TMEDA with some f block metals: Synthesis, DFT studies, spectral, thermal, cytotoxicity and antimetastatic properties

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 117938

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Heteroleptic complexes of cocaine/TMEDA with some f block metals: Synthesis, DFT studies, spectral, thermal, cytotoxicity and antimetastatic properties Nadia G. Zaki a, Walaa H. Mahmoud b, Ahmed M. El Kerdawy c,d,e, Abanoub Mosaad Abdallah a,⁎,1, Gehad G. Mohamed b,f a

Narcotic Research Department, National Center for Social and Criminological Research (NCSCR), Giza 11561, Egypt Chemistry Department, Faculty of Science, Cairo University, Giza 12613, Egypt c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo, P.O. Box 11562, Egypt d Molecular Modeling Unit, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo, P.O. Box 11562, Egypt e Department of Pharmaceutical Chemistry, Faculty of Pharmacy, New Giza University, Newgiza, km 22 Cairo–Alexandria Desert Road, Cairo, Egypt f Egypt Nanotechnology Center, Cairo University, El-Sheikh Zayed, 6th October City, Giza 12588, Egypt b

a r t i c l e

i n f o

Article history: Received 19 September 2019 Received in revised form 29 November 2019 Accepted 7 December 2019 Available online 09 December 2019 Keywords: Cocaine TMEDA Heteroleptic complexes TD-DFT Apoptosis p21 and p27 tumor suppressor proteins Antimetastatic effect Cell oxidative stress

a b s t r a c t A series of new three heteroleptic complexes of the general formula [Ln(Cn)(TMEDA)Cl(OH2)]·2Cl·xH2O, (where Ln = La(III), Er(III) and Yb(III), Cn = cocaine and TMEDA = N,N,N′,N′-tetramethylethylenediamine) were synthesized, structurally characterized by elemental analysis, spectroscopic methods, molar conductivity and mass spectrometry. Thermal properties of the synthesized complexes and their kinetic thermodynamic parameters were studied. Theoretical calculations including geometry optimization, electronic structure and electronic and thermal energies were carried out using DFT and TD-DFT calculations at B3LYP/LANL2DZ level of theory and the different quantum chemical parameters were calculated. The in vitro antiproliferative activity of the newly synthesized complexes was assessed by MTT assay on MCF-7 and HepG-2 cancer cell lines. Yb(III) complex showed promising cytotoxic activity comparable to that of cisplatin on both cell lines with minimum effect on human normal cells. Further molecular mechanistic investigations showed that Yb(III) complex is an apoptotic inducer as it raises the caspase-3 and caspase-9 cellular level in the MCF-7 cell line. Furthermore, it showed an elevating effect on the level of the tumor suppressor nuclear proteins P21 and P27 concentrations in MCF-7 cells. Moreover, Yb(III) complex hindered the cellular scavenger system of the reactive oxygen species through reducing the glutathione peroxidase (GPx) cellular level imperiling MCF-7 cells by unmanageable oxidative stress. In addition to its cytotoxic effect, Yb(III) complex showed antimetastatic properties as it decreased the cellular levels of matrix metalloproteinases MMP-3 and MMP-9. These results showed that the Yb(III) complex is a promising cytotoxic metal-based agent that exerts its action through various molecular mechanisms with minimum effects on normal cells and with additional antimetastatic properties. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Cancer is an uncontrolled cell multiplication with a successive abnormal increase in cell numbers in certain organs [1]. One of the hallmarks of cancerous tumors is metastasis which is the process by which primary cancer cells disseminate to a neighboring or distant location in the body through the hematopoietic and/or lymphatic systems and establish secondary cancers. Metastasis is the main cause of most cancer-related deaths among patients with solid tumors [2].

⁎ Corresponding author. E-mail address: [email protected] (A.M. Abdallah). 1 This work is abstracted from his PhD.

https://doi.org/10.1016/j.saa.2019.117938 1386-1425/© 2019 Elsevier B.V. All rights reserved.

Despite the remarkable progress made in its treatment and prevention, breast cancer is still the most frequent cancer among women worldwide causing the highest number of cancer-related deaths among them [3]. In Egypt, breast cancer amounted to 18.3% of the total cancer cases discovered in 2016 [4]. On the other hand, liver cancer is the fifth most common malignancy and ranks as the third prevalent cause of cancer-related deaths worldwide [5]. Since the discovery of cis-diamminedichloroplatinum (II) (cisplatin), metallopharmaceuticals have been extensively investigated as anticancer agents in the last few decades [6–10]. Moreover, heteroleptic complexes have gained particular attention as an important category of this class [11–16]. This type of complexes, with two different ligands, results in the formation of complexes with stepwise dissociation during the metal-DNA complex formation and so enhances the cellular uptake

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P27) level and effect on glutathione peroxidase (GPx) level in MCF-7 cells. 2. Experimental 2.1. Synthesis of the heteroleptic complexes

Fig. 1. Chemical structure of TMEDA.

and antitumor activity [17]. N,N,N′,N′-tetramethylethane-1,2-diamine (TMEDA), Fig. 1, has been widely involved in metal complexation [18–22] due to its ability to enhance the metalation rate of a variety of aromatic systems as well as improving the regiochemical outcome for these reactions [23]. Compounds containing the azabicyclo[3.2.1]octane skeleton (Fig. 2) exhibit desirable pharmacological effects such as anti-inflammatory, analgesic and anticancer effects and are occasionally used in drug molecules [24–31]. Cocaine (Cn) (Fig. 3a), an alkaloid extracted from the coca bush (Erythroxylum coca) leaves, is a naturally occurring azabicyclo[3.2.1]octane [32,33]. Owing to their high biological activities, low toxicity and applicability in DNA interaction for anticancer activity, lanthanide-based transition metal complexes have been extensively investigated as anticancer agents against various cancer cells [34–38]. Furthermore, among various lanthanide-metal complexes, La(III), Er(III) and Yb(III) complexes have been deeply examined due to their more apoptotic and antioxidant activities after coordination with ligands [39–42]. In the present work, La(III), Er(III) and Yb(III) heteroleptic complexes of cocaine with TMEDA were synthesized then structurally characterized by a variety of physicochemical tools such as elemental analysis, FT-IR, UV–Vis, mass spectrometry, molar conductance and thermal analysis techniques. Structural, spectroscopic, electronic and thermodynamic properties were also studied theoretically by performing density functional theory (DFT) and time-dependent density functional theory (TD-DFT) calculations to correlate between the experimental and theoretical results. The newly synthesized complexes were also investigated for their antiproliferative activity against human liver HepG-2 and breast MCF-7 cancer cell lines. Further, in vitro testing was carried out to study their antimetastatic properties, apoptosisinducing characteristics, effect on tumor suppressor proteins (P21 and

All chemicals and reagents utilized throughout the present study were of analytical reagent grade (Merck) without further purification. As it is sketched in Scheme 1, all the solid metal complexes were synthesized by adding a hot methanolic solution (60 °C) of the metal chloride (MCln·XH2O) (1.5 mmol; LaCl3·7H2O, 0.56 g; ErCl3·6H2O, 0.57 g; YbCl3·6H2O, 0.58 g) dropwisely to a stirred hot mixture (60 °C) of the methanolic solutions of the two ligands; cocaine (1.5 mmol; 0.51 g) and TMEDA (1.5 mmol; 0.23 mL). The reaction solution was stirred under reflux for 2–3 h and then cooled down to the room temperature, whereupon the solid complexes were precipitated. The formed solid complexes were filtered off, washed with methanol followed by diethyl ether and finally dried under vacuum. 2.2. Instruments The UV–vis measurements were performed using UVmini-1240 UV– vis Shimadzu spectrophotometer with matched 1 cm quartz cells within the 200–700 nm wavelength range. Infrared spectra were recorded using JASCO FT/IR-4100 spectrometer in the 4000–400 cm−1 region as KBr pellets. Elemental analyses (C, H, N and Cl) were carried out using Thermo Scientific Flash 2000 Organic Elemental Analyzer at the Microanalytical Center, Cairo University. Melting points' values were measured using the Stuart SMP30 instrument. Metal contents of the solid complexes were estimated by the dissolution in conc. HNO3 and dissolving the residue in deionized H2O. The metal content was performed using inductively coupled plasma atomic absorption spectrometry (ICP-AAS), Egyptian Petroleum Research Institute. Molar conductivities of the metal complexes were measured for their DMF solutions (10−3 M) at 25 ± 2 °C using Jenway 4010 conductivity meter. Mass spectra were recorded at the Microanalytical Center, National Research Center, Egypt, by EI ionization mode using MS-5988 GS-MS HewlettPackard instrument at 70 eV. Thermal analyses (TG and DTG) were performed using Shimadzu thermal gravimetric analyzer from room temperature to 1000 °C under N2 atmosphere with a heating rate of 10 °C min−1.

Fig. 2. Reported examples of azabicyclo[3.2.1]octane-containing compounds.

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Fig. 3. Optimized structures of (a) Cn and (b) the formed La(III) heteroleptic complex, H atoms were omitted in (b) for clarity.

2.3. Theoretical calculations The geometries of cocaine, TMEDA and their heteroleptic La(III) complex (as a representative example for the series) in the ground state in the gas-phase were fully optimized by carrying out DFT calculations using B3LYP functional [43] combined with the basis set LANL2DZ for all atoms [44] using Gaussian 03 package [45] and also the frequency calculations were determined at the same level of theory. Electronic spectra in DMF media were obtained by applying the time-dependent density functional theory (TD-DFT) calculations [46]. 2.4. Cell culture Human HepG-2 liver hepatocellular carcinoma, human MCF-7 breast adenocarcinoma and human normal non-malignant HFB4

melanocytes cell lines were obtained from American Type Culture Collection (ATCC) and routinely cultured in a Dulbecco's modified eagle medium (DMEM, Invitrogen/Life Technologies) supplemented with 10% fetal bovine serum (FBS, Hyclone), 1% penicillin-streptomycin and insulin (10 μg mL−1). All other chemicals and reagents were purchased from Sigma-Aldrich and Invitrogen. All cell lines were grown at 37 °C as adherent monolayers in a humidified atmosphere with 5% CO2. 2.5. Antiproliferative activity To investigate the cytotoxicity of the newly synthesized La(III) and Yb(III) complexes, the MTT assay [47] was utilized. The MTT assay is based on the reduction of the soluble 3-(4,5-methyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT) into a blue-purple formazan product, mainly by mitochondrial reductase activity inside living cells.

Scheme 1. Synthesis of [Ln(Cn)(TMEDA)Cl(OH2)]·2Cl·xH2O complexes.

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Cells were seeded in cell culture treated 96-well flat-bottomed plates at a density of 1.2–1.8 × 104 cells per well. Before adding the tested compounds, the plates were pre-incubated for 24 h in a drug-free medium at 37 °C and 5% CO2. Stock solutions (2 mM/L) of the tested compounds and cisplatin (as positive control) in DMSO were diluted in complete culture medium to obtain final concentrations in the range of 0.39–100 μM. Cells were treated with 100 μL aliquots of these dilutions (100, 25, 6.25, 1.56 and 0.39 μM) for a compound-exposure period of 48 h. Then, the MTT solution was added to each well and incubated further for 2 h. MTT solubilization solution was added to dissolve the resulting MTT formazan crystals. Cell metabolic activity was estimated spectrophotometrically by measuring the absorbance at 450/690 nm using ROBONIK P2000 Elisa Reader and the antiproliferative activity was quantified by comparison to positive (cisplatin) and negative (1% DMSO) controls. The results were expressed as the IC50 (μM), inducing a 50% inhibition of cell growth of treated cells when compared to the growth of untreated cells. IC50 values were determined using concentration-effect curves by interpolation. MTT assay was carried out for three independent experiments per concentration level. 2.6. Induction of apoptosis

18,000 ×g for 20 min at 4 °C. Cell lysate samples and standards (100 μL) were added to the human p21- or p27-specific antibodycoated wells followed by the antibody mix. After incubation for one hour at room temperature, the wells were washed by a wash buffer PT to remove unbound materials. TMB substrate (100 μL) was added and during incubation was catalyzed by HRP, generating a bluecolored product. This reaction was then stopped by the addition of 100 μL of the stop solution into each well completing any color change from blue to yellow. The optical density was generated proportionally to the amount of bound p21 or p27 proteins and the intensity was measured spectrophotometrically at 450 nm using ROBONIK P2000 Elisa Reader. 2.8. MMP-3 and MMP-9 level The cellular levels of matrix metalloproteinase-3 (MMP-3) and matrix metalloproteinase-9 (MMP-9) were determined using RayBio® human MMP-3 and MMP-9 ELISA kits from RayBiotech (Norcross, GA, USA). The procedures were carried out according to the protocols provided by the manufacturer. This assay employs an antibody specific for human MMP-3/MMP-9 coated on a 96-well plate. Cell lysate of standards and samples are pipetted into the wells and MMP-3/MMP-9 present in the sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human MMP-3/MMP-9 antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells. The color develops is in proportion to the amount of MMP-3/MMP-9 bound. The stop solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a ROBONIK P2000 spectrophotometer.

Induction of apoptosis was measured using DRG® Caspase-9 (human) ELISA (EIA-4860) kit (DRG International Inc., USA) and Invitrogen Caspase-3 (Active) (human) ELISA kit, Catalog # KHO1091 (96 tests) (Invitrogen Corporation, USA) to quantify the level of human active caspase-3 and caspase-9 proteins according to the manufacturer protocol. The human ELISA kit is a solid-phase sandwich ELISA. MCF-7 cells were treated with Yb(III) complex at IC50 concentration. The cells were harvested by scraping and gentle centrifugation and rinsed with PBS leaving an intact cell pellet (5 × 106 cells) which was then suspended in 1 mL of lysis buffer (1×) and incubated at room temperature for 1 h. Cells were then frozen at −80 °C and thawed with gentle shaking. Freeze/thaw cycle repeated for 3 times, then centrifuged at 1000 ×g for 15 min at 2–8 °C to remove cellular debris. 100 μL aliquots of the diluted cell lysate (standard or samples) were transferred to microwells that were precoated with primary antibodies specific to caspase-3 or caspase-9. Human caspase-3 or caspase-9 present in the aliquots binds to antibodies adsorbed to the microwells. Then, a detection (rabbit) antibody (50 μL) specific to the caspase captured by the primary antibody was added to bind to the caspase, forming a “sandwich” of the specific antibodies around it in the cell lysate which incubated for 2 h at room temperature. The unbound detection antibody was removed by washing the microwell plate twice with approximately 400 μL wash buffer solution (PBS with 1% Tween 20) per well. A 100 μL of diluted anti-rabbit-IgG-HRP (horseradish peroxidase-labeled anti-rabbit IgG) was then added to bind to the detection (rabbit) antibody and incubated further for 2 h at room temperature. The HRP domain was reacted with a tetramethyl-benzidine (TMB) substrate solution (100 μL) forming a colored product that was spectrophotometrically measured at 450 nm using ROBONIK P2000 Elisa Reader after terminating the reaction by the addition of the stop solution (1 M phosphoric acid, 100 μL). The optical density of this colored product is directly proportional to the concentration of human caspase present in the original cell lysate [48].

Glutathione peroxidase (GPx) activity was estimated calorimetrically using glutathione peroxidase assay kit from Abcam® (Cambridge, UK) by measuring the amount of NADPH consumed to reduce GSSG (oxidized form of glutathione) into GSH (reduced form of glutathione) at 340 nm which is directly proportional to the GPx activity. The procedure was performed according to the protocols provided by the manufacturer. Briefly, MCF-7 cells were treated with Yb(III) complex at IC50 concentration. The cells were harvested (2 × 106 cells), washed with cold PBS and resuspended in 200 μL of cold assay buffer. Cells were centrifuged at 10,000 ×g for 15 min at 4 °C to remove any insoluble material. 40 μL aliquots of reaction mix (33 μL assay buffer, 3 μL 40 mM NADPH, 2 μL GR and 2 μL GSH solutions) were transferred to sample (50 μL of MCF-7 cell lysate), positive control (10 μL GPx) and reagent control (50 μL of assay buffer) wells. The volume was adjusted to 50 μL per well with assay buffer before adding the reaction mix. Wells were mixed well and incubated at room temperature for 15 min to deplete all GSSG in the samples. 10 μL cumene hydroperoxide solution was added to start the GPx reaction, mixed well and incubated at 25 °C for 5 min before measuring at 340 nm using ROBONIK P2000 spectrophotometer.

2.7. Measurement of p21 and p27 expressions

3. Results and discussion

For quantitative determination of human p21 and p27 tumor suppressor nuclear proteins in cell lysates, human ab136945 p21 and ab195211 – p27 Kip1 ELISA kits from Abcam® (Cambridge, UK) were used, respectively. MCF-7 cells were treated with Yb(III) complex at IC50 concentration. The cells were harvested by scraping and gentle centrifugation and rinsed twice with PBS. After centrifugation, the pellet was solubilized at 2 × 107 cell mL−1 in chilled 1× cell extraction buffer PTR. The lysate was incubated on ice for 15 min, then centrifuged at

3.1. Elemental analysis

2.9. Cell Oxidative stress

Analytical and physical data of the studied solid complexes were collected and tabulated in Table 1. Elemental analysis results (C, H, N, Cl and Ln) were found to be in a good accordance with the values calculated for the suggested formulae of the [Ln(Cn)(TMEDA)Cl (OH2)]·2Cl·xH2O type with 1:1:1 metal–ligands stoichiometry in which both Cn and TMEDA ligands acted as a neutral bidentate ligand

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Table 1 Analytical and physical data of Cn, TMEDA and their synthesized heteroleptic complexes. MF (Mwt)

Cn [33] C17H21NO4 (g mol−1) TMEDA [56] C6H16N2 (116.24 g mol−1)

[La(Cn)(TMEDA)Cl(OH2)]Cl2·5.5H2O; C23H50Cl3N3O10.5La (781.97 g mol−1) [Er(Cn)(TMEDA)Cl(OH2)]Cl2·2.5H2O; C23H44Cl3N3O7.5Er (755.76 g mol−1) [Yb(Cn)(TMEDA)Cl(OH2)]Cl2·1.5H2O; C23H42Cl3N3O6.5Yb (744.11 g mol−1)

Color

Yield %

M.P. (°C)

White

–

Colorless liquid

–

White

Elemental analyses, % found (% calcd.)

Λm Ω−1 mol−1 cm2

C

H

N

Cl

Ln

96–98

–

–

–

–

–

–

–

–

–

–

–

–

86

−55 (M.P.) 121 (B.P.) 241–243

90

232–234

White

88

236–238

6.41 (6.45) 5.89 (5.86) 5.63 (5.69)

5.31 (5.37) 5.53 (5.56) 5.60 (5.65)

13.56 (13.60) 14.00 (14.06) 14.27 (14.29)

17.70 (17.76) 22.09 (22.12) 23.20 (23.26)

101.3

Pink

35.29 (35.33) 36.50 (36.53) 37.10 (37.13)

(Scheme 1). The complexes were stable in air at room temperature and commonly soluble in water, DMF, ethanol and methanol. 3.2. Molar conductance measurements Table 1 showed the molar conductance (Λm) values of DMF solutions of the studied complexes (1 × 10−3 mol L−1) at room temperature. The Λm values of La(III), Er(III) and Yb(III) complexes were found to be 101.3, 129.0 and 106.7 Ω−1 mol−1 cm2, respectively, suggesting their ionic natures (1:2 electrolytes) [49]. 3.3. Infrared spectra The FT-IR bands of the synthesized complexes were compared with those of the free Cn (Fig. S1) and the major bands were summarized and listed in Table S1 to confirm the complexation process and to determine the coordination sites. The sharp band attributed to ν(C\\N) in the free Cn (1272 cm−1) [50] did not exhibit any significant shift upon complex formation indicating that N atom of the azabicyclo–octane ring did not act as a site for coordination. The ν(C_O) stretching vibration of the carbonyl group attached to the methoxy group was observed at 1732 cm−1 in the FT-IR spectrum of free Cn while it shifted to lower wavenumbers (1720, 1716 and 1719 cm−1) in La(III), Er(III) and Yb(III) complexes' spectra, respectively, which is indicative of its participation in chelation. The ν(C_O) mode of the carbonyl group adjacent to the phenyl ring appeared at slightly lower wavenumbers, 1606, 1607 and 1607 cm−1 for La(III), Er(III) and Yb(III) complexes, respectively, (1610 cm−1 for free Cn). This slight shift can be attributed to the contribution of its neighboring etheric oxygen atom in the complexation. The coordination of N atom of TMEDA was supported by the appearance of νas(C\\N) of TMEDA (1338 cm−1) at 1379, 1357 and 1367 cm−1 in the IR spectra of the La(III), Er(III) and Yb(III) complexes, respectively [51]. The new weak bands detected in the range of 509–567 cm−1 in the FT-IR spectra of the studied complexes supported the formation of the M\\O bond [52,53]. Moreover, the new bands appeared at 992, 995 and 996 cm−1 for La(III), Er(III) and Yb(III) complexes, respectively, can be attributed to the coordinated water molecules [54] which were strongly confirmed by investigating the thermal analyses of these complexes. The coordination of the TMEDA nitrogen was also supported by the appearance of new metal-ligand weak bands ν(M\\N) at 463–481 cm−1. Therefore, it was concluded from the analysis of FT-IR data that Cn behaved as a neutral bidentate ligand and chelated to the metal center through the carbonyl oxygen of the (–COOCH3) ester and the etheric oxygen adjacent to the azabicyclo–octane ring.

129.0 106.7

3.4. Mass spectrometry The EI mass spectra of the formed complexes (Fig. S2) were carried out at 70 eV. The mass spectrum of La(III) complex, as a representative example, displayed a weak molecular ion peak, M+, at m/z 781 amu supporting the complex formation. Its fragmentation pattern (Scheme S1) showed the molecular ion peaks relative to the two ligands (Cn and TMEDA) at m/z 303 and 116 amu, respectively. The complex underwent sequential fragmentations led to the appearance of various peaks at different values of m/z which were very close to the calculated values. The most abundant peak was at m/z 58 amu and assigned to the trimethylamine. The molecular ion peak was in a good agreement with the molecular formula suggested from elemental and TG/DTG analyses. The Mass fragmentation pattern of TMEDA was also studied (Scheme S2) according to its mass spectrum [55].

3.5. Thermal analyses and kinetic studies Thermal properties of the synthesized complexes were investigated based on TG and DTG analyses (Fig. 4) within the temperature range of 30–1000 °C to get more information about their thermal stabilities, to investigate the nature of the water molecules and to provide a general scheme for their thermal decompositions. The obtained data were summarized and tabulated in Table 2. Free cocaine was thermally decomposed completely through two decomposition steps with maximum peaks at 221 and 521 °C (Fig. 4) while TMEDA completely decomposed through one step and began to evaporate at temperature before reaching its boiling point (121 °C) [56]. The TG/DTG curves of [La(Cn)(TMEDA)Cl(OH2)]·2Cl·5.5H2O complex (Fig. 4) exhibited five decomposition steps. The first three steps (45–315 °C) resulted in releasing of hydrated water molecules in addition to the coordinated water molecule and one and one-half moles of Cl2 gas with a mass loss of 28.40% (calcd. 28.58%). The fourth step (315–380 °C) involved the degradation of TMEDA moiety and a C9H9N molecule with a mass loss of 31.58% (calcd. 31.59%). The last step (380–815 °C) involved a 19.08% mass loss due to the decomposition of C8H12O2.5 molecule (calcd. 18.93%) leaving lanthanum oxide as a final residue with total mass loss of 79.06% (calcd. 79.10%). For [Er(Cn)(TMEDA)Cl(OH2)]·2Cl·2.5H2O complex (Fig. 4), its thermal decomposition passed through four steps with DTGmax of 106, 267, 494 and 718 °C with an overall estimated mass loss of 74.55% (calcd. 74.69). The first step (50–170 °C) resulted in the elimination of the hydrated water molecules with a mass loss of 6.04% (calcd. 5.95%). The next step (170–385 °C) was assigned to the releasing of the coordinated water molecule, one and one-half moles of Cl2 gas and a C2H2N molecule with a mass loss of 21.47% (calcd. 21.77%). The last two steps (615–1000 °C) involved a 47.04% mass loss due to the decomposition

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Fig. 4. TG and DTG curves of the studied complexes.

of C21H35N2O2.5 molecule (calcd. 46.97%) leaving erbium oxide as a final residue. The thermal decomposition of [Yb(Cn)(TMEDA)Cl (OH2)]·2Cl·1.5H2O complex (Fig. 4) displayed five decomposition steps maximized at 88, 302, 478, 605 and 892 °C with an overall mass loss of 73.61% (calcd. 73.45%). The first decomposition step (55–110 °C) correspond to releasing of hydrated water molecules with a mass loss of 3.88% (calcd. 3.63%). The next one (110–330 °C) involved a 31.01% mass loss due to the elimination of coordinated water molecule, one and one-half moles of Cl2 gas and C7H8N molecule

(calcd. 30.98%). The successive three steps (330–900 °C) brought the total mass loss up to 73.61% leaving ytterbium oxide as a final residue. The studying of rate-dependent thermodynamic kinetic parameters (such as activation energy ΔE⁎, enthalpy ΔH⁎, Gibbs free energy ΔG⁎ and entropy ΔS⁎) for solid-state non-isothermal decomposition processes by the analysis of TG curves has received a great interest in the literature [57]. Table S2 showed the kinetic parameters of Cn and the studied complexes calculated by using Coats–Redfern [58] and Horowitz–Metzger [59] methods (Fig. S3). The ΔE⁎ values were in the range of 24.90–360.7 kJ mol−1. The high values of ΔE⁎ indicated the thermal stability

Table 2 TG and DTG analyses of Cn, TMEDA and the formed complexes. Compound

TG range (°C)

n⁎

DTGmax (°C)

Mass loss, % Found (calcd.)

Assignment

Residue, % Found (calcd.)

Total mass loss, % Found (calcd.)

Cn (C17H21NO4) TMEDA [56] (C6H16N2) [La(Cn)(TMEDA)Cl(OH2)]Cl2·5.5H2O (C23H50Cl3N3O10.5La)

45–935

2

221, 521

99.99 (100.0)

–Loss of Cn

–

99.99 (100.0)

25–115

1

114

100.0 (100.0)

–Loss of TMEDA

–

100.0 (100.0)

45–315 315–380 380–815 50–170 170–385 385–865 55–110 110–330 330–940

3 1 1 1 1 2 1 1 3

79, 218, 247 360 796 106 267 494, 718 88 302 478, 605, 892

28.40 (28.58) 31.58 (31.59) 19.08 (18.93) 6.04 (5.95) 21.47 (21.77) 47.04 (46.97) 3.88 (3.63) 31.01 (30.98) 38.72 (38.84)

–Loss of 6.5 H2O, and 1.5 Cl2 –Loss of TMEDA, and C9H9N –Loss of C8H12O2.5 –Loss of 2.5 H2O –Loss of H2O, 1.5 Cl2, and C2H2N –Loss of C21H35N2O2.5 –Loss of 1.5 H2O –Loss of 1.5 Cl2, H2O, and C7H8N –Loss of C16H29N2O2.5

½ La2O3 20.94 (20.90)

79.06 (79.10)

½ Er2O3 25.45 (25.31)

74.55 (74.69)

½ Yb2O3 26.39 (26.55)

73.61 (73.45)

[Er(Cn)(TMEDA)Cl(OH2)]Cl2·2.5H2O (C23H44Cl3N3O7.5Er) [Yb(Cn)(TMEDA)Cl(OH2)]Cl2·1.5H2O (C23H42Cl3N3O6.5Yb) ⁎ n = number of decomposition steps.

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of the complexes. For a given complex, the ΔE⁎ values increased significantly on going from one decomposition step to the following one (54.20, 118.7 and 360.7 kJ mol−1, for La(III) complex) suggesting that the decomposition rate decreased in that order. The negative values of ΔS⁎ and ΔG⁎ indicated the spontaneous formation of these complexes and that they were highly ordered in activated states. The positive ΔH⁎ values suggested that the decomposition processes were endothermic in nature. The values of ΔG⁎ increased significantly for the subsequent thermal decomposition steps for a given complex as a result of the significant increases in the values of TΔS⁎ in the same order which may be ascribed to the structural rigidity of the residual part of the complex compared to its precedent complex which required more energy for its rearrangement before undergoing any structural change [60]. 3.6. Geometry optimization The optimized structures of Cn and the La(III) complex and numbering of atoms were shown in Fig. 3. Atomic coordinates of the optimized structures were listed (Table S3). Selected bond distances, angles and

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natural charges were tabulated (Table S4). The central La(III) atom adopted a distorted octahedral geometry (Fig. 3b), where Cn performed as a neutral bidentate ligand through the carbonyl oxygen of (– COOCH3) ester [La(68)–O(64) = 2.415 Å] and the etheric oxygen bonded to the bi-cycloalkane [La(68)–O(65) = 2.451 Å]. The two nitrogen atoms of TMEDA, [La(68)–N(66) = 2.531 Å] and [La(68)–N(67) = 2.484 Å] were lying in the molecular plane, while one chloride atom [La (68)–Cl(69) = 2.696 Å] and one water molecule [La(68)–O(70) = 2.879 Å] completed the octahedral geometry. The bond angles were deviated from 90° (Table S4) because of the rigidity of Cn and TMEDA which influence the regularity of the complex geometry. The charge on La(III) atom was +1.202e, which was reduced from the formal charge +3 due to the electron density donation by the active centers of the coordination sphere. Theoretical harmonic vibrational analyses were also carried out based on the optimized geometry. The scaling factor of 0.96 for the LanL2DZ level of theoretical calculation was used to correct the systematic errors such as the anharmonicity effects and the neglected part of electron correlation [61]. The practical FT-IR spectrum of the La(III)

Fig. 5. Calculated TD-DFT electronic transitions of Cn (in DMF).

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N.G. Zaki et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 117938

Fig. 6. Calculated TD-DFT electronic transitions of La(III) complex (in DMF), H atoms were omitted for clarity.

N.G. Zaki et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 117938

complex and its corresponding theoretical one are shown in Fig. S4. The most significant bands were theoretically calculated at close wavenumbers where they were practically observed. Table S5 represented the calculated values of electronic, thermal (vibrational, translational and rotational) and total energies (kcal mol−1) and the total dipole moments for Cn and the La(III) complex (gas phase) at 300 K. It was concluded from these values that the formed complex was highly stable than the free Cn (ΔE = −294,627.839 kcal mol−1). 3.7. Electronic structure Practically, the free ligand (Cn) displayed a single absorption band in DMF at 228 nm (5.44 eV) attributed to the π–π* electronic transition of the benzene ring and a shoulder at 272 nm (4.56 eV) assigned to the n–π* transition from the N atom of the azabicyclo-octane ring to the benzene ring, while those of the formed complexes displayed only one sharp band at 270 nm (4.60 eV) which was due to the intra-ligand π–π* electronic transition. These assignments were investigated by performing TD-DFT calculations with taking the lowest 50 spin-allowed singlet-to-singlet excitation states into consideration. The theoretical spectrum of Cn in DMF was characterized by three main bands at 277, 262 and 240 nm with oscillator strengths of 0.104, 0.018 and 0.473, respectively (Table S6). As shown in Fig. 5, the band at 277 nm (4.47 eV) was contributed primarily from HOMO → LUMO + 1 (68%) transition. The excitation energy at 262 nm (4.74 eV) was attributed to the HOMO − 1 → LUMO (54%) transition. The intense band at 240 nm (5.1659 eV) (f = 0.4738) was assigned to the HOMO − 2 → LUMO (68%) transition. The electronic spectrum of La(III) complex was characterized by one main band at 279 nm with a shoulder at 225 nm with oscillator strengths (f) of 0.161 and 0.054, respectively, (Table S6). As illustrated in Fig. 6, the transition at 279 (4.43 eV) nm was contributed from HOMO − 1 → LUMO + 2 (51%) and HOMO − 2 → LUMO + 2 (45%) transitions. The excitation energy at 225 nm (5.51 eV) was attributed to the combination of the HOMO − 13 → LUMO (28%), HOMO − 9 → LUMO (25%) and HOMO − 3 → LUMO + 1 (24%) electronic transitions. 3.8. Biological activity 3.8.1. In vitro antiproliferative activity Initially, the antiproliferative activity level of the newly synthesized complexes was estimated by determining their single-dose growth inhibitory activity. A dose of 100 μg mL−1 of each complex was used with the cell lines MCF-7 and HepG-2 for this purpose and was compared to the growth of untreated cells. La(III) complex showed 65% growth inhibition on the MCF-7 cell line with no significant growth inhibition on the HepG-2 cell line. On the other hand, Yb(III) complex showed 62% and 69% growth inhibition on MCF-7 and HepG-2 cell lines, respectively. Er(III) complex did not show any significant growth inhibition on both cell lines. IC50 values for metal complexes showing promising growth inhibitory activity were then determined in the corresponding cell line and were compared to cisplatin as a reference standard. As shown in Table S7, La(III) complex showed IC50 of 45.96 μM on the MCF-7 cell line much higher than that of cisplatin (IC50 = 2.99 μM) indicating its low cytotoxic activity. On the other hand, Yb(III) complex showed promising antiproliferative activity on both MCF-7 and HepG-2 cell lines with IC50 of 15.15 and 9.54 μM, respectively, with minimum effect on normal human cell line (IC50 = 73.36 μM). Whereas, cisplatin showed IC50 of 2.99 and 8.96 μM on MCF-7 and HepG-2 cell lines, respectively. This indicated the efficacy of the Yb(III) complex as an antiproliferative agent on MCF-7 and HepG-2 cell lines with high safety on normal cells.

9

Further studies were performed to study the effect of Yb(III) complex on the different processes in cancer pathophysiology. Thus, the effect of this complex on the cellular levels of the apoptosis markers caspase-3 and caspase-9 and the human tumor suppressor nuclear proteins p21 and p27 cellular levels was studied. Moreover, the influence of the complex on metastasis, one of the main problems of cancer, was investigated. Finally, its effect on the cellular oxidative stress of the cancer cells was also studied. 3.8.2. Apoptosis induction Apoptosis is an energy-dependent programmed cell death. A key step at the beginning of apoptosis is the activation of a cysteine protease family called caspases. Caspases activation induces apoptosis through activation of plasma membrane death receptors and mitochondrial dysfunction [62]. Caspases are either initiators or executioners of apoptosis [63,64]. Initiator caspases include caspases 9 whose activation results in activation of downstream or executioner caspases such as caspases 3 [65]. To study the apoptosis induction activity of the Yb(III) complex, the cellular levels of caspase-3 and caspase-9 were measured in MCF7 cell line upon treatment with the IC50 of the complex and cisplatin as a reference standard and compared to a negative control as shown in Table 3. Table 3 showed that there was a significant increase in the levels of caspase-3 and caspase-9 in Yb(III) complex-treated breast cancer cells after 24 h of incubation; 7.78 and 9.54 folds, respectively, relative to the negative control and comparable to that of cisplatin (9.48 and 10.86 folds, respectively). Therefore, these results showed that the Yb (III) complex is an apoptotic inducer. 3.8.3. P21 and P27 levels The tumor suppressor nuclear proteins P21 and P27 belong to the cyclin-dependent kinase (CDK) interacting protein/kinase inhibitory protein (CIP/KIP) family which is responsible for the regulation of the cyclin-dependent kinases (CDKs) induced cell-cycle progression [66,67]. One of the hallmarks in cancer pathophysiology is the hyperactivity of CDKs and the inactivation of their endogenous inhibitors such as P21 and P27 [68]. Upregulation of p21 and/or p27 causes growth inhibition in various cancer models [69–72]. Thus, compounds increase cellular P21 and P27 levels can show an antitumor effect in several cancer types. The effect of the Yb(III) complex on the cellular levels of P21 and P27 were determined in MCF-7 cell line upon treatment with the IC50 of the complex and compared to negative control and cisplatin as a reference standard as shown in Table 4. Table 4 showed that there was a significant increase in the cellular levels of P21 and P27 in Yb(III) complex-treated breast cancer cells after 24 h of incubation; 2.60 and 2.14 folds, respectively, relative to the negative control and comparable to that of cisplatin (3.10 and 2.77 folds, respectively). 3.8.4. Antimetastatic properties One of the main problems of malignant tumors is metastasis where cancer cells migrate and invade a distant organ or tissue [73]. A metalbased cytotoxic compound that can act against tumor metastasis will be an improvement on the conventional cytotoxic compounds. To possess antimetastatic properties, the antitumor agent should be able to inhibit enzymes that are contributing to tumor dissemination. Matrix Table 3 Caspase-3 and caspase-9 concentrations in MCF-7 cells after treatment with Yb(III) complex or cisplatin for 24 h and their n-fold increase in concentration relative to the negative control. Compound

Caspase-3 conc. (pg/mL) [fold]

Caspase-9 conc. (ng/mL) [fold]

Yb(III) complex Cisplatin Control

443.2 [7.78] 540.2 [9.48] 56.99 [1.00]

12.6 [9.54] 14.4 [10.9] 1.32 [1.00]

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N.G. Zaki et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 117938

Table 4 The tumor suppressor nuclear proteins P21 and P27 concentrations in MCF-7 cells after treatment with Yb(III) complex or cisplatin for 24 h and their n-fold increase in concentration relative to the negative control.

Table 6 Glutathione peroxidase (GPx) concentration in MCF-7 cells after treatment with Yb(III) complex or cisplatin for 24 h and its n-fold reduction in concentration relative to the negative control.

Compound

P21 conc. (ng/mL) [fold]

P27 conc. (ng/mL) [fold]

Compound

Glutathione peroxidase conc. (nM/mL) [fold]

Yb(III) complex Cisplatin Control

199.9 [2.60] 238.7 [3.10] 76.97 [1.00]

5.66 [2.14] 7.32 [2.77] 2.64 [1.00]

Yb(III) complex Cisplatin Control

19.1 [0.36] 34.1 [0.65] 52.4 [1.00]

metalloproteinases (MMPs) are a family of enzymes that are strongly involved in cancer metastasis, thus, compounds that can inhibit or downregulate this set of enzymes can efficiently prevent cancer metastasis [74]. In the present work, the effect of the Yb(III) complex on the cellular levels of representative matrix metalloproteinases, MMP-3 and MMP-9, were determined to study its antimetastatic properties (Table 5). From Table 5, it was revealed that there was a significant reduction in the MMP-3 and MMP-9 cellular levels in the breast cancer cells incubated with the IC50 concentration of Yb(III) complex for 24 h; 0.35 and 0.39 folds, respectively, relative to the negative control. The reduction was close to that caused by cisplatin (0.16 and 0.19 folds, respectively). Therefore, these results showed that the Yb(III) complex is not only a cytotoxic but also with antimetastatic properties. 3.8.5. Oxidative stress inducing characteristics Cancer cells show greater oxidative stress than normal cells due to oncogenic stimulation increased metabolic activity and mitochondrial malfunction [62]. Excessive reactive oxygen species can damage cellular macromolecules (proteins, lipids and DNA) leading to fatal lesions in cells and so, can induce apoptosis and cell death [75]. To overcome this, cancer cells activate the scavenging system for these reactive oxygen species represented by several reducing species and enzymes such as reduced glutathione-GSH and glutathione peroxidase (GPx). Agents hinder this scavenging system will induce unmanageable oxidative stress and, consequently, cell apoptosis and cancer cell death. To study the effect of the Yb(III) complex on the cellular oxidative stress, the level of the glutathione peroxidase (GPx) was determined at Yb(III) complex IC50 concentration (Table 6). Table 6 showed that there was a significant reduction in the cellular level of glutathione peroxidase (GPx) in Yb(III) complex-treated breast cancer cells after 24 h of incubation; 0.36 fold relative to the negative control and half that of cisplatin (0.65 fold) indicating that the newly synthesized complex is better than cisplatin in the induction of cellular oxidative stress.

carbonyl oxygen of the (–COOCH3) ester and the etheric oxygen adjacent to the azabicyclo[3.2.1]octane ring. Thermal properties of the synthesized complexes and their kinetic thermodynamic parameters were studied. Theoretical calculations including geometry optimization, electronic structure and electronic and thermal energies were carried out using DFT calculations at B3LYP/LANL2DZ level of theory and the different quantum chemical parameters were calculated. Electronic spectra of all synthesized complexes were characterized by one main band at 270 nm (4.60 eV) confirmed by the TD-DFT calculations. Initially, the in vitro antiproliferative activity of the newly synthesized complexes was assessed by MTT assay on MCF-7 and HepG-2 cancer cell lines using a single dose of 100 μg then compounds showed promising cytotoxic activity were further investigated to determine their IC50 on the corresponding cell lines. Yb(III) complex showed promising cytotoxic activity comparable to that of cisplatin on both cell lines (IC50 = 9.54 and 15.15 μM on HepG-2 and MCF-7, respectively) with minimum effect on human normal cells (IC50 = 73.36 μM). Further molecular mechanistic investigations showed that Yb(III) complex is an apoptotic inducer as it raises the caspase-3 and caspase-9 cellular level in MCF-7 cell line by 7.78 and 9.54 folds, respectively, relative to the negative control and comparable to that of cisplatin. Furthermore, it showed an elevating effect on the level of the tumor suppressor nuclear proteins P21 and P27 concentrations in MCF-7 cells by 2.60 and 2.14 folds, respectively, relative to the negative control and comparable to that of cisplatin. Moreover, Yb(III) complex hindered the cellular scavenger system of the reactive oxygen species through reducing the glutathione peroxidase (GPx) cellular level by 0.36 fold relative to the negative control better than that of cisplatin imperiling MCF-7 cells by unmanageable oxidative stress. In addition to its cytotoxic effect, Yb(III) complex showed antimetastatic properties as it decreased the cellular levels of matrix metalloproteinases (MMP) MMP-3 and MMP-9 by 0.35 and 0.39 folds, respectively, relative to the negative control, however, slightly less than that of cisplatin. These results showed that the Yb (III) complex is a promising cytotoxic metal-based agent that exerts its action through various molecular mechanisms with minimum effects on normal cells and with additional antimetastatic properties.

4. Conclusion A series of new three distorted octahedral heteroleptic complexes of the general formula [Ln(Cn)(TMEDA)Cl(OH2)]·2Cl·xH2O, (where Ln = La(III), Er(III) and Yb(III), Cn = cocaine and TMEDA = N,N,N′,N′tetramethylethylenediamine) with 1:1:1 metal–ligands stoichiometry in which both Cn and TMEDA ligands acted as a neutral bidentate ligand were synthesized, structurally characterized by elemental analysis, spectroscopic methods, molar conductivity and mass spectrometry. FT-IR data indicated that Cn chelated to the metal centers through the Table 5 Matrix metalloproteinase-3 and matrix metalloproteinase-9 concentrations in MCF-7 cells after treatment with Yb(III) complex or cisplatin for 24 h and their n-fold reduction in concentration relative to the negative control. Compound

MMP-3 conc. (ng/mL) [fold]

MMP-9 conc. (ng/mL) [fold]

Yb(III) complex Cisplatin Control

3.97 [0.35] 1.86 [0.16] 11.3 [1.00]

211.0 [0.39] 103.7 [0.19] 540.7 [1.00]

Author contribution All authors contributed equally to this work. Declaration of competing interest 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. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.saa.2019.117938. References [1] M. Schwab, Encyclopedia of Cancer, fourth ed. Springer-Verlag Berlin Heidelberg, Germany, 2017https://doi.org/10.1007/978-3-662-46875-3.

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