Rapid synthesis, characterization, anticancer and

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(AcAc), ethylcyanoacetate (ECA) and malanodinitrile. (MN) to get dinucleating ligands. The ligands were iso- lated, characterized and condensed with Ni (II), Cu ...
Rapid synthesis, characterization, anticancer and antimicrobial activity studies of substituted thiadiazoles and their dinucleating ligand metal complexes Anjali Jha, Y. L. N. Murthy, U. Sanyal & G. Durga

Medicinal Chemistry Research ISSN 1054-2523 Med Chem Res DOI 10.1007/s00044-011-9778-y

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Author's personal copy MEDICINAL CHEMISTRY RESEARCH

Med Chem Res DOI 10.1007/s00044-011-9778-y

ORIGINAL RESEARCH

Rapid synthesis, characterization, anticancer and antimicrobial activity studies of substituted thiadiazoles and their dinucleating ligand metal complexes Anjali Jha • Y. L. N. Murthy • U. Sanyal G. Durga



Received: 14 April 2011 / Accepted: 30 July 2011 Ó Springer Science+Business Media, LLC 2011

Abstract Synthesis of 2,5-disubstituted thiadiazoles was accomplished via a conventional method as well as microwave irradiation method. These substituted thiadiazoles were diazotized and coupled with 2,4-pentanedione (AcAc), ethylcyanoacetate (ECA) and malanodinitrile (MN) to get dinucleating ligands. The ligands were isolated, characterized and condensed with Ni (II), Cu (II) and Ru(III) chlorides. These compounds were screened on HL-60 Human leukemia cell Line and U-937 Lymphoma cell lines for anticancer activities. The antimicrobial activity of the ligands and their complexes against bacteria and fungi was also investigated. The effect of metal on the ligand activity is discussed. Keywords 2,5-disubstituted thiadiazoles  Dinucleating ligands  Ni(II)  Cu(II) and Ru(II) complexes  Anticancer activity  Antimicrobial activity

A. Jha (&) Department of Chemistry, GIS, GITAM University, Rushikonda, Visakhapatnam 530045, India e-mail: [email protected] Y. L. N. Murthy Department of Organic Chemistry and FDW, Andhra University, Visakhapatnam 530004, India U. Sanyal Chitaranjan National Cancer Institute, Kolkata, India G. Durga Department of Chemistry, St Josephs College for Women, Visakhapatnam 530004, india

Introduction Cancer is one of the leading causes of death in the world. With an improved understanding of the genes and pathways responsible for cancer initiation and progression, cancer drug development has undergone a paradigm change in recent years, from predominantly cytotoxic agent-based therapy to therapy aimed at molecular and genetic targets. Several five-membered aromatic systems having three hetero atoms at symmetrical position have been studied because of their interesting physiological properties (Hui et al., 2002 and Rudolph et al., 2001). It is also well established that various derivatives of 1,2,4-triazole and 1,3,4-thiadiazole exhibit a broad spectrum of pharmacological properties (Clerici, 2002). Thiazole derivatives are present in many natural and synthetic products displaying a wide range of activities, such as antibacterial (Tsuji and Ishikawa, 1994), antifungal (Sharma, 1967), antitumor (Crim John et al., 1967; Modi et al., 1970), anticancer (Gulsory and Guzeldemirci, 2007), antiinflammatory and (Sharma et al., 2009) activities. In addition, 1,3,4-thiadiazoles exhibit diverse biological activities possibly due to the presence of =N–C–S moiety (Holla et al., 2003). 2-aminothiazoles are well explored as useful clinical agents, and some of the derivatives of thiazoles have shown inhibition toward herpes simplex virus (Spector et al., 1998). 2-Aminothiazole can also be used as a thyroid inhibitor in the treatment of hyperthyroidism. Many studies (Crowe, 1988; Chohan et al., 2006) have described the biological role of metal chelation and have established the fact that anticancer drugs exhibit increased anticancer activity when administrated as their metal chelates. The connected metal centers in such molecules may involve different functions such as oxygen transport, DNA inhibition, enzymatic activity, electron transfer and locking

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Author's personal copy Med Chem Res

geometry. All these observations related to the essential role of metal linkages attracted our attention to commence a systematic research program to investigate the factors responsible for this relationship. Recently, organic transformation accelerated under solvent-free microwave irradiation condition has gained popularity due to many advantages, like enhanced reaction rates, high yields, improved selectivity and eco-friendly reaction condition in tune with green chemistry (Lindstrom et al., 2001; Lin et al., 2004; Mistry and Desai, 2006). Enthused by the interesting biological properties of metal complexes of NNS ligands and their potential cytotoxic and antimicrobial activities (Dholakiya and Patel, 2004), it was worthwhile to synthesize some new 2,5-disubstituted thiadiazoles by conventional as well as microwave-assisted (MW) method. These amines were further diazotized and condensed with active methylene groups to form dinucleating ligands. These ligands were thereafter condensed with metal chlorides. Ligands and metal complexes were characterized by various spectral methods and were subjected to activity studies. Copper complexes showed greater activity in antimicrobial studies while ruthenium complexes were found to be better anticancerous compounds.

Experimental Materials Reagents such as thiosemicarbazide, acetic acid, propionic acid, 2,4-pentanedione(AcAc), ethylcyanoacetate(ECA), malanodinitrile(MN) and metal chlorides were purchased from Across Ltd. and used as it is. All the solvents were of analytical grade and were distilled before use.

Synthesis of 2-amino-5-methyl/ethyl thiadiazole Substituted aminothiadiazole was prepared by conventional method as well as MW irradiation as outlined in Scheme 1. A mixture of carboxylic acid (acetic acid/propionic acid) (0.01 mol) and thiosemicarbazide (0.012 mol) in the presence of conc. H2SO4 (15 ml) was heated under reflux on water bath for 6 h, following the reported method (Pandey et al., 2003). Same ratio of carboxylic acid (acetic acid/propionic acid) and thiosemicarbazide in conc. H2SO4 (15 ml) was irradiated in microwave for 8 min. The resulting reaction mixture obtained from both the methods was cooled to room temperature and neutralized with ammonia solution. A dark brown solid that separated out was filtered and washed repeatedly with water. It was then recrystallized from ethanol. The substituted aminothiadiazoles were characterized by various spectral methods. L10 : (C3H5N3S): Yield (%): Conventional: 50, Microwave: 70; mp: 110°C; IR (mmax, KBr, cm-1): 3345, 3245 (NH2); 2987, 2941 (CH3); 2640 (CS);1555 (C=N of the ring), NMR(DMSO-d6dppm) 1H: 6.9 (NH2); 2.1 (CH3); 13 C: 168.45, 153.16 (C of thiadiazole ring in vicinity of NH2 and in vicinity of CH3, respectively); 20.1 CH3; Analysis: Calculated C, 31.30; H, 4.34; N, 36.52; Found: C, 31.28; H, 4.39; N, 36.60. L20 : (C4H7N3S): Yield (%): Conventional: 55, Microwave: 78; mp: 115°C; IR (mmax, KBr, cm-1): 3318, 3240 (NH2); 2987, 2941 (CH3); 2644 (CS); 1555 (C=N of the ring), NMR(DMSO-d6dppm) 1H: 7.6 (NH2); 3.2 (CH2); 1.8 (CH3); 13C: 168.45, 159.16 C of the thiadiazole ring in vicinity of NH2 and in vicinity of (CH2); 31.2 CH2, 20.1 CH3; Analysis: Calculated C, 37.20; H, 5.42; N, 32.55; Found: C, 37.28; H, 5.39; N, 32.58. Synthesis of dinucleating ligands

Measurements Melting points are uncorrected. IR spectra were recorded on Thermo Nicolet FTIR spectrophotometer at Andhra University, Visakhapatnam, and 1 H NMR and 13C spectra were taken on JEOL Model AL 400 NMR at RRL, Bhubaneswar, in DMSO-d6 using TMS as internal reference. The electronic spectra of the ligand and the complexes were recorded in DMSO solution using a Shimadzu UV-1700 spectrophotometer. ESR spectra were taken on Varian E 112 at room temperature and as well as liquid nitrogen temperature using DPPH as standard at SAIF, IIT Chennai. Microwave irradiation was carried out using a domestic microwave oven. Elemental analysis was carried out at Micro Analytical centre at Andhra University. The HPLC was recorded using Shimadzu LC 6A with Shimpack silica gel column.

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A solution of substituted amino-thiadiazoles (L10 and L20 ; 0.1 mol in separate reactions) in 10 ml conc. HCl was cooled between –5° and –10°C. To this solution, cold NaNO2 solution (1.5 g in 10 ml H2O) was added drop wise while stirring constantly (Scheme 2). The resulting reaction mixture was stirred for another 30 min. To this diazonium salt solution, a cold solution of acetyl acetone/

Conc H2SO4

RCOOH + NH2CSNHNH2 (a) Reflux,6 hrs R (b) MW,8min

N N S

NH2

R=CH3(L'1) =C2H5(L'2)

Scheme 1 Synthesis of 2-amino-5-methyl/ethyl thiadiazole

Author's personal copy Med Chem Res Scheme 2 Synthesis of dinucleating ligands

NaNO2/HCl

N N R

S

NH2

-5 to -10 ° C R

R' H2C R''

N N S

N N

+

N NCl -

R

NaOAc 20°C

S

R' N=N--HC R''

N N R

S

R' N--N=C R'' H

Where R=CH3 for L1,L2,L3 or R=CH 2CH3forL4,L5L6 R' & R''=COCH3=L1&L4 R'=CN & R''=COOC2H5=L2&L5 R' & R''=CN=L3&L6

ethylcyanoacetate/malanodinitrile (each 0.1 mol) containing sodium acetate (5 g) in water (10 ml) was added. Corresponding mixtures were further stirred for 3 h at 20°C and then refrigerated overnight. The solids thus obtained were filtered and washed several times with water and ethanol and then dried in vacuum. The crude products were crystallized in EtOH. The ligands (L1–L6) showed satisfactory elemental analysis and were further characterized by IR and 1H and 13C NMR spectra. L1: (C8H10N4SO2): Yield 65%, mp: 147°C; IR (mmax, KBr, cm-1): 3310 (NH); 2987, 2941 (CH3); 1680 (C=O); 1555 (C=N of the ring), NMR(DMSO-d6dppm) 1H: 5.4 (NH); 2.2 (CH3 of AcAc); 1.7 (CH3 of thiadiazole ring); 13 C: 210 (CO); 168.45, 158.16 (C of the thiadiazole ring); 56.2 (C of AcAc); 22.4 (CH3 of AcAc); 15.6 (CH3 of thiadiazole ring). Analysis: Calculated C, 42.47; H, 4.42; N, 24.77; Found: C, 42.51; H, 4.39; N, 24.72. L2: (C8H9N5SO2): Yield 55%, mp: 155°C; IR (mmax, KBr, cm-1): 3308 (NH); 2985, 2940 (CH3); 2540 (CS); 2230 (C:N); 1687 (C=O); 1555 (C=N of the ring); NMR(DMSO-d6dppm) 1H: 5.5 (NH); 2.8, 1.8 (CH2 & CH3 of ECA); 1.3 (CH3 of thiadiazole); 13C: 208 (CO); 169.45, 159.1 (C of thiadiazole ring); 105.2 (C:N of ECA); 55.2 (C of ECA); 38.2 (CH2), 20.4 (CH3 of ECA); 15.6 (CH3 of thiadiazole ring). Analysis: Calculated C, 40.16; H, 3.76; N, 29.28; Found: C, 40.11; H, 3.69; N, 29.22. L3: (C8H9N6S): Yield 60%, mp: 158°C; IR (mmax, KBr, cm-1): 3306 (NH); 2985, 2940 (CH3); 2538 (CS); 2226 (C:N); 1555 (C=N of the ring); NMR(DMSO-d6dppm) 1 H: 5.4 (NH); 1.2 (CH3 of thiadiazole); 13C: 169.45, 159.1 (C of thiadiazole ring); 115.4 (C:N of MN); 55.2 (C of MN); 15.6 (CH3 of thiadiazole ring). Analysis: Calculated C, 37.50; H, 2.08; N, 43.75; Found: C, 37.54; H, 2.11; N, 43.72. L4: (C9H12N4SO2): Yield 68%, mp: 165°C; IR (mmax, KBr, cm-1): 2987, 2941 (CH3); 2542(CS); 2238 (CN); 2238 (CN); 1685(C=O); 1555 (C=N of the ring);

NMR(DMSO-d6dppm) 1H: 5.8 (NH); 1.8 (CH3 of AcAc); 2.7 & 1.2 (CH2 & CH3 of thiadiazole); 13C: 208(CO); 169.45, 159.1 (C of thiadiazole ring); 55.2 (C of AcAc); 32.2, 15.6 (CH2, CH3 of thiadiazole ring); 22.4 (CH3 of AcAc). Analysis: Calculated C, 45.00; H, 5.0; N, 23.33; Found: C, 45.05; H, 5.06; N, 23.29. L5: (C9H11N5SO2): Yield 52%, mp: 182°; IR (mmax, KBr, cm-1): 3308(NH); 2987, 2941 (CH3); 2232 (C:N); 1680 (C=O); 1555 (C=N of the ring) NMR(DMSOd6dppm)1H: 5.3 (NH); 2.8, 1.8 (CH2 & CH3 of ECA); 2.4 & 1.1 (CH2 & CH3 of thiadiazole); 13C: 208 (CO); 169.45, 159.1 (C of thiadiazole ring); 110.1 (C:N of ECA); 58.2 (C of ECA); 39.2, 22.4 (CH2 & CH3 of ECA); 15.6 (CH3 of thiadiazole ring). Analysis: Calculated C, 42.68; H, 4.34; N, 27.66; Found: C, 42.65; H, 4.36; N, 27.69. L6: (C7H6N6S): Yield 67%, mp: 210°C; IR (mmax, KBr, cm-1): 3305 (NH); 2985, 2940 (CH3); 2544 (CS); 2228 (C:N); 1555 (C=N of the ring); NMR(DMSOd6dppm)1H: 5.5 (NH); 2.9, 1.5 (CH2 & CH3 of thiadiazole); 13C: 169.45, 159.1 (C of thiadiazole ring); 118.2 (C:N of MN); 57.8 (C of MN); 33.1 & 18.6 (CH2 & CH3 of thiadiazole ring). Analysis: Calculated C, 40.77; H, 2.91; N, 40.77; Found: C, 40.75; H, 2.95; N, 40.72. General procedure for the preparation of metal complexes An ethanolic solution of metal salts (2 mmol each NiCl26H2O, CuCl2H2O and RuCl33H2O) (10 ml) was added to the respective solution of ligands (L1–L6; 1 mmol each in 10 ml of ethanol) while stirring constantly (Mishra and Jha, 1996) followed by the addition of 2–3 drops of Et3N. The resulting solutions were then boiled under reflux while monitoring the progress of reaction periodically by TLC (thin layer chromatography) in 10% ethyl acetate and n-hexane. After 3 h, the reaction mixtures were cooled to room temperature and refrigerated overnight. The solids thus obtained were filtered and washed successively with ethanol and ether and dried in vacuum. The complexes

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Author's personal copy Med Chem Res Table 1 Analytical and physical data of complexes Sl. no.

1

Compounds

[Ni2(L1)Cl3(H2O)5]

Color/yield%

Found(Cacld)% C

H

N

Dark green

17.72

3.68

10.34

69

(17.78)

(3.70)

(10.37)

3.59

10.15

gk

g\

Ak RT (LNT)







2.241

2.023

165

2.

[Cu2(L1)Cl3(H2O)5]

Dark green

17.50

61

(17.46)

(3.63)

(10.18)

(2.322)

(2.144)

(160)

3.

[Ru2(L1)Cl3(H2O)5]

Dark brown

15.40

3.24

8.94







72

(15.37)

(3.20)

(8.96)

4.

[Ni2(L2)Cl3(H2O)5]

Dark green 65

17.39 (17.36)

3.47 (3.43)

12.64 (12.66)







5.

[Cu2(L2)Cl3(H2O)5]

Dark green

17.10

3.35

12.48

2.232

2.123

162

60

(17.06)

(3.37)

(12.44)

(2.332)

(2.116)

(165)

15.09

2.95

10.94











– 160

6.

[Ru2(L2)Cl3(H2O)5]

Dark brown 75

(15.05)

(2.98)

(10.98)

7.

[Ni2(L3)Cl3(H2O)5]

Dark green

14.27

2.72

16.56

65

(14.23)

(2.76)

(16.60)

8.

[Cu2(L3)Cl3(H2O)5]

Dark green

13.99

2.74

16.32

2.233

2.112

58

(13.96)

(2.71)

(16.29)

(2.323)

(2.210)

(162)

12.22

2.40

14.25













9.

[Ru2(L3)Cl3(H2O)5]

Dark brown 70

(12.19)

(2.37)

(14.22)

10.

[Ni2(L4)Cl3(H2O)5]

Dark green

19.51

3.99

10.15

68

(19.49)

(3.97)

(10.10)

11.

[Cu2(L4)Cl3(H2O)5]

Dark green

19.20

3.92

9.96

2.242

2.022

165

[Ru2(L4)Cl3(H2O)5]

60 Dark brown

(19.16) 16.94

(3.90) 3.47

(9.93) 8.79

(2.322) –

(2.114) –

(160) –

72

(16.91)

(3.44)

(8.77)

Dark green

19.09

3.74

12.37







61

(19.05)

(3.70)

(12.34)

Dark green

18.76

3.67

12.17

2.222

2.102

160

59

(18.73)

(3.64)

(12.14)

(2.343)

(2.204)

(160)

16.60

3.26

10.76













12. 13. 14.

[Ni2(L5)Cl3(H2O)5] [Cu2(L5)Cl3(H2O)5]

15.

[Ru2(L5)Cl3(H2O)5]

Dark brown 70

(16.57)

(3.22)

(10.74)

16.

[Ni2(L6)Cl3(H2O)5]

Dark green

16.19

3.10

16.19

66

(16.15)

(3.07)

(16.15)

Dark green

15.84

3.08

15.90

2.231

2.110

165

58

(15.86)

(3.02)

(15.86)

(2.302)

(2.116)

(165)

Dark brown

13.92

2.58

13.91







72

(13.89)

(2.60)

(13.89)

17. 18.

[Cu2(L6)Cl3(H2O)5] [Ru2(L6)Cl3(H2O)5]

showed satisfactory elemental analysis and were further characterized by various spectral methods (Table 1). Study of anticancer activity The anticancer screening of dinucleating ligands of substituted thiadiazoles and their metal complexes were done at Chitaranjan National Cancer Institute (CNCI), Kolkata, against HL-60 Human leukemia cell line and U-937 Lymphoma cell lines received from NCCS, Pune.

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Drug stock solutions were prepared in DMSO [20 mg of compound/1 ml of HYBRIMAX grade, Sigma, USA] to obtain working stock solutions. These were serially tenfold diluted with complete sterile growth medium (RPMI-1640 medium with 2 Mm glutamine, containing 10% fetal bovine serum and 1% antibiotics [10000 U penicillin/ml and 10,000 lg streptomycin/ml] prior to use to obtain different drug concentrations. Four clinical drugs such as Cis-Platinum, BCNU, Hydroxyurea and 5-fluoro uracil were included as standards. About 100 ll of cell

Author's personal copy Med Chem Res Table 2 In vitro cytotoxicity results of synthesized compounds Compounds

IC50 value (lM)

Table 3 Antifungal activity of synthesized compounds at concentration of 1 lg/ml Compound

Fungi

Leukemia HL-60

Lymphoma U-937

L1

23.01

24.67

L2

22.05

19.34

L1

12

18

6

4

L3

28.12

Inactive

L1–Ni

13

12

16

11

L1–Ni

16.50

17.96

L1–Cu

14.55

15.11

L1–Cu L1–Ru

16 17

17 16

19 19

14 12

L1–Ru

1.95

4.93

L2

11

17

7

NA

Cis–Platinum BCNU

7.0 30.50

3.2 12.3

L2–Ni

9

18

15

11

L2–Cu

12

22

21

15

4.7

L2–Ru

13

17

18

14

115.0

L3

12

15

8

NA

L3–Ni

12

16

18

NA

L3–Cu

15

19

19

14

L3–Ru

14

17

18

15

L4

10

14

7

6

L4–Ni

13

14

17

11

L4–Cu

18

21

22

14

L4–Ru

15

11

15

12

AF

5-FU Hydroxyurea

266.0 Inactive

IC50 = the cytotoxic dose at 50%, i.e. the concentration needed to reduce the growth of cancer cells by 50%. Generally, IC50 \ 5.0 means compound is strongly active, IC50 5.0–10.0 means moderately active and IC50 [ 25.0 means the compound is inactive

suspension from the stock 0.2 9 105 HL-60/U-937 was added to 96-well cell culture plates. Ten microliters of drug solutions of different concentrations was added to respective wells followed by the addition of the medium (90 ll) in triplicate (total volume 200 ll/well). All vehicle controls contained the same concentration of DMSO. Plates were incubated for 96 h at 37°C, 5% CO2/95% air with humidity. After removal of 100 ll of media from each well, 10 ll of a 5 mg/ml solution of MTT in Dulbecco’s PBS (GIBCO, BRL) was added to each well and the plate was incubated for 4 h at 37°. Subsequently, 100 ll of acid isopropanol solution (0.04 N HCl in isopropanol) was added to the wells to dissolve formed formazan crystals. The plate was read in a microplate reader at 540 nm. Curvefit software was used to calculate the IC50 value. The results are shown in Table 2. Study of antimicrobial assay The dinucleating ligands (L1–L6) and their metal complexes were examined for antimicrobial assay against eight bacteria [BS-Bacillus subtilis, EC-Ervinia carotovora, PV-Pseudomonas vulgaris, E coli-Escherichia coli, EF-Enterococcus faecalis, SF-Streptococcus faecalis, KP-Klebsiella pneumoniae and ML-Micrococcus luteus] and four fungi [AF-Aspergillus flavus, PE-Penicillium expansum, LT-Lasiodiplodia theobroma and RS-Rhizoctonia solani] using the well diffusion method (Odds, 1989). Two hundred milliliters of nutrient agar growth medium was dispensed into sterile conical flasks; these were then inoculated with 20 ll of cultures mixed gently and poured into sterile petri dish. After setting a borer with 6 mm diameter was properly sterilized by flaming and used to make three uniform wells in each petri dish. The wells were loaded

PE

LT

RS

L5

12

16

10

NA

L5–Ni L5–Cu

12 19

17 23

16 23

11 17

L5–Ru

17

12

16

14

L6

12

16

15

NA

L6–Ni

11

16

18

11

L6–Cu

20

22

23

18

L6–Ru

18

17

17

13

DMSO (control)

NA

NA

NA

NA

STD

31

35

NA

29

AF-Aspergillus flavus, PE-Penicillium expansum, LT-Lasiodiplodia theobroma, RS-Rhizoctonia solani, NA-not active, STD-ampicillin

with 50 ll of different investigated compounds. The solvent DMSO, used for reconstituting the solvent for diluting the compounds, was similarly analyzed for control. The plates were incubated at 37°C for 24 h. The above procedure is adopted also for fungal assays, and the medium is potato dextrose agar (instead of nutrient agar) and incubated at 27°C for 48 h. The zone of inhibition was measured with a Hi Antibiotic Zone Scale in mm, and the experiment was carried out in duplicate. The results are shown in Tables 3, 4.

Results and discussions Chemistry To reduce the reaction time and for better yields, substituted aminothiadiazole was prepared by conventional

123

Author's personal copy Med Chem Res Table 4 Antibacterial activity of synthesized compounds at concentration of 1 lg/ml Compound

Bacteria BS

EC

PV

Ecoli

EF

SF

KP

ML

L1

7

6

7

8

8

7

8

NA

L1–Ni

12

10

9

10

11

10

9

10

L1–Cu L1–Ru

14 13

12 10

11 10

14 13

12 11

12 10

12 9

13 11

L2

8

NA

8

7

7

6

7

NA

L2–Ni

11

10

8

8

11

10

11

9

L2–Cu

15

13

12

11

16

14

13

13

L2–Ru

12

10

11

12

13

12

10

11

L3

10

NA

10

6

9

6

8

7

L3–Ni

11

NA

9

10

9

8

NA

NA

L3–Cu

14

10

12

11

11

10

9

10

L3–Ru

11

9

11

10

12

9

9

11

L4

8

7

6

8

8

8

5

8

L4–Ni

10

NA

11

9

NA

NA

NA

NA

L4–Cu

13

11

13

12

10

NA

8

9

L4–Ru

10

9

11

8

NA

11

NA

NA

L5

9

NA

NA

9

6

5

7

6

L5–Ni L5–Cu

9 17

9 11

11 14

9 14

NA 14

11 14

9 17

10 12

L5–Ru

12

10

9

12

13

12

13

11

L6

11

7

NA

9

7

8

7

6

L6–Ni

11

7

8

8

9

9

10

9

L6–Cu

15

10

16

17

15

16

17

13

L6–Ru

11

9

11

12

12

13

11

12

DMSO (control)

NA

NA

NA

NA

NA

NA

NA

NA

STD

30

31

33

28

NA

30

29

32

AF-Aspergillus flavus, PE-Penicillium expansum, LT-Lasiodiplodia theobroma, RS-Rhizoctonia solani, BS-Bacillus subtilis, EC-Ervinia carotovora, PV-Pseudomonas vulgaris E coli-Escherichia coli, EF-Enterococcus faecalis, SF-Streptococcus faecalis KP-klebsiella pneumoniae, ML-Micrococcus luteus; NA-not active; STD-ampicillin

method as well as MW irradiation method. We had reduced the reaction time considerably in minutes (MW) from hours in conventional method. Identical TLC and 99% HPLC purity were found for the product obtained from both the methods. These substituted thiadiazoles were diazotized and coupled with active methylene groups to get dinucleating ligands. These ligands were further condensed with metal chlorides and characterized by various spectral methods. All the compounds were air stable. The ligands were soluble in organic solvents such as ethanol, acetonitrile, dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) while complexes were soluble in DMF and DMSO only. Analytical data as well as other physical properties of the complexes are listed in Table 1.

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The solid-state IR spectra of the 2,5-disubstituted thiadiazoles and free ligands were compared. The peaks for NH2 of thiadiazoles disappeared, and peaks at *3,300, *1,685 cm-1 in ligands L1, L2, L4 and L5 have been considered to arise from mNH and mC=O (Mishra et al., 1997) vibrations, respectively, whereas a peak observed in the range *2,640–2,620 is assigned to C–S of the ligands. Furthermore, strong peaks observed at *2,230 in the spectrum of L2, L3, L5 and L6 have been attributed to mC:N (Mishra and Jha, 1996). In the spectra of complexes, mNH peak disappeared while a new peak at 1,458–1,466 cm-1 assigned to N=N appeared, indicating that complexes exist in diazo form which is further supported by their 1H/13C NMR data. The lowering of mC=O from 1,680–1,690 to 1,644–1,655 cm-1 in the IR of metal complexes was indicative of metal ligand coordination. However, the peak observed in the range 1,548–1,518 cm-1 in the spectra of the complexes with all ligands were assigned to coordinated mC=N vibration of the ring (Tennant and Vevers, 1976). Additionally, the lower value by *15–20 cm-1 for C:N observed in the complexes with ligands L2, L3, L5 and L6 further supported the coordination with metal ions. This lowering of C:N, C=O, C=N of the ring upon complexation could be understood in view of the participation of their p-electrons in coordination with metal ions (Nakamoto, 2009). The bands at 550–450 cm-1 have been attributed to mM–N, mM–O and mM–Cl bonds; however, some overlapping bands of ligands are also present. The 1H and 13C NMR spectra of the 2,5-disubstituted thiadiazoles and free ligands were compared. The NH2 peaks at 6.9 and 7.6 ppm for methyl and ethyl thiadiazole were changed to NH *5.5 ppm for all the ligands and indicative of diazotization and further condensation with active methylene groups, viz. AcAc, ECA and MN. This was further supported by the presence of peaks at *2.2–2.9 for CH2 and *1.5–1.9 for CH3. The 13C values for all the ligands (L1 & L6) fitted well for the corresponding hydrazone form (Scheme 2). The poor solubility and paramagnetic nature of the metal complexes restricted us from recording their good NMR spectra. However, the 1 H NMR spectra of the Ni complexes showed no peaks due to OH or NH protons, which indicated the coordination of deprotonated ligand with the metal ions. Electronic spectral data of synthesized compounds were recorded in DMSO. The Cu (II) complexes exhibited two low-energy weak bands at 15,151–15,873 cm-1 and a strong high-energy band at 30,255–30,420 cm-1. The low-energy band in this position typically is expected for an octahedral configuration and may be assigned to 10Dq corresponding to the transition 2Eg ? 2T2g (Ballhausen, 1962; Lever, 1984),which was further supported by their ESR values. The strong high-energy band, in turn, is assigned to metal ? ligand charge transfer. Ni(II) complexes showed d–d bands

Author's personal copy Med Chem Res Fig. 1 Powder ESR spectra of [Cu2(L1)Cl3(H2O)5] at LNT

and O, O & N, O donors on the other could not be identified at this stage . Three ligands L1, L2 and L3 and nickel, copper and ruthenium complexes of ligand L1 were assayed for their cytotoxicity against HL-60 Human leukemia cell line and U-937 Lymphoma cell line received from NCCS, Pune, at the CNCI, Kolkata. The cytotoxic activity data are shown in Table 2 as IC50 value, i.e., the concentration that inhibits cell replication by 50% relative to control. Free ligands are rather less active in most of the cases. However, upon complexation, their activities enhanced significantly in almost all cases. Ruthenium complex of ligand L1 found to be most active against Leukemia HL-60 followed by Lymphoma U-937 cell line, while nickel and copper complexes of same ligand also showed considerable activity against these tumor cell lines. A comparative study of the ligands and their complexes (MIC values showed in Tables 3 and 4, graphical representation in Figs. 2, 3) indicated that all the complexes exhibited higher antibacterial activity than the free ligands. AF PE LT RS

20

15

Fungi

in the regions 24,390–25,000, 16,528–16,667 and 12,987– 13,333 cm-1. These are assigned to the spin-allowed transitions 3A2g(F) ? 3T2g(F), 3A2g(F) ? 3T1g(F) and 3 A2g(F) ? 3T1g(P), respectively, and in accordance with their well-defined octahedral configuration (Mohammed, 2001). The band at 29,815–30,335 cm-1 was assigned to metal ? ligand charge transfer. Three to four bands appeared in the region 15,625–40,000 cm-1 in the electronic spectrum of Ru complexes. The two spin-allowed transitions, 1A1g ? 1T1g (20,450 cm-1) and 1A1g ? 1T2g (18,800 cm-1), and a weak spin forbidden transition 1 A1g ? 3T1g (15,625 cm-1) are assigned to octahedral configuration of Ru(II) complexes (Karvembu and Natarajan, 2002). A strong intensity band at 37,700 cm-1 is probably due to charge transfer transition (T2g ? p*). The nature of the electronic spectra is similar to that observed for other octahedral Ru(II) complexes (Mishra and Sinha, 2002; Prasad et al., 2011). The ESR parameters for the Cu (II) complexes of all ligands at room temperature (RT) as well as liquid nitrogen temperature (LNT) are given in Table 1. The powder-state spectra of the complex [Cu2(L)Cl3(H2O)5] at RT and LNT showed four equally spaced lines as expected for Cu (II) ion showing g|| [ g\ with more clarity in LNT spectra (Fig. 1). The values are supportive of octahedral geometry around metal ion. Basic spectral characteristics at both temperatures are the same with slightly better resolution (Coucouvanis and Fackler Jr., 1967) at LNT. The half-field signal was not observed in any of these spectra, indicating that there is no Cu–Cu interaction between the complexes, hence supportive of dinuclear complexes. Additionally, the 14 N super hyperfine splitting showing five signals separated at 8G could easily be seen in the spectra of the complexes in solution state at both temperatures (Kumar et al., 2001). However, at LNT this splitting is better resolved showing two nitrogens around Cu (II) ions but the difference between the two coordination environments in the complex with L1, L2,L4 and L5 having N, N donor on the one side

10

5

0 L1 N C R L2 N C R L3 N C R L4 N C R L5 N C R L6 N C R

Compounds

Fig. 2 Antifungal activity of synthesized compounds. N=Ni, C=Cu, R=Ru complexes

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Author's personal copy Med Chem Res

BS EC PV ECOLI EF SF KP ML

18 16 14

Bacteria

12 10 8 6 4 2 0 L1 N C R L2 N C R L3 N C R L4 N C R L5 N C R L6 N C R

Compounds

Fig. 3 Antibacterial activity of synthesized compounds. N = Ni, C = Cu, R = Ru complexes

Cl

OH2 Cl M

N N R

N S

N

OH2 C

OH2 '

Cl

R

M R''

OH2

aminothiadiazole, which can be a viable alternative of conventional synthesis. The synthetic protocol has the inherent potential for future drug synthesis. A new series of mixed ligand complexes of nickel, copper and ruthenium were synthesized, and their octahedral geometry was inferred from their spectral data. Ru complexes showed promising activity against both the cell lines, while Ni and Cu complexes showed moderate activity. A comparative study of the MIC values of the ligands and their complexes indicates that complexes exhibit higher antimicrobial activity than the free ligands. However, Cu complexes showed more activity against almost all bacteria and fungi. In view of the structural formula of the complexes that exhibit anticancer as well as antimicrobial activity, metal moiety may play a significant role. From the results, it is also clear that these compounds would be better used in drug development to combat bacterial and fungal infections. Ru complexes may be better used as anticancer compounds. Acknowledgments Anjali Jha is thankful to the Department of Science and Technology (DST), Delhi, for financial support.

OH2 Where R=CH 3 for L1,L2,L3 or R=CH 2CH3forL4,L5L6 R' & R''=COCH 3=L1&L4 R'=CN & R''=COOC2H5=L2&L5 R' & R''=CN=L3&L6 M=Ni(II),Cu(II),Ru(II)

Fig. 4 Proposed structure of the complexes

Copper complexes were found to be more active against fungi. Such increased activity of the complexes can be explained on the basis of the Overtone concept and the Tweedy chelation theory. According to Overtone concept of cell permeability, the lipid membrane surrounding the cell favors the passage of only lipid-soluble materials, due to that, liposolubility is an important factor controlling the antimicrobial activity. On chelation, the polarity of the metal ion will be reduced to a greater extent due to the overlapping of the ligand orbital and partial sharing of positive charge of the metal ion with donor groups. Furthermore, the mode of action of the compound may involve the formation of a hydrogen bond through the azomethine group with the active center of cell constituents, resulting in interference with normal cell processes. Further studies are under process. Thus, on the basis of above analytical physical and spectral data, a tentative structure is given in Fig. 4. Conclusion We have developed very efficient microwave-assisted protocol for the green synthesis of 2,5-disubstituted

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