Zinc(II) complexes derived from imidazo[1,2-a]pyridin-2-ylacetic acid (HIP-2-ac): [Zn(IP-2-ac)2(H2O)] and unexpectedly, [Zn3(IP-2-ac)6(H2O)]·11H2O

Two zinc(II) complexes based on imidazo[1,2-a]pyridin-2-ylacetate (IP-2-ac), [Zn(IP-2-ac)2(H2O)] (1) and [Zn3(IP-2-ac)6(H2O)]·11H2O (2), were synthesized and characterized by single-crystal X-ray diffraction. In both 1 and 2, zinc(II) ions are five-coordinate with N2O3 donor set, best described as a distorted trigonal-bipyramidal geometry. In 1, two IP-2-ac ligands chelate zinc(II) through a N,O donor set, whereas in 2, both bidentate and μ-bridging binding modes of IP-2-ac are observed. The crystal of 1 comprises discrete Zn(IP-2-ac)2(H2O) coordination entities combined into layers by hydrogen bonds. Inter-layer stabilization of the 3-D crystal lattice is provided by weak C–H⋯O contacts and π⋯π interactions. The structure of 2 consists of discrete trinuclear Zn3(IP-2-ac)6(H2O) coordination entities joined into crystal lattice by multiple water molecules. Compound 1 was characterized by FTIR and FT-Raman spectroscopy, and in terms of thermal stability. Furthermore, its antibacterial activity was tested against selected gram-positive, gram-negative bacteria, and Candida albicans yeast and compared with activity of previously reported [M(IP-2-ac)2(H2O)2]·2H2O (M = Co, Ni, Mn, Cd) complexes. Graphical abstract

Surprisingly, the coordination chemistry of IP derivatives has received little attention. Reports are mostly devoted to stable anion radicals such as (bipo) ·and (obip) ·-, which have been used for the preparation of coordination polymers with interesting luminescence and magnetic properties [3,4]. Furthermore, organotin(IV) complexes of IP, and rhodium and iridium complexes of N-heterocyclic carbenes containing IP ring have been reported [10].
Recently, using imidazo[1,2-a]pyridin-2-ylacetatic acid (HIP-2-ac), we have synthesized a series of discrete isostructural [M(IP-2-ac) 2 (H 2 O) 2 ]·2H 2 O (M = Co, Ni, Mn, Cd) coordination compounds, in which M(II) ions are six-coordinate by two trans-oriented bidentate IP-2-ac anions and two water molecules [11]. Thermal analysis revealed that the studied compounds release crystallization and coordinated water in different manners, resulting in the lowest thermal stability of [Co(IP-2-ac) 2 (H 2 O) 2 ]·2H 2 O and the highest thermal stability of [Cd(IP-2-ac) 2 (H 2 O) 2 ]·2H 2 O. As an extension of the above mentioned work, [Zn(IP-2-ac) 2 (H 2 O)] (1) was prepared using previously described procedure and characterized by single-crystal X-ray diffraction, IR and Raman spectroscopy, thermogravimetric analysis (TG-DTA) and differential scanning calorimetry (DSC) thermal methods. Taking into account pharmaceutical importance of IP scaffold as well as numerous examples of antimicrobial activity of metal-based complexes with structurally similar ligands [12], we also decided to test antimicrobial activity of 1 and previously reported complexes against selected gram-positive and gram-negative bacteria, and Candida albicans yeast.

General information and materials
Imidazo[1,2-a]pyridin-2-ylacetic acid (HIP-2-ac) was synthesized according to previously described procedure [11]. All other reagents were obtained from commercial sources and used without purification. The C, H, and N microanalyses for 1 were carried out on an Elementar vario EL III analyzer. The resulting solution was inserted into a glass reactor and heated at 100-120°C for 6-8 h. Colorless crystals of 1 were collected by filtration, washed with cold distilled water, and allowed to dry in air (yield 40%). The same crystals can also be isolated when methylamine is replaced by 25% solution of ammonia (Anal. Calcd for C 18  Compound 2: On slow evaporation of filtrate remaining after separation of 1, small quantities of crystals different from 1 were unexpectedly isolated after ca. three weeks. X-ray single-crystal diffraction analysis revealed that highly hydrated trinuclear [Zn 3 (IP-2-ac) 6 (H 2 O)]·11H 2 O, 2 (stable in air for the duration of measurement), was formed. Unfortunately, our attempts to isolate sufficient amount of 2 for further characterization proved unsuccessful. Several crystal growth trials resulted mostly in crystals which decomposed within a few minutes; thus, this material remains unidentified.

X-ray crystallography
Crystallographic measurements were performed on a Kuma KM4-CCD (for 1) and Xcalibur R (for 2) automated four-circle diffractometers with graphite monochromated Mo Kα radiation at 100(2) K (1) or 296(2) K (2), using Oxford Cryosystems coolers. Data collection, cell refinement, data reduction, and analysis were carried out with CRYSALISCCD and CRYSALISRED, respectively [13]. Analytical (for 1) or multi-scan (for 2) absorption correction was applied to the data with use of CRYSALISRED. Structures were solved with direct methods using SHELXS-2013 [14] and refined by full-matrix least squares with SHELXL-2013 [14] with anisotropic thermal parameters for all non-H atoms, unless otherwise stated below. All hydrogens were initially located in difference Fourier maps (except for disordered ligand anions in 2, in which they were included from geometry), and in the final refinement cycles were treated as described below. All C-bound hydrogens were placed in calculated positions, with C-H = 0.93-0.99 Å, and refined with a riding model with U iso (H) = 1.2U eq (C). Water hydrogens in 1 were refined freely. In 2, water hydrogens were refined with U iso (H) = 1.5U eq (O), and with O-H and H⋯H distances restrained to 0.840(2) and 1.380(2) Å, respectively, and then constrained to ride on their parent atoms (AFIX 3 instruction in SHELXL-2013 [14]). During the refinement of 2, three of the water molecules were found to be disordered and were refined with O in two positions, each, and with occupancy factors of 0.6 and 0.4 (see the CIF file for details). Moreover, one of the ligand anions was disordered and refined over two locations, with 0.515(19) and 0.485(19) occupancies. In a second ligand anion, the acetate substituent was disordered, and refined over two sites with 0.75(4) and 0.25(4) occupancies. All of the disordered atoms in 2 were refined isotropically. Some restraints on geometry and anisotropic displacement parameters, as well as constraints on the fractional coordinates and ADPs (SAME, SIMU, EXYZ, EADP instructions in SHELXL-2013) were applied in the refinement procedure if appropriate. Selected crystallographic data and structure refinement parameters are summarized in table 1. All figures were made using DIAMOND [15].

Vibrational spectroscopy
ATR-FTIR (attenuated total reflection-infrared) measurements were made using a Bruker Vertex 70v Fourier transform infrared spectrometer equipped with a diamond ATR cell. The spectral data were collected at room temperature from 4000-400 cm −1 to 600-40 cm −1 with a resolution of 4 and 2 cm −1 , respectively.
FT-Raman spectra were collected on a Bruker MultiRam spectrometer (Nd:YAG laser with emitting radiation at a wavelength of 1064 nm) equipped with a liquid nitrogen cooled germanium detector. The spectra were accumulated with a resolution of 2 cm −1 and Raman laser power 50 mW.
Instrument control and initial data processing were performed using OPUS software (v. 7.0 Bruker Optics, Ettlingen, Germany).

Calculations
The normal vibrations of Zn(IP-2-ac) 2 (H 2 O) were calculated using the GAUSSIAN 09 package [16], employing the B3LYP functional [17] and LanL2DZ basis set [18]. The calculations were performed on the optimized structure, which corresponds well with that determined by single-crystal X-ray diffraction. A noticeable difference was found only for the coordinated water. O-H bond orientations are almost parallel to Zn-O bonds in the calculated structure, whereas a twist is observed in the crystal structure.
Normal vibrations of Zn(IP-2-ac) 2 (D 2 O) were also computed. Normal vibrations and corresponding IR and Raman bands were characterized on the basis of atom displacement  [20]. Theoretical spectra were generated by Chemcraft application, with Lorentzian band shape and 5 cm −1 half band width. For easier comparison of observed and calculated band positions, the calculated wavenumbers were scaled by 0.96 factor, typical for applied computation method [21].

Thermal analysis
TG-DTA was carried out using a Setaram SETSYS 16/18 thermogravimetric analyzer, operating under nitrogen, with a heating rate of 5°C/min −1 from 30 to 500°C (sample mass 4.312 mg). DSC measurements were performed on a Setaram DSC 92 instrument. Sample (mass 6.0 mg) was contained in an alumina pan in the presence of air as the furnace atmosphere with heating rate of 5°C/min −1 in a temperature range of 30-500°C.

Antimicrobial activity studies
The in vitro antimicrobial activities of studied ligand and its five complexes with zinc(II), nickel(II), cobalt(II), manganese(II), and cadmium(II) were tested against Bacillus subtillis, Staphylococcus aureus, Mycobacterium fortuitum, Enterococcus hirae, Escherichia coli, Pseudomonas aeruginosa, and C. albicans. Bacterial strains were obtained from the Polish Collection of Microorganisms located in the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences, Wrocław, Poland. Prior to use in tests, the cultures of bacteria were grown on nutrient broth at 37°C for 24 h. Antibacterial tests were carried out on solid samples. Compounds under consideration were milled with starch to give an active content of 10%. The resulting powders were pressed using a hydraulic press into 7 mm diameter pellets. Solid pellets were placed on agar plates with inoculated microorganisms and incubated at 37°C for 24 h, except for M. fortuitum for which incubation was carried out for 48 h. After this time, inhibition zones (if present) were measured in mm as r = (d2 -d1)/2 (where d1 is diameter of pellet and d2 is diameter of inhibition zone) and results were documented by digital photography. Pellets containing only starch were used as a control probe and those containing 10% of related M (II) nitrates in starch for comparison. All experiments were performed in duplicate and the average values are presented.

Electrospray mass spectrometry
ESI-MS experiment for saturated aqueous solution of crystalline solid isolated from postreaction mixture was performed on a Bruker MicrOTOF-Q spectrometer (Bruker Daltonic, Bremen Germany) equipped with an Apollo II electrospray ionization source with an ion funnel. The spectrum was acquired in a positive ion mode for 50 : 50 (v/v) MeOH/H 2 O sample. The instrumental parameters were as follows: the scan range m/z 200-2000, end plate offset −500 V, dry gas nitrogen (4 L/min), temperature 473 K, capillary voltage 4500 V, ion energy 5 eV. Data analysis was carried out with the use of Bruker Daltonics Data Analysis software v. 3.4.

Preparation of 1 and 2
As illustrated in scheme 1, [Zn(IP-2-ac) 2 (H 2 O)] (1) was prepared using previously reported procedure; however, it was isolated in lower yield as compared to previously synthesized compounds [11]. When filtrate remaining after isolation of 1 was left to stand at room temperature, a few crystals of 2 were unexpectedly and unprecedentedly isolated and one of them was characterized by single-crystal X-ray diffraction.
To understand why our attempts to obtain sufficient amount of 2 were unsuccessful (see experimental part), solid material isolated from post-reaction mixture was dissolved in water and subjected to ESI-MS analysis. ESI-MS is one of the most sensitive and useful techniques providing qualitative information on stoichiometric composition of metal complexes preformed in solution [22]. As can be seen in figure S1 (see online supplemental material at http://dx.doi.org/10.1080/00958972.2015.1048241), the spectrum exhibits two peaks at m/z = 437 and 453, respectively, attributed to [Zn(IP-2-ac) 2 13 Zn 3 ] + were identified. The complicated solution equilibrium involving mononuclear 1 and a variety of multinuclear zinc(II) species suggested by ESI-MS spectrum seems to explain the difficulty in reproducing the crystallization conditions suitable for growing crystals of 2. Furthermore, these results suggest that solid material isolated from post-reaction mixture may be inhomogeneous. In consequence, only 1 was subjected to further analysis by means of TG-DTA, DSC, and vibrational spectroscopy.

Molecular structure, crystal packing, and intermolecular interactions in 1
[Zn(IP-2-ac) 2 (H 2 O)] (1) crystallizes in the C2/c space group of the monoclinic crystal system. The molecular structure and atom numbering scheme for 1 are shown in figure 1. Selected geometrical parameters and hydrogen bonds are given in tables 2 and S1 (ESI), respectively. The structure of 1 consists of discrete Zn(IP-2-ac) 2 (H 2 O) coordination entities, in which the zinc(II) is five-coordinate by N1, O1, N1 i , and O1 i from two chelating IP-2-ac anions and O1W from a water molecule. Zinc(II) ion and coordinated O1W are located on a twofold axis; thus, the asymmetric unit comprises one-half of the complex. Coordination entities are additionally stabilized by C8-H8⋯O1 i contacts symmetry code: (i) −x + 1, y, −z + 1/2; for more details refer to table S1, enabling identification of an intramolecular hydrogen-bonded ring denoted by S(6) graph set notation [23].
In the crystal of 1, coordinated water molecules interact with the uncoordinated carboxylate oxygen of neighboring Zn(IP-2-ac) 2 (H 2 O) coordination entities by O1W-H1W⋯O2 ii hydrogen bonds to form a supramolecular chain that can be denoted by a C 4 4 (6) graph set. Chains are associated into R 4 4 (22) ring motifs, resulting in the formation of a molecular layer in the crystallographic ab plane, additionally stabilized by C10-H10B⋯O1 iv contacts (figure 2). Inter-layer stabilization of the 3-D arrangement in the crystal lattice is provided  by C7-H7⋯O2 iii contacts as well as π⋯π interactions, with the latter presented in figure 3 (geometrical parameters in table S1).

Molecular structure, crystal packing, and intermolecular interactions in 2
[Zn 3 (IP-2-ac) 6 (H 2 O)]·11H 2 O (2) crystallizes in the P2 1 /c space group of the monoclinic crystal system. The molecular structure and atom numbering scheme for 2 are shown in figures 4 and S2. Atoms of disordered ligands and water molecules refined with minor occupancies (see Section 2 for further details) were omitted for clarity in both figures and  discussion. Selected geometrical parameters and hydrogen bonds are given in tables 2 and S1, respectively.
As described above, the N1-C2-C10-C11 and C2-C10-C11-O1 torsion angles describe the conformation of IP-2-ac anions. The corresponding values for IP-2-ac chelating terminal Zn1 and Zn3 in 2 exhibit similar conformations to that present in 1 and previously reported complexes [11], whereas the bridging IP-2-ac anions adopt opposite orientations of the acetate group as related to the ligand backbone (table 2). An additional stabilization of 2 is provided by C-H⋯O-type contacts and π⋯π interactions (table S1).
The ZnN 2 O 3 coordination environments in 2 are best described by a distorted TB geometry with corresponding angles and bond lengths similar to those in 1 (table 2). The environment around Zn2 is very similar to Zn1 of 1 with a τ value of 0.86 [24]. For Zn1 and Zn3, the τ values equal 0.70 and 0.72, respectively, indicating larger deviation toward square pyramid as compared to Zn2. This suggests that Zn(IP-2-ac) 2 (H 2 O) may be regarded as a precursor in the formation of Zn 3 (IP-2-ac) 6 (H 2 O). A possible path for trinuclear entity formation deduced based on ESI-MS spectrum (figure S1) is given in figure S3.
In the crystal structure of 2, neighboring Zn 3 (IP-2-ac) 6 (H 2 O) coordination entities interact through an array of O-H⋯O hydrogen bonds formed between lattice water Figure 4. Molecular geometry, thermal ellipsoids (shown at 20% probability level), and partial numbering scheme for Zn 3 (IP-2-ac) 6 (H 2 O) in 2. C-bound hydrogens and ligand atoms refined with minor occupancies were omitted for clarity. For omitted species and numbering schemes of ligand backbones, refer to figure S2. molecules, O1W coordinated water, and carboxylate groups of IP-2-ac (table S1), resulting in a 3-D network. The 3-D crystal lattice is additionally stabilized by C-H⋯O contacts and π⋯π interactions (not shown herein, see table S1 for geometrical details). Packing in the unit cell of 2 is shown in figure 5.

Thermal stability of 1
Thermal behavior of 1 was assessed on crystalline samples by means of TG-DTA and DSC analyses carried out under nitrogen and air, respectively. There is no lattice water in the crystal of 1; therefore, its thermal stability was higher compared to previously reported [M(IP-2-ac) 2 (H 2 O) 2 ]·2H 2 O (M = Co, Ni, Mn, Cd) complexes, all of which decompose below 100°C [11]. Thermogram of 1 (figure S4) shows two steps of decomposition from 30-500°C. The first entropy-driven weight loss takes place above 139°C and corresponds to release of ca. one mole of coordinated water per mole of zinc (exp. 3.6%, calcd. 4.1%). Thereafter, subsequent endothermic event occurs (onset temperature 323°C, 48.6% weight loss) which can be attributed to decomposition of 1 by gradual elimination of coordinated ligands due to breaking coordination bonds. The DSC curve exhibits roughly similar endothermic event corresponding to dehydration of 1 (onset temperature 145°C, ΔH = 116.84 J/g, figure S5); however, further sample heating in oxidizing atmosphere leads to exothermic processes above 310°C (onset temperature 313°C, ΔH = −393 J/g). Similar behavior was observed for [M(IP-2-ac) 2 (H 2 O) 2 ]·2H 2 O (M = Co, Ni, Mn, Cd) complexes and attributed to simultaneous release of ligands and their thermal decomposition [11].

Vibrational analysis of 1
The presence of water molecule in the structure of 1 suggested the application of H/D isotope substitution for experimental detection of bands related to H 2 O and H 2 O-Zn vibrations. From the respective spectra shown in figure 6, deuteration caused much more spectral changes than expected. Apart from typical effects in the spectral region above 1800 cm −1 , Coupling is also expected between pyridine and imidazole ring vibrations as both rings have two common atoms. In order to assign the spectra as accurately as possible, DFT calculations were performed, which also included the deuteration effects. However, the results of calculations should be treated with caution because the computed molecular model does not fully reflect the environment of water ligand involved in intermolecular hydrogen bonds (figure 2 and table S1). In theoretical attempt, water molecule is considered to act as proton donor in the O 42 -H 44 ⋯O 33 intramolecular interaction (refer to figure S6 for atom numbering). In consequence, the calculated results were used mostly for skeletal and C-H group vibration assignments, whereas in the characterization of water-related bands, deuteration effects and literature data were applied first.
The results of such approach are reflected in table 3 presenting measured and calculated data together with proposed assignments. In order to minimize the size of table 3, the vibrations related to six-membered aromatic ring skeleton and C-H groups were omitted. These data can be found in supplementary table S2. Also, in the presentation of computed data, the theoretical band positions are given instead of normal vibration wavenumbers. Most of the calculated bands are generated by two normal modes of close wavenumbers and related to similar vibrations in two ligands present in the [Zn(IP-2-ac) 2 (H 2 O)] complex. For that    [2,4] 468 [5,9] δ(py-im)  [4,16] 128 [4,17] γ reason, also the PED terms for only one ligand are giventhe omitted ones, which usually are of similar values, can be amended using figure S6. The bending vibration of water molecule was assigned to the 1560 cm −1 strong shoulder of intense 1569 cm −1 IR band, together with ν(C=O) mode. This shoulder has no counterpart in the Raman spectrum, which is consistent with the spectroscopic criterion of bond polarity. After deuteration, the ν(C=O) vibration shifts to 1542 cm −1 while the δ(D 2 O) was attributed to band at 1128 cm −1 , which intensity significantly increases upon deuteration. This band was proposed based on δ(H 2 O)/δ(D 2 O) ratio which in the theoretical spectra is 1.36. Next, band sensitive to H 2 O/D 2 O substitution is observed at 1405 cm −1 (1394 cm −1 for deuterated complex). This band was assigned to calculated 1424 cm −1 mode with significant C-N py bond stretching, but also with small contribution of water bending vibration causing the mentioned isotope shift. Similar coupling with δ(H 2 O) (not reflected in calculations) probably causes the deuteration effects in the 1440-1300 cm −1 region, where respective changes are seen in the Raman spectra more clearly than in the IR ones. Taking into account the relative intensities, medium intensity Raman band at 1375 cm −1 shifts to 1321 cm −1 upon deuteration, whereas weak band at 1337 cm −1 appears as shoulder at 1329 cm −1 . Both vibrations show significant contributions of ν(C-N) modes in pyridine and imidazole rings, respectively.
The deformation modes (rocking, twisting, and wagging) of bonded water are usually strongly changed by hydrogen bonding; therefore, their assignment cannot be directly related to respective calculated modes. These vibrations were attributed to bands at 827 (rocking), 716, 702 (twisting), and 547 cm −1 (wagging) based on literature data [31,32] [11] were screened for their effect on microorganisms. The inhibition zones of microbial growth for ligand, 1, and starch used as a control probe are summarized in figure 7. Figure S7 presents analogous data for [M(IP-2-ac) 2 (H 2 O) 2 ]·2H 2 O complexes, and related photos are collected in table S3. Free ligand did not exhibit antimicrobial activity against the tested species. The nitrate salts were active against almost all species suggesting profound role of metal ions in inhibition of microbial species growth. Among five metal complexes studied, the best results were obtained for 1 and [Cd(IP-2-ac) 2 (H 2 O) 2 ]·2H 2 O, whereas related nickel(II), cobalt(II), and manganese(II) complexes were inactive under conditions of the experiment. Chelation of zinc(II) and cadmium(II) ions by IP-2-ac diminished effectiveness of inhibition of bacterial growth as compared to related nitrate salts ( figure S7). This can be explained as a result of slow dissociation of coordination compounds in the test environment, and consequent decrease in bioavailability of both cations as compared to simple salts.
All microbial species except for S. aureus and P. aeruginosa were susceptible when exposed to [Cd(IP-2-ac) 2

Conclusion
The reaction of HIP-2-ac with zinc(II) nitrate carried out under slightly alkaline conditions afforded two crystalline complexes, [Zn(IP-2-ac) 2