Synthesis, Structure and Antimicrobial Activity of New Co(II) Complex with bis -Morpholino/Benzoimidazole-s -Triazine Ligand

: A new Co(II) perchlorate complex of the bis -morpholino/benzoimidazole-s -triazine ligand, 4,4 (cid:48) -(6-(1 H -benzo[d]imidazol-1-yl)-1,3,5-triazine-2,4-diyl)dimorpholine ( BMBIT ), was synthesized and characterized. The structure of the new Co(II) complex was approved to be [Co(BMBIT) 2 (H 2 O) 4 ] (ClO 4 ) 2 *H 2 O using single-crystal X-ray diffraction. The Co(II) complex was found crystallized in the monoclinic crystal system and P2 1 /c space group. The unit cell parameters are a = 22.21971(11) Å, b = 8.86743(4) Å, c = 24.38673(12) Å and β = 113.4401(6) ◦ . This heteroleptic complex has distorted octahedral coordination geometry with two monodenatate BMBIT ligand units via the benzoimidazole N-atom and four water molecules as monodentate ligands. The hydration water and perchlorate ions participated signiﬁcantly in the supramolecular structure of the [Co( BMBIT ) 2 (H 2 O) 4 ](ClO 4 ) 2 *H 2 O complex. Analysis of d norm map and a ﬁngerprint plot indicated the importance of O ··· H, N ··· H, C ··· H, C ··· O, C ··· N and H ··· H contacts. Their percentages are 27.5, 7.9, 14.0, 0.9, 2.8 and 43.5%, respectively. The sensitivity of some harmful microbes towards the studied compounds was investigated. The Co(II) complex has good antifungal activity compared to the free BMBIT which has no antifungal activity. The Co(II) complex has good activity against B. subtilis , S. aureus , P. vulgaris and E. coli while the free BMBIT ligand has limited activity only towards B. subtilis and P. vulgaris . Hence, the [Co( BMBIT ) 2 (H 2 O) 4 ](ClO 4 ) 2 *H 2 O complex has broad spectrum antimicrobial action compared to the free BMBIT ligand.


Introduction
Heterocyclic rings are the main constituent of many compounds in natural and synthetic drugs.Therefore, the scientific exploration of new heterocyclic molecular systems has attracted many chemists in the field of inorganic and organic chemistry [1][2][3][4][5].In particular, s-triazine heterocycles are important nitrogen heterocyclic molecules.They are six-membered benzene rings incorporating three nitrogen atoms and three carbons.This interesting and exciting s-triazine scaffold has shown many possible applications which have been reviewed recently [6][7][8][9].In the medical field, many representative examples incorporating the symmetrical rigid structure of the s-triazine scaffold have been synthesized, characterized and explored in pharmacological applications against different targets [10].These applications include the use of s-triazine derivatives as anticancer [11,12], antimicrobial [13], antiviral [14], and antiprotozoal agents [15].
From this point of view, the incorporation of the benzimidazole scaffold as a very important pharmacophore with s-triazine in one molecule was explored and the results indicated the enhancement of biological activity in the case of the organic hybrid.Singa et al. reported a series of s-triazine molecules clubbed with benzimidazole scaffolds which were assessed against different cancer cells, including breast cancer, colon cancer, melanoma, leukemia, ovarian cancer, non-small cell lung cancer and central nervous system tumours.They exhibited a median growth inhibitory (GI 50 ) in the range of 1.91-2.72µM.The DNA intercalation and dihydrofolate reductase inhibition was optimized by the same research group [16,17].Kumar et al. demonstrated the anticancer reactivity and SAR for a set of compounds based on s-triazine with different heterocycles, such as benzimidazole and benzothiazole scaffolds.Interestingly, a noticeable enhancement in the cytotoxic activity was found due to benzimidazole moity and the IC 50 was found to be only 4.8 µM, indicating strong potency against breast cancer cell line MCF-7 [18].Another example was presented by Singa et al. in the form of s-triazine-benzimidazole derivatives.They constructed and tested s-triazine-benzimidazole derivatives for antiproliferative potential against a panel of cancer cell lines and for investigating the bovine serum albumin interaction, which exhibited the highest inhibiting potency [19].

Synthesis and Characterizations
The new bis-morpholino/benzoimidazole s-triazine (BMBIT) ligand was synthesized following the reported method by Matsuno et al. [39] as indicated in Scheme 1. Cyanuric chloride 1 was first reacted with benzoimidazole 2 in an acetone-water mixture (1:1) in presence of NaHCO 3 at 0 • C for two hours and crushed ice was added to obtain the dichlorobenzoimidazole s-triazine derivative 3 as a white solid in good yield and purity, as observed for its NMR spectra.The target bis-morpholino/benzoimidazole s-triazine (BMBIT) ligand was obtained from the reaction of 3 with two equivalents of morpholine in the presence of K 2 CO 3 using dimethylformamide as the solvent under heating conditions [39] (Scheme 1) to obtain the target product, 4,4 -(6-(1H-benzo[d]imidazol-1-yl)-1,3,5-triazine-2,4-diyl)dimorpholine (BMBIT), in excellent yield and purity, which was used directly in the complexation process (Scheme 2).Following this, self-assembly of the ethanolic solutions of the bismorpholino/benzoimidazole s-triazine (BMBIT) ligand and Co(ClO 4 ) 2 .6H 2 O was used to obtain the [Co(BMBIT) 2 (H 2 O) 4 ](ClO 4 ) 2 *H 2 O complex as a single-crystalline product (Scheme 2).The elemental analysis of the target complex confirmed the purity of the [Co(BMBIT) 2 (H 2 O) 4 ](ClO 4 ) 2 *H 2 O complex and its composition, which is further revealed by single-crystal X-ray structure analysis.The structural aspects of the [Co(BMBIT) 2 (H 2 O) 4 ](ClO 4 ) 2 *H 2 O complex were found in accordance with the FTIR spectra which confirm the coordination of the Co(II) ion with the azomethine nitrogen and also confirmed the involvement of the perchlorate ion in the structure (Figures S1 and S2 in Supplementary Materials).The FTIR spectra of the BMBIT ligand showed the ν (C=N) at 1600 and 1580 cm −1 and the ν (C=C) mode at 1498 cm −1 .In the case of the [Co(BMBIT) 2 (H 2 O) 4 ](ClO 4 ) 2 *H 2 O complex, the ν (C=N) mode was detected at lower wavenumbers of 1584 and 1575 cm −1 .On the other hand, the ν (C=C) mode was detected almost at the same wavenumber as for the free ligand.The ν (C=C) mode for the [Co(BMBIT) 2 (H 2 O) 4 ](ClO 4 ) 2 *H 2 O complex was detected at 1498 cm −1 .In addition, a broad splitted band at 1107 and 1073 cm −1 was assigned for the ν (Cl-O) vibrations, confirming the presence of the perchlorate anion.

X-ray Structure Description
The X-ray single-crystal structure of the newly synthesized complex was confirmed to be [Co(BMBIT) 2 (H 2 O) 4 ](ClO 4 ) 2 *H 2 O.The crystal system of this complex is monoclinic, and the space group is P2 1 /c.The unit cell parameters are a = 22.21971 (11) 2.

Analysis of Molecular Packing
The stability of the crystal structure is derived from many forces which keep the molecules arranged in a specific pattern to keep the crystal stable.The analysis of molecular packing with the help of Hirshfeld calculations provided all probable contacts in the crystal structure.The d norm , curvedness and shape index surfaces for the [Co(BMBIT) 2 (H 2 O) 4 ] (ClO 4 ) 2 *H 2 O complex are shown in Figure 5.The map of d norm gavea summary of all short contacts, which appear as red and white regions, indicating shorter and equal interaction distances than the vdWs radii sum of the interacting atoms.The longer contacts than the vdWs radii sum of the interacting atoms appeared as blue colored regions.Inspection of the d norm map indicated the importance of the O  3.
The Hirshfeld analysis predicts all possible intermolecular contacts and their percentages in the crystal structure of the [Co(BMBIT) 2 (H 2 O) 4 ](ClO 4 ) 2 *H 2 O complex.Hence, it not only presents a qualitative summary of molecular packing but also quantitatively provides the percentages of these non-covalent interactions (Figure 6).The percentages The fingerprint plot shows a graphical representation of the distance between the surface and an atom inside the surface (d i ) against the distance between the surface and an atom outside the surface (d i ), which not only provides a quantitative summary for all possible intermolecular contacts (Figure 6) but also reveal their importance (Figure 7).It is clear that the decomposed fingerprint plots of the O appear as sharp spikes.This pattern for the fingerprint plots is considered as strong evidence of a strong interaction occurring at short contact distances between the atoms.The different contacts that occurred at short distances are presented in Table 3.Interestingly, the Hirshfeld analysis detected the presence of anion-π stacking interactions as revealed by the presence of short C25

Antimicrobial Assay
The sensitivity of selected bacteria as Staphylococcus aureus and Bacillus subtilis (grampositive), Escherichia coli, Proteus vulgaris (gram-negative) and fungi such as Aspergillus fumigatus and Candida albicans towards the free ligand BMBIT and [Co(BMBIT) 2 (H 2 O) 4 ](ClO 4 ) 2 * H 2 O complex were examined.Recently, the antimicrobial activity of the free ligands DMPT and DPPT were compared with their metal (II) complexes [36][37][38].It was found that the majority of the studied metal (II) complexes have better antimicrobial activities than the free ligands [36][37][38].In accordance with our previous studies, it was observed that the free ligand BMBIT has no effect on both fungal species Aspergillus fumigatus and Candida albicans.In contrast, the [Co(BMBIT) 2 (H 2 O) 4 ](ClO 4 ) 2 *H 2 O complex has good activity against both microbes.The inhibition zone diameters are 13 and 15 mm, respectively, while the MIC values are 312 and 156 µg/mL, respectively.The results are relatively close to the antifungal agent Ketoconazole (Table 4).O complex compared to the free ligand BMBIT could be explained in terms of its ability to prevent the microbes from protein production, leading to the death of the treated microbes [40].

Physical Measurements
All chemicals were bought from their commercial sources and used without additional purifications (see Supplementary Materials).

Synthesis of 4,4 -(6-(1H-benzo
First, dichlorobenzoimidazole 3 was synthesized as follows: benzimidazole (0.1 mole, 11.8 g) was dissolved in acetone (15 mL) and added to a solution of cyanuric chloride (0.1 mole, 18.4 g) in acetone (100 mL) at 0-5 • C, and sodium bicarbonate solution (0.12 mole in 100 mL water) was added over a period of 10 min to adjust the pH of the reaction mixture.The reaction mixture was continued stirring for 2 h at 0-5 • C, crushed ice was added and the solid obtained was filtered and dried.The product was recrystallized from ethanol and the pure product 3 was obtained as an off-white precipitate with an 88% yield, mp 118-120 • C. Compound 3 was reacted with morpholine to obtain the target product as follows: 2-(benzimidazol-1-yl)-4,6,-dichloro-s-triazine 3 (10 mmol) was stirred with morpholine (20 mmol) in 10 mL DMF in the presence of K 2 CO 3 (22 mmol) at room temperature for 4 h and then heated at 80 • C overnight.The reaction mixture was poured into an ice-water mixture and the product was filtered, washed with ethanol and dried, to afford the target product BMBIT as a white solid with a 91% yield, mp.223-225

Crystal Structure Determination
The crystal of the Co(II) complex was determined as described Method S1 in Supplementary Materials [41][42][43][44].The crystallographic details are summarized in Table 5.

Hirshfeld Analysis
The Crystal Explorer Ver.3.1 program [45] was used to perform this analysis.

Figure 2 .
Figure 2. Structure of the coordination sphere of [Co(BMBIT)2(H2O)4](ClO4)2*H2O complex.One of the two perchlorate anions showed disorder and was omitted from this figure for better clarity.Figure 2. Structure of the coordination sphere of [Co(BMBIT) 2 (H 2 O) 4 ](ClO 4 ) 2 *H 2 O complex.One of the two perchlorate anions showed disorder and was omitted from this figure for better clarity.

Figure 2 .
Figure 2. Structure of the coordination sphere of [Co(BMBIT)2(H2O)4](ClO4)2*H2O complex.One of the two perchlorate anions showed disorder and was omitted from this figure for better clarity.Figure 2. Structure of the coordination sphere of [Co(BMBIT) 2 (H 2 O) 4 ](ClO 4 ) 2 *H 2 O complex.One of the two perchlorate anions showed disorder and was omitted from this figure for better clarity.

Table 1 .
Bond distances and angles (Å and • ) for the coordination environment of the [Co(BMBIT) 2 (H 2 O) 4 ](ClO 4 ) 2 *H 2 O complex.noting that the structure of the [Co(BMBIT) 2 (H 2 O) 4 ](ClO 4 ) 2 *H 2 O complex contains one hydration water molecule and two perchlorate counter anions which are not involved in the coordination with the Co(II) ion but are significantly involved in the molecular packing of this complex (Figure 3A).The packing view of the [Co(BMBIT) 2 (H 2 O) 4 ](ClO 4 ) 2 *H 2 O complex along the crystallographic ac-direction is clearly seen from Figure 3B.In the [Co(BMBIT) 2 (H 2 O) 4 ](ClO 4 ) 2 *H 2 O complex, molecular packing is controlled by a number of O•••H contacts.The coordinated and free water molecules are acting as H-bond donors while the perchlorate and the hydration water are H-bond acceptors.A list of the H-bond parameters is represented in Table 2.The majority of these intermolecular interactions are strong O-H•••O hydrogen bonds, and the donor to acceptor distances are in the range of 2.7011(16) Å (O2-H2B•••O6) to 3.082(3) Å (O4-H4A•••O14).The respective hydrogen to acceptor distances are 1.91(3) and 2.18(4) Å.
a R 1 =