1

Supporting Information

Synthesis of 2-acetylpyridine-N-substituted thiosemicarbazonates

of copper(II) with high antimicrobial activity against methicillin

resistant S.aureus, K. pneumonia 1 and C. albicans

Mani Kaushal,aTarlok S. Lobana*a, LovedeepNim,bRituBala,*aDaljit S. Arora,b Isabel

Garcia-Santos,c Courtney E. Duffdand Jerry P. Jasinskid

*Corresponding author (Tarlok S. Lobana) :tarlokslobana@yahoo.co.in

S1. Ligand details

Preparation of 2-acetylpyridine-N1-thiosemicarbazone (HL3H)

The solution of thiosemicarbazide (1 g, 10.9 mmol) in methanol was refluxed for 30 minutes

at 40˚C. To this solution, 2-acetylpyridine ketone (1.23 mL, 10.9 mmol) was added along

with 2-3 drops of glacial acetic acid and resulting solution was refluxed for seven hours.

After that, the solution was left for crystallization at room temperature. Slow evaporation of

the solution yielded orange crystals in 2- 3 days. Yield : 1.95g, 92%, m.p. 108-110ºC. 1H

NMR data (, ppm; DMSO-d6), 10.30 (s, 1H, N2H); 8.54 (dq, 1H, C7H); 8.39 (dt, 1H, C4H);

8.12 (s, 1H, N1H); 7.76 (ddd, 1H, C5H); 7.35 (m, 1H, C6H); 2.34 (s, 3H, CH3).Electronic

absorption spectrum, DMSO, λmax/nm, ε/L mol-1cm-1: [10-5M] 321 (8.53 × 104).

Preparation of 2-acetylpyridine-N1-methyl thiosemicarbazone (HL3Me)

The solution of 4-methyl-3-thiosemicarbazide (1 g, 9.5 mmol) in methanol was refluxed for

30 minutes at 40˚C. To this solution, 2-acetylpyridine ketone (1.07 mL, 9.5 mmol) was added

along with 2-3 drops of glacial acetic acid and resulting solution was refluxed for seven

hours. After that, the solution was left for crystallization at room temperature. Slow

evaporation of the solution yielded pale yellow crystalline product in 2- 3 days. Yield : 1.53g,

77%, m.p. 122-124 ºC. 1H NMR data (, ppm; DMSO-d6), 10.32 (s, 1H, N2H); 8.59 (d, 1H,

C7H); 8.53 (dq, 1H, N1H); 8.37 (dt, 1H, C4H); 7.78 (ddd, 1H, C5H); 7.35 (m, 1H, C6H); 3.01

(d, 3H, N1H-CH3); 2.34 (s, 3H, CH3).Electronic absorption spectrum, DMSO, λmax/nm, ε/L

mol-1cm-1: [10-5M] 320 (9.6 × 104).

Electronic Supplementary Material (ESI) for New Journal of Chemistry.This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019

2

Preparation of 2-acetylpyridine-N1-ethyl thiosemicarbazone (HL3Et)

The solution of 4-ethyl-3-thiosemicarbazide (1 g, 8.4mmol) in methanol was refluxed

for 30 minutes at 40˚C. To this, 2-acetylpyridine ketone (0.94 mL, 8.4mmol) was added

along with 2-3 drops of glacial acetic acid and resulting solution was refluxed for seven

hours. After that, the solution was left for crystallization at room temperature. Slow

evaporation of the solution yielded pale yellow crystalline product in 2- 3 days. Yield :

1.48g, 80%, m.p. 96-98 ºC. 1H NMR data (, ppm; DMSO-d6), 10.22 (s, 1H, N2H); 8.65 (t,

1H, N1H ); 8.54 (dq, 1H, C7H); 8.36 (dt, 1H, C4H); 7.78 (ddd, 1H, C5H); 7.35 (m, 1H, C6H);

3.59 (p, 2H, N1H-CH2); 2.34 (s, 3H, CH3); 1.12 (t, 3H, N1H-CH3). Electronic absorption

spectrum, DMSO, λmax/nm, ε/L mol-1cm-1: [10-5M] 319 (8.21 × 104).

Preparation of 2-acetylpyridine-N1-phenyl thiosemicarbazone (HL3Ph)

The solution of 4-phenyl-3-thiosemicarbazide (1 g, 5.9 mmol) in methanol was refluxed

for 30 minutes at 40˚C. To this, 2-acetylpyridine ketone (0.67 mL, 5.9 mmol) was added

along with 2-3 drops of glacial acetic acid and resulting solution was refluxed for seven

hours. After that, the solution was left for crystallization at room temperature. Slow

evaporation of the solution yielded pale yellow crystalline product in 2- 3 days. Yield : 1.27g,

79%, m.p. 98-100 ºC. 1H NMR data (, ppm; DMSO-d6), 10.64 (s, 1H, N2H); 10.17 (s, 1H,

N1H); 8.56 (dq, 1H, C7H); 8.50 (dt, 1H, C4H); 7.78 (ddd, 1H, C5H); 7.32-7.50 (m, 5H, N1H-

Ph); 7.19 (ddd, 1H, C6H); 2.42 (s, 3H, CH3).Electronic absorption spectrum, DMSO,

λmax/nm, ε/L mol-1cm-1: [10-5M] 322 (9.67 × 104).

S2.Electronic absorption spectra of ligands and complexes

Fig S1: UV Spectra of ligands (HL3R; R = H, Me, Et, Ph)

3

Fig S2: The electronic absorption spectra of complexes 1-12in 10-3 M solution

4

Fig S3: The electronic absorption spectra of complexes 1-12in 10-5 M solution

Magnetic field(gauss)

3000 3100 3200 3300 3400 3500

Inte

nsity

(a.u

)

-12000

-10000

-8000

-6000

-4000

-2000

0

2000

4000

6000

Fig. S4: EPR spectrum of [CuII(N,N,S-L3H)Cl] 1

5

Magnetic field(gauss)

2600 2800 3000 3200 3400 3600

Inte

nsity

(a.u

.)

-30000

-20000

-10000

0

10000

20000

30000

40000

Fig. S5: EPR spectrum of [CuII(N,N,S-L3Me)Cl] 2

Magnetic field(gauss)

2800 3000 3200 3400 3600 3800 4000

Inte

nsity

(a.u

.)

-8000

-6000

-4000

-2000

0

2000

4000

Fig. S6: EPR spectrum of [CuII(N,N,S-L3Ph)Cl] 4

6

Magnetic field(gauss)

3000 3100 3200 3300 3400 3500

Inte

nsity

(a.u

.)

-16000

-14000

-12000

-10000

-8000

-6000

-4000

-2000

0

2000

4000

6000

Fig. S7: EPR spectrum of [CuII(N,N,S-L3H)Br] 5

Magnetic field(gauss)

2000 2500 3000 3500 4000

Inte

nsity

(a.u

.)

-10000

-8000

-6000

-4000

-2000

0

2000

4000

6000

8000

10000

Fig. S8 EPR spectrum of [CuII(N,N,S-L3Me)Br] 6

7

Magnetic field(gauss)

2400 2600 2800 3000 3200 3400 3600 3800 4000

Inte

nsity

(a.u

.)

-25000

-20000

-15000

-10000

-5000

0

5000

10000

15000

Fig. S9 EPR spectrum of [CuII(N,N,S-L3Ph)Br] 8

Magnetic field(gauss)

1800 2000 2200 2400 2600 2800 3000 3200

Inte

nsity

(a.u

.)

-10000

-8000

-6000

-4000

-2000

0

2000

4000

6000

8000

10000

Fig. S10 EPR spectrum of [CuII(N,N,S-L3H)I] 9

8

Magnetic Field(Gauss)

2000 3000 4000 5000

Inte

nsity

(a.u

)

-40000

-20000

0

20000

40000

Fig. S11 EPR spectrum of [CuII(N,N,S-L3Me)I] 10

Magnetic field(gauss)

2800 3000 3200 3400 3600 3800

Inte

nsity

(a.u

)

-60000

-40000

-20000

0

20000

40000

60000

Fig. S12 EPR spectrum of [CuII(N,N,S-L3Et)I] 11

9

S3. ESI-mass studies

270.01371+

271.01731+

272.0113

273.01591+

274.01401+

+MS, 0.0-0.3min #1-18

269.99951+

271.00191+

271.99751+

272.99991+

273.99441+

CuC₉H₁₁N₄S, M, 269.9995

0

20

40

60

80

100

Intens.[%]

0

20

40

60

80

100

[%]270 271 272 273 274 m/z

270 271 272 273 274 m/z

Fig S13:The mass spectrum of fragment, [Cu(L3Me)]+ {chemical formula: CuC9H11N4S; m/z =

calc 269.99, obsd 270.01} with an isotopic pattern (complex 2)

575.00481+

576.00861+

577.00381+

578.00491+

579.00161+

580.01001+

581.00421+

+MS, 0.0-0.3min #1-18

575.97621+

576.97861+

577.97411+

578.97641+

579.97191+

580.97401+

581.96971+

Cu₂C₁₈H₂₂N₈S₂Cl, M+nH, 575.9762

0

10

20

30

40

Intens.[%]

0

20

40

60

80

100

[%]575 576 577 578 579 580 581 m/z

576 577 578 579 580 581 582 m/z

10

Fig S14: The mass spectrum of fragment,[Cu(L3Me)2CuCl + H]+{chemical formula:

Cu2C18H23N8S2Cl; m/z = calc 575.97, obsd 575.00} with an isotopic pattern (complex 2 )

284.01671+

285.02191+

286.01621+

287.01851+

288.01591+

+MS, 0.0-0.4min #2-21

284.01511+

285.01761+

286.01321+

287.01561+

288.01031+

C₁₀H₁₃CuN₄S, M, 284.0151

0

10

20

30

Intens.[%]

0

20

40

60

80

100

[%]284 285 286 287 288 289 m/z

284 285 286 287 288 289 m/z

Fig S15:The mass spectrum of fragment,[Cu(L3Et)]+{chemical formula: CuC10H13N4S; m/z =

calc284.01, obsd284.01} with anisotopic pattern (complex 3 )

603.00021+

603.99781+

604.99851+

605.99841+

606.99591+

608.00231+

608.99391+

610.00111+

+MS, 0.0-0.4min #2-21

602.99971+

604.00211+

604.99761+

605.99991+

606.99551+

607.99761+

608.99331+

C₂₀H₂₆Cu₂N₈S₂Cl, M, 602.9997

0

5

10

15

20

25

30

Intens.[%]

0

20

40

60

80

100

[%]603 604 605 606 607 608 609 610 611 m/z

603 604 605 606 607 608 609 610 611 m/z

11

Fig S16:The mass spectrum of fragment,[Cu(L3Et)2CuCl]+{chemical formula:

Cu2C20H26N8S2Cl; m/z = calc602.99, obsd603.00} with an isotopic pattern (complex 3 )

333.01581+

334.01981+

335.01561+

336.01391+

337.01411+

+MS, 0.0-0.5min #2-28

333.02301+

334.02561+

335.02111+

336.02371+

337.01911+

CuC₁₄H₁₃N₄S, M+nH, 333.0230

0

10

20

30

40

50

Intens.[%]

0

20

40

60

80

100

[%]333 334 335 336 337 m/z

333 334 335 336 337 m/z

Fig S17:The mass spectrum of fragment,[Cu(L3Ph) + H]+{chemical formula: CuC14H14N4S;

m/z = calc333.02,obsd333.01} with an isotopic pattern (complex 4)

12

700.99721+

701.99981+

702.99581+

703.99871+

704.99371+

705.99801+

+MS, 0.0-0.5min #2-28

700.00751+

701.01021+

702.00561+

703.00801+

704.00371+

705.00581+

Cu₂C₂₈H₂₆N₈S₂Cl, M+nH, 700.0075

0

10

20

30

40

Intens.[%]

0

20

40

60

80

100

[%]701 702 703 704 705 706 m/z

700 701 702 703 704 705 m/z

Fig S18:The mass spectrum of fragment,[Cu(L3Ph)2CuCl + H]+{chemical formula:

Cu2C28H27N8S2Cl; m/z = calc700.00,obsd700.99} with an isotopic pattern (complex 4)

255.98721+

256.98961+

257.98591+

258.98871+

259.98851+

+MS, 0.0-0.6min #2-36

255.98381+

256.98611+

257.98191+

258.98421+

C₈H₉CuN₄S, M, 255.9838

0

20

40

60

80

100

Intens.[%]

0

20

40

60

80

100

[%]256 257 258 259 260 261 262 m/z

256 257 258 259 260 261 262 m/z

13

Fig S19:The mass spectrum of fragment, [Cu(L3H)]+{chemical formula: CuC8H9N4S; m/z =

calc255.98, obsd255.98} with an isotopic pattern (complex 5 )

590.88411+

591.88591+

592.88191+

593.88551+

594.88071+

595.88241+

596.87771+

+MS, 0.0-0.6min #2-36

590.88661+

591.88881+

592.88461+

593.88681+

594.88271+

595.88471+ 596.8807

1+

597.88251+

C₁₆H₁₈Cu₂BrN₈S₂, M, 590.8866

0

5

10

15

20

Intens.[%]

0

20

40

60

80

100

[%]591 592 593 594 595 596 597 598 m/z

591 592 593 594 595 596 597 598 m/z

Fig S20:The mass spectrum of fragment,[Cu(L3H)2CuBr]+{chemical formula:

Cu2C16H18N8S2Br; m/z = calc590.88, obsd590.88} with an isotopic pattern (complex 5 )

270.00091+

271.00601+

271.99921+

273.00281+

273.99931+

274.91761+

+MS, 0.0-0.3min #2-19

269.99951+

271.00191+

271.99751+

272.99991+

273.99441+

C₉H₁₁CuN₄S, M, 269.9995

0

20

40

60

80

100

Intens.[%]

0

20

40

60

80

100

[%]270 271 272 273 274 275 276 m/z

270 271 272 273 274 275 276 m/z

14

FigS21:The mass spectrum of fragment,[Cu(L3Me)]+{chemical formula: CuC9H11N4S; m/z =

calc269.99, obsd270.00} with an isotopic pattern (complex 6 )

618.91751+

619.91681+

620.91551+

621.92071+

622.91321+

623.91251+

624.91201+

625.90001+

+MS, 0.0-0.3min #2-19

618.91791+

619.92021+

620.91591+

621.91821+

622.91401+

623.91621+

624.91211+

625.91391+

C₁₈H₂₂Cu₂BrN₈S₂, M, 618.9179

0

10

20

30

Intens.[%]

0

20

40

60

80

100

[%]619 620 621 622 623 624 625 626 m/z

619 620 621 622 623 624 625 626 m/z

Fig S22:The mass spectrum of fragment,[Cu(L3Me)2CuBr]+{chemical formula: Cu2C18H22N8S2Br; m/z = calc618.91, obsd618.91} with isotopic pattern (complex 6)

284.0166

285.1008

286.01731+

287.02111+

288.02671+

+MS, 0.0-0.3min #2-17

284.01511+

285.01761+

286.01321+

287.01561+

288.01031+

C₁₀H₁₃CuN₄S, M, 284.0151

0

20

40

60

80

100

Intens.[%]

0

20

40

60

80

100

[%]284 285 286 287 288 289 290 m/z

284 285 286 287 288 289 290 m/z

15

FigS23:The mass spectrum of fragment, [Cu(L3Et)]+{chemical formula: CuC10H13N4S; m/z = calc284.01, obsd284.01} with an isotopic pattern (complex 7)

646.97881+

647.97281+

648.96381+

649.96481+

650.95241+

651.95251+

+MS, 0.0-0.3min #2-17

646.94921+

647.95161+

648.94721+

649.94961+

650.94541+

651.94761+

C₂₀H₂₆Cu₂N₈S₂Br, M, 646.9492

0

10

20

30

40

50

Intens.[%]

0

20

40

60

80

100

[%]647 648 649 650 651 652 m/z

647 648 649 650 651 652 m/z

Fig S24:The mass spectrum of fragment, [Cu(L3Et)2CuBr]+{chemical formula: Cu2C20H26N8S2Br; m/z = calc646.94, obsd646.97} with an isotopic pattern (complex 7)

16

332.00151+

333.00221+

333.99961+

335.00221+

335.99521+

+MS, 0.0-0.2min #2-11

332.01511+

333.01781+

334.01331+

335.01581+

336.01121+

C₁₄H₁₃CuN₄S, M, 332.0151

0

10

20

30

Intens.[%]

0

20

40

60

80

100

[%]332 333 334 335 336 337 338 m/z

332 333 334 335 336 337 338 m/z

Fig S25:The mass spectrum of fragment, [Cu(L3Ph)]+{chemical formula: CuC14H13N4S; m/z = calc332.01, obsd332.00} with an isotopic pattern (complex 8)

743.4870

744.4932

745.4862

746.4876 747.4849

748.4898

+MS, 0.0-0.3min #2-17

743.95701+

744.95971+

745.95521+

746.95771+

747.95351+

748.95581+

749.95201+

750.95371+

C₂₈H₂₆Cu₂BrN₈S₂, M+nH, 743.9570

0.0

0.2

0.4

0.6

0.8

Intens.[%]

0

20

40

60

80

100

[%]743 744 745 746 747 748 749 750 m/z

743 744 745 746 747 748 749 750 751 m/z

Fig S26:The mass spectrum of fragment,[Cu(L3Ph)2CuBr + H]+{chemical formula: Cu2C28H27N8S2Br; m/z = calc743.95, obsd743.48} with an isotopic pattern (complex 8)

17

256.22431+

257.22871+

258.22381+

259.22821+

260.22991+

+MS, 0.0-0.3min #2-16

256.99171+

257.99391+

258.98971+

259.99201+

CuC₈H₉N₄S, M+nH, 256.9917

0

20

40

60

80

100

Intens.[%]

0

20

40

60

80

100

[%]256 257 258 259 260 m/z

257 258 259 260 261 m/z

Fig S27:The mass spectrum of fragment,[Cu(L3H) + H]+{chemical formula: CuC8H10N4S; m/z = calc256.99, obsd256.22} with an isotopic pattern (complex 9)

639.2521

640.2669

641.2476

643.2429

+MS, 0.0-0.3min #2-16

639.88051+

640.88281+

641.87871+

642.88081+

643.87671+

Cu₂C₁₆H₁₈N₈S₂I, M+nH, 639.8805

0

1

2

3

4

5

Intens.[%]

0

20

40

60

80

100

[%]639 640 641 642 643 m/z

640 641 642 643 644 m/z

FigS28:The mass spectrum of fragment,[Cu(L3H)2CuI + H]+{chemical formula:

18

Cu2C16H19N8S2I; m/z = calc639.88, obsd639.25} with an isotopic pattern (complex 9)

269.98851+

270.99071+

271.98621+

272.98851+

273.98311+

+MS, 0.1-0.4min #2-21

269.99951+

271.00191+

271.99751+

272.99991+

273.99441+

C₉H₁₁CuN₄S, M, 269.9995

0

20

40

60

80

100

Intens.[%]

-0.04

-0.02

0.00

0.02

0.04

[%]269 270 271 272 273 274 275 m/z

269 270 271 272 273 274 275 m/z

FigS29:The mass spectrum of fragment,[Cu(L3Me)]+{chemical formula: CuC9H11N4S; m/z = calc269.99, obsd269.98} with an isotopic pattern (complex 10)

19

666.88491+

667.88891+

668.88291+

669.88621+

670.8819

671.8846672.8814

+MS, 0.1-0.4min #2-21

666.90401+

667.90641+

668.90221+

669.90441+ 670.9003

1+

671.90211+

672.89821+

C₁₈IH₂₂Cu₂N₈S₂, M, 666.9040

0

5

10

15

Intens.[%]

0

20

40

60

80

100

[%]667 668 669 670 671 672 673 m/z

667 668 669 670 671 672 673 m/z

FigS30:The mass spectrum of fragment,[Cu(L3Me)2CuI]+{chemical formula: Cu2C18H22N8S2I; m/z = calc666.90, obsd666.88} with an isotopic pattern (complex 10)

284.01011+

285.01331+

286.00871+

287.01111+

288.00921+

+MS, 0.0-0.3min #1-20

284.01511+

285.01761+

286.01321+

287.01561+

288.01031+

C₁₀H₁₃CuN₄S, M, 284.0151

0

20

40

60

80

100

Intens.[%]

0

20

40

60

80

100

[%]284 285 286 287 288 289 290 291 m/z

284 285 286 287 288 289 290 m/z

Fig S31:The mass spectrum of fragment,[Cu(L3Et)]+{chemical formula: CuC10H13N4S; m/z = calc284.01, obsd284.01} with an isotopic pattern (complex 11)

20

694.91221+

695.92021+

696.91221+

697.92091+ 698.9104

1+

699.92231+

700.90511+

+MS, 0.0-0.3min #1-20

694.93531+

695.93781+

696.93351+

697.93581+ 698.9317

1+

699.93371+

700.92991+

C₂₀H₂₆Cu₂N₈IS₂, M, 694.9353

0

5

10

15

Intens.[%]

0

20

40

60

80

100

[%]695 696 697 698 699 700 701 702 703 m/z

695 696 697 698 699 700 701 702 703 m/z

FigS32:The mass spectrum of fragment, [Cu(L3Et)2CuI]+{chemical formula: Cu2C20H26N8S2I; m/z = calc694.93, obsd694.91} with an isotopic pattern (complex 11)

332.31491+

333.31841+

334.31421+

335.31691+

336.31321+

+MS, 0.0-0.3min #2-17

332.01511+

333.01781+

334.01331+

335.01581+

336.01121+

C₁₄H₁₃CuN₄S, M, 332.0151

0

20

40

60

80

100

Intens.[%]

-0.04

-0.02

0.00

0.02

0.04

[%]332 333 334 335 336 337 338 m/z

331 332 333 334 335 336 337 m/z

Fig. S33. The mass spectrum of fragment, [Cu(L3Ph)]+ {chemical formula: CuC14H13N4S; m/z = calc 332.01, obsd 332.31} with an isotopic pattern (complex 12)

21

791.42141+

792.42551+

793.42161+

794.42521+

795.42091+

796.42281+

+MS, 0.0-0.3min #2-17

791.94311+

792.94581+

793.94151+

794.94391+

795.94011+

796.94191+

Cu₂C₂₈H₂₆N₈S₂I, M+nH, 791.9431

0

5

10

15

20

25

Intens.[%]

0

20

40

60

80

100

[%]791 792 793 794 795 796 m/z

792 793 794 795 796 797 m/z

Fig. S34. The mass spectrum of fragment, [Cu(L3Ph)2CuI + H]+ {chemical formula: Cu2C28H27N8S2I; m/z = calc 791.94, obsd 791.42} with an isotopic pattern (complex 12)

S4.Test organisms and inoculum preparation

The reference strains of bacteria and yeasts were obtained from Microbial Type Culture

Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India while

the clinical isolate methicillin resistant Staphylococcus aureus (MRSA) was obtained from

Post graduate Institute of Medical Education and Research, (PGIMER), Chandigarh, India.

Reference strains included Gram positive bacteria: Staphylococcus aureus (MTCC-740),

Staphylococcus epidermidis (MTCC-435), Gram negative bacteria: Klebisella pneumoniae

(MTCC-530), Escherichia coli (MTCC-119), Salmonella typhimurium 1(MTCC-98),

Salmonella typhimurium 2(MTCC-1251), Shigella flexneri (MTCC-1457), and one yeast

strain: Candida albicans (MTCC-227). A loopful of isolated bacterial and yeast colonies

were inoculated into 5 mL of their respective medium and incubated at 37 °C and 25 °C,

22

respectively, for 4 h. This was used as inoculums after adjusting the turbidity as per 0.5

McFarland turbidity standard. This turbidity is equivalent to approximately 1 to 2 x 108

colony forming units per mL (CFU mL-1). The inoculums thus prepared were used for further

testing.

S4.1 Antimicrobial screening

A 100 µL of activated test organism (prepared as above) was inoculated onto suitable

medium plates by spread plate method. Wells measuring 8 mm in diameter were cut out in

the medium using sterilized stainless steel borer. Each well was filled with 0.1 mL of test

complex dissolved in DMSO and kept for incubation in an upright position for 18 – 24 h.

Sensitivity was measured in terms of diameter of the resultant zone of inhibition. Any

organism with a clear zone of inhibition ≤ 12mm was considered to be resistant to the

compound.

S4.2 Minimum inhibitory concentration (MIC)

Minimum inhibitory concentration of the selected complex compounds dissolved in

DMSO was worked out by the agar dilution method for their antimicrobial activity against

the sensitive microorganisms. A stock solution of a complex under investigation of

concentration (10 mg mL-1) was prepared and incorporated into Muller Hinton agar medium

for bacteria and yeast malt extract medium for yeast. The final concentration of the

compound in the medium containing plates ranged from (0.005–1 mg mL-1). These plates

were then inoculated with 0.1 mL of the activated bacterial and yeast strains by streaking

with a sterile tooth pick. The plates were incubated at 37ºC for bacteria and 25 ºC for yeast

for 24 h each. The minimum concentration of the extract causing complete inhibition of the

23

microbial growth was taken as MIC. The results were compared with that of control (i.e.

DMSO).

S4.3 Cellular toxicity testing using MTT assay

The biosafety of the test compounds was checked by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,

5-diphenyl tetrazolium bromide] assay [67]. Ten milliliter of sheep blood was taken

alongwith 3 mL of Alsever’s solution (anticoagulant) and transferred to sterile centrifuge

tubes. The blood was centrifuged at 16,000 rpm at room temperature (25°C) for 20 min so as

to separate the plasma from the cellular portion of the blood. The supernatant (plasma) was

discarded and to cellular part of blood, 6 mL phosphate buffer saline (1X PBS) was added

followed by centrifugation. The blood cells were washed thrice with 1X PBS by

centrifugation and the cellular pellet was re-suspended in 6 mL of PBS. Various dilutions of

these cells were prepared using PBS and counted with the help of a hemocytometer under a

light microscope so as to obtain cellular density equivalent to 1 × 105 cells/mL. One hundred

microlitre (100 µL) of this diluted suspension was added in each microtiter plate well and

incubated at 37 °C for overnight. The supernatant was removed carefully (discarded) and 200

µL of the sample under study (contains 10 mg/mL) was added to each well and incubated

further for 24 h. Supernatant was removed again and added 20 µL MTT solution (5 mg/mL)

to each well and incubated further for 3.5 h at 37 °C on orbital shaker at 60 rpm. After this

incubation, the supernatant was removed without disturbing the cells and 50 µL of DMSO

was added to each well to dissolve the formazan crystals. The absorbance was measured at

590 nm using an automated microplate reader (Biorad 680-XR, Japan). The well to which

DMSO in place of sample was added (untreated cells) served as control. Reduction of MTT

can occur only in metabolically active cells, as MTT is converted into formazan crystals.

The the viability of the cells can be calculated as follows:

24

% Cell viability = (OD of Treated / OD of Control) x 100.

4.4.Time kill assay

The time kill assay for the selected purified compounds was performed by the viable

cell count method (VCC). A stock solution of (1 mg mL-1 ) was prepared. Five mL of 4 h

grown inoculums was adjusted to 0.5 McFarland standards and serially diluted to 10-3 with

the respective double strength broth medium. An equal volume of each diluted inoculums and

the extract to be tested were mixed at their respective predetermined MIC values and

incubated at the respective temperature of 25°C for yeast and 37°C for bacteria. At different

time intervals viz. 0, 1, 2, 3, 4, upto 24 h, and 0.1 mL of the mixed suspension was spread on

suitable agar plates in duplicate and incubated for 24 h at suitable temperature. The mean

number of colonies was determined and compared with that of control, in which the

compounds were replaced with DMSO.

Table 1S. IR Spectral bands of the complexes 1-12

Complexes ν(N1-H){ν(N2-H)}

(C-H) Bands (I, II, III, IV)

Other bands

[CuII(L3H)Cl] 1

3446m 3292m

3164m 3067w

1493m (I)1320m (II)1097sb (III)786m (IV)

1632s, 1597m, 1560w, 1448s , 1363m, 1182s, 1160m,899vw, 810m , 736w, 647w, 615w, 567vw, 500w, 467m,

[CuII(L3Me)Cl] 2

3410m3312m

3069w, 3026m, 2994w,2936w, 2899w

1505s (I)1301s (II)1048m (III)782m (IV)

1596m, 1563m, 1527s, 1458m, 1398s, 1365s, 1331m, 1244m, 1187s, 1160m, 1143m, 1105s, 1077s, 823w, 744vw, 724vw, 649w, 621w, 564w, 471m, 413w

[CuII(L3Et)Cl] 3

3414m 3068w, 3024w, 2969w, 2929w, 2888w

1496s (I)1293w (II)1082s (III)785m (IV)

1628w, 1591w, 1562w, 1446s, 1371m, 1351w, 1328m, 1261m, 1235m, 1175m, 1159m, 832w, 806w, 745w, 720w, 663w, 648w, 614w, 565w, 466m, 412w

[CuII(L3Ph)Cl] 4

3320s 3124w, 3066w

1503s (I)1295m (II)1046m (III)

1597m, 1539m, 1458s, 1432s, 1368m, 1317m, 1249m, 1189m, 1158m, 1106m, 1086m, 898w,

25

773m (IV) 852w, 825w, 796w, 751m, 691w, 648w, 609w, 587w, 567w, 507w, 472w, 413w

[CuII(L3H)Br] 5

3447s3292m

3164m, 3067w

1492m (I)1318m (II)1103s (III)782m (IV)

1630s, 1596m, 1560w, 1444s, 1362m, 1261w, 1181s, 1159m, 808w, 734w, 646w, 613w, 567w, 498w, 465w, 430w

[CuII(L3Me)Br] 6

3319s 3075vw, 3017vw, 2953vw

1500m (I)1290w (II)1084s (III)770m (IV)

1628w, 1600w, 1564w, 1521s, 1459s, 1401s, 1371m, 1336w, 1249m, 1195s, 1162m, 827w, 739w, 646w, 614w, 562w, 469m, 440w

[CuII(L3Et)Br] 7

3345s 3064w, 3028w, 2968w2926w, 2867w

1504s (I)1294w (II)1082m (III) 779m (IV)

1635w, 1600w, 1561w, 1439s, 1384m, 1373m, 1351w, 1326m, 1269w, 1241s, 1178s, 1161m, 1128m, 1048m, 997w, 830w, 804w, 741w, 665w, 647w, 610w, 534w, 475w, 410w

[CuII(L3Ph)Br] 8

3311s 3124w, 3067m2912vw

1496s (I)1316s (II)1045w (III)775m (IV)

1598s, 1562m, 1535s, 1456s, 1431s, 1370m, 1248m, 1192m, 1156s, 1106m, 1083m, 996w, 895w, 850w, 825w,746m, 689m, 668w, 647w, 612w, 588w, 563w, 504w, 442w, 413w

[CuII(L3H)I] 9 3427m3289m

3148m, 3072m

1492s (I)1313m (II)1059m (III)779m (IV)

1628s, 1596s, 1560m, 1441s, 1358m, 1259w, 1178s, 1126m, 1101m, 1038m , 890vw, 807w, 732m, 674w, 644w, 614w, 565w, 494w, 463w, 416w

[CuII(L3Me)I] 10

3328s 3075vw, 2951w

1517s (I)1284m (II)1082m (III)767m (IV)

1631w, 1598w, 1564w, 1457s, 1400s, 1370m, 1335m, 1247m, 1194s, 1161s, 884vw, 825w, 737w, 643w, 612w, 551m, 472m, 438w, 413w

[CuII(L3Et)I] 11

3341s 3099vw, 3071vw, 2985w, 2972w, 2919w 2874w

1501s (I)1324m (II)1016w (III)776m (IV)

1598m, 1559vw, 1479m, 1441s, 1390m, 1372m, 1351m, 1295m, 1276w, 1241s, 1172s, 1158s, 1130m, 1107m, 10871m, 1044m, 999w, 894w, 830m, 807m, 742w, 665w, 646w, 636w, 566w, 531wm, 488w, 410w.

[CuII(L3Ph)I] 12

3342m3312m

3120w, 3069w,

1498s (I)1315m (II)

1596m, 1564w, 1534m, 1459s, 1433s, 1369m, 1251m, 1187m,

26

2911vw 1018w (III)747m (IV)

1171m,1156m, 1109w, 1084w, 1074w, 1045w, 996w, 895w, 849w, 823w, 795w, 775w, 689w, 647w, 625w, 606w, 585w, 564w, 505w, 442w, 412w

HL3H 3374s, 3261s{3185s}

3064m, 2983m

1502 s (I)1246 s (II)997 m (III)783m (IV)

1609s, 1584m, 1564 m, 1467s, 1431s, 1367m, 1316 m, 1151m, 1110s, 1086s, 1052m, 969w, 898vw, 849 m, 837m, 760w, 743w, 650m, 612w, 563m, 464w, 418w

HL3Me 3288s{3240s}

3064w, 3044w, 2967w,2943w, 2900w

1498s (I)1232s (II)990w (III)780m (IV)

1608vw, 1578m, 1539s, 1462s, 1434s, 1409m, 1364w, 1294m, 1149m, 1115m, 1074m, 1050m, 1038m, 967w, 904w, 834w, 743w, 683w, 644w, 622w, 575w, 542w

HL3Et 3349s, 3274m{3211s}

3055w, 2978w, 2932w2868w

1504s (I)1221s (II)992w (III)783m (IV)

1614vw, 1580m, 1561w, 1534s, 1471s, 1436m, 1368w, 1343w, 1311m, 1298m, 1155m, 1103s, 1084s, 932w, 903w, 824w, 748w, 668w, 640w, 619w, 588m, 552m, 470w, 432w

HL3Ph 3300s{3242m}

3050w, 2973w

1489s (I)1262m (II)1001w (III)783m (IV)

1583m, 1523s, 1467s, 1440s, 1360m, 1303m, 1189s, 1149m, 1113m, 1070m, 1026m, 971w, 928w, 896w, 844vw, 800w, 764w, 740m, 690m, 654w, 637w, 619w, 584w, 555m, 489w, 468w

*Note : Band I has contributions from δNH + δC-H + ʋC=N; Band II has contributions from ʋC-N + δNH + δC-H + ʋC=S; Band III has contributions from ʋC-N + ʋC-S; Band IV has contributions from ʋC-S

Table 2S: Important bond parameters for complexes 4-7 and 9-11

4 (Cl) 5 (Br) 6 (Br) 7 (Br)

5A 5B 7A 7B

Cu–NPy 2.021(3) 2.022(3) 1.994(3) 2.030(4) 2.010(3) 2.004(3)

27

Cu–N 1.958(3) 1.978(3) 1.977(3) 1.965(3) 1.974(3) 1.973(3)

Cu–S 2.2567(11) 2.2413(9) 2.2150(9) 2.2426(13) 2.2214(11) 2.2276(11)

Cu–X 2.2058(10) 2.3587(6) 2.3855(5) 2.3652(8) 2.3829(7) 2.3742(7)

S–C 1.752(3) 1.743(4) 1.733(3) 1.747(5) 1.750(4) 1.750(4)

Npy-Cu-S 164.67(9) 164.97(8) 165.80(8) 164.73(12) 164.03(10) 163.81(10)

N-Cu-S 84.03(8) 84.90(8) 84.85(8) 84.70(11) 84.74(10) 84.42(10)

Nazomethine-

Cu-X(Cl, Br)

177.80(9) 172.36(8) 175.98(8) 178.40(11) 171.92(10) 178.74(10)

Npy-Cu-N 80.65(12) 80.27(11) 80.97(11) 80.45(15) 80.64(14) 80.74(13)

N-Cu-X 97.53(9) 95.35(3) 99.81(8) 98.68(11) 98.64(10) 98.70(10)

S-Cu-X 97.78(4) 99.02(8) 94.39(3) 96.08(4) 96.74(4) 96.26(4)

9 10 11

9A 9B

Cu–NPy 2.006(8) 2.031(8) 2.027(3) 2.027(5)

Cu–N 1.978(8) 1.977(8) 1.955(3) 1.968(4)

Cu–S 2.213(3) 2.232(3) 2.2375(12) 2.2339(14)

Cu–I 2.5730(14) 2.5481(15) 2.5598(5) 2.5682(8)

S–C 1.736(10) 1.736(11) 1.747(4) 1.748(5)

Npy-Cu-S 166.0(3) 164.9(2) 164.42(9) 163.65(14)

N-Cu-S 84.7(2) 85.0(2) 84.59(10) 84.36(13)

Nazomethine-Cu-I 175.0(2) 173.2(3) 179.26(10) 179.18(12)

Npy-Cu-N 81.4(3) 80.0(3) 80.42(13) 80.40(18)

N-Cu-I 100.8(2) 100.7(2) 100.20(9) 100.39(13)

S-Cu-I 93.14(7) 94.02(8) 94.76(3) 94.84(4)