Aqeela Shaheen1,2, Shahbaz Akhtar3, Sajjad Ahmad1, Aziz-ur-Rehman1*, Abdul Saeed4, Adnan Sharif1, Mujahid Mustaqeem1Muhammad Nawaz5 and Syed Tahir Abbas Shah6

1Department of Chemistry, The Islamia University of Bahawalpur, 63100, Bahawalpur Pakistan.
2Department of Chemistry, Government Sadiq College Women University Bahawalpur, 63100, Bahawalpur Pakistan.
3Department of Biochemistry and Biotechnology, The Islamia University of Bahawalpur, 63100, Bahawalpur, Pakistan.
4Institute of Chemical Sciences, Bahauddin Zakariya University, Multan, 60800, Multan Pakistan.
5Department of Environmental Sciences, Bahauddin Zakariya University, Multan, 60800, Multan, Pakistan.
6Department of Biosciences, COMSATS Institute of Information Technology,
Islamabad. 45550, Islamabad Pakistan.

Abstract

An efficient and facile method for the synthesis of 2-hydroxy-N‘-{(E)-[6′-(2H-1″,2″,3″-triazol-2″-yl)pyridin-3′-yl]methylidene}benzohydrazide via N-arylation is described. The synthesized ligand was further coordinated with a variety of organotin(IV) salts. Structural elucidations of ligand and the complexes thus formed were made by CHN, IR, NMR and MS. The newly synthesized compounds were also screened for antifungal, antibacterial, cytotoxic and antiurease activities.Thecompounds 3,7,9 showed best antifungal activity against various strains while diorganotin(IV) moieties (7,8,10) showed excellent antibacterial activities. Compound 3 and 9 remained good antiurease and cytotoxic agents.

Keywords :N-arylation, organotin(IV), 1,2,3-triazol, 2-hydroxy-benzohydrazide.

Introduction

The ligand design, its synthesis and characterization plays a significant role in addressing and transporting the molecule to the target, interactions with biomolecules and pathogenic resistance[1]. This growing field has always been at the esteem of synthetic, biological and medicinal perspectives.Among the organic compounds/ligands, the Schiff bases particularly derived fromaroylhydrazonesare regarded as the most privileged class of ligands owing to their versatile synthesis, novel structural features & flexibility and superior metal chelating ability.Aroylhydrazones, mostly can exhibit keto-enoltautomerism(Figure-1), that is primarily due to the solvent/reagent effect, temperature change, UV irradiations or pH change. These isomers can impart diverse chelating properties [2] and are proved to be the best antimicrobial, antioxidant,anti-inflammatory,antitumour, antitubercular, anticoagulants, anticonvulsant agents[3-5].Also the metal complexes of hydrazones are proved to be the best luminescent probes[6], nonlinear optics, catalysts[7]and molecular sensors[8] etc.

     A                                                     B                                                     

R = substituted or non-substituted Alkyl or Aryl groups

Figure-1 : Possible tautomeric forms of aroylhydrazones and E/Z configuration with respect to the C=N double bond

 

The biocidal activities of the ligands increases upon chelation with suitable metal ions as it promotes their target oriented nature [2]. In this regard, organometallic derivatives/complexes of p-block elements, preferentiallybelonging to group 14, (e.g. tin(IV) and silicon(IV)), have been of significant interests.Organotin(IV) complexes with Schiff base ligands have fascinating chemical behavior, kinetically stable, relatively lipophilic in nature and less toxic than that of platinum drugs. Also the phenyl- and n-butyltin(IV) complexes display a larger array of biological activities as compared to their methyl-, hexyl- or octyltin(IV) analogs[9, 10]. Although there are several limitations of tin metal complexes used as pharmacological agents, like high cost, toxicity, immunogenicity, low lipid solubility, little penetration into cellsetc.But thesetherapeutic agents still include a plethora of compounds that are mostly antitumor, antimicrobial, antioxidant, antiflammatoryand radioprotectants[11].

On chelation the polarity of the tin metal declinesto a much greater extent, this is due to the overlap of the ligand orbital/orbitals, partial sharing of the positive charges of the metal ionsand delocalization This delocalization over the whole chelate ring enhances the lipophilic characters of the complexes. That actually promotes the penetration of the complexes into lipid layer of cell membrane and blocks the active sites on enzymes of microorganisms[12, 13]. As a result of which the macromolecular or DNA synthesis inhibition, reduction of mitochondrial energy metabolism or elevation in oxidative and DNA damage occurs. Therefore, the organotin(IV) compounds appear to be very promising as potential drugs particularly active against cancers.[14].

Experimental

Chemicals and reagents used in this study were research grade products and purchased from the commercial sources like Merck and Aldrich. These were used either without any further purification or purified where necessary. Analytical grade solvents were dried via methods cited in the literature before use. To determine melting points of the synthesized moieties, GallenKemp melting point apparatus was used and are uncorrected. Bruker Tensor 27 Fourier Transform Infra-Red Spectrophotometer was used in the range of 4000-400 cm-1 to collect IR spectral data. The elemental analyses (CHNS) were carried out on Carlo Erba 2400 automatic analyzer. Multinuclear NMR (1H and 13C) spectra were recorded on Varian MR Instrument at 300 MHz and/or 400 MHz in DMSO, CDCl3 or deutrated acetone using tetramethylsilane (TMS) as internal standard. The mass spectra were recorded on JEOL JMS 600-H series (EBE) MS spectrometer.

Synthesis of 6-(2′H-1′,2′,3′-triazol-2′-yl)pyridine-3-carbaldehyde(1)

N-arylation of 2H-1,2,3-triazole was co-reacted with 6-chloropyridine-3-carbaldehyde(Scheme-1), via pre-described method in literature [15]. Briefly, a mixture of 2H-1,2,3-triazole (0.1 mmol), anhydrous potassium carbonate (0.1 mmol), 6-chloropyridine-3-carbaldehyde(0.1 mmol), hexadecyltrimethylammonium bromide (20 µg) and dioxin (50 ml) were refluxed for the period of 18-22 hours. The progress of the reaction was monitored with the help of TLC. The precipitates of the required product were obtained in crushed ice. That were filtered, dried and recrystallized in suitable solvents like n-hexane etc.

Scheme-1 : Preparation and numbering scheme of 6-(2′H-1′,2′,3′-triazol-2′-yl)pyridine-3-carbaldehyde

Synthesis of 2-hydroxy-N‘-{(E)-[6′-(2H-1″,2″,3″-triazol-2″-yl)pyridin-3′-yl]methylidene}benzohydrazide (2)

 

The aldehyde (1) thus formed via N-arylation was further reacted with 2-hydroxybenzohydrazide following a procedure quoted in literature [16] (Scheme-2). Briefly, an equimolar (1mmol) mixture of aldehyde (1) and 2-hydroxybenzohydrazidein acetone was reflux for the period of 3-4 hours, few drops of acetic acid was added from the side wall. The reaction outcomes were monitored with TLC, the resulting solution was poured into crushed ice and left overnight. Fine crystalline product was obtained.

Scheme-2 : Preparation and numbering scheme of 2-hydroxy-N‘-{(E)-[6′-(2H-1″,2″,3″-triazol-2″-yl)pyridin-3′-yl]methylidene}benzohydrazide

 

General procedure for the synthesis of complexes (3-11)

The Na-salts of the ligand i.e. 2-hydroxy-N‘-{(E)-[6′-(2H-1″,2″,3″-triazol-2″-yl)pyridin-3′-yl]methylidene}benzohydrazide (2A) were further reacted with various organotin(IV) chlorides (Scheme-3) following method reported by Hussainet al., [17]. Suitable dry solvents like toluene, methanol, n-hexane etc. were used for reactions to complete. All the complexes thus isolated were crystalline or amorphous solids, stable at room temperature, non-hygroscopic and having sharp melting points. The products so obtained were soluble in organic solvents like benzene, chloroform, DMF and DMSO. Further, 1H, 13C NMR, IR and mass spectrometry were carried out for their confirmation

Scheme-2 : Preparation and numbering scheme of 2-hydroxy-N‘-{(E)-[6′-(2H-1″,2″,3″-triazol-2″-yl)pyridin-3′-yl]methylidene}benzohydrazide

 

General procedure for the synthesis of complexes (3-11)

The Na-salts of the ligand i.e. 2-hydroxy-N‘-{(E)-[6′-(2H-1″,2″,3″-triazol-2″-yl)pyridin-3′-yl]methylidene}benzohydrazide (2A) were further reacted with various organotin(IV) chlorides (Scheme-3) following method reported by Hussainet al., [17]. Suitable dry solvents like toluene, methanol, n-hexane etc. were used for reactions to complete. All the complexes thus isolated were crystalline or amorphous solids, stable at room temperature, non-hygroscopic and having sharp melting points. The products so obtained were soluble in organic solvents like benzene, chloroform, DMF and DMSO. Further, 1H, 13C NMR, IR and mass spectrometry were carried out for their confirmation

Scheme-3 : Preparation of di- and tri-oraganotin(IV)carboxylates through Na-salt of 2-hydroxy-N‘-{(E)-[6′-(2H-1″,2″,3″-triazol-2″-yl)pyridin-3′-yl]methylidene}benzohydrazide

 

Biological Activities:-

Pertinent literature confirmed that, the organotin(IV) complexes depict remarkable in vivo and in vitrobiocidal and antimicrobial activities, also these are frequently be used in cancer chemotherapy because of their best apoptotic properties. Usually, in vitroantibacterial or fungicidal activities of the organotin(IV) follow the general order of activity as: RSnX3<R2SnX2<R4Sn R3SnX, where the anionic X exhibit littleinfluence on activity[18, 19]. Hubert et al. reported that the geometry and stereochemistry around tin atom while Hadjikakouet al., confirmed that it is the only ligand type not the geometry that is responsible for the antitumor activity of the organtin(IV) compounds [20-22]. Hence, to explore the therapeutic effects of the synthesized compounds various biological activities were performed.

 

Procedure for biological activities

Antifungal assay

Antifungal assay of the ligands and their organotin(IV) complexes was carried out against various fungal strains i.e. C. albicans, A. flavus, M. canis using Miconazole as a standard drug.

Procedure at 96 –Well Plates

Antifungal bioassay was performed by pre-described method [15]. Briefly, the test compounds were dissolved in 2% DMSO and 20µg/well of this were loaded into wells of 96 well plates. In each well, 180µl overnight preserved fungal culture was poured.A volume in each well was retained up to 200 µl. The whole mixture was then incubated at 25oC for 24 hours by covering micro-plate with a lid to ensure the aseptic environment. After incubation, the absorbance of each well was measured at 405 nm using ELISA micro-plate reader.

The percentage inhibition was calculated by employing formula,

Percentage inhibition =   100(X-Y)/X

X = Absorbance for control with fungal culture without any antifungal agent

Y= Absorbance for test sample with fungus

Cytotoxicity Assay

Cytotoxicity assay of the ligand (2) and its corresponding organotin(IV) complexes (3-11) was performed by evaluating inhibitory effects on the growth of Nauplii (brine shrimps),by using brine shrimp lethality method [23].The method includes following steps. The hatching tray was filled with artificial sea water (i.e. salt solution 38g/l) and a porous membrane used to divide the tray into two halves. Brine shrimp eggs were sprinkled in one half of the tray and covered with a lid. The second half of the hatching tray was left uncovered, exposed under artificial light for 1-2days at 30 ±30C till the eggs hatched and the larvae moved towards the illuminated part of the tray via porous membrane. 10mM solutions of test compound were prepared in DMSO. From which, a specific volume of the test solution was poured in a tube and left overnight for evaporation. To this tube, the salt solution was added and the volume made up to 5ml. To this sample tube, 2-4 days old Shrimps, (10 in number) were added via Pasteur pipette. The tubes were incubated overnight and maintained 30 ±3 0C under illumination. Number of the survivors wasrecorded and the cytotoxicity was determined as the percentage of the dead larvae as given below:

 

The salt solution served as the negative control and Etoposide (standard drug) act as positive control in this study. Results of the assay are given in Figure.2.

Antiurease Activity:-

The antiurease activity test was modified from Berthelot assay [24][25]. Briefly explained here as, in each well of 96-well plate, 200µl assay volume containing 55µl of phosphate buffer (0.2 M) with pH 7 followed by the addition of 10µl test solution and 10µl enzyme mixture (0.015 units) was used. All the contents were incubated at 37ºC for 10 minutes. Then 10µl of urea stock solution (80mM) was added in each well and incubated at 37ºC for further 10 minutes. A pre-reading was taken at 630 nm. After that 115µl of phenol hypochlorite reagent was added in each well. The Phenol hypochlorite reagent was made freshly by combining 45µl phenol solution with 70µl alkali solution. After incubation at 37ºC for 10 minutes, the color appeared. And the absorbance was taken at 630 nm using 96-well plate reader.

The solution having no sample served as a negative control. Wells containing thiourea served as positive control.

Antibacterial Assay:-

Antibiotic sensitivity test was performed using a standard disc diffusion assay method by using E.coli, B. subtilisand S. aureusas indicator target strains[26] Briefly described, 50µl compound were loaded onto a sterile filter-paper disc (6 mm in diameter). A 50µl simple broth was used as a negative control, and imipenem(20µg/ml) was used as positive control. After application onto paper discs, the discs were air dried and placed onto the nutrient agar plate, which had been inoculated with a lawn of bacterial strain. After incubation for 24 h at 37oC, the antibacterial activity was evaluated by measuring the diameter of the growth-inhibition zones from the edge of each filter paper. The inhibitive radii mean (the clear zone radii in which the tested strains did not grow) was noted.

The percentage inhibition was calculated by the following formula

Spectral Data of Compounds 1-11

6-(2′H-1′,2′,3′-triazol-2′-yl)pyridine-3-carbaldehyde (1)

White amorphous solid, 63.3% yield; 1H-NMR (400 MHz, DMSO): δ 9.21 (s, 1H, H-2), 8.32 (d, 1H, J = 7.7Hz, H-4), 7.42 (d, 1H, J = 7.3Hz, H-5), 9.83 (s, H-7), 7.94 (d, 2H, J = 6.1Hz, H-4′,5′),; 13C-NMR (100 MHz, CDCl­3): δ 153.5 (C-2), 129.7 (C-3), 133.04 (C-4), 120.2 (C-5), 157 (C-6), 192.4 (C-7), 134.1 (C-4′,5′),IR (KBr, 4000-400cm-1): v1279(C=O), 2985(aromatic ring), 1579(C=C), 1342 (C-N)

M.P. 150-152 oC.Anal.Calcd. For C8H6N4O:C, 55.17; H, 3.47; N, 32.17; O, 9.19Found: C, 53.67; H, 5.1; N, 32.03; O, 9.19.

2-hydroxy-N‘-{(E)-[6′-(2H-1″,2″,3″-triazol-2″-yl)pyridin-3′-yl]methylidene}benzohydrazide (2)

 

Yellow crystalline solid, 47.5% yield; 1H-NMR (400 MHz, DMSO): δ 5.6 (s, 1H, OH-2), 6.1 (d, 1H, J = 7.1Hz, H-3), 7.21 (t, 2H, J = 7.8Hz, H-4,5), 7.62 (d, 1H, J = 7.1Hz, H-6), 7.31 (s, 1H, H-8), 8.01 (s, 1H, H-10), 9.01 (s, 1H, H-2′), 8.3 (d, 1H, J = 9.1Hz, H-4′), 7.51 (d, 1H,J = 7.7Hz, H-5′), 8.4 (d, 2H,J = 8.7Hz, H-4”,5”); 13C-NMR (100 MHz, CDCl­3): δ 120.3 (C-1), 160.2 (C-2), 115.4 (C-3), 135.4 (C-4), 124.3 (C-5), 126.6 (C-6), 165.1 (C-7), 145.2 (C-10), 154.7 (C-2′), 128.1 (C-3′), 133.7 (C-4′), 125.4 (C-5′), 156.1 (C-6′), 131.2 (C-4”,5”);IR (KBr, 4000-400cm-1): v 3342 (OH), 1617 (-C=N), 1279(C=O), 2985(aromatic ring), 1579(C=C); M.P. 184-185oC. Anal.Calcd. For C15H12N6O2 :C, 58.44; H, 3.92; N, 27.2; O, 10.38Found: C, 59.75; H, 2.87; N, 26.96; O, 10.38.

Tributyltin(IV)-2-hydroxy-N‘-{(E)-[6′-(2″H-1″,2″,3″-triazol-2″-yl)pyridin-3′-yl]methylidene}benzohydrazide (3)

1H-NMR (400 MHz, DMSO):δ 6.52 (d, 1H, J = 7.1Hz, H-3), 7.12 (t, 2H,J = 7.6Hz, H-4,5), 7.81 (d, 1H,J = 7.9Hz, H-6), 7.3 (s, 1H, H-8), 7.62 (s, 1H, H-10), 9.51 (s, 1H, H-2′), 8.6 (d, 1H,J = 9.1Hz, H-4′), 7.5 (d, 1H,J = 7.4Hz, H-5′), 7.92 (d, 2H,J = 8.2Hz , H-4”,5”), 1.43 (t, 6H, J = 2.3Hz, Hα,α’,α”), 1.37 (m, 6H, J = 1.9Hz, Hβ,β’,β”), 1.41 (m, 6H, J = 1.8Hz, Hϓ,ϓ ‘,ϓ “), 0.73 (t, 9H,J = 1.8Hz,Hδ,δ’,δ”);

13C-NMR (100 MHz, CDCl­3): δ 120.2 (C-1), 168.6 (C-2), 114.2 (C-3), 134.1 (C-4), 120.7 (C-5), 130.1 (C-6), 165.3 (C-7), 145.1 (C-10), 154.2 (C-2′), 126.1 (C-3′), 137.3 (C-4′), 125.2 (C-5′), 154.2 (C-6′), 133.6 (C-4”,5”), 18.4 (C α,α’,α”), 26.2 (C β,β’,β”), 29.3 (C ϓ,ϓ ‘,ϓ “), 15.4 (Cδ,δ’,δ”);IR (KBr, 4000-400cm-1): v 1622 (-C=N), 1285 (C=O), 2989 (aromatic ring), 451 (Sn-O), 494(Sn-C), 1589 (C=C);

Chlorodimethyltin(IV)-2-hydroxy-N‘-{(E)-[6′-(2″H-1″,2″,3″-triazol-2″-yl)pyridin-3′-yl]methylidene}benzohydrazide (4)

1H-NMR (400 MHz, DMSO):δ 6.73 (d, 1H,J = 6.9Hz, H-3), 7.51 (t, 2H, J = 7.6Hz, H-4,5), 7.82 (d, 1H,J = 8.2Hz, H-6), 8.01 (s, 1H, H-8), 7.34 (s, 1H, H-10), 9.6 (s, 1H, H-2′), 8.2 (d, 1H,J = 8.5Hz, H-4′), 7.63 (d, 1H,J = 7.8Hz, H-5′), 7.86 (d, 2H,J = 7.5Hz, H-4”,5”), 2.2 (s, 6H, Hα,α’);

13C-NMR (100 MHz, CDCl­3): δ 121.2 (C-1), 168.4 (C-2), 119.2 (C-3), 134.3 (C-4), 123.2 (C-5), 131.1 (C-6), 166.1 (C-7), 141 (C-10), 153.5 (C-2′), 127 (C-3′), 134.2 (C-4′), 125.3 (-5′), 155.1 (C-6′), 131.7 (C-4”,5”), 1.8 (Cα,α’);

IR (KBr, 4000-400cm-1): v 1626 (-C=N), 1279 (C=O), 2991 (aromatic ring), 446 (Sn-O), 489 (Sn-C), 1571 (C=C);

Dimethyltin(IV) bis [2-hydroxy-N‘-{(E)-[6′-(2″H-1″,2″,3″-triazol-2″-yl)pyridin-3′-yl]methylidene}benzohydrazide] (5)

1H-NMR (400 MHz, DMSO):δ 6.82 (d, 1H,J = 7.0Hz, H-3), 7.12 (t, 2H,J = 7.6Hz, H-4,5), 7.83 (d, 1H,J = 7.7Hz, H-6), 7.3 (s, 1H, H-8), 7.61 (s, 1H, H-10), 9.5 (s, 1H, H-2′), 8.6 (d, 1H,J = 8.9Hz, H-4′), 7.42 (d, 1H,J = 7.6Hz, H-5′), 9.1 (d, 2H, J = 9.4Hz. H-4”,5”), 1.8 (s, 6H, Hα);

13C-NMR (100 MHz, CDCl­3): δ 121.3 (C-1), 167.4 (C-2), 117.2 (C-3), 134.3 (C-4), 123.2 (C-5), 129 (C-6), 165.2 (C-7), 144 (C-10), 154.2 (C-2′), 125 (C-3′), 134.1 (C-4′), 125.3 (C-5′), 151 (C-6′), 136.2 (C-4”,5”), 2.4 (Cα,α);IR (KBr, 4000-400cm-1): v 1619 (-C=N), 1284 (C=O), 2995 (aromatic ring), 457 (Sn-O), 492 (Sn-C), 1584 (C=C);

Trimethyltin(IV)-2-hydroxy-N‘-{(E)-[6′-(2″H-1″,2″,3″-triazol-2″-yl)pyridin-3′-yl]methylidene}benzohydrazide (6)

1H-NMR (400 MHz, DMSO):δ  6.82 (d, 1H,J = 6.6Hz, H-3), 7.41 (t, 2H,J = 7.6Hz, H-4,5), 7.82 (d, 1H,J = 8.1Hz, H-6), 7.21 (s, 1H, H-8), 7.61 (s, 1H, H-10), 9.5 (s, 1H, H-2′), 8.5 (d, 1H,J = 8.2Hz, H-4′), 7.4 (d, 1H, J = 7.7Hz, H-5′), 8.1 (d, 2H,J = 8.6Hz, H-4”,5”), 2.1 (s, 9H, Hα, α’, α”);

13C-NMR (100 MHz, CDCl­3):  δ 122 (C-1), 168.3 (C-2), 117.2 (C-3), 135.5 (C-4), 124.3 (C-5), 130.1 (C-6), 165.1 (C-7), 145.2 (C-10), 153.2 (C-2′), 128 (C-3′), 137.1 (C-4′), 126.2 (C-5′), 154.3 (C-6′), 135.1 (C-4”,5”), 3.2 (Cα, α’, α”);IR (KBr, 4000-400cm-1): v 1610 (-C=N), 1273 (C=O), 2995 (aromatic ring), 446 (Sn-O), 487 (Sn-C), 1573 (C=C);

Chlorodiphenyltin(IV)-2-hydroxy-N‘-{(E)-[6′-(2″H-1″,2″,3″-triazol-2″-yl)pyridin-3′-yl]methylidene}benzohydrazide (7)

1H-NMR (400 MHz, DMSO):δ 6.52 (d, 1H,J = 6.1Hz, H-3), 7.42 (t, 2H,J = 7.7Hz, H-4,5), 7.81 (d, 1H,J = 8.2Hz, H-6), 7.2 (s, 1H, H-8), 7.61 (s, 1H, H-10), 9.42 (s, 1H, H-2′), 8.6 (d, 1H,J = 8.4Hz, H-4′), 7.1 (d, 1H,J = 7.2Hz, H-5′), 8.2 (d, 2H,J = 8.4Hz, H-4”,5”), 7.3 (m, 10H, J = 7.7Hz, Ph-H);

13C-NMR (100 MHz, CDCl­3): δ 122.1 (C-1), 164.3 (C-2), 117.3 (C-3), 136.3 (C-4), 124.2 (C-5), 130.2 (C-6), 161 (C-7), 146 (C-10), 154.2 (C-2′), 125 (C-3′), 138.1 (C-4′), 121.3 (C-5′), 154.3 (C-6′), 137.3 (C-4”,5”), 130.2 (Cα), 135.1 (Cβ,β’), 131 (Cϓ,ϓ’);IR (KBr, 4000-400cm-1): v 1623 (-C=N), 1280 (C=O), 2987 (aromatic ring), 444 (Sn-O), 493 (Sn-C), 1588 (C=C);

Diphenyltin(IV)bis [2-hydroxy-N‘-{(E)-[6′-(2″H-1″,2″,3″-triazol-2″-yl)pyridin-3′-yl]methylidene}benzohydrazide] (8)

1H-NMR (400 MHz, DMSO):δ 6.7 (d, 1H,J = 6.8Hz, H-3), 7.21 (t, 2H,J = 7.4Hz, H-4,5), 7.62 (d, 1H,J = 8.1Hz, H-6), 7.11 (s, 1H, H-8), 7.52 (s, 1H, H-10), 9.5 (s, 1H, H-2′), 8.61 (d, 1H,J = 8.8Hz, H-4′), 7.7 (d, 1H,J = 7.5Hz, H-5′), 8.01 (d, 2H,J = 8.3Hz, H-4”,5”), 7.23 (m, 10H, J = 7.9Hz, Ph-H);

13C-NMR (100 MHz, CDCl­3): δ 131.2 (C-1), 165.2 (C-2), 114.3 (C-3), 135.2 (C-4), 123.2 (C-5), 129.7 (C-6), 162.6 (C-7), 145.1 (C-10), 154.2 (C-2′), 127.1 (C-3′), 138.1 (C-4′), 126.3 (C-5′), 151 (C-6′), 136.6 (C-4”,5”), 130.2 (Cα), 136.5 (Cβ,β’), 124.7 (Cϓ,ϓ’);IR (KBr, 4000-400cm-1): v 1621 (-C=N), 1276 (C=O), 3004 (aromatic ring), 458 (Sn-O), 488 (Sn-C), 1576 (C=C);

Triphenyltin(IV)-2-hydroxy-N‘-{(E)-[6′-(2″H-1″,2″,3″-triazol-2″-yl)pyridin-3′-yl]methylidene}benzohydrazide (9)

1H-NMR (400 MHz, DMSO):δ 6.9 (d, 1H,J = 7.3Hz, H-3), 7.51 (t, 2H,J = 7.9Hz, H-4,5), 7.71 (d, 1H,J = 7.4Hz, H-6), 7.02 (s, 1H, H-8), 7.83 (s, 1H, H-10), 9.4 (s, 1H, H-2′), 8.7 (d, 1H,J = 8.2Hz, H-4′), 7.2 (d, 1H,J = 7.4Hz. H-5′), 8.3 (d, 2H,J = 8.8Hz, H-4”,5”), 7.8 (m, 15H, J = 8.3Hz, Ph-H);

13C-NMR (100 MHz, CDCl­3): δ 117.9 (C-1), 163.2 (C-2), 114.1 (C-3), 138.4 (C-4), 122.5 (C-5), 126.5 (C-6), 165.1 (C-7), 145.6 (C-10), 150.5 (C-2′), 128.3 (C-3′), 135.2 (C-4′), 124.5 (C-5′), 156.1 (C-6′), 133.6 (C-4”,5”), 130.2 (Cα), 139.4 (Cβ), 123.1 (Cϒ); IR (KBr, 4000-400cm-1): v 1615 (-C=N), 1270 (C=O), 2982 (aromatic ring), 442 (Sn-O), 480 (Sn-C), 1582 (C=C);

Dibutyltin(IV)bis [2-hydroxy-N‘-{(E)-[6′-(2″H-1″,2″,3″-triazol-2″-yl)pyridin-3′-yl]methylidene}benzohydrazide] (10)

1H-NMR (400 MHz, DMSO):δ 6.52 (d, 1H,J = 7.1Hz, H-3), 7.12 (t, 2H,J = 7.7Hz, H-4,5), 7.81 (d, 1H,J = 7.9Hz, H-6), 6.98 (s, 1H, H-8), 7.46 (s, 1H, H-10), 9.5 (s, 1H, H-2′), 8.6 (d, 1H,J = 8.9Hz, H-4′), 7.53 (d, 1H,J = 7.7Hz, H-5′), 8.22 (d, 2H,J = 8.6Hz, H-4”,5”), 1.42 (t, 4H, J = 1.7Hz. Hα,α’),  1.48 (m, 4H, J = 2.1Hz, Hβ,β‘), 1.5 (m, 4H, J = 2.2Hz, Hϓ,ϓ‘), 0.77 (t, 6H, J = 1.4Hz, Hδ,δ’);

13C-NMR (100 MHz, CDCl­3): δ 117.8 (C-1), 165.6 (C-2), 119.2 (C-3), 136.5 (C-4), 123.2 (C-5), 129.5 (C-6), 161.2 (C-7), 146.4 (C-10), 154.5 (C-2′), 127.3 (C-3′), 138.2 (C-4′), 122.5 (C-5′), 155.1 (C-6′), 131.3 (C-4”,5”), 27.4 (Cα,α’), 24.7 (Cβ,β‘), 29.3 (Cϓ,ϓ‘), 16.4 (Cδ,δ’);

IR (KBr, 4000-400cm-1): v 1627 (-C=N), 1283 (C=O), 2988 (aromatic ring), 450 (Sn-O), 499 (Sn-C), 1574 (C=C);

Tricyclohexyltin(IV)-2-hydroxy-N‘-{(E)-[6′-(2″H-1″,2″,3″-triazol-2″-yl)pyridin-3′-yl]methylidene}benzohydrazide (11)

1H-NMR (400 MHz, DMSO):δ 6.77 (d, 1H,J = 6.8Hz, H-3), 7.31 (t, 2H,J = 7.8Hz, H-4,5), 7.81 (d, 1H,J = 8.2Hz, H-6), 7.1 (s, 1H, H-8), 7.62 (s, 1H, H-10), 9.6 (s, 1H, H-2′), 8.61 (d, 1H, J = 8.3Hz, H-4′), 7.53 (d, 1H,J = 7.7Hz, H-5′), 8.3 (d, 2H,J = 8.5Hz. H-4”,5”), 1.5 (m, 3H, Hα), 1.6;1.4 (m, 12H, Hβ,β‘), 1.73;1.12 (m, 12H, Hϓ,ϓ‘), 1.42;1.41 (m, 6H, Hδ);

13C-NMR (100 MHz, CDCl­3): δ 119.6 (C-1), 167.6 (C-2), 118.2 (C-3), 134.6 (C-4), 122.4 (C-5), 127.8 (C-6), 165 (C-7), 144.2 (C-10), 152.7 (C-2′), 125 (C-3′), 136.7 (C-4′), 120.4 (C-5′), 156 (C-6′), 132.7 (C-4”,5”), 9.2 (Cα), 29.4 (Cβ,β‘), 33.2 (Cϓ,ϓ‘), 27.4 (Cδ);

IR (KBr, 4000-400cm-1): v 1611 (-C=N), 1286 (C=O), 3003 (aromatic ring), 449 (Sn-O), 495 (Sn-C), 1580 (C=C);

Results and Discussion :-

IR (4000-400 cm-1, KBr)

In free ligand 2, OH group is observed at 3342 cm-1 as a characteristic broad band. But the band vanished in the complexes (3-11) due to the deprotonation of the ligand. This gives a strong indication of the phenolic oxygen participation in coordination with the central tin atom.

Moreover, the stretching vibration of azomethine (-HC=N) for hydrazine appeared at 1617 cm-1 for ligand 2, as confirmed from the previous reports [27]. There is no significant decrease or increase in frequency (1610-1627 cm-1) of azomethine in almost all the spectra of complexes (3-11), this confirms the non-participation of nitrogen in complexation.

The carbonyl (-C=O) group showed its presence at 1279 cm-1 in 2, No significant change is detected in all the complexes (1270-1286 cm-1), it also confirms the non-participation of carbonyl oxygen in complexation.

Sn-C absorption bands appeared in the range of 487-499 cm-1 and an absorption band in the range of 442-458 cm-1 indicates the presence of a Sn-O bond in all the coordination compounds.

Antifungal activity profile of compound 2and its corresponding organotin(IV) complexes is graphed in figure-2. Ligand (2) reveals percent inhibition against all fungal strains in the range of 40.8-49.2%. Among its chelates, compound 3displayed highest value of 48.3% against C. albicans and the compound 9(55.3%) exhibits highest inhibition against A. flavus. While the compound 7 (59.3%) has shown highest value against M. canis. The result shows that the activity of the ligand enhanced upon chelation that is in accordance to the previous reports[28]. From the results, it is also clear that ll the compounds are moderately antifungal in nature.

Figure 2: Antifungal activity profile of ligand (2) and its corresponding organotin(IV) complexes by using 96 – well  plates method. Each bar represents the percentage inhibition (mm) for compounds and reference drug (10mM test concentration).

 

Antibacterial assay profile of ligand (2)and its corresponding organotin(IV) complexes is represented in figure-3. The ligand (2) has shown percent inhibition of 12.2-14.4 against E. coli, B. subtilisand S. aureus. Among tin complexes, compound 8 depicted maximum inhibitions of 19.1% against E. coli and the compound 10exhibited 21.6% against B. subtilis. While the compound 7 remained best inhibitor against S. aureus (17.8%). Compared with the inhibitory zones of the standard, the synthesized compounds are good to best antibacterial in nature. Also the results confirm the previous reports that the diorganotin(IV) complexes (7,8,10) are good antibacterial as compared to triorganotin(IV) complexes[29].

Figure 3: Antibacterial assay profile of ligand (2) and its corresponding organotin(IV) complexes by using disc diffusion method. Each bar represents the percentage inhibition (mm) for compounds and reference drug (10mM test concentration).

 

Figure-4 indicates that, compound 3(tributyltin(IV)) has shownmaximum antiureaseinhibition of 39.4% for 10µg and 44.8% for 100µg concentration. Khan et al., (2007) also confirmed that tributyltin(IV) complexes are usually moderate antiurease agents.

Figure 4: Antiurease assay profile of ligand (2) and its corresponding organotin(IV) complexes by using Berthelot (phenol-hypochlorite) method via 96-well plates. Each bar depicts the percentage inhibition (mm) for compounds and reference drug (10mM test concentration).

Figure-5 confirms that, the compound 9(triphenyltin(IV))has shownmaximum cytotoxicity of 39.6%. The literature also confirms its worth i.e. to be the best inhibitor[29].

Figure 5: Cytotoxicity assay profile of ligand (2) and its corresponding organotin(IV) complexes by using brine shrimp lethality method. Each bar reveals the percentage inhibition (mm) of compounds and reference drug (10mM test concentration).

. Acknowledgments

The authors are highly thankful to The Islamia University of Bahawalpur and HEC Pakistan  for financial support.

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