Coordination complexes constructed from pyrazole–acetamide and pyrazole–quinoxaline: effect of hydrogen bonding on the self-assembly process and antibacterial activity

Two mononuclear coordination complexes of N-(2-aminophenyl)-2-(5-methyl-1H-pyrazol-3-yl)acetamide (L1), namely [Cd(L1)2Cl2] (C1) and [Cu(L1)2(C2H5OH)2](NO3)2 (C2) and one mononuclear complex [Fe(L2)2(H2O)2](NO3)2·2H2O (C3), obtained after in situ oxidation of L1, have been synthesized and characterized spectroscopically. As revealed by single-crystal X-ray diffraction, each coordination sphere made of two heterocycles is completed either by two chloride anions (in C1), two ethanol molecules (in C2) or two water molecules (in C3). The crystal packing analysis of C1, C2 and C3, revealed 1D and 2D supramolecular architectures, respectively, via various hydrogen bonding interactions, which are discussed in detail. Furthermore, evaluation in vitro of the ligands and their metal complexes for their antibacterial activity against Escherichia coli (ATCC 4157), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923) and Streptococcus fasciens (ATCC 29212) strains of bacteria, revealed outstanding results compared to chloramphenicol, a well-known antibiotic, with a normalized minimum inhibitory concentration as low as 5 μg mL−1.


Introduction
There is growing interest in the development of new active antibacterial compounds as current clinical treatments remain insufficient to meet the challenge of the increasing emergence and spread of antimicrobial resistance. 1 In Europe, antibiotic resistance is responsible for approximately 33 000 deaths per year. 2 In the United States, more than 2.8 million people suffer from antibiotic-resistant infections, resulting in more than 35 000 deaths each year. 3 In 2019, the WHO identied 32 antibiotics in clinical development that address the WHO's list of priority pathogens, of which only six were classied as innovative. Furthermore, a lack of access to quality antimicrobials remains a major issue. Antibiotic shortages are affecting countries of all levels of development and especially in healthcare systems. 4 Therefore, there is an urgent need to develop new antimicrobial agents.
Nitrogen-based ligands are attracting growing attention due to their interesting properties in structural and inorganic chemistry. 5 Pyrazole derivatives are biologically active heterocyclic compounds. [6][7][8] This substance class has been the topic of numerous pharmaceutical studies being used for their medicinal properties such as anti-inammatory, 9 antidiabetic, 10,11 antiviral, 12 analgesic, 13 antitumor, 14 catecholase, 15 and antimicrobial properties. 16 On other hand, nitrogen systems have attracted more attention in recent years because of their interesting properties in coordination chemistry. [17][18][19] However, many reports on transition metal complexes explain their efficient bioactivity against a range of bacterial and fungal species. [20][21][22] In particular, heterocyclic metal complexes dominated medicinal chemistry due to their wide range of properties. [23][24][25] Metal complexes tethered with heterocyclic moieties like imidazole, pyrazole, 1,2,4-triazoles and benzimidazole have received remarkable interest as broad spectrum antibacterial, antifungal and antiviral agents. [26][27][28][29] Therefore, the antibacterial activity of many metal complexes has been demonstrated against several bacterial species both in vitro and in vivo, making it as promising antibacterial agents for use against these bacteria, pending a greater understanding of its safety upon systemic or topical administration in humans. [30][31][32][33][34][35][36] Recently, a study investigated organometallic compounds submitted to the Community for Open Antimicrobial Drug Discovery (CO-ADD) databank, established a classication based on the nature of their metal element, activity, as well as toxicity. 37 Metalcontaining compounds show actually a signicantly higher success rate (9.9%) compared to purely organic molecules (0.87%). Out of 906 compounds, 88 show activity against at least one of the tested strains, including fungi, while showing no cytotoxicity against mammalian cell lines or hemolytic properties. Amongst the metal complexes, cadmium, copper and iron, were the most frequent elements found in active 'non-toxic' compounds and show the highest overall success rate. 37 In order to search for new ligand candidates for assemblies of metal complexes, we considered the case of pyrazole acetamide ligands with O and N donor atoms. [38][39][40] These molecules are particularly interesting as ligands for the construction of polynuclear complexes as models for bioinorganic systems. 41,42 As a continuation of our research along this line, 43,44 we have Fig. 1 Structures of N-(2-aminophenyl)-2-(5-methyl-1H-pyrazol-3yl)acetamide (L 1 ) and 3-(5-methyl-1H-pyrazol-3-yl)quinoxalin-2(1H)one (L 2 ). Rened as an inversion twin Absolute structure parameter 0.267 (16) successfully synthesized three new Cd(II), Cu(II) and Fe(II) coordination complexes derived from the ligands, namely N-(2aminophenyl)-2-(5-methyl-1H-pyrazol-3-yl)acetamide (L 1 ) and 3-(5-methyl-1H-pyrazol-3-yl)quinoxalin-2(1H)-one (L 2 ) (Fig. 1). The molecular structures of the complexes were conrmed by single-crystal X-ray diffraction. All ligands and metal complexes were evaluated in vitro for their antibacterial activity against Escherichia coli (ATCC 4157), Pseudomonas aeruginosa (ATCC Scheme 1 Synthetic route for preparation of L 1 .  (1998)). The isomer shi is given with respect to a-Fe at room temperature. X-Ray powder diffraction patterns were recorded on a D8-Advance diffractometer (Bruker, Germany) working with a Cu Ka radiation (l ¼ 1.5148Å).

X-ray analysis
X-ray single-crystal data were collected on single crystals using Mo Ka (l ¼ 0.7107 A) radiation on a Bruker SMART APEX diffractometer equipped with CCD area detector. Unit cell renement data reduction (SAINT) and structure solution as well as renement (SHELXTL) 45 were carried out using the soware package of SMART APEX. The structures of C 1 , C 2 and C 3 were solved by direct method and rened in a routine manner. In both structures, non-hydrogen atoms were treated anisotropically. Molecular graphics were generated by using the sowares MERCURY 3.9 (ref. 46) and POV-Ray. The details of the X-ray crystal data and the structure solution as well as the renement are given in Table 1. CCDC 2095071-2095073 for C 1 , C 2 and C 3 , respectively contain the supplementary crystallographic data for these compounds.

Antibacterial activity
The antibacterial activity of the synthesized compounds was determined according to the method described in our previous work. 43

Synthesis of pyrazole-acetamide ligands L 1
Our strategy was to develop a simple, high-yield, synthetic procedure in a few steps to prepare the desired acetamide derivative. The development of the synthesis of L 1 is given in Scheme 1. The major product 3 47 was produced in good yield by condensation of o-phenylenediamine with dehydroacetic acid (DHA) in reuxing xylene for 4 h. The second step consists in the condensation of a stoichiometric amount of hydrazine monohydrate with the benzodiazepine compound 3 in reuxing ethanol for 2 h to give the ligand pyrazole-acetamide L 1 in good yield 48 (Scheme 1).

Synthesis of coordination complexes C 1 , C 2 and C 3
The three coordination complexes [Cd( were obtained as single crystals aer recrystallization from ethanol during the reaction carried out in an aqueous ethanolic solution involving pyrazole acetamide L 1 and Cd(II), Cu(II) and Fe(III) (metal/ligand ratio 1 : 2), respectively ( Table 2).
While classic coordination occurred for L 1 with cadmium and copper, an unexpected oxidation reaction followed by an intramolecular cyclization of the formed intermediate was presumably observed when iron nitrate was used as chelating agent. Thus, the iron(II) complex C 3 was synthesized in low yield by the reaction of Fe(NO 3 ) 3 $9H 2 O with L 1 in a 1 : 2 molar ratio using ethanol as a solvent. The low yield of the compound is attributed to the involvement of the L 1 ligand in the redox reaction with Fe(III) ion, where the Fe(III) ion was reduced to Fe(II) ion. The Fe(II) ion produced was complexed with the ligand L 2 formed by the oxidation of L 1 (Scheme 2). The rst Fe(III) promoted in situ oxidation of a thiazoline-2-thione to the corresponding hetero-disulphide with concomitant coordination to Fe(II) was reported by Raper et al., 49 Several studies have been conducted mainly to characterize the products of reactions between nitrogen-containing ligands and Fe(III) ions under aerobic conditions. [50][51][52][53] The presence of the electronwithdrawing groups increases the reduction potential of the Fe 3+ + e À 4 Fe 2+ redox couple, making the reduction product thermodynamically more stable. 54 A plausible mechanism (Scheme 2) is proposed to explain the original transformation of pyrazole acetamide L 1 into the new ligand L 2 . Thus, the participation of the ligand L 1 in redox reaction with Fe(III) ion, where Fe(III) ion was reduced to Fe(II) ion and L 1 was oxided to L 1 0 , is proposed. Aerwards, air oxygen reacted with the carbon-carbon double bond of the tautomeric form L 1 0 affording spiro oxetane pyrazole A which undergoes a ring opening of the oxetane moiety under the effect of a base to lead to the hydroxy pyrazoline B. The latter compound aromatizes by the loss of a water molecule to give the ketonic amide C which undergoes an intramolecular cyclization to afford aer a loss of a water molecule pyrazolyl quinoxaline acting as coordination compound towards Fe(II) ion produced by reduction of Fe(III). It should be noted that a similar oxidation reaction has already been observed in our previous work on 1,2,4-triazolo pyrimidines. The crystal structures of C 1 , C 2 and C 3 are shown in Fig. 2.
Crystallization of all the three coordination compounds was obtained by reaction of the ligand L 1 (for C 1 and C 2 ) or L 2 (for C 3 ) and metal salts in aqueous ethanolic solution (metal-: ligand ¼ 1 : 2) by slow evaporation.
. Orange colored single crystals of C 3 got crystalized in the centrosymmetric triclinic space group P 1 ( Table 1). The asymmetric unit comprises of one Fe(II) ion, two molecules of ligand L 2 , two water molecules (both water and L 2 are coordinated to Fe(II)), two nitrate counter anions and two solvated water molecules.  (6)Å] of nitrate with pyrazole ring of metal coordinated L 2 resulted in an twelve membered ring which contains the four donor and acceptor atoms with a graphset symbol R 4 4 (12) (Fig. 5a). This synthon is connected to the discrete [Fe(L 2 ) 2 (H 2 O) 2 ] complex unit node through nitrate anions, metal bound water molecule and quinoxalinone moiety  (Fig. 5a). The hydrogen bonding interactions through these two synthons resulted in the self-assembly of C2 along crystallographic axis "b" lead to the formation of one-dimensional hydrogen bonded chain (Fig. 5a). The self-assembly further follow through these two twelve membered ring synthons along crystallographic axis "a" resulted in a 2D hydrogen bonded sheet network structure (Fig. 5b and c). Interestingly, such 2D network structure further self-assembled through O-H-N hydrogen bonding [O-H/N ¼ 2.856(4)Å, :O-H-N ¼ 174 ] involving metal bound water and N atom of quinoxalinone (along crystallographic axis "c") lead to the formation of a 3D hydrogen bonded network (Fig. 5d). If the discrete [Fe(L 2 ) 2 (H 2 O) 2 ] complex, and twelve membered ring synthons are taken as nodes, the 3D hydrogen bonded network can be simplied to a 3D [3 2 $6-c]-connected net having point (Schläi) symbol of {4$8 2 } 2 {4 2 $6} 2 {4 2 $8 10 Â 10 3 } (Fig. 5e).

Hirshfeld surface analysis
To further explore the supramolecular interactions in the crystal structure of the coordination compounds, we have constructed their Hirshfeld surface and 2D-ngerprint plots by using Crystal Explorer program. 55 The surface where the electron density r int (r) of the molecules is larger than the electron density r ext (r) of the adjacent molecules is called the Hirshfeld surface. 56 Hirshfeld surfaces (HS) of the coordination compounds C 1 , C 2 and C 3 are shown in Fig. 6, displaying the surface map over the normalized contact distance (d norm ) in which the red and white colors indicating strong proximity and intermediate closeness of atoms to the HS from outside, respectively. We have used the following equation to calculate d norm from the values of d e (distance between the Hirshfeld surface and external molecule), d i (distance between the Hirshfeld surface and inside molecule) and van der Waals radii of the atoms (r vdw i or r vdw e ). From the value of d norm , we can easily determine the regions participating in the intermolecular interactions in the complexes.
In other words, these two colors indicating strong and intermediate hydrogen bonding interactions present between HS and neighboring atoms outside, respectively. The blue color regions in the HS meaning the longer distances than the van der Waals radii. The HS of C1 was generated by using a standard (high) surface resolution with 3D d norm surfaces mapped to a range À0.3180 to 1.5585 a.u. From the d norm mapping, it is Fig. 6 The d norm Hirshfeld surfaces of C 1 (a), C 2 (b), and C 3 (c) displaying hydrogen bonding interactions.
revealed that strong N-H/Cl hydrogen bonding interaction (between amide moiety of L 1 and chloride anion) was present in the crystal structure of C 1 , as observed from the bright red spots on the HS. The 3D d norm surfaced mapping of C 2 was done within the range of À0.5718 to 1.7268 a.u which showed bright red spots at amine and amide N-H (due to strong N-H/O hydrogen bonding with nitrate counter anion), and metal bound water molecule (O-H/O hydrogen bonding with nitrate). In the case of complex C 3 , the 3D d norm surfaced mapping (range of À0.7131 to 1.2768 a.u.) showed red spots near to metal bound water molecule and quinoxalinone moiety (due to strong O-H/O and O-H/N hydrogen bonding). We have also plotted the shape index and curvedness of the coordination complexes by using Crystal Explorer program; the red concave surface surrounded by the receptors and blue convex surface surrounding receptors on the HS in the shape index of the coordination complexes further conrm the presence of such hydrogen bonding ( Fig. S13 and S14, † ESI).
In order to quantify the contribution of various supramolecular interactions in the coordination complexes C 1 , C 2 and C 3 , we have plotted their 2D ngerprints by using Crystal Explorer program ( Fig. 7 and S15-S17, † ESI). The internal d i and external d e distances between the HS and atom contacts are given inÅ. We found that two strong hydrogen bonding N-H/O and N-H/Cl are presented in the crystal structure of  (Fig. 7).

Mössbauer spectroscopy
A powdered sample of C 3 was recorded at 298 K. The spectrum shows a quadrupole doublet with isomer shi d ¼ 0.346(3) mm s À1 and quadrupole splitting DE Q ¼ 0.72(1) mm s À1 (Fig. 8). Such a doublet is characteristic of high-spin Fe(III) species. Measurements were also recorded at high velocity up to v max ¼ 10 mm s À1 but no oxides were detected. This result contrasts with the one offered by single crystal X-ray diffraction which revealed Fe(II) species only. A microscope analysis shows that the powder contains few orange single crystals, whereas the majority of the powder is black. A crystal cell parameters analysis of orange crystals revealed similar parameters as those given in Table 1. This result is conrmed by powder X-ray diffraction of the powdered sample which shows an amorphous pattern plus diffraction peaks. These diffraction peaks correspond to the simulated ones from the cif le of C 3 (Fig. 9). Worth to note that the amount of C 3 detected in the powder was evaluated below the detection limit of Mössbauer spectroscopy, i.e. ca. 2%.

Antibacterial activity
The antibacterial activities of the synthesized molecules (3 and L) and metal complexes (C 1 , C 2 and C 3 ) were tested against E. coli and P. aeruginosa as Gram-negative and S. aureus and S. fasciens as Gram-positive microorganisms by the diffusion method disk. Table 3 reports the minimum inhibitory concentration (MIC) which is the lowest concentration for which no growth is detected for 24 h at 37 C. The results were compared with a standard, chloramphenicol, an antibiotic, e.g. used for the treatment of eyelid infection, 57 at various concentrations.
Overall the three complexes showed higher antibacterial activities against the four strains tested, compared to 3 and L 1 ligand, except in the case of S. fasciens with a remarkable MIC ¼ 5 mg mL À1 for 3 (Table 3). Such antibacterial activity of the C 1 -C 3 complexes compared to the ligand, could be due to the coordination of cadmium, copper and iron metal ions to the condensed ring system (as shown by single crystal X-ray diffraction), thus increasing the delocalization of p electrons throughout the chelated ring and improving the lipophilicity of the complexes and thus the penetration of the complexes into the lipid membrane and further limiting the multiplicity of microorganisms, following Overtone's concept on cell permeability, 58 and Tweedy's chelation theory. 59 Remarkably, C 1 reveals an outstanding activity against both Gram-negative (E. coli and P. aeruginosa) and Gram-positive (S. aureus) bacteria, compared to C 2 , C 3 complexes, with a MIC value of 5 mg mL À1 . This value is even better compared to chloramphenicol, a well-known antibiotic. Similarly, C 2 reveals Fig. 9 XRPD pattern of the black powder issued from the synthesis of C 3 compared to the computed XPRD pattern obtained from the cif file of C 3 .

Conclusions
In conclusion, three new Cd(II), Cu(II) and Fe(II) complexes formulated as [Cd(L 1 ) 2 Cl 2 ] (C 1 ), [Cu(L 1 ) 2 (C 2 H 5 OH) 2 ](NO 3 ) 2 (C 2 ) and [Fe(L 2 ) 2 (H 2 O) 2 ](NO 3 ) 2 $2H 2 O (C 3 ) have been synthesized and their crystal structures have been studied. Crystal structure and Hirshfeld Surface analysis have shown that the crystal lattices of all the complexes are inuenced for the presence of several intermolecular interactions, including hydrogen bonds. The N-H/O and N-H/Cl hydrogen bonding interactions benets to C 1 , which is the rst coordination complex with L 1 , to assemble to form a 1D chain as a primary supramolecular architecture. On the other hand, in complex C 2 , N-H/O and O-H/O hydrogen bonding play a role to self-assemble the crystallographically independent molecules of complexes and ethanol molecules to form a 2D corrugated hydrogen bonded sheet. The layers are joined by inversion-related C-H/O hydrogen bonds. On another hand in complex C 3 , the iron ion is coordinated by two chelating organic ligands and two water molecules with a slightly distorted octahedral geometry. O-H/ O, N-H/O and O-H/N hydrogen bonds and p-stacking interactions form layers of cations, anions and solvent water molecules. These are further linked into the full threedimensional structure by additional hydrogen bonds. Furthermore, the results of antibacterial activity testing reveal that C 1 , C 2 and C 3 complexes showed notable activity against all four strains of bacteria studied. Thus, the best result was shown by

Conflicts of interest
The authors declare no competing nancial interest.