The Scientific World Journal

Volume 2014 (2014), Article ID 404617, 9 pages

http://dx.doi.org/10.1155/2014/404617

Research Article

Microwave Assisted Synthesis, Spectral and Antifungal Studies of 2-Phenyl-N,N′-bis(pyridin-4-ylcarbonyl)butanediamide Ligand and Its Metal Complexes

Rayees Ahmad Shiekh,¹ Maqsood Ahmad Malik,² Shaeel Ahmed Al-Thabaiti,² Mohmmad Younus Wani,³ and Arshid Nabi⁴

¹Biomaterial Research Unit, School of Dental Sciences, Universiti Sains Malaysia, Kubang Kerian, 16150 Kelantan, Malaysia

²Department of Chemistry, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia

³Departamento de Quımica, FCTUC, Universidade de Coimbra, Rua Larga, 3004-535 Coimbra, Portugal

⁴School of Pharmaceutical Sciences, Lovely Faculty of Applied Medical Sciences, Lovely Professional University, Punjab-144411, India

Received 5 December 2013; Accepted 2 January 2014; Published 19 March 2014

Academic Editors: J.-P. Bouchara and S. A. El-Korashy

Copyright © 2014 Rayees Ahmad Shiekh et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

2-Phenyl-N,N′-bis(pyridin-4-ylcarbonyl)butanediamide ligand with a series of transition metal complexes has been synthesized via two routes: microwave irradiation and conventional heating method. Microwave irritation method happened to be the efficient and versatile route for the synthesis of these metal complexes. These complexes were found to have the general composition M(L)Cl₂/M(L)(CH₃COO)₂ (where M = Cu(II), Co(II), Ni(II), and L = ligand). Different physical and spectroscopic techniques were used to investigate the structural features of the synthesized compounds, which supported an octahedral geometry for these complexes. In vitro antifungal activity of the ligand and its metal complexes revealed that the metal complexes are highly active compared to the standard drug. Metal complexes showed enhanced activity compared to the ligand, which is an important step towards the designing of antifungal drug candidates.

1. Introduction

Nicotinamide is known as a component of the vitamin B complex as well as a component of the coenzyme, nicotinamide adenine dinucleotide (NAD). It is documented that heterocyclic compounds play a significant role in many biological systems, especially N-donor ligand systems being a component of several vitamins and drugs such as nicotinamide [1–3]. The presence of pyridine ring in numerous naturally abundant compounds is also of scientific interest. Nicotinamide itself plays an important role in the metabolism of living cells and some of its metal complexes are biologically active as antibacterial or insulin-mimetic agents [4]. Therefore, the structure of nicotinamide has been the subject of many studies [5–8]. Uses of metal ions in therapeutic agents are known to accelerate drug action and their efficacy enhances upon coordination with a metal ion [9, 10]. The classical coordination complex, cis-DDP or cisplatin (cis-diammine dichloroplatinum), has been the subject of much recent attention towards the metal-based chemotherapy, because of its beneficial effects in the treatment of cancer. These compounds present a great variety of biological activities, namely antitumour [11], antimicrobial [12, 13], anti-inflammatory, and antiviral activities. The inherent biological potential of sulphur/nitrogen donor ligands prompted us to undertake systematic studies with transition metals. In case of N and C based functionalized macrocyclic ligands, the mode of metal incorporation is very much similar to that of metalloproteins in which the requisite metal is bound in a macrocyclic cavity or cleft produced by the conformational arrangement of the protein [14]. The attachment of metal ions to proteins such as monoclonal antibodies can create new tools for use in biology and medicine [15]. These types of ligands have theoretical importance also because they are capable of furnishing an environment with controlled geometry and ligand field strength [16, 17]. The reagents used for such attachments are called bifunctional chelating agents [18]. The precise molecular recognition between macrocyclic ligands and their guest provides a good opportunity for studying key aspects of supramolecular chemistry, which are also significant in a variety of disciplines including chemistry, biology, physics, medicine and related science, and technology [19].

Candida albicans is an opportunistic and often deadly pathogen that attacks host tissues, undergoes a dimorphic shift, and then grows as a fungal mass in the kidney, heart, or brain. It is the fourth leading cause of hospital-acquired infection in the United States and over 95% of AIDS patients suffer from infections by C. albicans [20, 21]. Candida albicans is the predominant organism associated with candidiasis; but other Candida species, including C. glabrata, C. tropicalis, and C. krusei, are now emerging as serious nosocomial threats to patient populations [22]. The current antifungal therapy suffers from drug related toxicity, severe drug resistance, nonoptimal pharmacokinetics, and serious drug-drug interactions. The common antifungal drugs currently used in clinics belong to polyenes and azoles. Polyenes (amphotericin B and nystatin) cause serious host toxicity [23], whereas azoles are fungistatic and their prolonged use contributes to the development of drug resistance in C. albicans and other species [24]. Because of all these striking problems, there is an immediate need to develop novel antifungal drugs with higher efficiency, broader spectrum, improved pharmacodynamic profiles, and lower toxicity. In view of the importance of transition metal complexes in chemotherapy and as part of our continuing interest in metal-complexes, we report herein the synthesis, characterization, and in vitro antifungal study of 2-phenyl-N,N′-bis(pyridin-4-ylcarbonyl)butanediamide and its metal complexes.

2. Experimental

All the chemicals used were of analytical grade and were procured from Aldrich. Metal salts were purchased from E. Merck and were used as received. All solvents used were of standard/spectroscopic grade. All synthesis and handling were carried out under an atmosphere of dry and oxygen-free nitrogen using standard Schlenk techniques and samples for microanalysis were dried in vacuum to constant weight.

2.1. Physical Measurements

Elemental analyses were performed by a Perkin Elmer 2400 CHNSO Elemental Analyser. FT-IR spectra of solid samples were recorded on a Perkin Elmer Spectrum 100 FT-IR spectrometer (Universal/ATR Sampling Accessory). Bruker DPX-300 MHz spectrophotometer was used to record ¹H NMR spectra at room temperature with DMSO as solvent. The chemical shift is reported in parts per million (ppm) using tetramethylsilane as internal standard. Positive and negative ESI mass spectra were measured by Bruker (esquire 3000-00037) instrument. Magnetic susceptibility measurements were approved from a microanalysis laboratory by the Gouy method at room temperature. Electronic spectra were recorded on a spectro-UV-Vis Dual Beam 8 auto cell UVS-2700 LABOMED, Inc. US spectrophotometer using DMSO as solvent. Melting points (mp) were recorded on a Metrex melting point apparatus and the results are uncorrected.

2.2. Synthesis of Ligand (L)

2-Phenyl-N, -bis(pyridin-4-ylcarbonyl)butanediamide. Two different routes were employed for the synthesis of the ligand.

2.2.1. Microwave Assisted Synthesis

The ligand (L) was prepared by the condensation of phenylsuccinic acid (0.97 g, 5 mmoL) with nicotinamide (1.22 g, 10 mmoL). The reaction mixture was irradiated by the conventional microwave oven by taking 2-3 mL solvent. The reaction was completed in a short period (3-4 min). The resulting precipitate was then recrystallized from alcohol and dried under vacuum. These were characterized and analysed before use. Elemental analyses were conducted using the methods mentioned above and their results were found to be in good agreement with the calculated values. The structure of the ligand has been shown in Scheme 1.

Scheme 1: Synthesis of ligand and its metal complexes.

Yield 95%, mp. 180°C, IR (KBr, cm⁻¹): 3483 (N–H), 2985 (C–H), 1645 (C=O), 1580 (C–N), 1012, 863, 741; ¹H NMR (300 MHz,  ppm from TMS in DMSO–, 300 k): 8.01–7.17 (13H, Ar–C–H), 11.70–11.62 (2H, OC–NH), 2.88–2.63 (1H, OC–CH₂). ESI MS (m/z) 402 [M]⁺, 403 [M+1]⁺. Elem anal calcd. C 65.66, H 4.51, N 13.92%; found C 66.22, H 4.75, N 14.01.

2.2.2. Conventional Thermal Method

For comparison purposes, the above ligand was also synthesized by the thermal method. In this method, 100 mL of ethanol was used to dissolve the starting materials of the ligand and the contents were refluxed for nearly 6-7 h. The residue formed was separated out, filtered off, washed with water, recrystallized from ethanol, and finally dried in vacuum over fused calcium chloride (yield 82%), mp. 180°C. A comparison between thermal method and microwave method is given in Table 1.

Table 1: Comparison between microwave and thermal method.

Compound
Yield (%)
Solvent (mL)
Time

Thermal
Microwave
Thermal
Microwave
Thermal (h)
Microwave (min)

C₂₂H₁₈N₄O₄
82
95
100
2
7
3

CoLCl₂
75
87
40
4
5
5

CoL(CH₃COO)₂
76
86
40
3
5
6

NiLCl₂
78
85
40
5
5
5

NiL(CH₃COO)₂
72
82
40
4
4
6

CuLCl₂
69
79
40
4
5
7

CuL(CH₃COO)₂
73
83
40
3
4
6

L = (C₂₂H₁₈N₄O₄).

2.3. Synthesis of Metal Complexes

2.3.1. Microwave Assisted Synthesis

The ligand and metal salts were mixed in 1 : 2 (metal : ligand) ratio in a grinder. The reaction mixture was then irradiated by the microwave oven by taking 3–5 mL solvent. The reaction was completed in a short time (5–7 min) with higher yields. The resulting product was then recrystallized from ethanol and ether and finally dried under reduced pressure over anhydrous CaCl₂ in a desiccator. The progress of the reaction and purity of product was monitored by TLC using silica gel G.

2.3.2. Conventional Thermal Method

These complexes were also synthesized by the thermal method where instead of 5–7 min, reactions were completed in 4-5 h and the yield of the products was also less than that obtained by the microwave assisted synthesis.

2.3.3. [M(L)Cl₂/M(L)(CH₃COO)₂] Type of Complexes

M(L)Cl₂/M(L)(CH₃COO)₂ (where M = Cu(II), Co(II), Ni(II), and L = ligand).

A hot ethanolic (20 mL) solution of the corresponding metal salts (0.05 mmoL) was added to a hot ethanolic (20 mL) suspension of the macrocyclic ligand (0.10 mmoL). The mixture was stirred for 4-5 hours at 30°C and the solution was reduced to half of its volume. It was then allowed to stand overnight in a refrigerator. A coloured complex precipitated out, which was secluded by filtration under vacuum. It was washed systematically with cold ethanol and dried in vacuum over P₄O₁₀.

2.4. Antimicrobial Activity

2.4.1. Yeast Strains, Media, and Growth Conditions

The Candida strains were cultured in yeast extract, peptone, and dextrose (YEPD) broth and maintained on YEPD agar plates at 4°C and restreaked every 4–6 weeks. The culture was initiated with a loop full of cells maintained on YEPD slants into a 50 mL of appropriate medium (YEPD) and grown at 37°C in a rotary shaker at 150–170 rpm to the stationary phase (24 h) of growth and for experimental purposes 5 × 10⁶ cells (optical density = 0.1) were inoculated into the fresh media. Growth was followed for further 24–48 h and measured turbidometrically using LaboMed Spectrophotometer at 595 nm. For long term storage cultures were stored at −20°C with 1 : 1 glycerol as glycerol stocks.

2.4.2. Assessment of the Minimum Inhibitory Concentration (MIC₉₀)

Minimum inhibitory concentration (MIC₉₀) is defined as the lowest concentration (highest dilution) of the test agent that causes a 90% decrease in absorbance compared with that of control. The MICs of the ligand and its metal complexes against various Candida isolates were determined by the broth microdilution method, as described by the Clinical and Laboratory Standard Institute (CLSI) [25] (formerly the National Committee for the Clinical Laboratory Standards) (approved standard M27-A2, 2002). Stock solutions of the test compounds were prepared in DMSO. The cells were grown in YNB medium containing 2% glucose. The diluted cell suspensions were added to the wells of round-bottomed 96-well microtitre plates (100 μL/well) containing equal volumes of medium (100 μL/well) and different concentrations of test compounds. A drug-free control was also included. The plates were incubated at 35°C for 24 h. The MIC test endpoint was also evaluated both visually and by observing OD₅₉₅ in a microplate reader (BIO-RAD, i Mark, US) and is defined as the lowest compound concentration that gave ≥90% inhibition of growth compared with the growth of the controls.

2.4.3. WST-1 Cytotoxicity Assay

Colorimetric Assay for Quantification of Cellular Cytotoxicity & Proliferation. This is the accurate method for measuring the cellular toxicity [26]. In this experiment 2.5 mL assay buffer was added directly to each vial of water-soluble tetrazolium salt (WST-1) and cytoscan electron carrier (CEC). The obtained solution was stored at −20°C and protected from light. Equal volumes of the WST-1 and CEC solutions were mixed to prepare the assay dye solution before use and stored at −20°C and protected from light.

For a cytotoxicity assay, 5 × 10⁴–5 × 10⁵ cells were cultured per well of a 96-well plate with a final volume of 100 μL/well culture medium for 24 h. Following incubation, different concentrations of ligand and its metal complexes were exposed to Candida cells for a period of 1 h. At the end of the treatment, 10 μL WST-1/ CEC assay dye solution was added to each well and the plate was gently shaken to mix chemicals with medium. The plate was incubated for 30 mins in the incubator. It was then shaken for 1 minute on the shaker and absorbance was measured using a microplate reader at 450 nm and reference was set at wavelength 655 nm. All positive controls (varying concentrations of test compounds) were also included to subtract the reducing activity of test compounds towards tetrazolium from test results. The culture medium background was subtracted from assay results and percentage cytotoxicity was calculated with the following equation, using average absorbances for controls and experimental results as shown earlier [27].

Consider

3. Results and Discussion

On the basis of elemental analyses, the complexes were assigned the composition shown in Table 2. The analytical data of the complexes correspond well with the general formula [M(L)Cl₂/M(L)(CH₃COO)₂], where L = ligand and M = Co(II), Ni(II), Cu(II). The molar conductance indicates that all the complexes are 1 : 2 electrolytes in nature.

Table 2: Analytical data and physical properties of the ligand and complexes.

Complexes molecular formula
Colour
Molar conductance

(Ω⁻¹cm² mol⁻¹)
M.P.

(°C)
Mol. Wt.

found (Cal.) %
Molecular weight found (calculated) %

M
C
H
N
O

C₂₂H₁₈N₄O₄
Colourless

180
402 (401.45)

65.66 (66.02)
4.51 (4.75)
13.92 (14.01)
15.90 (15.98)

CoLCl2
Pink
263
220
531.83 (503.78)
11.08 (11.05)
49.63 (49.58)
3.38 (3.35)
10.52 (10.49)
12.03 (12.01)

CoL(CH₃COO)₂
Mauve pink
205
210
578.93 (577.95)
10.17 (10.14)
45.60 (45.55)
3.10 (3.08)
9.67 (9.64)
11.05 (11.02)

NiLCl₂
Green
220
215
531.59 (530.69)
11.04 (11.01)
49.66 (49.62)
3.38 (3.36)
10.53 (10.50)
12.03 (12.00)

NiL(CH₃COO)₂
Light green
202
208
578.69 (577.89)
10.14 (10.10)
45.62 (45.58)
3.11 (3.09)
9.67 (9.65)
11.05 (11.02)

CuLCl2
Royal blue
258
218
536.44 (535.24)
11.84 (11.80)
49.21 (49.18)
3.35 (3.31)
10.43 (10.40)
11.93 (11.90)

CuL(CH₃COO)₂
Greenish blue
198
212
583.54 (582.24)
10.88 (10.82)
45.24 (45.20)
3.08 (3.05)
9.59 (9.56)
10.96 (10.93)

L = (C₂₂H₁₈N₄O₄).

3.1. Infrared Spectra

Assignments of selected characteristic IR band positions provide significant indication for the formation of ligand (L) and its complexes. The appearance of new bands in the IR spectra at 1645 (C=O) amide I, 1580 (C–N) + (N–H) amide II, and 1255 (N–H) amide III are characteristic for amide groups (Table 3). A sharp band observed in the region of 3483 cm⁻¹ may be assigned to (N–H) of the amide group [28].

Table 3: Relevant IR spectral peaks (cm⁻¹) and their assignments.

Complexes
N–H
Amide-I C=O
Amide-II C–N, N–H
Amide-III N–H
M–N
M–Cl
M–(CH₃COO)

(asym)
(sym)

C₂₂H₁₈N₄O₄
3483
1645
1580
1255

CoLCl2
3250
1632
1565
1232
445
340
—

CoL(CH₃COO)₂
3165
1640
1552
1225
456
—
1564
1325

NiLCl₂
3153
1638
1558
1242
459
354
—

NiL(CH₃COO)₂
3147
1625
1569
1220
454
—
1595
1315

CuLCl2
3150
1630
1570
1238
425
350
—

CuL(CH₃COO)₂
3145
1628
1563
1222
432
—
1560
1330

L = (C₂₂H₁₈N₄O₄).

On complexation, the position of (N–H) and (C–N) bands shifting to a lower frequency compared to macrocyclic ligand indicates that the coordination takes place through the nitrogen [N₄] core and a new medium intensity band appearing at 420–465 cm⁻¹ attributed to (M–N) also designates that the flow of electron density towards the metal atom is through the nitrogen group [29]. Another medium intensity band in the region of 340–360 cm⁻¹ has been assigned to (M–Cl). The presence of bands in the regions of 1595–1560 cm⁻¹ and 1330–1315 cm⁻¹ are characteristic of asymmetric and symmetric COO⁻ stretching vibrations, respectively, with = ~250 cm⁻¹ [30].

3.2. ¹H NMR Spectra

¹H NMR spectral data of the ligand in DMSO shows the signals corresponding to the proposed structure, as it does not show any signal corresponding to the primary amine group and alcoholic proton. The ligand shows a multiplet in the region of 8.01–7.17 ppm Ar–CH (13H), due to the presence of aromatic ring protons. There is a sharp signal in the range of 11.70–11.62 ppm which is attributed to amide CO–NH, (2H) [31–33]. Another signal appearing in the range of 2.88–2.63 ppm has been ascribed to methylene protons OC–CH₂, (1H). These proton signals undergo down field shifting in all the metal complexes of the ligand because of the paramagnetic effect of metal (II) ions and hence support the coordination of the ligand towards the metal ions [34, 35].

3.3. Electrospray Ionization Mass Spectra (ESI MS)

The mass spectra of ligand (L) confirm the proposed formula by showing a peak at m/z 403 corresponding to the moiety [(C₂₂H₁₈N₄O₄)⁺] atomic mass m/z 402. The series of peaks in the range m/z 76, 120.7, 164.6, 230, 349, and so forth may be assigned to various fragments. Their intensity gives an idea of the stability of fragments. [M+2]⁺ peaks were observed in [CoL]Cl₂, [NiL]Cl₂, and [CuL]Cl₂ metal complexes, possibly due to the presence of isotopic chlorine in low quantities [35]. In some cases, the molecular ion peak was also associated with the solvent, water molecules, and some adduct ions from the mobile phase solution [36, 37] (Table 2).

3.4. Bands due to Anions

3.4.1. Cobalt(II) Complex

At room temperature the magnetic moment of cobalt(II) complexes lie in the range of 4.82–4.98 B.M. corresponding to three unpaired electrons [38]. The electronic spectra of cobalt(II) complexes exhibit absorption in the region 11,180–11,450 cm⁻¹, 14,710–16,680 cm⁻¹, 18,550–18,595 cm⁻¹, and 25,135–29,750 cm⁻¹. These bands may be assigned to ⁴T1g (F)→ ⁴T2g (F) (), ⁴T1g→ ⁴A2g (), and ⁴T1g (F)→ ⁴T1g (P) () transitions, respectively, and the fourth band may be due to charge transfer, suggesting an octahedral geometry around cobalt(II) ion [39].

3.4.2. Copper(II) Complex

The magnetic moment of all the Cu(II) complexes recorded at room temperature lie in the range 1.90–1.99 B.M. corresponding to one unpaired electron. The electronic spectra of the copper(II) complexes display bands in the range 10,263–11,486 cm⁻¹, 18,435–18,650 cm⁻¹, and 29,433–29,850 cm⁻¹. The first two bands may be assigned to the transitions: ²B1g→ ²A1g (d x −y2→d z2) () and ²B1g→ ²B2g (d x2−y2→d zy) (), respectively, and third band is due to charge transfer spectra [40].

3.4.3. Nickel(II) Complex

The magnetic moment of the Ni(II) complex at room temperature lie in the range of 2.75–2.90 B.M. corresponding to two unpaired electrons. The electronic spectra of Ni(II) complexes display three absorption bands in the region of 10,190–10,210 cm⁻¹, 18,738–18,757 cm⁻¹, and 20,410–21,550 cm⁻¹. These bands may be assigned to ³A2g(F)→ ³T2g(F)(), ³A2g(F)→ ³T1g(F)(), and ³A2g(F)→ ³T1g(P)(), respectively [41], showing six coordinated distorted octahedral geometries as shown in Scheme 1.

3.5. Antimicrobial Screening

3.5.1. Assessment of Minimum Inhibitory Concentration (MIC₉₀)

Figure 1 summarizes the in vitro susceptibilities of 3 fluconazole-sensitive Candida strains against ligand and its metal complexes. The data is reported as MIC which is defined as the lowest concentration required inhibiting 90% growth in comparison to control (absence of any test compound) for each isolate. The synthesized compounds were found to be active against all the tested Candida isolates. The MIC₉₀ of ligand [C₂₂H₁₈N₄O₄] against the sensitive isolates of different Candida isolates ranged 1300–1500 μg/mL, [CoL]Cl₂ ranged 900–1000 μg/mL, [CoL(CH₃COO)₂] ranged 1000–1100 μg/mL, [NiL]Cl₂ ranged 500–700 μg/mL, [NiL(CH₃COO)₂]Cl₂ ranged 700–900 μg/mL, [CuL]Cl₂ ranged 800–900 μg/mL, and that of [CuL(CH₃COO)₂] ranged 600–800 μg/mL, respectively.

Figure 1: Minimum inhibitory concentration (90%) of the synthesized ligand and its metal complexes against different clinical fluconazole-sensitive Candida isolates.

3.5.2. Growth Curve Studies

In the case of growth curve studies, the effect of increasing concentrations of the ligand and its complexes on the growth pattern of different fungal species has been studied. Control cells showed a normal pattern of growth with a lag phase of 4 h and an active exponential phase of 8–10 h before attaining stationary phase. An increase in the concentration of test compounds leads to a significant decrease in growth. NiCl₂ complex when treated against Candida albicans at a concentration of 40 μg/mL the growth pattern does not change, the lag phase is extended by 4 h, and the stationary phase does not reach the same level of cell growth as in the case of control, and at 60 μg/mL the lag phase is further extended by 2 h. At a concentration of 80 μg/mL (MIC₉₀ level), there is a total inhibition of growth showing a flat line (Figure 2). Fluconazole 20 μg/mL showed the lag phase further extended by 6 h with respect to control. A significant and pronounced effect is observed for all the synthesized complexes. Ni(II), Cu(II), and Co(II) complexes, in a concentration dependent manner, suppressed growth and delayed exponential phases. At MIC₉₀ values complete inhibition of growth was observed.

Figure 2: Effect of NiCl₂ complex in the concentration range of 40–80 μg/mL was studied on C. albicans STD 31. Growth curve plotted against absorbance at 595 nm and time (h) shows complete inhibition of growth at 80 μg/mL.

3.5.3. WST-1 Cytotoxicity Assay

The assay principle is based upon the reduction of the tetrazolium salt (WST-1) to formazan by cellular dehydrogenases that can be assessed visually and quantified spectrophotometrically. The generation of yellow coloured formazan is measured at 450 nm and is directly correlated to cell number. Table 4 gives % cytotoxicity of C. albicans STD 31, Candida glabrata STD 96, and Candida kruesi STD 116 at MIC concentrations of test compounds. All the test compounds show cytotoxicity for all the three fluconazole-susceptible Candida isolates used in this study. It was found that MIC₉₀ values of the test compounds showed pronounced cytotoxic effects. The average % cytotoxicity at MIC values of [CoL]Cl₂, [CoL(CH₃COO)₂], [NiL]Cl₂, [NiL(CH₃COO)₂]Cl₂, [CuL]Cl₂, and [CuL(CH₃COO)₂] was 44%, 35%, 86%, 69%, 59%, and 76% against the three types of species. Respective figures at the MIC values of ligand [C₂₂H₁₈N₄O₄] were 20% only. [NiL]Cl₂ at its MIC concentrations was found to be the most cytotoxic of all the compounds tested. An increase in cytotoxicity was observed with increase in concentration of the test compounds.

Table 4: % Cytotoxicity by MIC of ligand and its different complexes against three fluconazole-sensitive Candida isolates.

Complexes (µg/mL)
Control
Candida albicans STD 31
Candida glabrata STD 96
Candida kruesi STD 116

0
0
0

C₂₂H₁₈N₄O₄
MIC

CoLCl2
MIC

CoL(CH₃COO)₂
MIC

NiLCl₂
MIC

NiL(CH₃COO)₂
MIC

CuLCl2
MIC

CuL(CH₃COO)₂
MIC

L = (C₂₂H₁₈N₄O₄).

4. Conclusion

The synthesis and characterization of 2-phenyl-N,N′-bis(pyridin-4-ylcarbonyl)butanediamide ligand and its corresponding Cu(II), Co(II), and Ni(II) complexes have been carried out. The IR, ¹H-NMR, and ¹³C-NMR data were successfully used to elucidate the formation of the 2-phenyl-N,N′-bis(pyridin-4-ylcarbonyl)butanediamide ligand. On the basis of spectral studies an octahedral geometry for metal complexes has been assigned. All the fluconazole-susceptible Candida isolates investigated were found to be sensitive to the test compounds. The use of total mean MICs obtained gave a good indication of the overall antimicrobial effectiveness of each test compound. This may indicate that the yeast physiology may not be better equipped to counteract the antifungal properties of these compounds. The higher activity of [NiL]Cl₂ as compared to free ligand [C₂₂H₁₈N₄O₄] may be attributed to the increased lipophillicity that causes its efficient permeation through the lipid bilayers of the microbial cell membranes and a consequent cell death [42]. Cytotoxicity results obtained suggest that there is a drastic alteration in redox activity of cells and at higher values of metal chelates a maximum decrease in reduction of tetrazolium salt is seen. By combining the results of MIC studies and tetrazolium assays it can be concluded that metal chelates at their MIC₉₀ values show maximum effect either on growth or on metabolic activities of oxidases inside the cell. At higher concentrations, these metal chelates affected redox activity translates decreased growth which eventually leads to maximum growth inhibition at MIC₉₀ values.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

This Project was funded by the Saudi Basic Industries Corporation (SABIC) and the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under Grant no. MS/14/393/1433. The authors, therefore, acknowledge with thanks SABIC and DSR technical and financial support.

References

U. Brühlmann and E. Hayon, “One-electron redox reactions of water-soluble vitamins. I. Nicotinamide (vitamin B5) and related compounds,” Journal of the American Chemical Society, vol. 96, no. 19, pp. 6169–6175, 1974.

M. Kato and Y. Muto, “Factors affecting the magnetic properties of dimeric copper(II) complexes,” Coordination Chemistry Reviews, vol. 92, pp. 45–83, 1988.

R. Nagar, “Syntheses, characterization, and microbial activity of some transition metal complexes involving potentially active O and N donor heterocyclic ligands,” Journal of Inorganic Biochemistry, vol. 40, no. 4, pp. 349–356, 1990.

J. Eugen, K. Marian, M. Milan, and M. Jerzy, “Structural investigation of nickel(II)-nicotinamide-solvent interactions in solid complexes. Crystal structure of [NI(H₂O)₄(NA)₂](NO₃) ₂·2H₂O,” Journal of Coordination Chemistry, vol. 40, no. 3, pp. 167–176, 1996.

Y. Miwa, T. Mizuno, K. Tsuchida, T. Taga, and Y. Iwata, “Experimental charge density and electrostatic potential in nicotinamide,” Acta Crystallographica B, vol. 55, no. 1, pp. 78–84, 1999.

B. Vogelsanger, R. D. Brown, P. D. Godfrey, and A. P. Pierlot, “The microwave spectrum of a vitamin: nicotinamide,” Journal of Molecular Spectroscopy, vol. 145, no. 1, pp. 1–11, 1991.

P. Purcell, “Electric moment measurement of N-alkyl-substituted nicotinamides and calculation of aromatic amide group moments¹,” Journal of Physical Chemistry, vol. 68, no. 9, pp. 2666–2670, 1964.

W. P. Purcell and J. A. Singer, “Benzene and dioxane electric moments of N-alkyl-substituted nicotinamides from measurements in mixed benzene-dioxane solutions¹,” The Journal of Physical Chemistry, vol. 69, no. 12, pp. 4097–4101, 1965.

R. A. Sánchez-Delgado, K. Lazardi, L. Rincón, J. A. Urbina, A. J. Hubert, and A. N. Noels, “Toward a novel metal-based chemotherapy against tropical diseases. 1. Enhancement of the efficacy of clotrimazole against Trypanosoma cruzi by complexation to ruthenium in RuCl₂(clotrimazole)2,” Journal of Medicinal Chemistry, vol. 36, no. 14, pp. 2041–2043, 1993.

L. Tripathi, P. Kumar, and A. K. Singhai, “Role of chelates in treatment of cancer,” Indian Journal of Cancer, vol. 44, no. 2, pp. 62–71, 2007.

J. Patole, S. Padhye, S. Padhye, C. J. Newton, C. Anson, and A. K. Powell, “Synthesis, characterization and in vitro anti-cancer activities of semicarbazone and thiosemicarbazone derivatives of salicylaldehyde and their copper complexes against human breast cancer cell line MCF-7,” Indian Journal of Chemistry A, vol. 43, no. 8, pp. 1654–1658, 2004.

K. R. Ready, K. M. Reddy, and K. N. Mahendra, “Synthesis, characterization, antibacterial and anthelmentic activities of copper(II) complexes with benzofuran Schiff bases,” Indian Journal of Chemistry A, vol. 45, no. 2, pp. 377–381, 2006.

D. K. Demertzi, N. Kourkoumelis, M. A. Demertzis et al., “Trinuclear palladium(II) complexes with 2-hydroxy-4-methoxyacetophenone N ⁴-dimethylthiosemicarbazone: synthesis, spectral studies and crystal structure of a tripalladium complex,” European Journal of Inorganic Chemistry, no. 4, pp. 727–734, 2000.

E. Q. Gao, H. Y. Sun, D. Z. Liao, Z. H. Jiang, and S. P. Yan, “Synthesis of and magnetic interactions in binuclear Cu(II)-M(II) (M=Cu, Ni and Mn) complexes of macrocyclic oxamido ligands,” Polyhedron, vol. 21, no. 4, pp. 359–364, 2002.

C. F. Meares and T. G. Wensel, “Metal chelates as probes of biological systems,” Accounts of Chemical Research, vol. 17, no. 6, pp. 202–209, 1984.

J. Gao, A. E. Martell, and J. Reibenspies, “Novel Cu(II)Cd(II) macrocyclic complex that hydrolyzes an activated phosphate diester,” Inorganica Chimica Acta, vol. 329, no. 1, pp. 122–128, 2002.

S. Chandra, L. K. Gupta, and D. Jain, “Spectroscopic studies on Mn(II), Co(II), Ni(II), and Cu(II) complexes with N-donor tetradentate (N₄) macrocyclic ligand derived from ethylcinnamate moiety,” Spectrochimica Acta A, vol. 60, no. 10, pp. 2411–2417, 2004.

A. K. Mishra and J. F. Chatal, “Synthesis of macrocyclic bifunctional chelating agents: 1,4,7-tris(carboxymethyl)-10-(2-aminoethyl)-1,4,7,10-tetraazacyclododecane and 1,4,8-tris(carboxymethyl)-11-(2-aminoethyl)-1,4,8,11-tetraazacyclotetradecane,” New Journal of Chemistry, vol. 25, no. 2, pp. 336–339, 2001.

E. Labisbal, A. Sousa, A. Castiñeiras, J. A. García-Vázquez, J. Romero, and D. X. West, “Spectral and structural studies of metal complexes of isatin 3-hexamethyleneiminylthiosemicarbazone prepared electrochemically,” Polyhedron, vol. 19, no. 10, pp. 1255–1262, 2000.

H. W. Chi, Y. S. Yang, S. T. Shang et al., “Candida albicans versus non-albicans bloodstream infections: the comparison of risk factors and outcome,” Journal of Microbiology, Immunology and Infection, vol. 44, no. 5, pp. 369–375, 2011.

P. L. Fidel Jr., “Candida-host interactions in HIV disease: relationships in oropharyngeal candidiasis,” Advances in Dental Research, vol. 19, no. 1, pp. 80–84, 2006.

M. A. Pfaller and D. J. Diekema, “Rare and emerging opportunistic fungal pathogens: concern for resistance beyond Candida albicans and Aspergillus fumigatus,” Journal of Clinical Microbiology, vol. 42, no. 10, pp. 4419–4431, 2004.

B. Cohen, “Amphotericin B toxicity and lethality: a tale of two channels,” International Journal of Pharmaceutics, vol. 162, no. 1-2, pp. 95–106, 1998.

D. Sanglard, “Resistance of human fungal pathogens to antifungal drugs,” Current Opinion in Microbiology, vol. 5, no. 4, pp. 379–385, 2002.

Clinical and Laboratory Standard Institute (CLSI), Approved Standard M27-A2, Clinical Laboratory Standards Institute, Wayne, Pa, USA, 2002.

M. Ishiyama, M. Suiga, K. Sasamoto, M. Mizoguchi, and P. G. He, “A new sulfonated tetrazolium salt that produces a highly water-soluble formazan dye,” Chemical and Pharmaceutical Bulletin, vol. 41, no. 6, pp. 1118–1122, 1993.

R. A. Shiekh, S. Shreaz, L. A. Khan, and A. A. Hashmi, “Development and characterization of bioactive macrocyclic metal complexes, use as a potential drug,” Journal of Chemical and Pharmaceutical Research, vol. 2, no. 2, pp. 172–185, 2010.

K. Nakamoto, Infrared Spectra of Inorganic and Coordination Compounds, vol. 90, Wiley Internscience, New York, NY, USA, 1970.

M. Shakir, S. P. Varkey, and P. S. Hameed, “Divalent, cobalt, nickel, copper and zinc complexes of tetraaza macrocycles bearing polyamide groups: synthesis and characterization,” Polyhedron, vol. 12, no. 23, pp. 2775–2780, 1993.

M. Sebastian, V. Arun, P. P. Robinson et al., “Synthesis, structural characterization and catalytic activity study of Mn(II), Fe(III), Ni(II), Cu(II) and Zn(II) complexes of quinoxaline-2- carboxalidine-2-amino-5-methylphenol: crystal structure of the nickel(II) complex,” Polyhedron, vol. 29, no. 15, pp. 3014–3020, 2010.

P. S. Kalsi, Spectroscopy of Organic Compounds, New Age International, New Delhi, India, 4th edition, 1999.

R. A. Shiekh, I. A. Rahman, M. A. Malik et al., “Electrochemical and biological studies of nitrogen donor macrocyclic ligand and its transition metal complexes,” International Journal of Electrochemical Science, vol. 7, no. 12, pp. 12829–12845, 2012.

S. C. Rawle, A. J. Clarke, P. Moore, and N. W. Alcock, “Ligands designed to impose tetrahedral co-ordination: a convenient route to aminoethyl and aminopropyl pendant arm derivatives of 1,5,9-triazacyclododecane,” Journal of the Chemical Society, no. 18, pp. 2755–2757, 1992.

R. M. Silverstein and F. X. Webster, Spectroscopic Identification of Organic Compounds, vol. 482, John Wiley and Sons, New York, NY, USA, 6th edition, 1998.

W. Kemp, Organic Spectroscopy, Macmillan Press, New York, NY, USA, 1975.

R. A. Sheikh, S. Shreaz, M. A. Malik, L. A. Khan, and A. A. Hashmi, “Spectroscopic elucidation of new metal hetroscorpionates: a novel class of antifungal and antibacterial agents,” Journal of Chemical and Pharmaceutical Research, vol. 2, no. 3, pp. 133–146, 2010.

M. Mann, “Electrospray: its potential and limitations as an ionization method for biomolecules,” Organic Mass Spectrometry, vol. 25, no. 11, pp. 575–587, 1990.

S. Chandra and L. K. Gupta, “Spectroscopic characterization of tetradentate macrocyclic ligand: it's transition metal complexes,” Spectrochimica Acta A, vol. 60, no. 12, pp. 2767–2774, 2004.

S. Chandra and L. K. Gupta, “Spectroscopic studies on Co(II), Ni(II) and Cu(II) complexes with a new macrocyclic ligand: 2,9-dipropyl-3,10-dimethyl-1,4,8,11-tetraaza-5,7:12,14-dibenzocyclotetradeca-1, 3,8,10-tetraene,” Spectrochimica Acta A, vol. 61, no. 6, pp. 1181–1188, 2005.

Y. H. Su, J. Liu, J. Li, and X. Z. Si, “Synthesis, crystal structures and properties of two novel macrocyclic nickel(II) and copper(II) complexes,” Journal of Molecular Structure, vol. 837, no. 1–3, pp. 257–262, 2007.

S. Chandra and L. K. Gupta, “Mass, IR, electronic and EPR spectral studies on transition metal complexes with a new tetradentate 12-membered new macrocyclic ligand,” Spectrochimica Acta A, vol. 60, no. 13, pp. 3079–3085, 2004.

Z. H. Chohan, H. Pervez, A. Rauf, K. M. Khan, and C. T. Supuran, “Isatin-derived antibacterial and antifungal compounds and their transition metal complexes,” Journal of Enzyme Inhibition and Medicinal Chemistry, vol. 19, no. 5, pp. 417–423, 2004.