Hybrid MII-organic-diphosphate hydrates (MII = Mn, Ni and Zn): Structural characterization, hirshfeld surface analysis and antitumoral activity

Journal Pre-proof II II Hybrid M -organic-diphosphate hydrates (M = Mn, Ni and Zn): Structural characterization, hirshfeld surface analysis and antitumoral activity Intissar Moghdad, Aicha Mbarek, Ferdinando Costantino, Sabrina Nazzareni, Houcine Naїli PII:

S0022-2860(19)31435-8

DOI:

https://doi.org/10.1016/j.molstruc.2019.127326

Reference:

MOLSTR 127326

To appear in:

Journal of Molecular Structure

Received Date: 24 July 2019 Revised Date:

24 October 2019

Accepted Date: 30 October 2019

II Please cite this article as: I. Moghdad, A. Mbarek, F. Costantino, S. Nazzareni, H. Naїli, Hybrid M II organic-diphosphate hydrates (M = Mn, Ni and Zn): Structural characterization, hirshfeld surface analysis and antitumoral activity, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/ j.molstruc.2019.127326. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Hybrid MII-Organic-Diphosphate Hydrates (MII = Mn, Ni and Zn): Structural Characterization, Hirshfeld surface analysis and Antitumoral Activity

Intissar Moghdada, Aicha Mbareka, Ferdinando Costantinob, Sabrina Nazzarenic, Houcine Naїlid*

a

Laboratory of Advanced Materials, National School of Engineering, Box 1173, Sfax University, Sfax 3038, Tunisia; e-mail: [email protected] (I. Moghdad), [email protected] (A. Mbarek). b

Department of Chemistry, Biology and Biotechnology and CIRCC, University of Perugia Via Elce di Sotto 8, Perugia 06123,Italy; e-mail: [email protected] (F. Costantino). c

Department of Physics and Geology, University of Perugia, Perugia 06100, Italy; e-mail: [email protected] (S. Nazzareni).

d

LaboratoryPhysico Chemistry of the Solid State, Department of Chemistry, Faculty of Sciences ofSfax, BP 1171, Sfax University, Sfax 3000, Tunisia; e-mail: [email protected] (H. Naїli).

*Corresponding author Tel: +216 98 660 026; Fax: +216 74 274 437. E-mail address: [email protected].

1

Abstract Three inorganic-organic hybrid

diphosphates,

templated

by transition

metal

(2,6-

(CH3)2C6H3NH3)2[MII(H2P2O7)2(H2O)2] (MII = Mn, Ni, Zn), were successfully synthesized by low speed of evaporation conditions after using the ion exchange chemical procedure. Their crystal structures were solved from single-crystal X-ray diffraction data. All compounds are isotypic and crystallize in the triclinic system with space group Pī. The crystal structure is built from the alternatively arranged inorganic [MII(H2P2O7)2(H2O)2]2- anions and organic [(CH3)2C6H3NH3]+ cations, stabilized by O—H···O and N—H···O hydrogen bonds and π···π stacking. The complexes were investigated by using such techniques: IR, TGA/DTA, UVvisible characterization techniques (Ni(II), Mn(II) compounds) and 1H,

13

C, 31P NMR (Zn(II)

compound). Hirshfeld surface analyses of the studied salts were also carried out. Finally, in vitro anticancer activities of compounds were further determined with reference to three human cancer cell lines (Human cervical (A431), pancreatic (PSN1) and human ovarian cell lines (2008)). Hybrid complexes, based on manganese, nickel and zinc, displayed in vitro efficient antitumor activity compared with the positive reference drug cisplatin.

Keywords: Organic-inorganic hybrid; Transition metal; Diphosphate; Crystal growth; Antitumor activity.

2

1. Introduction The development of hybrid functional materials, constituting both inorganic and organic components, has paved the way for the development of innovative systems with complex architectures and interesting functions [1-3], particularly for biomedical or medicinal applications [4-9], such as drug-delivery systems possessing remarkable antiviral and antitumoral activities [10-12]. For medicinal applications, it would be highly desirable to develop concise functionalization strategies, allowing straight forward structural variations for stability, solubility and activity modulation. In this regards, great efforts have been conducted with respect to the development of new effective and target-specific transition metal anticancer complexes via the variation of coordination modes, metals or ligands [13-14]. Recently, the combination of phosphorus ligands (phosphites, phosphonates and phosphates) [15-17] with transition metals has represented one way to overcome the limitations pertaining to platinum drugs. A number of platinum, ruthenium and gold complexes with phosphine ligand have received considerable attention owing to their coordination chemistry and broad range of pharmacological properties, notable for anti parasital, antibacterial and antitumor activities [18-20]. It is particularly noteworthy that an Au-phosphine complex, auranofin (a thioglucose derivative of triethylphosphine gold (I)) into clinical use as an antiarthritic drug, shows significant anticancer activity [21]. It was reported that polyoxometalates, functionalized by bisphosphonate ligands, have been used clinically for decades to treat bone resorption diseases and some of them exhibit anti-tumor activity [22]. However, the use of metal complexes, bearing phosphates ligands as anticanceragents, has not been well studied. In this work, MII-organic-diphosphate hybrids, (C8H9NH3)2[MII(H2P2O7)2(H2O)2] with (MII = Mn, Ni, Zn), were successfully synthesized at ambient temperature and structurally characterized by various physicochemical techniques, including single-crystal X-ray diffraction, hirshfeld surface analysis, infrared, nuclear magnetic resonance and UV-visible spectroscopies. In

3

addition, their cytotoxicity effects on Human cervical, Pancreatic and Human ovarian cancer cell lines in vitro were demonstrated in detail. 2. Experimental section 2.1. Synthesis The single-crystals of (2,6-C8H9NH3)2[MII(H2P2O7)2(H2O)2] with MII = Mn, Ni and Zn, were grown at room temperature by slow evaporation from water solution containing a stoichiometric mixture of MIICl2.6H2O (MII = Mn, Ni, Zn), 2,6-dimethylaniline (C8H12N) and diphosphoric acid H4P2O7. The general equation for the reaction is shown in Scheme 1.

Scheme 1. Schematic outline of the synthesis of the complexes (M = Mn (II), Ni (II) or Zn (II)). The diphosphate ligand was obtained by using the ion exchange process [23] by mixing the starting material of sodium pyrophosphate Na4P2O7 and Amberlite resin IR 120. The beakers containing solutions were sealed with a perforated polythene sheet and kept in a dust-free atmosphere for slow evaporation at room temperature. After a period of 15 days, colorless and transparent crystals were obtained for the manganese and zinc complex and pale green crystals 4

for the nickel complex. Anal. Calcd (%) for C16H32MnN2O16P4: C, 27.9; H, 4.6; Mn, 8; N, 4.07; O, 37.2; P, 18. Found (%): C, 27.96; H, 4.96; Mn, 7.99; N, 4.08; O, 37.2; P, 18.03, for C16H32NiN2O16P4: C, 27.8; H, 4.6; Ni, 8.4; N, 4.05; O, 37.1; P, 17.9. Found (%): C, 27.81; H, 4.67; Ni, 8.49; N, 4.05; O, 37.05; P, 17.93 and C16H32ZnN2O16P4: C, 27.5; H, 4.5; Zn, 9.1; N, 4.02; O, 36.7; P, 17.8. Found (%): C, 27.5; H, 4.62; Zn, 9.37; N, 4.02; O, 36.69; P, 17.76. 2.2. Single-crystal X-ray data collection Small suitable single crystals of the title compounds (C8H12N)2[MII(H2P2O7)2(H2O)2] (MII= Mn (1), Ni(2), Zn (3)), with approximate dimensions of 0.14×0.07×0.05, 0.20×0.08×0.07 and 0.18×0.25×0.28 mm3 respectively, were cautiously selected under polarizing microscope. The X-ray single crystal structures data for the three compounds were collected on a four-circle Xcalibur, Sapphire2, CCD area-detector, Bruker D8-Venture and Kappa CCD diffractometers equipped with graphite monochromatic Mo Kα radiation (λ= 0.71073 Å) at 293 K. Therefore, a total of 6026 reflexions for compound (1), 9893 reflexions for compound (2) and reflexions 6042 for compound (3) were collected using the ω − 2θ scan technique, of which 2549, 4168 and 2460 for (1), (2) and (3), respectively, possess I>2σ(I), were used for the structures determination. Data collection, cell refinement and data reduction analysis were processed by means of CrysAlisCCD and CrysAlisRED [24]. Analytical absorption correction was applied to the data using CrysAlisRED [24]. Structures of (2,6-(CH3)2C6H3NH3)2[M(H2P2O7)2(H2O)2] (M = Zn, Mn, Ni) were solved with direct method using SHELXS-86 [25]. Program and the refinement were performed using SHELXL-2016 program [25] by a full-matrix least squares technique based on F2 with anisotropic thermal parameters for all non H-atoms. The aqua H atoms were located in a difference map and refined with O―H distance restraints of 0.85(2) Å and H―H restraints of 1.39(2) Å so that the H―O―H angle would be fitted to the ideal value of a water molecule and in the final refinement cycles of the C-bonded. H-atoms were placed in calculated positions, with C―H= 0.96 Å and refined with a riding model with Uiso(H) = 5

1.2Ueq(C). All figures were designed using DIAMOND program [26]. Crystallographic data together with experimental and refinement details are summarized in Table 2. 2.3. Hirshfeld surface analysis The percentage contributions for various intermolecular contacts in the crystals of manganese salt were obtained from Hirshfeld surface analyses, and 2D fingerprint plots were recorded on Crystal-Explorer (version 3.1) [27].A CIF input file is always used for surface analysis. The Hirshfeld surface is set using (dnorm). The measured connection distance (dnorm) is determined on the basis of de and di by



 −  
 −   = +    

Where rivdw and revdw are the van der Waals radii of the atoms. From the hirshfeld surface di and de correspond to the distances (Å) between the points and the nearest interior (di) and external (de) nuclei. A color code, blue to cyan, is applied to reflect an increasing density of overlapping points in the paths. The gray background contours correspond to the built-in plots for all types of contact. The value of dnorm identifies the regions of the intermolecular interactions with particular interest operated inside the molecule. The surface showed the colored according to the inner and the contacting atoms, although the intermolecular Vander Waals interactions. Red color indicates close proximity, however, white color represents medium proximity, and blue color shows little proximity of outside atoms [28, 29]. Fingerprint areas represent the coalescence of bi-directional de-di and di-de measures. The graph reveals that the areas, visible in the fingerprint, correspond to a single molecule, such as de>di and the other di>de. The 2-D fingerprint segment provides quantitative analysis of the molecular interconnections found in the molecule and displays such information in the form of a plot of color [30]. 2.4. Characterization methods

6

FT-IR measurements were recorded in the 400–4000 cm-1 region using a Perkin Elmer 1650 FT-IR Spectrophotometer. Samples were diluted with spectroscopic grade KBr and pressed into a pellet. The UV-Vis spectra of the complexes were also recorded using Perkin-Elmer lambda 35 spectrometer in the Wavelength region 200-800 nmusing a solution of low concentration (c = 2 10-3 mol.l-1) in 1 cm (quartz) tank. Thermal analysis was performed on a NETZSCH STA 449C TGA/DTA system with an UMX1 balance using an Al2O3 crucible. The thermogram was collected on 15.81, 21.82 and 24.28 mg of samples (1), (2) and (3) respectively, under air flow (100 ml/min) from room temperature to 1200 °C at a constant rate of 10 °C min-1. Our compounds were recorded in deuterated water by means of nuclear 1H, NMR spectroscopy. 1H,

31

P,

13

31

P,

13

C

C NMR spectra were performed on a Bruker AMX-300

instrument. 2.5. Experiments with human cells The suitable single crystals for the antitumor activity were ready to be dissolved in purified water just before the experiment. Besides, cisplatin was dissolved in 0.9% sodium chloride solution (NaCl). The growth inhibitory effect towards tumor cell lines was evaluated by means of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), SRB solution [SRB (4% w/v) in acetic acid solution], trichloroacetic acid (50%w/v), solution of tris(hydrosymethyl)aminomethane in 300ml of purified water and cisplatin, obtained from Sigma Chemical Co, St. Louis, USA. 2.5.1. Cell culture Human cervical (A431), pancreatic (PSN1) and human ovarian cell lines (2008) were kindly provided by Prof. G. Marverti (Dept. of Biomedical Science of Modena University, Italy). A431 human sensible and resistant squamous cervical carcinoma cells were kindly

7

supplied by Prof. F. Zunino (Division of Experimental Oncology B, Istituto Nazionale dei Tumori, Milan, Italy). Cell lines were incubated at 37°C in a 5% carbon dioxide atmosphere and were maintained using the following culture media containing 10% fetal calf serum (Euroclone, Milan, Italy), antibiotics (50 units/mL penicillin and 50µg/mL streptomycin) and 2mM l-glutamine and RPMI-1640 medium (Euroclone) for A431, PSN1 and 2008 cell lines. 2.5.2. Cytotoxicity assays The in vitro cytotoxicity assays supply a critical means of ranking chemical compounds for consideration with respect to drug discovery. The choice of using a specific activity or cytotoxicity test technology may be affected by particular research goals. The merits of such assays lie in offering the best combination of sensitivity, ease of performance, reproducibility and versatility. It was later applied in the determination of cytotoxicity in terms of screening of new drugs; assays measure cell viability by assessing the ability of the cell to reduce compounds and, the most commonly known, are MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) and SRB (Sulforhodamine B) tests with the use of 96-well plates. Numerous dilutions of different compounds can be tested rapidly. The anti-proliferative effect against tumor cell lines was assessed using the MTT assay in colorimetric microculture [31-32].
MTT Test The MTT assay is a calorimetric test for evaluating cell metabolic activity. In fact, the growth inhibitory effect towards tumor cell lines was assessed by means of the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The yellow tetrazole is reduced to purple Formazan in living cells [33]. Concisely, (3-8) × 103 cells/wells, relying upon the growth characteristics of the cell lines, were seeded in 96-well microplates in growth medium (100µL) and then incubated at 37°C in a 5% carbon dioxide atmosphere, for 24h.Subsequently, the medium was removed to be replaced with a fresh one, containing the commixture to be

8

tested at the appropriate concentration. Trinity cultures were established for each treatment. After 72h, fundamentally, the treated wells were treated with 10µL of a 5mg/mL MTT saline solution, following supplementary 5h of incubation, and then treated with 100µL of a sodium dodecyl sulfate (SDS) solution in HCl solution 0.01M. After an overnight incubation, the growth inhibitory effect induced by the compound to be studied was detected by measuring the absorbance of each well at 570nm using Bio-Rad 680 microplate reader. Mean absorbance for each drug dose was expressed as a percentage of the control and plotted vs. drug concentration. IC50 results, the drug concentrations that reduce the greedy absorbance of each well at 570nm to 50% of the above-mentioned in treated control wells, were calculated by means of four parameter logistic (4-PL) model (P<0.05).
SRB Test The solforhodamine B cell cytotoxicity assay was developed in 1990 [34].It is one of the most widely inexpensive methods used to detect cell viability or drug cytotoxicity, as well as being the method of choice for high cost-effective screenings [35]. The SRB test relies on the ability of sulforhodamine B solution to bind cellular proteins components and measure the total biomass. It is also endowed with applications to evaluate the effects of gene expression modulation. The Kit offers an excellent and efficient method for in vitro cytotoxicity studies, as well as high throughput drug screening. The protocol can be divided into four main steps, i.e. preparation of treatment, incubation of cells with treatment of choice, cell fixation and SRB staining, and absorbance measurement [36]. Bio vision’s SRB cell cytotoxicity assay Kit is simple, starting with the same step of the MTT assay cells, cultured and seeded in 96-well microplates in growth medium (100µL) and then incubated at 37°C in humidified incubator with 5% carbon dioxide atmosphere. After 24h, the medium was replaced with refresh one, containing the compound to be studied. After 72h, without removing the culture medium, 25µL of trichloroacetic acid was 9

added to each well to fix the cells and the microplates, which were incubated for 2h at 4°C in the refrigerator. In order to wash the cells carefully, all the liquid inside the wells was removed, then the cells were rinsed 3-5 times with 200µL of purified water. In fact, washing should be performed as gentle as possible to avoid disturbance of the cell monolayer. In each well, 50 µL of SRB solution was added [SRB at (4% w/v) in acetic acid solution (1% v/v)] and the microplatewas stained for 15-30min, at room temperature, in the dark. After removing the stained solution, the wells were washed with 200µL of acetic acid solution and the washed solutions were removed as much as possible. The wells were allowed to dry for 15-30min before being treated with 200µL 10µM of Tris solution. The plates were shaken on a shaker for 10min at room temperature. Finally, the absorbance of each well was measured at 570nm, if intense color was observed. Due to cell overload, we might use a suboptimal wavelength (490530 nm). 3. Results and discussion 3.1. Infrared spectra The title compounds (1), (2) and (3) were investigated by IR spectroscopy. Focus was placed on anti-symmetric and symmetric vibrations. Table 1 represents the major selected absorptions with their respective assignments (Fig.1), by analogy with their comparable compound [37]. The stretching vibrations of water molecule appeared as a broad band at 3568 cm-1. The strongest and medium bands of the spectra pertaining to the complex were tentatively assigned by comparing the complex spectra with the spectra of the ligand 2,6dimethylanilinium and the dihydrogendiphosphate [H2P2O7]2- anion [38-40]. The presence of the 2,6-dimethylanilinium was evidenced by the appearance of typical absorptions bands for the bending of the NH3+ group, giving rise to band at ~1538 cm-1. The stretching vibrations C―H were situated in the region 3000-3100 cm-1. However, A medium band ascribed to asymmetric deformation δas(CH3) is observed at 1476 cm-1 [41-44].The stretching C=C 10

vibration of the organic ring is detected at about 1485 cm-1. Band assignments for the fundamental modes of P2O72- anions are represented in Table 1. The band at lower frequency, centered near 500 cm-1, is assigned to the asymmetric υ4 bending vibrations of the O―P―O bonds [45] and the υ2 and υ3 modes appear at 460 cm-1 and 1183 cm-1, respectively in PO4 groups. Furthermore, the υP2O74-mode, appearing at778 cm-1, can be attributed to the increase of the symmetry pertaining to the P2O74- ion from C2v to D3h due to the P―O―P Bridge [46]. 130

Mn-(1)

120 110

Transmittance (au)

100 90

Ni-(2)

80 70

Zn-(3)

60 50 40 30 20 4000

3000

2000

1000 -1

Wavenumber (cm )

Fig. 1. FTIR spectra of (2.6-(CH3)2C6H3NH3)2[MII(H2P2O7)2(H2O)2] (MII = Mn (1), Ni (2), Zn (3)) crystals, respectively. 3.2. Crystal structure The structures of the new hybrid compounds (C8H12N)2[MII(H2P2O7)2(H2O)2] with (MII = Mn, Ni and Zn), reported in this work, possess similar crystalline structure. Hybrid materials, notably, are interesting not only for their chemical, physical and biomedical properties, but also owing to their unique structures [47]. At 293 K, the studied salts crystallize in the triclinic system, centrosymmetric space group Pī with Z = 2. Crystal and structure refinement data are presented in the Experimental Section. They are isostructural with the

11

cobalt-pyrophosphate equivalent (C8H12N)2[Co(H2P2O7)2(H2O)2] [48]. As the X-ray result demonstrated, all MII atoms lie in special positions on inversion centers, while all the other atoms occupy general positions. As shown in Fig.2(a), the asymmetric unit contains two crystallographically independent organic cations and [MII(H2P2O7)2(H2O)2] complex moiety. The packing of the different molecular entities is shown in Fig.2(b); the projection in (a,c) plane reveals a supramolecular layered structure made of an alternate stacking of inorganic layers, built of [MII(H2P2O7)2(H2O)2]2- anions, and organic protonated (C8H12N)+ cations, intercalated between the anionic sheets (Fig.3). The anionic and cationic parts, interconnected by two types of hydrogen bonds OW―H···O and N―H···O, give rise to a three-dimensional appearance to the structure (Table 3) and π stacking interactions between the aromatic rings of the amine molecules themselves. The interlayer space between two inorganic sheets in compound (1) is of 7.390 Å. Within the inorganic layers, each [MII(H2P2O7)2(H2O)2]2- anion exhibits a slightly distorted octahedral arrangement around MII with MII―O bonds, in the range 2.134 (3)-2.230 (4) Å,in manganese salt, between 2.036 (3)−2.091 (3) Å, in nickel compound and from 2.057 (3) to 2.251 (3) Å, in zinc sample. The O―MII―O bond angles range from 88.35 (11), 86.82 (14) and 89.93 (11) ° to 180°, respectively, for (1), (2) and (3). The bond distances and angles in MIIO6 with MII = Mn, Ni and Zn are adequately close to those found in the related MIIO6 complexes, featuring the chelating diphosphate ligand [49], for example, (C8H12N2)2[Co(H2P2O7)2(H2O)2]

[48],

(C7H10N)2[Co(H2P2O7)2(H2O)2]

[50],

(C8H12NO)2[Co(H2P2O7)2(H2O)2]·2H2O [51] and (C8H12NO)2[Mn(H2P2O7)2(H2O)2]·2H2O [52] and the metallic atoms are isolated from each other with a minimum distance Mn―Mn= 7.669Å, Ni―Ni= 7.290 Å and Zn―Zn= 7.572 Å, respectively, for (1), (2) and (3) compounds. This minimum intermetallic distance is comparable to that observed in their diphosphate based analogue compound, in which the shortest Mn―Mn distance is equal to 7.303 (5) Å. However, this latter adopts a different structure type and crystallizes in the space group P21/c [52]. The

12

coordination of MIIO6 octahedron is generated by two bidentate dihydrogendiphosphate ligands [H2P2O7]2- filling the equatorial positions to form a square plane around the MII atom and two symmetry equivalent oxygen atoms of water molecules, occupying the apical ones. The selected bond lengths and angles are presented in Table 4. The diphosphate ligand [H2P2O7]2is composed of two phosphorus atoms, which are tetrahedrally coordinated and covalently linked through O3 for (1) and (2) and O1 for (3) to form a P2O7 group with bent geometry (P1—O—P2 = 130°). The tetrahedra are slightly distorted as presented in Table 5 with P1―O and P2―O distances (1.477(3)
13

The analysis of hydrogen bonds within (1), (2) and (3), revealed an intricate network of O—H···O and N—H···O bonds, which, along with other interactions (electrostatic and Van der Waals), stabilize the whole structure. The O—H···O contacts link the complex anions, while the N—H···O bonds link the anions and the cations together. These H-bonds contribute in the cohesion of the three-dimensional network (Table 3, Fig.3).

Fig.2. (a) The asymmetric unit of 1 showing the atom-numbering scheme; with compounds (1)(3) having similar structures and (b) ORTEP view of the symmetric units of the hybrid samples. Turquoise ellipsoid represents MII with (MII = Mn, Ni and Zn). Black, blue, red and gray ellipsoids represent carbon, nitrogen, oxygen and hydrogen, respectively. The H-bonding involved.

14

Fig.3. View of the extended structures of (2.6-(CH3)2C6H3NH3)2[MII(H2P2O7)2(H2O)2] with (MII = Mn, Ni, Zn) in (b,c) plane. Green and purple polyhedra represent [PO4] and [MIIO6], respectively. The dotted lines indicate O―H···O and N―H···O hydrogen bonds. Offset-faceto-face interactions motifs (π···π stacking, broken lines). 3.3. Molecular Hirshfeld Surfaces calculations The Hirshfeld surface is a useful tool for describing the surface characteristics of molecules. The molecular Hirshfeld surfaces (dnorm, surface index and curvedness) are shown in Fig. 4, indicating the length of intermolecular contacts. In fact, the surface exhibited the colored according to the inner and the contacting atoms, whereas the intermolecular Vander Waals interactions are given in Fig. 4. The dnorm surfaces are mapped over a fixed color scale of -0.787 to 1.402 Å. The shape index was mapped in the color range of -1 to 1, and the curvedness was in the range of -4−0.4. The surfaces were shown to be transparent to allow the visualization of the molecular moiety in a similar orientation for all the structures, around which they were calculated. The dnorm values were mapped onto the Hirshfeld surface, using a red-blue-white color scheme as follows: red regions represented closer contacts and a negative dnorm value; blue regions represented longer contacts and a positive dnorm value; and white regions represented the distance of contacts equal to exactly the vdW separation with a dnorm value of zero. The shape index is highly sensitive to very subtle changes in the surface shape; the information conveyed by the shape index is consistent with 2D fingerprint plots. The curvedness is the measurement of “how much shape” there is in a crystal. In dnorm surfaces, the large circular deep red colored depressions indicate hydrogen bonding contacts and other spots which are due to C···C and O···O interactions.

15

Fig.4. Hirshfeld surfaces of (C8H12N)2[Mn(H2P2O7)2(H2O)2]: (a) 3D dnorm surface, (b) surface index and (c) curvedness. The 2D fingerprint plots can be deconstructed to highlight particular atom pair contacts. Fig.5 shows the two dimensional fingerprint plots of the studied (1) salt crystal, where various spikes point out diverse types of interactions between organic and inorganic molecules. The 2D fingerprint plots allow quantitative data to be obtained based on the percentage of element interactions contribution in the molecule. 58.1% improvement with pair of sharp spikes towards Hirshfeld surfaces were noticed from O···H, hydrogen bond contacts (Fig. 6).The fingerprint plots show the dominance of O···H contacts. However, 30.9% contributions arise from H···H contact, appearing as sharp spikes between two peaks of hydrogen bond over Hirshfeld surface of the studied salt (Fig. 6(b)), provided by the key role ascribed to the hydrogen bonds in creating a strong cohesion between molecules. In addition, the C···H, C···C and O···O contacts have low contributions of 5.3%, 1.8% and 3.7%, respectively, as presented in Fig. 6(c), 6(d) and 6(e) and Table 7. However, the void surface volume of the grown crystal is 503.27 Å3 per unit cell, and its surface area is of 441.05 Å2 , while from single crystal XRD analysis, the volume of the unit cell is of 703.13 (18) Å3 for the zinc sample.

16

Fig.5. Hirshfeld surface mapped dnorm. Dotted lines indicate hydrogen bonds and 2D fingerprint plots of (C8H12N)2[Mn(H2P2O7)2(H2O)2]. (a)

(b)

H….H

(c)

O….O

O….H

(e)

(d)

(e)

C….C

C….H

Fig. 6. 2D fingerprint plots of ionic salt, showing reciprocal contacts and resolved into: H….H (a), O….H/H….O (b), O…..O (c), C….C (d) and C….H/H….C (e) (di is the closest internal distance from a given point on Hirshfeld surface, and de is the closest contact point external to the surface). 3.4. Thermal decomposition 17

Thermogravimetric/differential thermal analyses (TG/DTA) were performed to check the thermal stability of each compound. The curves obtained during the decomposition of the three compounds 1-3, under flowing air with a heating rate of 10 °C/min between 12 and 800 °C, are reported, respectively, in Figs. 7 (a) and (b). The examination of the curves reveals that these complexes possess similar behavior of thermal stability. This demonstrates that the decomposition of the compounds is complex and takes place through two stages. First, the compounds start losing weight at about 82°C, 120°C and 140°C, respectively, accompanied by a weight loss of about 8.6%, 8.9% and 10% for compounds (1), (2) and (3), respectively. The main weight loss can be attributed to the loss of two water molecules, leading to the anhydrous diphosphate. Besides, the second stage starts at 109°C, 265°C and 132°C, respectively, for compounds (1), (2) and (3) and ends at about 1100°C for the first and second samples and about 900°C for the third sample. This result is evidenced by the calculated weight loss of 92% and 72.2% for compounds (1) and (2), respectively. Moreover, such decomposition process is accompanied by a significant endothermic peak. The endothermic character has already been explained by the destruction of the organic entities together with the compounds. Otherwise, the destruction of the organic rings of compound (3) underwent two steps, with a calculated weight loss of 54.9% and 29.37%.

(a)

(b) 4

100 3

80

2

Mn-(1) 1 -1

Heat flow (W.g )

Weight loss (%)

60 40 20

Mn-(1) 0 -20

Ni-(2)

-40

Zn-(3)

0

Ni-(2) -1 -2

Zn-(3)

-3 -4 -5 -6

-60 0

200

400

600

800

1000

1200

0

200

400

600

800

Temperature (°C)

Temperature (°C)

18

1000

1200

Fig.7. (a) Thermogravimetric analysis and (b) Differential thermal analysis scan for the decomposition of compounds (1), (2) and (3). 3.5. UV-vis spectra This study aimed to report the UV-visible spectra of 2,6-dimethylanilinium-MIIdiphosphate with MII = Mn, Ni and Zn, including the transition of the orbital electrons σ and π from the ground state to the higher energy states. The hybrids often have absorption bands in the UV-visible region. These bands characterize the electronic d-d and charge transfer (CT) transitions and have an unequal intensity. The d-d transitions could be simply assigned by using Tanabe-Sugano diagrams for octahedral sites [56]. Nevertheless, in the first approximation we could describe the distorted octahedron with four short bonds and two longest ones where Mn2+ ions occupy the octahedral sites. Fig. 8 shows the diffuse absorbance spectrum of (C8H12N)2[Mn(H2P2O7)2(H2O)2]. The d-d transitions with different spins (Mn2+) are very weak. The weak peaks observed between 340 and 700 nm in the diffuse absorbance spectrum of (C8H12N)2[Mn(H2P2O7)2(H2O)2] were labeled using the Tanabe–Sugano diagram for the d5 configuration. However, when six ligands approach in the octahedral coordination, the ground state term symbol becomes 6A1g in weak field. From tanabe-Sugano diagram, it can clearly be seen that there is no spin allowed d-d transitions in high-spin d5 Mn2+ complex (Laporteforbidden and spin-forbidden). The absorption peaks observed at 576 nm (17361 cm-1, ε= 0.51 102 L.mol-1.cm-1), 483 nm (20703cm-1, ε= 0.71 102 L.mol-1.cm-1), 392 nm (25510 cm-1, ε= 0.72 102 L.mol-1.cm-1) and 320 nm (31250 cm-1, ε= 0.51 102 L.mol-1.cm-1) are assigned to the d–d transitions in Mn2+ from the ground state 6A1g to the 4T1g(G), 4T2g(G), 4A1g/4Eg, (G), 4T2g(D), 4Eg(D) levels, respectively. The d-d transitions are more intense while ε is in the range of 102-103. At this state, the transitions from this ground state are expected to be spin-forbidden and the very low band's

19

intensities can be shown with the relaxation mechanism [57-58]. The strongest bands in the UV region appear in the (1): 206−231 nm (ε= 1.5 102-2.44 102 L.mol-1.cm-1) which can be attributed to the electrons pertaining’s excitation to aromatic ring systems intra ligand charge transfer (π-π*). The former absorption at 231−300 nm (ε= 4.28 102 L.mol-1.cm-1), is predominantly band due to the (n-π*) charge transfer. The UV–vis spectrum of the nickel complex exhibits two absorption bands at 202−238 nm and 238−283 nm. The former absorption at 238 nm (42016 cm-1) can be assigned to the π–π* transition of the aniline rings. The band around 283 nm (35335 cm-1) is relative to the n–π* transition. The visible spectrum of the complex displayed maxima in the 770 nm (12987 cm-1), 664 (15060 cm-1), 411nm (24330 cm-1) and 309 nm (32362 cm-1). The strongest band appears in the 320 nm, which is predominantly due to an intra-ligand charge transfer (π-π*). In addition, the absorption peaks observed at 770 nm (ε= 0.57 102), 664 (ε= 0.41 102), 411nm (ε= 0.96 102) arises due to the d–d transition from the ground state 3A2g(F) which is plotted along the x-axis from the tanabe-Sugano diagram, the first spin allowed transition is to the 3T2g(P) level, the second spin allowed transition is to the 3T1g(F) and the third transition is to the 3T2g(F) (Laporte -forbiden spin allowed d-d) [59]. The two bands in the wavelength region of the zinc complex: 202−240 nm and 240−295 nm, respectively, resulting from the excitation of electrons pertaining to aromatic ring systems and O2-―Zn2+ charge transfer. The molar extinction coefficient ε was obtained using the Beer–Lambert’s law: A = εlc (1) where A is the absorbance, l the path length of the cell filled with the sample (cm) and c the concentration of the compound in solution (mol.L−1).

20

1,0

0,7 0,2 0,2

0,6

Absorbance (a.u)

0,6

Absorbance (a.u)

0,5

Absorbance (u.a)

0,4 0,0 400

600

800

Wavelength (nm)

0,4

0,1

0,3 0,0 400

600

0,2

800

Wavelength (nm)

0,2

Ni-(2)

0,1

Mn-(1)

0,0

0,0 200

400

600

800

200

1000

400

600

800

Wavelength (nm)

Wavelength (nm)

0,8

1,0 0,6

Absorbance (a.u)

0,8

Absorbance (a.u)

Absorbance (u.a)

0,8

0,6

0,4

0,2

0,4 0,0 400

600

800

Wavelength (nm)

0,2

Zn-(3) 0,0 200

400

600

800

1000

Wavelength (nm)

Fig. 8. UV-visible spectra of (2,6-(CH3)2C6H3NH3)2[MII(H2P2O7)2(H2O)2] (MII= Mn (1), Ni (2), Zn (3)). 3.6. NMR spectra Fig. 9 displays the 1H,

13

C and

31

P NMR spectra obtained for the compound (3). At 4.69

ppm a singlet is due to the solvent. In each spectrum of (C8H12N)2[Zn(H2P2O7)2(H2O)2],the singlet chemical shift at 2.15 ppm is attributed to the protons of (CH3) functional group. The characteristic two doublets at 7.2-7.4 ppm belong to the protons of NH3 group and of aromatic ring pertaining to the 2.6-xylidine. The confirmation of the presence of C―N functional group in 2.6-dimethylaniline appears at 132.1272 ppm and the presence of aromatic carbon chemical shift is at 129.8121 and 129.2842 ppm. Based on the 31P NMR spectra, the presence of P2O7 is confirmed by a signal at -9.4637 ppm. Accordingly, all the characteristic functional groups of

21

1000

the organic-inorganic compounds were confirmed using the proton, carbon and phosphorus NMR analyses. (a)

(b)

(c)

Fig.

9:

1

H

NMR

(a),

31

P

NMR

(b)

and

13

C

NMR

spectra

of

2,6-

(CH3)2C6H3NH3)2[Zn(H2P2O7)2(H2O)2] in D2O. 3.7. Cytotoxicity Hybrid transition metal frameworks are the freshly synthesized compounds, which show conspicuous in vitro antitumor activity against a wide range of human tumor cell lines, borrowed from solid tumors by cytotoxicity assays. Cancer cell lines representative of a large number of different disease cells. The cytotoxicity parameters are shown in terms of IC50 (half maximal inhibitory concentration, a measure of the potency of a sample in inhibiting a specific 22

biological function, whose concentrations are calculated from dose-survival curves via MTT and SRB tests); after 72h the cytotoxicity parameters IC50 are listed in Tables 8 and 9. As a reference, the cytotoxicity of the cisplatin is the most commonly used anticancer drug that appraises under the same experimental conditions. It has manifested activity against several tumors and nowadays is used in clinical chemotherapy for the treatment of testicular, ovarian, head, neck and small cell lung cancers [60]. The aim of our study was to investigate the role of metal-drugs in the treatment of cisplatin resistant tumor cells in vitro. Indeed, in this study, all metal drugs were also tested for their in vitro antitumor activity on three human cell lines, derived from a variety of solid tumors. Accordingly, we used well-characterized human cervical cancer (A431), ovarian cancer cells (2008) and Pancreatic (PSN1) cell lines. During our treatment the cell lines we utilized displayed a regularly good response. The IC50 values range from less than 0.6 µM to 8 µM and the importance of our metallo-drugs is ascribed to the fact that the average IC50 values obtained are always near or lower than those found with the reference drug, which is the cisplatin and the half maximal inhibitory concentration tested by the MTT test, then confirmed by the SRB with the cell lines (A431). All the complexes exhibit a consistently good response towards 2 treatments. Table 8 illustrates the cytotoxicity parameters (the median growth inhibitory concentrations calculated from dose survival curves via MTT test) for the three complexes and cisplatin. Complex (1) was more effective than cisplatin that showed average IC50 values towards the cervical cell line of 3.92 µM. Similarly, complex (2) and cisplatin presented average IC50 values towards the cervical cell (A431) panel of 6.25 and 5.62 µM, respectively. Complex (3) was roughly the most promising and effective compared to cisplatin against human cervical A431 cell (mean IC50 values being 2.47 and 5.6 µM for (1) and cisplatin, respectively). Moreover, the cytotoxicity was assessed by the SRB assay against human cervical A431 cell

23

panel, complexes (1), (2) and (3), which were roughly 4- and 2-fold more effective than the reference drug with an average IC50 1.45, 3.5, 3.6 and 6.62 µM, respectively (see Table 9). Remarkably, against ovarian (2008) cell lines, the cytotoxic potency of complexes (1) and (2), were similarly effective compared to cisplatin with collected IC50 values 2.1, 1.8 and 2.28 µM, respectively. Interestingly, complex (3) showed the greatest anti-tumor efficacy with an average IC50 over ovarian cell type that was over 4 times lower than that of cisplatin (IC50 values being 0.65 and 2.28 µM for (3) and cisplatin, respectively). Table 8 shows the comparison of IC50 values detected by MTT assay in PSN1 cells treated with complexes (1), (2), (3) and cisplatin. Complex (1) was 3 times more effective than cisplatin with an average IC50 values, 1.88 and 5.62 µM, respectively. Complexes (2) and (3) have good response with mean IC50 values being 4.16, 2.76 and 5.62 µM, respectively. Complex (1) is about 1- and 2-fold more effective than (2) and (3), respectively. Usually, the mixed copper (I) complexes are the most useful and tested for the in vitro antitumor activity on the human cell lines because of the effect of the copper that is present in the human morphology, however, the innovation uses new metal-drugs with the same tests. 4. Conclusion We synthesized new hybrid template complexes (C8H12N)2[MII(H2P2O7)2(H2O)2] (with MII = Mn, Ni and Zn), which crystallize in a triclinic system with a centrosymmetric space group Pī. The crystalline packing of the complexes are stabilized by hydrogen bonding interactions O―H···O and N―H···O. Hence, the quantitative examination of the structures is observed by means of the analysis of the Hirschfield surface in the form of decomposed fingerprint plots, showing the intermolecular interactions in the structures. Based on NMR spectroscopy, the protonation of the NH2 group is confirmed. The TG/DTA studies confirmed the thermal stability of the crystals up to 102°C. The UV–visible absorption, recorded at room temperature, allowed their charge transfer. Finally, anti-tumor activity revealed that the 24

centrosymmetric structures of the title compounds (1)−(3) are so effective against human cervical (A431), ovarian (2008) and pancreatic (PSN1) cancer cell panels. All metal drugs were tested for their in vitro antitumor activity, using MTT and SRB assays.

Supplementary material Crystallographic data for CCDC-1938393 (Mn), CCDC-1938443 (Ni) and CCDC-1938444 (Zn) can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

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31

Table 1. Assignments of the bands of the infrared absorption spectra for (C8H12N)2[MII(H2P2O7)2(H2O)2] with (MII= Mn (1), Ni(2), Zn (3)).

Vibrational mode assignement (cm-1) υ2(PO4) P―O―P (υ P2O74-) υ3(PO4) Asymmetric stretching P―O―P C―N stretching C―C ring stretching NH3+ Symmetric bending mode Aromatic CH υ(O―H) coordinated water molecule

Compound (1) MII = Mn 494 768 950 1183

Compound (2) MII = Ni 493 770 953 1172

Compound (3) MII = Zn 492 768 961 1194

1294 1484 1529

1309 1475 1538

1299 1485 1530

2930 3569

3000 3578

3005 3579

32

Table 2. Crystal data, summary of intensity data collection and structure refinement.

Structural parameter Formula weight (g mol-1) Temperature (K) Crystal system Space group a(Å) b (Å) c (Å) α(°) β(°) γ(°) Z Crystal color/shape Crystal size (mm3) Diffractometer λ(MoKα) (Å) ρcal (mg m-3) Absorption correction µ(mm-1) Θ ranges (deg)

hkl range No. of reflection collected No. of independent reflection

Crystal data Compound (1) Compound (2) C16H32MnN2O16P4 C16H32NiN2O16P4 687.26 691.01 293(2) 293(2) Triclinic Triclinic Pī Pī 7.390(10) 7.290 (6) 7.669(10) 7.544(7) 13.373(19) 13.462(12) 85.305(12) 85.439 (3) 75.257(12) 75.623 (3) 73.595(12) 74.860 (3) 2 2 colorless/ Block green/ Block 0.14× 0.07 × 0.05 0.20 × 0.08 × 0.07 Intensity data collection Xcalibur Bruker D8-Venture 0.71069 0.71069 2.466 2.307 Analytical Analytical

Compound (3) C16H32ZnN2O16P4 697.71 293(2) Triclinic Pī 7.322(0) 7.572(0) 13.410(0) 85.390(0) 75.430(0) 74.570(0) 2 colorless/ Block 0.28 × 0.25 × 0.18 Kappa CCD 0.71069 1.671 Analytical

1.49 θmin= 2.9 θmax=26.1 −8 ≤ h ≤9 −9≤ k ≤9 −16 ≤ l ≤15 6026

1.01 θmin= 2.8 θmax=30.6 −9 ≤ h ≤10 −10≤ k ≤10 −17 ≤ l ≤19 9893

1.191 θmin= 1.6 θmax=28.7 −9 ≤ h ≤9 −10≤ k ≤10 −18 ≤ l ≤18 6042

2549

4168

3585

Structure refinement Programs system SHELXL-2016 and SHELXS-86 Refinementbased on F2 F (000) 355 358 R1 0.053 0.062 wR2 0.119 0.160 GooF 1.028 0.985 No. param. 205 194 Transmission factor Tmin = 0.863 Tmin = 0.734 Tmax = 1.000 Tmax = 1.000

33

360 0.058 0.165 1.028 194 Tmin = 0.620 Tmax = 0.783

Table 3. Hydrogen-bonding geometry (Å, °) for compounds (1), (2) and (3).

(C8H12N)2[Mn(H2P2O7)2(H2O)2] D—H···A d(D—H)(Å) d(H···A)(Å) d(D···A)(Å) ∠D—H···A(°) ii O3—H1···O5 0.82 1.75 2.533 (4) 159 O8—H3···O1iii 0.82 2.49 2.958 (4) 117 iii O8—H3···O5 0.82 2.52 3.336 (4) 175 O8—H3···Niv 0.82 2.57 3.167 (5) 119 v O8—H4···O6 0.79(5) 2.04(5) 2.821 (4) 169(5) N—H10···O5v 0.77(5) 2.08(5) 2.841 (5) 170(5) N—H9···O4iii 0.91(5) 1.90(5) 2.801 (5) 170(5) vi N—H8···O2 0.79(6) 2.40(6) 3.031 (5) 137(5) N—H8···O7iv 0.79(6) 2.37(6) 3.010 (5) 139(5) Symmetry codes: (ii) –x, -y+1, -z+1 ; (iii) x+1, y, z ; (iv) –x+2, -y, -z+1 ; (v) –x+1, -y, -z+1 ; (vi) x+1, y-1, z. (C8H12N)2[Ni(H2P2O7)2(H2O)2] D—H···A d(D—H)(Å) d(H···A)(Å) d(D···A)(Å) ∠D—H···A(°) ii O4—H4A···O6 0.82 1.71 2.523 (5) 173 O—HA···N7iii 0.85 1.97 2.818 (5) 172 iv O7—H7···O5 0.82 1.75 2.498 (5) 151 N1—H1A···O5iii 0.89(7) 1.91(7) 2.797 (5) 177 (6) N1—H1B···O6ii 0.80(6) 2.03(6) 2.821 (5) 172 (6) N1—H1C···O1i 0.79(7) 2.38(7) 3.012 (5) 138 (6) Symmetry codes: (i) -x, -y+1, -z; (ii) −x+1, −y+1, −z; (iii) x, y-1, z; (iv) −x, −y+2, −z. (C8H12N)2[Zn(H2P2O7)2(H2O)2] D—H···A d(D—H)(Å) d(H···A)(Å) d(D···A)(Å) ∠D—H···A(°) O3—H3···O5ii 0.82 1.75 2.533 (4) 159 iii O8—H8A···O1 0.82 2.49 2.958 (4) 117 O8—H8A···O5iii 0.82 1.52 3.336 (4) 175 iv O8—H8A···N 0.82 2.69 3.167 (5) 119 O8—H8B···O6v 0.79(5) 2.04(5) 2.821 (4) 169(5) N—HA···O5v 0.77(5) 2.05(4) 2.841 (5) 170(5) N—HB···O4iii 0.91(5) 1.90(5) 2.801 (5) 170(5) vi N—HC···O2 0.79(6) 2.40(6) 3.031 (5) 137 (5) N—HC···O7iv 0.79(6) 2.37(6) 3.050 (5) 139 (5) Symmetry codes: (ii) –x, -y+1, -z+1 ; (iii) x+1, y, z ; (iv) –x+2, -y, -z+1 ; (v) –x+1, -y, -z+1 ; (vi) x+1, y-1, z.

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Table 4. Selected bond distances (Å) and angles (°) of MIIO6 octahedron for compounds (1)-(3). Within the Mn (1) octahedron Mn1—O1 Mn1—O2 Mn1—O8 O1—Mn1—O2 O1—Mn1—O2i

2.134 (3) 2.141 (3) 2.230 (4) 88.30 (11) 91.70 (11)

O2—Mn1—O8 O2—Mn1—O8i O1—Mn1—O8 O1—Mn1—O8i

88.18 (14) 91.82 (14) 93.88 (14) 86.12 (14)

Ni1—O Ni1—O1 Ni1—O2 O—Ni1—O1 O—Ni1—O1ii

Within the Ni (2) octahedron 2.091 (3) O—Ni1—O2 2.036 (3) O—Ni1—O2ii 2.051 (3) O1—Ni1—O2 93.18 (14) O1—Ni1—O2ii 86.82 (14)

88.38 (13) 91.62 (13) 90.69 (12) 89.31 (12)

Within the Zn (3) octahedron Zn—O2 2.057 (3) O2—Zn—O8 91.82 (12) iii Zn—O7 2.060 (3) O2—Zn—O8 88.18 (12) Zn—O8 2.251 (3) O7—Zn—O8 86.57 (12) iii O2—Zn—O7 90.07 (11) O7—Zn—O8 93.43 (12) O2—Zn—O7iii 89.93 (11) Symmetry code: (i) –x, -y+2, -z+2, (ii) –x, -y+1, -z, (iii) –x+1, -y+1, -z+1.

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Table 5. Selected bond distances (Å) and angles (°) for [PO4] tetrahedrons, forming the H2P2O72- anion in compounds (1)-(3). Within the PO4 tetrahedron of Mn (1) P(1)O4 P1—O2 P1—O3 P1—O6 P1—O7 O2—P1—O3 O2—P1—O6 P(2)O4 P2—O1 P2—O3 P2—O4 P2—O5 O1—P2—O3 O1—P2—O4 P(1)O4 P1—O2 P1—O3 P1—O4 P1—O5 O2—P1—O3 O3—P1—O4 P(2)O4 P2—O1 P2—O3 P2—O6 P2—O7 O1—P2—O3 O1—P2—O6 P(1)O4 P1—O1 P1—O5 P1—O6 P1—O7 O1—P1—O5 O1—P1—O6 P(2)O4 P2—O1 P2—O2 P2—O3 P2—O4 O1—P2—O2

1.477 (3) 1.617 (3) 1.544 (3) 1.491 (1) 108.53 (18) 113.03 (19)

O2—P1—O7 O3—P1—O6 O3—P1—O7 O6—P1—O7

1.488 (3) O1—P2—O5 1.616 (3) O3—P2—O4 1.494 (3) O3—P2—O5 1.549 (3) O4—P2—O5 109.53 (17) 116.72 (19) Within the PO4 tetrahedron of Ni (2) 1.494 (3) 1.615 (4) 1.551 (3) 1.490 (3) 108.35 (17) 101.67 (19)

O4—P1—O5 O5—P1—O2 O2—P1—O4 O3—P1—O5

1.489 (3) O1—P2—O7 1.610 (3) O6—P2—O7 1.498 (3) O6—P2—O3 1.557 (3) O7—P2—O3 109.49 (17) 116.8 (2) Within the PO4 tetrahedron of Zn (3)

116.09 (18) 101.14 (17) 108.51 (18) 108.41 (19)

111.58 (18) 104.38 (17) 103.80 (19) 109.81 (18)

108.25 (19) 115.99(19) 102.55 (19) 109.0 (2)

111.52 (19) 109.5 (2) 104.82 (19) 103.6 (2)

1.605 (3) 1.486 (3) 1.547 (3) 1.483 (3) 104.78 (16) 104.02 (17)

O1—P1—O7 O5—P1—O6 O5—P1—O7 O6—P1—O7

109.45 (16) 109.55 (17) 116.81 (17) 111.29 (17)

1.613(3) 1.487 (3) 1.545 (3) 1.485 (3) 107.67 (16)

O1—P2—O4 O2—P2—O3 O2—P2—O4 O3—P2—O4 O1—P2—O3

108.71 (18) 113.41 (17) 116.78 (17) 107.58 (17) 101.54 (16)

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Table 6. Selected bond distances (Å) and angles (°) within the organic ring (C8H12N) for Compounds (1)-(3).

C1―C2 C2―C3 C3―C4 C4―C5 C5―C6 C6―C1 C6―C7 C2―C8 N―C1 C6―C1―N C2―C1―N C1―C2 C2―C3 C3―C4 C4―C5 C5―C6 C6―C7 C6―C1 C2―C8 C1―N1 C2―C1―N1 C6―C1―N1 C1―C2 C2―C3 C1―C4 C3―C5 C1―C6 C3―C7 C7―C8 C6―C8 C2―N C1―C2―N C3―C2―N

(C8H12N)2[Mn(H2P2O7)2(H2O)2] 1.389 (7) C1―C6―C5 1.388 (7) C1―C6―C7 1.367 (8) C5―C6―C7 1.376 (8) C6―C1―C2 1.388 (7) C1―C2―C3 1.385 (7) C1―C2―C8 1.504 (7) C3―C2―C8 1.505 (7) C4―C3―C2 1.481 (6) C4―C5―C6 117.3 (4) C3―C4―C5 119.0 (4) (C8H12N)2[Ni(H2P2O7)2(H2O)2] 1.387 (8) C1―C6―C5 1.399 (8) C1―C6―C7 1.362 (11) C5―C6―C7 1.376 (11) C2―C1―C6 1.400 (8) C1―C2―C3 1.402 (7) C1―C2―C8 1.484 (9) C3―C2―C8 1.503 (9) C4―C3―C2 1.472 (6) C3―C4―C5 119.7 (5) C4―C5―C6 116.4 (5) (C8H12N)2[Zn(H2P2O7)2(H2O)2] 1.381 (7) C2―C1―C6 1.406 (7) C2―C1―C4 1.485 (8) C6―C1―C4 1.497 (7) C1―C2―C3 1.401 (7) C7―C3―C2 1.382 (7) C7―C3―C5 1.370 (9) C2―C3―C5 1.365 (9) C8―C6―C1 1.475 (5) C8―C7―C3 119.5 (4) C6―C8―C7 116.4 (4)

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116.8 (5) 122.8 (5) 120.4 (5) 123.7 (5) 116.4 (5) 123.0 (5) 120.6 (5) 122.1 (6) 121.6 (6) 119.4 (6)

116.2 (6) 123.0 (5) 120.8 (6) 123.9 (5) 116.2 (6) 123.3 (5) 120.6 (6) 122.3 (7) 119.9 (6) 121.5 (6)

116.2 (5) 123.8 (5) 120.1 (5) 124.0 (4) 115.9 (5) 121.8 (5) 122.3 (4) 121.7 (6) 122.2 (6) 120.0 (5)

Table 7. Contributions (%) of intermolecular atom-atom contacts to the Hirshfeld surface.

Zinc compound

C····H/H····C 5.3%

O····H/H····O 58.1%

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O···O 3.7%

H····H 30.9%

C····C 1.8%

Table 8. In vitro antitumor activity of samples (1), (2) and (3). IC50 (µM) ± D.S.a Compound A431 2008 PSN1 (2.6-(CH3)2C6H3NH3)2[Mn(H2P2O7)2(H2O)2] (1) 3.92 2.1±0.35 1.88±0.28 (2.6-(CH3)2C6H3NH3)2[Ni(H2P2O7)2(H2O)2] (2) 6.25±1.63 1.8 4.16±1.36 (2.6-(CH3)2C6H3NH3)2[Zn(H2P2O7)2(H2O)2] (3) 2.47±0.31 0.65±0.07 2.76±0.85 Cisplatin 5.62±0.16 2.28±0.5 5.62±0.16 3 -1 Cells (3-8 × 10 mL ) were treated for 72h with increasing concentrations of the tested compounds. The cytotoxicity was assessed by the MTT assay. IC50 values were calculated by a four-parameter logistic model, 4-PL (P<0.05). aSD= standard deviation.

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Table 9. Cytotoxicity activity against A431 cancer cells with SRB assay of all samples. Compound A431 1.45 (2.6-(CH3)2C6H3NH3)2[Mn(H2P2O7)2(H2O)2] (1) 6.25±1.63 (2.6-(CH3)2C6H3NH3)2[Ni(H2P2O7)2(H2O)2] (2) 3.5±1.7 (2.6-(CH3)2C6H3NH3)2[Zn(H2P2O7)2(H2O)2] (3) 6.625±0.52 Cisplatin 3 -1 Cells (3-8 × 10 mL ) were treated for 72h with increasing concentrations of the tested compounds. The cytotoxicity was assessed by the SRB assay. IC50 values were calculated by a four-parameter logistic model, 4-PL (P<0.05). aSD= standard deviation.

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Highlights


Hybrid and centrosymmetric single crystals of (2.6-(CH3)2C6H3NH3)2[MII(H2P2O7)2(H2O)2] with (MII = Zn (1), Mn (2), Ni (3)) have been successfully synthesized and structurally characterized.
N/O―H···O hydrogen bonds and π···π interactions give rise to three-dimensional stable supramolecular networks.
The inorganic-organic hybrid diphosphates are ultra-efficient against human cervical (A431), ovarian (2008) and pancreatic (PSN1) cancer cell panels.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: