Synthesis, Structural Characterization, Antimicrobial Activity, and In Vitro Biocompatibility of New Unsaturated Carboxylate Complexes with 2,2′-Bipyridine

The synthesis, structural characterization, cytotoxicity, and antimicrobial properties of four new complexes formed by employing acrylate anion and 2,2′-bipyridine are reported herein. X-ray crystallography revealed the trinuclear nature of [Mn3(2,2′-bipy)2(C3H3O2)6] (1), meanwhile complexes with general formula [M(2,2′-bipy)(C3H3O2)2(H2O)x]∙yH2O ((2) M: Ni, x = 1, y = 0; (3) M: Cu, x = 1, y = 0; (4) M: Zn, x = 0, y = 1; 2,2′-bipy: 2,2′-bipyridine; C3H3O2: acrylate anion) were shown to be mononuclear. The lowest minimum inhibitory concentration (MIC) of 128 μg mL−1 was recorded for all four tested complexes against Candida albicans, for complex (3) against Escherichia coli, and for complex (4) against Staphylocococcus aureus. Compounds (3) and (4) were also potent efflux pumps activity inhibitors (EPI), proving their potential for use in synergistic combinations with antibiotics. Complexes (1)–(4) revealed that they were not cytotoxic to HCT-8 cells. They also proved to interfere with the cellular cycle of tumour HCT-8 cells by increasing the number of cells found in the S and G2/M phases. Taken together, these results demonstrate the potential of zinc and copper complexes for use in the development of novel antimicrobial and anti-proliferative agents.


Introduction
Carboxylate complexes have an extremely rich chemistry, due mostly to various coordination modes exhibited by carboxylate groups [1] and to the presence of various coligands found in the coordination sphere of the metallic ions [2][3][4].

Synthesis of the Complexes
In this paper we report the synthesis, structural characterization, and bioevaluation of four new complexes containing mixed ligands, i.e., the acrylate ion and 2,2 -bipyridine. The complexes' formulae have been established on the basis of chemical analysis, IR spectra, and X-ray diffraction analysis as follows: [Mn 3 (2,2 -bipy) 2  where 2,2 -bipy is 2,2 -bipyridine and C 3 H 3 O 2 is the acrylate anion. All complexes were obtained in two steps: firstly, metallic acrylates were obtained using raw materials such as carbonates or oxides; the second step consisted of the reaction of the metallic acrylates with 2,2 -bipyridine.
Bond distances and angles for the coordination spheres are collected in Table 2.
In the trinuclear complex with linear Mn arrangement, each pair of manganese atoms is bridged by three acrylato ligands, two of them coordinated in a bidentate fashion, whereas the third bridging acrylate coordinates in a unidentate mode via O5 and O11, respectively. The central manganese (Mn2) has a more regular octahedral environment formed exclusively by six oxygen atoms of acrylate ions and is located on a crystallographic inversion center. Mn2-O distances range from 2.1613 (15)  The coordination environments for Mn1 and Mn3 can be described as distorted octahedral, each coordination polyhedron being comprised from two nitrogen atoms provided by 2,2 -bipyridine, two oxygen atoms from one acrylate ion that acts as chelate and monoatomic bridge simultaneously, and two oxygen atoms given by two bridged acrylate ions. As noticed from Table 2, Mn1-O and  Mn3-O bonds are smaller than Mn2-O bonds, similar to those observed for manganese complexes with acetate [33] or chloroacetate [38], and 2,2 -bipyridine. No relevant π-πinteractions involving the bipyridine moieties were observed in the packing diagram. In the trinuclear complex with linear Mn arrangement, each pair of manganese atoms is bridged by three acrylato ligands, two of them coordinated in a bidentate fashion, whereas the third bridging acrylate coordinates in a unidentate mode via O5 and O11, respectively. The central manganese (Mn2) has a more regular octahedral environment formed exclusively by six oxygen atoms of acrylate ions and is located on a crystallographic inversion center. Mn2-O distances range from 2.1613(15) to 2.1862(14) Å, while O-Mn2-O angles range from 88.50(6)° to 91.66(6)°.
The linear arrangement of the trinuclear compound is sustained by the Mn1-Mn2-Mn3 angle of 178.11(1)°.
The linear arrangement of the trinuclear compound is sustained by the Mn1-Mn2-Mn3 angle of 178.11(1) • .
The crystal structure of compound (2) consists of neutral [Ni(2,2 -bipy)(C 3 H 3 O 2 ) 2 (H 2 O)] complexes ( Figure 3). The Ni(II) ion is six-coordinated by two bipyridine-nitrogen atoms, two oxygen atoms from one chelating acrylate, one oxygen atom from the other acrylate ion, and one oxygen from a coordinated water molecule, building a somewhat distorted octahedral surrounding. The acrylato ligands adopt two different coordination modes: one acts unidentate (Ni1-O3 = 2.034(2) Å) and the other is chelate bidentate (Ni1-O1 = 2.126(2) Å, Ni1-O2 = 2.147(3) Å). X-ray analysis indicates that the ethylene group in the chelating acrylate ligand is disordered over two crystallographic positions with occupancies of 75% and 25%, respectively. The shortest distance between the aromatic rings of bipyridine is about 3.527 Å, indicating the presence of weak π…πinteractions. The packing diagram ( Figure 4) presents the formation of dimers involving π…πinteractions and weak C-H…O hydrogen bonds between the bipyridine unit and the water oxygen atom (C4-H4…O5w 2.501 Å). These dimers are building linear chains along the a-axis through additional C-H…O hydrogen bonds between the chelate acrylato ligand and the vicinity bipyridine unit (C9-H9…O1 2.780 Å). The asymmetric unit of complex (3) presents two crystallographic independent mononuclear entities of [Cu(2,2′-bipy)(C3H3O2)2(H2O)] ( Figure 5). As the structural parameters are very similar, only molecule "A" is discussed below. Copper (II) ions are six-coordinated by two bipyridine-  The acrylato ligands adopt two different coordination modes: one acts unidentate (Ni1-O3 = 2.034(2) Å) and the other is chelate bidentate (Ni1-O1 = 2.126(2) Å, Ni1-O2 = 2.147(3) Å). X-ray analysis indicates that the ethylene group in the chelating acrylate ligand is disordered over two crystallographic positions with occupancies of 75% and 25%, respectively. The shortest distance between the aromatic rings of bipyridine is about 3.527 Å, indicating the presence of weak π…πinteractions. The packing diagram ( Figure 4) presents the formation of dimers involving π…πinteractions and weak C-H…O hydrogen bonds between the bipyridine unit and the water oxygen atom (C4-H4…O5w 2.501 Å). These dimers are building linear chains along the a-axis through additional C-H…O hydrogen bonds between the chelate acrylato ligand and the vicinity bipyridine unit (C9-H9…O1 2.780 Å). The asymmetric unit of complex (3) presents two crystallographic independent mononuclear entities of [Cu(2,2′-bipy)(C3H3O2)2(H2O)] ( Figure 5). As the structural parameters are very similar, only molecule "A" is discussed below. Copper (II) ions are six-coordinated by two bipyridine- The asymmetric unit of complex (3) presents two crystallographic independent mononuclear entities of [Cu(2,2 -bipy)(C 3 H 3 O 2 ) 2 (H 2 O)] ( Figure 5). As the structural parameters are very similar, only molecule "A" is discussed below. Copper (II) ions are six-coordinated by two bipyridine-nitrogen atoms, two oxygen atoms from one chelating acrylate, one oxygen atom from a unidentate acrylato ion, and an oxygen atom from a coordinated water molecule, building a slightly distorted octahedral geometry.
The geometry of complex (3) is almost identical to that of complex (2), but the coordination modes adopted by the chelating acrylato ligand in compound (3)  nitrogen atoms, two oxygen atoms from one chelating acrylate, one oxygen atom from a unidentate acrylato ion, and an oxygen atom from a coordinated water molecule, building a slightly distorted octahedral geometry. The geometry of complex (3) is almost identical to that of complex (2), but the coordination modes adopted by the chelating acrylato ligand in compound (3)   The presence of a coordinated water molecule and the acrylate coordination mode was alleged by IR spectra analysis. The distance between metallic ions in the asymmetric unit is 9.558 Å. The shortest distance between the aromatic rings of bipyridine is 3.467 Å, thus indicating π…π stacking interactions compared to those found for the Ni complex. The packing diagram of complex 3 (see Figure 6) is different from that found for compound 2 and presents the formation of linear chains along the b-axis involving strong O-H…O hydrogen bonds (O5W-H02…O2 1.890 Å) and π…π interactions with alternate distances (3.467 Å and 3.490 Å).  The presence of a coordinated water molecule and the acrylate coordination mode was alleged by IR spectra analysis. The distance between metallic ions in the asymmetric unit is 9.558 Å. The shortest distance between the aromatic rings of bipyridine is 3.467 Å, thus indicating π . . . π stacking interactions compared to those found for the Ni complex. The packing diagram of complex 3 (see Figure 6) is different from that found for compound 2 and presents the formation of linear chains along the b-axis involving strong O-H . . . O hydrogen bonds (O5W-H02 . . . O2 1.890 Å) and π . . . π interactions with alternate distances (3.467 Å and 3.490 Å).
Molecules 2018, 23, 157 7 of 18 nitrogen atoms, two oxygen atoms from one chelating acrylate, one oxygen atom from a unidentate acrylato ion, and an oxygen atom from a coordinated water molecule, building a slightly distorted octahedral geometry. The geometry of complex (3) is almost identical to that of complex (2), but the coordination modes adopted by the chelating acrylato ligand in compound (3)   The presence of a coordinated water molecule and the acrylate coordination mode was alleged by IR spectra analysis. The distance between metallic ions in the asymmetric unit is 9.558 Å. The shortest distance between the aromatic rings of bipyridine is 3.467 Å, thus indicating π…π stacking interactions compared to those found for the Ni complex. The packing diagram of complex 3 (see Figure 6) is different from that found for compound 2 and presents the formation of linear chains along the b-axis involving strong O-H…O hydrogen bonds (O5W-H02…O2 1.890 Å) and π…π interactions with alternate distances (3.467 Å and 3.490 Å).   Crystallographic investigation of complex (4) reveals a structure made up of two mononuclear units [Zn(2,2 -bipy)(C 3 H 3 O 2 ) 2 ] that are crystallographically independent in asymmetric cells.
The metallic ion is six-coordinated by two bipyridine-nitrogen atoms and four oxygen atoms provided by two acrylate ions, building a distorted octahedral environment. Similar to the cooper complex (3), only the molecule "A" is discussed below. Both acrylato ligands adopt an asymmetrical bidentate coordination manner with two short Zn-O bond lengths (Zn1-O1 1.994 (3) (Figure 7).
The bidentate coordination of acrylate was anticipated on IR spectra analysis, according to ∆ criterion [1,43].
Each acrylate ion participates at the equatorial plane with one oxygen atom, the other positions being occupied by nitrogen atoms. The apical positions are conquered by the remaining oxygen atoms of acrylate ions. The distance between metallic ions in the asymmetric unit is 7.987 Å. The shortest distance between the aromatic rings of bipyridine is 3.469 Å, indicating weaker π . . . π stacking interactions compared to the copper complex ( Figure 8).  (Figure 7).
The bidentate coordination of acrylate was anticipated on IR spectra analysis, according to ∆ criterion [1,43].
Each acrylate ion participates at the equatorial plane with one oxygen atom, the other positions being occupied by nitrogen atoms. The apical positions are conquered by the remaining oxygen atoms of acrylate ions. The distance between metallic ions in the asymmetric unit is 7.987 Å. The shortest distance between the aromatic rings of bipyridine is 3.469 Å, indicating weaker π…π stacking interactions compared to the copper complex ( Figure 8).   Crystallographic investigation of complex (4) reveals a structure made up of two mononuclear units [Zn(2,2′-bipy)(C3H3O2)2] that are crystallographically independent in asymmetric cells.
The bidentate coordination of acrylate was anticipated on IR spectra analysis, according to ∆ criterion [1,43].
Each acrylate ion participates at the equatorial plane with one oxygen atom, the other positions being occupied by nitrogen atoms. The apical positions are conquered by the remaining oxygen atoms of acrylate ions. The distance between metallic ions in the asymmetric unit is 7.987 Å. The shortest distance between the aromatic rings of bipyridine is 3.469 Å, indicating weaker π…π stacking interactions compared to the copper complex ( Figure 8).    The main bond lengths and angles for mononuclear complexes (2)-(4) are listed in Table 4.

Infrared Spectra
The bands that appear in IR spectra ( Figure S1, Supplementary Materials) in the range 1535-1540 cm −1 are characteristic of 2,2 -bipyridine and are shifted in comparison with the free ligand, indicating the chelate coordination of aromatic ammine. The presence of acrylate anions in the composition of complexes is sustained by the appearance of ν as (COO) and ν s (COO) bands in the IR spectra of all complexes.
The splitting of bands ν s (COO) or ν as (COO) that appear in complexes (1) and (2), respectively, may be associated according to literature studies [1,49], with different coordination modes adopted by the carboxylate ligand. This statement was confirmed by X-ray structure in the case of complex (2).
Also, it is known that the ∆ value (∆ = ν as (COO) − ν s (COO)) indicates the coordination mode associated with carboxylate [1]. Therefore, for complex (3), a ∆ value of 211 cm −1 indicates a unidentate coordination for acrylate, meanwhile for (4) a value of 183 cm −1 suggests a bidentate coordination mode [49]; the latter statement is proven by X-ray analysis for (3).
The presence of water molecules in complexes (2)-(4) generates a large band that appears in the range 3350-3460 cm −1 , and it is assigned to ν(OH) stretching vibrations [50]. Also, a medium intensity band that appears in the range 630-640 cm −1 for complexes (2) and (3) may be assigned to vibration mode ρ w (H 2 O) and suggests the coordinated nature of water molecules.

Electronic Spectra
The diffuse electronic reflectance spectra recorded for Mn(II), Ni(II), and Cu(II) ( Figure S2, Supplementary Materials) offered information regarding the coordination number and stereochemistry of complexes.
In the case of manganese complex [Mn(2,2 -bipy)(C 3 H 3 O 2 ) 2 ]·H 2 O (1), the electronic spectrum reveals a band at 375 nm that can be assigned to the 6 A 1g → 4 T 2g (D) spin-forbidden transition characteristic of d 5 high spin octahedral species.
For complex (3), the absorption maximum at 700 nm was assigned to d xz , d yz → d x 2 −y 2 transition, characteristic of octahedral distorted stereochemistry.
All complexes' spectra contain two strong bands in UV regions characteristic of π → π* intraligand transitions. These are shifted to lower energies compared with uncoordinated 2,2 -bipyridine bands.
Since the antimicrobial activity was carried out in dimethyl sulfoxide (DMSO) solutions, the complexes' stability in this media was investigated by UV-Vis spectroscopy. Changes observed over time for each complex solution indicated that all compounds are stable for 48 h ( Figure S3).

Biological Activity
In order to highlight potential applications of the obtained compounds, the antimicrobial and biocompatibility features of the obtained compounds have been investigated.

Antimicrobial Activity
In vitro screening of the antimicrobial activities of acrylate complexes (1)-(4) was performed by the broth microdilution method, in order to establish the minimum inhibitory concentration (MIC) against three microbial strains representative of infections with Gram-positive bacteria (Staphylocococcus aureus), Gram-negative (Escherichia coli) bacteria, and fungal (Candida albicans) strains (Table 6). Screening of the antimicrobial activity of complexes (1)-(4) revealed variable MIC values, ranging between 128 and 1024 µg mL −1 for the tested complexes, indicating a moderate to low antimicrobial potential. The antimicrobial activity of the tested complexes was improved by comparison with that of sodium acrylate (NaC 3 H 3 O 2 ), as demonstrated by the MIC values which were 4-39 times lower than that obtained for NaC 3 H 3 O 2 . The most susceptible microbial strain was C. albicans, towards which all four tested complexes exhibited the same MIC value (128 µg mL −1 ). Regarding the antibacterial effect, the most active compounds were (3) and (4), displaying an MIC value of 128 µg mL −1 against E. coli for complex (3) and against S. aureus for (4).
Complex (1)  . This behavior could be due to the octahedral stereochemistry of Ni(II) in complex (2) and its notorious preference for such surrounding, which was shown to have a low antimicrobial activity [26].

Flow Cytometry Analysis
Analysis of the microbial cell populations treated with sub-inhibitory concentrations of the tested compounds allowed us to formulate some hypotheses concerning the putative mechanisms of the antimicrobial activity of the tested compounds. Only the most susceptible strains and the most active compounds (i.e., those exhibiting MIC of 128 µg mL −1 ) have been tested.
Propidium iodide (PI) staining revealed the viability of the cells for all tested combinations, as shown by the negative FL3 signal recorded for the microbial suspensions treated with the tested compounds ( Figure S4), proving a microbiostatic rather than microbicidal activity of the tested compounds ( Figure S5). This could explain the relatively high MIC values obtained for the respective compounds.
In exchange, the tested compounds were confirmed as potent or moderate efflux pump activity inhibitors (EPI), as revealed by the increased percentage of cells showing cellular uptake of EB, materialized by the occurrence of increased FL2 fluorescence signal typical of EB, directly correlated with the EPI activity of the tested compounds ( Figure S6). The most potent EPI activity was noted for (4) against C. albicans and for (3) against E. coli ( Figure S7).

In Vitro Biocompatibility Assay
Cell cycle analysis of HCT-8 cells grown in the presence of different concentrations of the tested complexes highlighted that the tested compounds are not toxic, as the area under the G1 phase specific to apoptotic cells was absent ( Figure 9) and according trypan blue test (data not shown). Complexes (1) and (2)  antimicrobial potential. The antimicrobial activity of the tested complexes was improved by comparison with that of sodium acrylate (NaC3H3O2), as demonstrated by the MIC values which were 4-39 times lower than that obtained for NaC3H3O2. The most susceptible microbial strain was C. albicans, towards which all four tested complexes exhibited the same MIC value (128 μg mL −1 ). Regarding the antibacterial effect, the most active compounds were (3) and (4), displaying an MIC value of 128 μg mL −1 against E. coli for complex (3) and against S. aureus for (4). Complex (1) revealed moderate antimicrobial activity with an MIC of 256 μg mL −1 against the E. coli strain. The lowest antibacterial activity was shown by [Ni(2,2′-bipy)(C3H3O2)2(H2O)] (2), exhibiting the highest MIC against both the Gram-positive and Gram-negative bacterial strains (1024 μg mL −1 ). This behavior could be due to the octahedral stereochemistry of Ni(II) in complex (2) and its notorious preference for such surrounding, which was shown to have a low antimicrobial activity [26].

Flow Cytometry Analysis
Analysis of the microbial cell populations treated with sub-inhibitory concentrations of the tested compounds allowed us to formulate some hypotheses concerning the putative mechanisms of the antimicrobial activity of the tested compounds. Only the most susceptible strains and the most active compounds (i.e., those exhibiting MIC of 128 μg mL −1 ) have been tested.
Propidium iodide (PI) staining revealed the viability of the cells for all tested combinations, as shown by the negative FL3 signal recorded for the microbial suspensions treated with the tested compounds ( Figure S4), proving a microbiostatic rather than microbicidal activity of the tested compounds ( Figure S5). This could explain the relatively high MIC values obtained for the respective compounds.
In exchange, the tested compounds were confirmed as potent or moderate efflux pump activity inhibitors (EPI), as revealed by the increased percentage of cells showing cellular uptake of EB, materialized by the occurrence of increased FL2 fluorescence signal typical of EB, directly correlated with the EPI activity of the tested compounds ( Figure S6). The most potent EPI activity was noted for (4) against C. albicans and for (3) against E. coli ( Figure S7).

In Vitro Biocompatibility Assay
Cell cycle analysis of HCT-8 cells grown in the presence of different concentrations of the tested complexes highlighted that the tested compounds are not toxic, as the area under the G1 phase specific to apoptotic cells was absent ( Figure 9) and according trypan blue test (data not shown). Complexes (1) and (2) induced a slightly increase in the number of cells in the G0/G1 phases, correlated with a decrease in the number of cells in the S and G2/M phases, while complexes (3) and (4), which also exhibited the most intensive antimicrobial activity, induced an increase in the number of cells in the S and G2/M phases.
Chemical analysis of carbon, hydrogen, and nitrogen was performed using a Perkin Elmer PE 2400 analyzer (Perkin Elmer, Waltham, MA, USA). IR spectra were recorded in KBr pellets with a Tensor 37 spectrometer (Bruker, Billerica, MA, USA) in the 400-4000 cm −1 range. Diffuse reflectance electronic spectra were recorded at room temperature with a Jasco UV-VIS-NIR V670 spectrometer (Jasco, Easton, MD, USA) in the 200-2000 nm range, using MgO as reference. DMSO solution UV-VIS spectra were recorded on Jasco V530 spectrophotometer in the range of 250-650 nm (Jasco, Easton, MD, USA). X-ray data for complexes (2) and (4) was collected at room temperature on a STOE IPDS II diffractometer (STOE, Darmstadt, Germany). The structures were solved by direct methods and refined by full-matrix least squares techniques based on F2. The non-H atoms were refined with anisotropic displacement parameters. Calculations were performed using a SHELX-97 Figure 9. The effects of 10µg/mL (left histograms) or 1 µg/mL (middle histograms) compounds on the HCT8 cell cycle progression. In the right is represented the overlaid histograms of treated and untreated HCT8 cells.
Chemical analysis of carbon, hydrogen, and nitrogen was performed using a Perkin Elmer PE 2400 analyzer (Perkin Elmer, Waltham, MA, USA). IR spectra were recorded in KBr pellets with a Tensor 37 spectrometer (Bruker, Billerica, MA, USA) in the 400-4000 cm −1 range. Diffuse reflectance electronic spectra were recorded at room temperature with a Jasco UV-VIS-NIR V670 spectrometer (Jasco, Easton, MD, USA) in the 200-2000 nm range, using MgO as reference. DMSO solution UV-VIS spectra were recorded on Jasco V530 spectrophotometer in the range of 250-650 nm (Jasco, Easton, MD, USA). X-ray data for complexes (2) and (4) was collected at room temperature on a STOE IPDS II diffractometer (STOE, Darmstadt, Germany). The structures were solved by direct methods and refined by full-matrix least squares techniques based on F2. The non-H atoms were refined with anisotropic displacement parameters. Calculations were performed using a SHELX-97 crystallographic software package. Data sets for complexes (1) and (3) were collected with a Nonius Kappa CCD diffractometer (Nonius B. V., Delft, The Netherlands). Programs used: data collection, COLLECT [52]; data reduction, Denzo-SMN [53]; absorption correction, Denzo [54]; structure solution and refinement, SHELX-97 [55,56].
CCDC-1470787 (1), -1470788 (2), -1470789 (3), and -1470790 (4) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. The powder diffraction patterns for all complexes (Figures S8-S11, Supplementary Materials) matched well with those simulated from single crystal structure data indicating that bulk samples were isolated as pure phases.
Powder X-ray diffraction (XRD) patterns were recorded using an XRD-7000 diffractometer (Shimadzu, Kyoto, Japan) with Cu Kα radiation (λ = 1.5406 Å, 40 kV, 40 mA) at a step of 0.2 • and a scanning speed of 2 degrees min −1 in the 5-60 degrees 2θ range. A mixture formed from 0.6 g NiCO 3 ·2Ni(OH) 2 ·6H 2 O, 1 mL acrylic acid (ρ = 1.05 g mL −1 ) and 25 mL distilled water was stirred at room temperature for one hour. The reaction mixture was filtered off to eliminate excess carbonate. To the green filtrate a solution containing 0.43 g 2,2 -bipyridine and 10 mL ethanol was added. The blue colored solution was stirred at room temperature for 2 h and then left to evaporate slowly. After one week, light blue crystals were obtained which were filtered off, washed with ethanol, and air dried. Yield: 79% (1.75 g), Anal. A mixture formed from 0.44 g CuCO 3 ·Cu(OH) 2 , 0.6 mL acrylic acid (ρ = 1.05 g mL −1 ) and 25 mL distilled water was stirred at room temperature for one hour. The reaction mixture was filtered off in order to eliminate excess carbonate. To the blue filtrate a solution containing 0.3 g 2,2 -bipyridine and 10 mL ethanol was added, and the mixture was stirred at room temperature for 2 h. Then, the intense blue solution was allowed to evaporate slowly. After one week blue crystals were obtained which were filtered off, washed with ethanol, and air dried. Yield: 74% (1.12 g), Anal. A suspension containing 0.325 g ZnO, 0.6 mL acrylic acid (ρ = 1.05 g mL −1 ) and 25 mL distilled water was stirred at room temperature for one hour. The reaction mixture was filtered off. To the colorless filtrate a solution was added containing 0.62 g 2,2 -bipyridine and 10 mL ethanol. After stirring at room temperature for 2 h, the solution became pale yellow and then was allowed to evaporate slowly at room temperature. After one week pale yellow crystals were p which were filtered off, washed with ethanol, and air dried.

Synthesis of Complexes
Yield

Antimicrobial Activity Assay
The following microbial strains were used: Gram-positive (Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes) bacteria, Gram-negative (Escherichia coli, Salmonella enteritidis, Pseudomonas aeruginosa) bacteria, and one fungal strain (Candida albicans). Microbial suspensions of 1.5 × 10 8 CFU mL −1 (0.5 McFarland density) obtained from 15-18 h bacterial cultures developed on solid media were obtained. The compounds were suspended in DMSO to prepare a stock solution of 10 mg mL −1 concentration. The quantitative assay of the antimicrobial activity was performed by the liquid medium microdilution method in 96 multi-well plates. Two-fold serial dilutions of the compounds' solutions (ranging between 4 and 1024 µg mL −1 ) were performed in a 200 µL volume of broth, and each well was seeded with 50 µL of microbial inoculum. Positive controls for microbial cultures (wells containing culture medium seeded with the microbial inoculum) and standard antimicrobials (ciprofloxacin for bacteria and amphotericin B for fungi) were used. The influence of the DMSO solvent was also quantified in a series of wells containing DMSO, diluted accordingly with the dilution scheme. The plates were incubated for 24 h at 37 • C, and the MIC values were considered as the lowest concentration of the tested compound that inhibited the growth of the microbial overnight cultures, as compared to the positive control, revealed by a decreased value of absorbance at 600 nm (Apollo LB 911 ELISA reader, Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany) [57,58].

Flow Cytometry Assay for the Investigation of Putative Mechanisms of Antimicrobial Activity
Flow cytometry was carried out in order to evaluate the influence of the tested compounds on microbial efflux pump activity. For microbial cell staining, two intercalant fluorochromes with DNA affinity were used: propidium iodide (PI, 10 µg mL −1 ) and ethidium bromide (EB, 5 µg mL −1 ). PI was used for the determination of cellullar viability, as living cells are impermeable to this dye, and EB was used for the detection of bacterial efflux pump activity. Staining procedures were applied to the harvested cells grown in the presence of the tested compounds at the concentration MIC/2. The cells were centrifuged at 13,000 rpm for 3 min, washed 2 times, resuspended in phosphate buffered saline (PBS), stained with 10 µL PI or EB, and incubated for 10 min at 4 • C in the dark. Cells heat-treated for 30 min at 100 • C were used as positive controls and viable cells were used as negative controls. The samples were analyzed with a FACS Calibur instrument (Becton, Dickinson and Company, San Jose, CA, USA) equipped with a 488 nm Argon laser, using a 670 nm long pass filter for the samples stained with PI and a (585 ± 42) nm band pass filter for the samples stained with EB. The log scale was used for all measured parameters. The photomultiplier tube voltages were SSC (side scatter)-550 V, 670 nm long pass filter-480 V, and 585 ± 42 nm band pass filter-500 V. 10,000 events were collected in all runs.

Biocompatibility Assay
HCT-8 (human ileocecal adenocarcinoma) cells were cultivated in RPMI 1640 (Gibco, New York, NY, USA) supplemented with 10% heat-inactivated bovine serum and penicillin/streptomycin, at 37 • C, with 5% CO 2 , in the presence of two concentrations of the tested compounds: 10 µg mL −1 and 1 µg mL −1 , respectively. The 24 h monolayers were harvested, washed with PBS (pH 7.5), fixed in 70% cold ethanol, and maintained overnight at −20 • C. Each sample was washed in PBS, treated with 100 µg mL −1 RNase A for 15 min, and stained with 10 µg mL −1 PI by incubation at 3 • C for 1 h. After PI staining, events acquisition was performed using an Epics Beckman Coulter flow cytometer (Beckman Coulter, Indianapolis, IN, USA). The obtained data were analyzed using FlowJo software version 7.2.5 (FlowJo LLC, Ashland, OR, USA) and expressed as fractions of cells in different cell cycle phases [59].

Conclusions
Using the acrylate anion and 2,2 -bipyridine as ligands, four new complexes of Mn(II), Ni(II), Cu(II), and Zn(II) have been prepared and characterized. The trinuclear complex [Mn 3 (2,2 -bipy) 2 (C 3 H 3 O 2 ) 6 ] (1) presents a series of interesting features, such as the linear arrangement of metallic ions and various coordination modes of carboxylate ions (bridge through two oxygen atoms, monoatomic bridge, and chelate). In all complexes, metallic ions adopt an octahedral coordination geometry with different distortion degrees, while acrylato ligands exhibit different coordination modes. The packing diagram of complex (2) presents the formation of dimers involving π . . . π-interactions and additional weak C-H . . . O hydrogen bonds between the bipyridine unit and the water oxygen atom, while for complex (3) the formation of linear chains along the b-axis, involving strong O-H . . . O hydrogen bonds and π . . . π interactions with alternate distances was shown in the packing diagram. For complex (4), there was evidence of weaker π . . . π stacking interactions in comparison with complex (3).
Complexes (1)-(4) exhibited variable MIC values, ranging from 128 to 1024 µg mL −1 . All four complexes showed moderate antifungal activity. The most active antibacterial compounds were complex (3) against E. coli and (4) against S. aureus. Compounds (3) and (4) were also potent efflux pumps activity inhibitors (EPI), proving their potential for use in synergistic combinations with antibiotics. They also proved to interfere with the cellular cycle of the tumoral HCT-8 cells by increasing the number of cells found in the S and G2/M phases. Taken together, these results demonstrate the potential of zinc and copper complexes for use in the development of novel antimicrobial and anti-proliferative agents.
Supplementary Materials: The supplementary materials are available online.