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Modulating Anti-Cancer Potential by Modifying the Structural Properties of a Family of Zinc Metal-Organic Chains Based on 4-Nitro-1H-pyrazole Belen Fernandez, Ignacio Fernandez, Fernando J. Reyes-Zurita, Javier Cepeda, Marta Medina Odonnell, Eva E. Rufino-Palomares, Álvaro Raya-Barón, Santiago Gómez Ruiz, Amalia Pérez-Jimenez, Jose Antonio Lupiañez, and Antonio Rodriguez-Dieguez Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b01443 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 2017

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Crystal Growth & Design

Modulating Anti-Cancer Potential by Modifying the Structural Properties of a Family of Zinc MetalOrganic Chains Based on 4-Nitro-1H-pyrazole Belén Fernández,a Ignacio Fernández,b Fernando J. Reyes-Zurita,c,* Javier Cepeda,d Marta Medina Odonnell,c Eva E. Rufino-Palomares,c Álvaro Raya-Barón,b Santiago Gómez-Ruiz,e Amalia Pérez-Jiménez,c José Antonio Lupiáñezc and Antonio Rodríguez-Diéguez*,a a

Departamento Dept. of Inorganic Chemistry, C/ Severo Ochoa s/n, University of Granada,

18071, Granada, Spain. b

Department of Chemistry and Physics, Research Centre for Agricultural and Food

Biotechnology (BITAL), University of Almería, Ctra. Sacramento s/n, 04120 Almería, Spain c

Dept. of Biochemistry, Severo Ochoa s/n, University of Granada, 18071, Granada, Spain.

d

Department of Applied Chemistry, University of The Basque Country (UPV/EHU), 20018 San

Sebastián, Spain. e

Department of Biology and Geology, Physics and Inorganic Chemistry, Rey Juan Carlos

University, c/ Tulipán s/n, 28933, Móstoles (Madrid),Spain.

KEYWORDS: 4-Nitro-1H-pyrazole, coordination polymers, zinc, anti-cancer , solution NMR studies.

ACS Paragon Plus Environment

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Abstract

We have synthesized a novel family of metal-organic chains based on 4-nitro-1H-pyrazole linker and zinc as metal centre. We report the formation of these coordination polymers using hydrothermal routes with water as solvent. These materials display one-dimensional structures with major structural modifications from minimal synthetic variations. What is more interesting, we have carried out anti-tumor measurements and we have related these properties with the structural networks. To the best of our knowledge, these materials constitute the first examples of polymeric structures for 4-NO2-pz ligands. NMR and MS spectrometry were used to verify the characterization of the metal complexes in solution to corroborate that these materials are present in the biological assays.

Introduction

Since the discovery of coordination polymers (CPs), these materials have been intensively studied due to their potential applications in areas such as luminescence,1,2 gas adsorption,3,4 optical storage,5 magnetism6 and biology as drug-delivery systems7 or cytotoxic agents.8 Within these systems we could consider metal-organic chains (MOCs) as 1D-coordination polymers in which the combination of metal centres and organic ligands provides fantastic possibilities for the construction of materials with various structures and functionalities.9,10 In this sense, we and others have synthesized a large variety of coordination compounds based on triazolopyrimidine ligands with interesting biological properties.11,12 In particular, the study of these coordination


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compounds as anti-tumor materials has not been enough explored so far, though these systems with cheap and non-toxic metal centers can be essential in many biological processes. For these reasons, we decided to synthesize new MOCs by using zinc as metal ion in order to study some of the anti-cancer properties or activity exhibited by these materials. On another level, we selected 4-nitro-1H-pyrazole (µ-4-NO2-Hpz)13,14 as ligand, a scarcely explored molecule for CPs construction, given its potential capacity to fulfil the above mentioned structural and biological roles. Regarding its coordinating ability in metal-organic architectures, pyrazole based and related N-donor aromatic ligands such as tetrazole constitute a well established class of connectors that, combined with metal ions that render tetrahedral geometries, give rise to CPs with high thermal and chemical stability.15 Especially, those frameworks with zeolitic topologies are to be highlighted.16,17 The relative ortho disposition of the donor nitrogen atoms in the pyrazole ring gives this ditopic ligand the opportunity to show two possible coordination modes (Scheme I) according to its neutral or anionic nature. Despite its preference to act as a ditopic bridging linker by adopting coordination mode b, it is worth noticing the lack of polymeric architectures for this ligand among the scarce structural diversity governed by isolated complexes and metallacycles.18,19 As a consequence, we decided to exploit this interesting ligand as a potential linker to construct novel zinc based MOCs. The biological interest for complexes containing nitro substituted ligands, such as the deeply explored nitroimidazole based complexes,20,21 origins at their proven ability to mark hypoxic (oxygen deficient) cell regions, which are characteristic of solid tumors.22,23 In this sense, the slow metabolism of the nitro compounds and their ability to diffuse through nonvascular cell masses allow deep penetration, making them effective tools for marking the hypoxic cells. Moreover, these kinds of ligands may act as radiosensitizers owing to the electron affinity of the


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nitro group by dioxygen molecule, thus allowing drugs requiring dioxygen to be more effective.24 Bearing in mind the interest of our groups in the study of the cytotoxic anticancer properties of metal-organic chains and functionalized nanomaterials,25,26 we decided to study the cytotoxic properties of these novels Zn-MOCs which were prepared from 4-NO2-pz as ligand to establish a connection between structural properties and anti-cancer potential. Given that currently major commercially anticancer drugs exhibit low therapeutics efficacy due their inability to be accumulate preferentially in tumor tissues,27,28 and therefore are administrated at high doses in order to compensate for non-ideal biodistribution, which can lead to serious adverse effects,29,30 it is of great interest to search new drugs that can fight this disease effectively with minimal side effects. In these sense, the use of coordination polymers as promising chemotherapeutics anticancer agents has been recently reported.13,31,32 These new compounds exhibit several benefficial effects that improve their use as anticancer drugs, as for example have a high half life to allow their preferential tumor accumulation, due to their highest vascular permeability that permit their retention on tumor tissues.16 In this sense, we explored Zn-pyrazole complexes in order to enhance particle stability and reduce toxicity. Our main objective has been to search new compounds with high cytotoxicity and anti-tumor properties with biocompatible metal ions providing minimal side effects. In this article we studied the anti-cancer effects of three Zn-MOCs coordination complexes. Their cytotoxicity properties have been assayed on three different cancer cell lines, HT29 (colon cancer cells), HepG2 (hepatoma cells), and B16-F10 (melanoma cells). MOCs properties offer a new approach to new anticancer drug development, achieving less side effects and selective drugs delivery that could directly benefit cancer patients


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In summary, we report herein the synthesis, structure and anti-tumor properties of (4NO2-Hpz)4 (1), {[Zn2(µ-4-NO2-pz)3(µ-OH)]·H2O}n (2), {[Zn2(µ-4-NO2-pz)4]}n (3) and {[Zn3(µ4-NO2-pz)4(µ-ac)2(H2O)2]}n (4), demonstrating the potential of this pyrazolate linker to construct new MOCs with interesting physical and biological properties. Moreover, a remarkable structural feature of these compounds is the occurrence of one-dimensional arrays, which constitute the first examples of polymeric structures for 4-NO2-pz ligands.

Experimental Procedures

Materials and physical measurements All reagents were obtained from commercial sources and used as received. Elemental (C, H, and N) analyses were performed on a Leco CHNS-932 microanalyzer. IR spectra of powdered samples were recorded in the 400–4000 cm−1 region on a Nicolet 6700 FTIR spectrophotometer using KBr pellets.

Synthesis of {[Zn2(µ-4-NO2-pz)3(µ-OH)]•H2O}n (2). Compound 2 was obtained by hydrothermal routes through the following procedure: A mixture of ZnCl2 (0.068 g, 0.5 mmol), 4-nitro-1H-pyrazole (0.113 g, 1 mmol) and distilled water (10 mL) was sealed in a Teflon-lined acid digestion autoclave and heated at 120 ºC under autogenous pressure. After 12 h of heating, the reaction vessel was slowly cooled down to room temperature during a period of about 3 h. Yellow crystals of the compound under study were obtained. Yield: 43%, based on Zn. Anal. calcd C9H9N9O8Zn2: C 21.53, H 1.81, N 25.11. Found: C 21.42, H 1.76, N 25.32.


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Synthesis of {[Zn2(µ-4-NO2-pz)4]}n (3). A mixture of Zn(NO3)2·6(H2O) (0.149 g, 0.5 mmol), 4-nitro-1H-pyrazole (0.113 g, 1 mmol) and distilled water (10 mL) was sealed in a Teflon-lined acid digestion autoclave and heated at 120 ºC under autogenous pressure. After 12 h of heating, the reaction vessel was slowly cooled down to room temperature during a period of about 3 h. Yellow crystals of the compound under study were obtained. Yield: 55%, based on Zn. Anal. calcd C12H8N12O8Zn2: C 24.89, H 1.39, N 29.03. Found: C 24.69, H 1.31, N 29.24.

Synthesis of {[Zn3(µ-4-NO2-pz)4(µ-ac)2(H2O)2]}n (4). A mixture of zinc acetate (0.092 g, 0.5 mmol), 4-nitro-1H-pyrazole (0.113 g, 1 mmol) and distilled water (10 mL) was sealed in a Teflon-lined acid digestion autoclave and heated at 120 ºC under autogenous pressure. After 12 h of heating, the reaction vessel was slowly cooled down to room temperature during a period of about 3 h. Yellow crystals of the compound under study were obtained. Yield: 37%, based on Zn. Anal. calcd C16H18N12O14Zn3: C 24.06, H 2.27, N 21.05. Found: C 23.96, H 2.21, N 21.19.

Crystallographic refinement and structure solution Prismatic crystals for 1-4 were mounted on a glass fibre and used for data collection on a Bruker D8 Venture with Photon detector equipped with graphite monochromated MoKα radiation (λ=0.71073 Å). The data reduction was performed with the APEX233 software and corrected for absorption using SADABS.34 Crystal structures were solved by direct methods


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using the SIR97 program35 and refined by full-matrix least-squares on F2 including all reflections using anisotropic displacement parameters by means of the WINGX crystallographic package that provides a graphical user interface for other crystallographic programs, in particular for SHELXL.36, 37

Generally, anisotropic temperature factors were assigned to all atoms except for hydrogen atoms, which are riding their parent atoms with an isotropic temperature factor arbitrarily chosen as 1.2 times that of the respective parent. Final R(F), wR(F2) and goodness of fit agreement factors, details on the data collection and analysis can be found in Table 1. CCDC numbers are 1514766-1514769. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif

Solution NMR studies Solution NMR spectra were recorded using a Bruker Avance III spectrometer instrument operating at 1H Larmor frequency of 300 MHz and are referenced according to IUPAC recommendation, with 1H and 13C chemical shifts relative to TMS. NMR sample preparation: Compounds 3 and 4 were each of one placed in a J-Young NMR tube, which were connected to a vacuum/N2 line and evacuated by three N2/vacuum cycles. Then, 0.4 mL of anhydrous DMSO-d6 (dried over activated 4Å molecular sieves for 24 hours) were added to each sample. Each NMR tube was then inserted into the bore of the magnet and standard one-dimensional (1H,

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C and DEPT-135) and two-dimensional (HMQC and HMBC)

experiments were acquired.


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Drugs The different compounds used in cell treatment were dissolved before use at 5 mg/mL in DMSO. A stock solution was frozen and stored at 4°C. Prior to the experiments, this solution was diluted in cell-culture medium. All the experiments were measured and compared to the controls after 72 h of treatment.

Cell culture Human colorectal adenocarcinoma cell line HT29 (ECACC no. 9172201; ATCC no. HTB-38), human hepatocarcinome cell line Hep G2 (ECACC no. 85011430), and mouse melanoma cells B16-F10 (ATCC no. CRL-6475) were cultured in DMEM supplemented with 2 mM glutamine, 10% heat-inactivated FBS 10,000 units/mL of penicillin and 10 mg/mL of streptomycin, being incubated at 37C in an atmosphere of 5% CO2 and 95% humidity. Subconfluent monolayer cells were used in all experiments.

All cell lines used were provided by the cell bank of the University of Granada, Spain. B16F10 cells were derived from a melanoma from the skin of a C57BL/6 strain mouse, are adherent cells with fibroblast-like appearance, capable to producing melanin, and form metastatic tumor nodules in lung by injecting in mice [18]. The clon F10 of B16 cells is susceptible to lysis mediated by syngeneic lymphocytes [19]. HT29 cells were isolated from a colon cancer of a 44 years old Caucasian female, have tumorogenic capacity showed in nude mice, produce well differentiated adenocarcinoma, consistent with colon primary adenocarcinome (grade I). This cell line is positive for the expression of: myc+, ras+, myb+, fos+, p53+, abl-, ros-, src- (ATCC: HTB-38). Doubling time is around 48 h in normal culture conditions. Hep G2 cells were isolated


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from a liver biopsy of a male Caucasian aged 15 years, are consistent with a well differentiated hepatocellular carcinoma. This cell line not is tumorogenic , there is not evidence of a Hepatitis B virus genome in this cell line.

Cell-proliferation activity assay The cell proliferation effect after treatment with compounds 1, 2, 3 or 4 in B16-F10 murine melanoma cells, HT29 colon carcinoma cells, and Hep G2 hepatocarcinoma cells was measured using the MTT assay (Sigma, MO, USA), which is based on the ability of live cells to cleave the tetrazolium ring, thus producing formazan, which absorbs at 570 nm.

Cell viability was determined by measuring the absorbance of MTT dye staining of living cells. For this assay, 5·103 B16-F10 cells, 6·103 HT29 cells and15·103 Hep G2 cells were grown on a 96-well plate and incubated with different concentrations of compounds 1, 2, 3 and 4 (0-100 µg/ml). Lately, after 72 h of incubation, 100 µL of MTT solution (0.5 mg/mL) was added to each well. After 1.5 h of incubation the cells were washed twice with PBS, and formazan was resuspended in 100 µL DMSO. Relative cell viability, with respect to untreated control cells, was measured by absorbance at 550 nm on an ELISA plate reader (Tecan Sunrise MR20-301, TECAN, Austria).

Statistics Statistical and non lineal regression analyses were performed with the SigmaPlot 12.5 software. All quantitative data were summarized as the means ± standard deviation (SD). All


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data shown here were representative of at least two independent experiments performed in triplicate.

Results and Discussion

Description of the structures Structural description of 4-NO2-Hpz (1).

X-ray diffraction analyses of the ligand revealed that it crystallizes in the neutral form in good agreement with a previous structural solution reported by Llamas-Saiz et al.38 In the structure, there are three cyrstallographically (A, B, and C) independent 4-NO2-Hpz molecules, all of which are protonated at their N1 nitrogen atoms. A remarkable structural feature is that the three ligands are practically planar, finding only a subtle rotation for the nitro group of 0.90º– 1.74º with regard to the mean aromatic ring. Three A, B, and C ligands are displayed almost coplanar to each other in such a way that they are arranged into supramolecular trimers owing to the hydrogen bonding R33(9) ring established from N1–H···N2 interactions (Figure 1). In this line, it may be assumed that each ligand acts hydrogen bonding donor and acceptor and that protons are orderly located at N1 atoms according to the endocyclic N–N–C angles established within the aromatic ring of 4-NO2-Hpz ligands.

It is well-known that the protonation at N sites in similar cycles, such as nucleobases, does not modify substantially the bond distances but it enlarges the endocyclic C–N–C angles up to 4º as a consequence of the absence of the lone pair in those N atoms.39,40 In the present case,


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those angles may be compared to N–N–C angles of the pyrazole ring. In good agreement with last argument, a systematic enlargement of the latter angle is observed for the three ligands [110.8 vs 107.2º (A ligand), 110.4 vs 106.9º (B ligand), and 110.6 vs 107.8º (C ligand) for C5– N1–N2 and N1–N2–C3 angles, respectively], thus confirming the location of those hydrogen atoms.

Structural description of {[Zn2(µ-4-NO2-pz)3(µ-OH)]·H2O}n (2).

Compound 2 crystallizes in the monoclinic P21/c space group and its structure consists of a supramolecular packing of 1D double chains and lattice water molecules. The asymmetric unit contains two crystallographically independent zinc(II) atoms, three 4-NO2-pz ligands, a hydroxide anion, and a lattice water molecule. Zn1 and Zn2 atoms show tetrahedral environments established from three pyrazole nitrogen atoms and the hydroxide oxygen atom (Table 2). Continuous shape measurements performed by SHAPE program41,42 reveal that both of them exhibit a similar distortion with regard to ideal tetrahedra (ST of 0.88 and 0.39 for Zn1 and Zn2, respectively). Two of the three 4-NO2-pyz ligands (A and B) act as µ-κN1:κN2-bridges between Zn1 and Zn2 atoms and arrange them into dimeric [Zn2(µ-4-NO2-pz)2] cores in which a Zn···Zn distance of 3.68 Å is imposed. Hereafter, µ-hydroxide and µ-4-NO2-pz ligands (C molecules) emerge symmetrically from both zinc(II) atoms in an almost perpendicular arrangement to previous 4-NO2-pz linkers, thus joining adjacent dimeric cores one another into a one-dimensional double chain (Figure 2).


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Given the alternative bridges along the chain (µ-hydroxide and µ-4-NO2-pz), which impose slightly different Zn···Zn distances (3.45 and 3.84 Å, respectively), A and B 4-NO2-pz molecules do not arise completely coplanar to each other, but an angle of 3.45° is found between their aromatic rings. Nonetheless, this fact still permits establishing strong face-to-face intramolecular π–π interactions among consecutive A and B 4-NO2-pz ligands along the chain, which is propagated along the crystallographic [1 0 0] direction.

Lattice water molecules are anchored to the chains by acting as hydrogen bonding receptors of bridging hydroxide anions (Figure 3), while they also serve as a junction point among neighbouring chains. In particular, each of O1w molecules brings two additional chains together by establishing hydrogen bonds with free –NO2 groups, in such a way that the reference chain is surrounded by other six chains. In the overall packing, double chains are arranged with two relative dispositions with regard to the mean plane containing metal centres and C ligand in order to maximise the supramolecular interactions and afford cohesiveness to the crystal building. In this sense, it is worth mentioning that oxygen atoms pertaining to nitro groups of adjacent chains are forced to place at distances within the range of hydrogen bonding interactions, though it should be considered as a mere consequence of packing requirements.

Structural description of {[Zn2(µ-4-NO2-pz)4]}n (3).

Crystal structure of compound 3 builds up from single zig-zag chains established from the junction of the afore mentioned dimeric [Zn2(µ-4-NO2-pz)2] cores. Within these entities,


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crystallographically independent Zn1 and Zn2 atoms maintain tetrahedral environments formed solely by pyrazolate nitrogen atoms, a fact that generates a large difference between both metal centres (compared to those of compound 2) in terms of distortion with regard to ideal polyhedron (ST = 0.25 and 1.31 for Zn1 and Zn2). This geometrical feature affects the relative disposition between A and B bridging 4-NO2-pz ligands (Figure 4), which are slightly folded and acquire an angle of 24.8° between their aromatic rings. Hereafter, two additional centrosymmetric dimeric cores are emerged from each metal centre owing to the coordination of C and D ligands, which act as µ-κN1:κN2-bridges and bring the fusion of the dimers into a 1D array.

As a consequence, consecutive 4-NO2-pz ligands are arranged almost perpendicular to each other, minimising the sterical hindrance, while the corrugated chain is extended [Zn1···Zn2···Zn2(ii) of 144.6°] along the [0 –1 1] direction.

Despite of the aromatic nature of 4-NO2-pz ligands, only very weak intramolecular edgeto-face π–π interactions are allowed to happen along the chains given their relative arrangement (see Table S3). Instead, much more efficient π–π interactions are established between B ligands belonging to neighbouring chains arranged face-to-face one another, leading to layers spreading along the bc plane (Figure 5). These interactions are in turn reinforced by weaker R22(10) hydrogen bonding rings formed by C–H···Onitro bonds between almost parallel rings.43 These symmetric interactions, although of weaker nature, resemble base-pairings found in nucleobase based complexes, such as recurrent patterns established between Hoogsteen sides of two adenine molecules or Asn/Glu-adenine interaction in protein-nucleic acid complexes.44,45 Pilling of the layers of 1D chains along crystallographic a axis takes place due to the occurrence of additional


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supramolecular interactions, among which C–H···Onitro (with somewhat acute angles) as well as perpendicular Onitro•••ring-pz contacts. Further analysis of the crystal structure by means of Platon software46 reveals the presence of very small voids, in which even a water molecule does not enter (28.4 Å3 per unit cell volume, 2.9 %). Therefore, at first sight, one may anticipate the lack of strong cohesion in the overall crystal building of 3.

Structural description of {[Zn3(µ-4-NO2-pz)4(µ-ac)2(H2O)2]}n (4).

X-ray diffraction analysis of compound 4 reveals a similar structural nature of compound 3 since it also consists of polymeric chains. In fact, chains composing compound 4 may be considered as the result of the replacement of some 4-NO2-pz bridges by acetate linkers. Two independent zinc(II) atoms are distinguished in the asymmetric unit. Zn1 atom lies on an inversion centre and exhibits an almost ideal octahedral environment (SOC = 0.22), whereas Zn2 displays a less common pentacoordinated donor set formed by three nitrogen pyrazolate atoms and two chelating carboxylate oxygen atoms, which may be defined as a severely distorted trigonal bipyramid (STBPY = 2.44) (Table 4). The presence of such different environments may be attributed to the µ-ac ligand, which alternatively replaces a µ-4-NO2-pz bridge along the chain (Figure 6). Moreover, it is able to preserve the corrugated chain motif described for compound 3 by acquiring the µ-κ2O11,O12:κO12 bridging mode in order to accommodate to the Zn···Zn imposed by the 4-NO2-pz bridge. In this sense, the smaller steric hindrance imposed by acetate ligand favours the incorporation of two water molecules in the coordination shell of Zn1 so that it gains stability with regard to the initial coordination of acetate and pyrazolate moieties at the vertices of a square plane. Therefore, two different building units are to be distinguished:


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centrosymmetric [Zn2(µ-4-NO2-pz)2] cores and mixed [Zn2(µ-4-NO2-pz)(µ-ac)] dimers, both of which are joined into corrugated chains running along the c axis. However, a subtle difference is worth highlighting paying attention to the way in which these units are assembled, since in this case, the alternative propagation of both dimeric cores involves a Zn2(i)···Zn1···Zn2···Zn2(ii) sequence instead of previously observed Zn1(i)···Zn1···Zn2···Zn2(ii) one.

Packing of the chains is driven by the same kind of forces takes following a similar scheme to that previously observed for compound 3. First, strong face-to-face chains π–π (between A and B 4-NO2-pz ligands) as well as are hydrogen bonding interactions (involving coordination water molecules and Onitro groups) are established among moieties belonging to two adjacent chains, which generates well united layers spreading along ac plane (see Figure 7 and Supp. Info.).

Then, these layers are piled along the crystallographic c axis in a staggered fashion owing to additional hydrogen and π–π bonds occurring between intercrossing chains, in such a way that each chain interacts with four neighbouring ones. All these interactions give rise to the 3D compact architecture of 4.

Solution NMR studies

Apart from the crystallographic characterization and elemental analysis, except for compound 2, standard analytical methods such as NMR and MS spectrometry were used to verify the characterization of the metal complexes in solution to corroborate that species 3 and 4


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are present in the biological assays. In particular, MS (ESI) spectrometry from polar solvents such as dimethylsulfoxide gave evidence for the retention of the ligands and polymetallic scaffold in solution. In the coordination polymer 3, the negative ion mode revealed a parent ion corresponding to [Zn2(µ-4-NO2-pz)4(HO)]- with m/z of 591.81854, that only differs from the solid state in the incorporation of one hydroxide molecule. As expected, the observed isotope pattern matches well with the one calculated for the exact mass of C12H8N12O9Zn2 (Figure S14).

In complex 4 however, the molecular ion is [Zn3(µ-4-NO2-pz)3(µ-ac)(H2O)2]- with m/z of 622.91715, which corresponds to an ion that has kept their three zinc metals, but misses a pyrazolyl and an acetate ligand. These results evidenced that the trinuclear scaffold is strong enough to persist in the gas phase, but the ligand lability slightly differs from one complex to another. The latter statement is supported by other fragments observed in the mas spectrum that derived from the substitution of some pyrazolyl ligands by dimethylsulfoxide, the solvent of choice. Parent ions such as [Zn3(µ-4-NO2-pz)(DMSO)2(µ-ac)(H2O)2]- and [Zn3(µ-4-NO2pz)(DMSO)2(µ-ac)]- at 553.90308 and 516.91333, respectively, were also observed. Accordingly, their experimental isotope patterns match well with those calculated for C9H21N3O8S2Zn3 and C9H16N3O6S2Zn3, respectively (Figures S16 and 17).

The one-dimensional (1D) and two-dimensional (2D) NMR spectra of complexes 3 and 4 were recorded in anhydrous deuterated dimethyl sulfoxide, and are presented in the Supporting information (Figures S1–S12). The resonances found for both complexes are consistent with metallated pyrazole structures such as those obtained in the solid state.


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The room temperature 1H NMR spectrum for the coordination polymer 3 displays two broad singlets of different intensity for the 3,5-CHpz protons at δH 8.31 and 8.53 ppm. The broadened proton resonances obtained indicated a fluxional behavior between the magnetically different pyrazolyl ligands. We therefore performed high-temperature NMR measurements in order to increase the exchange rate of the fluxional process and get sharper signals. To our delight, upon heating a [D6]-DMSO solution of 3, we could observe how the signal sharpens at 323 K (W1/2 = 14.9 Hz), what proved the latter statement. In the 13C NMR spectrum, two signals were observed at δC 135.1 and 137.1 ppm, for the quaternary and methine carbons, respectively, what again matches with an average situation in solution, where all the pyrazolyl units are in fast exchange at this temperature.

Complex 4 shows in the 1H NMR spectrum two sharp signals located at δH 8.25 and 1.98 ppm assigned to 3,5-CHpz and acetate protons, respectively. Interestingly, the ratio between both signals is 4 to 3, what confirms the stoichiometry found in the solid state. At room temperature the exchange rate between pyrazolyl ligands is fast as it is deduced from the sharp signals observed in the spectrum. Again, the chemical shift of the pyrazole protons fit well with metallated ligands as observed in complex 3. The 13C NMR spectrum shows three resonances at δC 178.6, 136.8, 135.3 and 22.9, assigned to the carbonyl (acetate), methine (pyrazole), quaternary (pyrazole) and methyl (acetate) carbons, respectively.


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Potential antiproliferative activities

We assayed the proliferation effect of the ligand (Nitro-1H-pyrazole) and two Zn-MOCs (compounds 3 and 4) on three different cell lines, B16-F10 murine melanoma cells, HT29 colon cancer cells and HepG2 hepatome cells. Cell viability was tested in these three different cell lines by MTS assay at increased concentrations of compounds 1, 3 and 4 (range from 0 to 100 µg/mL). The viabilities (Figure 8) were determined by formazan dye uptake and expressed as percentage with respect to untreated control cells. IC50, which means products concentration required to inhibit cell growth at 50%, was determined for each compound on these three cell lines (Table 5). Also, we determinate the product concentration required for 20% and 80% growth inhibition (IC20 and IC80), to analyze the complete range of cytotoxicity. Both coordination complexes (compounds 3 and 4) show cytotoxicity properties at the conditions assayed (0 to 100 µg/mL) with IC50 data range between 42 to 53 µg/mL, IC20 data between 15 to 34 µg/mL, and IC80 data between 73 to 80 µg/mL. The effectiveness showed for these coordination complexes was different in each cell line assayed, however in all cases we have an improvement in the compounds cytotoxicity’s results compared to the ligand values; at IC50 concentrations, the lowest values was obtained in B16-F10 cell line (53%), and the highest one on HT29 (62%) and Hep G2 cells (81%). As conclusion, the IC50 cytotoxicity values are lower in Hep-G2 hepatome cells and HT29 colon cancer cells that in B16-F10 melanoma cells. The most effective compound was the compound 4, although with not significant differences with the other Zn complex. In all cell lines assayed, compound 4 show cytotoxic effects at lower concentrations than compound 3, especially in HT29 and B16-F10 cell lines (see IC20 values,


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Table 5). In Hep-G2 cell line the onset of the cytotoxic effect occurs in a similar way to both compounds, with concentrations close to 16µg/mL. For all complexes and in all cell lines assayed, the highest cytotoxic effect is reached at similar concentrations, around 75 µg/mL (see IC80 values, Table 5). In general terms, we found that complexes showed lower IC50 than 4-NO2-pz ligand. This ligand displayed a low cytotoxicity in all cells lines assayed and these cytotoxicities were increased with the formation of coordination complex. Currently, many anti-cancer drugs dosages are limited by side effects, which prevent the complete treatment of cancer in patients. For example, cisplatin is the most widely used chemotherapeutic drug for treatment in different cancer types. However, cisplatin produces many side effects, such as nephrotoxicity.14 Other coordination complexes do not exhibit adequate stability for in vivo applications. Our results showed the low cytotoxicity of the pyridazole by itself, which it enhance with the formation of coordination polymers. These results showed that the cytotoxic effect of Zn complexes on HT29 colon cancer cells, Hep-G2 hepatome cells and B16-F10 melanome cells present few variations with respect to cell type. There were also no significant differences among cytotoxicity influence of the complexes examined indicate that different substituent on the ligands not had a clear influence. Colon cancer cells seemed to be more sensitive to both Zn-pyridazole complexes, also this cell line showed lower IC50 value in the pyridazole treatment (Figure 9). The higher difference between coordination complexes treatment with respect to pyridazole treatment were found in the Hep-G2 cells, being approximately 5-fold more cytotoxic than the pyridazole ligand. Thus, these synthesized Zn-MOCs show significant advantages over existing system. These materials avoid the potential self-aggregation issue often encountered by other anticancer compound such


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as triterpenes47,48 and minimize nonspecific interactions suggesting that these compounds can be considered effective drugs for potential treatment from cancer.

Conclusions

In summary, we have developed novel 1D Zn-coordination complexes with interesting structural properties that show significant advantages over existing anti-tumor systems. The selftassembly of coordination complexes carried out under mild conditions, making complex highly stables. These compounds constitute the first examples of polymeric structures based on 4-NO2pyrazole ligands. Zn-coordination complex avoid the potential self-aggregation issue often encountered by other anticancer compounds such as triterpenes and minimize nonspecific interactions. NMR and MS spectrometry verified the presence of metal complexes in solution to corroborate that these materials are present in the biological assays. These results suggest that Zn-MOCs represent important novel and effective drugs for potential treatment from cancer. Futures studies will be necessary to characterize completely its anticancer effects.

ASSOCIATED CONTENT Supporting Information. Additional figures of Crystallographic data, FT-IR spectroscopy, NMR spectroscopy and High resolution mass spectrometry. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (F. J. R. Z.)


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*E-mail: [email protected] (A. R. D.)

ACKNOWLEDGEMENTS This work was supported by the Junta de Andalucía (FQM-1484, FQM-2668 and FQM-195); grants Group BIO 157 from the Technology and Innovation Council of the Andalucian regional government, the research contract ASOAN-C3650-00 under the program FEDERINNTERCONECTA from the Spanish Government and European Union FEDER funds and Guipuzcoana de Ciencia, Tecnolgía e Innovación (OF215/2016) and University of the Basque Country (GIU14/01, EHUA16/32). J. Cepeda thanks the University of the Basque Country (UPV/EHU) for the postdoctoral fellowship. A. R-B. thanks Plan Propio de Investigación of University of Almería for a PhD fellowship.

REFERENCES

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(19) Piñero, D.; Baran, P.; Boca, R.; Herchel, R.; Klein, M.; Raptis, R.G.; Renz, F.; Sanakis, Y. Inorg.Chem. 2007, 46, 10981-10989. (20) Kinzler, K.W.; Vogelstein, B. Nature, 1996, 379, 19-24. (21) Adams, G.E.; Stratford, I.J. Biochem. Pharmacol., 1986, 35, 71-76. (22) a) Fowler, J.W.; Adams, G.E.; Denekanp, J. Cancer Treatment. Rev. 1976, 3, 227-256. (23) Adams, G.E.; Fowler, J.F.; Dische, S.; Thomlinson, R.H. The Lancet 1976, 307, 186-188. (24) Adams, G.E. Br. Med. Bull., 1973, 29, 48-53. (25) Fernández, B.; Oyarzabal, I.; Fischer-Fodor, E.; Macavei, S.; Sánchez, I.; Seco, J.M.; Gómez-Ruiz, S.; Rodríguez-Diéguez, A. CrystEngComm 2016, 18, 8718-8721. (26) Parra, A.; Martin-Fonseca, S.; Rivas, F.; Reyes-Zurita, F.J.; Medina-O’Donnell, M.; Rufino-Palomares, E.; Martinez, A.; García-Granados, A.; Lupiañez, J.A.; Albericio, F. Comb. Sci., 2014, 16, 428-447. (27) Torchilin, V. Adv Drug Deliver Rev, 2011, 63, 131-135. (28) Jain, R.K.; Stylianopoulos, T. Nat Rev Clin Oncol, 2010, 7, 653-664. (29) Kelland, L. Nat Rev Cancer, 2007, 7, 573-584. (30) Davis, M.E.; Chen, Z.G.; Shin, D.M. Nat Rev Drug Discov, 2008, 7, 771-782. (31) Liu, D.M.; Poon, C.; Lu, K.D.; He, C.B.; Lin, W.B. Nat Commun, 2014, 5, 4182-4184. (32) Wang, A.Z.; Langer, R.; Farokhzad, O.C. Annu Rev Med, 2012, 63, 185-198. (33) Bruker Apex2, Bruker AXS Inc., Madison, Wisconsin, USA, 2004. (34) Sheldrick, G.M. SADABS, Program for Empirical Adsorption Correction, Institute for Inorganic Chemistry, University of Gottingen, Germany, 1996.


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(35) Altomare, A.; Burla, M.C.; Camilla, M.; Cascarano, G.L.; Giacovazzo, C.; Guagliardi, A.; Moliterni, A.G.G.; Polidori, G.; Spagna, R. J. Appl. Crystallogr. 1999, 32, 115-119. (36) Sheldrick, G.M. Acta Cryst. 2008, A64, 112-122. (37) Farrugia, L.J. J.Appl.Cryst. 2012, 45, 849-854. (38) Llamas-Saiz, A.; Foces-Foces, C.; Cano, F.H. Acta Cryst. 1994, B50, 746-762. (39) Marzotto, A.; Ciccarese, A.; Clemente, D.A.; Valle, G. J. Chem.Soc., Dalton Trans. 1995, 0, 1461-1468. (40) García-Terán, J.P.; Castillo, O.; Luque, A.; García-Couceiro, U.; Beobide, G.; Román, P. Dalton Trans. 2006, 902-911. (41) Llunell, M.; Casanova, D.; Cirera, J.; Bofill, J.M.; Alemany, P.; Alvarez, S.; Pinsky, M.; Avnir, D. SHAPE (1.7), University of Barcelona, Barcelona, 2010. (42) Cirera, J.; Alemany, P.; Alvarez, S. Chem. Eur. J. 2004, 10, 190-207. (43) Infantes, L.; Chisholm, J.; Motherwell, S. CrystEngComm 2003, 5, 480-486. (44) Pérez-Yáñez, S.; Castillo, O.; Cepeda, J.; García-Terán, J.P.; Luque, A.; Román, P. Eur. J. Inorg. Chem. 2009, 26, 3889-3899. (45) Cheng, A.C.; Frankel, A.D. J. Am. Chem. Soc. 2004, 126, 434-445. (46) Spek, A.L. J. Appl. Crystallogr. 2003, 36, 7-13. (47) Medina-O'Donnell, M.; Rivas, F.; Reyes-Zurita, F.J.; Martinez, A.; Martin-Fonseca, S.; Garcia-Granados, A.; Ferrer-Martin, R.M.; Lupiañez, J.A.; Parra, A. European Journal of Medicinal Chemistry, 2016, 118, 64-78. (48) Reyes-Zurita, F.J.; Medina-O'Donnell, M.; Ferrer-Martin, R.M.; Rufino-Palomares, E.E.; Martin-Fonseca, S.; Rivas, F.; Martinez, A.; Garcia-Granados, A.; Perez-Jimenez, A.; Garcia-


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Salguero, L.; Peragon, J.; Mokhtari, K.; Medina, P.P.; Parra, A.; Lupianez, J.A. Rsc Adv. 2016, 6, 93590-93601.


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For Table of Contents Use Only

Modulating Anti-Cancer Potential by Modifying the Structural Properties of a Family of Zinc Metal-Organic Chains Based on 4-Nitro-1H-pyrazole Belén Fernández,a Ignacio Fernández,b Fernando J. Reyes-Zurita,c,* Javier Cepeda,d Marta Medina Odonnell,c Eva E. Rufino-Palomares,c Álvaro Raya-Barón,b Santiago Gómez-Ruiz,e Amalia Pérez-Jiménez,c José Antonio Lupiáñezc and Antonio Rodríguez-Diéguez*,a

We have synthesized a novel family of metal-organic chains based on 4-nitro-1Hpyrazole and zinc as metal centre. These materials display one-dimensional structures with structural modifications from minimal synthetic variations. What is more interesting, we have related anti-tumor properties with the structural networks. To the best of our knowledge, these materials constitute the first examples of polymeric structures for 4NO2-pz ligands.


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FIGURES AND SCHEMES

Scheme I. 4-Nitro-1H-pyrazole (left) and the two different coordination modes presented by this linker (right: modes a and b).


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Figure 1. Excerpt of the supramolecular layer of compound 1 showing hydrogen bonding interactions within the central trimer and with neighbouring ones. Strong N–H···N (thick dashed orange line) and weak C–H···O (thine dashed orange line) interactions are drawn.


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Figure 2. Double 1D chains of compound 2 showing coordination environments of Zn1 and Zn2 and π–π interactions among 4-NO2-pz ligands (double dashed orange lines). Colour coding: C = grey, H = pink, N = blue, O = red, M = green.


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Figure 3. Packing of chains in 2 along crystallographic a axis. Hydrogen bonding scheme is detailed in the right side caption.


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Figure 4. One-dimensional chain of 3 established from the fusion of dimeric cores together with tetrahedral coordination environments. Numbering scheme for 4-NO2-pz ligands is shown.


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Figure 5. Crystal building of compound 3 detailing a summary of supramolecular interactions among 1D chains.


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Figure 6. Coordination environments of Zn1 and Zn2 atoms together with the corrugated chain of compound 4.


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Figure 7. Packing of compound 4 showing supramolecular interactions occurring among corrugated chains.


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Figure 8. Effects of compounds 1, 3 and 4, on the viability of B16-F10, HT29 and HepG2 cancer cells, after treatment with the compounds for 72h in a range of 0 to 100 µg/mL, each point represent the mean value ± S. D. of at least two independent experiments performed in triplicate.


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Figure 9. Growth-inhibitory effects (IC20, IC50 and IC80, µg/mL) of compounds 1, 2, 3 and 4, on the three cancer cell lines.


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TABLES Table 1. Crystallographic Data for compounds 1 - 4 Compound Chem. formula

1

2

3

4

C3H3N3O2

C9H9N9O8Zn2

C12H8N12O8Zn2

C16H18N12O14Zn3

CCDC number

1514767

1514768

1514769

1514766

113.08

501.99

579.04

798.53

100

100

100

100

cryst syst

Triclinic

Monoclinic

Triclinic

Monoclinic

space group

P-1

P21/c

P-1

C2/c

a/ Å

8.0288(16)

7.2679(3)

9.2248(4)

18.446(5)

b/ Å

9.6212(19)

23.1699(10)

10.0991(4)

13.740(5)

c/ Å

9.906(2)

9.9116(4)

11.4626(4)

10.814(5)

α/ º

74.395(3)

90

79.557(1)

90

β/ º

81.549(3)

92.337(2)

66.699(1)

109.872(5)

83.255(3)

90

87.241(1)

90

V/ Å Z

726.5(3)

1667.69(12)

964.21(7)

2577.6(17)

6

4

2

4

ρ(g cm-3)

1.551

1.999

1.994

2.058

µ(mm-1)

0.132

2.942

2.563

2.866

Unique reflections

2698

45276

22084

8194

R(int)

0.023

0.079

0.043

0.032

1.060

1.067

1.034

1.069

0.054

0.034

0.028

0.032

0.132

0.062

M/gmol T (K)

-1

σ/ º 3

GOF on F

2

R1 [I > 2σ(I)]

a

wR2 [I > 2σ(I)]

a a

0.058 2

R(F) = ||Fo| - |Fc||/|Fo|; wR(F ) =

[w(Fo2


2 2

0.074 4 1/2

– Fc ) /wF ]

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Table 2. Selected bond lengths for compound 2.a Zn1–N1A

2.013(2)

Zn2–N2A

1.987(2)

Zn1–N1B

1.991(2)

Zn2–N2B

1.997(2)

Zn1–N1C

1.986(2)

Zn2–N2C(i)

1.997(2)

Zn1–O1 1.927(2) Zn2–O1(ii) [a] Symmetries: (i) –x, –y, –z; (ii) –x – 1, –y, –z.

1.992(2)


2.010(2)

Zn2–N1D

1.987(2)

Zn1–N1B

1.997(2)

Zn2–N2A

1.986(2)

Zn1–N1C

2.000(2)

Zn2–N2B

2.036(2)

Zn1–N2C(i) 2.009(2) Zn2–N2D(ii) [a] Symmetries: (i) –x + 2, –y + 1, –z + 1; (ii) –x + 2, –y, –z + 2.

1.999(2)


2.023(2)

Zn2–N2A

2.036(2)

Zn1–N1A(i)

2.023(2)

Zn2–N1B

2.010(2)

Zn1–O11C

2.151(2)

Zn2–N2B(ii)

2.036(2)

Zn1–O11C(i)

2.151(2)

Zn2–O11C(i)

2.295(2)

Zn1–O1w

2.242(2)

Zn2–O12C(i)

2.063(2)

Zn1–O1w(i) 2.242(2) [a] Symmetries: (i) –x + 1/2, –y + 1/2, –z + 1; (ii) –x + 1/2, –y + 1/2, –z.


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Table 5. Growth-Inhibitory Effects of compounds 1, 2, 3 and 4 on the Three Cancer Cell Lines IC Ligb/ Cell line

Compd. #

IC20

IC Ligb/

IC of compound HT29

Hep-G2

B16-F10

IC Ligb/ IC80

IC50 IC of compound

IC of compound

1

56,5 ± 8,0

1,0

97,6 ± 4,9

1,0

216,5 ± 16,9

1,0

3

23,2 ± 4,9

2,4

46,7 ± 8,8

2,1

77,3 ± 13,7

2,8

4

18,8 ± 9,3

3,0

41,8 ± 15,0

2,3

75,2 ± 15,8

2,9

1

50,9 ± 16,4

1,0

210,6 ± 16,1

1,0

521,0 ± 15,9

1,0

3

17,4 ± 3,9

2,9

45,4 ± 1,9

4,6

78,0 ± 0,2

6,7

4

15,1 ± 2,6

3,4

45,8 ± 2,7

4,6

80,5 ± 1,6

6,5

1

82,7 ± 11,5

1,0

101,8 ± 1,1

1,0

113,6 ± 6,3

1,0

3

33,9 ± 6,3

2,4

52,6 ± 1,4

1,9

73,4 ± 2,4

1,5

4

15,9 ± 6,8

5,2

47,8 ± 4,9

2,1

78,3 ± 4,1

1,5


39

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