Mode of action of tin-based anti-proliferative agents: Biological studies of organotin(IV) derivatives of fatty acids

Mode of action of tin-based anti-proliferative agents: Biological studies of organotin(IV) derivatives of fatty acids

Journal of Photochemistry and Photobiology B: Biology 148 (2015) 88–100 Contents lists available at ScienceDirect Journal of Photochemistry and Phot...

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Journal of Photochemistry and Photobiology B: Biology 148 (2015) 88–100

Contents lists available at ScienceDirect

Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol

Mode of action of tin-based anti-proliferative agents: Biological studies of organotin(IV) derivatives of fatty acids Mala Nath a,⇑, Monika Vats a, Partha Roy b a b

Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, India Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, India

a r t i c l e

i n f o

Article history: Received 4 December 2014 Received in revised form 17 March 2015 Accepted 18 March 2015 Available online 3 April 2015

a b s t r a c t Some organotin(IV) carboxylates of the general formula RnSn(L)m [n = 3, m = 1, R = Me, Pr, Bu and Ph; n = 2, m = 2, R = Me, Bu and Oct; L = anion of lauric (HLA), stearic (HSA) and myristic acid (HMA)] have been synthesized and characterized by various spectroscopic studies. Tri- and diorganotin(IV) carboxylates adopt trigonal-bipyramidal and octahedral geometry around tin atom, respectively. They have been screened in vitro for anti-tumor activity against cancer cell lines of human origin, viz. MCF-7 (mammary), HEK-293 (kidney), PC-3 (prostate), HCT-15 (colon) and HepG-2 (liver). Enzyme assays viz. lipid peroxidase, glutathione peroxidase, glutathione reductase and total glutathione assay have been carried out to explore the cause of their cytotoxiciy. The results indicate that ROS (reactive oxygen species) generation may be responsible for their cytotoxicity but elevation in LDH (lactate dehydrogenase) suggests that necrosis cannot be excluded. Further, DNA (deoxyribonucleic acid) fragmentation, acridine orange and comet assay support the fact that the apoptosis is the main cause of cytotoxicity of organotin(IV) carboxylates, whereas the necrosis plays a minor role. The anti-inflammatory activity evaluation shows that the complexes possess moderate activity. Results of acute toxicity of the complexes have also been discussed. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction The serendipitous discovery of cis-platin (cis-diamminedichloroplatinum(II)), a chemotherapeutic drug used for treating solid malignancies, viz. ovarian, testicular, head/neck and bladder cancers [1–3], stimulated the search for other metal-based therapeutics. Development of new therapeutic modulities for cancer chemotherapy is the research area of immense interest owing to the fact that many present treatment regimes (platinum-based drugs) in chemotherapy have failed or fall short due to intrinsic acquired drug resistance, efficiency, patient compliance and toxicity problems associated with them [3–5]. Therefore, there is an exigency to design and identify effective metal-based therapeutics particularly, those that overcome inherent and acquired resistance to drug therapy and show improved therapeutic properties, stimulating the ongoing investigations of alternative molecular targeted metal-based drugs [6]. In view of this, attention has been shifted to non-platinum chemotherapeutics [7–24]; among these, gold and organotins attained particular interest because of their common activity on mitochondria and strong affinity to thiol groups of proteins and enzymes [25–27], and further, several organotins occupy ⇑ Corresponding author. Tel.: +91 9897135529. E-mail addresses: [email protected], [email protected] (M. Nath). http://dx.doi.org/10.1016/j.jphotobiol.2015.03.023 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.

an important place in cancer chemotherapy reports because of their apoptotic inducing character [28–32]. Organotin(IV) carboxylates, an important class of compounds having structural diversity ranging from monomeric, dimeric, tetrameric, oligomeric to polymeric motifs [14,33–36], display very promising antitumor activities [10,28–30,37–45]. Among these, several dibutyltin(IV) carboxylates have been found to be the most active, and in some cases they exhibit much higher activity than clinically used reference compounds such as cis-platin, doxorubicine and methotrexate [10,42]. Cancer chemotherapeutic drugs exert their cytotoxic mode of action by binding to nuclear DNA at the molecular level. A major challenge in cancer chemotherapy includes an efficient cellular drug delivery to the specific and appropriate site of action at the target site, which is a severe problem due to the charge, hydrophilic character or size of many therapeutic agents. Further, high-drug doses, necessary to compensate the reduced bioavailability, often cause strong adverse effects. This accounts for many therapeutic agents that have been tested in vitro to perform much less effectively in vivo [46]. Therefore, a rational drug design is required to achieve specific targeting features and to control toxicity (or other side effects) by controlling thermodynamic, kinetic processes of metal complexes [47] and their structural diversities responsible for more lipophilic character. Because the most lipophilic

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compounds are the most cytotoxic [48] and the ligands which possess biologically active pharmacophore and biocompatible properties are tethered to other ligands having multifunctional groups with N and O donor metal binding sites to obtain modulated ligand scaffold which can mute the potential toxicity of metallo-drugs [28–30]. Short-chain fatty acid (SCFA) anion, butyrate, has been found to be protective against colon carcinogenesis [49]. Lauric, myristic and stearic acids are naturally occurring fatty acids and exhibit various pharmacological applications [50–52]. Anti-tumor activity of organotin(IV) carboxylates and biological applications of fatty acids encouraged us to synthesize organotin(IV) derivatives of fatty acids. Some alkyltin derivatives of fatty acids including organocarboxystannoxanes (with Sn–O– Sn bond) have been reported to exhibit various industrial and biological applications [53–56]. To the best of our knowledge, anticancer assay of the titled organotin(IV) derivatives of fatty acids (molecular formula is depicted in Fig. 1) has not been investigated so far, and also there is a lack of systematic studies of their characterization, especially through multinuclear magnetic resonance. The main motive of the present study is to compare the cytotoxic behaviour of tri- and diorganotin(IV) carboxylates against five cancer cell lines of human origin, viz. MCF-7 (mammary), HEK-293 (kidney), PC-3 (prostate), HCT-15 (colon) and HepG-2 (liver). Enzyme assays such as lipid peroxidase, lactate dehydrogenase, glutathione peroxidase, glutathione reductase, and total glutathione have been performed in order to understand the possible cause of cytotoxicity and mode of action of the studied organotin(IV) carboxylates. DNA fragmentation assay, acridine orange assay and comet assay have also been done to provide the supportive evidences for the probable mode of action. The results of anti-inflammatory activity and toxicity (LD50) of the complexes have also been discussed in the manuscript. 2. Materials and methods 2.1. Reagents and materials Details of make and source of the reagents and materials used are described in supporting file (S-I). 2.2. Methods and instrumentation Details of various instruments used for characterization and the methods used for biological screening, enzyme assays, antiinflammatory and toxicity assay are presented in supporting file (S-I). 2.3. Synthesis 2.3.1. Synthesis of triorganotin(IV) and diorganotin(IV) derivatives of fatty acids by sodium salt formation method Fatty acid (2.0 mmol, HSA/HMA/HLA) was dissolved in the 20 mL of specially dried methanol and added to sodium methoxide which was prepared by reacting sodium (0.058 g, 2.5 mmol) with 10 mL of dry methanol (under dry nitrogen atmosphere). The mixture was stirred for half an hour (at room temperature) to give a clear solution of sodium salt of the acid and then refluxed for 4–6 h with constant stirring. 20 mL of hot methanol solution of dimethyltin(IV) dichloride (0.220 g, 1.0 mmol) or triphenyltin(IV) chloride (0.771 g 2.0 mmol) or tributyltin(IV) chloride (0.651 g, 2.0 mmol) or tripropyltin(IV) chloride (0.566 g, 2.0 mmol) or trimethyltin(IV) chloride (0.398 g, 2.0 mmol) was added to the solution of the preformed sodium salt of fatty acid. This solution was further refluxed for 24–36 h with constant stirring under dry nitrogen atmosphere. The solution was centrifuged and then

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Fig. 1. Molecular structure of fatty acids: where n = 10, lauric acid (HLA); n = 12, myristic acid (HMA) and n = 16, stearic acid (HSA).

filtered to remove the NaCl formed during the reaction. The excess of solvent was reduced under pressure. The solid/semi-solid products thus obtained were washed with methanol–hexane (1:3 v/v mixture) or methanol–petroleum ether (b.p. 40–60 °C) (1:3 v/v mixture). 2.3.2. Synthesis of dibutyltin(IV) and dioctyltin(IV) derivatives of fatty acids by the azeotropic removal of water method The titled complexes were prepared by drop wise addition of dry and hot methanol solution (10 mL) of dibutyltin(IV) oxide (0.498 g, 2.0 mmol) or dioctyltin(IV) oxide (0.722 g, 2.0 mmol) to a hot methanol solution (10 mL) of fatty acid (4.0 mmol, HSA/ HMA/HLA) under anhydrous nitrogen atmosphere. The above reaction mixture was refluxed for at least 24–26 h with constant stirring and water was removed azeotropically. The solvent was removed under reduced pressure and allowed to cool. The products were washed by either methanol–hexane or methanol–petroleum ether (b.p. 40–60 °C) mixture (1:3 v/v). 2.3.3. Pr3Sn(SA), CH3(CH2)16COOSnPr3 Physical state: Clear semi-solid; Yield: 66%; M. Wt. = 531. Anal. Calcd. for C27H56O2Sn: C, 61.01; H, 10.55, Sn, 22.40. Found: C, 61.06; H, 10.52, Sn, 22.42%. IR (KBr, cm1): 1652(s), 1461(s), 642(w), 550(w), 532(w). 1H NMR (d (ppm), CD3OD, 500.13 MHz): 2.91(t, 2H, CH2, J = 6.8 Hz); 1.92–1.62, 1.36–1.33 (mbr, 30H, CH2); 1.44 (m, 6H, b-H); 1.20(m, 9H, c-H); 1.11(t, 6H, a-H, J = 8.1 Hz); 1.06(t, 3H, CH3, J = 6.0 Hz). 13C NMR (d (ppm), CD3OD, 125.75 MHz): 180.10(OCO); 34.16–22.35(C1–C16); 25.11(C-b); 18.87(C-c); 15.38(C-a, 1J(13C–119Sn) = 552.1 Hz, h = 125.18°a); 12.41(CH3). 119Sn NMR (d (ppm), CD3OD, 186.50 MHz): 162.23. 2.3.4. Bu3Sn(SA), CH3(CH2)16COOSnBu3 Physical state: Clear semi-solid; Yield: 76%; M. Wt. = 573. Anal. Calcd. for C30H62O2Sn: C, 62.83; H, 10.84, Sn, 20.77. Found: C, 62.80; H, 10.82, Sn, 20.85%. IR (KBr, cm1): 1592(w), 1401(s), 648(w), 555(m), 525(w). 1H NMR (d (ppm), CD3OD, 500.13 MHz): 3.06(t, 2H, CH2, J = 7.1 Hz); 1.47(m, 6H, b-H); 1.38–1.32, 1.30– 1.26 (mbr, 30H, CH2); 1.25(m, 6H, c-H); 1.18(t, 6H, a-H, J = 8.0 Hz, 2J(1H–119Sn) = 66.2 Hz, h = 116.53°b); 0.95–0.92(t, 9H, d-H, J = 7.1 Hz); 0.80(t, 3H, CH3, J = 6.3 Hz). 13C NMR (d (ppm), CD3OD, 125.75 MHz): 181.10(OCO); 37.16–23.53(C1–C16); 16.88(C-a, 1J(13C–119Sn) = 534.2 Hz, h = 123.61°a); 26.87 (C-b, 2 13 J( C–119Sn) = 41.1 Hz); 25.19 (C-c); 15.21(CH3); 14.07 (C-d). 119 Sn NMR (d (ppm), CD3OD, 186.50 MHz): 156.0.

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2.3.5. Ph3Sn(SA), CH3(CH2)16COOSnPh3 Physical state: White solid; m.p.: 68–71 °C; Yield: 55%; M. Wt. = 633. Anal. Calcd. for C36H50O2Sn: C, 68.30; H, 7.94, Sn, 18.70. Found: C, 68.31; H, 7.89, Sn, 18.75%. IR (KBr, cm1): 1526(s), 1328(s), 449(w), 229(w), 227(w). 1H NMR (d (ppm), CD3OD, 500.13 MHz): 2.97(t, 2H, CH2, J = 7.0 Hz); 1.35–1.32(mbr, 12H, CH2); 1.31–1.29(mbr, 18H, CH2); 0.83(t, 3H, CH3, J = 6.0 Hz); 7.23(sbrc, 6H, a-H); 7.22–7.20(mbr, 9H, b-H, c-H). 13C NMR (d (ppm), CD3OD, 125.75 MHz): 175.00(OCO); 139.67(C-i, 1 13 J( C–119Sn) = 469.6 Hz, h = 117.95°a); 126.90–126.00(C-a, C-b, C-c); 31.70–23.07(C1–C16); 14.29(CH3). 119Sn NMR (d (ppm), CD3OD, 186.50 MHz): 172.50. 2.3.6. Bu2Sn(SA)2, (CH3(CH2)16COO)2SnBu2 Physical state: Pale viscous liquid; Yield: 75%; M. Wt. = 799. Anal. Calcd. for C44H88O4Sn: C, 66.08; H, 11.01, Sn, 14.89. Found: C, 66.04; H, 11.05, Sn, 14.82%. IR (KBr, cm1): 1655(s), 1462(m), 638(m), 559(w), 546(m). 1H NMR (d (ppm), CD3OD, 500.13 MHz): 2.85(m, 4H, CH2); 1.51–1.45(mbrc, 18H, CH2); 1.45–1.38(m, 8H, b-H, c-H); 1.38–1.34(mbr, 20H, CH2); 1.32–1.24(mbr, 18H, CH2); 1.22–1.19(mbr, 4H, CH2); 1.05–1.02(m, 4H, a-H); 0.92 (t, 6H, d-H, J = 6.3 Hz); 0.85–0.82(t, 6H, CH3, J = 6.2 Hz); 13C NMR (d (ppm), CD3OD, 125.75 MHz): 177.81(OCO); 35.11–23.03(C1–C16); 27.23(C-b, 2J(13C–119Sn) = 54.3 Hz); 26.77(C-c); 24.75(C-a, 1J (13C–119Sn) = 712.6 Hz, h = 139.26°a); 14.19(CH3); 13.28(C-d). 119 Sn NMR (d (ppm), CD3OD, 186.50 MHz): 271.71. 2.3.7. Oct2Sn(SA)2, (CH3(CH2)16COO)2SnOct2 Physical state: Clear semi-solid; m.p.: 20–25 °C; Yield: 57%; M. Wt. = 911. Anal. Calcd. for C52H104O4Sn: C, 68.49; H, 11.42, Sn, 13.06. Found: C, 68.45; H, 11.33, Sn, 13.13%. IR (KBr, cm1): 1661(s), 1469(m), 632(w), 550(w), 544(w). 1H NMR (d (ppm), CD3OD, 500.13 MHz): 2.46–2.32(tbr, 4H, CH2); 1.56–1.50(mbr, 20H, CH2); 1.52–1.48(mc, 10H, Oct-Sn); 1.48–1.41(mbrc, 24H, CH2); 1.41–1.36(mbrc, 16H, CH2); 1.23–1.17(mc, 8H, Oct-Sn); 0.94(t, 6H, CH3, J = 6.4 Hz); 0.94–0.90(mc, 10H, Oct-Sn); 0.87– 0.80(mc, 6H, x-H, Oct-Sn). 13C NMR (d (ppm), CD3OD, 125.75 MHz): 177.56(OCO); 36.84–23.21(C1–C16); 30.41(C-b); 29.72(C-c); 28.00(C-d); 27.30(C-h); 26.00(C-k); 24.26(C-l); 21.38(C-a); 17.11(C-x); 14.25(CH3). 119Sn NMR (d (ppm), CD3OD, 186.50 MHz): 216.14.

2.3.10. Bu3Sn(MA), CH3(CH2)12COOSnBu3 Physical state: Clear viscous liquid; Yield: 50%.; M. Wt. = 517. Anal. Calcd. for C26H54O2Sn: C, 60.35, H, 10.44, Sn, 23.30. Found: C, 61.18, H, 10.63, Sn, 22.61%. IR (KBr, cm1): 1632(m), 1446(m), 637(m), 527 (w), 525 (w). 1H NMR (d (ppm), CD3OD, 500.13 MHz): 3.01(t, 2H, CH2, J = 6.9 Hz); 1.36–1.30 (mbr, 14H, CH2); 1.53–1.40(m, 8H, CH2); 1.47(m, 6H, b-H); 1.25(m, 6H, c-H); 1.18(t, 6H, a-H, 8.0 Hz); 0.95–0.92(tbr, 9H, d-H); 0.80(t, 3H, CH3, J = 6.2 Hz). 13C NMR (d (ppm), CD3OD, 125.75 MHz): 179.20(OCO); 32.58–23.64(C1–C12); 27.07(C-b, 2J(13C–119Sn) = 3 13 44.8 Hz); 25.35(C-c, J( C–119Sn) = 75.6 Hz); 18.97(C-a, 1 13 119 J( C– Sn) = 458.2 Hz, h = 116.95°a); 13.21(C-d); 12.63(CH3). 119 Sn NMR (d (ppm), CD3OD, 186.50 MHz): 166.41. 2.3.11. Ph3Sn(MA), CH3(CH2)12COOSnPh3 Physical state: White solid; m.p.: >300 °C; Yield: 48%; M. Wt. = 577. Anal. Calcd. for C32H42O2Sn: C, 66.62, H, 7.30, Sn, 20.52. Found: C, 66.55, H, 7.27, Sn, 20.57%; IR (KBr, cm1): 1634(s), 1458(s), 567(m), 280(w), 227(w). 1H NMR (d (ppm), CD3OD, 500.13 MHz): 7.34(sbrc, 6H, a-H); 7.33–7.30(mbrc, 9H, b-H, c-H); 2.93(t, 2H, CH2, J = 7.2 Hz); 1.41–1.38(mbr, 10H, CH2); 1.38–1.35(mbr, 12H, CH2); 0.86(t, 3H, CH3, J = 6.1 Hz); 13C NMR (d (ppm), CD3OD, 125.75 MHz): 174.00(OCO); 139.03(C-i, 1 13 J( C–119Sn) = 469.6 Hz; h = 117.95°a); 127.11–126.07(C-a, C-b, C-c); 32.07–22.64 (C1–C12); 12.89(CH3). 119Sn NMR (d (ppm), CD3OD, 186.50 MHz): 190.40. 2.3.12. Me2Sn(MA)2, (CH3(CH2)12COO)2SnMe2 Physical state: White solid; m.p.: 70 °C; Yield: 49%; M. Wt. = 603. Anal. Calcd. for C30H60O4Sn: C, 59.70, H, 9.95, Sn, 19.73. Found: C, 59.19, H, 10.09, Sn, 19.21%. IR (KBr, cm1): 1656(s), 1472(s), 650(m), 552(w), 527(m). 1H NMR (d (ppm), CD3OD, 500.13 MHz): 2.90(t, 4H, CH2, J = 6.9 Hz); 1.55–1.48(mbr, 14H, CH2); 1.46–1.40(mbrb, 14H, CH2); 1.37–1.33(mbrb, 16H, CH2); 0.96(t, 6H, a-H, 2J (1H–119Sn) = 87.3 Hz, h = 140.82°b); 0.80(t, 6H, CH3, J = 6.1 Hz). 13C NMR (d (ppm), CD3OD, 125.75 MHz): 179.10(OCO); 33.11–22.75(C1–C12); 15.10(CH3); 8.12(C-a, 1J(13C–119Sn) = 726.6 Hz, h = 140.49°a). 119Sn NMR (d (ppm), CD3OD, 186.50 MHz): 230.11.

2.3.8. Me3Sn(MA), CH3(CH2)12COOSnMe3 Physical state: White solid; m.p.: 80–85 °C; Yield: 67%; M. Wt. = 391. Anal. Calcd. for C17H36O2Sn: C, 52.17; H, 9.21, Sn, 30.43. Found: C, 52.14, H, 9.25, Sn, 30.39%. IR (KBr, cm1): 1635(w), 1453(m), 644(w), 561(w), 527(m). 1H NMR (d (ppm), CD3OD, 500.13 MHz): 3.03(t, 2H, CH2, J = 6.9 Hz); 1.40–1.37(m, 6H, CH2); 1.37–1.34(sbr, 16H, CH2); 0.98(s, 9H, a-H, 2 1 J( H–119Sn) = 71.0 Hz, h = 120.84°b); 0.76(t, 3H, CH3, J = 6.1 Hz). 13 C NMR (d (ppm), CD3OD, 125.75 MHz): 178.60(OCO); 28.15– 23.26(C1–C12); 17.03(C-a, 1J(13C–119Sn) = 431.9 Hz, h = 114.64°a); 13.32(CH3); 119Sn NMR (d (ppm), CD3OD. 186.50 MHz): 160.30.

2.3.13. Bu2Sn(MA)2, (CH3(CH2)12COO)2SnBu2 Physical state: Clear viscous liquid; Yield: 76%; M. Wt. = 687. Anal. Calcd. for C36H72O4Sn: C, 62.88, H, 10.48, Sn, 17.32. Found: C, 61.86, H, 10.10, Sn, 17.11%. IR (KBr, cm1): 1640(s), 1453(s), 643(w), 570(m), 534(w). 1H NMR (d (ppm), CD3OD, 500.13 MHz): 2.94(t, 4H, CH2, J = 6.1 Hz); 1.54–1.49(mbr, 14H, CH2); 1.48– 1.44(mbr, 14H, CH2); 1.42–1.39(mbr, 16H, CH2); 1.36(m, 4H, b-H); 1.31(m, 4H, c-H); 1.15(t, 4H, a-H, J = 8.0 Hz, 2J(1H–119Sn) = 84.0 Hz, h = 136.12°b); 0.93(t, 6H, d-H); 0.86(t, 6H, CH3, J = 6.3 Hz). 13 C NMR (d (ppm), CD3OD, 125.75 MHz): 177.33(OCO); 35.61– 23.13(C1–C12); 27.41(C-b, 2J(13C–119Sn) = 46.0 Hz); 27.18(C-c, 3 13 1 13 J( C–119Sn) = 81.3 Hz); 25.25(C-a, J( C–119Sn) = 718.7 Hz, a 119 h = 139.80° ); 15.04(CH3); 13.16(C-d). Sn NMR (d (ppm), CD3OD, 186.50 MHz): 260.24.

2.3.9. Pr3Sn(MA), CH3(CH2)12COOSnPr3 Physical state: Clear semi-solid; Yield: 62%; M. Wt. = 475. Anal. Calcd. for C23H48O2Sn: C, 58.11, H, 10.08, Sn, 25.05. Found: C, 58.10, H, 10.10, Sn, 25.11%. IR (KBr, cm1): 1633(m), 1450(m), 640(w), 529(w), 493(w). 1H NMR (d (ppm), CD3OD, 500.13 MHz): 2.81(t, 2H, CH2, J = 7.1 Hz); 1.45(m, 4H, CH2); 1.40–1.36(mbr, 18H, CH2); 1.20(m, 6H, b-H); 1.14 (t, 9H, c-H, J = 7.2 Hz); 1.12(t, 6H, a-H, J = 7.0 Hz); 1.06(t, 3H, CH3, J = 6.0 Hz). 13C NMR (d (ppm), CD3OD, 125.75 MHz): 179.20(OCO); 27.24–23.24(C1–C12); 26.10(C-b); 18.15(C-c); 12.75(C-a, 1J (13C–119Sn) = 445.1 Hz, h = 115.8°a); 10.03(CH3). 119Sn NMR (d (ppm, CD3OD, 186.50 MHz): 153.09.

2.3.14. Oct2Sn(MA)2, (CH3(CH2)12COO)2SnOct2 Physical state: Clear semi-solid; m.p. 20–25 °C; Yield 77%; M. Wt. = 799. Anal. Calcd. for C44H88O4Sn: C, 66.08, H, 11.01, Sn, 14.89. Found: C, 65.09, H, 11.05, Sn, 14.84%. IR (KBr, cm1): 1622(s), 1439(s), 631(w), 545 (w), 543(w). 1H NMR (d (ppm), CD3OD, 500.13 MHz): 2.46–2.32(t, 4H, CH2); 1.56–1.50(mbr, 10H, CH2); 1.52–1.48(mc, 10H, Oct-Sn); 1.48–1.42 (mbr, 20H, CH2); 1.41–1.36(mbr, 14H, CH2); 1.23–1.17(mc, 6H, Oct-Sn); 0.95–0.89 (mc, 12H, Oct-Sn); 0.94(t, 6H, CH3, J = 6.3 Hz); 0.87–0.80(mc, 6H, x-H, Oct-Sn). 13C NMR (d (ppm), CD3OD, 125.75 MHz): 177.56(OCO); 36.84–23.21 (C1–C12); 30.41(C-b); 29.72(C-c);

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28.00(C-d); 27.30(C-h); 26.00(C-k); 24.26(C-l); 21.38(Ca,1J(13C–119Sn) = 920.5 Hz, h = 157.50°a); 17.11(C-x); 14.25(CH3). 119 Sn NMR (d (ppm), CD3OD, 186.50 MHz): 222.51.

2.3.19. HSA See supporting file (S-I).

2.3.15. Bu3Sn(LA), CH3(CH2)10COOSnBu3 Physical state: Clear liquid; Yield: 56%; M. Wt. = 489. Anal. Calcd. for C24H50O2Sn: C, 58.90, H, 10.22, Sn, 24.33. Found: C, 58.17, H, 10.49, Sn, 24.62%. IR (KBr, cm1): 1615(m), 1418(s), 647(w), 560(w), 535(w). 1H NMR (d (ppm), CD3OD, 500.13 MHz): 2.85 (t, 2H, CH2, J = 6.9 Hz); 1.37–1.33 (mbr, 18H, CH2); 1.37–1.32 (m, 6H, b-H); 1.20 (m, 6H, c-H); 1.13 (t, 6H, a-H, J = 8.0 Hz); 1.05 (t, 9H, d-H, J = 6.8 Hz); 0.91 (t, 3H, CH3, J = 6.1 Hz). 13C NMR (d (ppm), CD3OD): 178.31 (OCO); 31.20–21.98(C1–C10); 27.92(C-b, 2J (13C–119Sn) = 44.7 Hz); 25.42 (C-c); 19.05 (C-a, 1J (13C–119Sn) =

2.3.20. HMA See supporting file (S-I).

472.2 Hz, h = 118.18°a); 13.69(C-d); 12.91(CH3). (ppm), CD3OD, 186.50 MHz): 142.01.

119

Sn NMR (d

2.3.16. Ph3Sn(LA), CH3(CH2)10COOSnPh3 Physical state: White solid; m.p.: 120 °C; Yield: 52%; M. Wt. = 571. Anal. Calcd. for C30H38O2Sn: C, 64.02, H, 6.78, Sn, 21.02. Found: C, 65.57, H, 6.29, Sn, 21.62%. IR (KBr, cm1): 1629(m), 1430(s), 545(m), 279(w), 227(w). 1H NMR (d (ppm), CD3OD, 500.13 MHz): 7.23(sbrc, 6H, a-H); 6.79(mbr, 9H, b-H, c-H). 3.03(t, 2H, CH2, J = 6.8 Hz); 1.37–1.30(mbr, 18H, CH2); 0.83(t, 3H, CH3, J = 6.0 Hz); 13C NMR (d (ppm), CD3OD): 176.88(OCO); 31.70–22.77(C1–C10); 139.54(C-i, 1J(13C–119Sn) = 469.6 Hz, h = 117.95°a); 127.01–126.00(C-a, C-b, C-c); 119 13.09(CH3); Sn NMR (d (ppm), CD3OD): 184.60. 2.3.17. Bu2Sn(LA)2, (CH3(CH2)10COO)2SnBu2 Physical state: Yellow semi solid; m.p.: 22–24 °C; Yield: 51%; M. Wt. = 631. Anal. Calcd. for C32H64O4Sn: C, 60.86, H, 10.14, Sn, 18.85. Found: C, 60.08, H, 10.10, Sn, 18.87%. IR (KBr, cm1): 1613(m), 1420(s), 633(w), 568 (w), 541(w). 1H NMR (d (ppm), CD3OD, 500.13 MHz): 2.81(t, 4H, CH2); 1.64–1.53(mbr, 20H, CH2); 1.44– 1.37(m, 8H, b-H, k-H); 1.34–1.30(mbr, 16H, CH2); 0.86(t, 6H, CH3, J = 6.1 Hz); 1.10–1.07(m, 4H, a-H, 2J(1H–119Sn) = 85.0 Hz, h = 137.54°b); 0.95(t, 6H, d-H). 13C NMR (d (ppm), CD3OD, 125.75 MHz): 178.94(OCO); 30.07–21.79(C1–C10); 27.27(C-b); 27.19(C-c); 25.55(C-a, 1J(13C–119Sn) = 729.6 Hz, h = 140.76°a); 13.76 (CH3); 13.30 (C-d). 119Sn NMR (d (ppm), CD3OD, 186.50 MHz): 266.10. 2.3.18. Oct2Sn(LA)2, (CH3(CH2)10COO)2SnOct2 Physical state: White semi-solid; Yield: 77%; M. Wt. = 743. Anal. Calcd. for C40H80O4Sn: C, 64.60, H, 10.76, Sn, 16.02. Found: C, 64.89, H, 10.70, Sn, 15.97%. IR (KBr, cm1): 1615(s), 1421(s), 629(w), 565(w), 548(m). 1H NMR (d (ppm), CD3OD, 500.13 MHz): 2.98(t, 4H, CH2); 1.58–1.52(mbr, 20H, CH2); 1.50–1.46(mc, 12H, Oct-Sn); 1.43–1.38(mbr, 16H, CH2); 1.25–1.20(mc, 8H, Oct-Sn); 0.92– 0.86(mc, 8H, Oct-Sn); 0.90(t, 6H, CH3, J = 6.4 Hz); 0.85–0.79(mc, 6H, x-H, Oct-Sn). 13C NMR (d (ppm), CD3OD, 125.75 MHz): 180.27(OCO); 32.50–24.82(C1–C10); 25.73(C-a, 1J (13C–119Sn) = 721.4 Hz, h = 140.04°a); 30.62(C-b); 28.42(C-c); 28.04(C-d); 27.02(C-h); 26.07(C-k); 23.56(C-l); 14.02(C-x); 13.63(CH3). 119Sn NMR (d (ppm), CD3OD, 186.50): 220.12.

91

2.3.21. HLA See supporting file (S-I). IR: s: strong; w: weak; m: medium; br: broad; sh: shoulder; NMR: s: singlet; d: doublet; t: triplet; m: multiplet; br: broad; a angle h calculated from Eq. (1J(|13C–119Sn)|) = 11.4h–875; bangle h calculated from Eq.: \C–Sn–C (h) = 0.0161|2J|2  1.32|2J| + 133.4; c strong over-lapping.

Details of methods used for cytotoxicity assay (MTT assay), enzyme assays (such as lipid peroxidise, glutathione reductase, glutathione peroxidise, total glutathione, lactate dehydrogenase), DNA fragmentation assay, acridine orange assay, comet assay, anti-inflammatory activity, toxicity and statistical analysis are described in supporting file (S-I). 3. Results and discussion 3.1. Synthetic aspects The reaction of triorganotin(IV) chloride(s) with sodium salt of the acid (Eq. (1)) led to the formation of triorganotin(IV) carboxylate(s) (1:1 ratio, Eq. (2)) and the reaction of dimethyltin(IV) dichloride with sodium salt of the acid led to the formation of dimethyltin(IV) carboxylate (1:2 ratio, Eq. (3)). The reaction of diorganotin oxide(s) with the acid resulted in the formation of diorganotin(IV) carboxylate(s) (1:2 ratio, Eq. (4)).

HL þ MeONa ! NaL þ MeOH

ð1Þ

HL = CH3(CH2)10C(O)OH (HLA), CH3(CH2)12C(O)OH (HMA) and CH3(CH2)16C(O)OH (HSA) 1:1

R3 SnCl þ NaL ! R3 SnðLÞ þ NaCl MeOH

ð2Þ

R = Me, Pr, Bu and Ph 1:2

R2 SnCl2 þ 2NaL ! R2 SnðLÞ2 þ 2NaCl MeOH

ð3Þ

R = Me MeOH

R2 SnO þ 2LH ! R2 SnðLÞ2 þ H2 O 1:2

ð4Þ

R = Bu and Oct The reaction in Eq. (1) required 4–6 h of refluxing. The reactions in Eqs. (2), (3) required refluxing for 24–36 h and Eq. (4) required 24–26 h of refluxing. The resulting products were obtained in 49–77% yield and found to be sensitive towards air and moisture, except triphenyltin(IV) derivatives which were stable for more than six months. The complexes are soluble in methanol, ethanol and DMSO, but show low solubility in other organic solvents. The molar conductance values of 103 M solutions (in methanol) of the

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synthesized organotin(IV) complexes are in the range 10–17 ohm1 cm2 mol1, suggesting their non-electrolytic nature. 3.2. Infrared and far-infrared spectral studies The mas(OCO) shifted towards lower frequencies, whereas the ms(OCO) frequencies either remained same or shifted towards lower frequencies in comparison to the free acid (data of free acids are given in S-I). For the studied organotin(IV) carboxylates, the Dm (199–176 cm1 < 200 cm1) is comparable to that of sodium salt of the corresponding fatty acid (Dm = 196–180 cm1), indicating that the carboxylate group is behaving as bidentate ligand, involved in bridging [57–59]. A band in the region 570–449 cm1 has been assigned to m(Sn–O), which supports the bonding of carboxylate group to the tin atom [28,29]. The observed mas(Sn–C) and ms(Sn–C) frequencies in di-/trialkyltin(IV) derivatives (629– 650 cm1 and 525–552 cm1, respectively) and triphenyltin(IV) derivatives (229–280 cm1 and at 227 cm1, respectively) correspond to the reported values [28,29]. 3.3. Multinuclear (1H,

13

C and

Table 1 ESI-MS data of triorganotin(IV) derivatives of fatty acids. Complex

ESI-MS: m/z

Pr3Sn(SA)

532 [M + H]+ (100%), 554 [M + Na]+, 248 [M-L]+, 779 [M + SnPr3]+, 206 [HSnPr2]+, 164 [H2SnPr]+ 574 (100%) [M + H]+, 596 [M + Na]+, 290 [M-L]+, 863 [M + SnBu3]+, 234 [HSnBu2]+, 178 [H2SnBu]+ 634 [M + H]+, 656 [M + Na]+, 350 (100%) [M-L]+, 983 [M + SnPh3]+, 274 [HSnPh2]+, 198 [H2SnPh]+ 392 [M+H]+ (100%), 414 [M + Na]+, 164 [M-L]+, 555 [M + SnMe3]+, 150 [HSnMe2]+, 136 [H2SnMe]+ 476 [M+H]+ (100%), 498 [M + Na]+, 248 [M-L]+, 723 [M + SnPr3]+, 206 [HSnPr2]+, 164 [H2SnPr]+ 518 (100%) [M + H]+, 540 [M + Na]+, 290 [M-L]+, 807 [M + SnBu3]+, 234 [HSnBu2]+, 178 [H2SnBu]+ 578 [M + H]+, 600 [M + Na]+, 350 (100%) [M-L]+, 927 [M + SnPh3]+, 274 [HSnPh2]+, 198 [H2SnPh]+ 490 (100%) [M + H]+, 512 [M + Na]+, 290 [M-L]+, 779 [M + SnBu3]+, 234 [HSnBu2]+, 178 [H2SnBu]+ 572 [M + Na]+, 350 (100%) [M-L]+, 899 [M+SnPh3]+, 274 [HSnPh2]+, 198 [H2SnPh]+

Bu3Sn(SA) Ph3Sn(SA) Me3Sn(MA) Pr3Sn(MA) Bu3Sn(MA) Ph3Sn(MA) Bu3Sn(LA) Ph3Sn(LA)

119

Sn) NMR spectral studies

1 H and 13C NMR spectra of the fatty acids and the studied complexes were recorded in CD3OD. Absence of resonance of acidic proton (OH signal at d 12.29–12.60 ppm) in the spectra of studied complexes suggests deprotonation of the acid (HSA/HMA/HLA) upon complexation. The 2J(1H–119Sn) and 1J(13C–119Sn) for triorganotin(IV) carboxylates (having well resolved satellites) lie in the range 66.15–71.00 Hz and 431.91–552.10 Hz, respectively, which correspond to penta-coordinated tin atom in the complexes. The C–Sn–C angles calculated with the help of 2J(1H–119Sn) and 1 13 J( C–119Sn) using Lockhart and Manders equation [60,61] lie in the range 116.53–120.84° and 114.64–125.18°, respectively. These values further support the trigonal-bipyramidal geometry around tin atom with the three organic groups in equatorial plane and the electronegative oxygen atoms of carboxylate group in axial positions. The observed 2J(1H–119Sn) (87.27–84.00 Hz) and 1J(13C–119Sn) (920.49–712.55 Hz) for diorganotin(IV) carboxylates and the calculated values of \C–Sn–C using 2J(1H–119Sn) and 1J(13C–119Sn) values in Lockhart and Manders equation [60,61] in the range 140.87– 136.12° and 157.50–139.26°, respectively, indicate a distorted octahedral geometry around the tin atom with trans organic groups disposed in a distorted linear skeleton of carbon–tin– carbon and more electronegative (O) atoms in the equatorial positions. Only one 119Sn resonance has been observed in the spectra of tri- and diorganotin(IV) carboxylates, which rules out the existence of two different tin species. The observed 119Sn resonances for triand diorganotin(IV) carboxylates lie in the ranges 142.01 to 190.40 ppm and 216.14 to 271.71 ppm, respectively, which further support five-coordinated (distorted trigonal-bipyramidal) and six-coordinated (distorted octahedral) geometry around the tin atom for triorganotin(IV) carboxylates and diorganotin(IV) carboxylates, respectively [28,29].

Table 2 ESI-MS data of diorganotin(IV) derivatives of fatty acids. Complex

ESI-MS: m/z

Bu2Sn(SA)2

800 [M+H]+ (100%), 822 [M + Na]+, 516 [M-L]+ = [Bu2SnL]+, 234 [M + H-2L]+ = [HSnBu2]+, 178 [H2SnBu]+ 912 [M+H]+ (100%), 934 [M + Na]+, 628 [M-L]+ = [Oct2SnL]+, 346 [M + H-2L]+ = [HSnOct2]+, 234 [H2SnOct]+ 604 [M+H]+ (100%), 626 [M + Na]+, 376 [M-L]+ = [Me2SnL]+, 150 [M + H-2L]+ = [HSnMe2]+, 136 [H2SnMe]+ 688 [M+H]+ (100%), 710 [M + Na]+, 460 [M-L]+ = [Bu2SnL]+, 234 [M + H-2L]+ = [HSnBu2]+, 178 [H2SnBu]+ 800 [M+H]+ (100%), 822 [M + Na]+, 572 [M-L]+ = [Oct2SnL]+, 346 [M + H-2L]+ = [HSnOct2]+, 234 [H2SnOct]+ 632 [M+H]+ (100%), 654 [M + Na]+, 432 [M-L]+ = [Bu2SnL]+, 234 [M + H-2L]+ = [HSnBu2]+, 178 [H2SnBu]+ 744 [M+H]+ (100%), 766 [M + Na]+, [M-L]+ = [Oct2SnL]+, 346 [M + H-2L]+ = [HSnOct2]+, 234 [H2SnOct]+

Oct2Sn(SA)2 Me2Sn(MA)2 Bu2Sn(MA)2 Oct2Sn(MA)2 Bu2Sn(LA)2 Oct2Sn(LA)2

([SnR3]+) ion peaks have been observed due to loss of one mole of ligand, involving the cleavage of weak Sn–O bond yielding two complementary ions, where the cationic part of the complex is measured in positive mode [62]. The peaks corresponding to [M + SnR3]+ have also been observed which provide additional proof for five-coordinated linear polymeric structure of the triorganotin(IV) carboxylates [63]. The subsequent fragment ions [HSnR2]+ and [H2SnR]+ resulting from [M-L]+ ([SnR3]+) are well observed in ESI-MS spectra of triorganotin(IV) carboxylates, which have also been reported earlier in the literature [63]. The [M-L]+ ion peaks formed by the elimination of ligand from the complex ions have been observed in the spectra of diorganotin(IV) carboxylates, which have also been reported in literature [64]. The fragments corresponding to [HSnR2]+ and [H2SnR]+ have been observed in ESI-MS spectra of diorganotin(IV) carboxylates. Probable structures for tri- and diorganotin(IV) derivatives of fatty acids (proposed on the basis of IR, NMR and ESI-MS) have been presented in Fig. 2.

3.4. ESI-MS spectrometric studies 3.5. Cytotoxicity assay (MTT assay) The ESI-MS spectral data for organotin(IV) carboxylates were recorded in the range m/z 50–1000, using methanol as solvent and further diluting with acetonitrile and water mixture. The observed data has been presented in Tables 1 and 2. The tri- and diorganotin(IV) derivatives of fatty acids yield the molecular ion peaks corresponding to [M + Na]+ and [M + H]+, which have been used to determine molecular weight of the complexes [62–64]. In the ESI-MS spectra of triorganotin(IV) carboxylates, [M-L]+

The studied tri- and diorganotin(IV) carboxylates have been screened for in vitro anti-tumor activity against a panel of five human cancer cell lines, viz. MCF-7 (mammary), HEK-293 (kidney), PC-3 (prostate), HCT-15 (colon) and HepG-2 (liver). The IC50 values of the studied complexes along with those of the standard reference drugs, cis-platin (CPT) and 5-fluorouracil (5-FU) have been reported in Table 3.

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Fig. 2. Probable molecular structures for (a) triorganotin(IV) carboxylates and (b) diorganotin(IV) carboxylates, where R = Ph, Oct, Bu, Pr and Me; n = 10 (lauric acid, HLA); n = 12 (myristic acid, HMA) and n = 16 (stearic acid, HSA).

Table 3 Anti-tumor screening data (IC50 in lg/mL ± SEM) of tri- and diorganotin(IV) derivatives of fatty acids. Complex

MCF-7

HEK-293

PC-3

HCT-15

HepG-2

Pr3Sn(SA) Bu3Sn(SA) Ph3Sn(SA) Bu2Sn(SA)2 Oct2Sn(SA)2 Me3Sn(MA) Pr3Sn(MA) Bu3Sn(MA) Ph3Sn(MA) Me2Sn(MA)2 Bu2Sn(MA)2 Oct2Sn(MA)2 Bu3Sn(LA) Ph3Sn(LA) Bu2Sn(LA)2 Oct2Sn(LA)2

16.91 ± 0.90 9.31 ± 1.00 95.10 ± 5.30 17.23 ± 0.60 30.99 ± 0.30 23.46 ± 0.90 16.12 ± 0.61 9.51 ± 0.12 577 ± 9.30 26.86 ± 1.60 15.29 ± 1.50 27.24 ± 0.30 0.093 ± 0.01 0.141 ± .001 11.37 ± 0.20 26.39 ± 3.30

16.45 ± 0.90 6.69 ± 0.40 76.08 ± 2.50 18.63 ± 4.70 31.82 ± 0.30 27.13 ± 0.80 23.90 ± 2.64 8.91 ± 1.12 1154 ± 10.10 28.32 ± 0.80 11.45 ± 0.20 25.43 ± 0.30 0.15 ± 0.08 0.254 ± 0.02 7.94 ± 0.54 25.41 ± 0.60

21.40 ± 1.90 7.41 ± 0.40 63.4 ± 3.90 21.84 ± 4.20 22.96 ± 0.80 24.85 ± 6.80 16.84 ± 5.20 10.11 ± 1.02 577 ± 7.30 29.78 ± 1.10 15.72 ± 0.20 22.52 ± 0.50 0.085 ± 0.01 0.084 ± 0.06 13.14 ± 0.6 24.93 ± 2.90

21.56 ± 1.70 9.03 ± 0.60 76.08 ± 1.40 17.96 ± 3.90 19.80 ± 0.60 19.55 ± 3.70 15.11 ± 1.10 8.16 ± 1.14 2885 ± 9.70 30.13 ± 0.50 9.78 ± 0.60 31.72 ± 1.60 0.98 ± 0.04 1.69 ± 0.10 9.18 ± 0.53 24.58 ± 2.23

25.75 ± 2.90 8.62 ± 0.90 95.10 ± 1.40 18.77 ± 2.30 34.58 ± 0.30 21.56 ± 1.10 15.25 ± 1.30 13.60 ± 0.36 7.50 ± 0.80 28.14 ± 1.10 16.44 ± 0.90 41.16 ± 6.40 1.38 ± 0.62 2.26 ± 0.10 15.91 ± 0.30 28.81 ± 3.49

CPT 5-FU

13.98 ± 3.90 0.48 ± 0.08

15.44 ± 5.12 2.06 ± 0.23

18.67 ± 6.90 0.92 ± 0.30

5.04 ± 1.40 3.93 ± 2.05

9.46 ± 1.90 0.48 ± 0.10

Among the triorganotin(IV) carboxylates, Bu3Sn(LA) exhibited the highest activity against all cell lines followed by Ph3Sn(LA), whereas Ph3Sn(MA) exhibited the lowest activity against all cell lines except HepG-2. Among the diorganotin(IV) carboxylates, Bu2Sn(LA)2 exhibited the highest activity followed by Bu2Sn(MA)2. The complexes, viz. Bu3Sn(SA) (against MCF-7, HEK293, PC-3 and HepG-2), Pr3Sn(MA) (against PC-3), Bu3Sn(MA) (against MCF-7, HEK-293 and PC-3), Bu2Sn(MA)2 (against HEK293 and PC-3), and Bu2Sn(LA)2 (against MCF-7, HEK-293 and PC3) are more active in comparison to cis-platin. Bu3Sn(LA) and Ph3Sn(LA) are more active than cis-platin as well as 5-fluorouracil against all cell lines. Whereas, Pr3Sn(SA) (against HEK-293) and Bu2Sn(MA)2 (against MCF-7) are slightly less active in comparison to cis-platin. Classification of organotin(IV) carboxylates according to the extent of their cytotoxicity has been summarised in Table 4. The structure–activity correlation of the studied tri- and diorganotin(IV) derivatives of fatty acids reveals that (a) triorganotin(IV) derivatives are more active than diorganotin(IV) derivatives, (b) phenyl derivatives are less active than alkyl derivatives, (c) order of activity of alkyl derivatives is Bu > Pr > Me  Oct. Similar results for organotin(IV) complexes have also been reported earlier [10,33,58,68]. Activity of organotin(IV) compounds depend on few structural features, including availability of coordination positions around the tin atom (R3SnL > R2SnL), the Sn–O bond stability, relative stability of the Sn–alkyl and Sn–aryl bonds towards hydrolytic cleavage, conformation adopted by the ligand in the cellular fluid and balance between lipophilic and

hydrophilic properties [10,23,24,28–30,68]. Lipophilic nature is required for crossing the cell membrane and hydrophilic character is required to display activity in an aqueous environment [10,68]. Thus, transportation of organotin ions (R3Sn+/R2Sn2+ moiety formed by the hydrolysis of Sn–O bond) or organotin(IV) carboxylate as a whole as a single entity across the cellular membrane may be responsible for the activity of organotin(IV) carboxylates. 3.6. Enzymatic assays The results of enzymatic assays for Ph3Sn(LA), Bu3Sn(LA) and Bu2Sn(LA)2 against MCF-7 are presented in Tables 5 and 6 (graphically represented in Figs. 3a–3f). Necrosis and apoptosis are two ways through which cell death can occur. Cell death observed with in short interval of time, due to membrane toxicity (non-specific necrosis), leads to sudden and marginal increase of lactate dehydrogenase (LDH) concentration. A marginal increment in LDH activity (1.18–1.10-fold) has been observed in the studied complexes treated MCF-7 cells in comparison to control or untreated cells (Figs. 3a, 3c and 3d), therefore, a minor role of necrosis cannot be excluded. Generation of reactive oxygen species (ROS) in the cells, as indicated by a substantial fold increase in induction of antioxidant enzymes’ activity, suggests that oxidative stress may be responsible for cell death. Lipid peroxidation assay is a widely accepted assay to determine oxidative damage. Treatment of MCF-7 cells with Ph3Sn(LA) and Bu3Sn(LA) results in rapid increase (Figs. 3b and 3e) in lipid

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Table 4 Classification of tri- and diorganotin(IV) derivatives of fatty acids on the basis of their Anti-tumor activity. Complex

Anti-tumor activitya

Pr3Sn(SA) Bu3Sn(SA) Ph3Sn(SA) Bu2Sn(SA)2 Oct2Sn(SA)2 Me3Sn(MA) Pr3Sn(MA) Bu3Sn(MA) Ph3Sn(MA) Me2Sn(MA)2 Bu2Sn(MA)2 Oct2Sn(MA)2 Bu3Sn(LA) Ph3Sn(LA) Bu2Sn(LA)2 Oct2Sn(LA)2

MCF-7, PC-3, HEK-293, HCT-15, MCF-7, PC-3, HEK-293, HCT-15, – MCF-7, PC-3, HCT-15, HEK-293, PC-3, HCT-15 MCF-7, HEK-293, HepG-2 MCF-7, PC-3, HCT-15, HEK-293, MCF-7, HEK-293, PC-3, HCT-15, HepG-2 MCF-7, HEK-293, HepG-2 MCF-7, PC-3, HEK-293, HCT-15, MCF-7, PC-3, HEK-293, HCT-15, MCF-7, PC-3, HEK-293, HCT-15, MCF-7, PC-3, HEK-293, HCT-15, MCF-7, PC-3, HEK-293, HCT-15, MCF-7, PC-3, HCT-15, HEK-293,

Active HepG-2 HepG-2 HepG-2

HepG-2 HepG-2

HepG-2 HepG-2 HepG-2 HepG-2 HepG-2 HepG-2

Moderate

Inactive

– – MCF-7, PC-3, HEK-293, HCT-15, HepG-2 – MCF-7, HEK-293, HepG-2 PC-3, HCT-15 – – – PC-3, HCT-15 – – – – – –

– – – – – – – – MCF-7, PC-3, HCT-15, HEK-293 – – – – – –

a According to the NCI guideline, a compound is active when the IC50 6 30 lg/mL, moderately active when the IC50 values within the range of 30–100 lg/mL and inactive when the IC50 P 100 lg/mL [65–67].

Table 5 Enzymatic assays of Ph3Sn(LA) against MCF-7. Enzymes

Control/untreated a

Lipid peroxidase Glutathione peroxidaseb Total glutathionea Glutathione reductaseb Lactate dehydrogenaseb a

3.50 ± 0.001  10 5.66 ± 0.1  101

4

3.70 ± 0.001  103 1.62 ± 0.02  101 5.59 ± 1.1

Treated 7.45 ± 0.01  10 15.82 ± 0.9

IF 3

5.60 ± 0.001  102 3.54 ± 1.1 6.59 ± 1.1

24.00 27.90 18.00 21.00 1.18

lM/lg protein.

b

IU/mg protein; Induction fold (IF): mean of treated/mean of untreated; the experiment was performed in triplicate and mean ± SEM of the three experiments are shown here.

peroxidation, which stimulates the oxidative reactions, ensuring the generation of reactive oxygen species (ROS) in the cells. Drugs like doxorubicin [69], gingerol [70], curcumin [71] and resveratrol [72] are known to generate reactive oxygen species (ROS), which affect the intracellular mitochondrial membrane potential. Thus, disturbance in mitochondrial membrane potential through complex induced ROS generation may be responsible for cytotoxicity of organotin(IV) carboxylates. An increase in glutathione peroxidase activity by 27-fold (Fig. 3a), and in glutathione reductase (GR) activity by 21-fold (Fig. 3a) have been observed after the treatment with Ph3Sn(LA). Bu3Sn(LA) also induces increase in glutathione peroxidase activity by 3.02-fold, and glutathione reductase (GR) activity by 1.89-fold (Fig. 3c). Increase in total glutathione content by 18-fold (Fig. 3b) and 1.12-fold (Fig. 3f) has also been observed in the complexes, Ph3Sn(LA) and Bu3Sn(LA) treated cells, respectively. Glutathione

peroxidase and glutathione reductase are known for regulating the redox system of cells [73]. Any change in concentration of these enzymes affects the redox system of cells, as a result of which the cells undergo oxidative stress [74]. It is reported that the oxidative stress plays a vital role in apoptosis [75,76]. Apoptosis due to increase in glutathione peroxidase activity induced by ellagic acid has also been reported in literature [77]. There are various mechanisms through which cell death may be induced. One of them is cell membrane damage (necrosis) which leads to the leakage of LDH, GST and GSH resulting in cell death. ROS generation and alteration in mitochondrial potential may also be responsible for cytotoxicity. Oxidative stress is the other possible way which may affect cell survival and hence inhibiting cell growth. Alternatively, Ph3Sn(LA) and Bu3Sn(LA) might have induced peroxides generation, which would have been metabolized by glutathione peroxidase, consequently forming oxidized glutathione from reduced form of glutathione, affecting the mitochondrial activity. Organotins are also known to influence [Ca2+] level, generating reactive oxygen species and affecting mitochondrial activity, ultimately resulting in oxidative stress in tumor cells leading to apoptosis [78–81]. The results indicate that Ph3Sn(LA) and Bu3Sn(LA) are exhibiting the cytotoxic activity due to apoptosis and as well as due to necrosis. Many anti-cancer drugs like cis-platin, cladribine, doxorubicin, phyllanthus and 5-fluorouracil, have been reported earlier, which lead to cell death through both apoptotic and necrotic effects [82,83]. It is reported that trimethyltins also exhibit cell damage by both apoptosis and necrosis [84]. Treatment of MCF-7 cells with Bu2Sn(LA)2 results in marginal increase (1.92-fold, Fig. 3e) in lipid peroxidase activity, resulting in ROS generation in very small amount. Increase in total glutathione

Table 6 Enzymatic assays of Bu3Sn(LA) and Bu2Sn(LA)2 against MCF-7.

a b

Enzymes

Control/untreated

Treated with Bu3Sn(LA)

IF

Treated with Bu2Sn(LA)2

IF

Lipid peroxidasea Glutathione peroxidaseb Total Glutathionea Glutathione reductaseb Lactate dehydrogenaseb

2.137 ± 0.003 3.091 ± 0.014 0.0037 ± 0.001 2.208 ± 0.024 5.129 ± 0.015

15.420 ± 0.02 9.343 ± 0.063 0.00414 ± 0.0035 4.169 ± 0.060 5.660 ± 0.060

7.21 3.02 1.12 1.89 1.10

4.103 ± 0.010 3.008 ± 0.029 0.00533 ± 0.0019 2.263 ± 0.029 5.640 ± 0.052

1.92 0.97 1.44 1.02 1.10

lM/lg protein.

IU/mg protein; induction fold (IF): mean of treated/mean of untreated; the experiment was performed in triplicate and mean ± SEM of the three experiments are shown here.

M. Nath et al. / Journal of Photochemistry and Photobiology B: Biology 148 (2015) 88–100

Fig. 3a. Estimation of enzymatic activity of glutathione peroxidase (GPx); glutathione reductase (GR) and lactate dehydrogenase (LDH) in response to Ph3Sn(LA) in MCF-7. The data is expressed as mean ± SEM. ⁄, ⁄⁄ and ⁄⁄⁄ represent the significant difference in glutathione peroxidase, glutathione reductase and lactate dehydrogenase activity with respect to their respective controls/untreated cells (p < 0.05), respectively.

95

Fig. 3d. Estimation of enzymatic activity of glutathione peroxidase (GPx); glutathione reductase (GR) and lactate dehydrogenase (LDH) in response to Bu2Sn(LA)2 in MCF-7. The data is expressed as mean ± SEM. ⁄⁄⁄ Represents the significant difference in lactate dehydrogenase activity with respect to their controls/untreated cells (p < 0.05), respectively.

Fig. 3b. Lipid peroxidation and total glutathione content in MCF-7 after the treatment with Ph3Sn(LA). The data is expressed as mean ± SEM. Treated cells show the significant increase in lipid peroxidation and total glutathione content, with respect to their respective controls/untreated cells (p < 0.05), for MCF-7. Fig. 3e. Lipid peroxidation content in MCF-7 after the treatment with Bu3Sn(LA) and Bu2Sn(LA)2 (mean ± SEM). The results suggest a significant increase in lipid peroxidation with respect to the control/untreated (p < 0.05), ⁄⁄ and ⁄⁄⁄ represent the significant increase in lipid peroxidation in Bu3Sn(LA) and Bu2Sn(LA)2 treated MCF-7 cells, respectively.

Fig. 3c. Estimation of enzymatic activity of glutathione peroxidase (GPx); glutathione reductase (GR) and lactate dehydrogenase (LDH) in response to Bu3Sn(LA) in MCF-7. The data is expressed as mean ± SEM. ⁄⁄, ⁄⁄⁄ and ## represent the significant difference in glutathione peroxidase, glutathione reductase and lactate dehydrogenase activity with respect to their controls/untreated cells (p < 0.05), respectively.

content (1.44-fold, Fig. 3f) has also been observed. However, glutathione peroxidase activity (0.97-fold) and glutathione reductase activity (1.02-fold) remained almost unaltered in Bu2Sn(LA)2 treated MCF-7 cells (Fig. 3d). The results suggest that along with

Fig. 3f. Total glutathione content in MCF-7 after the treatment with Bu3Sn(LA) and Bu2Sn(LA)2 with respect to control (untreated cells). The data is expressed as mean ± SEM.

necrosis and ROS generation, there might be some other mechanism responsible for its cytotoxicity. To determine if apoptosis is the main cause of cell death, DNA fragmentation assay, acridine orange assay and comet assay have also been performed.

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Fig. 4. (a) Formation of DNA ladder in MCF-7 cells in response to Ph3Sn(LA) through gel electrophoresis; lane 1: treated with Ph3Sn(LA); lane 2: treated with cis-platin; lane 3: control (extracted DNA from MCF-7); lane 4: DNA ladder, (b) Formation of DNA ladder in MCF-7 cells in response to Bu3Sn(LA) through gel electrophoresis; lane 6: treated with cis-platin; lane 7: control (extracted DNA from MCF-7); lane 8: treated with Bu3Sn(LA); lane 9: DNA ladder and (c) Formation of DNA ladder in MCF-7 cells in response to Bu2Sn(LA)2 through gel electrophoresis; lane 10: DNA ladder; lane 11: control (extracted DNA from MCF-7); lane 12: treated with Bu2Sn(LA)2; Lane 13: treated with cis-platin.

3.7. DNA fragmentation assay DNA fragmentation assay is one of the most reliable method which has been used to distinguish apoptosis from necrosis. Fig. 4 represents the results obtained after the treatment of Ph3Sn(LA), Bu3Sn(LA) and Bu2Sn(LA)2 to MCF-7 cells with respect to control (untreated) and cis-platin. The sequence in Fig. 4 is as follows: DNA extracted from MCF-7 cells were taken as control (lane 3, lane 7 and lane 11); cis-platin (lane 2, lane 6 and lane 13); DNA ladder (lane 4, lane 9 and Lane 10); DNA-extracted from MCF-7 cells treated with Ph3Sn(LA) (lane 1); DNA extracted from MCF-7 cells treated with Bu3Sn(LA) (lane 8) and DNA extracted from MCF-7 cells treated with Bu2Sn(LA)2 (lane 12). The image was taken with the help of Gel Doc software. Fig. 4(a) and (b) clearly show the formation of DNA–ladder (smearing) (when MCF-7 cells treated with Ph3Sn(LA) and Bu3Sn(LA), respectively) which is significantly higher than that with cis-platin. The results indicate that DNA fragmentation (break) has occurred, suggesting that the cytotoxic effect of Ph3Sn(LA) and Bu3Sn(LA) is selectively mediated through the induction of apoptosis. Fig. 4(c) indicates that Bu2Sn(LA)2 treated cells do not show significant fragmentation (in comparison to cisplatin), suggesting that apoptosis is occurring only to a small extent and is less efficient than Ph3Sn(LA) and Bu3Sn(LA) (Fig. 4(a) and (b)). 3.8. Acridine orange assay Morphological changes have been observed by acridine orange fluorescent staining assay [28,29]. The MCF-7 cells were incubated for 24 h with IC50 value of organotin compounds and cis-platin, and nuclear staining was visualized under 200 objective of fluorescent microscope (Zeiss, Axiovert 25, Germany). As shown in Fig. 5, the induction of apoptosis has been observed, followed by membrane blebbing and chromatin condensation in Bu3Sn(LA) treated MCF-7 cells, which has not been significantly visible in MCF-7 cells treated with Bu2Sn(LA)2. Live cells and apoptotic cells can be easily distinguished by the percentage uptake of AO:EB. In view of the fact that AO permeates all live cells, and hence appear green. EB is taken up only when the cells have lost their cytoplasmic membrane integrity due to

apoptosis and hence appear red. Bu3Sn(LA) induced cell death has been considered to be apoptotic by observing the typical apoptotic morphological change by AO/EB staining, whereas Bu2Sn(LA)2 treated cells do not show significant changes. In Fig. 5 the live cells have green colour1 (shown by unfilled arrows), while apoptotic cells have red/orange nucleus (shown by unfilled arrows). 3.9. Comet assay Cytotoxicity leading to DNA damage has also been validated by single cell gel electrophoresis. It is an uncomplicated and sensitive technique for the detection of DNA damage to the level of the individual cell [28,29]. For this assay the nuclei were stained with ethidium bromide and visualized under fluorescent microscope. The arrows in the Fig 6(b) and (c) indicate the comet tail (DNA break), which further supports that apoptosis is the main cause of cell death, whereas Fig. 6(d) shows no such comet formation and similarly control (untreated cell) in Fig 6(a) shows no prominent tailing. A comparatively higher comet tailing has been observed in Bu3Sn(LA) treated MCF-7 cell when compared to cisplatin treated MCF-7 cell. Fig. 7 represents the % DNA in comet tail in Bu3Sn(LA) treated MCF-7 cells in comparison to cis-platin and control. Comet tail length has been calculated as the distance between the end of nuclei heads and end of each tail. Tail moments were defined as the product of the % DNA in each tail, and the distance between the mean of the head and tail distributions [85] with the help of the following equation:

% DNA ¼ TA  TAI=½ðTA  TAIÞ þ ðHA  HAIÞ  100 where TA = tail area, TAI = tail area intensity, HA = head area and HAI = head area intensity. 3.10. Anti-inflammatory activity The anti-inflammatory and anti-cancer activities are interrelated. Some non-steroidal compounds (inhibit the over-expression of prostaglandin cascade and cox-2) are anti-inflammatory drugs 1 For interpretation of color in Fig. 5, the reader is referred to the web version of this article.

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Fig. 5. Acridine orange assay: (a) Control (MCF-7), (b) MCF-7 treated with Cis-platin, (c) MCF-7 treated with Bu3Sn(LA) and (d) MCF-7 treated with Bu2Sn(LA)2. Unfilled arrows and filled arrows indicate live cells and apoptotic cells, respectively. The experiment was performed in triplicate and a representative experiment is presented.

Fig. 7. Percentage of DNA in comet tail in Bu3Sn(LA) (A) treated MCF-7 cells in comparison to cis-platin (B) and control (C); ⁄⁄ represents the significant increase in comet tail with respect to cis-platin and control.

Fig. 6. Comet assay: (a) Negative control, (b) Cis-platin treated MCF-7 cell, (c) Bu3Sn(LA) treated MCF-7 cell and (d) Bu2Sn(LA)2 treated MCF-7 cell. The DNA breaks from individual cell (MCF-7) were visualized as comet tails (marked by arrows). The experiment was performed in triplicate and a representative experiment is presented.

and they also exhibit potential anti-cancer activity [86,87]. Therefore, it becomes indispensible to evaluate the anti-inflammatory activity of the titled complexes. The anti-inflammatory

activity data (% inhibition) of organotin(IV) derivatives of fatty acids are presented in Table 7. The results obtained indicate the following order for the anti-inflammatory activity of the studied complexes: for organotin(IV) derivatives of lauric acid, Ph3Sn(LA) > Bu3Sn(LA) > Oct2Sn(LA)2 > Bu2Sn(LA)2; for organotin(IV) derivatives of myristic acid, Ph3Sn(MA) > Oct2Sn(MA)2 > Bu3Sn(MA) > Bu2Sn(MA)2 > Pr3Sn(MA) > Me3Sn(MA) > Me2Sn(MA)2, and for organotin(IV) derivatives of stearic acid, Ph3Sn(SA) > Bu3Sn(SA) > Oct2Sn(SA)2 > Pr3Sn(SA) > Bu2Sn(SA)2. Ph3Sn(LA) exhibited good anti-inflammatory activity (31.4% inhibition) followed by Ph3Sn(MA) (26.6% inhibition). The highest activity of Ph3Sn(LA) may be due to the easy formation and frequent transportation of [Ph3Sn(IV)]+ moiety across the cell membrane as a part of its mode of action (mechanism) [24,28].

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Table 7 Anti-inflammatory activity and toxicity of tri- and diorganotin(IV) derivatives of fatty acids. Complexa/standard drug

Anti-inflammatory activity dose (50 mg/kg) Paw vol. before (A) (mean ± SEM)

Paw vol. after (B) (mean ± SEM)

Paw vol. difference (mean ± SEM))

p Value w.r.t. indomethacin

% Inhibition

Pr3Sn(SA) Bu3Sn(SA) Ph3Sn(SA) Bu2Sn(SA)2 Oct2Sn(SA)2 Me3Sn(MA) Pr3Sn(MA) Bu3Sn(MA) Ph3Sn(MA) Me2Sn(MA)2 Bu2Sn(MA)2 Oct2Sn(MA)2 Bu3Sn(LA) Ph3Sn(LA) Bu2Sn(LA)2 Oct2Sn(LA)2 Ibuprofen Indomethacinb Ph3SnCl Bu3SnCl Pr3SnCl Me3SnCl Me2SnCl2 Bu2SnO Oct2SnO

9.00 ± 0.30 8.80 ± 0.20 9.76 ± 0.31 8.72 ± 0.30 8.68 ± 0.16 10.08 ± 0.07 9.06 ± 0.09 8.66 ± 0.25 8.60 ± 0.14 9.20 ± 0.23 9.36 ± 0.24 9.32 ± 0.22 8.70 ± 0.28 9.74 ± 0.31 9.42 ± 0.21 9.30 ± 0.33 8.02 ± 0.15 8.48 ± 0.19 9.76 ± 0.11 9.46 ± 0.16 8.86 ± 0.12 9.70 ± 0.23 9.92 ± 0.11 9.94 ± 0.48 10.12 ± 0.14

10.88 ± 0.30 10.55 ± 0.17 11.58 ± 0.17 10.62 ± 0.29 10.54 ± 0.24 12.04 ± 0.18 10.86 ± 0.19 10.26 ± 0.18 10.14 ± 0.13 11.18 ± 0.24 11.14 ± 0.17 10.16 ± 0.70 10.28 ± 0.36 11.18 ± 0.19 11.18 ± 0.32 11.04 ± 0.25 9.26 ± 0.21 9.24 ± 0.29 11.68 ± 0.19 11.46 ± 0.31 10.82 ± 0.17 11.60 ± 0.18 11.98 ± 0.22 11.92 ± 0.48 12.06 ± 0.15

1.88 ± 0.02 1.75 ± 0.23 1.82 ± 0.16 1.90 ± 0.03 1.86 ± 0.20 1.96 ± 2.00 1.80 ± 0.12 1.60 ± 0.13 1.54 ± 0.02 1.98 ± 0.04 1.78 ± 0.15 1.64 ± 0.02 1.58 ± 0.12 1.44 ± 0.17 1.76 ± 0.31 1.74 ± 0.18 1.24 ± 0.09 0.76 ± 0.09 1.92 ± 0.08 2.00 ± 0.07 1.96 ± 0.09 1.90 ± 0.05 2.06 ± 0.12 1.98 ± 0.06 1.94 ± 0.09

<0.0010 <0.0045 0.0007 <0.0001 0.0014 0.0002 0.0002 0.0002 <0.0001 <0.0001 0.0006 <0.0001 0.0002 0.0078 0.0015 0.0020 – – – – – – – – –

10.5 12.5 13.3 9.5 11.4 6.6 14.3 17.5 26.6 5.7 15.2 21.9 18.6 31.4 16.2 17.1 36.08 60.82 8.57 4.76 6.66 9.52 1.91 5.71 7.83

3.11. Toxicity The LD50 values of the studied organotin(IV) derivatives of fatty acids are found to be in wide range varying from >50 to >800 mg kg1 and are presented in Table 7. Octyltin(IV) derivatives are found to be less toxic and safer than other alkyltin(IV) and phenyltin(IV) derivatives.

4. Conclusions Organotin(IV) derivatives of fatty acids have been synthesized and characterized. IR, 1H, 13C and 119Sn NMR spectral and ESI-MS studies of all the synthesized tri- and diorganotin(IV) carboxylates suggest a distorted trigonal-bipyramidal and distorted octahedral geometry, respectively. Anti-cancer screening of these carboxylates suggests that Bu3Sn(LA) is the most active complex among the studied title complexes, which exhibits potent anticancer activity against all the cell lines. Triorganotin(IV) carboxylates are more potent than diorganotin(IV) carboxylates. Significant increase in glutathione peroxidise activity and lipid peroxidation assay support that cancer cells undergo oxidative stress. Marginal increase in LDH suggests that necrosis may also occur to a small extent. Furthermore, DNA ladder formation, AO and comet assays indicate that these complexes inhibit the cancer cell growth through apoptosis. Taken together, the results indicate that the complexes possess dual capability of cell death: necrosis and apoptosis. However, further experiments are needed to establish the fact more strongly. An anti-cancer agent is expected to possess an anti-inflammatory activity. It has been observed that titled complexes show good anti-inflammatory activity. In vitro cytotoxicity assays, and in vivo anti-inflammatory activity suggest titled compounds as potential anti-tumor drugs. More clear view of the mode of action responsible for the cytotoxicty induced by organotins can be established firmly through other biological experiments and assays. Therefore, further studies are required to understand the exact or precise mechanism of organotin(IV)

c

Toxicity LD50 mg/kg

>200 >200 >100 >400 >800 >100 >100 >200 >200 >200 >400 >800 >150 >200 >50 >800 >2000 >1000 >400 >400 >200 >200 >400 >400 >800

carboxylates which may be responsible for their activity and further, clarify the cross-talk among various apoptotic pathways to make organotins as anti-tumor drugs. 5. Abbreviations

HLA HMA HSA

lauric acid myristic acid stearic acid

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