Introduction

Thiourea compounds are important ligands in coordination chemistry, indispensable intermediates for the preparation of organocatalysts or ancillaries in chemosensors, and important part of anticorrosion compounds [1, 2]. They are widely recognized for their biological and antibacterial properties, with increasing use as antitubercular or antitumor agents, insecticides, antiprotozoal agents, and herbicides [35]. In addition, morpholine derivatives possess anti-inflammatory, antimicrobial and central nervous system activities [6]. The morpholine nucleus incorporated in a wide variety of therapeutically important drugs like linezolid, which belongs to the oxazolidinone class of anti-infectives and is used for the treatment of infections caused by Gram-positive bacteria [7]. On the other hand, the complexation capacity of thiourea derivatives and the biological activities of thiourea complexes are well recognized for different biological systems [8,9,10].

Among the transition metal, cobalt, nickel, and silver ions have been attracted the biologists attention due to their individual characteristics. A large number of reports on the antibacterial characteristics of cobalt complexes have developed in the literature and selecting Co complexes as the main subjects for the study in order to find effective antibacterial agents still being continued [8, 11]. Nickel is an essential trace element for bacteria, plants, animals, and humans [12]. There is spectroscopic evidence for nickel–methyl complexes in CODH of anaerobic bacteria [13]. For instance, the nickel transport system in E. coli composed of five proteins, NiKABCDE.

On the other hand, silver deposition has been found in the skin, the lungs, the gingiva, mucous membranes, and gastrointestinal tract [14, 15]. Essentially complex structures of silver-protein are take in within the body but have no physiological or biochemical roles [14, 16, 17]. Ag complexes have a very broad range of antibacterial activity and annihilate both Gram-positive and Gram-negative bacteria. Silver is a more lethal element to microorganisms than many other metals [18,19,20]. Actually, silver destructs the structure of bacterial cell membrane, prevent the cell respiration, and increase or diminish the expression abundance of some enzymes [20]. Indeed, the mechanism for the antibacterial activity of silver complex is not correctly recognized; although it is accepted that they intervene with cell growth via interruption of cell metabolism, restraint of transport actions in the cell wall, and interaction with DNA [14].

In this research, we report the synthesis of a novel thiourea derivative, N-(4-chlorophenyl)morpholine-4-carbothioamide (HL) and its Co(III), Ni(II), and Ag(I) complexes (1a, 1b, and 1c) and their characterization by FT-IR, 1H NMR, and UV-vis spectroscopy. For HL and its 1c complex, crystal structures were determined using X-ray single-crystal diffraction methods. We also evaluated the antibacterial activities of these compounds using three methods, macro dilution (MIC), colony count and well diffusion. The relationship between the structures of these specimens and their antimicrobial activity was prospected based on density functional theory (DFT) calculations.

Results and discussion

Synthesis

The reaction between 4-chlorophenyl isothiocyanate and morpholine in acetone resulted in the HL ligand (Scheme 1). This ligand was recrystallized by ether diffusion into the N,N-Dimethylformamide solution of the ligand.

Scheme 1
scheme 1

.Synthetic route to HL ligand, 1a, 1b, and 1c complexes

According to the Scheme 1, the Co(III), Ni(II), and Ag(I) complexes, 1a, 1b, and 1c were prepared by adding a hot methanolic solution (50 °C) of the respective metal nitrate to a solution of the HL ligand in the molar ratio of 1:3 for 1a and 1:2 for 1b and 1c. The synthesized Co(III), Ni(II), and Ag(I) complexes are soluble in CHCl3, DMF, and DMSO. These compounds were characterized by UV-vis, 1H NMR, FT-IR spectroscopy and the single-crystal X-ray diffraction analysis.

FT-IR spectral studies

In the FT-IR spectra of HL ligand, the band at 3159 cm−1 could be attributed to the stretching frequencies of the N–H group. Stretching vibrations of C = S bond was observed at 1323 cm−1. Symmetric and asymmetric stretching vibrations of (-CH2) protons appeared at 2861–2908 cm−1. The absence of ν(S-H) band at about 2570 cm−1 and the presence of the ν(N-H) band at about 3159 cm−1 indicated formation of the thione tautomer in the solid state (Figure S1a in the supplementary content). After complexation of the HL ligand to the Co(III) and Ni(II) the N-H stretching vibrations disappeared due to the loss of the H bonded to the N atom of thioamide group (Figure S1b and c, in the supplementary content). Also, the vibrational frequencies corresponding to C = S group in the free ligand were shifted towards down fields upon the complexation, confirming that the HL ligand is coordinated to the cobalt and nickel ions via the nitrogen and sulfur atoms in a form of neutral complexes (1a and 1b) [21, 22].

The FT-IR spectrum of the 1c complex demonstrates bands similar to those of ligand with a slight shift to the lower/upper wavenumbers owing to its coordination with Ag(I) metal center in the complex. For example, the characteristic band at 3208 and 1304 cm−1 can be assigned to N-H and C-S stretching vibration of 1c complex. Therefore, the presence of the ν(N-H) band and shifting of the ν(C-S) band towards lower wavenumber confirmed that the HL coordinates to the Ag(I) via S atom, unlike Co(III) and Ni(II) complexes. The infrared absorption band of the nitrate anion in this complex exhibits that the nitrate isn’t coordinated to Ag(I) center. This fact was deduced from the strong new band revealed at 1343 cm−1 (Figure S1d in the supplementary content) [14, 23].

NMR spectral studies

The 1H NMR spectra were recorded at room temperature at 300 MHz in DMSO-d6 for ligand, Co(III) and Ag(I) complexes and in CDCl3 for Ni(II) complex (Figure S2 a-d, in the supplementary content). The formed thione tautomer of HL instead of thiol tautomer was proved by presence of NH resonances at δ 9.42 ppm and absence of SH resonances. The phenyl protons and the diastereotopic hydrogens of -CH2 (morpholine moiety) were observed at δ 7.32–7.39 and δ 3.65–3.92 ppm, respectively.

Based on the results, complexation of HL to Ag(I) through S atom is approved by the presence of the N–H signal at δ 10.01 ppm. Indeed, pursuant to the Lewis acids and bases theory, the soft Ag(I) metal center tends to coordinate to the sulfur atom of the ligand than the nitrogen or oxygen atoms. In the spectrum of 1c, each peak is shifted toward upfield from its normal peak position corresponding to HL. The diastereotopic hydrogens of -CH2 (morpholine moiety) and phenyl protons are observed at δ 3.72–3.75, δ 3.91–3.94 and δ 7.19–7.46 ppm, respectively.

On the other hand, the loss of NH resonances in the 1a and 1b metal complexes confirmed that the chelation of HL ligand to the Co(III) and Ni(II) occurs via N and S atoms. The diastereotopic hydrogens of -CH2 morpholine moiety appeared at δ 3.06–3.78 and δ 3.14–3.86 ppm for 1a and 1b, respectively. Similarly, the characteristic signal of the phenyl protons corresponding to 1a and 1b was observed at δ 6.40–7.64 and δ 6.55–7.26 ppm, respectively. Both FT‐IR and NMR data are in agreement with the proposed structures [24].

Electronic absorption spectroscopy

CHCl3 solutions of the thiourea ligand HL and its metal complexes 1a, 1b, and 1c were prepared and their electronic absorption spectra were recorded in the range of 200–800 nm. For HL, the observed absorption bands at 240 and 270 nm were assigned to intra-ligand charge transfer transitions ππ* (phenyl ring) and nπ* corresponding to C = S bond [25, 26].

As expected from the results of FT-IR spectroscopy, no significant shifts in the ππ* transitions were observed after complexation but a bathochromic shift that corresponding to the n–π* transitions of C-S bond were recognized, which demonstrated that the ligand coordinated to the Co(III) and Ni(II) through the N and S atoms. Accordingly, in the 1a and 1b, bands above 340 nm are attributed to charge transfer processes, probably from the ligand to the metal. Absorption bands at higher wavelengths in the visible region (650 and 635 nm for 1a and 1b, respectively) were due to d–d transitions. In the 1c complex, the absorption band corresponding to the n–π* transition of C = S is demonstrating a bathochromic shift (265 nm) due to the complexation of thione sulfur atoms to metal ion. Generally, in electronic absorption spectra of 1c complex, no d–d transition was observed due to d10 electronic configuration of Ag(I) ion [27,28,29].

Description of crystal structures

Crystal structure of HL

The structure of HL was affirmed by the results of single-crystal X-ray structure determination. The molecule of this compound is shown in Fig. 1, details about crystal data collection and structure refinement and important bond lengths and angles are summarized in Table 1. The bond length of thiocarbonyl (C7-S1 = 1.685(5) Å) lies between values of a single and a double bond [30, 31]. On the other hand, due to the delocalization of electron density in the ligand, the mentioned C–N bonds, C7-N1 = 1.365(1) Å, C7-N2 = 1.353(3) Å, become shorter in comparison with the typical single C–N bond length of 1.479 Å [C8-N2 = 1.466(9) Å and C11-N2 = 1.470(8) Å] [32, 33]. This delocalization is also confirmed by values of the angles N1-C7-N2 = 114.87(7)°, N1-C7-S1 = 122.73(6)° and N2–C7-S1 = 122.35(0)° indicating sp2 hybridization on the C7 atom [34].

Fig. 1
figure 1

ORTEP diagram of HL ligand. CCDC number is 1884234

Table 1 Summary of X-ray diffraction data and selected bond lengths (Å), angles (°), torsion angles (°) for HL ligand and Ag(I) complex (1c)

Crystal structure of 1c

Figure 2 shows that the Ag center is coordinated to two ligands in a neutral monodentate (S) binding mode, giving rise to a nearly linear geometry around Ag [S1a-Ag1-S1b = 175.007(19)°]. The crystal structure also contains a free disordered NO3 anion and two partially occupied crystal water molecules, one of them being weakly coordinated to Ag (Ag1-O2w = 2.5115(216) Å). A summary of selected bond lengths and bond angles for the 1c complex is given in Table 1. The C–S bond lengths for 1c are longer than that for HL [35, 36]. Other bond lengths and angles are in good agreement. For other newly synthesized complexes (1a and 1b) several attempts failed to obtain a single-crystal suitable for X-ray crystallography.

Fig. 2
figure 2

ORTEP diagram of 1c complex. CCDC number is 1884216. (The hydrogens of water molecules and nitrate ion were omitted for more clarity)

Antibacterial activities

The bactericidal properties of HL, 1a, 1b, 1c, and the reference antibiotics amikacin (AMK-30 µg) and gentamicin (GEN-10 µg) were examined against two Gram-negative; Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) and two Gram-positive; Staphylococcus aureus (ATCC 25923) and Enterococcus faecalis (ATCC11700) bacteria. DMSO was employed as a negative control while amikacin and gentamicin were utilized as positive controls for Gram-positive and Gram-negative antibacterial activity, respectively. Silver sulfadiazine (AgSD) is regarded as reference Ag(I) specimen for susceptibility testing [14, 37]. Macro dilution, colony count, and well diffusion methods were employed to characterize the antimicrobial activity of these reagents.

Minimum inhibitory concentration (MIC) assay

In our investigation, the metal salts all presented moderate MIC values against the studied bacteria strains (MIC values below 0.1 mg/mL are considered significant, between 0.1 and 0.5 mg/mL moderate, between 0.5 and 1.0 mg/mL weak, and above 1.0 mg/mL negligible) [38,39,40]. On the other hand, although HL ligand also showed a moderate MIC value against E. coli, P. aeruginosa, and S. aureus, its activity against E. faecalis was weak. As for the metal complexes, a significant increase of the bactericidal activity was observed in comparison to the free ligand. The order by antimicrobial activity was 1c > 1b > 1a > HL. The enhanced activity of these complexes in comparison with the free ligand can be explained by the Ovetone’s concept explaining the relationship between the bactericidal activity and lipophilicity. As we know, the lipid membrane that envelopes the cell favors the passage of lipid-soluble substances [41,42,43,44,45,46,47]. Furthermore, the presence of the chlorine moiety in the structure of these reagents may also contribute to their antibacterial effect due to the hypochlorous acid formation. This acid crumbles forming hydrochloric acid and oxygen, and the oxygen damages microorganisms through oxidizing cellular parts. Chlorine compounds can also interact and combine with proteins and enzymes of membranes and as a result, destroy the microbes or prevent their growth by obstruction of their active sites [48]. Meanwhile, the presence of the chlorine atom and the morpholine group increases the lipophilicity and the electron-withdrawing effect on these samples, and this can also improve the bactericidal effects [49, 50]. Although antimicrobial activity of all complexes was observed to have the notable effect versus all tested Gram-negative and Gram-positive bacteria, they exhibited lower activity compared to standard antibiotics amikacin and gentamicin.

Well diffusion assay

The results of well-diffusion method for antimicrobial activity of synthesized samples show the highest zone of inhibition (19.7 mm) for 1c against E. coli. The results (Table 2) show the studied compounds possess antibacterial activity against the above-mentioned Gram-negative and Gram-positive bacteria, and the activity against the Gram-negative bacteria is larger. The efficacy of these compounds is controlled by the cell-wall thickness and the polarity of the inner cell membrane, so the permeation into the bacterial strains of some agents can be facilitated [51,52,53,54,55,56]. Gram-negative bacterium has a thin peptidoglycan layer that an outer protein-phospholipid-lipopolysaccharide membrane covers that, whereas a Gram-positive bacterium has a thick peptidoglycan layer that contains teichoic and lipoteichoic acid. As a result, the cell wall of Gram-negative bacteria is more polar [57, 58]. As a result, the bactericidal properties of HL ligand and its complexes against P. aeruginosa and E. coli is higher than against S. aureus and E. faecalis [59]. On the other hand, for all tested bacterial strains, the antibacterial activity of complexes was higher than for the free ligand. Indeed, the coordination of metal ions to the ligand enhanced the antibacterial activities. This is maybe caused by a synergistic effect involving Co(III), Ni(II), and Ag(I) ions and the thiourea ligand.

Table 2 Results of minimal inhibitory concentrations (MIC, mg/mL) and zones of inhibition (ZI, diameter/mm) of all compounds against the studied bacterial strains

Colony count assay

As shown in Fig. 3, the consequences achieved from the colony count assay corroborate the results of MIC as well as of well diffusion. The 1c complex demonstrates stronger activity than the other specimens and inhibits all bacteria strains, E. coli, and S. aureus, in the lowest time.

Fig. 3
figure 3

Plot of the survival number of a E. coli ATCC 25922 and b S. aureus ATCC 25923 with HL ligand, 1a, 1b, and 1c complexes in colony count method

Overall, the 1c complex has more ability than the other reagents to penetrate the cell wall or the lipid layer of the cell membrane. This is caused by relative lability of this complex compared with the Co and Ni complexes, Therefore, 1c interacts more adequately with biological ingredients inside the cytoplasm, interlinks to bacterial surface and as a result, frustrates the microorganism activities by either changing the cell wall or the membrane function or barricade replication [60]. On the other hand, the lower activity of the 1a, and 1b complexes can be attributed to low lipid solubility. Therefore, the metal ion cannot reach the desired site of action of the cell wall [61, 62].

Structure-activity relationship

The molecular structures of HL, 1a, 1b, and 1c compounds were optimized by using the DFT method (B3LYP) at 6‐31 G* and LANL2DZ basis set level. After that, DFT results were obtained for the frontier molecular orbital energies. Fig. 4(a) demonstrates the frontier molecular orbitals (FMOs) of these samples.

Fig. 4
figure 4

a Frontier molecular orbitals of the studied specimens; b The linear relations of the differences between the band gap and the zone of inhibitions for the studied bacteria strains in HL, 1a, 1b, and 1c

FMOs have a substantial role in the advent of optical and electrical properties. The band gap is also associated with antimicrobial properties. The narrower band gap facilitates the electron transfer from the electron-donor groups to the electron-acceptor groups, that can cause interruption of the cellular respiration process, deactivation of several cellular enzymes and denaturation of one or more cellular proteins and ultimately lead to cell death [63, 64].

The inverse relationship between the size of the band gap (ΔEL–H) and the bactericidal activities of these samples were approved by these conclusions. i.e., if the difference between HOMO and LUMO energies is smaller, the greatness of the antibacterial activity gets more [31].

Also, the linear relationship between the thickness of zone of inhibitions for above-named standard bacterial strains and band gap values can be found by an accurate examination of the computational conclusions. Pursuant to Fig. 4(b), the 1c complex has the narrowest band gap, which corresponds to its strongest bactericidal activity among the investigated samples [8].

Conclusion

In the present research, we synthetized a novel thiourea ligand and its Co(III), Ni(II) and Ag(I) complexes. Their structures were characterized by FTIR, UV-vis, 1H NMR. For HL and 1c, we also performed single-crystal X-ray diffraction analysis. These characterizations proposed the HL coordinated to the Co(III), Ni(II) ions through N and S atoms and constructed a four-membered chelate rings, while to Ag(I) it coordinated via S atom as a monodentate ligand. For all of the samples, antimicrobial activity was examined against Gram-negative (E. coli and P. aeruginosa) and Gram-positive (S. aureus and E. faecalis) bacteria by three methods, showing that they have good bactericidal activity in the order of 1c > 1b > 1a > HL. While the HL ligand did not demonstrate a remarkable antibacterial activity, its related complexes, especially 1c, had the best performance versus all tested strains.

Materials and methods

Experimental instruments

Infrared spectra were recorded on KBr pellets using a PerkinElmer 781 spectrophotometer FTIR. 1H NMR was recorded in DMSO-d6 and CDCl3 solvents on a BRUKER 300 MHz AVANCE III spectrometer with tetramethylsilane as internal reference. The melting points were measured on an Electro-thermal 9100 apparatus. Single-crystal X-ray data were collected on Gemini diffractometer of Rigaku Oxford Diffraction using the graphite monochromated MoKα radiation from a sealed X-ray tube and CCD detector Atlas S2 at 120 K. For structure plots, Diamond 4.5.2 was used.

All solvents and chemical materials were reagent grade and were purchased from Merck and Fluka, including 4-chlorophenyl isothiocyanate, Co(NO3)2.6H2O, Ni(NO3)2.6H2O, AgNO3, morpholine, methanol, and acetone.

Preparation of the N-(4-chlorophenyl)morpholine-4-carbothioamide (HL)

The thiourea ligand was synthesized by dissolving 0.01 mol 4-chlorophenyl isothiocyanate and 0.01 mol morpholine in acetone. This mixture was refluxed for 1 h. After that, this solution was poured into 250 mL distilled water, resulting a white precipitate that was filtered off and washed with distilled water. The colorless crystals were obtained by ether diffusion into the DMF solution of ligand. Yield (87%). Mp: 158–160 °C. Selected FT-IR data (ν, cm−1): 3159 (N-H), 2953–3097 (Ph-H), 2861–2908 (C-H), 1324 (C = S). 1H NMR (DMSO-d6; δ, ppm): 9.42 (s, 1 H, N-H), 7.31–7.41 (m, 4 H, Ph), 3.89–3.92 (t, 4 H, O-CH2), 3.65–3.69 (t, 4 H, N-CH2).

Preparation of Co complex (1a)

The desired complex was prepared by addition of the methanolic solution of Co(NO3)2.6H2O in 50 °C to the solution of HL ligand in 1:3 molar ratio and the resulting mixture was refluxed for 3 h. The dark green precipitates of the complex were filtered off and dried in vacuo. Yield (78%). Mp: 138–140 °C. Selected FT-IR data (ν, cm−1): 2918–2965 (Ph-H), 2848 (C-H), 1233 (C = S). 1H NMR (DMSO-d6; δ, ppm): 6.40–7.46 (m, 12 H, Ph), 3.06–3.78 (m, 24 H, -CH2).

Preparation of Ni complex (1b)

A similar procedure was carried out using HL (2 mmol) and Ni(NO3)2.6H2O (1 mmol) and refluxed for 3 h. The green precipitates were filtered off and dried in vacuo. Yield (75%). Mp: 280–282 °C. Selected FT-IR data (ν, cm−1): 2919–3026 (Ph-H), 2848–2892 (C-H), 1241 (C = S). 1H NMR (CDCl3; δ, ppm): 6.55–7.26 (m, 8 H, Ph), 3.14–3.86 (m, 16 H, -CH2).

Preparation of Ag complex (1c)

In a similar procedure, the Ag complex was obtained by the reaction of AgNO3 with the ligand (molar ratio of 1:2) in methanol. The gray precipitates of the complex were filtered off and dried in vacuo. Yield (88%). Mp: 170–172 °C. Selected FT-IR data (ν, cm−1): 3208 (N-H), 3044–3165 (Ph-H), 2858–2968 (C-H), 1343 (NO3-), 1304 (C = S). 1H NMR (DMSO-d6; δ, ppm): 10.01 (s, 2 H, N-H), 7.19–7.46 (m, 8 H, Ph), 3.91–3.94 (t, 8 H, -CH2), 3.72–3.75 (t, 8 H, -CH2).

X-ray crystallographic analysis

The measurement of HL was performed at 120 K on a Gemini diffractometer of Rigaku Oxford Diffraction using the graphite monochromated MoKα radiation from a sealed X-ray tube, and CCD detector Atlas S2. The measurement of 1c was performed at 95 K on a SuperNova diffractometer of Rigaku Oxford Diffraction using the mirror-collimated CuKα radiation from a micro-focus sealed X-ray tube, and CCD detector Atlas S2. CrysAlis PRO software was utilized for correction of reduction and absorption data [65]. The structures were solved using charge flipping methods and refined using Jana2006 [66, 67]. The residual electron density maps were plotted using MCE [68]. All hydrogen atoms could have been distinguished in difference Fourier maps and could have been refined to a reasonable geometry. According to common practice, H atoms bonded to C were kept in ideal positions with C–H 0.96 Å while positions of H atom bonded to N were refined with restrained bond lengths. In both cases, Uiso(H) was set to 1.2Ueq(C,N). All non-hydrogen atoms were refined using harmonic refinement.

Hydrogen atoms of partially occupied crystal water molecules (0.270(10) for O1w and 0.064(6) for O2w] in 1c could not be determined because they were not present in a difference Fourier map. Moreover, 1c contained a disordered NO3 anion with two close positions occupied 0.774(12) and 0.226(12). These positions were refined using the rigid body approach of Jana2006, and their occupancies were restricted to keep the full total occupancy.

In vitro antibacterial studies

The in vitro antimicrobial tests were examined against P. aeruginosa, ATCC 27853, and E. coli, ATCC 25922 (Gram-negative) and S. aureus, ATCC 25923, and E. faecalis, ATCC 11700 (Gram-positive) bacteria. Nutrient broth (NB) medium for both macro dilution (MIC) and colony count methods and Nutrient agar (NA) medium was used for the well-diffusion method. The bacterial strains were retained on agar slant at 4 °C and were sub-cultured on the fresh appropriate agar plate in incubators for 18–24 h prior to any antimicrobial analysis. To promote the solubility of these samples in water, they were dissolved in 10% DMSO solution. The same solution (without reagents) was used as negative control. The amikacin and gentamicin were used as positive control for Gram-positive and Gram-negative bacteria, respectively. Comparative investigation of the bactericidal activities of these samples was determined by using three following methods:

Macro-dilution (tube) broth method

According to the literature, 1 mL of Mueller-Hinton broth was added to the desired quantity of sample tubes (10 sample tubes) [69]. Then 1 mL stock solution of any compounds (HL, 1a, 1b, and 1c) dissolved in 10% DMSO solution and added to the first test tubes. After that, different concentration of the ligand and its complexes (93.68, 46.83, 23.42,11.71, 5.85, 2.93, 1.46 μM) were prepared from initial solutions. 1 mL of the suspension of bacteria that had been adjusted to 0.5 McFarland in sterile peptone water, was added to each sample tubes. After mixing, the sample tubes were incubated at 37 °C for 24 h. Then, 100 μL samples were taken from each tube and were applied on the specified amount of the Nutrient Agar and incubated at 37 °C for 24 h. The lowest concentration of test compounds eliminating bacterial growth indicates the MIC (Minimum Inhibitory Concentration).

Colony count method

The bacteria strains have been streaked in nutrient broth medium (NB) contained HL ligand, 1a, 1b, and 1c complexes suspension at the concentration of 20 μg/mL for measuring the rate of bacteria growth. These samples were appended into 10 mL NB medium which was added 200 μL bacteria at the concentration of 1.5 × 105 CFU. The same condition was used for preparing the blank samples by growth of the culture on the ligand and metal complexes free medium. Then a shaker platform was used to wiggle these samples at a speed of 200 rpm. After that, 100 μL of each bacteria suspension was dispersed on the Nutrient Agar medium. Finally, after 24 h of incubation at 37 °C the number of colonies forming units (CFUs) was counted by considering the dilution factor [33].

Agar well diffusion test

In vitro antimicrobial activity of all synthesized HL ligand and its Co(III), Ni(II), and Ag(I) complexes was investigated in an agar diffusion test. In a common method, the test bacteria were counted by dilution plate method. The cultured bacteria were spread on the agar medium by an appropriate sterile swab. After drying the plates, a well was created on the agar medium by sterilized cork borer. DMSO stock solutions of all compounds were diluted with sterile water to the test concentration of 100 µg/mL, were added (100 μL) to these wells by using a micropipette and then were incubated with different test bacteria. During this step, the growth of the inoculated bacteria was impressed by diffusion of the test solution. Subsequently, antimicrobial activity was assessed by measuring the diameter of the inhibition zones around the hole. The size of inhibition zones was determined visually. If the inhibition zone was greater than 6 mm the compounds were regarded as active [70,71,72].