Crystal data and structure refinement for complex 2.

<p>Crystal data and structure refinement for complex 2.</p

The result of the analysis of SQUID magnetometry results by fitting to the Curie-Weiss law.

<p>The result of the analysis of SQUID magnetometry results by fitting to the Curie-Weiss law.</p

<i>In vitro</i> and <i>in vivo</i> anti-inflammatory active copper(II)-lawsone complexes

<div><p>We report <i>in vitro</i> and <i>in vivo</i> anti-inflammatory activities of a series of copper(II)-lawsone complexes of the general composition [Cu(Law)₂(L_N)_x(H₂O)_(2-x)]·<i>y</i>H₂O; where HLaw = 2-hydroxy-1,4-naphthoquinone, <i>x</i> = 1 when L_N = pyridine (<b>1</b>) and 2-aminopyridine (<b>3</b>) and <i>x</i> = 2 when L_N = imidazole (<b>2</b>), 3-aminopyridine (<b>4</b>), 4-aminopyridine (<b>5</b>), 3-hydroxypyridine (<b>6</b>), and 3,5-dimethylpyrazole (<b>7</b>). The compounds were thoroughly characterized by physical techniques, including single crystal X-ray analysis of complex <b>2</b>. Some of the complexes showed the ability to suppress significantly the activation of nuclear factor <i>κ</i>B (NF-<i>κ</i>B) both by lipopolysaccharide (LPS) and TNF-alpha (complexes <b>3</b>–<b>7</b> at 100 nM level) in the similar manner as the reference drug prednisone (at 1 μM level). On the other hand, all the complexes <b>1</b>–<b>7</b> decreased significantly the levels of the secreted TNF-alpha after the LPS activation of THP-1 cells, thus showing the anti-inflammatory potential <i>via</i> both NF-<i>κ</i>B moderation and by other mechanisms, such as influence on TNF-alpha transcription and/or translation and/or secretion. In addition, a strong intracellular pro-oxidative effect of all the complexes has been found at 100 nM dose <i>in vitro</i>. The ability to suppress the inflammatory response, caused by the subcutaneous application of λ-carrageenan, has been determined by <i>in vivo</i> testing in hind-paw edema model on rats. The most active complexes <b>1</b>–<b>3</b> (applied in a dose corresponding to 40 μmol Cu/kg), diminished the formation of edema simalarly as the reference drug indomethacine (applied in 10 mg/kg dose). The overall effect of the complexes, dominantly <b>1</b>–<b>3</b>, shows similarity to anti-inflammatory drug benoxaprofen, known to induce intracellular pro-oxidative effects.</p></div

<i>In vitro</i> and <i>in vivo</i> anti-inflammatory active copper(II)-lawsone complexes - Fig 1

<p>The general formula of 1,4-naphthoquinone showing most usual positions of substitutions, as indicated by arrows (A), and the formulas of selected 1,4-naphthoquinone derivatives isolated from natural sources: lawsone (B), juglone (C), and lapachol (D).</p

The pro-oxidative effect of complexes 1–7, reference aqua-complex and lawsone determined by dichlorofluorescein method.

<p>THP1-XBlue™-MD2-CD14 cell line were treated with the complexes, or standard antioxidant Trolox® at the final concentration of 100 nM for 1 h (left diagram) and 24 h (right diagram). The data represent the relative increase of fluorescence intensity after the addition of DCFH-DA in comparison with the untreated control. The results are expressed as the mean values ± S.E.M. for three independent experiments.</p

The general pathway of the preparation of complexes 1–7.

<p>The general pathway of the preparation of complexes 1–7.</p

Effect of complexes 1–7 on the NF-<i>κ</i>B activity.

<p>THP1-XBlue™-MD2-CD14 cell line were pre-treated with the complexes (100 nM, dissolved in PBS), [Cu(Law)₂(H₂O)₂]·0.5H₂O, HLaw and prednisone (1 μM, dissolved in DMSO) for 1 h. Subsequently, LPS (1 <b>μ</b>g/mL) (A) or TNF-α (10 ng/mL) (B) or Pam3CSK4 (100 ng/mL) (C) was added [except for the <i>control</i> cells (basal)] to trigger the activation of NF-<i>κ</i>B. After 24 h, the activity of NF-<i>κ</i>B was evaluated based on the amount of the secreted alkaline phosphatase measured spectrophotometrically. The results are expressed as the mean ± S.E.M. for six independent experiments. ** Indicates a significant difference in comparison with the vehicle-treated cells <i>p</i> < 0.01, and **** indicates a significant difference in comparison with the vehicle-treated cells <i>p</i> < 0.0001.</p

Selected bond lengths and angles (Å, °) for 2 and two reference complexes selected from CSD.

<p>Selected bond lengths and angles (Å, °) for 2 and two reference complexes selected from CSD.</p

The effect of the tested complexes on the secretion of TNF-α.

<p>THP-1 macrophages were pre-treated with the compounds <b>1</b>–<b>7</b>, [Cu(Law)₂(H₂O)₂]·0.5H₂O (aqua-complex), HLaw (100 nM, dissolved in PBS) and prednisone (1 <b>μ</b>M, dissolved in DMSO) for 1 h. Subsequently, LPS (1 <b>μ</b>g/mL) was added [except for the <i>control</i> cells (basal)] to trigger the secretion of the pro-inflammatory cytokine TNF-α. After 24 h, the amount of the secreted TNF-α was evaluated by ELISA. The results are expressed as the mean values ± S.E.M. for three independent experiments. * Indicates a significant difference in comparison with the vehicle-treated cells <i>p</i> < 0.05, ** indicates a significant difference in comparison with the vehicle-treated cells <i>p</i> < 0.01, *** indicates a significant difference in comparison with the vehicle-treated cells <i>p</i> < 0.001, and **** indicates a significant difference in comparison with the vehicle-treated cells <i>p</i> < 0.0001.</p

The result of temperature-dependent SQUID magnetometry measurements for complex 5.

<p>The curves represent the best fit of experimental data according to the Curie-Weiss law (the best-fit parameters are <i>g</i>_<i>iso</i> = 2.18, <i>C</i> = 5.585×10⁻⁶ m³.mol^-1, and θ <b>=</b> -0.181 K with a temperature-independent paramagnetism term a_TIP = 3.40×10⁻⁸ m³.mol^-1). C₀ = 4.7141997×10⁻⁶ m³.mol^-1.</p