Synthesis and biological potentials of dioxomolybdenum(VI) complexes with ONS and ONN chelating thiosemicarbazones: DNA-binding, antioxidant and enzyme inhibition studies

Synthesis and biological potentials of dioxomolybdenum(VI) complexes

with ONS and ONN chelating thiosemicarbazones: DNA-binding,

antioxidant and enzyme inhibition studies

Songül Eg˘lence-Bakır

a

, Musa S

_ßahin

a,⇑

, Muhammad Zahoor

b

, Eylem Dilmen-Portakal

c

, Bahri Ülküseven

c a

Department of Chemistry, Istanbul University, 34134, Beyazit, Istanbul, Turkey b

Department of Biochemistry, Malakand University, Chakdara, Dir Lower, 18800 KPK, Pakistan c

Department of Chemistry, Istanbul University-Cerrahpasßa, 34320 Avcilar, Istanbul, Turkey

a r t i c l e i n f o

Article history: Received 14 July 2020 Accepted 13 August 2020 Available online 17 August 2020

Keywords: Thiosemicarbazone dioxomolybdenum (VI) Antioxidant Enzyme inhibition DNA binding

a b s t r a c t

In this study dioxomolybdenum (VI) complexes of 5-methoxysalicylidene N- or S-alkyl substituted thiosemicarbazones {where alkyl is N-methyl (L1), N-octyl (L2), S-methyl (L3) or S-octyl (L4)} were syn-thesized, characterized by different spectroscopic techniques (UV, IR,1_{H NMR). The structure of the}

com-plex with S-methyl-substituted thiosemicarbazone (comcom-plex 3) was also determined by X-ray diffraction method. The compounds were evaluated for their antibacterial, antioxidant, anticholinesterase, antidia-betic, and DNA interaction potentials. K. pneumonia was more potently inhibited by ligand L3 (29 ± 0.025 mm zone of inhibition) while E. coli and S. typhi by complex 2 with zone of inhibition of about 28 ± 0.082 and 26 ± 0.245 mm respectively. Complex 2 more potently scavenged DPPH free radical with IC50of 231lg/mL while ABTS by ligand L1 (IC50= 350lg/mL). Complex 4 with S-octyl was found to have

high percent inhibition of acetylcholinesterase with IC50of 104lg/mL. Complex 4 strongly inhibited a-amylase with an IC50value of 153mg/ml while ligand L4 with IC50value of 285mg/ml was more potent

inhibitor ofa-glucosidase. DNA interaction studies revealed the noncovalent interaction of these com-pounds with DNA. Highest binding constant among these comcom-pounds was recorded for ligand L2 with blue shifts and hyperchromism while lowest for L3.

Ó 2020 Elsevier Ltd. All rights reserved.

1. Introduction

The first medical applications of thiosemicarbazones were reported in 1950 s with the discovery of their biological activities against tuberculosis and leprosy[1–2]. In 1960 s, antiviral effects of this class of compounds were also reported[3]. Until now, a con-siderable amount of literature has been published on thiosemicar-bazones and their derivatives[4–6]. The ligand potencies of the thiosemicarbazones are due to nitrogen and sulfur atom while additionally some other donor atoms[7–9]. The reported metal complexes of this class have exhibited a broad spectrum of biolog-ical activities such as antiviral[10], antibacterial[11], antitumor

[12], antioxidant [13], anticancer [14] etc. A number of studies revealed that biological activities of thiosemicarbazones depend on the type of metal center and the groups attached to the sulfur atom, hydrazine, and amide nitrogen atoms, and even the sub-stituents on these groups[15–16].

Molybdenum is as an oligo element. It has exhibited some bio-logical applications and is an important cofactor that catalyzes redox reactions[17–18]. It is of great interest to have a coordina-tion site suitable for substrate binding in thiosemicarbazone-molybdenum complexes and to become a potential catalyst when the coordination molecule is displaced by the activated enzyme molecule. As it plays very important role in enzyme systems, molybdenum chelates may form very convenient model for the catalyst systems[19–20].

The pharmaceutical applications of dioxomolybdenum com-plexes like anticancer [21], antitumor[22], antioxidant[23], and DNA interaction/cleavage activities[24]have been reported in lit-erature. These features have increased the interest of scientists in cis-MoO2(VI) chelates in recent years and many dioxomolybdenum

complexes have been investigated regarding their various aspects. This study was aimed to synthesize ONS and ONN donor diox-omolybdenum (VI) complexes of alkyl substituted thiosemicar-bazones (Fig. 1). The compounds were characterized by using elemental analysis, IR, 1_{H NMR spectroscopies. The structure of}

complex 3 was determined by X-ray diffraction method. The

https://doi.org/10.1016/j.poly.2020.114754

0277-5387/Ó 2020 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.

E-mail address:musahin@istanbul.edu.tr(M. Sßahin).

Contents lists available atScienceDirect

Polyhedron

compounds were examined for their antibacterial, antioxidant, enzyme inhibitory and DNA binding potentials.

2. Experimental section

2.1. Materials and physical measurements

All chemicals used in the study were of reagent grade and were used without purification. The elemental analyses were performed on a Thermo-Finnigan Flash EA 1112 Series Elementary Analyzer. UV–Vis spectra of the compounds were recorded at 10-5_M

concen-tration in CHCL3 solution with a Shimadzu 2600 UV–Vis Spectrom-eter. Infrared spectra of the complexes were recorded on a Mattson 1000 FTIR spectrophotometer in range of 4000 to 400 cm
1 at

room temperature.1H NMR spectra were recorded on a Bruker

Avance-500 model spectrometer relative to SiMe4using

deuter-ated chloroform as solvent at 25 ± 2°C. The X-ray intensity data were recorded on a Bruker D8 VENTURE system equipped with a

multilayer monochromator and a Mo K

a

sealed tube

(k = 0.71073 Å).

2.2. Synthesis of the ligands

In the first step, N4_{-octyl thiosemicarbazide and MoO} 2(acac)2,

were synthesized as reported in literature[23,25]. The ONS donor thiosemicarbazone ligands were obtained using method men-tioned previously[26];

For synthesis of N1_{-5-methoxysalicylidene-N}4

-methyl-thiosemicarbazone (L1), N4_{-metylthiosemicarbazide (0.50 g,}

2 mmol) was dissolved in ethanol with gentle heating. About 5 ml of ethanolic solution of 5-methoxysalicylaldehyde (0.50 ml, 2 mmol) were added to it, and the mixture was refluxed for an hour. The yellow precipitates obtained were washed several times with cold water and ethanol. The ligand was recrystallized from ethanol and dried under vacuum. The other ONS ligand (L2) was prepared by the same procedure.

To prepare the ONN ligand, N1

-5-methoxysalicylidene-S-methyl- thiosemicarbazone (L3), thiosemicarbazide (0.50 g, 5 mmol) and methyl iodide (0.33 ml, 5 mmol) were refluxed in 10 ml ethanol for two hours. Five milliliters of ethanolic solution

of 5-methoxysalicylaldehyde (0.70 ml, 5 mmol) was added to the colorless mixture. Then sufficient amount of aqueous NaHCO3

solution was added to neutralize the hydrogen iodide formed. The separated yellow precipitate was filtered, washed several times with cold ethanol and dried under vacuum. The S-octyl thiosemicarbazone ligand (L4) was prepared similarly.

L1: Yellow, m.p. 144–145°C, yield 89%. Anal. Calc. for C10H13N3

-O2S (239.29 g/mol): C, 50.19; H, 5.48; N, 17.56; S, 13.40. Found: C,

50.37; H, 5.27; N, 17.33; S, 13.33%. UV–Vis (nm, log

e

in parenthe-sis): 250 (4.24); 310 (4.11); 358 (4.32). IR (ATR, cm
1):ʋ(OH) 3377; ʋ(NH) 3281; ʋ(CH)aliph.3013, 2937, 2832;ʋ(C = N1) 1550.1H NMR

(DMSO d6): 11.41 (s, 1H, OH); 8.26 (s, 1H, CH = N1); 9.45 (s, 1H,

N2_{H); 8.44 (m, 1H, N}4_{H); 7.49 (d, 1H, c); 7.07 (d, 1H, b); 6.78 (d,}

1H, a); 3.72 (s, 3H, –OCH3); 3.01 (s, 3H, N4-C1H3).

L2: Yellow, m.p. 132–133_{°C, yield 84%. Anal. Calc. for C}17H27N3

-O2S (337.48 g/mol): C, 60.50; H, 8.06; N, 12.45; S, 9.50. Found: C,

60.51; H, 8.06; N, 12.40; S, 9.51%. UV–Vis (nm, log

e

in parenthe-sis): 256 (4.33); 311 (4.36); 358 (4.85). IR (ATR, cm
1): _ʋ(OH) 3408;ʋ(NH) 3142; ʋ(CH)aliph.3006, 2916, 2849;ʋ(C = N1) 1576. 1_{H NMR (DMSO d} 6): 11.34 (s, 1H, –OH); 8.32 (s, 1H, CH = N1); 9.45 (s, 1H, N2H); 8.45 (t, 1H, N4H); 7.46 (d, 1H, c); 7.10 (d, 1H, b); 6.78 (d, 1H, a); 3.72 (s, 3H, –OCH3); 3.53 (m, 2H, N4-C1H2); 1.58–1.23 (m, 12H, –C2-7_H 2); 0.87 (t, 3H, -C8H3).

L3: Yellow, m.p. 122–123°C, yield 87%. Anal. Calc. for C10H13N3

-O2S (239.29 g/mol): C, 50.19; H, 5.48; N, 17.56; S, 13.40. Found: C,

50.10; H, 5.32; N, 17.48; S, 13.28%. UV–Vis (nm, log

e

in parenthe-sis): 255 (3.41); 305 (3.55); 358 (3.67). IR (ATR, cm
1):ʋ(OH) 3423; ʋ(NH2) 3308;ʋ(CH)aliph.3002, 2940, 2836;ʋ(C = N1) 1647;ʋ(C = N2) 1571.1_{H NMR (DMSO d} 6): 11.06, 10.20 (s, i:2/1, 1H, –OH); 8.43, 8.30 (s, i:2/1, 1H, CH = N1_{); 7.21 (d, 1H, c); 7.05 (d, 1H, b); 6.81} (d, 1H, a); 6.78 (d, 2H, N4_H 2); 3.72 (s, 3H, –OCH3); 2.37 (s, 3H, S-C1_H 3).

L4: Yellow, m.p. 103–104 °C, yield 84%. Anal. Calc. for

C17H27N3O2S (337.48 g/mol): C, 60.50; H, 8.06; N, 12.45; S, 9.50.

Found: C, 60.49; H, 8.12; N, 12.70; S, 9.63%. UV–Vis (nm, log

e

in parenthesis): 255 (3.84); 305 (3.90); 358 (4.10). IR (ATR, cm
1):ʋ (OH) 3399;ʋ(NH2) 3319;ʋ(CH)aliph.3006, 2926, 2855;ʋ(C = N1) 1653;ʋ(C = N2_{) 1561.}1_{H NMR (DMSO d} 6): 11.17 (s, 1H, –OH); 8.41, 8.30 (s, i:1/1, 1H, CH = N1_{); 7.20 (d, 1H, c); 7.03 (d, 1H, b);} 6.84 (d, 1H, a); 6.81 (s, 2H, N4_H 2); 3.71 (s, 2H, –OCH3); 2.98 (m, 2H, S-C1_H 2); 1.62–1.24 (m, 12H, –C2-7H2); 0.83 (m, 3H, -C8H3).

CH

O

N

N

X

Mo

O

O

Z

O

R

2

CH3

H

H

₃

CO

R

1

a

b

c

a

b

c

CH

OH

N

N

X

Z R

2

H

₃

CO

R

1

+ MoO

2

(acac)

2

- 2 acacH

+ CH

3

OH

Ligand

Complex

Donor set

X

Z

R

1

_R

2

L1

1

ONS

S

N

-

methyl

L2

2

ONS

S

N

-

octyl

L3

3

ONN

N

S

H

methyl

L4

4

ONN

N

S

H

octyl

2.3. Synthesis of the complexes

To synthesize cis-dioxo-(N1

-5-methoxysalicylidene-N4_{-methylthiosemicarbazonato)(O,N,S)-methanol-molybdenum(VI)}

(complex 1), N1_{-5-methoxysalicylidene-N}4

-methyl-thiosemicar-bazone (L1), (0.24 g, 1.0 mmol) was dissolved in acetonitrile (4 ml) at about 45–50_{°C. The 2 ml methanolic solution of MoO}2

(acac)2(0.36 g,1.1 mmol) was added to the hot solution. The

reac-tion mixture was stirred for one hour. The orange precipitates were collected by filtration and washed with cold methanol. Recrystal-lization of the product from methanol gave the analytical grade pure compound. Other ONS coordinated complex 2 was prepared by the same procedure. The ONN coordinated complexes (3–4) were synthesized by the little modification in which only methanol was used as a solvent. The colours, m.p. (°C), yields (%), elemental analysis, UV–Vis, IR and1H NMR data for the compounds are given below.

1: Orange, m.p. 160–161 °C, yield 65%. Anal. Calc. for

C11H15MoN3O5S (397.26 g/mol): C, 33.26; H, 3.81; N, 10.58; S,

8.07. Found: C, 33.06; H, 3.88; N, 10.50; S, 8.11%. UV–Vis (nm, log

e

in parenthesis): 258 (4.51); 313 (4.12); 355 (4.54); 435 (3.78). IR (ATR, cm
1):ʋ(OH) 3341; ʋ(NH) 3246; ʋ(CH)aliph.3006,

2933, 2834;_{ʋ(C = N}1_{) 1589;} ʋ(C = N2_{) 1556;} ʋs,ʋas(MoO2) 934, 891. 1_{H NMR (DMSO d} 6): 8.54 (s, 1H, CH = N1); 7.46 (m, 1H, N4_{H); 7.22 (d, 1H, c); 7.11 (d, 1H, b); 6.79 (d, 1H, a); 3.79 (s, 3H,} –OCH3); 3.16 (d, 3H, N4-C1H3); 4.08 (s, 1H, –OH); 2.80 (s, 3H, –CH3).

2: Orange, m.p. 182–183°C, yield 69%. Anal. Calc. for C18H29

-MoN3O5S (495.45 g/mol): C, 43.64; H, 5.90; N, 8.48; S, 6.47. Found:

C, 43.61; H, 5.85; N, 8.43; S, 6.40%. UV–Vis (nm, log

e

in parenthe-sis): 259 (4.59); 303 (4.52); 335 (4.59); 435 (3.77). IR (ATR, cm
1): ʋ(OH) 3386; ʋ(NH) 3315; ʋ(CH)aliph.3004, 2924, 2853;ʋ(C = N1) 1599;ʋ(C = N2_{) 1559;ʋ} s,ʋas(MoO2) 935, 892.1H NMR (DMSO d6): 8.50 (s, 1H, CH = N1_{); 7.22 (t, N}4_{H); 7.01 (d, 1H, c); 6.87 (d, 1H, b);} 6.78 (d, 1H, a); 3.79 (s, 3H, –OCH3); 3.21 (m, 2H, N4-C1H2); 1.53–1.23 (m, 12H, –C2-7_H 2); 0.86 (t, 3H, -C8H3); 4.06 (s, 1H, –OH); 3.18 (s, 3H, –CH3).

3: Orange, m.p. 144–145 °C, yield 70%. Anal. Calc. for

C11H15MoN3O5S (397.26 g/mol): C, 33.26; H, 3.81; N, 10.58; S, 8.07. Found: C, 33.00; H, 3.77; N, 10.48; S, 8.07%. UV–Vis (nm, log

e

in parenthesis): 257 (4.33); 300 (4.25); 342 (4.36); 447 (3.80). IR (ATR, cm
1):ʋ(OH) 3398 ʋ(N4_{H) 3259;ʋ(CH)} aliph.2994, 2950, 2830; ʋ(C = N1 ) 1654;ʋ(C = N2 ) 1595;ʋs,ʋas(MoO2) 930, 885.1_{H NMR (DMSO d} 6): 8.47 (s, 1H, CH = N1); 9.05 (s, 1H, N4H); 7.20 (d, 1H, c); 7.07 (d, 1H, b); 6.75 (d, 1H, a); 3.15 (s, 3H, – OCH3); 2.45 (s, 3H, S-C1H3); 4.08 (s, 1H, –OH); 2.87 (d, 3H, –CH3).

4: Orange, m.p. 123–124°C, yield 62%. Anal. Calc. for C18H29

-MoN3O5S (495.45 g/mol): C, 43.64; H, 5.90; N, 8.48; S, 6.47. Found:

C, 43.61; H, 5.85; N, 8.43; S, 6.40%. UV–Vis (nm, log

e

in parenthe-sis): 257 (4.33); 304 (4.33); 345 (4.04); 442 (4.55). IR (ATR, cm
1): ʋ(OH) 3399; ʋ(N4_{H) 3319;ʋ(CH)} aliph.3006, 2926, 2855;ʋ(C = N1) 1653;ʋ(C = N2_{) 1561;ʋ} s,ʋas(MoO2) 928, 891.1H NMR (DMSO d6): 8.44 (s, 1H, CH = N1); 9.04 (s, 1H, N4H); 7.20 (d, 1H, c); 7.10 (d, 1H, b); 6.74 (d, 1H, a); 3.32 (s, 3H, –OCH3); 3.03 (t, J:6.83 J:7.32 2H, S-C1_H 3); 1.65–1.24 (m, 12H, –C2-7H2); 0.86 (m, 3H, -C8H3); 4.07 (m, 1H, –OH); 3.16 (d, 3H, –CH3). 2.4. Antibacterial activities

The antibacterial activities of the compounds were determined by agar well diffusion method. Muller-Hinton agar was used as growth media. Petri dishes and agar solution were sterilized at 121 °C for 15 min. The hot agar solution was poured into petri dishes under in a limner-flow. The prepared agar plates were kept in an incubator for 24 h to check them for the contamination of unwanted bacteria. Four holes were made in each plate with sterile crock borer. In one hole standard antibiotic solution was poured

while in the other three the synthetic compound solutions. Each plate was inoculated with different bacteria. Then these plates were kept in incubator for 24 h at 37°C. The mean diameter of zone of inhibition in each plate was measured.

2.5. Antioxidant potentials 2.5.1. DPPH assay

For the determination of free radical scavenging ability of the compounds 2,2- Diphenyl-1-picrylhydrazyl (DPPH) assay modified by Brand-Williams et al with some modification was used[27].

In methanol, oxidized form of DPPH gives deep violet color. The donation of electron by antioxidant compound to DPPH radical causes its color change from deep violet to yellow. DPPH solution was prepared by dissolving 20 mg in 100 ml of methanol (stock solution) and then from this solution 3 ml was taken and adjusted its absorbance at 0.75 at 515 nm (control solution). This stock solu-tion was then covered with aluminum foil and kept in dark place for 24 h for the formation of free radical in it. To prepare com-pounds stock solution, 5 mg of each compound were dissolved in 5 ml of methanol (5000mg/ 5 ml). From this stock solution different diluted solution such as 1000, 500, 250, 125, 62.5mg/ml were pre-pared through serial dilution. From these diluted solution 2 ml was mixed with 2 ml solution of DPPH and incubated for 15 min dark. The percent scavenging abilities of the compounds were estimated using the following formula:

%Radicalsca

v

engingacti

v

ity¼A
B

A X100 ð1Þ

Where A is the absorbance in oxidized form of pure DPPH and B is the absorbance of sample which is taken after 15 min of reaction with DPPH.

For the determination of IC50values of the compounds, from

standard ascorbic acid solution (5 mg/5 ml) similar dilutions as mentioned above were prepared and treated with DPPH solution. 2.5.2. ABTS assay

The 2,2-azinobis-[3-ethylbenzthiazoline]-6-sulfonic acid (ABTS) free radicals scavenging assay was also used to determine the antioxidant potential of the compounds. For the preparation of ABTS and potassium per sulphate solutions, 7 mM ABTS and 2.45 mM K2S2O8 solutions were prepared in 100 ml methanol.

These two solutions were mixed thoroughly and kept in dark over night for producing free radicals. After incubation, 3 ml were taken and by the addition of 50% methanol, its absorbance was adjusted to 0.75 at 745 nm. About 300

l

l of test samples were taken and mixed well with 3 ml of ABTS solution and incubated for 15 min at 25°C. The absorbance of the obtained solution was measured using a double beam spectrophotometer at 745 nm. The same pro-cedure was followed for the preparation of different dilution of ascorbic acid used as positive control. The data was recorded in triplicate and percent.

2.6. Anticholinesterase activities

Acetyl cholinesterase (AChE) was used to examine the enzyme inhibitory potential of the compounds using Ell men’s assay

[28–29].

The hydrolysis of acetylthiocholineiodide or butyrylthiocholine iodide caused by AChE results in the formation of thiocholine which make complex with an anion formed from DTNB (5-thio-2-nitrobenzoate) resulting in a coloured complex which is detected after reaction time of 15 min by spectrophotometer. About 1 ml from each dilution as mentioned above, was mixed with 100ml of DNTB and AChE). The mixture was incubated for 15 min at room temperature. Then 100ml of substrate (acetylcholine iodide) was

put in each sample and incubated for additional 15 min. The absor-bance of the mixtures after incubation were recorded at 412 nm. Equations(2)–(4)were used to calculate enzyme inhibition (Ei).

Rateofreaction¼

D

_D

A

T ð2Þ

Eað%Þ ¼ Rateofreaction

Maximumrateofreactionx100 ð3Þ

Eið%Þ ¼ 100
%Ea ð4Þ

Where, Ea = Enzyme activity, Ei = Enzyme inhibition,

A = Absorbance, V = reaction rate in the presence of inhibitor, and Vmax= reaction rate without inhibitor.

2.7. Alpha-amylase inhibition

The

a

-amylase inhibition was estimated from the decrease in the amount of reducing sugar (maltose equivalent) liberated under the assay conditions. A modified dinitrosalicylic acid (DNS) method was used in this study[28–29]. About 1 ml of complex solutions were pre-incubated with

a

-amylase (1 U/ml) for 30 min and there-after 1 ml (1% w/v) starch solution was added. The mixture was incubated at 37°C for 10 min. The reaction was stopped by adding 1 ml DNS reagent (12.0 g of sodium potassium tartrate tetrahy-drate in 8 ml of 2 M NaOH and 96 mM 3, 5- dinitrosalicylic acid solution) and the contents were heated in a boiling water bath for 5 min. A blank was prepared without compounds and another without the amylase enzyme, replaced by equal quantities of buf-fer (20 mM Sodium phosphate bufbuf-fer with 6.7 mM Sodium chlo-ride, pH 6.9 at 20°C). The absorbance was measured at 540 nm. The reducing sugar released from starch was estimated as maltose equivalent from a standard graph. Acarbose was used as positive control. The compounds were diluted in buffer to give a final con-centration of 5, 7, and 9 mg/mL. The anti-diabetic potential was calculated using relation:

%inhibition ¼ ðmaltoseÞtest

ðmaltoseÞcontrolX100 ð5Þ

2.8. Alpha-glucosidase inhibition

The yeast alpha glucosidase was dissolved in 100 mM phos-phate buffer (pH 6.8) and was used as the enzyme extract.

P-nitro-phenyl-

a

-D-glucopyranoside was used as the substrate.

Compounds were used in the concentration ranging from 125 to 1000

l

g/ml. Different concentrations of compounds were mixed with 320

l

l of 100 mM phosphate buffer at 30°C for 5 min. About 3 ml of 50 mM sodium hydroxide was added to the mixture and the absorbance was recorded at 410 nm. The control samples were prepared without compounds. The % inhibition was calculated using following relation:

Inhibitionð Þ ¼% Controlabsorbanceat410
Sapleabsorbanceat410 Controlabsorbanceat410

ð6Þ

Acarbose was used as the reference alpha glucosidase inhibitor. 2.9. DNA binding activities

Fixed concentration of the compounds were mixed with differ-ent concdiffer-entrations of DNA in the presence of sodium dihydrogen phosphate buffer (pH = 7.5).

Two beaker marked as A and B were taken and each one was charged with 14 ml solution of the respective complex. Their pH

were maintained at 7.5 by addition of 1 ml phosphate buffer. About 5 ml DNA solution was added to beaker A while 5 ml dis-tilled water to beaker B. The volume in both beakers were kept similar (20 ml). About 5 ml from beaker A having DNA was trans-ferred to 5 ml volumetric flask and was labeled as solution 1. To keep the volume same in both beakers, about 2.5 ml was trans-ferred from beaker B into beaker A as a result the volume in both beaker become equal to 17.5 ml. Now again 5 ml from beaker A was transferred to 5 ml volumetric flask and was labeled as solu-tion 2. In the mensolu-tioned manner 2.5 ml was transferred from beaker B into beaker A to keep the volume same (15 ml). The same procedure was repeated and a total eight solutions (1–8) were prepared. Each solution having different concentration of DNA in such a way that solution 1 has maximum DNA and solu-tion 8 has no DNA. After preparasolu-tion of all solusolu-tions their absor-bance was determined with UV–Vis spectrophotometer in the range of 200 to 800 nm.

3. Results and discussion

3.1. Some physical properties of the compounds

The colour of free ligands, obtained as crystalline powders, are light yellow. They are soluble in common solvents such as alcohols and DMSO. The reaction of the ligands with MoO2(acac)2in

metha-nol yielded diamagnetic complexes, [MoO2(L)CH3OH]. The

com-plexes are soluble in methanol, chloroform and dichloromethane. The orange colored complexes are stable in the air. They transform to black coloured amorphous materials at reflux temperature of solvent a few hours later.

3.2. IR and1_{H NMR spectra}

The spectroscopic data of the thiosemicarbazone ligands and their dioxomolybdenum (VI) complexes clearly proved the forma-tion of the molybdenum centered ONS and ONN chelate complexes containing methanol in 6th coordination site.

For ONS type complexes (1 and 2), the new C = N2 _band

appeared at 1556–1559 cm
1 supports that the sulfur atom

participates in coordination after conjugation. Characteristic ʋs

andʋasbands of the cis-MoO22+group were observed in the range

of 885–892 and 928–934 cm
1, respectively[23,30].

In 1_{H NMR spectra of thiosemicarbazones, the proton of the}

phenolic hydroxyl group appeared as a singlet between 11.41 and 11.06 ppm. The absence of this peak in complex spectra shows that the coordination to the molybdenum center is via phenolic oxygen. The imine group (CH = N) of the thiosemicarbazones appeared at 8.26–8.43 ppm.

The signals at 9.45 ppm and 8.44–8.45 ppm can be assigned to the protons of N2H and N4H groups in thiosemicarbazones having the ONS donor set (L1 and L2) , respectively. The disappearance of the signal belonging to the N2_{H group in the complex spectra}

point-ing toward the coordination of thiosemicarbazones via sulfur atom,

leading to the formation of N2 _{= C due to the thion-thiol}

tautomerism[31].

When the spectra of the ligands with ONN donor set (L3 and L4) were examined, the chemical shift values of the N4_{H proton was}

recorded at 6.78 and 6.81 ppm. With complexation, the deprotoniza-tion of the N4_{H group and the shifting to lower field (9.04 and}

9.05 ppm) reveals that the ligand system is coordinated to the diox-omolybdenum (VI) center via the nitrogen atom of amide moiety. New signals have been observed for –OH and –CH3group of

coordi-nated methanol molecule at 4.06–4.08 ppm and 2.80–3.18 ppm, respectively

3.3. Crystallography

The solid-state structure of complex 3 have been determined using single crystal X-ray analysis. The ortep diagram of the non-centrosymmetrical dimer-like structure is given in Fig. 1, while

Fig. 2 showing hydrogen bonds and unit cell. Crystal data and structure refinement details are summarized in Table 1, while the bond lengths and angles for atoms that contribute to the hydrogen bond interactions are given inTable 2. Crystallographic studies indicates that the dioxomolybdenum (VI) complex has a triclinic crystal systemFig. 3.

The unit cell consists of two non-centrosymmetrical dimer-like structures equal to four complexes. There are significant hydrogen bonding interactions between the molecules that constitute this unsymmetrical dimer-like structure. The strongest hydrogen bond between the two molecules are O8-H10 N2 with HA with a dis-tance of 2.11 Å. In addition, the weaker interactions of C1-H9CO8 and trifurcated hydrogen bondings including O2 atom also hold the two molecules of dimer-like system together. As can be seen in

Fig. 2, the strongest hydrogen bonding interactions between the two dimeric systems are O3-H2N5 with HA having distance of 2.12 Å and N1-H3O1 with HA having distance of 2.30 Å. The others interactions of weakest type were observed between C12-H17BO3 and trifurcated hydrogen bondings including O6 atom.

The coordination environment of the molybdenum center can be described as distorted octahedral geometry. The thiosemicar-bazone molecule coordinates to the center of cis-MoO2 through

the oxygen atom of the phenolic oxygen, amidic nitrogen atom and oxygen atom of methanol molecule. The angle values of

Fig. 2. ORTEP diagram of non-centrosymmetrically dimer-like complex 3 showing the numbered atoms. Thermal ellipsoids are at 30% probability level. Table 1

Crystal data and structure refinement for complex 3. Parameters

CCDC deposition no. 1,945,704

Color/shape Orange/rod

Chemical formula C11H15MoN3O5S Formula weight (g.mol
1) 397.28 Temperature (K) 296(2) Wavelength (Å) 0.71073 Crystal system triclinic

Space group P
1

Unit cell parameters

a, b, c (Å) 7.5778(3), 12.0202(5), 16.2909(6) a,b,c(°) 90.0030(10), 89.9880(10), 81.6650 (10) Volume (Å3 ) 1468.21(10) Z 4 Dcalc(g.cm
3) 1.797 Absorption correction multi-scan Tmin, Tmax 0.9010, 0.9890 F(0 0 0) 800 Crystal size (mm) 0.010 0.030  0.100 Diffractometer/measurement method Index ranges h,k,l
9h10,
15k15,
21l21 h Range for data collection (°) 5.000° < 2h < 55.43°

Reflections collected 52,889 Independent reflections 7236

Rint 0.0453

Refinement method Full-matrix least-squares on F2 Data/restraints/parameters 7236 / 0 / 394

Goodness-of-fit on F2

1.960

Final R indices [I > 2 s (I)] R1= 0.0319, wR2= 0.0583 R indices (all data) R1= 0.0597, wR2= 0.0668

O6-Mo1-O7 and O2-Mo-O1 were 105.89 and 105.87 respectively, proving the cis nature of structure as has been observed in similar studies[31,32].

3.4. Antibacterial activities

The synthesized compounds were tested for their antibacterial potentials by agar well diffusion method. Three bacterial strains E. coli, K. pneumonia and S. typhi were used in this study. The results have been shown inTable 3.

Comparatively all the compounds were more potent against S. typhi as an appreciable zone of inhibition has been produced.

Againt S. typhi complex 2 (28 ± 0.326 mm) and 4

(23 ± 0.082 mm) were more potent. Amongst the ligands, L3 exhib-ited the highest antibacterial spectrum against K. pneumonia (29 ± 0.025 mm) followed by L1 (27 ± 0.653 mm).

The attachment of metal to ligand has only enhanced the activ-ities in case of ligand L2 (22 ± 0.045 mm) and L4 (16 ± 0.008 mm) as the corresponding complexes 2 (28 ± 0.326 mm) and 4 (23 ± 0.082 mm) have shown enhanced activity against K. Pneumo-nia. In case of the rest of ligands a decrease in antibacterial spec-trum with metal attachment was observed.

All these compounds were also effective against E. coli. Amongst complexes, complex 2 exhibited highest zone of inhibition of about

28 ± 0.082 mm followed by complex 1. The ligand L1

(25 ± 0.816 mm) was more potent against this bacteria followed by L4 (24 ± 0.326 mm). Only in case of L2 enhanced activity were observed on complexation with metal. A decreased activity in case of the rest of ligands on complexation with metal have been observed. Comparatively the S. typhi was inhibited to lesser extent by these compounds. L2 (25 ± 0.408 mm) and complex 2 (26 ± 0.245 mm) were the more potent inhibitors of S. typhi. Here also the enhanced activities have been observed on complexation of metal with ligand.

3.5. Free radical scavenging activities

Reactive oxygen species (ROS) are constantly produced during metabolism of aerobic cells that can damage bio-molecules like proteins, lipids, enzymes, DNA and RNA. They are involved in the progression of different chronic diseases like cancer, atherosclero-sis, cardiovascular diseases, diabetes mellitus, rheumatism,

nephri-tis, ischemic, Alzheimer’s disease (AD) and Parkinson’_s

neurodegenerative disease[33–35].

Table 2

Hydrogen bond parameters (Å ando

) of compound 3.

D—HA D—H HA DA D—HA

N1-H3O1 0.72(3) 2.30(2) 2.99(3) 163(3) O3-H2N5 0.66(3) 2.12(3) 2.77(3) 173(4) C4-H1BO2 0.960 2.643 3.359(4) 131.7 C14-H13O2 0.931 2.690 3.404(3) 134.1 C16-H14O2 0.930 2.710 3.403(3) 132.0 C12-H17BO3 0.959 2.704 3.375(3) 127.5 C7-H6O6 0.930 2.709 3.405(3) 132.4 C22-H11AO6 0.960 2.628 3.356(4) 132.9 C3-H7O6 0.929 2.692 3.406(3) 134.2 O8-H10N2 0.66(3) 2.11(3) 2.76(3) 172(3) C1-H9CO8 0.960 2.703 3.37(3) 127.4

Fig. 3. The molecular packing arrangement of complex 3 showing hydrogen-bond network depicted as dotted lines and unit cell. Table 3

Antibacterial activities of compounds against selected bacterial strains. Compound Mean of zone of diameter of inhibition (mm)

E. coli K. pneumonia S. typhi

L1 25 ± 0.816 27 ± 0.653 19 ± 0.489 L2 20 ± 1.630 22 ± 0.045 25 ± 0.408 L3 22 ± 0.163 29 ± 0.025 19 ± 1.630 L4 24 ± 0.326 16 ± 0.008 18 ± 0.735 1 23 ± 0.408 21 ± 0.044 19 ± 0.008 2 28 ± 0.082 28 ± 0.326 26 ± 0.245 3 19 ± 0.245 14 ± 0.045 17 ± 0.489 4 22 ± 0.489 23 ± 0.082 19 ± 0.816 Ofloxacin (Standard) 30 ± 0.735 33 ± 0.163 290.653

The antioxidant potentials of the compounds determined through DPPH and ABTS methods are shown inTable 4.

The antioxidant potential of these compounds are moderate. The lowest IC50in case of complexes was obtained with complex

2 (IC50= 231

l

g/mL) where the metal attachment have enhanced

the antioxidant potential of L2. Ligand L1 (IC50 = 250

l

g/mL)

more potently inhibited DPPH free radical followed by L2 (IC50= 392

l

g/mL). Against the ABTS free radical, L1 (IC50=

350-l

g/mL) and complex 2 ((IC50 = 375

l

g/mL) were comparatively

more potent than the other tested compounds. The antioxidant potential of L2 and L4 on complexation increased with metal com-plexation. The differences amongst IC50values of ABTS and DPPH

assays are due the fact that these two radical are dissolved in dif-ferent solvents and the solvent effect cannot be neglected as both methods are based on the transfer of an electron from the depro-tonated antioxidant to the probe with mechanism known as sequential proton loss electron transfer. The number of exchanged

electrons in the reactions with chromogenic radicals is dependent on solvent composition, pH of reaction media, length of assay, and chemical structure of the antioxidant. The number exchangeable electron in different media are different.

3.6. Cholinesterase inhibition

Alzheimer’s disease is special type of dementia. In most of dementia patients the enhanced activities of acetylcholinestarases leads to deficiency of acetylcholine. The strategy followed to treat such complication is the inhibition of acetylcholinesterases. About 5 inhibitors of this enzymes are used as clinical practices to treat dementia [29,36–39]. The anticholinesterase activities of the complexes are given inTable 5.

Amongst ligands L4 (IC50= 149

l

g/mL) and L2 (IC50= 200

l

g/mL)

were potent inhibitors of the selected enzyme. The more powerful inhibitor of this enzyme was the corresponding complex of ligand L4 (complex 4) which more potently inhibited AChE with IC50of

104

l

g/mL. Here also enhanced activities with metal complexation have been observed.

3.7. Antidiabetic potentials

Diabetes mellitus (DM) is a chronic metabolic disorder charac-terized by hyperglycemia and impaired carbohydrates, lipids and proteins metabolism [27,28]. In diabetes mellitus either insuffi-cient amounts of insulin is secreted by pancreatic islet cells of Langerhans or there is insulin resistance leading to increase in blood glucose level. It is a major chronic diseases in human after cancer and cardiovascular diseases[29,36–39].

Table 4

% DPPH and ABTS radical scavenging potential of the compounds. Samples Concentrations (lg/mL) DPPH Percent inhibition (mean ± S.E. M) DPPH IC50 (lg/ mL) ABTS percent inhibition (mean ± S.E. M) ABTS IC50 (lg/ mL) 1000 69.15 ± 0.99 250 63.37 ± 2.45 350 500 63.21 ± 1.52 57.33 ± 0.67 L1 250 49.41 ± 0.92 43.00 ± 1.00 125 43.27 ± 1.2 36.33 ± 0.77 62.5 35.55 ± 0.90 23.45 ± 1.52 1000 66.51 ± 0 0.83 392 57.26 ± 1.03 680 500 50.66 ± 1.34 44.05 ± 0.77 L2 250 35.68 ± 1.47 28.30 ± 2.33 125 28.45 ± 0.78 21.97 ± 1.09 62.5 23.03 ± 0.69 13.10 ± 0.50 1000 65.18 ± 0.67 395 57.15 ± 0.44 473 500 51.50 ± 0.55 48.33 ± 2.33 L3 250 45.02 ± 0.57 37.64 ± 0.58 125 28.37 ± 0.65 23 0.62 ± 0.87 62.5 11.56 ± 2.38 06.03 ± 1.89 1000 54.27 ± 0.72 820 46.50 ± 0.76 978 500 32.49 ± 1.58 29.00 ± 1.08 L4 250 13.04 ± 1.88 08.16 ± 0.70 125 9.31 ± 0.67 05.06 ± 2.38 62.5 4.41 ± 1.45 1.04 ± 1.54 1000 60.55 ± 0.71 398 52.84 ± 0.47 477 500 54.32 ± 0.60 47.33 ± 0.78 1 250 42.22 ± 1.66 36.22 ± 0.46 125 31.05 ± 1.53 22.01 ± 0.52 62.5 12.12 ± 0.64 07.25 ± 0.36 1000 72.45 ± 1.45 231 65.03 ± 0.88 375 500 66.07 ± 0.57 60.66 ± 0.81 2 250 45 0.04 ± 1.46 43.02 ± 0.44 125 31.08 ± 0.73 24.11 ± 0.66 62.5 15.50 ± 3.45 09.64 ± 2.39 1000 55.94 ± 1.86 600 49.67 ± 0.78 497 500 47.29 ± 0.79 42.45 ± 0.96 3 250 34.93 ± 0.45 29.34 ± 2.16 125 23.90 ± 0.48 16.90 ± 0.85 62.5 09.88 ± 0.32 03.12 ± 1.99 1000 58.44 ± 0.86 596 50.67 ± 1.73 640 500 52.29 ± 0.79 44.45 ± 0.96 4 250 46.93 ± 0.45 41.34 ± 2.16 125 32.90 ± 0.48 26.90 ± 0.85 62.5 17.88 ± 0.32 10.78 ± 01.76 Ascorbic acid 1000 91.50 ± 0.23 87.41 ± 0.34 500 88.65 ± 0.45 83.65 ± 0.65 250 84.35 ± 0.88 25 76.78 ± 1.41 45 125 70.56 ± 1.16 65.80 ± 1.45 62.5 52.65 ± 0.87 49.88 ± 0.56 Table 5

Acetyl cholinesterase inhibition of the compounds. Sample Concentration mg/ml Percent AChE (mean ± SEM) AChE IC50 (mg/ml) 1000 62.43 ± 1.32 L1 500 54.65 ± 0.75 400 250 39.56 ± 1.95 125 31.54 ± 0.82 1000 67.34 ± 1.45 200 L2 500 63.56 ± 0.13 250 51.83 ± 0.32 125 43.41 ± 1.43 1000 70.14 ± 1.18 300 L3 500 51.91 ± 0.19 250 47.21 ± 0.19 125 32.84 ± 0.69 1000 80.97 ± 0.75 149 L4 500 71.21 ± 0.98 250 57.41 ± 0.56 125 44.13 ± 0.81 1000 60.12 ± 0.73 496 1 500 49.15 ± 1.04 250 31.75 ± 2.13 125 20.21 ± 1.11 1000 72.91 ± 0.04 220 2 500 63.89 ± 1.13 250 51.18 ± 0.97 125 41.30 ± 1.42 1000 58.92 ± 1.83 390 3 500 55.68 ± 0.12 250 41.02 ± 0.15 125 36.90 ± 0.12 1000 81.12 ± 1.11 104 4 500 70.91 ± 0.63 250 57.01 ± 1.20 125 50.18 ± 0.54 1000 90.56 ± 0.34 Galantamine 500 83.76 ± 0.98 45 250 76.88 ± 0.36 125 66.77 ± 0.85

The antidiabetic potential of the ligands and complexes (L1-4 and 1–4) were tested using

a

-amylase and glucosidase as marker enzymes.Table 6shows the inhibitory effects of the compounds on

a

-amylase and glucosidase. Comparatively these compounds were more potent inhibitors of

a

-amylase as compared to

a

-glu-cosidase. Amongst complexes with an IC50value of 153mg/ml

com-plex 4 was most powerful inhibitor of

a

-amylase. Amongst ligands L2 and L4 with IC50value of 210 and 255mg/ml respectively were

more potent inhibitors of alpha amylase. Enhancement in antiamy-lase activities have been observed for L1 and L4 upon complexa-tion with metal.

Against glucosidase the inhibition was moderate and amongst ligands L4 with IC50value of 285mg/ml was more potent inhibitor

while amongst complexes, complex 2 (IC50= 295mg/ml) was the

more potent inhibitor of this enzyme. The activities of L1 and L2 have been enhanced by metal complexation.

3.8. DNA interactions

The way through which the compounds interacts with DNA was determined through UV–Visible spectroscopy in the present study. In this technique the change in both; wavelength and absorbance are monitored after the addition DNA. The vales of Kb(binding

con-stant) are obtained using the Benesi-Hildebrand equation[39]:

Ao A
Ao¼

e

G



H
G



Gþ



G



H
G



G X 1 Kb½DNA ð7Þ

Where, A0= absorbance’s of pure drug, A = absorbance’s of

DNA-complex, Kbis complex binding constant,ƐGandƐH–G= absorption

coefficients of drug and drug–DNA complex respectively. The hyp-sochromic shifts along with binding constants are given inTable 7. The values of Kb in between 103 to 106 shows non covalent

interactions of types; electrostatic, groove binding and intercala-tion[39–43]. InTable 7all values are in between 103_{to 10}6

there-fore these compounds noncovalently interacts with DNA. Ligand L2 exhibited more effective interaction with DNA (binding constant value of 7.09 105_M
1_{) while weakest interaction was observed}

for L3 amongst the ligands. Almost similar interactions were observed for all the synthesized complexes.

Usually, with increase in DNA concentration red or blue shifts are observed with hypo or hyperchromism. Blue shift with hypo-chromism indicates electrostatic interactions while with

hyper-chromism indicates mix intercalation and groove binding

interactions and red shift with hypochromism indicate intercala-tion mode of interacintercala-tions while with hyperchromism shows elec-trostatic mode of interactions. Red shift with hypochromism

Table 6

Alpha amylase and glucosidase inhibition of the compounds.

Sample Concentrationmg/ml Percent Amylase (mean ± SEM) IC50(mg/ml) Percent Glucosidase (mean ± SEM) IC50(mg/ml)

1000 51.13 ± 1.02 480 55.73 ± 1.23 487 L1 500 44.14 ± 0.05 50.47 ± 1.95 250 37.49 ± 1.93 37.57 ± 0.69 125 32.04 ± 1.12 28.13 ± 0.89 1000 68.19 ± 1.41 210 61.42 ± 0.57 302 L2 500 60.99 ± 0.37 54.97 ± 0.68 250 56.16 ± 3.47 47.76 ± 2.47 125 39.13 ± 1.03 39.78 ± 1.48 1000 71.13 ± 2.11 300 63.89 ± 0.97 395 L3 500 65.91 ± 1.97 56.12 ± 1.49 250 44.44 ± 1.42 41.34 ± 2.10 125 39.43 ± 2.10 35.65 ± 0.99 1000 73.12 ± 0.48 255 65.43 ± 0.76 285 L4 500 56.10 ± 1.35 58.87 ± 2.65 250 49.11 ± 3.60 49.43 ± 1.40 125 35.13 ± 2.56 42.98 ± 1.93 1000 75.12 ± 1.13 282 55.98 ± 1.46 479 1 500 61.49 ± 1.24 49.64 ± 2.93 250 44.91 ± 2.13 32.68 ± 2.57 125 35.19 ± 1.11 15.49 ± 1.87 1000 66.11 ± 1.94 395 61.49 ± 1.84 295 2 500 51.15 ± 1.30 53.32 ± 1.98 250 46.01 ± 1.31 42.67 ± 1.68 125 31.33 ± 1.06 24.76 ± 2.54 1000 60.74 ± 1.11 500 55.65 ± 0.72 798 3 500 52.66 ± 2.53 47.63 ± 2.78 250 44.72 ± 1.23 24.56 ± 0.77 125 35.68 ± 0.64 18.54 ± 1.95 1000 82.93 ± 1.53 153 56.94 ± 0.66 491 4 500 74.02 ± 2.75 44.20 ± 2.94 250 68.41 ± 0.15 35.01 ± 1.53 125 54.11 ± 1.84 19.98 ± 2.62 1000 68.40 ± 0.22 6.34 92.76 ± 0.52 35 Acarbose 500 62.76 ± 0.76 83.34 ± 0.77 250 58.12 ± 0.60 73.98 ± 0.44 125 46.78 ± 0.66 0.14 Table 7

DNA interaction parameters of complexes.

Sample Observed shift atkmax(nm) Binding constant Kb(M
1)

L1 630 9.20x104 L2 595 7.09 105 L3 588 5.60x103 L4 658 7.58x104 1 580 3.60 104 2 585 4.67x104 3 618 2.04x104 4 650 3.03 104

have been observed for L1, L4 and complex 3 while the rest of com-plexes blue shifts with hyperchromism have been observed. 4. Conclusion

Four new dioxomolybdenum (VI) complexes, [MoO2(L)CH3OH],

with ONS and ONN chelating thiosemicarbazone ligands were syn-thesized with appreciable yield. The compounds were investigated in context of effect of the donor set and N- or S- substituents, methyl and octyl, and biological efficiencies. In this context, the antibacterial, enzyme inhibition, DNA binding, and antioxidant capabilities of ligands and complexes were determined. The ligands and complexes were quite effective against selected bacte-rial strains. As antibactebacte-rial agent ligand L3 was highly active against K. pneumonia while complex 2 against E. coli and S. typhi. In cholinesterase inhibition results, IC50value of complex 4

signif-icantly distinguished from the others and approximated to that of standard Galantamine. In

a

-amylase and glucosidase inhibition experiments, the ligands and complexes bearing octyl chain exhib-ited the better IC50values than the methyl-substituted ones. The

antioxidant and antidiabetic potentials of them were moderate however, anticholinesterase activities were comparable. Spectro-scopic data indicated non-covalent interaction between DNA and the ligand and complexes. Interestingly, DNA binding constants of the structures containing N- and S-octyl were in higher values than the others. Considering the enzyme inhibition and DNA bind-ing results, this interaction tendency of the octyl-substituted thiosemicarbazone-based compounds with the studied macro-bio-molecules may be evaluated as a valuable output for drug researches. Further, in vivo studies are needed to confirm the bio-logical potentials observed in this study about these complexes. Declaration of Competing Interest

The authors declare that they have no known competing finan-cial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

This study was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project Number: 216Z044). Appendix A. Supplementary data

Supplementary data to this article can be found online at

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