Introduction

The field of medicinal inorganic chemistry were explored widely [1]. Metal complexes with biologically active heterocyclic ligands enhance its applications [2]. Design and synthesis of such compounds having less side effects and more potentiality has been a primary step towards exploring its application in biological medicinal chemistry [3,4,5]. Transition metals have been studied widely since they often alter and bind with the DNA nucleic bases [6, 7]. The key area of interest for the chemist have been developing drugs which provide resistance to diseases, more effective and less toxic or having less side effects [8, 9]. Design of heterocyclic ligands which fit with metal ions geometrically is equally important [10]. Substitution on heterocyclic rings enhance biological potentiality of ligand as well as its metal ion based complex [11]. The substituents having ability to enhance liphophilicity, stabilize the oxidation state of metal ion based complex and stabilize the complex making it kinetically inert and thermodynamically stable in important aspect of designing such compounds concluded by structural and activity relationship [12].

DNA has been a primary target of biomolecules to cure diseases genetically by interaction with base pairs via electrostatic, Vander Waal and hydrophobic forces of attractions. Complexes with octahedral geometry have been found to be kinetically more inert since releasing of bioactive ligand on specific DNA site is of prime importance. Quinoline based heterocycles have been proved to have good binding affinity than ciprofloxacin drug [13]. Binding affinity of quinoline heterocycles are highly stabilized by hydrogen bonding interactions concluded by docking studies [14, 15]. Quinoline class of heterocycles have been proved to possess greater cytotoxicity activities when explored to study on wide cancer cell lines [16, 17].

Quinoline nucleus-based complexes have been synthesized and studied and their biological assessment have been proved to be impactful and superior than ligands [18]. Osmium metal ion readily bind with -N, O donor ligands [19,20,21,22]. Osmium metal ion-based complexes have been explored to in cytotoxic assays and varied cell lines [23]. These have been also in doing interaction with DNA and adhering its mechanism which puts it into plethora of vast medicinal synthetic chemistry [24, 25]. Osmium metal ion complexes often prefer to bind via intercalation mode with DNA macromolecule Osmium metal ion-based complexes have been also proved effective in playing role in DNA binding and cleavage agent [26].

Present report includes, synthesis of six different substituted pyrazole nucleus-based -N,O donor ligands and Os(IV) complexes. The compounds have been well characterized and justified using various techniques. All the compounds have undergone cytoxicity assays, bacteriostatic study, DNA binding and cleavage activities.

Experimental

Materials and Reagents

Phosphorous oxychloride, dimethyl formamide used in reaction were purchased from SDFCL, SD-Fine chemicals ltd. 4-hydroxide benzhydrazide, potassium tertiary butoxide, osmium tetroxide (Crystals), hydrobromic acid, ammonium bromide was purchased from Sigma Aldrich, Artemia cyst eggs were purchased from local aquarium. Luria broath, ethidium bromide and TAE buffer solution were purchased from Himedia(India). Bacterial culture was borrowed from MTCC, Chandigarh (India). Herring sperm DNA solution was purchased from Sigma Aldrich.

Physical Measurement

Liquid crystal mass spectroscopy, waters model with micromass technology were used for mass analysis. Electronic spectra were taken using UV (160A) spectrophotometer. Elemental analysis were carried out using Euro elemental analyzer. Melting point of compounds were taken using Thermo Cal10. Melting point apparatus. Magnetic property of complexes were measured using Guoy’s method. The smeared images of cytotoxicity assays were visualized by AlphaDigDoc software (California). Heteronuclear spectra were taken using Bruker Avance NMR spectrophotometer (400 MHz). Infra-red spectra were taken using Shimadzu instrument (400–4000 cm−1).

Characterization

General Method for Synthesis of Ligands

2-Chloro-6-methoxyquinoline-3-carbaldehyde have been synthesized using Vilsmeier–Haack reaction (Scheme 1) [27]

Scheme 1
scheme 1

General reaction scheme for synthesis of ligands L1-L6 and complexes C1-C6

Full size image

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Synthesis and Characterization of (5-(2-Chloro-6-Methoxyquinolin-3-Yl)-3-(4-Chlorophenyl)-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone (L1)

Ligand L1 have been synthesized by Claisen Schmidt condensation reaction between 2-chloro-6-methoxyquinoline-3-carbaldehyde (2 g, 9 mmol) and 4-chloro acetophenone (1.39 g, 9 mmol) in presence of potassium hydroxide (0.2 g, 4 mmol) as base. The chalcone formed (2 mmol, 0.42 g) have been cyclized with (1 mmol, 0.15 g) in presence of potassium tertiary butoxide (4 mmol, 0.22 g) to form ligand L1. Empirical formula: C26H19Cl2N3O3, Color: Yellow, yield: 90%, mol. Wt: 492.36 g/mol, m.p.: 285 °C; anal. Cal. (%) for: C, 63.43; H, 3.89, N, 8.53; Found (%), C, 63.22; H, 3.80, N, 8.20; Mass m/z(%): 425[M]+. 1H-NMR (400 MHz, DMSO-d6) δ/ppm: 2.404 (S, 3H, OCH3), 3.248(dd, 1H, H4a, 2J1 = 5.2 Hz, 3J2 = 5.2 Hz), 4.005(dd, 1H, H4b, 2J1 = 11.6 Hz, 3J2 = 11.6 Hz), 6.269(t, 1H, H5), 7.244–8.175(m, 14H, Ar-H), 9.644(s, 1H, OH) 13C-NMR (100 MHz, DMSO-d6) δ/ppm: 189.65, (C=O, Cquat); 152.35, (C5, Cquat); 146.35, (C1’, Cquat); 145.06, (C4’, Cquat); 142.32, (C2”, Cquat); 132.36, (C3”, Cquat); 132.35, (C2”, C6”, -CH); 132.05 (C3’, C5’, -CH); 131.43 (C4”, CH); 131.38, (C5”, CH); 131.37 (C7”, CH); 131.36 (C4a’, Cquat); 131.12, (C8”, Cquat); 130.88, (C8a’, Cquat); 128.67, (C2”’, C6”, CH); 128.27 (C6”’, Cquat); 126.76, (C3”, C5”, CH); 126.43, (C1”, Cquat); 126.42, (C4”, Cquat); 55.67, (methoxy, CH3); 44.59 (C4, CH2); 36.46 (C3, CH). (UV-Vis (DMSO, c = 10−4 mol dm−3): λmax (ɛ) = 232.00 nm (29,350) mol- 1dm3 cm−1). IR (KBr, cm−1): 3099 ν(=C-H); 1540 ν(C-N); 1312 ν(C-H); 1666 ν(C=C); 1610 ν(C=O).

Synthesis and Characterization of (5-(2-Chloro-6-Methoxyquinolin-3-Yl)-3-(4-Hydroxyphenyl)-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone (L2)

It was synthesized using 4-hydroxy acetophenone (9 mmol, 1.22 g) using procedure mentioned in the synthesis of ligand L1. Empirical formula: C26H20ClN3O4, Color: Yellow, yield: 88%, mol. Wt: 473.91 g/mol, m.p.: 281 °C; anal. Cal. (%) for: C, 65.90; H, 4.25, N, 8.87; Found (%),C, 65.85; H, 4.12, N, 8.80; Mass m/z(%): 473.00 [M]+. 1H-NMR (400 MHz, DMSO-d6) δ/ppm: 2.412 (S, 3H, OCH3), 3.260(dd, 1H, H4a, 2J1 = 5.6 Hz, 3J2 = 2.8 Hz), 4.017(dd, 1H, H4b, 2J1 = 7.6 Hz, 3J2 = 12.8 Hz), 6.276(t, 1H, H5), 7.489–8.183(m, 14H, Ar-H), 9.743(s, 1H, OH) 13C-NMR (100 MHz, DMSO-d6) δ/ppm: 189.70, (C=O, Cquat); 152.40 (C5, Cquat); 146.40 (C1’, Cquat); 145.11, (C4’, Cquat); 142.37, (C2”, Cquat); 132.41, (C3”, Cquat); 131.84, (C2’, C6’, CH); 131.83, (C3’, C5’, CH); 131.53, (C4”, CH); 130.91, (C5”, CH); 130.86, (C7”, CH); 130.85, (C4a’, Cquat); 129.37, (C8”, CH); 129.13, (C8a’, Cquat): 126.92 (C6”, Cquat); 126.52 (C2”’, C6”’, CH); 124.01 (C1”, Cquat); 124.84, (C3”’, C5”’); 124.67, (C4”’, Cquat); 54.52, (methoxy, OCH3); 42.84 (C4, CH2); 34.71 (C3, CH). (UV-Vis (DMSO, c = 10−4 mol dm−3): λmax (ɛ) = 230.50 nm (29,350) mol- 1dm3 cm−1). IR (KBr, cm−1): 3067 ν(=C-H); 1521 ν(C-N); 1320 ν(C-H); 1678 ν(C=C); 1625 ν(C=O).

Synthesis and Characterization of (5-(2-Chloro-6-Methoxyquinolin-3-Yl)-3-Phenyl-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone (L3)

It was synthesized using acetophenone (9 mmol, 1.08 g) using procedure mentioned in the synthesis of ligand L1. Empirical formula: C26H20ClN3O3, Color: Yellow, yield: 82%, mol. Wt: 457.91 g/mol, m.p.: 283 °C; anal. Cal. (%) for: C, 68.20; H, 4.40, N, 9.18; Found (%), C, 68.08; H, 4.30, N, 9.11; Mass m/z(%):457.00 [M]+. 1H-NMR (400 MHz, DMSO-d6) δ/ppm: 2.418 (S, 3H, OCH3), 3.265(dd, 1H, H4a, 2J1 = 5.6 Hz, 3J2 = 8.4 Hz), 4.027(dd, 1H, H4b, 2J1 = 10.4 Hz, 3J2 = 9.2 Hz), 6.278(t, 1H, H5), 7.489–8.188(m, 14H, Ar-H), 9.883(s, 1H, OH) 13C-NMR (100 MHz, DMSO-d6) δ/ppm: 189.75, (C=O, Cquat); 152.45, (C5, Cquat); 146.45, (C1’, Cquat); 145.16, (C4’, Cquat); 142.42, (C2”, Cquat); 132.46, (C3”, Cquat); 131.89, (C2’, C6’, CH); 131.88, (C3’, C5’, CH); 131.58, (C4”, CH); 130.96, (C5”, CH); 130.91, (C7”, CH); 130.90, (C4a’, Cquat); 129.42, (C8”, CH); 128.18, (C8a’, Cquat); 126.97, (C2”’, C6”’, CH), 126.57, (C6”, CH); 126.06, (C1”, CH); 124.89, (C3”’, C5”’, CH); 124.72, (C4”’, Cquat); 56.20, (methoxy, OCH3); 42.89, (C4, CH2); 34.46, (C4, CH). (UV-Vis (DMSO, c = 10−4 mol dm−3): λmax (ɛ) = 238.50 nm (29,350) mol- 1dm3 cm−1). IR (KBr, cm−1): 3055 ν(=C-H); 1543 ν(C-N); 1335 ν(C-H); 1680 ν(C=C); 1632 ν(C=O).

Synthesis and Characterization of (5-(2-Chloro-6-Methoxyquinolin-3-Yl)-3-(4-Fluorophenyl)-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone (L4)

It was synthesized using 4-floro acetophenone (9 mmol, 1.24 g) using procedure mentioned in the synthesis of ligand L1. Empirical formula: C26H19ClFN3O3, Color: Yellow, yield: 84%, mol. Wt: 475.90 g/mol, m.p.: 280 °C; Mass m/z(%):475.00 [M]+. (UV-Vis (DMSO, c = 10−4 mol dm−3): λmax (ɛ) = 267.50 nm (29,350) mol- 1dm3 cm−1). 1H-NMR (400 MHz, DMSO-d6) δ/ppm: 2.422(S, 3H, OCH3), 3.271(dd, 1H, H4a, 2J1 = 5.2 Hz, 3J2 = 4.8 Hz), 4.029(dd, 1H, H4b, 2J1 = 11.6 Hz, 3J2 = 10.4 Hz), 6.286(t, 1H, H5), 7.494–8.195(m, 14H, Ar-H), 9.443(s, 1H, OH) 13C-NMR (100 MHz, DMSO-d6) δ/ppm: 189.81, (C=O, Cquat); 152.51, (C5, Cquat); 146.50, (C1’, Cquat); 144.22, (C4’, Cquat); 142.48, (C2”, Cquat); 132.52, (C3”, Cquat); 131.95, (C2’, C6’, CH); 131.94, (C3’, C5’, CH); 131.64, (C4”, CH); 130.02, (C5”, CH); 130.97, (C7”, CH); 130.96, (C8”, CH); 129.48, (C2”’, C6”’); 128.24, (C4a’, Cquat); 126.63, (C8a’, Cquat); 126.03, (C8a’, Cquat); 125.12, (C1”, Cquat); 124.95, (C3”’, C5”’, CH); 124.78, (C4”’, CH); 56.43, (Methoxy, OCH3); 42.95, (C4, CH2); 34.82, (C3, CH). (UV-Vis (DMSO, c = 10−4 mol dm−3): λmax (ɛ) = 239.50 nm (29,350) mol- 1dm3 cm−1). IR (KBr, cm−1): 3046 ν(=C-H); 1533 ν(C-N); 1342 ν(C-H); 1687 ν(C=C); 1646 ν(C=O).

Synthesis and Characterization of 7(3-(4-Bromophenyl)-5-(2-Chloro-6-Methoxyquinolin-3-Yl)-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone (L5)

It was synthesized using 4-bromo acetophenone (9 mmol, 1.79 g) using procedure mentioned in the synthesis of ligand L1. Empirical formula: C26H19BrClN3O3, Color: Yellow, yield: 80%, mol. Wt: 536.81 g/mol, m.p.: 292 °C; anal. Cal. (%) for: C, 58.17; H, 3.57, N, 7.83; Found (%),C, 58.10; H, 3.45, N, 7.75; Mass m/z(%): 536.00[M]+. 1H-NMR (400 MHz, DMSO-d6) δ/ppm: 2.435 (S, 3H, OCH3), 3.277(dd, 1H, H4a, 2J1 = 5.2 Hz, 3J2 = 5.6 Hz), 4.032(dd, 1H, H4b, 2J1 = 8.8 Hz, 3J2 = 12.4 Hz), 6.288(t, 1H, H5), 7.511–8.210(m, 14H, Ar-H), 9.646(s, 1H, OH). 13C-NMR (100 MHz, DMSO-d6) δ/ppm: 189.56, (C=O, Cquat); 152.26, (C5, Cquat); 146.26, (C1’, Cquat); 144.97, (C4’, Cquat); 142.23, (C2”, Cquat); 132.27, (C3”, Cquat); 131.70, (C2’, C6’, CH); 131.69, (C3’, C5’, CH); 131.39, (C4”, Cquat); 130.77, (C5”, CH); 130.72, (C7”, CH); 130.71, (C4a’, Cquat); 129.23, (C8”, CH), 128.99, (C8a’, Cquat); 126.78, (C2”’, C6”’, CH); 126.38, (C6”, Cquat); 124.87, (C1”, Cquat); 124.70, (C3”’, C5”’, CH); 124.53, (C4”, Cquat); 54.60, (Methoxy, OCH3); 42.70, (C4, CH2); 34.57, (C3, CH). (UV-Vis (DMSO, c = 10−4 mol dm−3): λmax (ɛ) = 230.50 nm (29,350) mol- 1dm3 cm−1). IR (KBr, cm−1): 3032 ν(=C-H); 1520 ν(C-N); 1352 ν(C-H); 1653ν(C=C); 1648ν(C=O).

Synthesis and Characterization of (3-(2-Bromophenyl)-5-(2-Chloro-6-Methoxyquinolin-3-Yl)-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone (L6)

It was synthesized using 3-bromo acetophenone (9 mmol, 1.79 g) using procedure mentioned in the synthesis of ligand L1. Empirical formula: C26H19BrClN3O3, Color: Yellow, yield: 85%, mol. Wt: 536.81 g/mol, m.p.: 280 °C; anal. Cal. (%) for: C, 58.17; H, 3.57, N, 7.83; Found (%),C, 58.10; H, 3.10, N, 7.70. Mass m/z(%): 536.00 [M]+. 1H-NMR (400 MHz, DMSO-d6) δ/ppm: 2.438 (S, 3H, OCH3), 3.281(dd, 1H, H4a, 2J1 = 5.2 Hz, 3J2 = 6.8 Hz), 4.042(dd, 1H, H4b, 2J1 = 8.4 Hz, 3J2 = 12.8 Hz), 6.298(t, 1H, H5), 7.519–8.218(m, 14H, Ar-H), 9.748(S, 1H, OH) 13C-NMR (100 MHz, DMSO-d6) δ/ppm: 189.61, (C=O, Cquat); 152.31, (C5, Cquat); 146.31, (C1’, Cquat); 145.02, (C2’, Cquat); 142.28, (C2”, Cquat), 132.32, (C3”, Cquat); 131.75, (C3’, CH); 131.74, (C4’, CH); 131.44, (C5’, CH); 130.39, (C6’, CH); 130.37, (C4”, CH); 130.33, (C4a’, Cquat); 129.85, (C5”, CH), 128.61, (C8a’, Cquat), 126.46, (C6”, Cquat); 126.00 (C1”, Cquat); 124.49, (C7”, CH), 124.32, (C8”, CH); 124.18, (C2”’, C6”’, CH); 124.15, (C4”, Cquat); 119.80, (C3”’, C5”’, CH); 53.12, (Methoxy, OCH3); 42.32, (C4, CH2); 34.19, (C3, CH). (UV-Vis (DMSO, c = 10−4 mol dm−3): λmax (ɛ) = 231.50 nm (29,350) mol- 1dm3 cm−1). IR (KBr, cm−1): 3024 ν(=C-H); 1517 ν(C-N); 1361 ν(C-H); 1690 ν(C=C); 1628 ν(C=O).

Synthesis and Characterization of Metal Salt and Complexes

Synthesis of Ammonium Hexabromoosmate(IV)

(NH4)2OsBr6 have been synthesized using osmium tetroxide and hydrobromic acid as starting reactants (Scheme 1) [28, 29].

Synthesis and Characterization of Tetrabromido(5-(2-Chloro-6-Methoxyquinolin-3-Yl)-3-(4-Chlorophenyl)-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone)Osmium(IV) (C1)

Tetrabromido(5-(2-chloro-6-methoxyquinolin-3-yl)-3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(4-hydroxyphenyl)methanone)osmium(IV) have been synthesized using (NH4)2OsBr6 (0.60 g, 0.085 mmol) and ligand L1 (0.41 g, 0.085 mmol). Color: Brownish black, yield: 60%, mol. Wt: 1000.63 g/mol, m.p.: >300 °c. Cal. (%) for: Os,18.98; Found (%), Os,18.68;. ESI-MS (70 eV): 1002.20, 922.30, 842.39, 762.49, 682.59, 492.59 m/z: Magnetic Moment: 2.70 B.M. (UV-Vis (DMSO, c = 10−4 mol dm−3): λmax (ɛ) = 284.00 nm (29,350) mol- 1dm3 cm−1). Conductance: 36 ℧ mol−1 cm2. IR (KBr, cm−1): 3112 ν(=C-H); 1562 ν(C-N); 1318 ν(C-H); 1680 ν(C=C); 1618 ν(C=O); 556 ν(N-Os); 611 ν(N-Os-Br).

Synthesis and Characterization of Tetrabromido(of (5-(2-Chloro-6-Methoxyquinolin-3-Yl)-3-(4-Hydroxyphenyl)-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone)Osmium(IV) (C2)

It was synthesized using (NH4)2OsBr6 (0.60 g, 0.085 mmol) and ligand L2 (0.40 g, 0.085 mmol). Color: Brownish black, yield: 60%, mol. Wt: 983.76 g/mol, m.p.: >300 °c. Cal. (%) for: Os,19.34; Found (%),Os,19.20; (UV-Vis (DMSO, c = 10−4 mol dm−3): λmax (ɛ) = 239.50 nm (29,350) mol- 1dm3 cm−1). Conductance: 32 ℧ mol−1 cm2. IR (KBr, cm−1): 3098 ν(=C-H); 1534 ν(C-N); 1342 ν(C-H); 1686 ν(C=C); 1634 ν(C=O); 562 ν(N-Os); 580 ν(N-Os-Br).

Synthesis and Characterization of Tetrabromido(5-(2-Chloro-6-Methoxyquinolin-3-Yl)-3-Phenyl-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone)Osmium(IV) (C3)

It was synthesized using (NH4)2OsBr6 (0.60 g, 0.085 mmol) and ligand L3 (0.38 g, 0.085 mmol). Color: Brownish black, yield: 60%, mol. Wt: 967.76 g/mol, m.p.: >300 °c. Cal. (%) for: Os,19.66; Found (%),Os,19.48; (UV-Vis (DMSO, c = 10−4 mol dm−3): λmax (ɛ) = 237.50 nm (29,350) mol- 1dm3 cm−1). Conductance: 28 ℧ mol−1 cm2. IR (KBr, cm−1): 3068 ν(=C-H); 1558 ν(C-N); 1344 ν(C-H); 1710 ν(C=C); 1667 ν(C=O); 575 ν(N-Os); 624 ν(N-Os-Br).

Synthesis and Characterization of Tetrabromido(5-(2-Chloro-6-Methoxyquinolin-3-Yl)-3-(4-Fluorophenyl)-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone)Osmium(IV) (C4)

It was synthesized using (NH4)2OsBr6 (0.60 g, 0.085 mmol) and ligand L4 (0.40 g, 0.085 mmol). Color: Brownish black, yield: 60%, mol. Wt: 985.75 g/mol, m.p.: >300 °c. Cal. (%) for: Os,19.30; Found (%),Os,19.13; (UV-Vis (DMSO, c = 10−4 mol dm−3): λmax (ɛ) = 256.50 nm (29,350) mol- 1dm3 cm−1). Conductance: 26 ℧ mol−1 cm2. IR (KBr, cm−1): 3057 ν(=C-H); 1543 ν(C-N); 1348 ν(C-H); 1693 ν(C=C); 1653 ν(C=O); 586 ν(N-Os); 617 ν(N-Os-Br).

Synthesis and Characterization of Tetrabromido(3-(4-Bromophenyl)-5-(2-Chloro-6-Methoxyquinolin-3-Yl)-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone)Osmium(IV) (C5)

It was synthesized using (NH4)2OsBr6 (0.60 g, 0.085 mmol) and ligand L5 (0.45 g, 0.085 mmol). Color: Brownish black, yield: 60%, mol. Wt: 1046.66 g/mol, m.p.: >300 °c. Cal. (%) for: Os,18.18; Found (%),Os,18.02; (UV-Vis (DMSO, c = 10−4 mol dm−3): λmax (ɛ) = 225.50 nm (29,350) mol- 1dm3 cm−1). Conductance: 24 ℧ mol−1 cm2. IR (KBr, cm−1): 3047 ν(=C-H); 1538 ν(C-N); 1362 ν(C-H); 1661 ν(C=C); 1653 ν(C=O); 592 ν(N-Os); 627 ν(N-Os-Br).

Synthesis and Characterization of Tetrabromido(3-(2-Bromophenyl)-5-(2-Chloro-6-Methoxyquinolin-3-Yl)-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone)Osmium(IV) (C6)

It was synthesized using (NH4)2OsBr6 (0.60 g, 0.085 mmol) and ligand L5 (0.45 g, 0.085 mmol). Color: Brownish black, yield: 60%, mol. Wt: 1046.66 g/mol, m.p.: >300 °c. Cal. (%) for: Os,18.18; Found (%),Os,17.98; Conductance: 28 ℧ mol−1 cm2. (UV-Vis (DMSO, c = 10−4 mol dm−3): λmax (ɛ) = 223.00 nm (29,350) mol- 1dm3 cm−1). Conductance: 36 ℧ mol-1 cm2. IR (KBr, cm−1): 3044 ν(=C-H); 1526 ν(C-N); 1377 ν(C-H); 1706 ν(C=C); 1634 ν(C=O); 565 ν(N-Os); 610 ν(N-Os-Br).

Biological Studies

Hydrodynamic Volume Measurement for Complex to HS-DNA Interaction

Mode of intercalation between compounds and DNA solution have been determined with the help of hydrodynamic volume measurement study. Different concentration of compounds have been mixed with DNA solution keeping its concentration at 200 uM. The flow time of incubated solution have been calculated in triplicate manner keeping it in static environment at temperature 27 ± 0.5 °C, and phosphate buffer pH 7.2 in digital viscometric bath [30].

Molecular Docking with B-DNA

The PDB files of compounds formatted with the help of CHIMERA 1.5.1 software have been interacted with B-DNA PDB file using HEX 6.0 software by activating docking process [31].

Absorption Titration Study of Complex to HS- DNA

HS-DNA solution have been dissolved in phosphate buffer solution keeping pH 7.2 in dimethyl sulfoxide at low temperature of 4 °C. With the fixed concentration of compounds and varying concentration of DNA solution, titration study have been carried out [32].

Fluorescence Quenching Study

The binding ability and the number of active sites on biomacromolecules on which compounds are effective have been determined with the help of fluorescence study. Depending upon the ability of Os(IV) complexes to displace EB-DNA, relative change in intensity is observed [33].

Gel Electrophoresis

The photolytic cleavage property of Os(IV) complexes have been determined by gel electrophoresis technique [34].

Bacteriostatic Activity

MIC activity have been carried out by growing bacterial culture in Luria broath solution. The bacterial culture have been allowed to grow keeping it in orbital shaker at optimum revolution and temperature [35].

In-Vitro Cytotoxicity

The varying concentration of compounds have been treated with pre-cultured S. Pombe cell’s DNA previously grown in liquid yeast media to calculate percentage cell viability of compounds [36].

In-Vivo Cytotoxicity

Brine shrimp lethality assay have been carried out by using protocol of Mayer et al., to calculate LC50 values of compounds [37].

Cytotoxicity Using HCT-116 Cell Line

MTT assay have been used to carry out screening of test compounds using HCT-116 cell line. DMSO have been used to solubilize the test compounds with different concentration ranging from 10 to 1000 μg/mL with 1% DMSO as vehicle control [38].

Results and Discussion

Characterization

1H NMR and 13C-APT Characterization of Compounds

All the ligands were well characterized using 1H-NMR and 13C-APT spectroscopy. In ligand L1, H4a proton represents in dd pattern at 3.248 δ/ppm whereas its analogues proton H4b shows same pattern at 4.005 δ/ppm. Further, H3 proton falls at 6.269 δ/ppm as triplet. Aromatic region is justified between 7.244–8.175 δ/ppm. Hydroxy proton and methoxy proton are obtained as singlet at 9.644 and 2.404 δ/ppm respectively. Carbon skeleton of ligand L1 were justified using 13C-APT. Carbonyl carbon shows its presence at 189.65 δ/ppm whereas methoxy carbon shows its primary carbon peak at 55.67 δ/ppm. 1H NMR and 13C-APT characterization of all compounds were given in Supplementary Material 1 and 2.

LC-MS and ESI-MS Characterization of Compounds

Compounds were well justified using LC-MS and ESI-MS spectroscopy. Base peak of ligand L1 is obtained at 358 m/z as highly intense peak whereas molecular ion peak is obtained at 492 m/z with lower intensity. Complex C1 were well characterized using ESI-MS spectroscopy. Molecular ion of C1 represents at 1002.20 m/z with [M+], [M + 2], [M + 4], [M + 6] and [M + 8] peaks whereas ligand detached from molecule shows its present at 492.59 m/z. Relative isotopic abundance pattern of bromine atoms were reflected at 922.30, 842.39, 762.49 and 682.59 m/z showing quintet, quartet, triplet and doublet pattern with relative intensity of 1:3:5:3:1, 1:2:2:1, 1:3:1 and 1:3 respectively. LC-MS of ligands L1-L6, ESI-MS spectrum and ICP-OES plot of complex C1 are given in Supplementary Material 3 and 4.

IR Spectra, Electronic Spectra, Magnetic and Conductance Measurement of Compounds

IR spectroscopy justify the formation of ligand and metal complexation. ν(=C-H) stretching band marks its presence at 3099 cm−1 in ligand L1. Consequently, ν(C-N), ν(C-H), ν(C=C), and ν(=C-O) stretching bands are observed at 1340, 1312, 1666 and 1610 cm−1, respectively. Upon the complexation in case of Os(IV) pyrazole compound C1, disturbance in vibrational bands caused marks presence of bands viz. ν(=C-H), ν(C-N), ν(C-H), ν(C=C), and ν(=C-O) at 3112, 1562, 1318, 1680, and 1618 cm−1, respectively. The shift in frequency is observed due to the complexation of osmium metal ion with relative coordinating sites of pyrazole nucleus ligand L1. The band at 556 cm−1 represents the coordinate covalent bond formed between nitrogen atom of ligand and osmium metal ion. The complex has octahedral geometry, consequently the expected ν(N-Os-Br) linkage vibrational stretching band is observed at 611 cm−1. Electronic spectra of ligands fall in wavelength of 223.00–284.00 nm whereas that of Os(IV) complexes shows peaks at 230.50–267.50. Complex C1 shows bands at 284.00, 339.50 and 458.50 nm justifying n- π*, π- π* and d-d transition bands accounting for (t2g)4(eg)0 electronic configuration representing octahedral behavior. Magnetic measurement of all the complexes justify paramagnetic behavior of Os(IV) complexes. All the complexes show non-electrolytic behavior confirmed by conductance measurements. IR spectrum of all the compounds are given in Supplementary Material 5. CHNS data of all the ligands have been given in Supplementary Material 6.

Biological Studies

Viscosity Measurement

Mode of interaction of compounds were well concluded with the help of hydrodynamic volume measurement study. The HS-DNA solution were incubated prior to run with the compound’s concentration. The solution was poured into viscometer held in digital viscometric bath in static environment. Flow time of solution were measured in triplicate manner in order to get the accurate results. All the compounds show intercalative mode of binding [39]. Figure shows increase in viscosity of compounds kept w.r.t. ethidium bromide taken as reference material. The viscosity measurement plot of compounds is given in Fig. 1.

Fig. 1
figure 1

Plots represent relative viscosity of HS-DNA upon increasing concentration of compounds

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Molecular Docking

Molecular docking is phenomenon of calculating effective docking energy upon interaction of molecules with the macro biomolecules. The molecules were allowed to run in ChemBio3D software to refine the molecule according to most stable conformational arrangement. The molecules were formatted as PDB files and were allowed to interact with B-DNA in HEX 6.0 software. The molecules and specific sites of DNA tend to refine, adjust according to conformational changes and interact with each other by activating docking process. The effective energy of interaction in which molecule befit with DNA were counted as its docking energy.. Docking energy of complexes C1, C2, C3, C4, C5 and C6 are −326.49, −318.71, −312.94, −292.30, −292.30 and − 292.30 kJ/mol whereas, their respective ligands show docking interaction energy of −295.49, −300.25, −289.39, −287.06, −290.79 and − 293.84 kJ/mol respectively showing effective intercalation [40, 41]. Docked structure of complex C1 is given in Fig. 2. Docked images of all the compounds are given in Supplementary Material 7.

Fig. 2
figure 2

Docking of Os(IV) compound C1 with B-DNA

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Absorption Titration Study of Complex to HS- DNA

Absorption titration is UV-visible spectrophotometer-based activity to calculate binding constant of compounds upon interaction with HS-DNA. Compounds were titrated in absence and presence of DNA solution. With fixed concentration of compound and varying concentration of DNA solution, the experiment was carried out. The relative shifts in DNA treated and untreated compounds helps in predicting percentage hypochromism shift. The effective binding constant were calculated with the help of equation given by Wolfe et al., Table 1. shows binding constant and percentage hypochromism shift of compounds. Absorption titration curve of complex C1 were represented in Fig. 3. All Os(IV) complexes have better binding constant compared to reported some Co(III) complexes with different 2-oxo-1,2-dihydrobenzo[h]quinoline-3-carbaldehyde moiety based ligands. While Os(IV) complexes C2, C5 and C6 show selectively better strength compare to Co(III) complex with 2-Oxo-1,2-dihydro-benzo[h]quinoline-3-carbaldehyde N-phenylthiosemicarbazone ligand [42].

Table 1 Intrinsic binding constant (Kb) values and percentage hypochromism shift values of all the synthesized compounds
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Fig. 3
figure 3

Absorption titration plot of varying concentration of HS-DNA with Os(IV) compounds. Inscribed graph represents change in absorption

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Fluorescence Quenching Study

Phenomenon of quenching deals with decrease in intensity of compound in presence of suitable quenchers. Chemically, bromide ion was used as chemical quenching agent taking ethidium bromide. Ethidium bromide was used as fluorophore material since its an effective intercalating agent. Intense emission bands were observed at 610 nm when allowed to excite at wavelength of 470 nm due to formation of EB-DNA bond. Upon addition of varying complex concentration, the ethidium bromide slowly gets displaced from bonding with macromolecule resulting in decrease in fluorescence intensity. Binding constant, effective binding sites and quenching constant were evaluated from fluorometric titration and are given in Table 2. The data were in good agreement with the absorption titration concluding effectiveness of intercalation mode of binding. Plots have been represented in Fig. 4 and mechanism of fluorescence quenching were presented in Fig. 5. Complexes C2 and C5 have quenching constant greater than reported complexes Pd(II) and their respective ligand [43].

Table 2 Stern-Volmer quenching constant (Ksv), binding sites (n), and binding constant (Kf) values
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Fig. 4
figure 4

Plot of fluorescence titration whereas, graphs represent Stern-Volmer equation parameters to calculate quenching constant (Ksv), binding sites (n) and binding constant (Kf)

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Fig. 5
figure 5

Fluorescence quenching observed with Os(IV) compound

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Gel Electrophoresis

Gel electrophoresis is a well-known technique for separation of charged particles based on difference in polarity to attract towards opposite poles of gel electrophoresis plate. 1% agarose gel were used in carrying out photolytic cleavage study. The solutions of compounds were run over casting apparatus set at optimum voltage required to run the gel. Upon addition of varying concentration of compounds on the solution of S. Pombe cell’s DNA. The degradation of DNA were observed resulting in the unwinding of its helix by rupturing the bonds formed between its base pairs. DNA converts to single strand from double stranded structure. The unwinded helical structure were allowed to move over mobile phase of gel under specific voltage which stretch the helix more compactly resulting in smeared image when observed under spectrophotometer. The RNA remains intact, whereas DNA gets as smeared forming intense band. Intensity of smearing depends upon the potency of compound at specific concentration to cleave the macromolecule. Photographed image of treated compounds with S. Pombe cell’s DNA under gel electrophoresis are represented in Supplementary Material 8.

Bacteriostatic Activity

This activity was carried out using Gram+ve and Gram-ve bacteria. All the compounds in different concentration range were treated with pre-grown bacterial culture. The compounds having substituents with electron delocalizing tendency tend to show better MIC values since they rupture the cell walls of bacteria there by resisting the growth. The minimal effective dosage of DMSO solubilized compound which resist the growth of bacteria is noted as its MIC value. Os(IV) complexes have better bacteriostatic activity compare to their respective ligands. MIC of all the compounds were represented in Fig. 6. MIC of complexes are found to be 72.5–100 μM, whereas that of ligands fall at about 122.5–150 μM. MIC of Os(IV) complexes are better than reported quinoline based quinazolinone-4-thiazolidinone heterocycles (5a-5 k) [44].

Fig. 6
figure 6

MIC values of compounds in terms of μM upon exposure to series of Gram(+ve) and Gram(−ve) bacteria

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Cellular Level in-Vivo Cytotoxicity

This assay were carried out using S. Pombe cell’s DNA. Cell culture have been pre-grown 17 h before treatment with the test compounds. Varying concentration of compounds have been treated with the cell culture keeping it in conical flask in laminar air flow cabinet in presence of FV light. The solutions were kept for 24 h overnight before taking readings. The treated solutions were centrifuged to collect the culture. The collected culture was kept in incubation with tryptan blue dye and dead cells were counted under digital microscope. The dye allows dead cells to penetrate into it whereas it resists live cells. Percentage cell viability of Os(IV) complexes were found better than that of respective ligands. Table 3 shows percentage cell viability of compounds. Percentage cell viability of Os(IV) complexes is more effective compare to reported oxovanadium(IV) complexes [45].

Table 3 Percentage viability on S. Pobme cell’s DNA presented with standard deviation for three independent experiment
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In-Vitro Cytotoxicity against Artemia Cyst Lethality Bioassay

Meyer et al.’s protocol was implemented to carry out the assay. This is primary assay to judge molecule for the pharmaceutical importance. Nauplii hatched from brine shrimp eggs have been treated with different concentration of compounds. The death of larvae is noted as lethality result. It is concluded that with increase in concentration of compounds, percentage mortality was increased. Cytotoxic value of ligands and Os(IV) complexes are respectively. Os(IV) complexes possess better LC50 than synthesized ligands. LC50 count of ligands fall in the range of 16.22–17.28 μg/mL whereas that of Os(IV) fall in the range of 4.87–5.87 μg/mL. LC50 of Os(IV) compounds show better value compare to reported oxovanadium (IV) complexes with 2-thiophene carboxylic acid hydrazide ligand [46]. LC50 of compounds are represented in Fig. 7.

Fig. 7
figure 7

Bar graph represents LC50 of osmium(IV) compounds and ligands

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Cytotoxicity Using HCT-116 Cell Line

Cytotoxicity of complexes were screened over HCT-116 cell line. Figure 8 Show plot of percentage proliferation vs. concentration (μg/mL) with different concentration of test compounds taken with respect to vehicle and control media. Complexes C1, C2, C4 and C5 have IC50 of 423, 752, 520 and 666 μg/mL while others show moderate impact. IC50 of Os(IV) C1 dominate over reported Re(I) with 5-(4-Chlorophenyl)-1-(4-nitrophenyl)-3- (thiophen-2-yl)-4,5-dihydro-1H-pyrazole ligand [47]. Complex C1 represents least IC50 amongst the complexes since it possess chlorine functionality which put an inductive effect in dominance.

Fig. 8
figure 8

Plots of % cell proliferation vs. concentration represents IC50 of Os(IV) compounds

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Conclusion

Various pyrazole nucleus-based ligands and their Os(IV) complexes were synthesized and well characterized using different spectroscopic techniques viz. 1H-NMR, 13C-APT, LC-MS, ESI-MS, ICP-OES, IR spectroscopy, electronic spectroscopy, conductance and magnetic measurements. The synthesized compounds were subjected for biological screening. All the compounds show intercalation mode of binding when interacted with HS-DNA justified with docking energy and DNA interaction study. Docking energy of ligands fall at about −287.06 to −300.25 kJ/mol. Whereas, complexes fall at about −292.30 to −326.49 kJ/mol showing effective intercalation. Binding constant of Os(IV) complexes are found to be 8.1 to 9.2 × 104 M−1.All Os(IV) complexes have better binding constant compared to reported Co(III) complexes with different substituted 2-oxo-1,2-dihydrobenzo[h]quinoline-3-carbaldehyde moiety based ligands. Binding constant have been further justified with fluorescence quenching study. Quenching constant of complexes fall at about 1.1 to 8.1 × 103 M−1. Complexes C2 and C5 have quenching constant better than reported Pd(II) complexes. MIC of complexes are found to be 72.5–100 μM, whereas that of ligands fall at about 122.5–150 μM. MIC of Os(IV) complexes are better than reported quinoline based quinazolinone-4-thiazolidinone heterocycles. Percentage cell viability of Os(IV) complexes is more effective compare to reported oxovanadium(IV) complexes. LC50 of Os(IV) compounds show better value compare to reported oxovanadium (IV) complexes with 2-thiophene carboxylic acid hydrazide ligand. Os(IV) complexes showed intense and impactful smearing of S. Pombe cell’s DNA when allowed to run over gel electrophoresis. IC50 of Os(IV) C1 dominate over reported Re(I) with 5-(4-chlorophenyl)-1-(4-nitrophenyl)-3- (thiophen-2-yl)-4,5-dihydro-1H-pyrazole ligand.