Complexes
Supplementary Information for
In vitro and In vivo Anticancer Activity of Copper Bis(thiosemicarbazone) Complexes Duraippandi Palanimuthu,† Sridevi Vijay Shinde,‡ Kumaravel Somasundaram,*‡ and Ashoka G. Samuelson*†
†
Duraippandi Palanimuthu and Prof. A. G. Samuelson, Department of Inorganic and Physical
Chemistry, Indian Institute of Science, Bangalore, India 560012, Fax: (+91) 80-2360-1552. e-mail: [email protected]. ‡Sridevi Vijay Shinde and Prof. Kumaravel Somasundaram, Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India 560012, Fax: (+91) 80-2360-2697. e-mail: [email protected] Table of contents
Page
General scheme for the ligand synthesis
S2
Crystallographic data (Table S1)
S3
Cyclic voltammogram of Cu(GTSC)
S4
In vitro DNA binding study for Cu(GTSCHCl)
S5
Viscometric titration
S6
pBBR322 DNA cleavage by Cu(GTSC)
S7
pBBR322 DNA cleavage by Cu(ATSM), Cu(BTSC) and Cu(BTSCH)ClO4
S8
pBBR322 DNA cleavage by Cu(ATSC) Cu(ATSCH)ClO4
S9
pBBR322 DNA cleavage by Cu(GTSCHCl) under aerobic and anaerobic condition
S10
Topoisomerase IIα relaxation assay for copper complexes
S11
S1
LIGANDS
R1
R2
R3
BTSCH2
CH2-CH2
CH2-CH2
Ph
ATSCH2
CH2-CH2
CH2-CH2
CH3
GTSCH2
CH3
Ph
H
ATSMH2
CH3
H
CH3
Scheme S1. General scheme for the synthesis of ligands.
S2
Table S1. Selected crystallographic data for complexes Cu(BTSC) 0.5CH2Cl2, Cu(GTSC) and Cu(ATSC). Crystal identification
Cu(BTSC) 0.5CH2Cl2
Cu(GTSC)
Cu(ATSC)
Chemical Formula Molecular weight Crystal color Crystal dimension (mm) Crystal system Space group a (Å) b (Å) c (Å) α (deg) β(deg) γ(deg) V(Å3 ) Z Density(g.cm-3) µ(mm-1) F (000) Radiation (λ=0.71073Å) Temperature (K) Scan type θ range (deg) H ,k, l range
C49H54N12S4Cl2Cu2 1137.26 Dark brown 0.92 × 0.41 × 0.29 Monoclinic P21/c 15.1825(9) 11.7338(6) 29.6982(17) 90.000 100.534(4) 90.000 5201.5(5) 4 1.452 1.129 2352 Mo-Kα 293 (2) ω-2θ 1.36 to 25.50 -18 ≤ h ≤ 17 -14 ≤ k ≤ 14 -35 ≤ l ≤ 35 81895 9691 [Rint= 0.0900] Full-matrix leastsquares on F2 1.000 R1= 0.0543, wR2= 0.1380 R1= 0.1208, wR2= 0.1587
C18H18N6S2Cu 446.04 Dark brown 0.9 × 0.2 × 0.2 Orthorhombic Pbcn 31.3720(3) 7.5677(8) 7.9918(9) 90.000 90.000 90.000 1897.3(4) 4 1.561 1.387 916 Mo-Kα 293 (2) ω-2θ 1.30 to 25.50 -38 ≤ h ≤ 38 -9 ≤ k ≤ 9 -9≤ l ≤9 22949 1771 [Rint= 0.0695] Full-matrix leastsquares on F2 1.050 R1= 0.0363, wR2= 0.0903 R1= 0.0499, wR2= 0.0963
C28H48N12S4Cu2 808.10 Dark brown 0. 9 × 0.3 × 0.2 Triclinic P-1 7.580(5) 12.830(5) 18.298(5) 98.489(5) 99.605(5) 91.102(5) 1733.6(4) 2 1.548 1.508 836.0 Mo-Kα 293 (2) ω-2θ 1.61 to 30.10 -10 ≤ h ≤10 -17 ≤ k ≤18 -25 ≤ l ≤24 34695 10219 [Rint= 0.0526] Full-matrix leastsquares on F2 0.954 R1= 0.0528, wR2= 0.1020 R1= 0.1684,
0.961 and -0.624
0.342 and -0.340
0.342 and -0.340
851285
851286
851284
No. of reflections measured No. of unique reflections Refinement method Goodness-of-fit on F2 Final R indices [I >2σ(I)] R indices (all data)
Largest diff. peak and hole (e.Å-3) CCDC number
wR2 = 0.1362
S3
Figure S1. Representative cyclic voltammogram of Cu(GTSC) complex (5 mM) in dry DMF (5 mL) at the scan rate of 10 mVs-1.
S4
Figure S2. Representative pictures are showing the in vitro DNA binding behavior of copper complexes measured by ethidium bromide displacement assay. A) Addition of Cu(GTSCHCl) complex to DNA-EB mixture in 400 mM NaClO4, with increasing concentration, quenching the fluorescence. A plot of F/F0 vs complex concentration at 598 nm was allowed to calculate the apparent binding constant from 50% quenching.
S5
Figure S3. Viscometric analysis illustrating the effect of increasing concentration of copper complexes on the relative viscosities of CT-DNA at 37 °C in 5 mM Tris-HCl buffer (5 mM NaCl) at pH 7.2. Ethidium bromide, a known DNA intercalator, and Hoechst 33258, a minor groove binder, were used as controls. All data shown with ± SD and are the average of three experiments.
S6
Figure S4. Effect of Cu(GTSC) complex on pBR322 DNA. Lane 1, DNA control in 20 mM HEPES buffer (pH 7.2); lane 2, DNA + 3.8% DMF; lane 3 and 4, DNA + [Cu(phen)2](NO3)2 (20 µM); lanes 4 to 16, DNA + Cu(GTSCHCl) with indicated concentration. Experiment was carried for 4 h at 37 °C and the percentage of DNA cleavage is given in the form of nicked circular (% NC). Reaction of copper complex with DNA was duplicated in each concentration.
S7
Figure S5. Treatment of Cu(BTSC) and Cu(BTSCH)ClO4 complexes with pBR322 DNA for 4 h at 37 °C at 100 µM concentration. Lane 1, DNA control in 20 mM HEPES buffer (pH 7.2); lane 2, DNA + 3.8% DMF; lane 3 and 4, DNA + [Cu(phen)2](NO3)2 (20 µM); lanes 5 and 6, DNA + Cu(BTSC) (100 µM) and lanes 7 and 8, DNA + Cu(BTSC)ClO4 (100 µM). Reaction of copper complex with DNA was duplicated in each concentration.
S8
Figure S6. Agarose gel electrophoresis of pBR322 DNA incubated with different concentrations of Cu(ATSC) and Cu(ATSCH)ClO4 for 4 h at 37 °C. Lane 1, DNA control in 20 mM HEPES buffer (pH 7.2); lane 2, DNA + 5 % DMF; lane 3 and 4, DNA + [Cu(phen)2](NO3)2 (20 µM); lanes 5 and 6, DNA + Cu(ATSC) (10 µM); lanes 7 and 8, DNA + Cu(ATSC) (100 µM) lanes 9 and 10, Cu(ATSC) (500 µM); lanes 11 and 12, Cu(ATSCH)ClO4 (10 µM); lanes 13 and 14, Cu(ATSCH)ClO4 (100 µM); lanes 15 and 16, Cu(ATSCH)ClO4 (500 µM). Reaction of copper complex with DNA was duplicated in each concentration.
S9
Figure S7. Representative agarose gel for the cleavage of pBR322 DNA by Cu(GTSCHCl) complex under anaerobic (argon) or aerobic conditions, to determine the mechanism of DNA cleavage. Lane 1, DNA control in 20 mM HEPES buffer (pH 7.2); lane 2, DNA + 3.8% DMF; lane 3, DNA + [Cu(Phen)2](NO3)2 (20 µM) (under argon); lane 4, DNA + [Cu(Phen)2](NO3)2 (20 µM) (under oxygen); reactions were carried under oxygen or argon atmosphere at the indicated concentration.
S10
Figure
S8.
DNA
relaxation
assay
for
topoisomerase
IIα
inhibition
by
copper
bis(thiosemicarbazone) complexes. The pBR322 plasmid (lane 1) was incubated with topoisomerase IIα in the absence (lane 2) or in the presence of 100 µM of complexes for 30 min at 37 °C (lane 5 to lane 14). Adriamycin (5 µM) was used a positive control (lane 3 and 4). Reactions of copper complex with DNA-topoisomerase IIα mixture were duplicated. The supercoiled and nicked circular DNA were indicated by I and II.
S11
In vitro and In vivo Anticancer Activity of Copper Bis(thiosemicarbazone) Complexes Duraippandi Palanimuthu,† Sridevi Vijay Shinde,‡ Kumaravel Somasundaram,*‡ and Ashoka G. Samuelson*†
†
Duraippandi Palanimuthu and Prof. A. G. Samuelson, Department of Inorganic and Physical
Chemistry, Indian Institute of Science, Bangalore, India 560012, Fax: (+91) 80-2360-1552. e-mail: [email protected]. ‡Sridevi Vijay Shinde and Prof. Kumaravel Somasundaram, Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India 560012, Fax: (+91) 80-2360-2697. e-mail: [email protected] Table of contents
Page
General scheme for the ligand synthesis
S2
Crystallographic data (Table S1)
S3
Cyclic voltammogram of Cu(GTSC)
S4
In vitro DNA binding study for Cu(GTSCHCl)
S5
Viscometric titration
S6
pBBR322 DNA cleavage by Cu(GTSC)
S7
pBBR322 DNA cleavage by Cu(ATSM), Cu(BTSC) and Cu(BTSCH)ClO4
S8
pBBR322 DNA cleavage by Cu(ATSC) Cu(ATSCH)ClO4
S9
pBBR322 DNA cleavage by Cu(GTSCHCl) under aerobic and anaerobic condition
S10
Topoisomerase IIα relaxation assay for copper complexes
S11
S1
LIGANDS
R1
R2
R3
BTSCH2
CH2-CH2
CH2-CH2
Ph
ATSCH2
CH2-CH2
CH2-CH2
CH3
GTSCH2
CH3
Ph
H
ATSMH2
CH3
H
CH3
Scheme S1. General scheme for the synthesis of ligands.
S2
Table S1. Selected crystallographic data for complexes Cu(BTSC) 0.5CH2Cl2, Cu(GTSC) and Cu(ATSC). Crystal identification
Cu(BTSC) 0.5CH2Cl2
Cu(GTSC)
Cu(ATSC)
Chemical Formula Molecular weight Crystal color Crystal dimension (mm) Crystal system Space group a (Å) b (Å) c (Å) α (deg) β(deg) γ(deg) V(Å3 ) Z Density(g.cm-3) µ(mm-1) F (000) Radiation (λ=0.71073Å) Temperature (K) Scan type θ range (deg) H ,k, l range
C49H54N12S4Cl2Cu2 1137.26 Dark brown 0.92 × 0.41 × 0.29 Monoclinic P21/c 15.1825(9) 11.7338(6) 29.6982(17) 90.000 100.534(4) 90.000 5201.5(5) 4 1.452 1.129 2352 Mo-Kα 293 (2) ω-2θ 1.36 to 25.50 -18 ≤ h ≤ 17 -14 ≤ k ≤ 14 -35 ≤ l ≤ 35 81895 9691 [Rint= 0.0900] Full-matrix leastsquares on F2 1.000 R1= 0.0543, wR2= 0.1380 R1= 0.1208, wR2= 0.1587
C18H18N6S2Cu 446.04 Dark brown 0.9 × 0.2 × 0.2 Orthorhombic Pbcn 31.3720(3) 7.5677(8) 7.9918(9) 90.000 90.000 90.000 1897.3(4) 4 1.561 1.387 916 Mo-Kα 293 (2) ω-2θ 1.30 to 25.50 -38 ≤ h ≤ 38 -9 ≤ k ≤ 9 -9≤ l ≤9 22949 1771 [Rint= 0.0695] Full-matrix leastsquares on F2 1.050 R1= 0.0363, wR2= 0.0903 R1= 0.0499, wR2= 0.0963
C28H48N12S4Cu2 808.10 Dark brown 0. 9 × 0.3 × 0.2 Triclinic P-1 7.580(5) 12.830(5) 18.298(5) 98.489(5) 99.605(5) 91.102(5) 1733.6(4) 2 1.548 1.508 836.0 Mo-Kα 293 (2) ω-2θ 1.61 to 30.10 -10 ≤ h ≤10 -17 ≤ k ≤18 -25 ≤ l ≤24 34695 10219 [Rint= 0.0526] Full-matrix leastsquares on F2 0.954 R1= 0.0528, wR2= 0.1020 R1= 0.1684,
0.961 and -0.624
0.342 and -0.340
0.342 and -0.340
851285
851286
851284
No. of reflections measured No. of unique reflections Refinement method Goodness-of-fit on F2 Final R indices [I >2σ(I)] R indices (all data)
Largest diff. peak and hole (e.Å-3) CCDC number
wR2 = 0.1362
S3
Figure S1. Representative cyclic voltammogram of Cu(GTSC) complex (5 mM) in dry DMF (5 mL) at the scan rate of 10 mVs-1.
S4
Figure S2. Representative pictures are showing the in vitro DNA binding behavior of copper complexes measured by ethidium bromide displacement assay. A) Addition of Cu(GTSCHCl) complex to DNA-EB mixture in 400 mM NaClO4, with increasing concentration, quenching the fluorescence. A plot of F/F0 vs complex concentration at 598 nm was allowed to calculate the apparent binding constant from 50% quenching.
S5
Figure S3. Viscometric analysis illustrating the effect of increasing concentration of copper complexes on the relative viscosities of CT-DNA at 37 °C in 5 mM Tris-HCl buffer (5 mM NaCl) at pH 7.2. Ethidium bromide, a known DNA intercalator, and Hoechst 33258, a minor groove binder, were used as controls. All data shown with ± SD and are the average of three experiments.
S6
Figure S4. Effect of Cu(GTSC) complex on pBR322 DNA. Lane 1, DNA control in 20 mM HEPES buffer (pH 7.2); lane 2, DNA + 3.8% DMF; lane 3 and 4, DNA + [Cu(phen)2](NO3)2 (20 µM); lanes 4 to 16, DNA + Cu(GTSCHCl) with indicated concentration. Experiment was carried for 4 h at 37 °C and the percentage of DNA cleavage is given in the form of nicked circular (% NC). Reaction of copper complex with DNA was duplicated in each concentration.
S7
Figure S5. Treatment of Cu(BTSC) and Cu(BTSCH)ClO4 complexes with pBR322 DNA for 4 h at 37 °C at 100 µM concentration. Lane 1, DNA control in 20 mM HEPES buffer (pH 7.2); lane 2, DNA + 3.8% DMF; lane 3 and 4, DNA + [Cu(phen)2](NO3)2 (20 µM); lanes 5 and 6, DNA + Cu(BTSC) (100 µM) and lanes 7 and 8, DNA + Cu(BTSC)ClO4 (100 µM). Reaction of copper complex with DNA was duplicated in each concentration.
S8
Figure S6. Agarose gel electrophoresis of pBR322 DNA incubated with different concentrations of Cu(ATSC) and Cu(ATSCH)ClO4 for 4 h at 37 °C. Lane 1, DNA control in 20 mM HEPES buffer (pH 7.2); lane 2, DNA + 5 % DMF; lane 3 and 4, DNA + [Cu(phen)2](NO3)2 (20 µM); lanes 5 and 6, DNA + Cu(ATSC) (10 µM); lanes 7 and 8, DNA + Cu(ATSC) (100 µM) lanes 9 and 10, Cu(ATSC) (500 µM); lanes 11 and 12, Cu(ATSCH)ClO4 (10 µM); lanes 13 and 14, Cu(ATSCH)ClO4 (100 µM); lanes 15 and 16, Cu(ATSCH)ClO4 (500 µM). Reaction of copper complex with DNA was duplicated in each concentration.
S9
Figure S7. Representative agarose gel for the cleavage of pBR322 DNA by Cu(GTSCHCl) complex under anaerobic (argon) or aerobic conditions, to determine the mechanism of DNA cleavage. Lane 1, DNA control in 20 mM HEPES buffer (pH 7.2); lane 2, DNA + 3.8% DMF; lane 3, DNA + [Cu(Phen)2](NO3)2 (20 µM) (under argon); lane 4, DNA + [Cu(Phen)2](NO3)2 (20 µM) (under oxygen); reactions were carried under oxygen or argon atmosphere at the indicated concentration.
S10
Figure
S8.
DNA
relaxation
assay
for
topoisomerase
IIα
inhibition
by
copper
bis(thiosemicarbazone) complexes. The pBR322 plasmid (lane 1) was incubated with topoisomerase IIα in the absence (lane 2) or in the presence of 100 µM of complexes for 30 min at 37 °C (lane 5 to lane 14). Adriamycin (5 µM) was used a positive control (lane 3 and 4). Reactions of copper complex with DNA-topoisomerase IIα mixture were duplicated. The supercoiled and nicked circular DNA were indicated by I and II.
S11
