Cobalt(II) and magnesium(II) complexes with 1,3-pdta-type of ligands: influence of an alkyl substituent at 1,3-propanediamine chain on the structural and antimicrobial properties of the complex

Abstract To investigate how modification in the structure of 1,3-propanediamine chain of 1,3-pdta (1,3-propanediamine-N,N,N′,N′-tetraacetate) ligand affects the structural and biological properties of the corresponding metal complexes, two new octahedral complexes, [Co(H2O)5Co(2,2-diMe-1,3-pdta)]·H2O (1) and [Mg(H2O)5Mg(2,2-diMe-1,3-pdta)]·1.5H2O (2) (2,2-diMe-1,3-pdta = 2,2-dimethyl-1,3-propanediamine-N,N,N′,N′-tetraacetate), were synthesized and characterized by IR spectroscopy and single-crystal X-ray diffraction analysis. Additionally, UV-Vis and NMR spectroscopic methods were applied for the characterization of 1 and 2, respectively. Crystallographic data indicate that these complexes contain 2,2-diMe-1,3-pdta coordinated to the metal ion through 2 N and 4 O atoms forming [M(H2O)5M′(2,2-diMe-1,3-pdta)] complex unit (M, M′ = Co(II), Co(II) (1) and M, M′ = Mg(II), Mg(II) (2)), which is composed of [M′(2,2-diMe-1,3-pdta)]2− and [M(H2O)5O]2+ octahedra bridged by one of the axial carboxylate groups. The antimicrobial activities of 1 and 2 were evaluated against different bacteria and Candida spp., while their cytotoxic effect was tested on the normal human lung fibroblasts (MRC-5). The ability of 1 and 2 to inhibit formation of C. glabrata biofilms was also assessed. The obtained structural parameters and biological properties of the two complexes were compared to Co(II) and Mg(II) complexes with 1,3-pdta ligand. Graphical Abstract

The conformational behavior of the ligand backbone is expected to vary when the two hydrogen atoms of the central methylene group are replaced with two geminal methyl groups (Thorpe-Ingold effect) [15,16]. A simple aminopolycarboxylate ligand with the 2,2-dimethyl-1,3-propanediamine backbone is the 1,3-pdta analogue 2,2dimethyl-1,3-propanediamine-N,N,N 0 ,N 0 -tetraacetate (2,2-diMe-1,3-pdta). We have recently synthesized this ligand and used it for the synthesis of seven-coordinate manganese(II) and cadmium(II) complexes of formula fBa[M(2,2-diMe-1,3pdta)]Á3H 2 Og n (M ¼ Mn(II) or Cd(II)) [ Magnesium(II) is one of the most abundant divalent metal cations in both prokaryotic and eukaryotic cells [20]. Recently, new interest on this cation arose due to its antimicrobial properties. Thus, it was found that antibiotic activity was enhanced in the presence of Mg(II) ion [21]. This enhancement was explained by the fact that the presence of Mg(II) affects the curvature of the bacterial membrane, increasing the vulnerability of bacteria and efficiency of the antibiotic. Moreover, it was demonstrated that Mg(II) and Ca(II) ions disrupt model Staphylococcus aureus membranes and kill stationary-phase S. aureus cells, indicating their membrane activity [22]. More recently, it was shown that surfaces coated with magnesium or its compounds are more effective in prevention of bacteria adherence, as well as biofilm formation [23].
Cobalt is also an element of biological interest. Its biological role is mainly focused on its presence in the active center of vitamin B 12 , which indirectly regulates the synthesis of DNA. Many cobalt(II) complexes have been reported to show activity against different microbial species [24][25][26]. Thus, in vitro antibacterial activity of cobalt(II) complexes with a series of amino acids was evaluated against different Gram-positive and Gram-negative bacteria. The complexes with leucine and histidine are more active than the parent free ligands, while a moderate activity was observed for complexes with methionine and phenylalanine. However, lower antibacterial activity was observed for cobalt(II) complexes with lysine and valine [24]. Mixed ligand complexes of cobalt(II) with different thiosemicarbazones and N-phthaloyl derivative of D,L-glycine, L-alanine and L-valine against various bacterial and fungal strains showed better antimicrobial activity than the parent ligands [25]. Recent study of antibacterial activity of cobalt(II) complexes with the quinolone antimicrobial agent enrofloxacin showed that these complexes were more active than free enrofloxacin [26]. A remarkable antifungal activity against Candida albicans was observed for the coordination polymer of cobalt(II) with indole-3-carboxylic acid, while only moderate antibacterial activity was shown by complexes of this metal ion with oxydiacetate anions [27,28].

Materials and measurements
2,2-Dimethyl-1,3-propanediamine and chloroacetic acid were obtained from Acros Organics. All other common chemicals were of reagent grade and used without purification.
All pH measurements were performed at room temperature using the pH meter S220 SevenCompact TM pH/Ion, Mettler Toledo, which was calibrated with buffer solutions of pH 4.01 and 7.00. Elemental analyses for carbon, hydrogen and nitrogen were performed by the Microanalytical Laboratory, Faculty of Chemistry, University of Belgrade. IR spectra were recorded on a Perkin-Elmer Spectrum One FT-IR spectrometer using KBr pellets from 4000 À 450 cm À1 . UV-Vis spectra were recorded at room temperature from 1100 À 200 nm on a Shimadzu double-beam spectrophotometer after dissolving 1 in water immediately after its dissolution. The concentration of the solution used for this measurement was 5.0 Â 10 À2 M. The NMR spectra of 2 were recorded on a Varian Gemini 2000 spectrometer at 200 MHz ( 1 H) and 50 MHz ( 13 C) using standard Varian software. 5.0 mg of the complex was dissolved in 0.6 mL of D 2 O and transferred into a 5 mm NMR tube. TSP (sodium 3-(trimethylsilyl)propionate) was used as the internal reference. Chemical shifts, r, are expressed in ppm (parts per million) and scalar couplings, J, are reported in Hz (Hertz).

Synthesis of [Co(H 2 O) 5 Co(2,2-diMe-1,3-pdta)]ÁH 2 O (1)
CoSO 4 Á7H 2 O (0.7028 g, 2.5 mmol) was dissolved in 15 mL of H 2 O at 70 C. Solid Ba 2 (2,2-diMe-1,3-pdta)Á2H 2 O (2.5 mmol, 1.6024 g) was added and the reaction mixture was heated at 70 C with stirring for 30 min. The precipitated BaSO 4 was removed by filtration. Solid CoSO 4 Á7H 2 O (2.5 mmol, 0.7028 g) was added to the dark pink filtrate obtained after removing BaSO 4 and the mixture was stirred with heating for 20 min at 60 C. Deposited BaSO 4 was filtered off and volume of the filtrate was reduced to 5 mL. After cooling at room temperature, this solution was mixed with 5 À 6 mL of ethanol and the obtained mixture was allowed to stand in a refrigerator for several days at 4 C.

Synthesis of [Mg
MgSO 4 Á7H 2 O (1.2324 g, 5 mmol) was dissolved in 40 mL of H 2 O. Solid Ba 2 (2,2-diMe-1,3pdta)Á2H 2 O (2.5 mmol, 1.6024 g) was added and the reaction mixture was stirred with heating for 9 h at 90 C. During this time, the volume of the reaction mixture was kept at 40 mL by adding distilled water. Deposited BaSO 4 was filtered off and the volume of filtrate was reduced to 5 mL. After one week of standing at room temperature, colorless crystals of [Mg(H 2 O) 5

Crystallographic data collection and refinement of the structures
Single crystals of 1 and 2 were selected and mounted on a loop with inert oil on a Stoe STADIVARI diffractometer. The crystals were kept at 250(2) K during data collection. Using Olex2 [33], the structures were solved with the ShelXT [34] structure solution program using Intrinsic Phasing and refined with the ShelXL [35] refinement package using Least Squares minimization. Hydrogen atom positions were calculated geometrically and refined using the riding model. Crystal data and details of the structure determinations are given in Table S1. CCDC 2170842-2170843 contains the supplementary crystallographic data for this article. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via https://www.ccdc.cam.ac. uk/data_request/cif. All analyzed compounds were dissolved in DMSO at a final concentration of 50 mM, and the highest tested concentration was 500 mM. The inoculums were 5 Â 10 5 colony-forming units, cfu/mL, for bacteria and 1 Â 10 5 cfu/mL for Candida species. The MIC value was recorded as the lowest concentration that completely inhibited the growth after 24 h at 37 C using the Epoch Microplate Spectrophotometer, BioTek Instruments, Inc.

Anti-biofilm activity assessment on C. glabrata ATCC 2001
An anti-biofilm assay was conducted using the previously published methodology [36]. Briefly, the assay was carried out in 96-well round-bottom polystyrene microtiter plates. Cells were harvested from overnight grown cultures, washed twice with sterile phosphate-buffered saline (PBS; Sigma-Aldrich, Munich, Germany), and resuspended in RPMI 1640 medium (Sigma-Aldrich) containing 2% glucose (w/v) to give a final concentration of 1 Â 10 5 cfu/mL. Candida suspension was incubated with compounds (concentrations started from MIC and lower, six dilutions in total) in 200 lL final volume per well for 48 h at 37 C to allow biofilm formation. Biofilm growth was analyzed by crystal violet (CV) staining of adherent cells and the absorbance at 590.0 nm was read on an Epoch Microplate Spectrophotometer, BioTek Instruments, Inc.

Cytotoxicity
In vitro cytotoxicity was determined as an antiproliferative activity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [37] on human lung fibroblasts (MRC-5) obtained from American Type Culture Collection (ATCC). The cells, cultured in the complete RPMI 1640 medium as a monolayer (1 Â 10 4 cells per well), were incubated with the investigated compounds at concentrations ranging from 1 to 250 mM, in a humidified atmosphere of 95% air and 5% CO 2 at 37 C, and the cell viability was measured after 48 h. The extent of MTT reduction was measured spectrophotometrically at 540.0 nm using the Epoch Microplate Spectrophotometer, BioTek Instruments, Inc., and cell survival was expressed as a percentage of the control (DMSO treated cells) arbitrarily set to 100%. Cytotoxicity is expressed as the concentration of the compound inhibiting growth by 50% (IC 50 ).
To examine the impact of substitution of the hydrogen atoms with methyl groups from the central 1,3-propanediamine carbon of the ligand backbone on the strain of M-1,3-pdta system, we performed a strain analysis of related series of [M 0 (1,3-pdta)] 2À and [M 0 (2,2-diMe-1,3-pdta)] 2À (M 0 ¼ Mg(II), Co(II) and Ni(II)) complexes. The major contributions to strain are considered to be: (i) the octahedral angles around the metal ion, (ii) the ring angle sums of the various types of rings, (iii) the M-O-C bond angles, and (iv) the bond angles that the chelating nitrogen atom makes with its connectors. The results of strain analysis are given in Table 1. In general, the [M 0 (2,2-diMe-1,3pdta)] 2À complexes show greater octahedral strain than the corresponding complexes of the 1,3-pdta ligand, as indicated by the calculated P D(O h ) values for the series of Mg(II), Co(II) and Ni(II) complexes. As expected, the five-membered glycinate rings of the G type (girdle, or in-plane with respect to the diamine ring) are much more strained than those of the R type (relaxed, or out-of-plane with respect to the diamine ring). For the [M 0 (1,3-pdta)] 2À complexes, the total deviation from the ideal chelate ring bond angle sum (538.5 ) is þ1 and from À11 to À10 for the R and G type of the glycinate rings, respectively. For the [M 0 (2,2-diMe-1,3-pdta)] 2À complexes, the total deviation from the ideal chelate ring bond angle sum is from À2 to À1 and from À14 to À13 for the R and G rings, respectively, indicating the greater strain in the corresponding glycinate rings of [M 0 (2,2-diMe-1,3-pdta)] 2À system compared to that of [M 0 (1,3-pdta)] 2À . Important source of strain in this kind of chelates is bonding geometry made by the chelating nitrogen atoms. Each N atom makes four bonds with six ideally 109.5 bond angles. The total deviation about the chelating N atoms in M(II)- 1,3-pdta complexes is 11 for Ni(II) and Co(II) complexes and 13 for Mg(II), and for M(II)-2,2-diMe-1,3-pdta complexes, this deviation is 18 for Ni(II) and Co(II) and 21 for Mg(II) complex.

Infrared (IR) spectra of cobalt(II) and magnesium(II) complexes
IR carboxylate stretching frequencies for 1 and 2 are summarized in Table S4 and are compared with those for Co(II) [4] and Mg(II) [5] complexes with 1,3-pdta ligand. Both 1 and 2 show one very strong and sharp band at 1614 and 1618 cm À1 , respectively, indicating that all carboxylate groups of 2,2-diMe-1,3-pdta ligand in these complexes are coordinated [38]. These bands are almost identical in the shape and absorption intensity, and they do not show any tendency for splitting. However, in the same region, one very strong and broad band (1587 and 1597 cm À1 ) with clear tendency for splitting on the lower energy side (1675 and 1687 cm À1 ) was observed for cobalt(II) and magnesium(II) complexes with 1,3-pdta ligand, respectively (Table S4). The part of the IR spectra due to the symmetric stretching vibrations of the coordinated carboxylate groups for 1 and 2 is slightly complex compared to those for cobalt(II) and magnesium(II) complexes with 1,3pdta. Based on the abovementioned facts, it can be concluded that the difference between carboxylate stretching frequencies of cobalt(II) and magnesium(II) complexes with 2,2-diMe-1,3-pdta and those for the complexes of these metal ions with 1,3-pdta might result from the presence of two methyl groups at the central carbon atom of 1,3propanediamine ring of the 2,2-diMe-1,3-pdta complexes.   pdta)]Á2H 2 O [5], were recorded in D 2 O. The spectrum of 2 shows signals of one wellresolved AB pattern centered at 3.35 ppm, which belongs to the methylene protons from two pairs of equivalent R (out-of-plane) and G (in-plane) glycinate rings (J ¼ 16.76 Hz). This finding is opposite to those previously reported for the cobalt(III) complexes with hexadentate 2,2-diMe-1,3-pdta and 1,3-pdta ligands which showed two well-resolved AB patterns belonging to two pairs of nonequivalent R and G glycinate rings [19,39] Figure S1 and corresponding numerical data are given in Table S5. The crystal structure of the latter complex was previously reported [5] and herein the synthesis of this complex was repeated to compare its 13 C NMR spectrum with that for 2. In respect to the spectrum of [Mg(H 2 O) 6 ][Mg(1,3-pdta)]Á2H 2 O, all signals in that of 2 are shifted downfield. The largest shifting was observed for carbon atoms of 1,3-propanediamine ring (C1/C3 72.73 and 59.94, C2 40.65 and 25.52 ppm for 2,2-diMe-1,3-pdta and 1,3-pdta, respectively; see Figure  S1 and Table S5), what is consequence of the presence of two methyl groups attached at the central carbon atom of 2,2-diMe-1,3-pdta ligand. Only one signal was observed for the methylene carbon of glycinate rings for both complexes. Only one signal for the methylene carbon of glycinate rings was also observed in the spectrum of [Co(2,2-diMe-1,3-pdta)]complex, while two signals for these carbon atoms appeared in the spectrum of [Co(1,3-pdta)]complex due to its two pairs of nonequivalent axial (R) and equatorial (G) glycinate rings [19]. Difference in the 13 C NMR spectra between [Mg(1,3-pdta)] 2and [Co(1,3-pdta)]could result from different size of Mg(II) and Co(III) ions.

Antimicrobial properties of cobalt(II) and magnesium(II) complexes
There is a need for new antimicrobial compounds to combat the spread of antibiotic resistance. Metal complexes are currently used for treatment of different diseases, with many of them investigated as potential antimicrobial therapeutics [41,42]. In this study, the antimicrobial potentials of 1 and 2, the metal salts used for their synthesis and cobalt(II) and magnesium(II) complexes with 1,3-pdta ligand [4,5] were evaluated against two Gram-positive bacteria Staphylococcus aureus and methicillin-resistant S. aureus (MRSA), two Gram-  negative bacteria Pseudomonas aeruginosa and Klebsiella pneumoniae, and six Candida spp., including two C. albicans, C. parapsilosis, C. glabrata, C. krusei and C. auris (Table 3). Overall, increased activity of complexes against a range of Candida spp., in comparison to the tested bacterial strains, was observed (  [20][21][22][23]. Contrary to the presently reported results, chromium(III) and cobalt(III) complexes, Na[Cr(2,2-diMe-1,3-pdta)]Á3.75H 2 O and Na[Co(2,2-diMe-1,3-pdta)]Á3.88H 2 O, did not inhibit the growth of the tested microorganisms, even when the concentration of 500 mM was applied [19]. From these results, it can be concluded that the oxidation state of metal ion has an influence on the antimicrobial activity of the complexes with 2,2-diMe-1,3-pdta ligand. Thus, metal(II) complexes with this ligand show remarkable and selective antifungal activity, while the analogue metal(III) species penetrate through the microbial cell wall less effectively, resulting in a decrease of antimicrobial activity.
The cytotoxic effect of the investigated compounds against human lung fibroblasts (MRC-5) was assessed. Low toxicity is observed for cobalt(II) and magnesium(II) salts as well as 1, while complex 2 and 1,3-pdta complexes showed high toxic effects against MRC-5 cells (Table 3). This further implies that 1 with a positive value of selectivity index (a ratio between IC 50 and MIC values) against the tested Candida species, especially C. glabrata, could be considered as an antifungal therapeutic.

Anti-biofilm activity of cobalt(II) and magnesium(II) complexes
Antimicrobial resistance is especially connected with the presence of biofilms, which represent micro-structured consortia in which microbial cells are enclosed in a self- produced extracellular polymeric matrix composed of carbohydrates, proteins and extracellular DNA. The concentration of microbial cells in biofilms is 100 À 1000 times higher than in planktonic phases, and in this form, microbes commonly show up to 1000 times higher resistance to clinically used antimicrobial agents [43,44]. The ability of 1 and 2, the metal salts used for their synthesis and cobalt(II) and magnesium(II) complexes with 1,3-pdta ligand [4,5] to inhibit the formation of C. glabrata ATCC 2001 biofilms was assessed ( Figure 3). As can be seen from this figure, 2 shows a slightly higher efficiency in the anti-biofilm activity in comparison to 1. The highest inhibition percentage was 66% for 2 in a concentration of 0.98 mM and the comparable effect was reached from 3.9 to 1.95 mM, while for 1, it was 68% in a concentration of 3.9 mM (Figure 3a). In comparison to this, no inhibition of the biofilm formation was observed for  Figure 3b). From these results, it can be drawn that the introduction of two methyl substituents at a central 1,3-propanediamine carbon atom significantly increases the inhibitory activity against microorganisms in both planktonic and biofilm forms.

Conclusion
We have demonstrated that coordination of 2,2-diMe-1,3-pdta ligand, which differs from 1,3-pdta by containing two methyl substituents at the central 1,3-propanediamine carbon atom, with Co(II) and Mg(II) ions leads to [M(H 2 O) 5  Ni(II)) has shown that all 2,2-diMe-1,3-pdta complexes have greater octahedral strain than the corresponding 1,3-pdta complexes. The structural diversification between [M 0 (1,3pdta)] 2À and [M 0 (2,2-diMe-1,3-pdta)] 2À was also confirmed by spectroscopic measurements for the corresponding cobalt(II) and magnesium(II) complexes. The structural difference between 1,3-pdta and 2,2-diMe-1,3-pdta complexes is attributed to the presence of two methyl groups at the central carbon atom of 1,3-propanediamine chain of the 2,2-diMe-1,3-pdta ligand. Complexes 1 and 2 showed increased activity against a range of Candida spp. in comparison to the tested bacterial strains, with most potent antifungal activity of 2. Also, this complex shows a slightly higher efficiency in the anti-biofilm activity in comparison to complex 1. Nevertheless, 1 has a positive value of selectivity index against the tested Candida species, especially C. glabrata, and could be further considered as potential antifungal agent.

Disclosure statement
No potential conflict of interest was reported by the authors.