SYNTHESIS AND CHARACTERIZATION OF 2-(PYRIDIN-2-YL)GUANIDINE DERIVATIVES AND THEIR METAL COMPLEXES AS POTENTIAL ANTIBACTERIAL AGENTS USING PHOSPHORYL CHLORIDE

SYNTHESIS AND CHARACTERIZATION OF 2-(PYRIDIN-2-YL)GUANIDINE DERIVATIVES AND THEIR METAL COMPLEXES AS POTENTIAL ANTIBACTERIAL AGENTS USING PHOSPHORYL CHLORIDE

Hoda Pasdar ^*, Bahare Hedayati Saghavaz, Ramona Khadivi, Mehran Davallo and Naser Foroughifar

Department of Chemistry, Tehran North Branch, Islamic Azad University, Tehran, Iran.

ABSTRACT: Guanidines were prepared by the condensation reaction of primary amine (-NH₂) with urea derivatives (R₂NCONR₂) in the presence of POCl₃. In this research guanidine-pyridine, hybrid derivatives were synthesized by substituted urea derivatives namely 1,1,3,3-tetramethylurea and N,  N′-dimethylurea with 2-aminopyridine. The metal complexes were obtained by reaction of metal(II) chloride with guanidine ligands in the molar ratio 1:1 and 1:2 (M:L). The structure of synthesized compounds was assessed by nuclear magnetic resonance (¹H-NMR), Fourier-transform infrared spectroscopy (FT-IR), ultraviolet-visible (UV-Visible) mass spectroscopy studies and molar conductance. The measured molar conductance values indicated that the complexes were non-electrolytic. An octahedral geometry is proposed for the cobalt complexes but, square-planar geometry is suggested for both copper and nickel complexes. The synthesized compounds were also subjected to antibacterial study. Evaluation of antibacterial activities indicated that the metal complexes more inhibited the Gram-positive and Gram-negative bacteria strains as compared to the parent ligands. Among all the metal complexes the CuL₂′ showed highest antibacterial activity with zone inhibition diameter of 29 mm and MIC value of 15.62 µg/ml based on broth dilution method and 31.25 µg/ml based on agar dilution method.

Guanidine, Guanidine-pyridine, Antibacterial study, Zone inhibition diameter, Broth dilution, Agar dilution

INTRODUCTION: It is known that resistance to most antibacterial agents is a serious health concern ^{1, 2}. Further, the growth of multidrug resistance, among both Gram-negative and Gram-positive pathogens, has been identified as a major problem in health care systems in recent decades ³. Finding antibacterial agents that can offer new modes of actions has always been an important and often difficult task ⁴.

Developing materials that can limit bacteria colonization on their surface is known as an approach to control infections to prevent the threatening growth of bacteria strains ⁵.

Chelation and its relationship with different biological processes seem to be a promising research area aiming at designing a novel therapeutic methodology for discovering new antibiotic compounds to control and prevent the growth of drug-resistant bacterial strains worldwide ^6-8. Guanidine functionalities can be seen in a wide range of natural compounds, which are either in cyclic form or terminal groups of pendent substituents ^{9, 10}. Compounds containing CN₃ unit belong to the group of compounds named guanidine ^11-13.

They can be found in important antibiotic drugs including Streptomycin, Trimethoprim, Chlorhexidine, Polyhexamethylene bi-guanidine, etc. ^14-17 Pyridine ring unit is known as a common structural feature employed in numerous pharmaceutically significant molecules ¹⁸. It has been demonstrated by a large number of literature reports that there can be many medicinal applications of guanidine-pyridine hybrid compounds ^{19, 20}. Accordingly, both pyridine and guanidine moiety can display considerable biological features ^21-23.

Regardless of the biological significance of guanidine derivatives, a large number of methods have been employed in recent years to make these compounds ^24-27. The use of urea derivatives as starting material is one of such methods ²⁸. The reaction of urea derivatives with different amines can be achieved using POCl₃ ²⁹. In this way, a wide range of substituents can be readily considered for the guanidine moiety, representing the stepwise synthesis. These substituents are likely to have the additional donor atoms; these atoms are likely to play important roles in the coordination of guanidine toward the metal center ^30-32.

In this paper, the synthesis, spectroscopic characterization and biological activities of the new Co(II), Ni(II) and Cu(II) complexes of two different guanidine-pyridine hybrid ligands were carried out to evaluate the antibacterial activities of these new series of metal-organic compounds.

MATERIALS AND METHODS: All the reagents and solvents were bought from Aldrich and Merck, and used without further purification. Solvents were distilled from the drying agents before use. Infrared Spectra of ligands and metal complexes were taken as 1% dispersion in KBr pellets using Shimadzu 300 spectrometer. Melting points were obtained on an Electrothermal type 9100 melting point apparatus. UV-Visible spectra were recorded on a Varian Cary 100 UV-Vis spectrophotometer using a 1 cm path length cell. ¹H-NMR spectra of ligands were recorded on a Brucker AMX 250 MHz spectrometer at ambient temperature in DMSO-d₆ with tetramethylsilane (TMS) as an internal standard. The splitting of proton resonances in the reported ¹H NMR spectra are defined as s= singlet, d= doublet, t= triplet, q= quartet, and m= complex pattern; coupling constants are reported in Hz. The molar conductance of the complexes in DMSO (1×10^-3 M solution) was recorded at 25 °C using Oakton ECTester 11 dual-range, conductivity tester. The Mass spectra were run at 70 eV at 230 °C with Agilent technologies. The completion of reactions was monitored by thin layer chromatography (TLC) on silica gel polygram SILG/UV 254 nm plates. All the bacteria strains were maintained as stock strains in Microbank cryovials and kept at -80 °C until further used.

Synthesis of the Hybrid Guanidine Ligands (L, L′): A solution of phosphoryl chloride (0.3 mmol) in dry toluene was added drop-wise under constant stirring to an ice-cooled solution of urea derivative (0.1 mmol). The resulting suspension was stirred at room temperature overnight, then a solution of 2-aminopyridine (0.1 mmol) in dry toluene was added to it in several portions. After 16-20 h at 100ºC under reflux condition, an aqueous solution of 25 wt. % NaOH (20 ml) was added to bring the solution pH to about 14. The solution was then extracted into the toluene phase (3 ´ 20 ml). The organic layer obtained was dried with MgSO₄ and after filtration; the solvent was evaporated under reduced pressure. Finally, the resulting residue was recrystallized from ethanol to prepare the corresponding compounds.

1, 1, 3, 3-tetramethyl-2-(pyridin-2-yl) guanidine (L): Brown solid; m.p. 120-122 ºC; Yield: 87%; FT-IR (KBr, cm^-1): 3100, 2927, 1567, 1386, 1012; ¹H NMR (250 MHz, DMSO-d₆): δ ppm 8.11 (t, 1H, J = 5.0 Hz, ArH), 7.44 (d, 1H, J = 2.5 Hz), 6.69 (t, 1H, J = 12.5 Hz, Ar-H), 6.57 (d, 1H, J = 7.5 Hz, Ar-H), 2.63 (s, 12H, CH₃); UV/Vis (DMSO): λ_max [nm] = 280, 310.

1,3-dimethyl-2-(pyridin-2-yl)guanidine (L′): Pale yellow solid; 85%; m.p. 175-177 ºC; FT-IR (KBr, cm^-1): 3483, 3006, 2941, 1641, 1509, 1378, 1063; ¹H NMR (250 MHz, DMSO-d6): δ ppm 7.86 (d, 1H, J = 5.0 Hz, Ar-H), 7.32 (m, 1H, Ar-H), 6.45 (t, 2H, J = 15 Hz, Ar-H), 5.84 (s, 1H, N-H), 3.80 (s, 6H, CH₃); UV/Vis (DMSO): λ_max [nm] = 320.

General Procedure for Synthesis of Metal Complexes: Reaction of guanidine ligands with metal(II) ions in the molar ratio 1:1 and 2:1 afforded the corresponding stoichiometry transition metal complexes. A solution of metal(II) chloride (1 mmol) in methanol (20 mL) was added to the solution of the corresponding amount of ligand, i.e., for mono complexes, 1 metal: 1 ligand and bis complexes 1 metal: 2 ligands. The solutions were stirred for 30 min and heated under reflux for 2-3 h and 5-6 h for mono and bis-complexes, respectively. The resulting precipitate was filtered off, then washed with methanol and finally dried in a vacuum desiccator.

Mono (1, 1, 3, 3- tetramethyl- 2- (pyridin-2-yl) guanidine)copper (II)(CuL): Dark brown solid; 85%; m.p. >300 ºC; FT-IR(KBr, cm^-1): 3350, 3100, 2900, 1664, 1634, 1550, 1048, 561; UV-Vis: λ_max = 290, 380, 420 nm; Mass: [m/z]⁺ = 326, Molar conductance (Ω^-1cm²mol^-1) in DMSO: 12.

Mono (1, 1, 3, 3- tetramethyl- 2- (pyridin-2-yl) guanidine)cobalt(II) (CoL): Dark blue solid; 73%; m.p. >300 ºC; FT-IR (KBr, cm^-1): 3424, 2924, 1635, 1317, 558; UV-Vis: λ_max = 280, 310, 600, 690 nm; Mass: [m/z]⁺ = 357, Molar conductance (Ω^-1cm²mol^-1) in DMSO: 16.

Mono (1, 1, 3, 3- tetramethyl- 2- (pyridin-2-yl) guanidine)nickel(II) (NiL): Green solid; 56%; m.m. >300 ºC; FT-IR (KBr, cm^-1): UV-Vis: λ_max = 270, 410 nm; Mass: [m/z]⁺ = 321, Molar conductance (Ω^-1cm²mol^-1) in DMSO: 20.

Bis (1, 1, 3, 3- tetramethyl- 2- (pyridin- 2-yl) guanidine)copper (II) (CuL₂): Dark green solid; 79%; m.p. >300 ºC; FT-IR (KBr, cm^-1): 3448, 2996, 1913, 1658, 1437, 1018, 670, 532; UV-Vis: λ_max = 290, 380, 420 nm; Mass: [m/z]⁺ = 447, Molar conductance (Ω^-1cm²mol^-1) in DMSO: 22.

Bis (1, 1, 3, 3- tetramethyl- 2- (pyridin- 2- yl) guanidine)cobalt(II) (CoL₂): Blue solid; 80%; m.p. >300 ºC; FT-IR(KBr, cm^-1): 3407, 2900, 1634, 1423, 1049, 561; UV-Vis: λ_max =  260, 290, 605, 690 nm; Mass: [m/z]⁺ = 514, Molar conductance (Ω^-1cm²mol^-1) in DMSO: 10.

Bis (1, 1, 3, 3- tetramethyl- 2- (pyridin- 2- yl) guanidine)Nickel(II) (NiL₂): Pale green solid; 63%; m.p. >300 ºC; FT-IR(KBr, cm^-1): 3437, 2996, 2913, 1656, 1437, 1028, 670, 524; UV-Vis: λ_max = 290, 450 nm; Mass: [m/z]⁺ = 443, Molar conductance (Ω^-1cm²mol^-1) in DMSO: 14.

Mono (1, 3- dimethyl- 2-(pyridin-2-yl)guanidine) copper (II) (CuL′): Green solid; 75%; m.p. 92-94 ºC; FT-IR (KBr, cm^-1): 3436, 2997, 2913, 1565, 1312, 669; UV-Vis: λ_max = 580 nm; Mass: [m/z]⁺ = 297, Molar conductance (Ω^-1cm²mol^-1) in DMSO: 14.

Mono (1, 3- dimethyl- 2-(pyridin-2-yl)guanidine) cobalt(II) (CoL′): Blue solid; 83%; m.p. 98-100 ºC; FT-IR (KBr, cm^-1): 3431, 2997, 2913, 1663, 1313, 520; UV-Vis: λ_max = 490, 570 nm; Mass: [m/z]⁺ = 329, Molar conductance (Ω^-1cm²mol^-1) in DMSO: 14.

Mono (1, 3- dimethyl- 2-(pyridin-2-yl)guanidine) nickel(II) (NiL′): Pale green solid; 56%; m.p. 85-87 ºC; FT-IR (KBr, cm^-1): 3436, 2997, 2913, 1658, 1312, 669; UV-Vis: λ_max = 560 nm; Mass: [m/z]⁺ = 292, Molar conductance (Ω^-1cm²mol^-1) in DMSO: 20.

Bis (1, 3- dimethyl- 2- (pyridin- 2- yl) guanidine) copper (II)) (CuL₂′): Dark green solid, 70%; 102-105 ºC; FT-IR (KBr, cm^-1): 3436, 2996, 2912, 1565, 1312, 669; UV-Vis: λ_max = 570 nm; Mass: [m/z]⁺ = 389, Molar conductance(Ω^-1cm²mol^-1) in DMSO: 20.

Bis (1, 3- dimethyl- 2- (pyridin- 2- yl) guanidine) cobalt(II) (CoL₂′): Dirty green solid; 70%; 108-111 ºC; FT-IR (KBr, cm^-1): 3434, 2998, 2913, 1656, 1313, 670; UV-Vis: λ_max = 410, 490, 580 nm; Mass: [m/z]⁺ = 456, Molar conductance (Ω^-1cm²mol^-1) in DMSO: 20.

Bis (1, 3- dimethyl- 2- (pyridin- 2- yl) guanidine) nickel(II) (NiL₂′): Pale green solid; 67%; 95-97 ºC; FT-IR (KBr, cm^-1): 3428, 2997, 2913, 1659, 1312, 669; UV-Vis: λ_max = 550 nm; Mass: [m/z]⁺ = 384, Molar conductance (Ω^-1cm²mol^-1) in DMSO: 20.

Biological Studies: A variety of laboratory methods can be used to evaluate the in-vitro antimicrobial activity of compounds. The most known and basic methods are disc diffusion and broth or agar dilution methods. In this study in-vitro antibacterial activity of ligands and their metal complexes were investigated by applying agar dilution and disc diffusion methods. The Minimal Inhibitory Concentration (MIC) values of synthesized compounds were determined by serial broth dilution and agar dilution tests. The inhabitation zone of each test compound was measured via the disc diffusion method. The tests were performed using the methodology described in the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) ³³. In this study, two Gram-positive (S. aureus ATCC: 6838, B. subtilis ATCC: 6633) and two Gram-negative (E. coli ATCC: 25922, S.marcescens ATCC: 13880) microorganisms were used. Each of the bacterial strain was cultured onto Muller-Hinton agar (MHA) plate and incubated for 18-24 h at 35 °C to obtain colony counts. Each test was repeated three times.

Disc Diffusion Method: The solution of the synthesized compounds was prepared at 20 mg/mL in DMSO (which contained no antibacterial activity) under sterile conditions. Amikacin was used as a standard drug reference. Suspensions of each bacteria strain were prepared from their 24 h cultures to obtain approximately 1.5 × 10⁸ colony forming units (CFU) per ml. Paper discs of 8 mm diameter were impregnated individually with a constant amount (100 μg/ml) of the compounds and then were allowed to dry. Next, the dried discs were placed on the inoculated agar surface and incubated for 18-24 h at 35 °C. The antibacterial activity was indicated by the presence of clear inhibition zones around the discs.

Broth Micro-Dilution Test: The MIC values of compounds were determined by serially diluted of 1 ml of the antibacterial agent (stock solution 2000 µgml-1) added to 1 ml of Muller Hinton Broth (MHB) medium and 0.1 ml of bacteria suspension (1.5 × 10⁸ CFU/ml). Amikacin used as reference antibacterial drug was dissolved in DMSO. The two-fold dilution of the compounds and amikacin were prepared in the tubes ranging from 1000 to 1.95 µgml^-1. The negative control tube did not contain bacterial strain, and the positive control tube was free of an antibacterial compound. The tubes were incubated for 18-24 h at 35 ºC. The MIC is defined as the endpoint where no visible turbidity could be detected concerning controls.

Agar Dilution Test: Dilution of each compound was freshly prepared in DMSO for each experiment. Different dilutions of the antibacterial stock solutions were incorporated into Muller Hinton agar medium. An agar plate without antibacterial compound was established as positive control plate. After solidification of the agar, the plates were inoculated with a bacterial suspension of 1.5 × 10⁸ CFU/mL by a sterile inoculation loop. After the plates were incubated for 18-24 h, the MIC values were recorded as the lowest concentration of tested compound that inhibited the growth of microorganism.

RESULTS AND DISCUSSION:

Characterization: The guanidine ligands were prepared by the condensation of 2-aminopyridine and urea derivatives in the molar ratio 1:1 in the presence of POCl₃ Fig. 1. To optimize the reaction conditions to obtain the highest yield, the reaction of N, N′-dimethylurea, and 2-aminopyridine was carried out using various reaction parameters such as solvent type and amount of phosphoryl chloride as shown in Table 1. According to the results given in Table 1, when POCl₃ was not added to the reaction mixture, no compound was produced. By increasing the POCl₃ level in the reaction mixture using toluene compared to benzene resulted in the higher percent yield.

TABLE 1: THE REACTION OF N,N′-DIMETHYLUREA (0.1 mmol) AND 2-AMINOPYRIDINE (0.1 mmol) IN THE PRESENCE OF POCl₃ IN DIFFERENT CONDITIONS

The optimum value was obtained when the amount of POCl₃ reached 0.3 mmol which was found to be 85%. However, by increasing POCl₃>0.3 mmol the percent yield of the reaction decreased to 53% and 35% using toluene and benzene, respectively.

FIG. 1: PREPARATION OF GUANIDINE LIGANDS

The molar conductivity of metal complexes was calculated using Equation (1):

Ʌ_m = K / C..........(1)

Where  is the measured conductivity and C is the concentration of the solutions. The molar conductance values of all the complexes were in the range 8-22 (Ω^-1cm²mol^-1) which indicated these complexes were non-electrolytes. Therefore, in all of the metal complexes, the chlorine atoms are present in the inner sphere of these complexes.

As can be seen in Table 2, the percent yields of metal complexes were in the range of 56-85%. Among these metal complexes, the highest yield was achieved for CuL metal complex (85%) with molar conductance of 20 (Ω^-1cm²mol^-1) whereas NiL and NiL′' metal complexes showed the lowest percent yield of 56% with molar conductance of 16 and 20 (Ω^-1cm²mol^-1), respectively. The proposed structures of metal complexes are shown in Fig. 2.

As presented in Table 2, in all of the complexes derived from L′ ligand, there is a significant decrease in the melting point compared to the free ligand. This is due to the presence of N-H groups is forming hydrogen bonds in this ligand. However, these hydrogen bonds are eliminated by attaching to the ligand thereby resulting in the reduction of the melting point in these metal complexes.

TABLE 2: PHYSICAL PROPERTIES OF LIGANDS AND THEIR METAL COMPLEXES

FIG. 2: THE PROPOSED STRUCTURE OF METAL COMPLEXES

The ligands, L, L′, and their metal complexes were characterized by FT-IR, ¹H NMR, and UV-Vis and mass spectroscopies. The important FT-IR data and assignments for free ligands and their metal complexes are reported in Table 3. In the FT-IR spectra of ligands, two important bands were observed. The first one was associated with the disappearance of the stretching frequency of NH₂ band in the region 3285 and 3267 cm^-1 of 2-aminopyridine, as well as stretching frequency of carbonyl groups, ʋ(C=O) of 1,1,3,3-tetramethylurea or N, N′-dimethylurea in 1723 cm^-1. The second one was assigned to the appearance of intensive bands at 1632 cm^-1 and 1641 cm^-1, and at 1012 cm^-1 and 1063 cm^-1 corresponding to azomethine groups (C=N), and C-N groups for L and L′, respectively.

In the ¹H-NMR spectra of ligands the absence of NH₂ group confirmed the condensation of the amine and carbonyl groups. The signals due to the methyl groups (CH₃) and pyridine ring observed at 2.95 ppm and 8.11-6.54 ppm and at 3.80 ppm and 7.84-6.39 ppm for L and L′, respectively. The spectra of L′, display signal due to the N-H protons at 5.84 ppm.

TABLE 3: IMPORTANT FT-IR DATA OF LIGANDS AND THEIR METAL COMPLEXES

In the electronic spectra of L-ligand, band at 280 nm can be assigned to p®p*  transition of the aromatic ring, while the more intensive band at 310 nm can be assigned to –C=N group. In the electronic absorption of L′- ligand, one intensive band at 320 nm is observed which is due to the p®p* transition. As can be observed in Table 3, in the FT-IR spectra of metal complexes the broadband in the range 3495-3359 cm^-1 and 3100-2996 cm^-1 are assigned to the ʋ (H₂O) and ʋ (ArCH), respectively. The CH₃ groups stretching are observed in the range of 2900-2912 cm^-1. The bands at 1664-1620 cm^-1 and 1048-1026 cm^-1 are attributed to ʋ (C=N) and ʋ (C-N) resonance of guanidine unit, respectively.

The position of the (C=N) and (C-N) bands shifted to the lower frequencies when compared to the free ligands, indicating that the coordination occurs through the nitrogen atoms to the metal ions. The bonding of the nitrogen atoms with metal ions observed in the range 520-670 cm^-1 is assigned to the ʋ (M-N) mode of bonding.

Table 4 represents all peaks derived from UV-Visible spectra of ligands and Cu(II), Co(II) and Ni(II) complexes, as well as the geometry surround the metal ions. The UV-Vis absorption spectra of metal complexes were recorded in DMSO solution at ambient temperature. The UV-Visible spectra of metal complexes were compared to the spectra of the free ligands. By observing the spectra of metal complexes, the intra-ligand bands (p®p*and n®p*) have shifted to the lower frequencies upon the coordination of ligand to the metal ions.

According to the d-d transition for both Cu(II) and Ni(II) complexes, a square-planner geometry is proposed, but octahedral geometry is suggested for Co(II) metal complexes using L and L′ ligands.

The mass spectrum of the synthesized metal complexes was recorded, and the results are in good agreement with the proposed structures. The mass spectrum of NiL′₂ complex is shown in Fig. 3. In the mass spectrum of this complex a molecular ion peak at m/z = 384.3 (calc. M. Wt. = 385) with an intensity of 36.35% which is equal to its molecular weight. The other peaks in the mass spectrum are attributed to the fragmentation of the molecule obtained from the rapture of different bond in the molecule. In addition, in the mass spectrum of other complexes molecular ion peak observed at m/z = 326, 357, 321, 447, 514, 443, 297, 329, 292, 389 and 456 are attributed to CuL, CoL, NiL, CuL₂, CoL₂, NiL₂, CuL′, CoL′, NiL′, CuL′₂ and CoL′₂, respectively.

FIG. 3: THE MASS SPECTRUM OF NiL′₂ COMPLEX

TABLE 4: UV-VISIBLE DATA OF LIGANDS AND THEIR METAL COMPLEXES

Biological Study: The antibacterial activities of synthesized compounds were carried out with the following microorganisms under the disc diffusion, broth dilution, and agar dilution methods:

• Staphylococcus aureus
• Bacillus subtilis
• Escherichia coli
• Serratia marcescens

Both the broth dilution and the agar dilution tests are known as standard in-vitro susceptibility tests which are recommended by the National Committee for Clinical Laboratory Standards (NCCLS). However, it should be noted that these two tests have advantages and disadvantages. The first one, the broth dilution test, is simple, easily manageable, and easy to handle, as well as yielding more reproducible results. Also, this test can be performed in both test tubes and microplates. On the other hand, the agar dilution test takes a long time to carry out; it is also very laborious, and many factors may influence the obtained results. The advantage of agar dilution test is that several bacterial strains on the same plate can be examined, and the growth conditions of the bacteria can be monitored.

From a theoretical point of view, both tests can be applied. However, in the case of the agar dilution test, the antibacterial powder is gradually deposited at the bottom of the plate in the course of preparation, due to insolubility. The resulting heterogeneity in the medium can influence the results to a great extent. For regarding the broth dilution test, while antibacterial solutions could be converted to homogeneous suspensions using shaking, avoiding sediments could be very problematic when the solution stopped shaking after inoculation. Consequently, antibacterial agents may not be able to react fully with the bacteria. To tackle this problem, a stirrer could be employed to prevent the sedimentation of antibacterial agents; however, the effect of stirring on the growth of bacteria should be considered.

The evaluation of the antibacterial effect of ligands and their metal complexes against pathogenic strains based on the disc diffusion test was conducted by measuring the diameter of growth inhibition are shown in Table 5, and the minimal inhibitory concentration (MIC) was examined based on the broth microdilution method and agar dilution method are listed in Table 6 and 7, respectively.

The antibacterial activities of complexes were compared to standard drug amikacin, as well as to free ligands. Table 6 revealed that the guanidine ligands had poorer antibacterial activity against all the examined bacteria strains compared to standard drug amikacin and metal complexes with zone inhibition in the range 8-12 mm. The other compounds displayed medium to excellent inhibition against both Gram-positive and Gram-negative bacteria. Among all the synthesized compounds, the highest antibacterial activity was performed by CuL′₂-complex against Gram-positive microorganism (S. aureus) with zone inhabitation of 29 mm, followed by CuL₂, CuL′ and CuL compound with the same zone inhibition of 20 mm against B. subtilis, S. aureus, and S. marcescens, respectively. It should be noted that antibacterial activity was not observed for DMSO. As can be seen from Table 6 and 7 the results of the broth dilution test are significantly lower than those of the agar dilution test for both ligands and metal complexes. This implied that antibacterial powders deposited in the former test could still react with the bacteria by releasing some effective ingredients, whereas, in the later test the releasing ingredients into the agar medium may have been reduced, hence resulting in lower values of MIC in agar dilution test. Also, for the same antibacterial agent, no correlation between the results from these two methods was found. These findings further suggest that the distribution of antibacterial powders in the agar dilution test was probably uneven compared to the broth dilution test. Thus, the broth dilution test appears to be more suitable for testing insoluble inorganic antibacterial agents.

TABLE 5: INHIBITION ZONE (mm) OF LIGANDS AND THEIR METAL COMPLEXES AGAINST PATHOGENIC STRAINS BASED ON DISC DIFFUSION TEST

* Not active

The data listed in Table 6 and 7 showed that metal complexes had better antibacterial activities when compared to parent ligands. This can be explained on the basis of Overton’s concept and chelation theory ^34-36. The chelation reduces the polarity of metal to some extent due to the overlap of the ligand orbital and partial sharing of the positive charge of the metal ion. The chelation increases the delocalization of p-electrons over the whole chelate ring and enhances the lipophilicity of the complexes which in turn, increases the penetration of the complexes into lipid membranes, and results in blockage of metal sites in the enzymes of the microorganisms. In addition, metal complexes hinder the respiration process of the cell and, block the synthesis of proteins and prevent further growth of the organism. The results given in Table 6 suggested that the MIC values of compounds are in the order CuL₂ = CoL₂ < CuL = NiL = CoL′ < CoL = CuL′₂ = CoL′₂ < NiL₂ = CuL′ = NiL′ against B. subtilis, CuL′₂ < CuL′ < CoL′ = NiL′ = CoL′₂ < CoL = NiL = CuL₂ = CoL₂ < CuL = NiL = NiL′₂ against S. aureus, CuL = CoL′ < CoL′₂ < NiL = CuL_{2 =} CuL₂ = NiL₂ = CuL′₂ < CoL = CuL′ = NiL′ against E. coli and CuL < CuL₂ = NiL₂ = CoL′ = CuL′₂ = CoL′₂ < NiL = CoL₂ = CuL′ < CoL = NiL ′= NiL′₂ against S. marcescens bacteria. Whereas, the data given in Table 7 showed that the MIC values of compounds are in the order CoL < CuL = NiL = CuL₂ = CoL′ < CuL′₂ = CoL′₂ < CoL = CuL′ < NiL₂ = NiL′ against B. subtilis, CuL′₂ < CuL′ < CuL = NiL₂ < CoL′ = < CoL′₂ < CoL = NiL = CoL₂ = CuL₂ = NiL′₂ against S. aureus, CoL′ < CoL′₂ < NiL = CuL₂ = CoL₂ = CuL′₂ < CuL = CoL = NiL₂ = CuL′ < NiL′ against E. coli and CoL′ < CuL = NiL = CuL′₂ = CoL′₂ < NiL = CuL₂ = NiL₂ = CoL′₂ < CuL′ = CuL′₂ < CoL₂ = NiL′ < CoL = NiL′₂ against S. marcescens bacteria. From the results obtained in Table 5-7 it may be deduced that CuL′₂ complex had the highest inhibition activity against S. aureus bacteria with zone inhibition diameter of 29 mm and MIC value of 15.62 µg/ml (based on broth dilution test) and 31.25 µg/ml (based on agar dilution test). Hence, the MIC value for CuL′₂ complex suggested that this compound had much stronger antibacterial activity compared to other synthesized compounds and therefore it may be used as antibacterial drug.

TABLE 6: MINIMAL INHIBITORY CONCENTRATIONS (µg/ml) OF LIGANDS AND THEIR METAL COMPLEXES AGAINST PATHOGENIC STRAINS BASED ON BROTH MICRO-DILUTION METHOD

TABLE 7: MINIMAL INHIBITORY CONCENTRATIONS (µg/ml) OF LIGANDS AND THEIR METAL COMPLEXES AGAINST PATHOGENIC STRAINS BASED ON AGAR DILUTION METHOD

CONCLUSION: With the growing public health concerns of the pathogenic effect by microorganisms, there is an increasing need for new antibacterial agents in many applications such as medical and dental equipment and services, water purifications, food packing and storage, and hospitals. In this study, two new guanidine ligands were synthesized by the reaction of 2-aminopyridine and urea derivatives in optimum conditions. The metal complexes were derived from the reaction of metal ions with different ligands in a molar ratio 1:1 and 2:1. The ligands were coordinated to the metals through nitrogen atoms as bidentate ligands, forming a six-membered ring. The coordination number of Ni and Cu complexes is four with square-planner geometry while the Co complexes have octahedral with a coordination number of six.

The presence of water in the outer sphere of complexes was confirmed by the results of FTIR spectrum in all of the complexes. The antibacterial activities of the ligands and their metal complexes were carried out against gram-positive and gram-negative bacteria such as B. subtilis, S. aureus, E. coli and S. marcescens by disc diffusion, agar dilution, and broth dilution methods.

The synthesized compounds were prepared in DMSO which had no antibacterial activity. In general, metal complexes had better biological activities when compared to the parent ligands. In comparison, CuL₂′ complex showed the highest antibacterial activity than other metal complexes with zone inhibition diameter of 29 mm and MIC value of 15.62 µg/ml against S. aureus.

ACKNOWLEDGEMENT: The authors are grateful to the Chemistry Department, Tehran North Branch, Islamic Azad University for the financial support given in this project.

CONFLICT OF INTEREST: The authors declare that there is no conflict of interest regarding the publication of this article.

REFERENCES:

1. Pasdar H, Saghavaz HB, Foroughifar N and Davallo M: Synthesis, characterization and antibacterial activity of novel 1,3-diethyl-1,3-bis(4-nitrophenyl)urea and its metal(ii) complexes. Molecules 2017; 22: 2125.
2. Kalkundri TA and Dinnimath BM: design, development, and evaluation of a new polyherbal mouth wash for antibacterial potency against oral bacteria. International Journal of Pharmaceutical Sciences and Research 2018; 9(12): 5301-07.
3. Fathi H, Gholipour A, Ebrahimzadeh MA, Yasari E, Ahanjan M and Parsi: In-vitro evaluation of the antioxidant potential, total phnolic and flavonoid contents and antibacterial activity of Lamium album extracts 2018; 9(10): 4210-19.
4. Afradi M, Foroughifar N, Pasdar H and Moghhanian M: Facile green one-pot synthesis of novel thiazolo[3,2-a]pyrimidine derivatives using Fe₃O₄ L-argainine and their biological investigation as potent antimicrobial agents. App Organ Chem 2016; 1-16.
5. Gholami-Dehbalaei M, Foroughifar N, Khajeh-Amiri A and Pasdar H: N-propylbenzoguanamine sulfonic acid-functionalized magnetic nanoparticles: A novel and magnetically retrievable catalyst for the synthesis of 1,4dihydropyridine derivatives. J Chin Chem Soc 2018; 1-14.
6. Ammar RA, Alghaz RA, Abdel-Naser MA, Zayed ME and Al-Bdair LA: Synthesis, spectroscopic, molecular structure, antioxidant, antimicrobial and antitumor behavior of Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) complexes of O₂N type tridentate chromone-2-carboxaldehyde Schiff's base ligand. J Mol Struct 2017; 1141: 368
7. Ishimoto K, Nagata T, Murabayashi M and Ikemoto T: Oxidative cyclization of 1-(pyridin-2-yl)guanidine derivatives: a synthesis of [1,2,4]triazolo[1,5-a]pyridin-2-amines and an unexpected synthesis of [1,2,4]triazolo[4,3-a]pyridin-3-amines. Tetrahedron 2015; 71(3): 407-18.
8. Khadivi R, Pasdar H, Foroughifar N and Davallo M: Synthesis and structural characterization of metal complexes derived from substituted guanidine-pyridine as potential antibacterial agents. Biointerface Research in Applied Chemistry 2017; 7(6): 2238-42.
9. Beytoot E, Alca A and Gakce HI: Pathological and immunohistochemical evaluation of the effects of interferon gamma (IFN-γ) and aminoguanidine in rats experimentally infected with Fasciola hepatica. Turk J Vet Anim Sci 2011; 35(4): 243-53.
10. Strassl F, Hoffmann A, Grimm-Lebsanft B, Rukser D, Biebl F, Tran MA, Metz F, Rübhausen M and Herres-Pawlis S: Fluorescent bis(guanidine) copper complexes as precursors for hydroxylation catalysis. Inorganics 2018; 6: 114.
11. Issa RM, Azim SA, Khedr AM and Draz DF: Synthesis, characterization, thermal, and antimicrobial studies of binuclear metal complexes of sulfa-guanidine Schiff bases. J Coord Chem 2009; 62(11): 1859.
12. Pattarawarapan M, Jaita S, Wangngae S and Phakhodee W: Ultrasound-assisted synthesis of substituted guanidines from thioureas. Tetrahedron Lett 2016; 57(12): 1354.
13. Wangngae S, Pattarawarapan M and Phakhodee W: Ph3P/I2-Mediated Synthesis of N,N′, N″-Substituted Guanidines and 2-Iminoimidazolin-4-ones from Aryl Isothiocyanates. J Org Chem 2017; 82(19): 10331.
14. Cafieri F, Fattorusso E, Mangani A and Tagliakatela-Scafati O: Dispacamides, anti-histamine alkaloids from Caribbean Agelas sponges. Tetrahed Lett 1996; 37: 3587.
15. Hensler ME, Bernstein G, Nizet V and Nefzi A: Pyrrolidine bis-cyclic guanidines with antimicrobial activity against drug-resistant Gram-positive pathogens identified from a mixture-based combinatorial library. Bioorganic & Med Chem Lett 2006; 16: 5073.
16. Kratzer C, Tobudic S, Graninger W, Buxbaum A and Georgopoulos A: In-vitro antimicrobial activity of the novel polymeric guanidine Akacid plus. J Hospital Infection 2006; 63: 316.
17. Qian L, Guan Y, He B and Xiao H: Modified guanidine polymers: Synthesis and antimicrobial mechanism revealed by AFM. Polymer. Polymer 2008; 49: 2471.
18. Zhang LJ, Zhang JA, Zou XZ, Liu YJ, Li N, Zhang ZJ and Li Y: Studies on antimicrobial effects of four ligands and their transition metal complexes with 8-mercapto quinoline and pyridine terminal groups. Bioorganic & Med Chem Lett 2015; 25: 1778.
19. Aikman B, Wenzel MN, Mósca AF, Almeida A, Klooster WT, Coles SJ, Several G and Casini A: Gold(III) Pyridine-benzimidazole complexes as aquaglyceroporin inhibitors and antiproliferative agents. Inorganics 2018; 6: 123.
20. Murtaza G, Rauf MR, Badshah A, Ebihara M, Said M, Gielen M, Vos DD, Dilshad E and MirzaEur B: Synthesis, structural characterization and in-vitro biological screening of some homoleptic copper(II) complexes with substituted guanidines. J Med Chem 2012; 48: 26.
21. Klein PJ, Chomet M, Mataxas A, Christiaana JA, Kooijman E, Schuit RC, Lammertsma AA, Van Berckel BN and Windhast AD: Synthesis, radiolabeling and evaluation of novel amine guanidine derivatives as potential positron emission tomography tracers for the ion channel of the N-methyl-d-aspartate receptor. Eur J Med Chem 2016; 118: 143.
22. Hoffmann A, Bienemann A, Vieira IDS and Herres-Pawlis S: New guanidine-pyridine copper complexes and their application in ATRP. Polymers 2014; 6: 995-07.
23. Liu MC, Lin TS, Cory JG, Cory AH and Sartorelli AC: Synthesis and biological activity of 3- and 5-amino derivatives of pyridine-2-carboxaldehyde thiosemi-carbazone. J Med Chem 1996; 39(13): 2586.
24. Guo ZX, Cammidge AN and Horwell DC: A convenient and versatile method for the synthesis of protected guanidines. Synth Comm 2000; 30: 2933.
25. Vaidyanathan G and Zalutsky MR: A new route to guanidines from bromoalkanes. J Org Chem 1997; 62: 4867.
26. Powell DA, Ramsden PD and Batey RA: Phase-transfer-catalyzed alkylation of guanidines by alkyl halides under biphasic conditions:  a convenient protocol for the synthesis of highly functionalized guanidines. J Org Chem 2003; 68: 2300.
27. Dodd DS and Kozikowski AP: Conversion of alcohols to protected guanidines using the mitsunobu protocol. Tetrahedron Lett 1994; 35: 977.
28. Castiglia A, El Sehrawi HM, Orbegozo T, Spitzner D, Claasen B, Frey W, Kantlehner W and Jager V: Synthesis and characterization of chiral guanidines und guanidinium salts derived from 1-phenylethylamine. Z Naturforsch 2012; 67b: 337.
29. Foroughifar N, Leffek KT and Lee YG: Synthesis and basicity of 2-N-quinolyl- and 2-N-isoquinolyl1,1,3,3-tetramethylguanidines. Can J Chem 1992; 70: 2856.
30. Tahir S, Badshah A and Hussain RA: Guanidines from ‘toxic substances’ to compounds with multiple biological applications- detailed outlook on synthetic procedures employed for the synthesis of guanidines. Bioorg Chem 2015; 59: 39.
31. Prasad KS, Kumar LS, Shekar CS, Prasad M and Revanasiddappa HD: Oxovanadium complexes with bidentate n, o ligands: synthesis, characterization, DNA binding, nuclease activity and antimicrobial studies. Chem Sci 2011; 12: 1.
32. Thirunarayanan G: Green approach using water-mediated Diels-Alder reaction for the synthesis of some 2-(4-bromo-1-naphthyl)-3-(aryl)bicyclo[2.2.1]hept-5-ene methanones and evaluation of their antimicrobial and antioxidant activities. Macedonian Journal of Chemistry and Chemical Engineering 2017; 36(1): 1-14.
33. Hindi KM, Panzner MJ, Tessier CA, Cannon CL and Young W: The medicinal applications of imidazolium carbene-metal complexes. J Chem Rev 2009; 109: 3859.
34. Shaygan S, Pasdar H, Foroughifar N, Davallo M and Motiee F: Cobalt (II) complexes with Schiff base ligands derived from terephthalaldehyde and ortho-substituted anilines: synthesis, characterization and antibacterial activity. Appl Sci 2018; 8(3): 385.
35. Pasdar H, Foroughifar N, Hedayati-Saghavaz B and Davallo M: Synthesis, characterization and biological activity of novel zirconium, zinc and cadmium complexes derived from substituted tetramethylguanidine. Int J Chem 2017; 9(3): 49-53.
36. Hedayati-Saghavaz B, Pasdar H and Foroughifar N: Novel dinuclear metal complexes of guanidine-pyridine hybrid ligand: synthesis, structural characterization and biological activity. Biointerface Research in Applied Chemistry 2016; 6(6): 1842-46.
    

    ---

    How to cite this article:

    Pasdar H, Saghavaz BH, Khadivi R, Davallo M and Foroughifar N: Synthesis and characterization of 2-(pyridin-2-yl)guanidine derivatives and their metal complexes as potential antibacterial agents using phosphoryl chloride. Int J Pharm Sci & Res 2019; 10(9): 4304-14. doi: 10.13040/IJPSR.0975-8232.10(9).4304-14.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---