Journal of Inorganic Biochemistry 143 (2015) 20–33

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Structural and theoretical studies on rhodium and iridium complexes with 5-nitrosopyrimidines. Effects on the proteolytic regulatory enzymes of the renin–angiotensin system in human tumoral brain cells Nuria A. Illán-Cabeza a, Sonia B. Jiménez-Pulido a, María J. Ramírez-Expósito b, Antonio R. García-García a, Tomás Peña-Ruiz c, José M. Martínez-Martos b, Miguel N. Moreno-Carretero a,⁎ a b c

Departamento de Química Inorgánica y Orgánica, Universidad de Jaén, 23071 Jaén, Spain Departamento de Ciencias de la Salud, Universidad de Jaén, 23071 Jaén, Spain Departamento de Química Física y Analítica, Universidad de Jaén, 23071 Jaén, Spain

a r t i c l e

i n f o

Article history: Received 4 September 2014 Received in revised form 14 November 2014 Accepted 14 November 2014 Available online 22 November 2014 Keywords: Antitumor activity Crystal structure Uracil Violuric acid Renin–angiotensin-system

a b s t r a c t The reactions of [RhCl(CO)(PPh3)2], [RhCl(CO)2]2 and [IrCl(CO)(PPh3)2] with different 5-nitrosopyrimidines afforded sixteen complexes which have been structurally characterized by elemental analysis, IR and NMR (1H and 13C) spectral methods and luminescence spectroscopy. The crystal and molecular structures of [RhIIICl(VIOH−1)2(PPh3)], [RhIIICl(DVIOH−1)2(PPh3)] and [RhII(DVIOH−1)2(PPh3)2] have been established from single crystal x-ray structure analyses. The three complexes are six-coordinated with both violurato ligands into an equatorial N5,O4-bidentate fashion, but with different mutually arrangements. Theoretical studies were driven on the molecular structure of [RhIIICl(VIOH−1)2(PPh3)] to assess the nature of the metal–ligand interaction as well as the foundations of the cis–trans (3L–2L) isomerism. An assortment of density functional (SOGGA11-X, B1LYP, B3LYP, B3LYP-D3 and wB97XD) has been used, all of them leading to a similar description of the target system. Thus, a topological analysis of the electronic density within AIM scheme and the study of the Mulliken charges yield a metal–ligand link of ionic character. Likewise, it has been proved that the cis–trans isomerism is mainly founded on that metal–ligand interaction with the relativistic effects playing a significant role. Although most of the compounds showed low direct toxicity against the human cell lines NB69 (neuroblastoma) and U373-MG (astroglioma), they differently modify in several ways the renin–angiotensin system (RAS)-regulating proteolytic regulatory enzymes aminopeptidase A (APA), aminopeptidase N (APN) and insulin-regulated aminopeptidase (IRAP). Therefore, these complexes could exert antitumor activity against both brain tumor types, acting through the paracrine regulating system mediated by tissue RAS rather than exerting a direct cytotoxic effect on tumor cells. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Cancer figures among the leading cause of death worldwide and it is expected that the annual cancer cases will rise to 22 million within the next two decades [1]. Among chemotherapeutic agents, cisplatin and the other platinum-based drugs have occupied an important position [2–4]. Even today, despite limitations and new therapeutic strategies, about two thirds of cancer patients are getting a platinum-based drug during their treatment. Considerable research efforts have been made to develop novel metal-based antitumor complexes with the aim of

⁎ Corresponding author. Tel.: +34 953212738; fax: +34 953211876. E-mail address: [email protected] (M.N. Moreno-Carretero).

http://dx.doi.org/10.1016/j.jinorgbio.2014.11.004 0162-0134/© 2014 Elsevier Inc. All rights reserved.

improving effectiveness and reducing the severe side effects (such as emetogenesis, neuro-, hepato- and nephrotoxicity) and development of drug resistance mechanisms (intrinsic and acquired) [5–8]. Owing to the large variety of coordination geometries and modes of interaction with their ligands, metal complexes give access to a different field of pathways in cancer treatment than do organic compounds. Ruthenium(II) and (III) compounds have received considerable attention [9–13], as possible alternatives to Pt(II) anticancer agents. In contrast to this, the group 9 elements rhodium and iridium have attracted less interest as potential anticancer agents [14–16], in particular in their +3 oxidation state. Although the first mention of antitumor properties for a rhodium(III) complex, RhCl3 · 3H2O [17] appeared in 1953, it is only in the past five years that the structure–activity relationships (SARs) and cellular effects of cytotoxic Rh(III) and Ir(III) compounds have been studied. In this way, Rh(I) and Rh(II) complexes have aroused more interest, in particular dirhodium(II) carboxylate complexes [14–16,18,19] because of their notable antitumor activity and limited side effects. Recent studies

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since 2002 on rhodium and iridium anticancer complexes in the oxidation states +1 to +3 have been summarized in three review articles [14–16]. Although it is only very recently that they have received significant attention, Rh and Ir complexes exhibit a variety of properties that make them interesting as prospective anticancer drugs. Their reactivities, binding preferences and cellular uptake are strongly dependent on their ligand combination and coordination geometry and this allows for considerable scope in drug design development. The chemistry of pyrimidines and related N-containing heterocyclic derivatives has been of great interest for many years [20–22] since they play a key role in many biological processes and are important intermediates for the design and synthesis of fused polycyclic pharmaceutical targets, such as purines, pteridines and nucleoside analogues [23,24]. 5-Nitrosopyrimidines have been extensively used as ligands due to their similarities with uracil and they have been used for analytical and biological purposes [25]. The renin–angiotensin system (RAS) including angiotensin receptors is widely distributed in the brain [26–28] and classically has been described as an important regulator of blood pressure, electrolyte balance, and fluid homeostasis [29]. This system comprises the inactive peptide angiotensinogen, which is converted to angiotensin I and then the active peptide angiotensin II (Ang II) through the action of renin and angiotensin-converting enzyme (ACE) [30]. Angiotensin II is the most important modulator of cardiovascular function and exerts a pronounced hypertensive effect [31] although this and other angiotensins have been also involved in angiogenesis and tumor growth [32]. Proteolytic cleavage of Ang II by glutamyl aminopeptidase A (APA) and membrane alanyl aminopeptidase N (APN) results in the sequential removal of single aminoacids residues from the N-terminal end of the peptide, to form Ang III and Ang IV [33]. Ang IV plays a central role in the brain and it has been suggested that the insulin-regulated aminopeptidase (IRAP) is the major target for Ang IV in the brain [31]. Besides this angiotensinase function, APN and APA are directly involved in the tumoral process [34]. We have previously described a strong relationship between RASregulating aminopeptidase activities and tumor growth in a rat model of experimental glioma. Thus, we found a time-dependent significant decrease in APN activity, and a significant increase of APA activity concomitantly with tumor growth in tumor tissue [35]. Herein, new Rh(III), Rh(II), Rh(I) and Ir(I) complexes with different 5-nitrosopyrimidines derivatives were synthesized, and the physicochemical, structural, luminescence properties and biological activity have been studied. To investigate their activity against tumoral process, we have centered the study on their effects on RAS-regulating proteolytic regulatory enzymes APA, APN and IRAP on the human tumor brain cell lines NB69 (neuroblastoma) and U373-MG (astroglioma).

2. Experimental 2.1. Instrumentation C, H, N microanalyses were performed on a Thermofinnigan Flash 1112 Series elemental analyzer. Molar conductivities were measured on a Radiometer CMD2e apparatus (10− 3 M dimethylformamide solutions). IR spectra were measured on a Bruker FT-IR Tensor-27 (4000–400 cm−1, KBr pellets) and Vector-22 (600–220 cm−1, polyethylene pellets) spectrophotometers. 1H and 13C-NMR spectra were recorded using a Bruker Avance 400 MHz apparatus (DMSO-d6 solutions, δ(TMS, tetramethylsilane) = 0.0 ppm). Solid-state 13C CP/TOSS (cross polaryzed total sideband suppression) NMR spectra were recorded with a Bruker Avance 500 MHz spectrometer with TOSS pulse sequence to eliminate spinning side bands. Fluorescence excitation and emission spectra were measured with a Cary Elipse fluorescence spectrophotometer in CH3 CN at room temperature. Chemicals were purchased from ABCR and Alfa-Aesar and used without further purification.

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2.2. Crystallography Details of the crystallographic data collection and refinement parameters are given in Table 1. The measurements were performed on a Bruker-Nonius Kappa CCD diffractometer with graphite monochromated Mo-Kα (λ = 0.71073 Å) radiation. The temperature (120 K) was controlled employing an Oxford Cryosystem low-temperature device. Lorentz, polarization and multiscan absorption corrections were applied with SADABS [36]. The structure was solved by direct methods and refined using SHELXL97 program [37] inside the WinGX package [38] employing full-matrix least-squares methods on F2. All non-H atoms were refined anisotropically; all hydrogen atoms were placed in calculated positions following riding models. All calculations were carried out with PLATON [39] and graphics were drawn with MERCURY [40].

2.3. Computational details Gaussian 09, Revision D.01, suite of programs has been used to produce the data and wave functions necessary to analyze the bonding of the complex [RhCl(VIOH−1)2(PPh3)] and to study of the cis–trans isomerism [41]. Five different density functionals has been chosen to achieve the previous objectives on the basis of a study by Luo and Truhlar [42] on the performance of an assortment of density functionals in order to treat different multiplicities and ionization States of 4d transition metals. Thus, three meta-hybrid GGA density functionals have been selected, SOGGA11-X [43], B1LYP [44] and B3LYP [45] along with the range-separated hybrid GGA-D one, ωB97X-D [46] that includes dispersion. In addition, empirical dispersion has been considered with B3LYP density functional within Grimme's D3 model [47] (B3LYP-D3, empiricaldispersion = gd3). It is important to note that SOGGA11-X, B1LYP and B3LYP are placed in positions 1, 2 and 5 respectively in the ranking yielded by Luo and Truhlar's research [42]. The integration grid for the two-electron integrals and their derivatives was pruned to 99 radial shells and 590 angular points per shell (ultrafine). In addition, Hartree–Fock calculations were conducted for comparative purposes. To obtain the wavefunctions used to make the topological analysis of the electronic density for the target complex as well as its molecular orbitals and Mulliken charges, single point calculations were accomplished on the x-ray diffraction geometry of the system by using the previous density functionals implementing the basis sets aug-ccpVTZ-DK [48,49] on Rh, Cl and P atoms; aug-cc-pVDZ-DK [50] on both oxygen and nitrogen atoms of the octahedral coordination sphere and 6-31++G(d,p) on the remaining atoms [51,52]. As for the study of the cis–trans isomerism, two prototypes designed to account for the metal–ligand interactions were optimized with the previously commented density functionals and the basis sets aug-cc-pVTZ-PP [48] on Rh, that includes a small core relativistic pseudopotential; augcc-pVTZ [34] on Cl and P atoms, aug-cc-pVDZ [50] on both oxygen and nitrogen atoms of the octahedral coordination sphere and 631++G(d,p) on the remaining atoms. In a second stage, single point calculations were conducted on the optimized geometries by using the basis set used for the full complex (FC). Second order scalar relativistic effects within Douglas–Kress–Hall scheme [53] were considered for the single point calculations of the complex and the prototypes as well. The topology of the electron density surface was analyzed within the premises of Bader's Atoms in Molecules theory (AIM) [54] implemented in the code AIM2000 [55].

2.4. Ligands Commercial grade chemicals were used without further purification. The organic precursors were prepared by the methods described in our earlier reports [56,57]. The structure of the ligands is depicted in Fig. 1.

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Table 1 Crystal data and structure refinement for complexes, [RhCl(VIOH−1)2(PPh3)] · 2H2O (3), [RhCl(DVIOH−1)2(PPh3)] · CH3CN (5) and [Rh(DVIOH−1)2(PPh3)2)] (6).

CCDC number Empirical formula Formula weight Crystal system Space group a (Å) b (Å) c (Å) α (°) β (°) γ (°) Volume (Å3) Z Dcalc (g cm−3) μ (mm−1) F(000) θ (°) Limiting indices

Data/restrains/parameters Goodness-of-fit on F2 R1/wR2 [I N 2σ(I)] R1/wR2 (all data) Δρ (e Å−3)

[RhCl(VIOH−1)2(PPh3)] · 2H2O

[RhCl(DVIOH−1)2(PPh3)] · CH3CN

[Rh(DVIOH−1)2(PPh3)2)]

929237 RhC26H19N6O10PCl 744.80 Triclinic P-1 11.272(1) 16.306(2) 17.868(3) 92.35(1) 90.57(1) 109.77(1) 3087.0(6) 4 1.603 0.756 1496 3.80–27.50 −14 ≤ h ≤14 −21 ≤ k ≤21 −23 ≤ l ≤23 14141/1/847 1.066 0.0597/0.1410 0.0994/0.1683 1.945/−1.540

929236 RhC32H30N7O8PCl 809.98 Monoclinic P21/n 10.644(1) 12.431(2) 25.532(4) 90 101.486(7) 90 3310.7(8) 4 1.625 0.708 1648 3.82–27.50 −13 ≤ h ≤13 −16 ≤ k ≤16 −33 ≤ l ≤33 7585/0/532 1.105 0.0401/0.0867 0.0636/0.0997 1.614/−0.906

929235 RhC48H42N6O8P2 995.75 Monoclinic P21/n 10.363(2) 17.753(2) 11.701(1) 90 94.81(1) 90 2145.1(5) 2 1.542 0.538 1022 5.00–27.50 −13 ≤ h ≤13 −23 ≤ k ≤23 −15 ≤ l ≤15 4904/0/295 1.091 0.0424/0.0897 0.0735/0.1061 1.574/−0.750

2.5. Preparation of the complexes 2.5.1. RhCl(MANUH − 1 ) 2 (PPh 3 ) 2 (1), RhCl(DANUH − 1 ) 2 (PPh 3 ) (2), [RhCl(VIOH − 1 ) 2 (PPh 3 )] · 2H 2 O (3), RhCl(MVIOH − 1 ) 2 (PPh 3 ) 2 (4), [RhCl(DVIOH− 1)2(PPh3)] · CH3CN (5) Compounds 1, 3 and 5 were prepared by solvothermal reaction using a Teflon-lined stainless-steel reactor (Parr) of 0.125 mmol of ligand and 0.125 mmol of [RhCl(CO)(PPh3)2] in 10 mL of CH3CN at 70 °C for 72 h. The complex 2 was obtained following a similar procedure but in a L:M molar ratio 2:1, whereas compound 4 was synthesized in MeOH medium (L:M = 1:1). Solids precipitate for 1, 2 and 4 which were filtered off, washed with EtOH and Et2O and air dried. Crystals suitable for x-ray diffraction were obtained for [RhCl(VIOH−1)2(PPh3)] · 2H2O (3) and [RhCl(DVIOH−1)2(PPh3)] · CH3CN (5); this one loses the solvent molecule easily, so the remaining structural characterization have been done with [RhCl(DVIOH−1)2(PPh3)] complex. RhCl(MANUH−1)2(PPh3) 2 (1), Anal. Found: C, 55.35; H, 4.77; N, 10.85. Calc. for RhC46H40N8O6ClP2 (1001.24): C, 55.18; H, 4.03; N, 11.12, ΛM = 83.2 ohm −1 cm2 mol −1. RhCl(DANUH−1)2(PPh3) (2), Anal. Found: C, 47.24; H, 4.04; N, 14.54. Calc. for RhC30H29N8O6ClP (767.00): C, 46.98; H, 3.81; N, 14.61, ΛM = 2.1 ohm− 1 cm2 mol− 1. [RhCl(VIOH− 1)2(PPh3)] · 2H2O (3), Anal. Found: C, 37.87; H, 3.17; N, 11.79. Calc. for RhC22H23N6O10ClP (700.84): C, 37.70; H, 3.31; N, 11.99, ΛM = 43.5 ohm −1 cm2 mol −1. RhCl(MVIOH− 1)2(PPh3)2 (4), Anal. Found: C, 54.97; H, 4.04; N, 8.40. Calc. for RhC46H38N6O8ClP2 (1003.20): C, 55.06; H, 3.82; N, 8.38, ΛM =

111.1 ohm −1 cm2 mol −1. [RhCl(DVIOH−1)2(PPh3)] · CH3CN (5), Anal. Found: C, 46.88; H, 3.75; N, 10.86. Calc. for RhC30H27N6O8ClP (768.96): C, 46.85; H, 3.55; N, 10.93, ΛM = 2.3 ohm −1 cm2 mol −1. 2.5.2. [Rh(DVIOH−1)2(PPh3)2] (6) The compound was prepared by reaction of [RhCl(CO)(PPh3)2] (0.125 mmol) with DANU (0.25 mmol) and KOH (0.25 mmol) in 20 mL of CH3OH at 50 °C for 3 h; during the process, DANU underwent hydrolysis to give DVIO. Crystals suitable for x-ray diffraction with formula [Rh(DVIOH−1)2(PPh3)2] were obtained. [Rh(DVIOH−1)2(PPh3)2] (6), Anal. Found: C, 57.89; H, 4.25; N, 8.44. Calc. for RhC48H42N6O8P2 (995.79): C, 57.16; H, 4.51; N, 8.68, ΛM = 8.2 ohm −1 cm2 mol −1. 2.5.3. RhCl(CO)(MANU) · 3H 2O (7), RhCl(CO)(DANU) · 3H 2O (8), RhCl(CO)(2MeOANU) · 3H2 O (9), RhCl(CO)(VIO) · 3H2 O (10), RhCl(CO)(DVIO) · 2H2O (11) The synthesis of the complexes were carried out by reacting 0.125 mmol of the ligand with 0.125 mmol of [RhCl(CO)2]2 in acetone. The mixture was stirred and heated for 3 h until almost dryness. Then, hexane was added and complexes precipitated. The solids were filtered off, washed with EtOH and Et2O and air dried. RhCl(CO)(MANU) · 3H2O (7), Anal. Found: C, 18.64; H, 2.42; N, 14.55. Calc. for RhC6H12N4O7Cl (390.58): C, 18.45; H, 3.10; N, 14.35, ΛM = 8.6 ohm −1 cm2 mol −1. RhCl(CO)(DANU) · 3H2O (8), Anal. Found: C, 20.70; H, 3.00; N, 14.14. Calc. for RhC7H14N4O7Cl (404.61): C, 20.78; H, 3.49; N, 13.85, ΛM =

Fig. 1. More stable tautomeric structure and nomenclature of the ligands: (1) R = R′ = H, X = O, 6-amino-5-nitrosouracil (ANU, 6-amino-5-nitroso-1H-pyrimidin-2,4-dione); R = CH3, R′ = H, X = O, 6-amino-1-methyl-5-nitrosouracil (MANU); R = R′ = CH3, X = O, 6-amino-1,3-dimethyl-5-nitrosouracil (DANU); (2) 6-amino-2-methoxy-5-nitroso-3H-pyrimidin-4one (2MeOANU); (3) R = R′ = H, violuric acid (VIO, 5-hydroxyimino-pyrimidine-2,4,6(1H,3H,5H)-trione); R or R′ = CH3, methylvioluric acid (MVIO); R = R′ = CH3, dimethylvioluric acid (DVIO).

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4.4 ohm −1 cm2 mol −1. RhCl(CO)(2MeOANU) · 3H2O (9), Anal. Found: C, 18.57; H, 3.05; N, 14.18. Calc. for RhC6H12N4O7Cl (390.58): C, 18.45; H, 3.10; N, 14.35, ΛM = 6.6 ohm −1 cm2 mol −1. RhCl(CO)(VIO) · 3H2O (10), Anal. Found: C, 15.97; H, 2.09; N, 11.77. Calc. for RhC5H9N3O8Cl (377.53): C, 15.91; H, 2.40; N, 11.13, ΛM = 16.6 ohm −1 cm2 mol −1. RhCl(CO)(DVIO) · 2H2O (11), Anal. Found: C, 21.88; H, 2.88; N, 10.83. Calc. for RhC7H11N3O7Cl (387.54): C, 21.69; H, 2.84; N, 10.85, ΛM = 7.9 ohm −1 cm2 mol −1. 2.5.4. IrCl(CO)(ANU)(PPh3)2 · 2H2O (12), IrCl(CO)(MANU)(PPh3)2 · 2H2O (13), IrCl(CO)(DANU)(PPh 3 ) 2 · 2H 2 O (14), IrCl(CO)(2MeOANU) (PPh3)2 · 2H2O (15), IrCl(CO)(DVIO)(PPh3)2 (16) A suspension of the ligand (0.125 mmol) in hot MeOH (30 mL) was reacted with [IrCl(CO)(PPh3)2] (0.25 mmol). The mixture was stirred and heated for three hours (50 °C). The resulting solids were filtered off, washed with EtOH and Et2O and air dried. IrCl(CO)(ANU)(PPh3)2 · 2H2O (12), Anal. Found: C, 50.74; H, 4.19; N, 5.84. Calc. for IrC41H38N4O6ClP2 (972.54): C, 50.64; H, 3.95; N, 5.76, ΛM = 20.2 ohm −1 cm2 mol −1. IrCl(CO)(MANU)(PPh3)2 · 2H2O (13), Anal. Found: C, 51.48; H, 4.19; N, 5.57. Calc. for IrC42H40N4O6ClP2 (986.47): C, 51.13; H, 4.10; N, 5.68, ΛM = 7.3 ohm −1 cm2 mol −1. IrCl(CO)(DANU)(PPh3)2 · 2H2O (14), Anal. Found: C, 51.39; H, 4.59; N, 5.75. Calc. for IrC43H42N4O6ClP2 (1000.50): C, 51.62; H, 4.24; N, 5.60, ΛM = 10.5 ohm −1 cm2 mol −1. IrCl(CO)(2MeOANU)(PPh3)2 · 2H2O (15), Anal. Found: C, 50.99; H, 3.36; N, 5.78. Calc. for IrC42H40N4O6ClP2 (986.47): C, 51.13; H, 4.10; N, 5.68, ΛM = 6.5 ohm −1 cm2 mol −1. IrCl(CO)(DVIO)(PPh3)2 (16), Anal. Found: C, 53.66; H, 4.77; N, 4.20. Calc. for IrC43H37N3O5ClP2 (965.44): C, 53.49; H, 3.87; N, 4.35, ΛM = 5.7 ohm −1 cm2 mol −1. Yields are 40%–60% for rhodium compounds and 70%–80% for iridium complexes. In the supplementary material, detailed IR and NMR data are available. Although the elemental analyses data for certain atoms for compounds 6, 7, 10, 15 and 16 are somewhat unsatisfactory, the crystal structure analysis (compound 6) or NMR (1H, 13C) and IR spectroscopic analysis data reasonably support their formula. 2.6. Biological studies 2.6.1. Cell culture Human neuroblastoma NB69 and astroglioma U373-MG cells were grown in 5% fetal bovine serum (FBS)-supplemented DMEM medium without antibiotics. Cells were incubated at 37 °C in a modified atmosphere of 5% CO2/95% air until reaching confluence. Freedom from mycoplasma contamination was checked regularly by testing with Hoechst 33528.

2.6.4. Insulin-regulated aminopeptidase activity assay IRAP was measured fluorometrically in whole cells using leucyl-βnaphthylamide (LeuNNap) as substrate. After removal of the culture medium, the cells were incubated by triplicate for 30 minutes at 37 °C with 100 μL of the substrate solution containing the reported compounds at a range of concentrations (1–5 μM), 100 μM of LeuNNap and 0.65 mM DTT in artificial cerebrospinal fluid (NaCl 116 mM, KCl 5.4 mM, MgCl2 0.9 mM, CaCl2 1.8 mM, NaHCO3 25 mM, glucose 10 mM) pH 7.2. The reactions were stopped by addition of 100 μL of acetate buffer 0.1 M (pH 4.2) and the amount of β-naphthylamine released as the result of the enzymatic activities was measured fluorometrically at 412 nm emission wavelength with and excitation wavelength of 345 nm. Specific enzyme activities were expressed as picomoles of the corresponding aminoacyl-β-naphthylamide hydrolyzed per min per 106 cells, by using a standard curve prepared with the latter compound under corresponding assay conditions. 2.6.5. Statistics We used one-way analysis of variance (ANOVA) to analyze differences between groups. Post-hoc comparisons were made using Newman–Keuls test P-values below 0.05 were considered significant. 3. Results and discussion A brief description of the synthetic pathway for the isolation of complexes is depicted in Fig. 2. Several pyrimidine compounds were used in this report to study their interaction with rhodium and iridium compounds. Solvothermal reaction of the ligands with [RhCl(CO)(PPh 3 ) 2] afforded the Rh(III) monomeric compounds with general formula RhCl(LH − 1 ) 2 (PPh3 ) x (x = 1 or 2) (1–5) where rhodium has undergone a two-electron oxidation during the synthetic reaction and oxygen might act as oxidizing agent. The CO molecule was displaced by the organic ligands which were deprotonated during the reaction. However, the different conditions and the presence of KOH originates the hydrolysis of DANU and the oxidation of Rh(I) to Rh(II) in [Rh(DVIOH− 1)2(PPh3)2] (6). The Rh(I) carbonyl complexes were prepared through a bridge-splitting reaction of the rhodium carbonyl dimer (7–11). Finally, the reaction with [IrCl(CO)(PPh3)2] yielded the complexes IrCl(CO)(L)(PPh3)2 (12–16). All the complexes show a non-electrolyte nature except RhCl(MANUH − 1 ) 2 (PPh 3 ) 2 (1) and RhCl(MVIOH − 1 ) 2 (PPh 3 ) 2 (4) whose character is 1:1, suggesting that chloride is not bound to the metal ion [58].

2.6.2. Aminopeptidase A activity assay APA activity was analyzed in whole cells, using glutamyl-βnaphthylamide (GluNNap) as substrate. After removal of the culture medium, the cells were incubated by triplicate for 30 min at 37 °C with 100 μL of the assay medium containing the reported compounds at a range of concentrations (1–5 μM), 100 μM of GluNNap and 0.65 mM dithiothreitol (DTT), in artificial cerebrospinal fluid (NaCl 116 mM, KCl 5.4 mM, MgCl2 0.9 mM, CaCl2 1.8 mM, NaHCO3 25 mM, glucose 10 mM) pH 7.2. 2.6.3. Aminopeptidase N assay APN was also measured fluorometrically in whole cells using alanylβ-naphthylamide (AlaNNap) as substrate. After removal of the culture medium, the cells were incubated by triplicate for 30 minutes at 37 °C with 100 μL of the substrate solution containing the title compounds at a range of concentrations (1–5 μM), 100 μM of AlaNNap and 0.65 mM DTT in artificial cerebrospinal fluid (NaCl 116 mM, KCl 5.4 mM, MgCl 2 0.9 mM, CaCl2 1.8 mM, NaHCO3 25 mM, glucose 10 mM) pH 7.2.

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Fig. 2. Synthetic pathways for the title complexes.

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3.1. Crystallographic studies Selected interatomic distances and angles useful to describe the metal coordination sphere as well as the tautomeric form (keto-oxime, ketonitroso or nitroso-enol) of the ligands are included in Table 2. In the molecular unit of complexes [RhCl(VIOH−1)2(PPh3)] · 2H2O (3) and [RhCl(DVIOH−1)2(PPh3)] · CH3CN (5) (Fig. 3), the Rh(III) ion displays a distorted octahedral geometry with two equatorial N5-O4 bidentate violurato ligands and a chloride and a phosphorus atom from a triphenylphosphine in the apical positions. The pyrimidine ligands coordinate through two five-membered chelate rings with a narrow bite angle of 80°; the M–N and M–O distances fall within normal limits and are similar to average values in other structurally characterized Rh(III) complexes (ca. 2.0–2.1 Å) [59–61]. The average Rh–Cl distances are similar to those of other analogous complexes [59,60]. The RhIII-P distances are shorter than the usual ones found for other rhodium(III)–PPh3 compounds (ca. 2.4 Å) [59,60]. The P–Rh–Cl angles, around 177°, are somewhat lower than the ideal angle 180°. The coordination of the two organic ligands results in two nearly coplanar five-membered chelate rings (Rh–N5–C5–C4–O4) with dihedral angles of 15° in [RhCl(VIOH− 1)2(PPh3)] · 2H2O and 3.2° in [RhCl(DVIOH− 1)2(PPh3)] · CH3CN. For [M(bidentate)2(monodentate)2] octahedral six-coordinate compounds in which the two ends of bidentate ligands are chemically dissimilar, cis or trans stereoisomers can be found. The energy gap between them is small and it would be expected that all isomers would be possible [62]. In the two complexes, the two carbonyl oxygens O4 and the nitroso nitrogens N5 are mutually cis. The absolute configuration of the rhodium(III) in both complexes must be described, following the Bailar's nomenclature, as the 3L stereoisomer [63], in contrast with the usual absolute configuration 2L centrosymmetric stereoisomer

found in similar compounds [64–67] and the compound [Rh(DVIOH−1)2 (PPh3)2] described below. The atoms O5 and O6 from the same violurato ligand and the O5 atoms from the two violurato units show distances of around 2.8 Å which may indicate a weak interaction between these atoms which stabilizes the molecule. The single crystal x-ray diffraction study of [Rh(DVIOH−1)2(PPh3)2] (6) revealed that Rh(II) center is lying at the center of a square plane from two (O4,N5)-dimethylviolurato ligands in a six-coordinated distorted octahedral geometry (Fig. 4). Both triphenylphosphine ligands have taken up the two axial positions and are mutually trans. Distortion becomes evident from the small bite angle N5–Rh–O4 of 79.4(1)°. The Rh–P distances are about 15% longer that the Rh–N and Rh–O ones and also slightly longer than the average Rh–P distance found in [RhCl(DVIOH−1)2(PPh3)] · CH3CN; steric requirements of the PPh3 ligands and the different oxidation state of the rhodium might be causes of these values. Both pyrimidine and chelate planes are quasi-coplanar (0.9(1)°) and the two carbonyl O4 oxygens and the nitroso N5 nitrogens are mutually trans (2L stereoisomer). The hydrolysis of DANU to give DVIO can be clearly observed in the structure on comparing the distance C4–O4 with the one found in [RhCl(DVIOH−1)2(PPh3)] · CH3CN which are very similar (1.269(4) and 1.271(4) Å, respectively); also, if we compare with structures containing the deprotonated DANU, it can be observed that the C–N distances associated with the hydrolyzed amino group are always larger [68]. Upon coordination, some changes in the bond lengths of the organic precursors VIO and DVIO are observed. The violuric acid ligands contain one acidic proton involved in a keto-oxime ⇄ nitroso-enol tautomeric equilibrium which is lost in the coordination to the rhodium center. XRD experiments clearly confirmed the nitroso-enolato form of the ligands. Thus, in [RhCl(VIOH−1)2(PPh3)] · 2H2O the N5–O5 and C5–N5 distances are shorter and longer, respectively, than those found in the

Table 2 Relevant distances (Å) and angles (°). [RhCl(VIOH−1)2(PPh3)] · 2H2Oa,b

Rh–N5 Rh–O4 Rh–P Rh–Cl C2–O2 C4–O4 C5–N5 N5–O5 C6–O6 N5–Rh–O4 O4–Rh–P N5–Rh–P O4–Rh–Cl N5–Rh–Cl O4–Rh–O4' N5–Rh–N5' N5–Rh–O4' P–Rh–Cl a b

1

2

2.002(4) 1.994(4) 2.087(3) 2.087(3) 2.313(1) 2.397(1) 1.222(6) 1.221(6) 1.256(6) 1.259(6) 1.344(6) 1.351(7) 1.228(5) 1.222(6) 1.229(6) 1.211(7) 80.4(2) 80.6(2) 88.7(1) 92.5(1) 94.5(1) 94.8(1) 89.6(1) 86.3(1) 86.6(1) 86.9(1) 99.0(1) 99.6(2) 172.9(2) 176.5(2) 177.71(5)

1.990(5) 1.997(4) 2.083(3) 2.097(3) 2.327(2) 2.390(1) 1.214(6) 1.229(6) 1.264(6) 1.259(6) 1.346(7) 1.375(6) 1.237(6) 1.226(6) 1.220(7) 1.218(6) 80.7(2) 81.3(2) 91.5(1) 85.8(1) 97.9(2) 94.9(1) 86.9(1) 88.5(1) 87.8(1) 86.7(1) 98.8(1) 98.7(2) 176.3(3) 173.6(2) 173.73(6)

Two different molecules per asymmetric residual unit. For each parameter, the second value corresponds to the L′ ligand.

[RhCl(DVIOH−1)2(PPh3)] · CH3CNb

[Rh(DVIOH−1)2(PPh3)2]

1.993(3) 1.992(3) 2.078(2) 2.082(2) 2.3090(8) 2.3926(8) 1.211(4) 1.213(4) 1.271(4) 1.264(4) 1.358(4) 1.359(4) 1.234(3) 1.238(3) 1.211(4) 1.212(4) 80.0(1) 80.2(1) 86.51(6) 87.99(6) 94.55(8) 92.86(8) 91.26(6) 91.13(6) 86.27(8) 89.34(8) 98.59(8) 101.3(1) 177.01(9) 178.6(1) 177.46(3)

2.028(2) 2.075(2) 2.3451(8) – 1.214(4) 1.269(4) 1.327(4) 1.252(3) 1.237(4) 79.4(1) 87.30(7) 87.06(7) – – – – – –

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25

Fig. 3. ORTEP drawing (50% probability ellipsoids) and atom-labeling scheme for [RhCl(DVIOH−1)2(PPh3)] · CH3CN.

free ligand (around 0.112 Å and 0.052 Å, respectively) [69]. So, the N5–O5 bond is strengthened and N5–C5 bond is weakened suggesting the presence of a 5-nitroso group (−N = O) over a 5-oxime one (=N–O). The C2–O2 and C6–O6 distances correspond to a double bond (ca. 1.22 Å) whereas C4–O4 distance is longer (ca. 1.25 Å) as consequence of both deprotonation and coordination effects. A similar behavior for the Rh-coordinated DVIOH− 1 anion has been found in complexes 5 and 6, in a comparable way than the analogous VIOH−1 complex and other related coordination compounds [64,70]. The most important intermolecular contacts are shown in Table 3. There are π–π stacking interactions contributing to stabilize the structures which involve uracil and phenyl (from PPh3 molecules) rings

and σ–π interactions (X–H · · · Cg and Y–X · · · Cg) to form a threedimensional network [71]. 3.2. Computational studies of the complex [RhCl(VIOH−1)2(PPh3)] 3.2.1. Bonding analysis The nature of the bonding scheme around the metal center of fullcomplex (FC) is initially assessed by analysis of the electronic density within the Atoms in Molecules theory (AIM). Thus, Fig. 5 shows different sections of that property for the coordination sphere of the Rh(III) atom. As it is observed, the topology is correct since a bond critical point (BCP) appears between each couple of interacting atoms as well as the

Fig. 4. Molecular structure of [Rh(DVIOH−1)2(PPh3)2] (50% probability ellipsoids).

26

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Table 3 Geometrical features of the intermolecular interactions. π · · · π interactions

d(c1–c2) (Å)a

α (°)b

β (°)c

γ (°)c

Slippage

[RhCl(DVIOH−1)2(PPh3)] · CH3CN (5) DVIO · · · phenyl

3.424(2)

9.7

13.8

11.1

0.74

[Rh(DVIOH−1)2(PPh3)2] (6) Phenyl · · · phenyl (−x,−y,1 − z)

3.673(2)

0

13.5

13.5

0.86

Y–X · · · Cg

d(X · · · Cg) (Å)d

δ (°)e

∠(Y-X · · · Cg) (°)

[RhCl(VIOH−1)2(PPh3)] · 2H2O (3) C2–O2 · · · phenyl (−x,1 − y,−z) C2–O2 · · · uracil (1 − x,2 − y,1 − z) C2–O2 · · · uracil (1 − x,1 − y,−z) N5–O5 · · · phenyl C12-H12(phenyl) · · · phenyl C16-H16(phenyl) · · · phenyl (−x,1 − y,1 − z)

3.515(5) 3.413(4) 3.436(6) 3.819(5) 2.89 2.88

15.1 27.8 27.3 27.0 27.9 14.5

78.4(3) 106.6(3) 82.7(4) 81.6(3) 139 99

[RhCl(DVIOH−1)2(PPh3)] · CH3CN (5) C4–O4 · · · phenyl (5⁄21 − x,½ + y,5⁄21 − z) N5–O5 · · · phenyl C1S-N1S · · · uracil (½ + x,½1 − y,½ + z) C3A-H3A · · · phenyl (3⁄21 − x,½ + y,5⁄21 − z)

3.771(3) 3.753(3) 3.010(5) 2.93(4)

8.80 26.0 11.6 5.2

141.6(2) 76.2(2) 161.9(4) 135(3)

[Rh(DVIOH−1)2(PPh3)2](6) C2–O2 · · · phenyl (−½ + x,½1 − y,−½ + z) C22-H22 · · · phenyl (½1 − x,½ + y,½1 − z)

3.689(3) 2.66

14.7 5.5

91.6(2) 149

a b c d e

Distance between centroids of rings involved in π-interactions (only those shorter than 4 Å are given). Angle between planes of both centroids. Slipping angles between the centroid-centroid vector and the normal to each stacked ring plane. Distance between the X atom and the centroid of the ring. Slipping angle between the X-centroid vector and the normal to the ring

corresponding bonding path (BP) and the interatomic surface (IAS). The positive values of the laplacian (Table 4) reveal that the interactions X-Rh(III) (X = Cl,P,N,O) are of non-covalent type. Likewise, it is observed that all the DFT (density functional theory) methods describe the topology of the electronic density in a common way, yielding similar values for the different parameters (Table 4). Therefore, the lowest value of ρ (electron density) is obtained for the Rh · · · Cl interaction and highest one for both Rh · · · N interactions. The order of increasing electronic density is Rh · · · Cl b Rh · · · O b Rh · · · P b Rh · · · N irrespective of the DFT approach. As for ∇ (laplacian of the electron density), all methods follow the pattern Rh · · · P b Rh · · · Cl b Rh · · · N b Rh · · · O but for HF. The only minor discrepancy among the different density functionals concerns the ε (ellipticity: anisotropy of the electron density at the BCP), where it is observed its value is higher for Rh · · · Cl than for Rh · · · P in the case of B1LYP, B3LYP and B3LYP-D3 but the situation is reversed for SOGGA11X and ωB97XD. The previous observations on the non-covalent nature of the metal– ligand are further supported by an analysis of Mulliken charges for the atoms involved in the coordination sphere. Thus, a positive charge in the range 2.4–3.2 is allocated on the rhodium atom by the DFT approximations which is in agreement with the presence of Rh3+. As regards the atoms that surround the metal cation, negative charges are allocated on all of them. The amount of negative charge follows the pattern N b Cl b P b O, for all methods (including HF) with a discrepancy in the case of ωB97XD that yields a charge for P similar to that of the O atoms. This separation between positive and negative charges suggests a strong contribution of ionic bonding in the metal–ligand bond. Detailed data about Mulliken charges are given in supplementary material. Anyway, a study of the molecular orbitals can account for electronsharing contributions to the interactions metal–ligand. As a general observation, the contributions of Cl, P, O4, O4D, N5 and N5D to the molecular orbitals (MO) that link them to Rh(III) are low, 0.05-0.20 (base 1.00), and highly distributed among different MOs. This is in accordance with the non-covalent character yielded by the AIM calculations. The most significant orbitals are described in Table S3 in supplementary material.

3.2.2. Other intramolecular interactions There exist other interactions that stabilize the target complex; data are supported in Table 4. Therefore, a non-covalent interaction N = O · · · O = N can be observed (Fig. 6b). A BCP is found between the two oxygen atoms along with the bonding path and the interatomic surface. Likewise, a ring critical point (RCP) is observed as corresponds with the structural motif involved in this interaction. Also, another two contacts of the type C = O · · · O = N could exist inside the violurate units since a BCP appears between the oxygen atoms but in these cases, BCPs are very close to RCPs. As both a BCP and a RCP coalesce they annihilate each other and no interaction can be characterized, then the existence of these interactions must be considered with caution. In addition two C-H · · · O = C hydrogen bonds have been characterized (Table 4) according to Koch and Popelier criteria [72]. Both hydrogen bonds involve the same hydrogen atom, H6B, and the oxygen atoms, O4 and O4D. The topology can be observed in Fig. 6a and shows the compulsory elements, BCP, bond path and planar interatomic surface. These hydrogen bonds satisfy the eight criteria proposed by Koch and Popelier (only electronic density and laplacian are reported in Table 4).

3.2.3. Cis–trans isomerism As it was explained in Section 3.1, these complexes could show cis–trans isomerism (or 3L–2L following Bailar's nomenclature [63]). As 2L structures are more common than 3L [64–67], then the existence of a 3L structure in complexes [RhCl(VIOH−1)2(PPh3)] · 2H2O (3) and [RhCl(DVIOH− 1)2(PPh3)] · CH3CN (5) deserves further attention. To afford this study, prototypes of both 3L and 2L stereoisomers of [RhCl(VIOH−1)2(PPh3)] (please, see Fig. 7) have been built up. These prototypes mainly involve the atoms of the coordination sphere and have two important advantages. First of all, they concern only the metal–ligand and N = O · · · O = N interactions, avoiding other intramolecular and/or intermolecular contacts that could contribute to stabilize one of the isomers. Secondly, the computational effort is significantly reduced what makes possible to optimize the two prototypes and therefore,

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27

Fig. 6. Topology of other interactions: (a) C-H · · · O; (b) N–O · · · O-N. Symbols and colors as in Fig. 5.

the corresponding wave functions and energies with their different components. Those calculations have included scalar second order relativistic effects. As a first stage, it is necessary to validate our prototype 3L model since it is possible to compare it with the full complex. The property used to achieve this goal has been the topology of the electronic density analyzed within AIM scheme. Table 5 reports the data of the metal–ligand interactions for the full complex (FC), the non-optimized 3L prototype (N3LP) that makes use of FC geometry for the coordination sphere, the optimized 3L prototype (O3LP) and the optimized 2L prototype (O2LP). The comparison of FC with N3LP reveals no significant differences for the values of ρ; the data concerning ∇ is similar but for the contact Rh · · · P where an increase of this parameter is observed for N3LP; however, the pattern Rh · · · P b Rh · · · Cl b Rh · · · O b Rh · · · N is fulfilled for both systems; the ellipticity follows order Rh · · · Cl b Rh · · · P b Rh · · · O b Rh · · · N

Fig. 5. Topology of the interactions metal–ligand: (a) Rh–N and Rh–O; (b) Rh–Cl, Rh–P. Black triangles: atoms. Red squares: bond critical points (BCP). Black lines linking atoms across a BCP: bond paths (BP). Black lines (pseudo)perpendicular to BPs: Interatomic surfaces (IAS).

to obtain a reasonable proposal for the non-existing 2L complex. Both prototypes were optimized by using the basis set aug-cc-pVTZ-PP on the rhodium atom that includes a small relativistic pseudopotential [48]. Later on, all-electron single-point calculations have allowed to obtain

Table 4 Electronic density (ρ), laplacian of the electron density (∇) and ellipticity (ε, anisotropy of the electron density at the BCP) for the coordination sphere and other stabilizing interactions of rhodium full complex (atomic units). Type

Interaction

B1LYP

B3LYP

B3LYP-D3

SOGGA11X

WB97XD

HF

ρ

∇

ε

ρ

∇

ε

ρ

∇

ε

ρ

∇

ε

ρ

∇

ε

ρ

∇

ε

Metal–ligand Rh–Cl Rh–P Rh–O4 Rh–O4D Rh–N5D Rh–N5

0.078 0.106 0.089 0.087 0.134 0.134

0.172 0.087 0.432 0.426 0.418 0.417

0.019 0.022 0.054 0.038 0.101 0.066

0.078 0.106 0.089 0.088 0.134 0.134

0.171 0.086 0.430 0.424 0.412 0.411

0.017 0.024 0.041 0.026 0.096 0.063

0.078 0.106 0.089 0.088 0.134 0.134

0.171 0.086 0.430 0.424 0.412 0.411

0.017 0.024 0.041 0.026 0.096 0.063

0.078 0.107 0.088 0.086 0.134 0.134

0.178 0.086 0.443 0.437 0.428 0.425

0.023 0.018 0.076 0.058 0.122 0.080

0.078 0.105 0.088 0.087 0.134 0.134

0.175 0.097 0.440 0.434 0.418 0.416

0.024 0.017 0.051 0.034 0.102 0.066

0.077 0.114 0.084 0.083 0.129 0.129

0.205 0.092 0.467 0.461 0.534 0.527

0.047 0.018 0.372 0.360 0.247 0.169

Other N–O5D · · · O5–N interactions C = O6D · · · O5D–N C = O6 · · · O5–N O4 · · · H6B O4D · · · H6B

0.011 0.014 0.013 0.012 0.009

0.037 6.408 3.146 0.051 0.037

0.032 0.362 0.549 0.162 0.085

0.011 0.014 0.013 0.012 0.009

0.036 6.354 3.166 0.050 0.037

0.024 0.392 0.598 0.158 0.079

0.011 0.014 0.013 0.012 0.009

0.036 6.354 3.166 0.050 0.037

0.024 0.392 0.598 0.158 0.079

0.012 0.014 0.013 0.012 0.009

0.038 5.785 2.090 0.051 0.037

0.072 0.283 0.412 0.192 0.102

0.012 0.014 0.013 0.012 0.087

0.037 4.609 1.500 0.440 0.434

0.037 0.374 0.558 0.171 0.034

0.011 0.013 0.012 0.012 0.008

0.041 7.085 2.899 0.054 0.039

0.018 0.155 0.256 0.201 0.154

28

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Fig. 7. Prototypes of [RhCl(VIOH−1)2(PPh3)] built up to study the 3L–2L isomerism.

for FC and N3LP but for some exception. These results show that the chemical environment of the coordination sphere does not influence its electronic density if the experimental structure of FC is preserved. As for the comparison between N3LP and O3LP, the previously commented patterns of behavior for ρ, ∇ and ε are equivalent for both systems irrespective of the density functional with the minor exception of the electronic density for the contact Rh · · · Cl that is increased in the optimized prototype and exhibits a value close to that of the Rh · · · O interactions. Thus, it can be concluded that the proposed optimized 3L prototype is an acceptable model to simulate the coordination sphere of the target complex. As expected, the comparison of the topology of O3LP and O2LP reveals no remarkable differences for the contacts Rh · · · Cl and Rh · · · P since they are not involved in the 3L–2L interchange. The main differences are observed for the Rh · · · O and Rh · · · N interactions (Table 5), being the electronic density of the former increased in O2LP respect to O3LP, and the corresponding of the later decreased. These modifications of the electronic density are associated to a logical lengthening/shortening of the Rh · · · N/Rh · · · O distances (~0.03–0.06 Å) in O2LP respect to O3LP due to the decrease/increase of the covalent contribution of the bonding. Finally, once the proposed prototypes were tested, the relative stability of the 3L–2L isomers and its causes were analyzed by using the simple energy partition scheme that separates the energy in both components kinetic (KE) and potential (PE), being the potential term split in nuclear-nuclear repulsion (NN), electron–electron repulsion (EE) and nucleus–electron attraction (EN) (Eq. (1)). E ¼ KE þ PE ¼ KE þ NN þ EE þ EN

ð1Þ

Table 6 reports the relative values in Hartrees for the components of the previous equation along with the final value of energy. Relativistic as well as non-relativistic all electron data are supported. 3L prototype has been chosen as reference, then negative relative values for NN, EE and KE mean this components have higher absolute values for O3LP.

Likewise, positive relative values for EN, PE and E have the same interpretation. Focusing on the relativistic data, it is observed that O3LP is much stable than O2LP. Therefore, due to the structural motifs involved in the prototypes, the over stabilization of the cis (3L) isomer respect to the trans (2L) is motivated by the metal–ligand interactions along with the N = O · · · O = N one. The repulsion terms NN and EE are higher for O3LP than for O2LP. The EN contribution is higher for O3LP but it is not able to compensate both repulsion components, what makes PE more favorable to O2LP than to O3LP. Thus, a conclusion arises, the N = O · · · O = N interaction, despite it stabilizes the 3L structure, cannot account for the higher stability of the O3LP structure, as far as PE is concerned. So the main feature that contributes to that stability is the electronic kinetic energy, which is lower for the electrons of the O3LP than for the O2LP ones. The non-relativistic data show an opposite behavior for the PE and KE terms. Thus, despite the NN and EE contributions exhibit similar values to the relativistic data, the EN attraction is much more favorable for O3LP than for O2LP but the opposite behavior of KE over compensates PE and provides higher stability for O2LP respect to O3LP. This feature reveals the great importance of the relativistic effects in the target systems. 3.3. IR spectra and NMR spectroscopy IR spectroscopy has been used in order to explore the deprotonation and the binding mode of the 5-nitrosopyrimidines derivatives in complexes. The complexes show intense ν(C = O) absorption bands over 1700–1600 cm−1. The carbonyl stretching frequencies could decrease on coordination of O4 to metallic center and/or deprotonation in N6 amino group in 6-amino-5-nitrosopyrimidine ligands. In the latter case, there are also significant changes in the ν(N–H) bands with the appearance of a very sharp and strong band, at around 3300–3400 cm− 1 [65,73]. In general, ν(N = O) nitroso bands are shifted to lower wavenumber. Complexes with violuric acids undergo a tautomeric change from the oxime metal-free form to the coordinated-nitroso one, in both deprotonated and neutral forms [56, 68]. In complexes IrCl(CO)L(PPh3)2, the carbonyl stretching vibration is shifted to a lower wavenumber (between 20–80 cm− 1) whereas ν(N = O) remains almost unchanged. As the structure is unknown, no fairly sure statement about the type of interaction between the pyrimidine ligands and the metal fragment can be suggested. For RhCl(CO)L and IrCl(CO)L(PPh3)2 compounds, a band assignable to ν(C ≡ O) around 2050–2100 cm− 1 indicates a terminal carbonyl group [74,75]. Complexes RhCl(LH− 1)2(PPh3)2, [Rh(DVIOH− 1)2(PPh3)2] and IrCl(CO) L(PPh3)2 show bands indicative of the presence of PPh3 (3050, 1435, 1090 and 700 cm−1) [76]. The ν(M-Cl) for Rh(III) complexes is observed around 315 cm− 1, whereas for Rh(I) compounds appears over 280 cm−1. The MX vibrations are shifted to higher frequencies as

Table 5 Electronic density (ρ), laplacian of the electronic density (∇) and ellipticity (ε, anisotropy of the electron density at the BCP) for the full complex (FC), non-optimized 3L prototype (N3LP), optimized 3L prototype (O3LP) and optimized 2L prototype (O2LP). DFT

SOGGA11X

B3LYP

Atomic units.

Interaction

Rh–Cl Rh–P Rh–O4 Rh–O4D Rh–N5D Rh–N5 Rh–Cl Rh–P Rh–O4 Rh–O4D Rh–N5D Rh–N5

FC

N3LP

O3LP

O2LP

ρ

∇

ε

ρ

∇

ε

ρ

∇

ε

ρ

∇

ε

0.078 0.107 0.088 0.086 0.134 0.134 0.078 0.106 0.089 0.088 0.134 0.134

0.178 0.086 0.443 0.437 0.428 0.425 0.171 0.086 0.430 0.424 0.412 0.411

0.023 0.018 0.076 0.058 0.122 0.080 0.017 0.024 0.041 0.026 0.096 0.063

0.079 0.102 0.089 0.088 0.134 0.134 0.078 0.101 0.091 0.090 0.134 0.134

0.169 0.121 0.433 0.428 0.422 0.419 0.162 0.117 0.420 0.414 0.408 0.405

0.009 0.013 0.073 0.073 0.111 0.076 0.001 0.008 0.045 0.048 0.084 0.058

0.091 0.099 0.089 0.089 0.130 0.130 0.085 0.097 0.087 0.087 0.125 0.125

0.195 0.145 0.427 0.426 0.402 0.402 0.174 0.137 0.388 0.388 0.371 0.371

0.016 0.007 0.063 0.064 0.109 0.109 0.010 0.022 0.053 0.053 0.091 0.091

0.091 0.099 0.100 0.101 0.120 0.119 0.085 0.097 0.097 0.100 0.115 0.114

0.193 0.144 0.475 0.468 0.384 0.398 0.172 0.136 0.435 0.437 0.352 0.370

0.027 0.021 0.084 0.096 0.105 0.094 0.020 0.037 0.077 0.072 0.097 0.077

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Table 6 Relative values (Hartrees) of the contributions to the energy of the complex (E). Type

DFT

NN

EE

EN

PE

KE

E

Relativistic

B1LYP B3LYP B3LYP-D3 SOGGA11X WB97XD HF B1LYP B3LYP B3LYP-D3 SOGGA11X WB97XD HF

−4.453 −4.410 −4.880 −5.395 −5.510 −3.473 −4.453 −4.410 −4.880 −5.395 −5.510 −19.835

−5.263 −5.228 −5.701 −6.054 −6.271 −4.090 −5.001 −4.954 −5.437 −5.967 −6.096 −20.237

6.933 6.904 7.849 8.503 8.997 4.355 14.314 14.160 15.232 16.645 16.863 42.743

−2.782 −2.734 −2.733 −2.946 −2.784 −3.208 4.860 4.795 4.915 5.283 5.257 2.671

5.389 5.342 5.340 5.553 5.387 5.842 −5.041 −4.973 −5.097 −5.485 −5.457 −2.770

2.607 2.607 2.608 2.606 2.603 2.634 −0.181 −0.177 −0.182 −0.202 −0.199 −0.099

Non-relativistic

NN: nuclear repulsion. EE: electronic repulsion. EN: nucleus–electron attraction. PE: potential energy. KE: kinetic energy.

the oxidation state of the metal is higher [76]. Carbonyl complexes exhibit a ν(M-CO) band at around 410 cm− 1 (RhCl(CO)L complexes) and 436 cm−1 (IrCl(CO)L(PPh3)2 compounds). Most relevant 1H and 13C assignments from NMR spectra of the title complexes are given in Table 7. Even after long acquisition times, it was not possible to get information from 1H and 13C-NMR solution spectra (DMSO-d6) in RhCl(CO)L and IrCl(CO)L(PPh3)2 complexes. The 1 H-NMR spectra in complexes derived from [RhCl(CO)(PPh3)2] indicate the deprotonation of the ligands; the signal at 14 ppm assigned to hydroxyimine hydrogen of the free violuric acids [57] does not appear. Free 6-amino-5-nitrosouracil derivatives show two signals at around 12 and 10 ppm due to a strong intramolecular H-bond between the N6 amino group and the oxygen atom from the nitroso group whereas in the monodeprotonated form (complexes 1 and 2) only a signal over 9.60 ppm is detected. The 13C spectra of rhodium and iridium complexes in solid state with neutral 6-amino-5-nitrosouracil derivatives show a downfield shift of the signal of the C4 atom (ca. 17 ppm) whereas the C5 signal is slightly upfield shifted as consequence of the charge redistribution after coordination process. If these ligands act in deprotonated form, changes in C6 signal are observed (6-amino deprotonation and coordination to metallic center). Moreover, the coordination and change to the nitroso-enolato tautomeric form in complexes with violuric acids induce a significant deshielding of C4 and C5 signals (around 17 and 3 ppm, respectively). In conclusion, octahedral structures for rhodium(III) complexes (1–5) and [Rh(DVIOH−1)2(PPh3)2] (6) may be proposed with the organic ligand acting in a bidentate mode (N5 and N6 atoms in complexes 1 and 2 and O4 and N5 atoms in complexes 3–6); compounds 1, 4 (electrolytes 1:1) and 6 complete their coordination sphere with two PPh3 molecules whereas compounds 2, 3 and 5 (non-electrolyte) with a Cl atom and

one PPh3 molecule. For compounds RhCl(CO)L (7–11) a square-planar structure can be suggested where neutral ligands act in a bidentate mode through O4 and N5 atoms; the non electrolyte nature of these compounds suggest the coordination of Cl atom. Finally, for iridium(I) complexes (12–16), the changes observed in IR and NMR indicates the possible interaction through O4; perhaps, the existence of an adduct between IrClCO(PPh3)2 and the pyrimidine moiety could be suggested although further XRD studies are required to know how the bond between them takes place. 3.4. Luminescence properties Table 8 summarizes the emission data of the title compounds in CH3CN solution at room temperature (10− 5 M). Figures with luminescence data are supplied in Supplementary material. The ligands exhibit an absorption band in the range 220–240 nm whereas only weak emissions were observed for compounds MANU and DVIO which are assigned to π–π* transitions. The precursor compound [RhCl(CO)(PPh3)2] presents two emission bands at 290 and 340 nm which are associated to a phenyl-localized 1ππ* and 3ππ* states, respectively [77] which are also observed in the rhodium violuric derivatives. Compound RhCl(MANUH−1)2(PPh3)2 (1) displays a band assigned to an intraligand π–π* transition which is shifted to a lower energy (40 cm−1) respect to MANU with increase of the fluorescence intensity. The emission spectrum recorded for RhCl(DANUH− 1)2(PPh3) (2) shows two bands at 290 and 390 nm consistent with a phenyl ππ* and an intraligand transition, respectively. In the same way, the photoexcitation of iridium complexes derived from [IrCl(CO)(PPh3)2] at 225 nm leads to emissions at 290 and 385 nm (phenyl-localized 1ππ* and 3ππ* transitions). Also, intraligand influence, in the second emission

Table 7 Main features of 1H, 13C and 13C CP/TOSS NMR spectra (δ, ppm) of complexes. Compound

RhCl(MANUH−1)2(PPh3)2 (1) RhCl(DANUH−1)2(PPh3) (2) [RhCl(VIOH−1)2(PPh3)] · 2H2O (3) RhCl(MVIOH−1)2(PPh3)2 (4) [RhCl(DVIOH−1)2(PPh3)] (5) RhCl(CO)(MANU) · 3H2O (7) RhCl(CO)(DANU) · 3H2O (8) RhCl(CO)(2MeOANU) · 3H2O (9) RhCl(CO)(VIO) · 3H2O (10) RhCl(CO)(DVIO) · 2H2O (11) IrCl(CO)(ANU)(PPh3)2 · 2H2O (12) IrCl(CO)(MANU)(PPh3)2 · 2H2O (13) IrCl(CO)(DANU)(PPh3)2 · 2H2O (14) IrCl(CO)(2MeOANU)(PPh3)2 · 2H2O (15) IrCl(CO)(DVIO)(PPh3)2 (16) In brackets, chemical shift 13C-NMR in DMSO-d6 solution. a Not observed.

1

13

–NH2

C2

C4

C5

C6

9.50 9.72

167.18 –a 156.86 148.67 148.37 (148.02) 160.81 159.06 166.57 152.01 148.97 155.64 158.77 156.86 168.70 148.67

176.59 170.82 (170.20) 182.05 (173.64) 173.25 (171.01) 177.19 177.22 180.23 172.64 172.64 –a 178.75 –a 179.62 171.43

–a –a 138.05 141.39 138.96 134.71 139.81 136.23 138.36 137.44 –a –a –a –a –a

149.58 148.76 (147.40) 176.28 152.01 –a 151.10 151.19 154.74 162.02 159.90 –a 150.80 148.97 155.65 155.65

H-NMR

–a –a –a –a

C-NMR

30

N.A. Illán-Cabeza et al. / Journal of Inorganic Biochemistry 143 (2015) 20–33

Table 8 Luminescent properties of title compounds. Compound

λmax excitation (nm)

λmax emission (nm)

Ligands ANU MANU DANU 2MeOANU VIO MVIO DVIO

223 241 243 241 224 225 225

295, 340 350 No emission No emission 290, 340 290, 340 290, 340

Precursors [RhCl(CO)(PPh3)2] [RhCl(CO)2]2 [IrCl(CO)(PPh3)2]

226 229 226

290, 340 390 290,385

Complexes RhCl(MANUH−1)2(PPh3)2 RhCl(DANUH−1)2(PPh3) [RhCl(VIOH−1)2(PPh3)] · 2H2O RhCl(MVIOH−1)2(PPh3)2 [RhCl(DVIOH−1)2(PPh3)] RhCl(CO)(MANU) · 3H2O RhCl(CO)(DANU) · 3H2O RhCl(CO)(2MeOANU) · 3H2O RhCl(CO)(VIO) · 3H2O RhCl(CO)(DVIO) · 2H2O IrCl(CO)(ANU)(PPh3)2 · 2H2O IrCl(CO)(MANU)(PPh3)2 · 2H2O IrCl(CO)(DANU)(PPh3)2 · 2H2O IrCl(CO)(2MeOANU)(PPh3)2 · 2H2O IrCl(CO)(DVIO)(PPh3)2

240 224 225 224 226 225 225 224 225 227 225 225 225 225 226

390 290, 390 290, 340 290, 335 290, 340 390 390 385 340, 390 300, 390 290, 385 290, 385 290, 385 290, 385 290, 385

of the organic ligands MANU and DVIO can be suggested in compounds IrCl(CO)(MANU)(PPh3)2 · 2H2O (13) and IrCl(CO)(DVIO)(PPh3)2 (16). Finally, compounds [RhCl(CO)(L)] display an emission band centered at 390 nm derived from a M → π*(CO) charge-transfer transition [78]. 3.5. Biological studies Data with renin–angiotensin system-regulating specific aminopeptidase activities in NB69 and U373-MG tumoral cell lines after the treatment with the title rhodium and iridium compounds are given in Tables 9, 10 and 11. To visualize them, bar plots are available in the Supplementary Material. Table 9 shows the effects of complexes 1–5 on APA, APN and IRAP activities in NB69 and U373 cells. Whereas RhCl(MANUH−1)2(PPh3)2 and [RhCl(VIOH−1)2(PPh3)] · 2H2O did not modify APA activity in either NB69 or U373 cells, RhCl(DANUH− 1)2 (PPh3) showed and inhibitory effect in both cell types, although U373 cells were more sensitive than NB69 cells. On the contrary, RhCl(MVIOH−1)2(PPh3)2 did not show any effect in U373 cells, but inhibited APA activity in NB69 cells. Finally, [RhCl(DVIOH−1)2(PPh3)] did not show any effect on NB69 cells but significantly increased APA activity in U373 cells. However, RhCl(MANUH−1)2(PPh3)2 promoted a dose-dependent increase in APN activity in both cell types. RhCl(DANUH−1)2(PPh3) showed a biphasic effect on U373 cells, depending on the concentration used, or a concentration-dependent increase in NB69 cells. This biphasic effect on U373 cells was also shown with RhCl(MVIOH−1)2(PPh3)2, whereas NB69 cells were not affected. Similarly, [RhCl(VIOH−1)2(PPh3)] · 2H2O did not modify APN in NB69 cells but significantly increased in a concentration-dependent manner APN activity in U373 cells. Finally, [RhCl(DVIOH−1)2(PPh3)] did not affect APN activity in either NB69 or U373 cells. Regarding IRAP activity, although [RhCl(DVIOH−1)2(PPh3)] · CH3CN did not show any effect in this enzyme activity either in U373 or NB69 cells, RhCl(MANUH−1)2 (PPh3)2, RhCl(DANUH− 1)2(PPh3), [RhCl(VIOH− 1)2(PPh3)] · 2H2O and RhCl(MVIOH−1)2(PPh3)2 showed a significant concentrationdependent inhibitory effect in IRAP activity in a similar degree on both cell types.

Table 9 Renin–angiotensin system-regulating specific aminopeptidase activities in human neuroblastoma and glioma cell lines NB69 and U373-MG after the treatment with complexes 1–5. Dose (μM)

Aminopeptidase A (APA) NB69

Aminopeptidase N (APN)

U373-MG NB69

Insulin-regulated aminopeptidase (IRAP)

U373-MG

NB69

20 28 18 32 19 48

1970 2553 2835 2834 2803 2806

± ± ± ± ± ±

86 38 46 86 90 69

747 746 750 646 561 467

± ± ± ± ± ±

16 7 6 1 7 2

2790 2798 2756 2219 1976 1771

± ± ± ± ± ±

59 79 41 63 58 30

± ± ± ± ± ±

15 11 16 13 12 17

1934 2337 2741 2443 2302 1971

± ± ± ± ± ±

22 36 39 50 84 56

736 740 747 628 551 490

± ± ± ± ± ±

29 12 15 9 3 11

3096 2854 2612 2244 1911 1652

± ± ± ± ± ±

68 47 26 67 26 74

[RhCl(VIOH−1)2(PPh3)] · 2H2O (3) Control 141 ± 3 679 ± 16 615 1 142 ± 3 679 ± 19 641 2 144 ± 5 683 ± 17 663 3 151 ± 3 693 ± 16 696 4 148 ± 5 700 ± 19 696 5 142 ± 1 682 ± 11 681

± ± ± ± ± ±

15 34 15 18 19 18

1595 2208 2632 2544 2586 2583

± ± ± ± ± ±

36 30 28 98 60 59

778 767 750 702 671 536

± ± ± ± ± ±

5 10 30 2 17 27

2596 2592 2445 2193 1880 1873

± ± ± ± ± ±

73 26 33 90 67 45

RhCl(MVIOH−1)2(PPh3)2 (4) Control 163 ± 1 651 ± 1 156 ± 1 655 ± 2 153 ± 4 653 ± 3 144 ± 2 652 ± 4 133 ± 4 656 ± 5 128 ± 5 633 ±

17 12 12 13 16 19

641 654 644 651 660 667

± ± ± ± ± ±

17 11 15 18 17 25

1937 2314 2451 2427 2157 1944

± ± ± ± ± ±

39 42 42 77 79 31

730 727 709 624 551 490

± ± ± ± ± ±

25 14 13 6 18 17

3439 3236 2511 2292 1962 1701

± ± ± ± ± ±

66 37 58 40 41 67

[RhCl(DVIOH−1)2(PPh3)] (5) Control 145 ± 5 643 ± 1 145 ± 1 660 ± 2 144 ± 9 675 ± 3 148 ± 5 679 ± 4 157 ± 3 719 ± 5 161 ± 6 757 ±

16 14 13 13 14 18

405 409 408 397 388 393

± ± ± ± ± ±

25 15 13 10 13 12

1540 1520 1561 1671 1647 1666

± ± ± ± ± ±

57 60 26 52 14 31

720 721 728 719 739 724

± ± ± ± ± ±

26 26 6 12 16 24

2648 2677 2606 2613 2664 2660

± ± ± ± ± ±

93 39 81 87 92 81

RhCl(MANUH−1)2(PPh3)2 (1) Control 157 ± 9 701 ± 1 156 ± 1 708 ± 2 155 ± 2 726 ± 3 155 ± 3 715 ± 4 156 ± 3 725 ± 5 147 ± 1 733 ±

13 14 20 13 14 15

395 555 714 662 672 680

± ± ± ± ± ±

RhCl(DANUH−1)2(PPh3) (2) Control 159 ± 1 733 ± 1 155 ± 2 707 ± 2 145 ± 5 693 ± 3 143 ± 3 652 ± 4 146 ± 1 663 ± 5 136 ± 2 669 ±

24 13 13 18 16 16

394 596 625 665 667 660

U373-MG

Values are expressed in picomoles of the corresponding aminoacyl-β-naphthylamide hydrolyzed per minute and per milligram of protein (mean ± SEM; n = 4).

Table 10 shows the effects of complexes 7–11 on APA, APN and IRAP activities in NB69 and U373 cells. None of the complexes modified APA activity in U373 cells, whereas RhCl(CO)(2MeOANU) · 3H2O and RhCl(CO)(DVIO) · 2H2O showed and inhibitory effect in APA activity on NB69 cells, being RhCl(CO)(DVIO) · 2H2O more potent than RhCl(CO)(2MeOANU) · 3H2O. On the contrary, although RhCl(CO)(MANU) · 3H2O did not modify APN activity either in NB69 or U373 cells, RhCl(CO)(DANU) · 3H2O, RhCl(CO)(2MeOANU) · 3H2O and RhCl(CO)(DVIO) · 2H2O showed a biphasic effect on APN activity in U373 cells, depending on the concentration used. This biphasic effect could also be observed for RhCl(CO)(DANU) · 3H2O and, RhCl(CO)(2MeOANU) · 3H2O in NB69 cells, whereas RhCl(CO)(DVIO) · 2H2O showed a concentration-dependent effect on APN activity in NB69 cells. Finally, RhCl(CO)(VIO) · 3H2O also showed a concentrationdependent effect on APN activity in NB69 cells, although did not show any effect on U373 cells. Regarding IRAP activity, all the complexes showed an inhibitory effect of this activity in a concentrationdependent manner in U373 cells. Similarly, RhCl(CO)(MANU) · 3H2O, RhCl(CO)(DANU) · 3H2 O and RhCl(CO)(VIO) · 3H 2 O showed an inhibitory effect on IRAP activity in NB69 cells. However, RhCl(CO)(2MeOANU) · 3H2O and RhCl(CO)(DVIO) · 2H2O did not modify IRAP activity in NB69 cells.

N.A. Illán-Cabeza et al. / Journal of Inorganic Biochemistry 143 (2015) 20–33 Table 10 Renin–angiotensin system-regulating specific aminopeptidase activities in human neuroblastoma and glioma cell lines NB69 and U373-MG after the treatment with complexes 7–11. Dose (μM)

Aminopeptidase A (APA) NB69

Aminopeptidase N (APN)

U373-MG NB69

Insulin-regulated aminopeptidase (IRAP)

U373-MG

NB69

U373-MG

15 19 15 12 16 11

582 590 602 653 646 587

± ± ± ± ± ±

24 18 18 14 27 28

1874 1821 1826 1840 1876 1831

± ± ± ± ± ±

59 34 33 38 52 83

645 648 644 640 583 514

± ± ± ± ± ±

18 39 17 19 22 15

3462 3203 2862 2594 2053 1650

± ± ± ± ± ±

RhCl(CO)(DANU) · 3H2O (8) Control 147 ± 5 752 ± 1 151 ± 1 760 ± 2 145 ± 5 770 ± 3 153 ± 3 760 ± 4 148 ± 3 750 ± 5 168 ± 4 753 ±

11 15 13 10 9 13

330 583 613 500 437 309

± ± ± ± ± ±

18 16 13 23 14 13

1607 2149 2256 1796 1437 1119

± ± ± ± ± ±

29 8 88 99 60 15

737 616 596 438 366 276

± ± ± ± ± ±

5 29 24 8 20 2

2914 2732 2064 1502 1172 967

· 3H2O (9) 685 ± 14 679 ± 12 682 ± 13 689 ± 19 663 ± 17 681 ± 16

355 493 620 723 714 683

± ± ± ± ± ±

15 20 14 29 16 28

2751 2956 3556 3537 3316 3083

± ± ± ± ± ±

36 26 97 75 41 48

639 698 700 701 640 650

± ± ± ± ± ±

24 11 15 5 5 12

RhCl(CO)(2MeOANU) Control 164 ± 2 1 164 ± 1 2 161 ± 1 3 156 ± 4 4 149 ± 3 5 149 ± 3

Table 11 Renin–angiotensin system-regulating specific aminopeptidase activities in human neuroblastoma and glioma cell lines NB69 and U373-MG after the treatment with complexes 12–16. Dose (μM)

· 3H2O (7) 9 685 ± 11 653 ± 5 652 ± 8 678 ± 5 670 ± 7 665 ±

RhCl(CO)(MANU) Control 165 ± 1 163 ± 2 151 ± 3 152 ± 4 143 ± 5 140 ±

31

Aminopeptidase A (APA) NB69

Aminopeptidase N (APN)

U373-MG NB69

Insulin-regulated aminopeptidase (IRAP)

U373-MG

NB69

17 18 16 18 25 29

2041 2491 2897 3248 3236 3208

± ± ± ± ± ±

65 54 10 38 54 23

677 682 668 655 665 659

± ± ± ± ± ±

2 5 3 4 3 2

2355 2349 2165 2101 1948 1772

± ± ± ± ± ±

32 19 10 48 19 31

± ± ± ± ± ±

13 25 14 16 14 29

2405 2436 2468 2482 2485 2495

± ± ± ± ± ±

84 35 19 69 42 34

630 640 640 650 648 631

± ± ± ± ± ±

7 6 13 7 4 4

2953 2810 2571 2147 2020 1616

± ± ± ± ± ±

49 23 27 65 61 59

± ± ± ± ± ±

16 16 23 11 23 11

1932 1988 1912 1945 1916 1927

± ± ± ± ± ±

31 49 94 51 31 40

615 603 592 532 528 446

± ± ± ± ± ±

4 10 16 8 3 14

2355 2343 2059 1826 1619 1457

± ± ± ± ± ±

49 59 38 29 46 79

12 33 79 81 30 20

IrCl(CO)(ANU)(PPh3)2 Control 142 ± 5 1 143 ± 4 2 142 ± 2 3 155 ± 7 4 156 ± 1 5 159 ± 4

· 2H2O (12) 581 ± 12 361 641 ± 14 532 705 ± 14 549 719 ± 10 654 747 ± 11 658 745 ± 12 658

± ± ± ± ± ±

± ± ± ± ± ±

18 17 11 12 16 15

IrCl(CO)(MANU)(PPh3)2 · 2H2O (13) Control 167 ± 2 706 ± 14 360 1 167 ± 6 703 ± 12 485 2 153 ± 1 654 ± 16 601 3 140 ± 4 630 ± 13 666 4 133 ± 1 646 ± 17 664 5 131 ± 6 600 ± 19 675

2618 2480 2336 2138 1892 1747

± ± ± ± ± ±

15 16 15 18 14 13

IrCl(CO)(DANU)(PPh3)2 · 2H2O (14) Control 139 ± 5 642 ± 9 364 1 141 ± 6 643 ± 10 463 2 141 ± 6 650 ± 12 528 3 142 ± 8 651 ± 16 595 4 146 ± 5 653 ± 15 623 5 150 ± 4 659 ± 17 621

U373-MG

· 3H2O (10) ±1 649 ± ±1 659 ± ±1 658 ± ±1 670 ± ±6 660 ± ±4 660 ±

13 12 17 15 17 18

373 496 560 606 601 603

± ± ± ± ± ±

16 11 21 12 23 13

2293 2264 2293 2376 2276 2238

± ± ± ± ± ±

45 31 84 13 35 33

640 643 583 514 457 414

± ± ± ± ± ±

3 16 18 18 5 24

2556 2450 2042 1765 1728 1401

± ± ± ± ± ±

17 40 11 15 18 14

IrCl(CO)(2MeOANU)(PPh3)2 · Control 150 ± 4 669 ± 1 153 ± 1 662 ± 2 154 ± 4 663 ± 3 154 ± 6 664 ± 4 146 ± 1 669 ± 5 140 ± 5 670 ±

2H2O (15) 17 346 ± 11 481 ± 13 614 ± 10 617 ± 19 611 ± 13 615 ±

17 18 23 15 17 17

2317 2359 2343 2363 2355 2326

± ± ± ± ± ±

40 39 19 29 80 95

640 648 653 652 649 641

± ± ± ± ± ±

12 29 15 18 10 18

3125 2844 2627 2209 1964 1708

± ± ± ± ± ±

28 29 22 19 27 18

RhCl(CO)(DVIO) · 2H2O (11) Control 164 ± 3 654 ± 1 161 ± 3 648 ± 2 146 ± 2 647 ± 3 137 ± 2 653 ± 4 137 ± 3 653 ± 5 134 ± 5 657 ±

11 19 16 16 18 13

377 510 594 630 628 620

± ± ± ± ± ±

15 19 13 10 17 17

2482 2741 3150 3209 3053 2679

± ± ± ± ± ±

21 17 80 30 17 22

657 686 688 662 647 550

± ± ± ± ± ±

12 10 12 26 15 15

2860 2834 2404 2126 1823 1579

± ± ± ± ± ±

14 10 32 41 16 39

IrCl(CO)(DVIO)(PPh3)2 (16) Control 140 ± 2 653 ± 1 147 ± 6 650 ± 2 154 ± 5 659 ± 3 150 ± 3 658 ± 4 149 ± 3 653 ± 5 150 ± 6 658 ±

17 17 15 15 10 9

17 27 23 10 15 12

1754 2279 2672 2692 2792 2848

± ± ± ± ± ±

50 45 12 24 19 25

604 608 613 606 600 591

± ± ± ± ± ±

23 15 19 9 23 29

2724 2315 2134 1921 1884 1664

± ± ± ± ± ±

17 14 22 14 12 13

RhCl(CO)(VIO) Control 160 1 149 2 152 3 155 4 155 5 154

334 442 572 595 607 592

± ± ± ± ± ±

Values are expressed in picomoles of the corresponding aminoacyl-β-naphthylamide hydrolyzed per minute and per milligram of protein (mean ± SEM; n = 4).

Values are expressed in picomoles of the corresponding aminoacyl-β-naphthylamide hydrolyzed per minute and per milligram of protein (mean ± SEM; n = 4).

Table 11 shows the effects of complexes 12–16 on APA, APN and IRAP activities in NB69 and U373 cells. Although IrCl(CO)(ANU)(PPh3)2 · 2H2O promoted an increased effect on APA activity in both U373 and NB69 cells, IrCl(CO)(MANU)(PPh 3)2 · 2H2O showed an inhibitory effect in both cell types in a similar degree. On the other hand, IrCl(CO)(DANU)(PPh3)2 · 2H2O, IrCl(CO)(2MeOANU)(PPh3)2 · 2H2O and IrCl(CO)(DVIO)(PPh3)2 did not modify APA activity either in NB69 or U373 cells. Regarding APN activity, IrCl(CO)(MANU)(PPh3)2 · 2H2O, IrCl(CO)(DANU)(PPh3)2 · 2H2O and IrCl(CO)(2MeOANU)(PPh3)2 · 2H2O did not show any effect on this activity in U373 cells, but significantly increased APN activity in a concentration-dependent manner in NB69 cells. Similarly, IrCl(CO)(ANU)(PPh3)2 · 2H2O and IrCl(CO)(DVIO)(PPh3)2 significantly increased, in a concentrationdependent manner, APN activity in both U373 and NB69 cells. Regarding IRAP activity, only IrCl(CO)(DANU)(PPh3)2 · 2H2O showed an inhibitory effect on this activity in NB69 cells, whereas the other complexes did not modify IRAP activity in this cell type. On the contrary, all complexes of this group showed a potent significant concentration-dependent effect on IRAP activity in U373 cells. At the concentrations used, most of the complexes show important and differential effects on RAS (renin–angiotensin system)-regulating proteolytic regulatory enzymes, which support the idea that their effects

on cell growth/proliferation could be more related to the paracrine functions of the peptides which regulates than a direct citotoxic effect. In this sense, our results show that rhodium complexes modify RAS-regulating enzyme activities in several ways. Thus, RhCl(MANUH−1)2(PPh3)2, [RhCl(VIOH − 1 ) 2 (PPh 3 )] · 2H 2 O, RhCl(CO)(DANU) · 3H 2 O and RhCl(CO)(VIO) · 3H2O mainly increase APN activity and decrease IRAP activity, showing no effects on APA activity. RhCl(DANUH−1)2(PPh3), RhCl(MVIOH − 1 ) 2 (PPh 3 ) 2 , RhCl(CO)(2MeOANU) · 3H 2 O and RhCl(CO)(DVIO) · 2H2O inhibit APA and IRAP activities, showing a biphasic effect on APN activity depending on the concentration used. Finally, only [RhCl(DVIOH− 1)2(PPh3)] acts increasing exclusively APA activity and RhCl(CO)(MANU) · 3H2O acts decreasing exclusively IRAP activity. In most cases, the potency and sensibility of the different complexes vary depending on the cell type. In RAS cascade, the first group of complexes would increase the conversion of AngIII to AngIV, whereas the catabolism of AngIV would be diminished, which would enhance the functions of AngIV. In the second group of complexes, AngII is preponderant, whereas AngIII/AngIV have a pivotal role depending on the concentration used due to their biphasic effect. Also, [RhCl(DVIOH− 1)2(PPh3)] · CH3CN would act promoting the role of AngIII and RhCl(CO)(MANU) · 3H2O acts enhancing the functions of AngIV.

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Regarding iridium complexes, a similar pattern of effects can be found. Thus, IrCl(CO)(ANU)(PPh3)2 · 2H2O increases APA and APN activities and decreases IRAP activity. IrCl(CO)(MANU)(PPh3)2 · 2H2O inhibit APA and IRAP activities and increases APN activity. Finally, IrCl(CO)(DANU)(PPh3)2 · 2H2O, IrCl(CO)(2MeOANU)(PPh3)2 · 2H2O and IrCl(CO)(DVIO)(PPh3)2 increase APN and decrease IRAP activity, showing no effects on APA activity. As stated before, the potency and sensibility of the different iridium complexes also vary depending on the cell type. Also considering the RAS cascade, IrCl(CO)(ANU)(PPh3)2 · 2H2O would enhance the functions of AngIV, IrCl(CO)(MANU)(PPh3)2 · 2H2O enhances the functions of AngII and AngIV and the rest of complexes would increase the conversion of AngIII to AngIV, whereas the catabolism of AngIV would be diminished, which would enhance the functions of AngIV. It indicates that these new bioactive molecules act through specific mechanisms of cell growth in which the RAS is involved, and also may act through the angiogenic and invasive processes mediated by angiotensin peptides. It has been shown that some neurons or glial cells in normal tissue express several of the components of the RAS and their receptors, although the participative mechanisms are unclear [79–82]. However, the actual knowledge about the role of RAS-regulating proteolytic regulatory enzymes in tumor tissue of brain origin indicates that APA was upregulated and enzymatically active in blood vessels of human tumors, but was not detected in normal blood vessels [83]. Using an animal model of experimental glioma, the existence of a time-dependent significant decrease in the specific activity of APN, and a concomitant increase in the specific activity of APA with tumor growth have been described [35]. In addition, in the transplacental N-ethyl-nitrosourea (ENU)induced model of glioma tumors, an increase in APA activity and a decrease in APN and IRAP activities were also found (unpublished results). This suggests a predominant action of AngIII in tumor cells proliferation, because in RAS cascade, APA degrades AngII to form AngIII, but also angiotensinogen to form des-Asp-AngI, which in turn is converted also to AngIII by ACE. As APN is the enzyme responsible for the degradation of AngIII to form AngIV, its inhibition further promotes AngIII accumulation [84]. Furthermore, APA expression was found on dysplastic cells and was increased in precancerous lesions and invasive cervical cancer [85]. These data and others about expression of APA on prostate cancer cells [86], suggest that APA may play a regulatory role in neoplastic transformation and disease progression in various types of cancer. By other hand, others authors have reported that several kinds of carcinomas including those of colon, kidney, breast and lung, exhibited little expression of APN [87,88]. The importance of the multiple effects of some of the tested complexes on several RAS-regulating proteolytic regulatory enzymes is that these changes on the bioavailability of the different angiotensins could modify the ratio between them, which could be reflected in changes on cell growth and angiogenesis [86]. In fact, it is known that gliomas are accompanied by extensive angiogenesis, essential for tumor growth and invasiveness [89]. We can conclude that the analysis in vitro of the antiproliferative efficacy and the effects on RAS-regulating APA, APN and IRAP specific activities of the new rhodium and iridium complexes on the human neuroblastoma and glioma cell lines NB69 and U373-MG allow us to propose some of them (mainly those which antagonizes the activities of the RAS-regulating enzymes in tumor tissue) as complexes with antitumor activity against both brain tumor types, acting through the paracrine regulating system mediated by tissue RAS rather than exerting a direct cytotoxic effect on tumor cells. Acknowledgments Financial support of “Plan de Apoyo a la Investigación, al Desarrollo Tecnológico y a la Innovación” of the University of Jaén and PAIDI Junta de Andalucía (FQM273, FQM337 and BIO296) is acknowledged. Also, thanks are due to Centro de Servicios de Informática y Redes de Comunicaciones (CSIRC) of University of Granada for computational resources.

Appendix A. Supplementary data Full NMR and IR data, tables with Mulliken charges and molecular orbital contributions for [RhCl(VIOH-1)2(PPh3)], figures with luminescence data and figures with renin–angiotensin system-regulating specific aminopeptidase activities in human neuroblastoma and glioma cell lines NB69 and U373-MG after the treatment with the reported compounds are available. All atomic coordinates, anisotropic displacement parameters for non-hydrogen atoms and all interatomic distances and bond angles for compounds [RhCl(VIOH- 1)2(PPh3)] · 2H2O, [RhCl(DVIOH- 1)2(PPh3)] · CH3CN and [Rh (DVIOH-1)2(PPh3)2] have been deposited in the Cambridge Crystallographic Data Centre (CCDC numbers 929237, 929236 and 929235). These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html; email: [email protected]. Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j. jinorgbio.2014.11.004. These data include MOL files and InChiKeys of the most important compounds described in this article. References [1] The World Health Organization, website http://www.who.int/mediacentre/ factsheets/fs297/en/. [2] R.A. Alderden, M.D. Hall, T.W. Hambley, J. Chem. Educ. 83 (2006) 728–734. [3] L. Kelland, Nat. Rev. Cancer 7 (2007) 573–584. [4] J. Reedijk, Eur. J. Inorg. Chem. (2009) 1303–1312. [5] Z.H. Siddik, Oncogene 22 (2003) 7265–7279. [6] Y. Jung, S.J. Lippard, Chem. Rev. 107 (2007) 1387–1407. [7] M. Galanski, B.K. Keppler, Anti-Cancer Agents Med. Chem. 7 (2007) 55–73. [8] P.C.A. Bruijnincx, P.J. Sadler, Curr. Opin. Chem. Biol. 12 (2008) 197–206. [9] M.A. Jakupec, M. Galanski, V.B. Arion, C.G. Hartinger, B.K. Keppler, Dalton Trans. (2008) 183–194. [10] M.J. Clarke, Coord. Chem. Rev. 232 (2002) 69–93. [11] Y.K. Yan, M. Melchart, A. Habtemariam, P.J. Sadler, Chem. Commun. (2005) 4764–4776. [12] A. Levina, A. Mitra, P.A. Lay, Metallomics 1 (2009) 458–470. [13] G. Süss-Fink, Dalton Trans. 39 (2010) 1673–1688. [14] I. Haiduc, C. Silvestru, Cobalt, rhodium and iridium, Organometallics in Cancer Chemotherapy, CRC Press, Boca Raton, Florida, 1990, pp. 169–207. [15] N. Katsaros, A. Anagnostopoulou, Crit. Rev. Oncol. 42 (2002) 297–308. [16] Y. Geldmacher, M. Oleszak, W.S. Sheldrick, Inorg. Chim. Acta 393 (2012) 84–102. [17] A. Taylor, N. Carmichael, Cancer Stud. 2 (1953) 36–79. [18] P. Qu, H. He, X. Liu, Prog. Chem. 18 (2006) 1646–1651. [19] R.G. Hughes, J.L. Bear, A.P. Kimball, Proc. Am. Assoc. Cancer Res. 13 (1972) 120–125. [20] K.D. Karlin, R.W. Cruse, Y. Gultneh, A. Farooq, J.C. Hayes, J. Zubieta, J. Am. Chem. Soc. 109 (1987) 2668–2679. [21] N. Kitajima, T. Koda, S. Hashimoto, T. Kitagawa, Y. Morooka, J. Am. Chem. Soc. 113 (1991) 5664–5671. [22] S.W. Lai, T.C. Cheung, M.C.W. Chan, K.K. Cheung, S.M. Peng, C.M. Che, Inorg. Chem. 39 (2000) 255–262. [23] D.T. Hurst, An Introduction to the Chemistry and Biochemistry of Pyrimidines, Purines and Pteridines, Wiley, Chichester, 1980. [24] D.J. Brown, The Pyrimidines, Interscience Publishers, New York, 1994. [25] I. Votruba, A. Holy, K. Jost, FEBS Lett. 22 (1972) 287–288. [26] D. Ganten, G. Speck, Biochem. Pharmacol. 27 (1978) 2379–2389. [27] M. Bader, J. Peters, O. Baltatu, D.N. Müller, F.C. Luft, D. Ganten, J. Mol. Med. 79 (2001) 76–102. [28] O.C. Baltatu, L.A. Campos, M. Bader, Peptides 32 (2011) 1083–1086. [29] D.M. Shah, Curr. Hypertens. Rep. 8 (2005) 144–152. [30] W.R. Welches, K.B. Brosnihan, C.M. Ferrario, Life Sci. 52 (1993) 1461–1480. [31] H. Andersson, M. Hallberg, Int. J. Hypertens. (2012) (2012) (Article ID 789671). [32] J.M. Martínez-Martos, M. del Pilar Carrera-González, B. Dueñas, M.D. Mayas, M.J. García, M.J. Ramírez-Expósito, Breast 20 (2011) 444–447. [33] R. Ardaillou, D. Chansel, Kidney Int. 52 (1997) 1458–1468. [34] M.J. Ramírez-Expósito, M. del Pilar Carrera-González, M.D. Mayas, B. Dueñas, J. Martínez-Ferrol, J.M. Martínez-Martos, Maturitas 72 (2012) 79–83. [35] M.D. Mayas, M.J. Ramírez-Expósito, M.P. Carrera, M. Cobo, J.M. Martínez-Martos, Anticancer Res. 32 (2012) 3675–3682. [36] G.M. Sheldrick, SADABS 2.10, 2003. [37] G.M. Sheldrick, SHELXL-97, University of Göttingen, Germany, 1997. [38] L.J. Farrugia, WINGX 1.70.01, University of Glasgow, Scotland, 2005. [39] A.L. Spek, PLATON A Multipurpose Crystallographic Tool, Utrecht University, Utrecht, the Netherlands, 2003. [40] Cambridge Crystallographic Data Centre MERCURY (2002). Cambridge, England. [41] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K.

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