogalla, c Andrzej

Wojtczak, d Agnieszka Lewińska, a Jerzy Sitkowski, e,f Joanna Wiśniewska, d Iwona

Łakomska, d and Jerzy Gubernatorc

a Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wrocław, Polandb Institute of Low Temperature and Structure Research, PAS, Okólna 2, 50-422 Wrocław, Polandc Department of Lipids and Liposomes, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14a,

50-383 Wroclaw, Polandd Faculty of Chemistry, Nicolaus Copernicus University in Toruń, Gagarina 7, 87-100 Toruń, Polande National Institutes of Medicines, Chełmska 30/34, 00-725 Warszawa, Polandf Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warszawa, Poland

*Corresponding authors e-mail:

m.fandzloch@intibs.pl

anna.jaromin@uwr.edu.pl

† Both authors contributed equally to this manuscript.

Electronic Supplementary Material (ESI) for Dalton Transactions.This journal is © The Royal Society of Chemistry 2020

2

Table of Contents

Table S1

Figure S1

Figure S2

Figure S3

Figure S4

Figure S5

Figure S6

Figure S7

Figure S8

Figure S9

Figure S10

Figure S11

Figure S12

Figure S13

Figure S14

Figure S15

Figure S16

Figure S17

Figure S18

Figure S19

Figure S20

Figure S21

Figure S22

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S4

S5

S5

S6

S6

S7

S7

S8

S8

S9

S9

S10

S10

S11

S11

S12

S13

S13

S14

S14

S15

S16

S16

3

Figure S23

Figure S24

Figure S25

Figure S26

Figure S27

Figure S28

Figure S29

Figure S30

Figure S31

Figure S32

Figure S33

Figure S34

Table S2

Table S3

Table S4

Table S5

Figure S35

Figure S36

Figure S37

Figure S38

Figure S39

Figure S40

Figure S41

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S17

S17

S18

S19

S19

S20

S20

S21

S21

S22

S22

S23

S24

S25

S26

S27

S28

S29

S30

S30

S31

S32

S33

4

Table S1. Crystal data and structure refinement for cis,fac-[RuCl2(dmso)3(ibmtp)]·0.5CH3OH (3·0.5CH3OH), cis,cis,cis-[RuCl2(detp)2(dmso)2] (4) and cis,cis,cis-[RuCl2(dbtp)2(dmso)2]·CH3OH (5·CH3OH).

(3·0.5CH3OH) (4) (5·CH3OH)Empirical formula C16.50H34Cl2N4O3.50RuS3 C22H36Cl2N8O2RuS2 C31H56Cl2N8O3RuS2Formula weight 612.62 680.68 824.92Temperature; K 293(2) 293(2) 293(2) Wavelength; Å 0.71073 0.71073 0.71073 Crystal system Monoclinic Monoclinic MonoclinicSpace group P21/n Cc C2/cUnit cell dimensions; Å, °

a = 14.7951(4) a = 8.7701(7) a = 33.4606(14)

b = 11.7116(3) b = 21.7923(15) b = 17.0002(4) c = 15.5540(4) c = 15.5674(10) c = 17.9715(9) = 90 = 90 = 90= 105.289(3) = 99.426(7) = 120.104(6) = 90 = 90 = 90

Volume; Å3 2599.74(11) 2935.1(4) 8844.0(8) Z 4 4 8Density (calculated); Mg/m3

1.565 1.540 1.239

Absorption coefficient; mm-1 1.076 0.894 0.607F(000) 1260 1400 3456Crystal size; mm 0.611 x 0.456 x 0.181 0.242 x 0.147 x 0.094 0.390 x 0.207 x 0.087 Theta range for data collection

2.214 to 28.130°. 2.292 to 28.574°. 2.268 to 28.080°.

Index ranges -19<=h<=18, -15<=k<=15, -17<=l<=19

-11<=h<=11, -28<=k<=27, -20<=l<=20

-43<=h<=43, -22<=k<=21, -21<=l<=22

Reflections collected 16524 10137 29286Independent reflections 5694 [R(int) = 0.0306] 4957 [R(int) = 0.0768] 9669 [R(int) = 0.0605]Completeness to theta 26.000° 99.9 % 25.242° 100.0 % 25.000° 99.9 % Absorption correction Analytical Analytical AnalyticalMax. and min. transmission

0.855 and 0.642 0.943 and 0.867 0.956 and 0.818

Refinement method Full-matrix least-squares on F2

Full-matrix least-squareson F2

Full-matrix least-squareson F2

Data/restraints/parameters 5694 / 1 / 280 4957 / 24 / 354 9669 / 38 / 438Goodness-of-fit on F2 1.046 1.012 0.967Final R indices [I>2sigma(I)]

R1 = 0.0306, wR2 = 0.0825

R1 = 0.0588, wR2 = 0.1206

R1 = 0.0522, wR2 = 0.1396

R indices (all data) R1 = 0.0402, wR2 = 0.0861

R1 = 0.1098, wR2 = 0.1492

R1 = 0.1047, wR2 = 0.1588

Absolute structure ParameterExtinction coefficient

n/a -0.11(7)

n/a

n/a

Largest diff. peak and hole; e.Å-3

1.007 and -0.438 0.629 and -0.498 0.902 and -0.488

5

389.

6942

4.60

440.

5746

9.60

496.

69

576.

79593.

7662

0.56

678.

8971

9.51

764.

48806.

40

905.

3591

7.47

938.

8797

9.53

1008

.03

1091

.90

1194

.95

1251

.72

1272

.20

1295

.20

1304

.87

1356

.36

1403

.55

1522

.52

1552

.34

1614

.12

(1)

45

50

55

60

65

70

75

80

85

90

95

100

%T

600 800 1000 1200 1400 1600 1800 Wavenumbers (cm-1)

Fig. S1 FT-IR spectrum for cis,fac-[RuCl2(dmso)3(tp)] (1).

380.

5639

0.38

423.

1544

8.07

485.

91

532.

10

602.

72

661.

9067

5.09

720.

73

773.

97

815.

6584

4.19

865.

65937.

1197

1.28

1020

.00

1082

.51

1200

.94

1263

.32

1291

.19

1307

.09

1373

.07

1430

.01

1552

.41

1624

.39

(2)

35

40

45

50

55

60

65

70

75

80

85

90

95

100

%T

600 800 1000 1200 1400 1600 1800 Wavenumbers (cm-1)

Fig. S2 FT-IR spectrum for cis,fac-[RuCl2(dmso)3(dmtp)] (2).

6

383.

0839

6.61

422.

8144

8.71

494.

57

608.

07647.

2866

3.28

681.

7772

0.12

775.

65

927.

99

978.

4610

22.4

3

1072

.42

1086

.11

1104

.69

1196

.0312

69.1

312

99.8

8

1391

.0414

12.7

014

28.9

514

64.8

0

1546

.19

1618

.96

(3)

55

60

65

70

75

80

85

90

95%

T

600 800 1000 1200 1400 1600 1800 Wavenumbers (cm-1)

Fig. S3 FT-IR spectrum for cis,fac-[RuCl2(dmso)3(ibmtp)] (3).

391.

9142

4.68

481.

70593.

01618.

5965

8.92

681.

8072

0.64

915.

59

973.

0310

15.8

0

1082

.78

1197

.7812

57.5

912

93.9

7

1369

.16

1394

.98

1459

.08

1547

.90

1624

.40

1643

.37

(4)

62

64

66

68

70

72

74

76

78

80

82

84

86

88

90

92

94

96

98

%T

600 800 1000 1200 1400 1600 1800 Wavenumbers (cm-1)

Fig. S4 FT-IR spectrum for cis,fac-[RuCl2(dmso)3(ibmtp)] (4).

7

389.

1242

1.01

441.

83

518.

19

607.

1662

1.31

648.

3467

7.91

711.

82

758.

4978

3.64

832.

0085

6.20

884.

8491

0.18

929.

4496

6.56

1011

.13

1037

.5810

85.6

210

98.6

8

1128

.71

1178

.99

1201

.78

1218

.69

1254

.98

1267

.6713

01.9

913

38.0

113

69.3

413

98.8

5

1465

.09

1533

.79

1543

.66

1613

.71

1792

.26

1934

.78

(5)

60

65

70

75

80

85

90

95

100%

T

600 800 1000 1200 1400 1600 1800 Wavenumbers (cm-1)

Fig. S5 FT-IR spectrum for cis,cis,cis-[RuCl2(dbtp)2(dmso)2] (5).

425.

1744

1.95

485.

08

601.

9761

8.14

635.

8265

4.55

665.

5069

4.23

721.

2475

9.46

777.

73

863.

72

923.

4395

4.31

1011

.80

1071

.48

1093

.02

1145

.67

1197

.34

1239

.44

1264

.33

1302

.33

1377

.82

1440

.39

1491

.98

1545

.47

1574

.73

1611

.83

(6)

55

60

65

70

75

80

85

90

95

100

%T

600 800 1000 1200 1400 1600 1800 Wavenumbers (cm-1)

Fig. S6 FT-IR spectrum for cis,cis,cis-[RuCl2(dmso)2(dptp)2] (6).

8

Fig. S7 1H NMR spectrum recorded for cis,fac-[RuCl2(dmso)3(tp)] (1) in CDCl3.

Fig. S8 1H-15N HMBC spectrum recorded for cis,fac-[RuCl2(dmso)3(tp)] (1) in CDCl3.

9

Fig. S9 1H-13C HSQC spectrum recorded for cis,fac-[RuCl2(dmso)3(tp)] (1) in CDCl3.

Fig. S10 1H-13C HMBC spectrum recorded for cis,fac-[RuCl2(dmso)3(tp)] (1) in CDCl3.

10

Fig. S11 1H NMR spectrum recorded for cis,fac-[RuCl2(dmso)3(dmtp)] (2) in CDCl3.

Fig. S12 1H-15N spectrum recorded for cis,fac-[RuCl2(dmso)3(dmtp)] (2) in CDCl3.

11

Fig. S13 13C NMR spectrum recorded for cis,fac-[RuCl2(dmso)3(dmtp)] (2) in CDCl3.

Fig. S14 1H-13C HSQC spectrum recorded for cis,fac-[RuCl2(dmso)3(dmtp)] (2) in CDCl3.

12

Fig. S15 1H-13C HMBC spectrum recorded for cis,fac-[RuCl2(dmso)3(dmtp)] (2) in CDCl3.

13

Fig. S16 1H NMR spectrum recorded for cis,fac-[RuCl2(dmso)3(ibmtp)] (3) in CDCl3.

Fig. S17 1H-15N HMBC spectrum recorded for cis,fac-[RuCl2(dmso)3(ibmtp)] (3) in CDCl3.

14

Fig. S18 13C NMR spectrum recorded for cis,fac-[RuCl2(dmso)3(ibmtp)] (3) in CDCl3.

Fig. S19 1H-13C HSQC spectrum recorded for cis,fac-[RuCl2(dmso)3(ibmtp)] (3) in CDCl3.

15

Fig. S20 1H-13C HMBC spectrum recorded for cis,fac-[RuCl2(dmso)3(ibmtp)] (3) in CDCl3.

16

Fig. S21 1H NMR spectrum recorded for cis,cis,cis-[RuCl2(detp)2(dmso)2] (4) in CDCl3.

Fig. S22 1H-15N HMBC spectrum recorded for cis,cis,cis-[RuCl2(detp)2(dmso)2] (4) in CDCl3.

17

Fig. S23 13C NMR spectrum recorded for cis,cis,cis-[RuCl2(detp)2(dmso)2] (4) in CDCl3.

Fig. S24 1H-13C HSQC spectrum recorded for cis,cis,cis-[RuCl2(detp)2(dmso)2] (4) in CDCl3.

18

Fig. S25 1H-13C HMBC spectrum recorded for cis,cis,cis-[RuCl2(detp)2(dmso)2] (4) in CDCl3.

19

Fig. S26 1H NMR spectrum recorded for cis,cis,cis-[RuCl2(dbtp)2(dmso)2] (5) in CDCl3.

Fig. S27 1H-15N HMBC spectrum recorded for cis,cis,cis-[RuCl2(dbtp)2(dmso)2] (5) in CDCl3.

20

Fig. S28 13C NMR spectrum recorded for cis,cis,cis-[RuCl2(dbtp)2(dmso)2] (5) in CDCl3.

Fig. S29 1H-13C HSQC spectrum recorded for cis,cis,cis-[RuCl2(dbtp)2(dmso)2] (5) in CDCl3.

21

Fig. S30 1H NMR spectrum recorded for cis,cis,cis-[RuCl2(dmso)2(dptp)2] (6) in CDCl3.

Fig. S31 1H-15N HMBC spectrum recorded for cis,cis,cis-[RuCl2(dmso)2(dptp)2] (6) in CDCl3.

22

Fig. S32 13C NMR spectrum recorded for cis,cis,cis-[RuCl2(dmso)2(dptp)2] (6) in CDCl3.

Fig. S33 1H-13C HSQC spectrum recorded for cis,cis,cis-[RuCl2(dmso)2(dptp)2] (6) in CDCl3.

23

Fig. S34. 1H-13C HMBC spectrum recorded for cis,cis,cis-[RuCl2(dmso)2(dptp)2] (6) in CDCl3.

24

Table S2. Chemical shifts (13C NMR in CDCl3) for (1-6) [inppm]

Complex C2 C3a C5 C6 C7 Ra** CH3(dmso)

cis,fac-[RuCl2(dmso)3(tp)] (1)(*)

160.8(4.5)

153.9(-1.3)

155.0(0.2)

111.4(1.1)

137.8(1.8) -

48.045.947.5

cis,fac-[RuCl2(dmso)3(dmtp)] (2)(*)

159.5(4.4)

153.7(-1.3)

165.4 (0.8)

111.8 (1.3)

148.8 (2.3)

24.7 (-0.1)16.9 (-0.1)

47.645.848.0

cis,fac-[RuCl2(dmso)3(ibmtp)] (3)(*)

159.3(4.1)

153.8(-1.6)

165.3(0.6)

111.5(1.0)

151.7(2.0)

24.8 (-0.02)22.6 (0.02)39.4 (-0.02)26.1 (-0.01)

47.945.847.6

cis,cis,cis-[RuCl2(detp)2(dmso)2] (4)(*)

159.2(3.9)

154.9(-0.5)

169.6(-0.1)

108.4(0.8)

159.5(7.8)

23.4 (-8.3)10.4 (-2.3)23.6 (-0.1)10.3 (0)

45.8, 47.745.6, 46.0

cis,cis,cis-[RuCl2(dbtp)2(dmso)2] (5)(*)

158.6(4.4)

155.1(-0.8)

176.2(0.5)

103.7(0.3)

158.9(1.5)

38.5(-0.3)29.0 (-0.7)36.6 (0.4)27.2 (0.2)

45.1, 47.845.6, 46.1

cis,cis,cis-[RuCl2(dmso)2(dptp)2] (6)(*)

160.5(5.7)

.155.8(0.5)

160.6-1.9)

105.0-2.2

149.0(0.6)

128 - 132(-0.1 - 0.3)

47.5, 45.845.9, 45.8

= complex - ligand ** Ra = H5 and H7 for tp, CH3(5) and CH3(7) for dmtp, CH3(5) and (CH3)2(7)CH(7)CH2(7) for ibmtp, C(CH3)3(5) and(CH3)3(7) for dbtp, CH3(5)CH2(5) and CH3(7)CH2(7) for detp, C6H5(5) and C6H5(7) for dptp.

25

Table S3. Hydrogen bonds for (3) [Å and °].

D-H···A d(D-H) d(H···A) d(D···A) <(DHA)C71-H71A...O91#1 0.97 2.63 3.571(9) 163.5C11-H11A...N4 0.96 2.38 3.204(4) 143.3C11-H11B...O21 0.96 2.65 3.331(4) 127.9C12-H12A...N4 0.96 2.58 3.344(4) 137.0C21-H21C...Cl1#2 0.96 2.78 3.718(4) 165.7C22-H22A...O31 0.96 2.25 3.063(5) 141.7C22-H22B...O11 0.96 2.42 3.043(4) 122.4C22-H22C...Cl2#3 0.96 2.65 3.516(3) 150.8C31-H31A...Cl2 0.96 2.73 3.376(4) 125.3C31-H31B...O11#4 0.96 2.29 3.245(4) 172.8C31-H31C...Cl1 0.96 2.70 3.181(4) 111.4C32-H32B...O11 0.96 2.48 3.157(5) 127.2O91-H91A...O31#5 0.82 2.09 2.823(8) 149.1C92-H92B...O31#5 0.96 2.64 3.110(14) 110.3

Symmetry transformations used to generate equivalent atoms:

#1 x-1/2,-y+1/2,z+1/2 #2 -x+1,-y,-z #3 x+1/2,-y+1/2,z+1/2

#4 -x+3/2,y-1/2,-z-1/2 #5 -x+3/2,y+1/2,-z-1/2

26

Table S4. Hydrogen bonds for (4) [Å and °].

D-H···A d(D-H) d(H···A) d(D···A) <(DHA)C2-H2A···Cl2 0.93 2.78 3.233(14) 111.0C2-H2A···Cl2 0.93 2.78 3.233(14) 111.0C6-H6A···Cl1#1 0.93 2.99 3.617(13) 126.4C26-H26A···Cl1#2 0.93 2.86 3.712(16) 152.4C29-H29B^a···Cl2#2 0.96 2.87 3.76(2) 153.5C31-H31C^b···N1#2 0.98 2.68 3.55(2) 148.4C41-H41A···Cl1 0.96 2.61 3.260(16) 125.4C43-H43A···Cl2 0.96 2.72 3.371(18) 125.7C43-43B···O1 0.96 2.33 3.05(2) 131.0C44-H44A···Cl2 0.96 2.95 3.556(18) 121.9

Symmetry transformations used to generate equivalent atoms:

#1 x+1/2,-y+3/2,z-1/2 #2 x-1/2,-y+3/2,z-1/2

27

Table S5. Hydrogen bonds for (5) [Å and °].

D-H···A d(D-H) d(H···A) d(D···A) <(DHA)C2-H2A···Cl1 0.93 2.79 3.231(5) 110.5C22-H22C···N4 0.96 2.65 3.440(7) 139.6C23-H23A···Cl1 0.96 2.75 3.390(6) 124.5C23-H23B···O22 0.96 2.44 3.145(8) 129.9C24-H24A···Cl2O(94)-H(94O)...O(21)#2

0.960.82

2.592.50

3.259(6)3.309(11)

127.0170.0

Symmetry transformations used to generate equivalent atoms:

#1 -x+1,y,-z+1/2 #2 -x+1,-y+1,-z

28

Fig. S35 The hemolysis assay for free (5) (1-100 µM), (5)-loaded liposomes at a concentration of (5) corresponding to 1 µM and unloaded liposomes.

(5) Concentration (µM) (5)-loaded unloaded positive negative

1 10 50 100 liposomes liposomes control control

29

0 1000 2000 3000 4000 50000,815

0,820

0,825

0,830

0,835

0,840

0,845

0,850

0,855

0,860Ab

sorb

ance

Time (min)

Fig. S36 Kinetic trace for the hydrolysis of cis,cis,cis-[RuCl2(dbtp)2(dmso)2] (5). Experimental conditions: [RuII] = 4.6 10-4 M, 100 mM NaCl, = 270 nm, l = 0.94 cm, T = 37 C.

30

200 300 400 500 600 700 8000

0.5

1

1.5

2

2.5

3

Wavelength (nm)

Abs

orba

nce

Fig. S37 The electronic spectra of cis,cis,cis-[RuCl2(dbtp)2(dmso)2] (5) after mixing with 0.1 M phosphate buffer solution (1:1) (red line) and after 4 days (black line); [RuII] = 4.6 10-4 M, pH = 6.5, T = 37 C.

200 300 400 500 600 700 8000

0.5

1

1.5

2

2.5

3

Wavelength (nm)

Abs

orba

nce

Fig. S38 The electronic spectra of cis,cis,cis-[RuCl2(dbtp)2(dmso)2] (5) after mixing with water (black line), after 1 day (red line), after 2 days (green line), after 3 days (dark blue line) and after 4 days (light blue line); [RuII] = 4.6 10-4 M, T = 37 C.

31

2

m/z425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 825 850 875 900 925 950 975

%

0

100umk_jw2313_NaCl_neg 10 (0.226) Cm (9:38-(2:3+48:49)) 1: TOF MS ES-

9.69e5794.42

560.59

558.59

502.63

500.64

444.67442.68

440.68

446.67

498.64

448.66

504.63

506.62 556.59

508.62

562.59

792.42

564.58

618.54

616.55566.58

614.55590.57

678.51676.51

622.54

648.52

646.53674.51

790.42

680.50736.46

734.46682.50

732.46

765.44

763.44

796.42

798.41

970.29

968.29

912.33824.40 852.38

826.39

827.39

910.34

882.35

880.36

879.36909.34

914.33966.30

941.31

972.29

974.29

Fig. S39 Negative ion mode ESI-MS spectrum of cis,cis,cis-[RuCl2(dbtp)2(dmso)2] (5) analysed directly after mixing in 100 mM NaCl. Experimental conditions: a Synapt G2-S mass spectrometer, spray voltage 1.8 kV, source temperature 150 °C, ion transfer capillary voltage 20 V, tube lens offset 0 V.

[RuCl2(dbtp)2(dmso)2][RuCl2(dbtp)2(dmso)2 − dbtp]

32

2

m/z805 810 815 820 825 830 835 840 845 850 855 860 865 870 875

%

0

100umk_jw2313_analiza_po_2dniach_neg 36 (0.725) Cm (9:36-(2+49)) 1: TOF MS ES-

4.79e4829.17

827.17

811.21809.21

808.21

807.20

806.21

803.21

826.17

819.24

818.24813.21

817.24

821.24

825.17

847.18

845.18

831.17

844.19

843.18832.17

833.17 842.19

837.20

849.18

850.19

871.20856.19 870.20

857.20 868.20

872.20875.20

Figure S40. Negative ion mode ESI-MS spectrum of cis,cis,cis-[RuCl2(dbtp)2(dmso)2] (5) complex analysed after two days in 100 mM NaCl. Experimental conditions: a Synapt G2-S mass spectrometer, spray voltage 1.8 kV, source temperature 150 °C, ion transfer capillary voltage 20 V, tube lens offset 0 V.

[RuCl2(dbtp)2(dmso)2] + Cl-

[RuCl2(dbtp)2(dmso)2] + Cl- + H2O[RuCl(OH)(dbtp)2(dmso)2] + Cl-

33

2

m/z590 600 610 620 630 640 650 660 670 680 690 700 710 720 730 740 750 760 770 780 790 800 810

%

0

100umk_jw2313_analiza_po_2dniach 11 (0.243) Cm (9:36-(2:3+44:48)) 1: TOF MS ES+

3.76e5757.24

756.24

755.24

754.24

751.24599.20 679.22606.57

664.53617.21724.48

720.25

759.24

760.24

761.24

782.44

778.44

784.44

786.44

788.43

Fig. S41 Positive ion mode ESI-MS spectrum of cis,cis,cis-[RuCl2(dbtp)2(dmso)2] (5) analysed after two days in 100 mM NaCl. Experimental conditions: a Synapt G2-S mass spectrometer, spray voltage 1.8 kV, source temperature 150 °C, ion transfer capillary voltage 20 V, tube lens offset 0 V.

[RuCl(OH)(dbtp)2(dmso)2− OH]+