Organotin(IV) carboxylate derivatives as a new addition to anticancer and antileishmanial agents: design, physicochemical characterization and interaction with Salmon sperm DNA

Abstract

This article demonstrates the synthesis and characterization of novel organotin(IV) carboxylate complexes and their medicinal applications. Metal complexes and organometallic compounds have growing importance in medicine, particularly in oncology. Organotin derivatives have caught much attention during the last two decades for their potential biocidal activities. In recent years several organotin compounds have been synthesized, some with interesting cytotoxic properties. Here we will focus on the relevance of organotin derivatives as very promising potential candidates in anticancer therapy. Ten new organotin(IV) complexes were synthesized and characterized by using FT-IR, NMR (¹H, ¹³C and ¹¹⁹Sn), elemental analysis, mass spectrometry and single crystal X-ray techniques. The results of infrared spectroscopy of the sodium salt and complexes showed that the coordination took place through the oxygen atoms of the carboxylate group. Crystallographic data for complexes 1 and 3 showed that the tin has a distorted trigonal bipyramidal geometry with the three alkyl groups in the trigonal plane and the two oxygen atoms in the equatorial plane. In the case of complex 4 the geometry is tetrahedral due to the steric hindrance of the bulky phenyl groups. The compounds were screened for in vitro antiproliferative activity against lung carcinoma (H-157) and kidney fibroblast (BHK-21) cell lines and revealed significant anticancer activity. They were also tested for antileishmanial activity against the promastigote form of leishmania major and exhibited IC₅₀ value 0.98 ± 0.06 μM (Compound 10) as compared to amphotericin B (IC₅₀ value 0.29 ± 0.05 μM). The synthesized compounds act as a potent intercalator of SS-DNA and insert themselves into the nitrogenous bases of the DNA resulting in hypochromism and bathochromic shift.

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1. Introduction

The use of metal complexes as chemotherapeutic agents in the treatment of illness, which is a major public health concern, appears as a very attractive alternative. The discovery of cis-platin for the treatment of testicular and ovarian cancer attracted researchers’ attention to other metal-based antineoplastic agents. Metal-based compounds are of particular interest due to their physical and chemical properties. Properties such as ligand exchange rates, redox properties, oxidation states, coordination affinities, solubility, biodisponibility, and biodistribution could be modified in order to enhance the therapeutic effect while significantly decreasing the side effects.¹ One approach that could produce successful results involves the metal coordination of ligands with well-known biological activity. In this way, the designed metal-based compound combines ligands with important biological activity and pharmacologically active metals in a single chemical moiety. This strategy could produce enhanced efficiency and reduced toxic or side effects while lowering the therapeutic doses and/or overcoming drug resistance mechanisms. Additionally the metal could act as a carrier and/or stabilizer of the drug until it is able to reach the target. At the same time, the organic ligand with well-known biological activity could transport and protect the metal, then avoiding side reactions on its route to the potential targets. The metal–ligand combined effects may result in a significant improvement in the activity of the resulting coordination compounds.^1–3

Cancer chemotherapy based on metal complexes has gained momentum after the serendipitous discovery of cis-platin, and remains a front line treatment for most aggressive solid tumors.⁴ Therefore, novel treatments are urgently needed for cancer therapy. The fight against cancer is the main and primary target concerning this research. The initial efforts in the evaluation of platinum-based anticancer drugs have been shifted to non-platinum metal-based agents.⁵ Thus, an intensive study of other metals (Ti, Ga, Ge, Pd, Au, Co, Ru and Sn) is being carried out and is helping to improve the problems associated with the use of platinum compounds as anticancer drugs.⁶ Recent studies have shown very promising in vitro antitumor properties of organotin compounds against a wide panel of tumor cell lines of human origin.⁷ In some cases, organotin(IV) derivatives have also shown acceptable antiproliferative in vivo activity as new chemotherapy agents.⁸

Leishmaniasis has been defined by the World Health Organization as a group of diseases that severely affects 12 million people residing in the warm areas of the world.⁹ In most cases, patients cannot survive if proper treatment is not provided during development of this sand-fly mediated parasitic disease. Several antileishmanial agents have already been reported^10,11 but none of these proved to be the ultimate choice of drug due to varying degrees of efficacy and toxicity. Among these, however, pentavalent antimonials are recognized to be the most useful drug for treatment of visceral leishmaniasis caused by Leishmania donovani.¹² Discovery of antimony salt resistant pathogenic strains has made the situation worse.¹³ Miltefosine, an orally active phosphocholine analogue, also appeared to be effective in the treatment of the disease.¹⁴ However, there is still a need to identify new chemotherapeutic agents for effective therapy of the visceral form of leishmaniasis, commonly known as kala-azar.¹⁵

In continuation of our previous research work, we report here the synthesis and biological applications of organotin(IV) carboxylate derivatives of N-[(2-methoxy-5-nitrophenyl)]-4-oxo-4-[oxy]butanamide. All the synthesized compounds were successfully characterized by FT-IR, multi NMR (¹H, ¹³C, ¹¹⁹Sn), elemental analysis (CHN), mass spectrometry and single crystal X-ray analysis. They were tested for in vitro antiproliferative and antileishmanial activities and got excellent results.

2. Experimental section

2.1. Materials and methods

Reagents Me₃SnCl, n-Bu₃SnCl, Ph₃SnCl, Cy₃SnCl, Me₂SnCl₂, n-Bu₂SnCl₂, tert-Bu₂SnCl₂, Ph₂SnCl₂, n-Oct₂SnO, 2-methoxy-5-nitroaniline, succinic anhydride were obtained from Aldrich (USA) and were used without further purification. All the solvents purchased from E. Merck (Germany) were dried before use according to the literature procedures.¹⁶ Sodium salt of Salmon sperm DNA (SS-DNA) (Arcos) was used as received. The melting points were determined in a capillary tube using a Gallenkamp (UK) electrothermal melting point apparatus. IR spectra in the range 4000–100 cm⁻¹ were obtained on a Thermo Nicolet-6700 FT-IR Spectrophotometer equipped with a DTGS (deuterated triglycine sulphate) detector. Elemental analysis was carried out using a CE-440 Elemental Analyzer (Exeter Analytical, Inc) and the experimentally found values are given in parentheses in the experimental part. ¹H, ¹³C and ¹¹⁹Sn NMR were recorded on a 400 MHz JEOL ECS instrument, using DMSO as an internal reference [¹H (DMSO-d₆) = 2.50 ppm and ¹³C (DMSO-d₆) = 39.5 ppm]. For ¹¹⁹Sn NMR the measurement was recorded at a working frequency of 37.718749 MHz. Chemical shifts are given in ppm and coupling constant (J) values are given in Hz. The multiplicities of the signals in ¹H NMR are given with chemical shifts; (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of a doublet). The absorption spectra were measured on a Shimadzu 1800 UV-visible Spectrophotometer. X-ray data for complexes 3 and 4 were collected at 150 (2) K on a Bruker Apex II CCD diffractometer. Details are given in Table 2. All the non-hydrogen atoms were refined using anisotropic atomic displacement parameters, and hydrogen atoms bonded to carbon were inserted at calculated positions using a riding model. Hydrogen atoms bonded to O or N were located from difference maps and their coordinates refined. SHELXS-97¹⁷ was used to solve and SHELX2012¹⁸ to refine the structures. The mass spectra were recorded on a Thermo Scientific executive (orbitrap) utilizing an Advion TriVersa ™NanoMate sample introduction system. The m/z values were evaluated assuming that H = 1, C = 12, N = 14, O = 16, Cl = 35, and Sn = 120.

2.2. Synthesis

Synthesis of sodium salt of ligand: sodium N-[(2-methoxy-5-nitrophenyl)]-4-oxo-4-[oxy]butanamide (NaL). An aqueous solution of sodium hydrogen carbonate (NaHCO₃) was added to a suspension of the ligand, N-[(2-methoxy-5-nitrophenyl)]-4-oxo-4-[oxy]butanamide, in distilled water. The mixture was stirred at room temperature to get a clear solution, which was then rotary evaporated to get the desired sodium salt of the ligand.¹⁹ The chemical reaction is shown in Scheme 1.

Scheme 1 Systematic route for the synthesis of NaL and organotin(IV) complexes.

IR (4000–400 cm⁻¹): 3357 ν (NH); 1680 ν (amide C=O); 1537 ν (COOasym); 1259 ν (COOsym); 278 (Δν).

Synthesis of organotin(IV) complexes. Organotin(IV) carboxylates were synthesized by refluxing a mixture of R₃SnCl (5 mmol)/R₂SnCl₂ (2.5 mmol) and the sodium salt of ligand NaL (5 mmol) in dry toluene for 8 h (Scheme 1).¹⁹ The refluxed solution was kept overnight at room temperature. The NaCl precipitate was removed by filtration and the solvent was removed under reduced pressure. The product was purified by recrystallization from chloroform at room temperature. The numbering of the ligand, N-[(2-methoxy-5-nitrophenyl)]-4-oxo-4-[oxy]butanamide (HL), and alkyl groups attached to Sn is given in Scheme 2.

Scheme 2 Numbering pattern for HL and organic moieties attached to Sn atom.

Trimethylstannyl 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoate (1). Yield: 85%: m.p. 172–174 °C: mol. wt.: 431.0: anal. calc. for C₁₄H₂₀N₂O₆Sn: C, 39.0 (38.8); H, 4.7 (4.8); N, 6.3 (5.9): IR (4000–400 cm⁻¹): 3358 ν (NH); 1691 ν (amide C=O); 1526 ν (COOasym); 1366 ν (COOsym); 160 (Δν); 550 ν (Sn–C); 461 ν (Sn–O): ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.55 (t, 2H, H2, ³J[¹H, ¹H] = 6.8 Hz); 2.32 (t, 2H, H3, ³J[¹H, ¹H] = 6.8 Hz); 9.47 (s, 1H NH); 8.98 (d, 1H, H6, ⁴J[¹H, ¹H] = 2.8 Hz); 7.98 (dd, 1H, H8, ⁴J[¹H, ¹H] = 2.8 Hz); 7.21 (d, 1H, H9, ³J[¹H, ¹H] = 9.2 Hz); 3.94 (s, 3H, H11); 0.32 (s, 3H, Hα, ²J[¹¹⁹Sn–¹Hα] = 69 Hz): ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 172.5 (C1); 33.1 (C2); 31.5 (C3); 176.4 (C4); 128.6 (C5); 111.4 (C6); 140.8 (C7); 115.8 (C8); 120.4 (C9); 154.6 (C10); 57.2 (C11); 0.7 (Cα, ¹J[¹¹⁹Sn–¹³Cα = 520 Hz): ¹¹⁹Sn NMR (DMSO-d6, 400 MHz) δ (ppm): −10.8: ESI-MS, m/z (%): [C₁₄H₂₀N₂O₆SnNa]⁺, m/z = 455 (51.4); [C₁₄H₂₀N₂O₆Sn]⁺, m/z = 432 (28); [C₁₁H₁₁O₆N₂]⁺, m/z = 267 (3); [C₁₀H₁₁N₂O₄]⁺, m/z = 223 (2); [C₃H₉Sn]⁺, m/z = 165 (100); [C₂H₆Sn]⁺, m/z = 150 (2); [CH₃Sn]⁺, m/z = 135 (32); [Sn]⁺, m/z = 120 (13); [C₁₃H₁₇N₂O₆Sn]⁺, m/z = 417 (5); [C₁₂H₁₇N₂O₄Sn]⁺, m/z = 373 (24); [C₁₁H₁₄N₂O₄Sn]⁺, m/z = 358 (31); [C₁₀H₁₁N₂O₄Sn]⁺, m/z = 343 (70).

Triethylstannyl 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoate (2). Yield: 80%: m.p. 124–126 °C: mol. wt.: 473.1: anal. calc. for C₁₇H₂₆N₂O₆Sn: 43.0 (43.0); H, 5.5 (5.5); N, 5.9 (5.7): IR (4000–400 cm⁻¹): 3419 ν (NH); 1734 ν (amide C=O); 1520 ν (COOasym); 1365 ν (COOsym); 155 (Δν); 546 ν (Sn–C); 486 ν (Sn–O): ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.54 (t, 2H, H2, ³J[¹H, ¹H] = 6.8 Hz); 2.33 (t, 2H, H3, ³J[¹H, ¹H] = 6.8 Hz); 9.50 (s, 1H NH); 8.97 (d, 1H, H6, ³J[¹H, ¹H] = 9.2 Hz); 7.98 (dd, 1H, H8, ⁴J[¹H, ¹H] = 2.8 Hz); 7.23 (d, 1H, H9, ⁴J[¹H–¹H] = 2.8 Hz); 3.93 (s, 3H, H11); 1.02 (q, 2H, Hα, ²J[^119/117Sn–¹Hα] = 76, 74 Hz); 1.20 (t, 3H, Hβ, ³J[¹H, ¹H] = 8.0 Hz): ¹³C NMR (DMSO-d6, 100 MHz) δ (ppm): 172.4 (C1); 33.0 (C2); 31.2 (C3); 176.4 (C4); 128.5 (C5); 111.4 (C6); 140.8 (C7); 115.8 (C8); 120.3 (C9); 154.5 (C10); 57.1 (C11); 11.1 (Cα, ¹J[¹¹⁹Sn–¹³Cα] = 580 Hz); 10.5 (Cβ, ²J[¹¹⁹Sn–¹³Cβ] = 34 Hz): ¹¹⁹Sn NMR (DMSO-d₆, 400 MHz) δ (ppm): −16.2: ESI-MS, m/z (%): [C₁₇H₂₆N₂O₆SnNa]⁺, m/z = 497 (30); [C₁₇H₂₆N₂O₆Sn]⁺, m/z = 432 (15); [C₁₁H₁₁N₂O₆]⁺, m/z = 267 (34); [C₁₀H₁₁N₂O₄]⁺, m/z = 223 (13); [C₆H₁₅Sn]⁺, m/z = 207 (100); [C₄H₁₀Sn]⁺, m/z = 178 (35); [C₂H₅Sn]⁺, m/z = 149 (40); [Sn]⁺, m/z = 120 (35); [C₁₅H₂₁N₂O₆Sn]⁺, m/z = 445 (16); [C₁₄H₂₁N₂O₄Sn]⁺, m/z = 401 (11); [C₁₂H₁₆N₂O₄Sn]⁺, m/z = 372 (13); [C₁₂H₁₆N₂O₄]⁺, m/z = 252 (15).

Tributylstannyl 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoate (3). Yield: 85%: m.p. 115–117 °C: mol. wt.: 557.3: anal. calc. for C₂₃H₃₈N₂O₆Sn: C, 49.6 (48.1); H, 6.9 (7.3); N, 5.0 (4.9): IR (4000–400 cm⁻¹): 3358 ν (NH); 1738 ν (amide C=O); 1526 ν (COOasym); 1373 ν (COOsym); 153 (Δν); 540 ν (Sn–C); 489 ν (Sn–O): ¹H NMR (DMSO-d6, 400 MHz) δ (ppm): 2.56 (t, 2H, H2, ³J(¹H, ¹H) = 6.4 Hz); 2.34 (t, 2H, H3, ³J[¹H, ¹H] = 6.4 Hz); 9.43 (s, 1H NH); 9.03 (d, 1H, H6, ⁴J[¹H, ¹H] = 2.8 Hz); 7.95 (dd, 1H, H8, ⁴J[¹H, ¹H] = 2.8 Hz); 7.20 (d, 1H, H9, ³J[¹H, ¹H] = 9.2 Hz); 3.93 (s, 3H, H11); 0.99 (t, 2H, Hα, ³J[¹H, ¹H] = 8.0 Hz); 1.53 (m, 2H, Hβ); 1.26 (m, 2H, Hγ); 0.79 (t, 3H, Hδ, ³J[¹H, ¹H] = 7.2 Hz): ¹³C NMR (DMSO-d6, 100 MHz) δ (ppm): 172.4 (C1); 33.2 (C2); 31.4 (C3); 176.4 (C4); 128.8 (C5); 111.2 (C6); 140.8 (C7); 115.6 (C8); 120.2 (C9); 154.4 (C10); 57.1 (C11); 21.4 (Cα, [¹¹⁹Sn–¹³Cα] = 384 Hz); 28.2 (Cβ, [¹¹⁹Sn–¹³Cβ] = 27 Hz); 27.0 (Cγ, [¹¹⁹Sn–¹³Cγ] = 75 Hz); 14.1 (Cδ): ¹¹⁹Sn NMR (DMSO-d₆, 400 MHz) δ (ppm): −17.3: ESI-MS, m/z (%): [C₂₃H₃₈N₂O₆SnNa]⁺, m/z = 581 (5); [C₂₃H₃₈N₂O₆Sn]⁺, m/z = 558 (15); [C₁₁H₁₁N₂O₆]⁺, m/z = 267 (5); [C₁₀H₁₁N₂O₄]⁺, m/z = 223 (10); [C₁₂H₂₇Sn]⁺, m/z = 291 (100); [C₈H₁₈Sn]⁺, m/z = 234 (35); [C₄H₉Sn]⁺, m/z = 177 (25); [Sn]⁺, m/z = 120 (10); [C₁₉H₂₉N₂O₆Sn]⁺, m/z = 501 (11); [C₁₈H₂₉N₂O₄Sn]⁺, m/z = 457 (41); [C₁₄H₂₀N₂O₄Sn]⁺, m/z = 400 (12); [C₁₄H₂₀N₂O₄Sn]⁺, m/z = 343 (23).

Triphenylstannyl 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoate (4). Yield: 85%: m.p. 161–163 °C: mol. wt.: 617.2: anal. calc. for C₂₉H₂₆N₂O₆Sn: C, 56.4 (55.6); H, 4.3 (4.2); N, 4.5 (4.0): IR (4000–100 cm⁻¹): 3306 ν (NH); 1739 ν (amide C=O); 1536 ν (COOasym); 1324 ν (COOsym); 212 (Δν); 541 ν (Sn–C); 439 ν (Sn–O): ¹H NMR (DMSO-d6, 400 MHz) δ (ppm): 2.53 (t, 2H, H2, ³J[¹H, ¹H] = 6.4 Hz); 2.35 (t, 2H, H3, ³J[¹H, ¹H] = 6.4 Hz); 9.42 (s, 1H NH); 9.01 (d, 1H, H6, ⁴J[¹H, ¹H] = 2.8 Hz); 7.98 (dd, 1H, H8, ⁴J[¹H, ¹H] = 3.2 Hz); 7.19 (d, 1H, H9, ³J[¹H, ¹H] = 9.2 Hz); 3.98 (s, 3H, H11); 7.45 (d, 1H, Hβ, ³J[¹H, ¹H] = 6.4 Hz); 7.81 (m, 1H, Hγ); 7.34 (m, 1H, Hδ): ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 172.2 (C1); 32.9 (C2); 31.5 (C3); 176.2 (C4); 128.9 (C5); 111.3 (C6); 140.8 (C7); 115.7 (C8); 120.3 (C9); 154.4 (C10); 57.1 (C11); 143.7 (Cα, [¹¹⁹Sn–¹³Cβ] = 580 Hz); 136.7 (Cβ, [¹¹⁹Sn–¹³Cβ] = 45 Hz); 128.7 (Cγ, [¹¹⁹Sn–¹³Cγ] = 61 Hz); 129.2 (Cδ): ¹¹⁹Sn NMR (DMSO-d₆, 400 MHz) δ (ppm): 224.1, −257.5: ESI-MS, m/z (%): [C₂₉H₂₆N₂O₆SnNa]⁺, m/z = 641 (6); [C₂₉H₂₆N₂O₆Sn]⁺, m/z = 618 (19); [C₁₁H₁₁N₂O₆]⁺, m/z = 267 (17); [C₁₀H₁₁N₂O₄]⁺, m/z = 223 (10); [C₁₈H₁₅Sn]⁺, m/z = 351 (100); [C₁₂H₁₀Sn]⁺, m/z = 274 (35); [C₄H₉Sn]⁺, m/z = 197 (25); [Sn]⁺, m/z = 120 (20); [C₂₃H₂₁N₂O₆Sn]⁺, m/z = 541 (21); [C₂₂H₂₁N₂O₄Sn]⁺, m/z = 497 (34); [C₁₆H₁₆N₂O₄Sn]⁺, m/z = 420 (28); [C₁₀H₁₁N₂O₄Sn]⁺, m/z = 343 (34).

Tricyclohexylstannyl 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoate (5). Yield: 83%: m.p. 129–131 °C: mol. wt.: 635.4: anal. calc. for C₂₉H₄₄N₂O₆Sn: C, 54.8 (54.7); H, 7.0 (7.5); N, 4.4 (4.2): IR (4000–400 cm⁻¹): 3327 ν (NH); 1738 ν (amide C=O); 1531 ν (COOasym); 1365 ν (COOsym); 166 (Δν); 540 ν (Sn–C); 440 ν (Sn–O): ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.61 (t, 2H, H2, ³J[¹H, ¹H] = 6.4 Hz); 2.21 (t, 2H, H3, ³J[¹H, ¹H] = 6.4 Hz); 9.48 (s, 1H NH); 9.07 (d, 1H, H6, ⁴J[¹H, ¹H] = 2.8 Hz); 7.96 (dd, 1H, H8, ⁴J[¹H, ¹H] = 2.8 Hz); 7.20 (d, 1H, H9, ³J[¹H, ¹H] = 8.8 Hz); 3.94 (s, 3H, H11); 1.54 (t, 1H, Hα, ³J[¹H, ¹H] = 6.4 Hz); 1.79 (m, 2H, Hβ); 1.50 (m, 2H, Hγ), 1.40 (m, 2H, Hδ): ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 172.3 (C1); 33.1 (C2); 31.1 (C3); 176.8 (C4); 128.8 (C5); 111.2 (C6); 140.8 (C7); 115.5 (C8); 120.2 (C9); 154.4 (C10); 57.1 (C11); 27.1 (Cα, ¹J[¹¹⁹Sn–¹³Cα] = 599 Hz); 29.2 (Cβ, ²J[¹¹⁹Sn–¹³Cβ] = 72 Hz); 31.1 (Cγ); 36.4 (Cδ): ¹¹⁹Sn NMR (DMSO-d₆, 400 MHz) δ (ppm): −70.9: ESI-MS, m/z (%): [C₂₉H₄₄N₂O₆SnNa]⁺, m/z = 659 (20); [C₂₉H₄₄N₂O₆Sn]⁺, m/z = 636 (30); [C₁₁H₁₁N₂O₆]⁺, m/z = 267 (18); [C₁₀H₁₁N₂O₄]⁺, m/z = 223 (22); [C₁₈H₃₃Sn]⁺, m/z = 369 (100); [C₁₂H₂₂Sn]⁺, m/z = 287 (43); [C₆H₁₁Sn]⁺, m/z = 197 (29); [Sn]⁺, m/z = 120 (32); [C₂₃H₃₃N₂O₆Sn]⁺, m/z = 553 (26); [C₂₂H₃₃N₂O₄Sn]⁺, m/z = 509 (29); [C₁₆H₁₆N₂O₄Sn]⁺, m/z = 426 (12); [C₁₀H₁₁N₂O₄Sn]⁺, m/z = 343 (16).

Dimethylstannanediyl bis(4-(2-methoxy-5-nitrophenylamino)-4-oxobutanoate) (6). Yield: 75%: m.p. 181–183 °C: mol. wt.: 683.2: anal. calc. for C₂₄H₂₈N₄O₁₂Sn: C, 42.2 (42.1); H, 4.1 (4.0); N, 8.2 (8.0): IR (4000–400 cm⁻¹): 3351 ν (NH); 1738 ν (amide C=O); 1501 ν (COOasym); 1366 ν (COOsym); 135 (Δν); 539 ν (Sn–C); 464 ν (Sn–O): ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.63 (t, 2H, H2, ³J[¹H, ¹H] = 6.4 Hz); 2.47 (t, 2H, H3, ³J[¹H, ¹H] = 6.4 Hz); 9.52 (s, 1H NH); 8.97 (d, 1H, H6, ⁴J[¹H, ¹H] = 2.4 Hz); 7.97 (dd, 1H, H8, ⁴J[¹H, ¹H] = 2.8 Hz); 7.21 (d, 1H, H9, ³J[¹H, ¹H] = 9.2 Hz); 3.94 (s, 3H, H11); 0.73 (s, 3H, Hα, [¹¹⁹Sn–¹Hα] = 100 Hz): ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 172.0 (C1); 32.2 (C2); 31.2 (C3); 174.5 (C4); 128.5 (C5); 111.4 (C6); 140.8 (C7); 115.9 (C8); 120.5 (C9); 154.6 (C10); 57.22 (C11); -0.1 (Cα, ¹J[¹¹⁹Sn–¹³Cα] = 800 Hz): ¹¹⁹Sn NMR (DMSO-d6, 400 MHz) δ (ppm): −303.7: ESI-MS, m/z (%): [C₂₄H₂₈N₄O₁₂SnNa]⁺, m/z = 707 (80); [C₂₄H₂₈N₄O₁₂Sn]⁺, m/z = 684 (25); [C₂₃H₂₅N₄O₁₂Sn]⁺, m/z = 669 (6); [C₁₂H₁₄N₂O₆Sn]⁺, m/z = 402 (12); [C₁₁H₁₁N₂O₆Sn]⁺, m/z = 387 (12); [C₁₁H₁₁N₂O₆]⁺, m/z = 267 (32); [C₁₀H₁₁N₂O₄]⁺, m/z = 223 (23); [C₂H₆Sn]⁺, m/z = 150 (10); [CH₃Sn]⁺, m/z = 135 (14); [Sn]⁺, m/z = 120 (6); [C₁₃H₁₇N₂O₆Sn]⁺, m/z = 417 (100); [C₁₂H₁₇N₂O₄Sn]⁺, m/z = 373 (17); [C₁₁H₁₄N₂O₄Sn]⁺, m/z = 358 (20); [C₁₀H₁₁N₂O₄Sn]⁺, m/z = 343 (17); [C₂₂H₂₅N₄O₁₀Sn]⁺, m/z = 625 (8).

Dibutylstannanediyl bis(4-(2-methoxy-5-nitrophenylamino)-4-oxobutanoate) (7). Yield: 84%: m.p. 149–151 °C: mol. wt.: 767.4: anal. calc. for C₃₀H₄₀N₄O₁₂Sn: C, 46.9 (46.2); H, 5.2 (5.5); N, 7.3 (7.5): IR (4000–400 cm⁻¹): 3300 ν (NH); 1738 ν (amide C=O); 1504 ν (COOasym); 1366 ν (COOsym); 138 (Δν); 538 ν (Sn–C); 439 ν (Sn–O): ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.65 (t, 2H, H2, ³J[¹H, ¹H] = 6.4 Hz); 2.46 (t, 2H, H3, ⁴J[¹H, ¹H] = 6.4 Hz); 9.01 (s, 1H NH); 7.96 (d, 1H, H6, ⁴J[¹H, ¹H] = 2.4 Hz); 7.23 (dd, 1H, H8, ⁴J[¹H, ¹H] = 2.4 Hz); 7.21 (d, 1H, H9, ³J[¹H, ¹H] = 9.2 Hz); 3.94 (s, 3H, H11); 1.18 (t, 2H, Hα, ³J[¹H, ¹H] = 7.2 Hz); 1.43 (m, 2H, Hβ); 1.31 (m, 2H, Hγ); 0.74 (t, 3H, Hδ, ³J[¹H, ¹H] = 7.2 Hz): ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 171.9 (C1); 32.2 (C2); 31.2 (C3); 176.6 (C4); 128.6 (C5); 111.3 (C6); 140.8 (C7); 115.8 (C8); 120.4 (C9); 154.5 (C10); 57.2 (C11); 29.7 (Cα); 27.3 (Cβ); 26.2 (Cγ); 14.0 (Cδ): ¹¹⁹Sn NMR (DMSO-d₆, 400 MHz) δ (ppm): −306.5: ESI-MS, m/z (%): [C₃₀H₄₀N₄O₁₂SnNa]⁺, m/z = 791 (21); [C₃₀H₄₀N₄O₁₂Sn]⁺, m/z = 768 (10); [C₂₆H₃₁N₄O₁₂Sn]⁺, m/z = 711 (9); [C₁₅H₂₀N₂O₆Sn]⁺, m/z = 444 (12); [C₁₁H₁₁N₂O₆Sn]⁺, m/z = 387 (10); [C₁₁H₁₁N₂O₆]⁺, m/z = 267 (8); [C₁₀H₁₁N₂O₄]⁺, m/z = 223 (9); [C₈H₁₈Sn]⁺, m/z = 234 (16); [C₄H₉Sn]⁺, m/z = 177 (100); [Sn]⁺, m/z = 120 (5); [C₁₉H₂₉N₂O₆Sn]⁺, m/z = 501 (80); [C₁₈H₂₉N₂O₄Sn]⁺, m/z = 457 (7); [C₁₄H₂₀N₂O₄Sn]⁺, m/z = 400 (8); [C₁₀H₁₁N₂O₄Sn]⁺, m/z = 343 (20); [C₂₅H₃₁N₄O₁₀Sn]⁺, m/z = 667 (9).

Diphenylstannanediyl bis(4-(2-methoxy-5-nitrophenylamino)-4-oxobutanoate) (8). Yield: 85%: m.p. 152–154 °C: mol. wt.: 807.4: anal. calc. for C₃₄H₃₂N₄O₁₂Sn: C, 50.6 (50.9); H, 4.0 (4.4); N, 7.0 (6.9): IR (4000–100 cm⁻¹): 3304 ν (NH); 1738 ν (amide C=O); 1510 ν (COOasym); 1363 ν (COOsym); 147 (Δν); 538 ν (Sn–C); 440 ν (Sn–O): ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.66 (t, 2H, H2, ³J[¹H, ¹H] = 6.4 Hz); 2.48 (t, 2H, H3, ³J[¹H, ¹H] = 6.4 Hz); 9.42 (s, 1H NH); 9.01 (d, 1H, H6, ⁴J[¹H, ¹H] = 2.8 Hz); 7.98 (dd, 1H, H8, ⁴J[¹H, ¹H] = 3.2 Hz); 7.19 (d, 1H, H9, ³J[¹H, ¹H] = 9.2 Hz); 3.95 (s, 3H, H11); 7.29 (d, 1H, Hβ, ³J[¹H, ¹H] = 6.4 Hz); 7.43 (m, 1H, Hγ); 7.12 (m, 1H, Hδ): ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 171.3 (C1); 31.5 (C2); 29.3 (C3); 174.3 (C4); 128.6 (C5); 111.5 (C6); 140.8 (C7); 116.0 (C8); 120.6 (C9); 154.6 (C10); 57.2 (C11); 143.5 (Cα, ¹J[¹¹⁹Sn–¹³Cα] = 1236 Hz); 136.1 (Cβ); 128.1 (Cγ); 129.4 (Cδ): ¹¹⁹Sn NMR (DMSO-d₆, 400 MHz) δ (ppm): −304.9: ESI-MS, m/z (%): [C₃₄H₃₂N₄O₁₂SnNa]⁺, m/z = 831 (4); [C₃₄H₃₂N₄O₁₂Sn]⁺, m/z = 808 (12); [C₂₈H₂₇N₄O₁₂Sn]⁺, m/z = 731 (7); [C₁₇H₁₆N₂O₆Sn]⁺, m/z = 464 (8); [C₁₁H₁₁N₂O₆Sn]⁺, m/z = 387 (8); [C₁₁H₁₁N₂O₆]⁺, m/z = 267 (11); [C₁₁H₁₁N₂O₆]⁺, m/z = 269 (100); [C₁₀H₁₁N₂O₄]⁺, m/z = 223 (13); [C₁₂H₁₀Sn]⁺, m/z = 274 (21); [C₆H₅Sn]⁺, m/z = 197 (230); [Sn]⁺, m/z = 120 (8); [C₂₃H₂₁N₂O₆Sn]⁺, m/z = 541 (90); [C₂₂H₂₁N₂O₄Sn]⁺, m/z = 497 (11); [C₁₆H₁₆N₂O₄Sn]⁺, m/z = 420 (21); [C₁₀H₁₁N₂O₄Sn]⁺, m/z = 343 (30); [C₂₇H₂₇N₄O₁₀Sn]⁺, m/z = 687 (21).

Di-tert-butylstannanediyl bis(4-(2-methoxy-5-nitrophenylamino)-4-oxobutanoate) (9). Yield: 80%: m.p. 173–175 °C: mol. wt.: 767.4: anal. calc. for C₃₀H₄₀N₄O₁₂Sn: C, 46.9 (45.6); H, 5.2 (5.3); N, 7.3 (7.1): IR (4000–400 cm⁻¹): 3360 ν (NH); 1739 (amide C=O); 1510 ν (COOasym); 1366 ν (COOsym); 144 (Δν); 539 ν (Sn–C); 462 ν (Sn–O): ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.70 (t, 2H, H2, ³J[¹H, ¹H] = 6.4 Hz); 2.57 (t, 2H, H3, ³J[¹H, ¹H] = 6.4 Hz); 9.58 (s, 1H NH); 8.93 (d, 1H, H6, ⁴J[¹H, ¹H] = 3.6 Hz); 7.98 (dd, 1H, H8, ⁴J[¹H, ¹H] = 3.6 Hz); 7.21 (d, 1H, H9, ³J[¹H, ¹H] = 6.8 Hz); 3.94 (s, 3H, H11); 1.23 (s, 9H, Hβ): ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 171.7 (C1); 32.0 (C2); 30.6 (C3); 175.8 (C4); 128.4 (C5); 111.5 (C6); 140.8 (C7); 116.2 (C8); 120.6 (C9); 154.7 (C10); 57.2 (C11); 31.2 (Cα, ¹J[¹¹⁹Sn–¹³Cα] = 837 Hz); 29.9 (Cβ, ²J[¹¹⁹Sn–¹³Cβ] = 133 Hz): ¹¹⁹Sn NMR (DMSO-d6, 400 MHz) δ (ppm): −292.1: ESI-MS, m/z (%): [C₃₀H₄₀N₄O₁₂SnNa]⁺, m/z = 791 (55); [C₃₀H₄₀N₄O₁₂Sn]⁺, m/z = 768 (30); [C₂₆H₃₁N₄O₁₂Sn]⁺, m/z = 711 (22); [C₁₅H₂₀N₂O₆Sn]⁺, m/z = 444 (13); [C₁₁H₁₁N₂O₆Sn]⁺, m/z = 387 (16); [C₁₁H₁₁N₂O₆]⁺, m/z = 267 (3); [C₁₁H₁₁N₂O₆]⁺, m/z = 269 (40); [C₁₀H₁₁N₂O₄]⁺, m/z = 223 (5); [C₈H₁₈Sn]⁺, m/z = 234 (17); [C₄H₉Sn]⁺, m/z = 177 (27); [C₃H₆Sn]⁺, m/z = 162 (1); [C₂H₃Sn]⁺, m/z = 147 (1); [Sn]⁺, m/z = 120 (17); [C₁₉H₂₉N₂O₆Sn]⁺, m/z = 501 (56); [C₁₈H₂₉N₂O₄Sn]⁺, m/z = 457 (3); [C₁₄H₂₀N₂O₄Sn]⁺, m/z = 400 (5); [C₁₀H₁₁N₂O₄Sn]⁺, m/z = 343 (33); [C₂₅H₃₁N₄O₁₀Sn]⁺, m/z = 667 (21).

Dioctylstannanediyl bis(4-(2-methoxy-5-nitrophenylamino)-4-oxobutanoate) (10). Yield: 86%: m.p. 115–117 °C: mol. wt.: 879.6: anal. calc. for C₃₈H₅₆N₄O₁₂Sn: C, 51.9 (51.8); H, 6.4 (6.4); N, 6.4 (6.2): IR (4000–400 cm⁻¹): 3304 ν (NH); 1736 (amide C=O); 1507 ν (COOasym); 1380 ν (COOsym); 127 (Δν); 538 ν (Sn–C); 455b ν (Sn–O): ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.66 (t, 2H, H2, ³J[¹H, ¹H] = 6.0 Hz); 2.46 (t, 2H, H3, ³J[¹H, ¹H] = 6.0 Hz); 9.51 (s, 1H NH); 9.0 (d, 1H, H6, ⁴J[¹H, ¹H] = 2.8 Hz); 7.96 (dd, 1H, H8, ⁴J[¹H, ¹H] = 2.8 Hz); 7.21 (d, 1H, H9, ³J[¹H, ¹H] = 9.2 Hz); 3.94 (s, 3H, H11); 1.24 (t, 2H, Hα, ³J[¹H, ¹H] = 6.8 Hz); 1.50 (bs, 6H, Hβ,γ,δ); 1.04 (b, 6H, Hα′,β′,γ′); 0.76 (t, 3H, Hδ′, ³J[¹H, ¹H] = 6.8 Hz): ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 171.8 (C1); 33.2 (C2); 31.8 (C3); 177.9 (C4); 128.6 (C5); 111.4 (C6); 140.8 (C7); 115.8 (C8); 120.4 (C9); 154.5 (C10); 57.2 (C11); 24.9 (Cα); 29.5 (Cβ, ²J [¹¹⁹Sn–¹³Cβ] = 17 Hz); 34.3 (Cγ); 29.2 (Cδ); 29.6 (Cδ); 29.1 (Cα′); 31.8 (Cβ′); 22.6 (Cγ′); 14.4 (Cδ′): ¹¹⁹Sn NMR (DMSO-d₆, 400 MHz) δ (ppm): −271.7: ESI-MS, m/z (%): [C₃₈H₅₆N₄O₁₂SnNa]⁺, m/z = 903 (33); [C₃₈H₅₆N₄O₁₂Sn]⁺, m/z = 880 (2); [C₃₀H₃₉N₄O₁₂Sn]⁺, m/z = 767 (1); [C₁₉H₂₈N₂O₆Sn]⁺, m/z = 500 (2); [C₁₁H₁₁N₂O₆Sn]⁺, m/z = 387 (2); [C₁₁H₁₁N₂O₆]⁺, m/z = 267 (3); [C₁₀H₁₁N₂O₄]⁺, m/z = 223 (4); [C₁₆H₃₄Sn]⁺, m/z = 346 (3); [C₈H₁₇Sn]⁺, m/z = 233 (2); [Sn]⁺, m/z = 120 (1); [C₁₆H₃₄]⁺, m/z = 226 (32); [C₁₅H₃₁]⁺, m/z = 213 (100); [C₂₇H₄₅N₂O₆Sn]⁺, m/z = 613 (70); [C₂₆H₄₅N₂O₄Sn]⁺, m/z = 569 (12); [C₁₈H₂₈N₂O₄Sn]⁺, m/z = 456 (8); [C₁₀H₁₁N₂O₄Sn]⁺, m/z = 343 (5); [C₂₉H₃₉N₄O₁₀Sn]⁺, m/z = 723 (8).

Cell lines and cultures. Lung carcinoma (H-157), (ATCC CRL-5802) and kidney fibroblast (BHK-21), (ATCC CCL-10) cell lines were kept in RPMI-1640 [including heat-inactivated fetal bovine serum (10%) glutamine (2 mM), pyruvate (1 mM), 100 U mL⁻¹ penicillin and 100 μg mL⁻¹ streptomycin] in T-75 cm² sterile tissue culture flasks in a 5% CO₂ incubator at 37 °C.²⁰ 96-well plates were used for growing H-157 and BHK-21 cells by inoculating 10⁴ cells per 100 μL per well. For experiments, both cell lines were grown in 96-well plates by inoculating 10⁴ and 5 × 10⁴ cells per 100 μL per well, respectively, and plates were incubated at 37 °C in a 5% CO₂ incubator. Within 24 h, a uniform monolayer was formed, which was used for experiments.
Cytotoxicity analysis by sulforhodamine B (SRB) assays. To perform a cytotoxicity assay with H-157 and BHK-21 cells, a previously described method by Skehan et al.²¹ was adopted with some modifications. Briefly, cells were cultured in different 96-well plates for 24 h. The compounds, in different concentrations (100, 10, 1 and 0.1 μM), were inoculated into test wells, while control and blank wells were also prepared containing the standard drug (vincristine) and culture media with cells, respectively. The plates were then incubated for 48 h. After that the cells were fixed with 50 μL of 50% ice cold trichloroacetic acid solution (TCA) at 4 °C for 1 h. The plates were washed 5 times with phosphate-buffered saline (PBS) and air dried. The fixed cells were further treated with 0.4% w/v sulforhodamine B dye prepared in 1% acetic acid solution and left at room temperature for 30 min. After that the plates were rinsed with 1% acetic acid solution and allowed to dry. In order to solubilize the dye, the dried plates were treated with 10 mM Tris base solution for 10 min at room temperature. Absorbance was measured at 490 nm, subtracting the background measurement at 630 nm.²²

2.3. Antileishmanial activity

Parasite and culture. Leishmania major promastigotes were cultured at 25 ± 1 °C to logarithmic phase in D-MEM/F-12 medium (Gibco BRL) without phenol red, supplemented by 10% heat inactivated fetal bovine serum (FBS), 100 IU per mL penicillin and 100 μg mL⁻¹ streptomycin, then washed 3 times with PBS by centrifugation at 1500 rpm for 10 min at room temperature and resuspended at a concentration of 2.5 × 10⁶ parasites per mL in medium.

Antileishmanial activity assays (MTT assay). The antileishmanial activity of the compounds was evaluated in vitro against the promastigote forms of leishmania major using a MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide)-based microassay as a marker of cell viability. The MTT assay used was based on that originally described by Mosmann (1983),²³ modified by Niks and Otto (1990).²⁴ A stock solution of MTT (Sigma Chemical Co., St. Louis, Mo.) was prepared by dissolving in PBS at 5 mg mL⁻¹ and storing in the dark at 4 °C for up to 2 weeks before use. For the antileishmanial activity assays, 100 μL per well of the culture, which contained 2.5 × 10⁶ cells per mL promastigotes, was seeded in 96-well flat-bottom plates. Then 10 μL per well from various concentrations of compounds was added to triplicate wells and plates were incubated for 72 h at 25 ± 1 °C. The first well of 96 wells was a blank well that only contained 100 μL culture medium without any compound, drug or parasite. Amphotericin B was used as the standard drug. At the end of incubation, 10 μL MTT was added to each well and plates were incubated for 3 h at 25 ± 1 °C. Enzyme reaction was then stopped by the addition of 100 μL 50% isopropanol and 10% sodium dodecyl sulfate. The plates were incubated for an additional 30 min, under agitation at room temperature. Relative optical density (OD) was then measured at a wavelength of 570 nm using a 96-well microplate reader (Bio-Tek ELx 800TM, Instruments, Inc. USA). The background absorbance of plates was measured at 690 nm and subtracted from 570 nm measurement. The absorbance of the formazan produced by the action of mitochondrial dehydrogenases of metabolically active cells is shown to correlate with the number of viable cells. All experiments were repeated at least three times. Results reported are the mean of three independent experiments (±SEM) and expressed as percentage inhibitions calculated by the formula:

IC₅₀ values of inhibitors were determined with the help of the Graph Pad prism 5.0 Software Inc., San Diego, California, USA.

2.4. DNA interaction study assay by UV-visible spectroscopy

SS-DNA (50 mg) was dissolved by overnight stirring in deionized water (pH = 7.0) and kept at 4 °C. Deionized water was used to prepare buffer (20 mM phosphate buffer (NaH₂PO₄–Na₂HPO₄), pH = 7.2). A solution of (SS-DNA) in the buffer gave a ratio of UV absorbance at 260 and 280 nm (A₂₆₀/A₂₈₀) of 1.8, indicating that the DNA was sufficiently free of protein.^25,26 The DNA concentration was determined via absorption spectroscopy using a molar absorption coefficient of 6600 M⁻¹ cm⁻¹ (260 nm) for SS-DNA^27,28 and was found to be 1.4 × 10⁻⁴ M. The compound was dissolved in ethanol at a concentration of 1 mM. The UV absorption titrations were performed by keeping the concentration of the compound fixed while varying the SS-DNA concentration. Equivalent solutions of SS-DNA were added to the complex and reference solutions to eliminate the absorbance of DNA itself. Compound–DNA solutions were allowed to incubate for about 10 min at room temperature before measurements were made. Absorption spectra were recorded using cuvettes of 1 cm path length at room temperature (25 ± 1 °C).

3. Results and discussion

3.1. FT-IR

The IR spectra of the NaL and its organotin(IV) carboxylates were recorded using a Thermo Nicolet-6700 FT-IR Spectrophotometer in the range 4000–400 cm⁻¹ and comparison was made to assign the IR bands. The far-infrared spectra of Ph₃SnCl/Ph₂SnCl₂ derivatives were recorded in the range 400–100 cm⁻¹. Generally, ν (Sn–C) vibration bands for Ph–Sn complexes were observed in the far-infrared region (lower wavelength) compared to the ν (Sn–C) of dialkyltin(IV) or trialkyltin(IV) complexes, which may be due to the mass effect of the Sn-Ph.²⁹ Details of the FT-IR data of the synthesized ligand and its complexes are given in the Experimental section.

FT-IR data of organotin(IV) derivatives reveal valuable information about the structure of the complexes in the solid state. The peaks of interest to us are ν OH, ν NH, ν C=C, ν CO, ν Sn–C and ν Sn–O of the precursors and complexes.

The formation of NaL was confirmed by FT-IR in which the OH peak at 3264 cm⁻¹ was absent, showing the displacement of H of HL with Na. NaL was then used for complexation as a starting material. Another important factor is Δν [ν (COOasym) − ν (COOsym)]. When there is interaction between the oxygen of the carboxylate group and tin, the asymmetric vibration ν (COOasym) decreases and the symmetric vibration ν (COOsym) increases, so the difference [Δν(COO)] decreases.³⁰ In the IR spectra of NaL the ν (COOasym) appears at 1537 cm⁻¹ and ν (COOsym) at 1259 cm⁻¹, while in complexes 1–10 the carboxylate bands are observed in the characteristic regions of 1536–1501 cm⁻¹ for ν (COOasym) and 1380–1324 cm⁻¹ for ν (COOsym). When the coordination changes from four to five or a higher number, the ν (COOasym) shifts to lower frequencies, while ν (COOasym) shifts to higher frequencies, causing a decrease in the Δν value of the compounds.³¹ The magnitude of Δν for the reported complexes is less than 200 cm⁻¹, which reflects either chelating or bidentate nature of the ligand. The Δν value was also calculated from single crystal XRD data by using the following equation:^32,33

Δν = 1818.1δr + 16.47(θOCO − 120) + 66.8

where δr is difference between the two C–O bond lengths (Å) and θOCO is the O–C–O angle (°).

A good correlation was found between the Δν value calculated from FT-IR data for complex 4 (212) and single crystal XRD data (216). In the case of complex 3 the distances C1–O1 and C1–O2 are the same (1.258 Å), which means that the carboxylate moiety symmetrically bridges the two Sn atoms.

Similarly, the appearance of new peaks for ν (Sn–C) in the range 550–538 cm⁻¹ (for alkyl-Sn), 281–277 cm⁻¹ (for phenyl-Sn) and for ν (Sn–O) in the range of 489–439 cm⁻¹, respectively, confirms the synthesis of the new complexes.³⁴

3.2. Multinuclear (¹H, ¹³C and ¹¹⁹Sn) NMR spectroscopy

NMR spectroscopy is one of the most widely used techniques for the characterization of organotin(IV) complexes. It is also helpful to predict the geometry of the complexes. The parameters ⁿJ(¹¹⁹Sn,¹H), ⁿJ(¹¹⁹Sn,¹³C) and δ(¹¹⁹Sn) are determined by this technique and give information about the geometry of the organotin(IV) complexes in the solution state.

3.2.1. ¹H NMR spectroscopy. The ¹H and ¹³C NMR spectra for the synthesized ligand and its complexes were recorded in DMSO using tetramethylsilane as an external standard and the data are given in the experimental section. The ¹H NMR spectrum of complex 1 (as a representative one) is shown in Fig. 1a. The ¹H NMR studies provide further support for the formation of compounds. The aliphatic protons at positions 2 (H2) and 3 (H3) appear as a triplet with ³J bond coupling in all complexes. Protons 6 (H6) and 7 (H7) give doublets, while proton 8 (H8) gives a doublet of doublets. There is no significant change in the position of the NH signal in the spectra of the complexes, indicating that nitrogen is not involved in coordination to tin.

Fig. 1 (a) ¹H NMR spectrum of complex 1. (b) ¹³C and ¹¹⁹Sn NMR spectrum of complex 1. (c) ¹¹⁹Sn NMR spectrum of complex 4.

According to the literature, the coordination pattern of tin(IV) in di- and trimethyltin(IV) derivatives with the ^1,2J coupling constant values are as follows: in tetracoordinated tin compounds (θ ≤ 112°) ¹J values are predicted to be smaller than about 400 Hz, whereas ²J values should be below 59 Hz; for pentacoordinated tin (θ = 115–130°), ¹J values fall in the range 450–670 Hz and ²J values fall in the range 65–80 Hz; finally, for hexacoordinated tin (θ = 129–176°) ¹J and ²J values are generally larger than 670 and 83 Hz, respectively.^35,36 The methyl protons in trimethyltin(IV) complex (1) give a characteristic signal at 0.32 ppm with ²J[¹¹⁹Sn–¹ H] = 69 Hz, which falls in the range of 5-coordinate trigonal bipyramidal geometry around the tin atom in the solution state.³⁷ The protons of triethyltin(IV) complex (2) give two peaks: a quartet for Hα with ²J[^119/117Sn–¹H] = 76, 74 Hz and a triplet for Hβ. In the case of the butyl proton (complex 3 and 7), two triplets (one for Hα and one for Hδ) and two multiplets (one for Hβ and one for Hγ) were observed. The aromatic protons in Ph-Sn (complex 4 and 8) appear as one doublet (Hβ) and two triplets (one for Hγ and one for Hδ). The protons of the cyclohexyl derivatives (complex 5) generally give a triplet for Hα, a multiplet for Hβ and broad signals for Hγ and Hδ in the aliphatic regions. The methyl protons in the dimethyltin(IV) complex (6) give a signal at 0.73 ppm, and ²J[¹¹⁹Sn–¹H] for dimethyltin(IV) derivatives was found to be 100 Hz, which falls in the range of 6-coordinate octahedral geometry. The methyl protons of the tertiary butyl group in complex 9, {C(CH₃)₃}, appear as a singlet at 1.23 ppm. In the case of octyltin(IV) complex (10), a somewhat different and complex pattern is observed for the methylene protons. The terminal protons give a triplet while the remaining protons give broad and complex signals. The observed ²J[¹¹⁹Sn–¹H] coupling constants for the methyl and ethlytin(IV) derivatives were used to calculate the C–Sn–C values while using Lockhart’s equation³⁸ and fall in the range of 5-coordinate trigonal bipyramidal geometry for the triorganotin(IV) derivatives and 6-coordinate octahedral geometry for the diorganotin(IV) derivatives. The values of C–Sn–C are given in Table 1.

Table 1 (C–Sn–C) angles (°) based on NMR parameters of selected organotin(IV) derivatives^abcde

Comp. no.	^1J(^119Sn, ^13C) (Hz)	^2J(^119Sn, ^1H) (Hz)	Angle (°)
			^1J	^2J
a Numbering of compounds is according to Scheme 1.b θ = 0.0105|^2J(^119Sn–^1H)|^2 − 0.799 |^2J(^119Sn–^1H)| + 122.4 (for methyl and ethyl derivatives).c |^1J(^119Sn–^13C)| = 11.4θ − 875 (for methyl and ethyl derivatives).d |^1J(^119Sn–^13C)| =(9.99 ± 0.73)θ − (746 ± 100) (for butyl derivatives).e |^1J(^119Sn–^13C)| = (15.56 ±0.84)θ − (1160 ± 101) (for phenyl derivatives).
1	520	69	122	117
2	580	76	128	122
3	384	—	113	—
4	580	—	112	—
5	559	—	111	—
6	800	100	147	147
8	1236	—	154	—
10	137	—	88	—

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3.2.2. ¹³C NMR spectroscopy. Details of the ¹³C NMR data of the ligand and its organotin(IV) derivatives are given in the Experimental section. The ¹³C NMR spectra of complex 1 (as a representative one) are shown in Fig. 1b. The values were assigned to each carbon atom of the ligand and its complexes on the basis of incremental methods and by comparison with literature values.

The C=O resonances of the complexes were shifted downfield compared to those in the free ligand. This downfield shift is due to the decrease of electron density at the carbon atoms when oxygen is bonded to the electropositive tin atom.³⁷ This observation provides further evidence that the complexation occurred through the oxygen atoms of the carboxylate group. However, the signals for the –C–N– and –CH₂–CH₂– peaks, for the alkyl as well as aryl groups attached to the tin atom appeared in their specific regions.³⁹ The positions of the phenyl carbons of the ligand undergo minor changes in the complex as compared to those observed in the free ligand. The ipso carbon (Cα) of the phenyl group attached to the tin atom appears in the range 143.5–143.7 ppm, which lies in the range of 5/6-coordination geometry.^40,41

By substituting the value of ¹J[¹¹⁹Sn–¹³C] in Lockhart’s equation,³⁸ a C–Sn–C bond angle can be calculated for the methyl and ethyl derivatives. The ¹J[¹¹⁹Sn–¹³C] values for the butyl and phenyltin(IV) derivatives were determined using the relationships proposed by Howard et al.,⁴² and are given in Table 1.

3.2.3. ¹¹⁹Sn NMR spectroscopy. According to the literature the δ(¹¹⁹Sn) values of four-coordinate complexes fall in the range +200 to −60 ppm; the five-coordinate complexes fall between −90 and −190 ppm, and the six- and seven-coordinate complexes fall between −210 and −400 ppm.⁴³ However, chemical shifts δ (¹¹⁹Sn) of sthe ix-coordinate chelate complexes lie in the range −260 to −404.6 ppm. The tin chemical shift δ (¹¹⁹Sn) values indicated the coordination number of tin and thus provided information about the geometry of the organotin(IV) complexes. The δ (¹¹⁹Sn) values were found to depend on the nature of the group attached to Sn. However, it must be carefully analyzed since tin resonance is strongly dependent upon other factors, such as electronegativity of the ligands, temperature and concentration employed in the experiments. In the case of the electron donating group the Sn atom becomes progressively more shielded and the δ (¹¹⁹Sn) value moves to a higher field. The nature of the ligand (X) in organotin(IV) complexes (R_4−nSnX_n) also influences the values of δ (¹¹⁹Sn). The electronegativity of the coordinating group of the ligand played a key role in the δ (¹¹⁹Sn) value. Generally a higher electronegativity of the coordinate ligand shifts the δ (¹¹⁹Sn) value to lower fields.^19,44

The low field displacement of tin chemical shifts in the case of R₃Sn (R = CH₃, C₂H₅ and C₄H₉) derivatives is caused by the influence of electron withdrawing and donating ability of the groups attached to the ligand, as well as electronegativity of the substituent at the tin atom. If the substituent has lone-pair electrons or π electrons of multiple bonds, then an increase in ¹¹⁹Sn shielding will be observed due to the partial filling of the empty 5d orbital of the tin atom (d–p or d_π–p_π interaction). The combination of these competitive effects leads to the tin shielding minimum for the series Sn(CH₃)₃, Sn(C₂H₅)₃, Sn(C₄H₉)₃.^45,46 For complex 5, Ph₃SnL, two peaks were observed, i.e. at −226.1 ppm and −257.7 ppm, respectively, which represent the presence of the two species. The peak at −226.1 ppm may be due to the presence of the 4-coordinate tetrahedral species, while the other peak is due to the presence of the 5-coordinate species. This was further conformed by the single crystal X-ray result in which the tin atom is present both in the 4-coordinated and 5-coordinated environments. In the case of diorganotin(IV) derivatives the ¹¹⁹Sn NMR values range from −271.7 ppm to −306.5 ppm, falling in the range of 6-coordinate geometry. The ¹¹⁹Sn NMR spectra of the complexes 1 and 4 are given in Fig. 1b and c, respectively.

3.3. X-ray crystallography

The perspective diagrams with unit cell packings for complexes 1, 3 and 4 are given in Fig. 2–4, respectively. Crystal data and structure refinement parameters are shown in Table 2, while the selected bond lengths and bond angles are given in Tables 3 and 4, respectively. The presence of a bidendate ligand, 4-(2-methoxy-5-nitrophenylamino)-4-oxobutanoate, leads to the formation of the polymeric structure. In other words, the central R₃Sn (R = CH₃ (1), C₄H₉ (3)) group bridges the two neighboring 4-(2-methoxy-5-nitrophenylamino)-4-oxobutanoate ligands via carboxylate moieties to form a one-dimensional polymeric chain.⁴⁷ In complex 4 the geometry around the Sn atom is distorted tetrahedral, due to steric hindrance of the three bulky phenyl groups. The values of τ (τ = (β − α)/60, where β is the largest basal angle around the tin atom while α is the next largest angle around the tin atom) for complexes 1 and 3 are 0.87 and 0.92, respectively, which are typical for distorted trigonal bipyramidal geometry.⁴⁸ The three R groups occupy the equatorial positions with essentially identical bond distances [Sn–C = 2.081–2.142 Å]. The O–Sn–O angle is approximately linear [O–Sn–O = 173.9–175.8°]. The C–Sn–C and O–Sn–C angles are within the expected range of values for 5-coordinated geometry around the tin atom [C–Sn–C = 114.4°–125.5° and O–Sn–C = 85.9–96.4°]⁴⁹ for complexes 1 and 3. The sum of the C–Sn–C angles in the equatorial plane equals 359.1° and 359.5°, respectively, for complexes 1and 3, indicating slight distortion. This distortion from the ideal trigonal bipyramidal geometry is found in the axial angle [O–Sn–O]. In the case of complex 4 the C–Sn–C = 108.2°–114.9°, which falls in the range of 4-coordinated tetrahedral geometry around the tin atom. In complex 4 two molecules are present in the structure. In Fig. 4c, it was found that the Sn atoms are present in different environments, i.e. Sn1 is present in the 5-coordinated environment while Sn2 is present in the 4-coordinated environment. The bond distance of Sn1–O2 (2.605 Å) is longer than the normal Sn–O1 (2.054 Å) bond distance but due to weak interaction the O2 participate in coordination to Sn atom resulting in 5-coordinated geometry. The asymmetric Sn–O separations are reflected in the associated C1–O1 and C1–O2 distances of 1.276(9) Å, 1.236(9) Å and 1.258(8) Å, 1.258(8) Å, 1.210(2) Å, 1.224(6) Å, respectively for complexes 1 and 3.⁵⁰ In this study complex 1 has a difference only 0.04 Å, which is probably due the chelating nature of the carboxylate ligand. The anisobidentate bidentate carboxylate has a difference of 0.058 Å between its C–O bonds while for the bidentate carboxylate this difference is only 0.021 Å; the variations in the C–O bond distances suggest charge delocalization over the carboxylate group COO. The different modes of bonding of the acetates, i.e. bridging or chelating, are thus easily differentiated by the relevant bond lengths.⁵¹ There is an intramolecular C–H⋯O interaction within the polymeric chain. Furthermore, C–H⋯π interactions and intramolecular hydrogen bonds stabilize the polymeric chains in a zigzag manner, which is linked into a three-dimensional network via C–H⋯π interactions. The polar imino hydrogen atom of the amide derivative participates in intramolecular hydrogen bonds (N1–H1⋯O3). Complexes are self-assembled via π → π, C–H⋯π and stacking interactions. Extended networks of O–Sn–O, C–H–O and C–H⋯π contacts lead to aggregation and a supramolecular assembly. The details of intramolecular H-bonds existing within the structure are given in Table 5.

Fig. 2 (a) A perspective view of the complex 1 showing the crystallographic numbering scheme. All H-atoms have been omitted for clarity. (b) Packing diagram with unit cell of complex 1 viewed along the a-axis.

Fig. 3 (a) A perspective view of the complex 3 showing the crystallographic numbering scheme. All H-atoms have been omitted for clarity. (b) Packing diagram with unit cell of complex 3 viewed along the b-axis.

Table 2 Crystal data and structure refinement parameters for its organotin(IV) complexes

Parameters	Complex 1	Complex 3	Complex 4
Empirical formula	C14H20N2O6Sn	C23H38N2O6Sn	C29H26N2O6Sn
Formula weight	431.01	557.24	617.21
Temperature/K	296(2)	150(2)	150(2)
Crystal system	Triclinic	Monoclinic	Monoclinic
Space group	P1	P21/c	P21
a/Å	7.9109(5)	15.853(3)	9.837(4)
b/Å	7.9252(5)	10.0376(18)	13.099(5)
c/Å	14.3797(10)	16.700(3)	21.032(8)
α, β, γ/°	88.257(4), 83.081(4), 74.025(3)	90, 98.974(3), 90	90, 77.875(7), 90
Volume/Å^3	860.41(10)	2624.8(8)	2649.7(17)
Z	2	4	4
ρcalc mg/mm^3	1.664	1.410	1.547
m/mm^−1	1.515	1.011	1.011
F(000)	432.0	1152.0	1248.0
Crystal size/mm^3	0.32 × 0.22 × 0.2	0.46 × 0.16 × 0.06	0.27 × 0.23 × 0.08
2θ range for data collection	2.852 to 55.924°	4.75 to 56.528°	1.98 to 54.708°
Radiation MoKα (λ)	0.71073	0.71073	0.71073
Reflections collected	13 549	35 017	25 489
Independent reflections	6976 [Rint = 0.0431, Rsigma = 0.0723]	6473 [Rint = 0.1039, Rsigma = 0.0782]	11 790 [Rint = 0.1680, Rsigma = 0.2008]
Data/restraints/parameters	6976/3/423	6473/0/294	11 790/1/686
Goodness-of-fit on F^2	0.943	1.042	0.935
Final R indexes [I ≥ 2σ(I)]	R1 = 0.0389, wR2 = 0.0583	R1 = 0.0675, wR2 = 0.1496	R1 = 0.0855, wR2 = 0.1839

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Table 3 Selected bond lengths (Å) for complexes 1, 3, 4

Bond lengths for complex 1
O1–Sn1	2.208(6)	C12–Sn1	2.119(7)
O8–Sn1	2.364(5)	C13–Sn1	2.112(9)
C1–C2	1.493(10)	C14–Sn1	2.125(8)
C1–O1	1.276(9)	C1–O2	1.236(9)
Bond lengths for complex 3
O1–Sn1	2.351(5)	C12–Sn1	2.138(8)
O2–Sn1	2.217(5)	C16–Sn1	2.114(8)
C1–C2	1.508(9)	C20–Sn1	2.081(10)
C1–O1	1.258(8)	C1–O2	1.258(8)
Bond lengths for complex 4
O1–Sn1	2.054(13)	C12–Sn1	2.142(16)
C1–O1	1.34(2)	C18–Sn1	2.116(19)
C1–O2	1.22(2)	C24–Sn1	2.117(19)
O7–Sn2	2.052(12)	C41–Sn2	2.136(17)
C30–O7	1.30(2)	C47–Sn2	2.105(19)
C30–O8	1.227(19)	C53–Sn2	2.104(18)

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Table 4 Selected bond angles (°) for complexes 1, 3, 4

Bond angles for complex 1
O1–Sn1–C12	94.2(3)	C12–Sn1–O8	88.9(3)
O1–Sn1–C13	94.6(4)	C13–Sn1–O8	86.0(4)
O1–Sn1–C14	90.7(3)	C4–Sn1–O8	85.4(3)
O1–Sn1–O8	175.8(2)	C12–Sn1–C13	123.8(4)
O1–C1–O2	121.0(7)	C12–Sn1–C14	116.2(3)
O1–C1–C2	116.7(7)	C13–Sn1–C14	119.1(4)
Bond angles for complex 3
O1–Sn1–C12	85.9(3)	C12–Sn1–O2	88.8(3)
O1–Sn1–C16	90.9(2)	C16–Sn1–O2	94.0(3)
O1–Sn1–C20	86.1(3)	C20–Sn1–O2	93.9(3)
O1–Sn1–O2	173.9(15)	C12–Sn1–C16	114.6(4)
O1–C1–O2	122.5(6)	C12–Sn1–C20	119.4(5)
O1–C1–C2	120.2(6)	C16–Sn1–C20	125.5(4)
Bond angles for complex 4
O1–Sn1–C12	94.6(6)	O7–Sn2–C41	112.2(6)
O1–Sn1–C18	116.9(6)	O7–Sn2–C47	102.0(6)
O1–Sn1–C24	109.7(6)	O7–Sn2–C53	90.7(6)
C12–Sn1–C18	110.4(7)	C41–Sn2–C47	116.7(6)
C18–Sn1–C24	114.9(6)	C41–Sn2–C53	118.2(7)
C12–Sn1–C24	108.2(6)	C47–Sn2–C53	112.9(7)
O1–C1–O2	118.0(16)	O7–C30–O8	121.6(15)

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Fig. 4 (a) A perspective view of the complex 4 showing the crystallographic numbering scheme. All H-atoms have been omitted for clarity. (b) Packing diagram with unit cell of complex 4 viewed along the b-axis. (c) View of complex 4 showing two different coordination environments.

Table 5 Hydrogen-bond angles and bond lengths (Å, °) for complexes 1, 3, 4^a

D–H⋯A	D–H	H⋯A	D⋯A	D–H⋯A
a Symmetry transformations used to generate equivalent atoms for complex:^1 −1 + x, +y, 1 + z. Symmetry transformations used to generate equivalent atoms for complex:^3 (i) +x, −1/2 − y, −1/2 + z, complex:^4 (i) 1 + x, +y, +z; (ii) −x, 1/2 + y, 1 − z.
Complex 1
C6–H6⋯O3	0.93	2.29	2.864(11)	119.6
C9–H9⋯O9^i	0.93	2.62	3.291(12)	129.7
N3–H3⋯O7	0.86	2.31	3.028(9)	141.2
C20–H20⋯O9	0.93	2.19	2.785(11)	120.7
Complex 3
N1–H1⋯O6^i	0.88	2.44	3.225(8)	149.1
C6–H6⋯O3	0.95	2.24	2.835(8)	119.5
Complex 4
N1–H1⋯O9^i	0.88	2.26	3.138(18)	174.1
C6–H6⋯O3	0.95	2.26	2.83(2)	117.1
C8–H8⋯O8^ii	0.95	2.37	3.23(2)	149.2
C32–H32A⋯O3	0.99	2.44	3.32(2)	147.4
N3–H3⋯O3	0.88	2.28	3.137(18)	165.8
C35–H35⋯O9	0.95	2.30	2.85(2)	116.4

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3.4. Mass spectrometry

Electrospray ionization (ESI) method was used to obtain the mass spectral data for the ligand and complexes. The data are given in the experimental section along with m/z and % intensity. The resultant fragments are in good agreement with the expected structures of the compounds. The general fragmentation patterns for tri- and diorganotin(IV) complexes are shown in Scheme 3 and 4, respectively.

Scheme 3 General mass fragmentation pattern for triorganotin(IV) derivatives.

Scheme 4 General mass fragmentation pattern for diorganotin(IV) derivatives.

In the mass spectra of all the complexes, each fragment ion occurs in a group of peaks as a result of tin isotopes. For simplicity the mass spectral fragmentation data reported here are related to the principle isotope ¹²⁰Sn.⁵² The low-intensity molecular ion peaks, M⁺ are observed in all synthesized organotin carboxylates (1–10). Also the [MNa]⁺ fragment is observed in all spectra. The fragmented ions are in good agreement with the expected structure of the compounds and consistent with the literature.^53,54

In triorganotin compounds, three primary fragmentation patterns are proposed, based on the observed m/z in their spectra. Elimination of different groups like COOR′ and R gave [Sn]⁺ as an end product in one of the pathways. The other two pathways after primary elimination of [R]⁺ and [R₃Sn]⁺ groups and then elimination of COO and successive R (in one of the pathway) results in the formation of [R′]⁺ (Scheme 3), which shows a similar pattern for the further elimination of different groups.⁵⁵

A slightly different scheme for the mass fragmentation pattern has been suggested for the diorganotin compounds (Scheme 4) but these pathways end up in a similar way to those suggested for the triorganotin compounds. In addition, the following ions: [C₄H₉]⁺, [C₆H₅]⁺, [C₇H₇]⁺, and [C₈H₁₇]⁺ are also observed with reasonable intensities in the mass spectra of all organotin(IV) derivatives.⁵⁵

3.5. Anticancer activity

The mortality rate due to cancer is increasing worldwide due to the rapid growth of the world population as well as the adoption of cancer-causing behaviors. The search for novel anticancer drugs continues. Agents that can eliminate the cancerous cells via programmed cell death but do not affect the normal cells may have a therapeutic advantage for the elimination of cancer cells. In the present study, we provide evidence that investigated compounds act as a potential antiproliferative agent.⁵⁶ At present, only a few agents are known to possess the potential for selective/preferential elimination of cancer cells without affecting the normal cells.⁵⁷ The discovery of cis-platin as an anticancer drug and recently its improved analogs (carboplatin, lobaplatin, nedaplatin, oxaliplatin) as marketing chemotherapeutic drugs lead to interest in the exploration of metal-based anticancer drugs.⁵⁸

The biological significance of the synthesized compounds is well renowned, owing to their anticancer activity. Cytotoxicity evaluation of the investigated compounds explored another biological feature of the synthesized compounds as being strong anticancer agents. We examined the effects of the investigated compounds on the growth and proliferation of cancerous lung carcinoma (H-157) and kidney fibroblast (BHK-21) cell lines using an MTT assay to compare their behavior.⁵⁹ All the compounds showed remarkable cytotoxicity when checked at different concentrations. Vincristine was used as a standard drug and the IC₅₀ values are shown in Table 6. The results revealed that all the tested compounds showed good cytotoxicity against H-157 and BHK-21 cell lines in a dose dependent manner. NaL demonstrated less cytotoxicity while compounds 1–5 exhibited moderate cytotoxic potency for both the cell lines. Compounds 6–10 showed good anticancer activity against both the cell lines and exhibited IC₅₀ values near to the standard drug, vincristine.

Table 6 Anticancer activity of NaL and its organotin(IV) derivatives^a against BHK-21 and H-157 cell lines

IC50 ± SEM (μM)
Comp. no.	BHK-21	H-157
a Numbering of compounds is according to the Scheme 1.b Standard drug.
NaL	4.96 ± 0.48	4.32 ± 0.11
1	2.53 ± 0.14	2.93 ± 0.07
2	2.44 ± 0.16	2.56 ± 0.27
3	3.29 ± 0.33	3.88 ± 0.34
4	2.95 ± 0.12	3.01 ± 0.36
5	2.95 ± 0.42	3.47 ± 0.03
6	1.33 ± 0.21	1.29 ± 0.06
7	1.27 ± 0.13	1.52 ± 0.05
8	1.62 ± 0.11	1.54 ± 0.04
9	1.24 ± 0.25	1.73 ± 0.49
10	1.82 ± 0.13	1.99 ± 0.15
Vincristine^b	1.08 ± 0.09	1.03 ± 0.04

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These results suggest that these compounds may be a good choice for cancer treatment after in vivo and other clinical studies. However, additional studies are needed to determine the exact mechanism(s) of apoptosis of the investigated compounds in cancer cells versus normal cells. Also, to establish a broader understanding, additional studies are needed to verify these data in other normal cells and cancer cell types and to assess the effectiveness of the synthesized compounds in an in vivo model system.

3.6. Antileishmanial activity

MTT assay was used to examine the in vitro antileishmanial activity of the synthesized compounds against the promastigote forms of leishmania major. The investigated compounds produced a significant reduction in viable promastigotes as shown in Table 7. All the synthesized compounds exhibit strong antileishmanial activity when checked at different concentrations. Amphotericin B was used as standard. The IC₅₀ values shown in Table 7 revealed that compound 10 was the most potent against promastigote forms of leishmania major and exhibited an IC₅₀ value of 0.98 ± 0.06 μM as compared to amphotericin B, which has an IC₅₀ value of 0.29 ± 0.05 μM. Compounds 7–9 also showed remarkable potency against leishmanias, while the rest of the compounds exhibited moderate antileishmanial activity. Their antileishmanial activity may be due to interference with the function of parasite mitochondria. This study therefore demonstrated the potential use of these compounds as a source of novel agents for the treatment of leishmaniasis.

Table 7 Antileishmanial activity data of NaL and its organotin(IV) derivatives^a

Comp. no.	IC50 ± SEM (μM)
a Numbering of compounds is according to the Scheme 1.b Standard drug.
NaL	4.21 ± 0.65
1	4.23 ± 0.14
2	3.23 ± 0.28
3	5.12 ± 0.44
4	4.62 ± 0.61
5	4.74 ± 0.46
6	2.26 ± 0.08
7	1.93 ± 0.02
8	1.83 ± 0.19
9	1.23 ± 0.07
10	0.98 ± 0.06
Amphotericin B^b	0.29 ± 0.05

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3.7. DNA binding study by UV-visible spectroscopy

Electronic absorption spectra were initially used to examine the interaction between the compounds and SS-DNA. Fig. 5–9 show the UV-visible spectra observed when representative compounds (1, 2, 3, 4 and 7) interact with different concentrations of DNA. It was observed that all compounds except 7 have two strong absorption peaks at 254–257.6 nm and 291.80–296.40 nm, which are probably due to π–π* (due to the aromatic group) and n–π* (due to either the NO₂ or CO groups) transitions, respectively. In the case of compound 7 a strong absorption peak at 439.50 nm appeared. After interaction with increasing amounts of DNA, all the peaks decreased gradually (indicating the hypochromic effect) and there was a minor red shift of up to 5 nm for all compounds. Long et al.⁶⁰ have pointed out that the peak shifts of the small molecules after they interacted with DNA could be clues to judge the binding mode between the small molecules and DNA: If the binding involves a typical intercalative mode, a hypochromism effect coupled with obvious bathochromism for the characteristic peaks of the small molecules will be found due to the strong stacking between the chromophore and the base pairs of DNA.⁶¹ Therefore, based on this viewpoint, the interaction between compounds and SS-DNA could be noncovalent intercalative binding. After intercalating the base pairs of DNA, the π* orbital of the intercalated ligand could couple with the π orbital of the base pairs, thus decreasing the π–π* transition energy, and further resulting in bathochromism. On the other hand, the coupling of a π orbital with partially filled electrons decreases the transition probabilities and hence results in the hypochromic shift. Hypochromism due to π–π* stacking interactions may appear in the case of the intercalative binding mode, while bathochromism (red-shift) may be observed when the DNA duplex is stabilized.⁶² The stability of the DNA–compound complex was also checked after 24 h and 48 h and got the same result, which means that the DNA–compound complex is stable.

Fig. 5 Absorption spectrum of 1 mM compound 1 in the absence (a) and presence of 9 (b), 18 (c), 27 (d), 36 (e), 45 (f), 54 (g), 63 (h), 72 (i), 81 (j) and 90 (k) μM DNA. The arrow indicates the increasing conc. of DNA. The inset graph represents the plot of A_o/A − A_o vs. 1/[DNA] (μM)⁻¹ for the calculation of binding constant (K) and Gibb’s free energy (ΔG).

Fig. 6 Absorption spectrum of 1 mM compound 2 in the absence (a) and presence of 9 (b), 18 (c), 27 (d), 36 (e), 45 (f), 54 (g), 63 (h), 72 (i), 81 (j) and 90 (k) μM DNA. The arrow indicates the increasing conc. of DNA. The inset graph represents the plot of A_o/A − A_o vs. 1/[DNA] (μM)⁻¹ for the calculation of binding constant (K) and Gibb’s free energy (ΔG).

Fig. 7 Absorption spectrum of 1 mM compound 3 in the absence (a) and presence of 9 (b), 18 (c), 27 (d), 36 (e), 45 (f), 54 (g), 63 (h), 72 (i), 81 (j) and 90 (k) μM DNA. The arrow indicates the increasing conc. of DNA. The inset graph represents the plot of A_o/A − A_o vs. 1/[DNA] (μM)⁻¹ for the calculation of binding constant (K) and Gibb’s free energy (ΔG).

Fig. 8 Absorption spectrum of 1 mM compound 4 in the absence (a) and presence of 9 (b), 18 (c), 27 (d), 36 (e), 45 (f), 54 (g), 63 (h), 72 (i), 81 (j) and 90 (k) μM DNA. The arrow indicates the increasing conc. of DNA. The inset graph represents the plot of A_o/A − A_o vs. 1/[DNA] (μM)⁻¹ for the calculation of binding constant (K) and Gibb’s free energy (ΔG).

Fig. 9 Absorption spectrum of 1 mM compound 7 in the absence (a) and presence of 9 (b), 18 (c), 27 (d), 36 (e), 45 (f), 54 (g), 63 (h), 72 (i), 81 (j) and 90 (k) μM DNA. The arrow indicates the increasing conc. of DNA. The inset graph represents the plot of A_o/A − A_o vs. 1/[DNA] (μM)⁻¹ for the calculation of binding constant (K) and Gibb’s free energy (ΔG).

Based upon the variation in absorbance, the intrinsic binding constant of the compound with DNA was determined according to Benesi–Hildebrand’s equation:⁶³

where K is the association/binding constant, A_o and A are the absorbances of the compound and its complex with DNA, respectively, and ε_G and ε_H–G are the absorption coefficients of the compound and the compound–DNA complex, respectively. The association constants were obtained from the intercept-to-slope ratios of A_o/(A − A_o) vs. 1/[DNA] plots. The Gibbs free energy (ΔG) was determined from the equation:

ΔG = −RT ln K

where R is the general gas constant (8.314 JK⁻¹ mol⁻¹) and T is the temperature (298 K). The values of K and ΔG are given in the inset graph of the corresponding figures. The order of interaction with DNA is: 2 > 1 > 3 > 4 ∼ 7.

DNA and enzymes represent the most targeted bioreceptors for small molecules in various regulatory processes such as gene expression, gene transcription, mutagenesis, carcinogenesis.⁶⁴ Most anticancer drugs bind to DNA and proteins either in a reversible or irreversible manner, suggesting a direct relationship between their interactions with macromolecules, leading to their therapeutic effect.⁶⁵ It was proposed that the compounds interact with nitrogenous bases of nucleotides of nucleic acid and inhibit the cell division by interfering the replication and transcription of DNA molecules. The compounds may also affect the multienzyme complexes responsible for replication and transcription of DNA, thus causing an end to proliferation of the cells.⁶⁶ From the interaction study with SS-DNA it can be concluded that the synthesized compounds may have potential as anticancer drugs.

4. Conclusions

Sodium salt of N-[(2-methoxy-5-nitrophenyl)]-4-oxo-4-[oxy]butanamide was obtained in good yield and was characterized by FT-IR. Multinuclear NMR results showed that triorganotin(IV) derivatives exhibit distorted trigonal-bipyramidal geometry, both in the solution and solid states, while in the case of diorganotin(IV) derivatives the proposed geometry around the tin atom is octahedral. A good correlation was found between the Δν value calculated from the FT-IR data and the single crystal XRD data. From the single crystal X-ray structural analysis it is revealed that these compounds have packing diagrams like dendrimers, with characteristics that make them useful for numerous biological applications. The synthesized compounds exhibited significant cytotoxic and antileishmanial activity. These results may lead to development of a new drug against cancer cells and leishmanias after in vivo studies. Binding studies of small molecules to DNA are very important in the development of DNA molecular probes and new therapeutic reagents. UV-visible spectroscopic results show that the synthesized compounds bind to DNA via the intercalative mode of interaction, resulting in hypochromism and minor red shift.

Acknowledgements

M. Sirajuddin gratefully acknowledges the Higher Education Commission (HEC) Islamabad, Pakistan, for financial support.

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Footnote

† CCDC 984493–984495. For crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ra10487k

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