In this method, Arylamine was treated with ammonium thiocyanate to prepare phenylthiourea. For this purpose, 13.8 g/12.3 g (0.1 mol) p-nitroaniline / p-methoxy aniline was heated in a 250 ml three-necked flask with stirrer, dropping funnel and reflux condenser with a mixture of 9 ml (6N HCl) and 25 ml water at a temperature of 32°C on water bath till the aniline hydrochloride was formed. The solution now obtained is allowed to cool at room temperature and then 7.6 g (1 mol) ammonium thiocyanate was added to it. The reaction mixture was refluxed for about four hours on a water bath. After cooling the solid separated out was filtered, washed with cold water, dried and then recrystallized with ethanol (Schemes 1 and 2).

Scheme 1 Synthesis of phenylthiourea of from 4-nitro aniline.

Scheme 2 Synthesis of phenylthiourea of from 4-methoxy aniline.

Copper palmitate was prepared by mixing 1g of palmitic acid into 25 ml ethyl alcohol, shake the mixture in hot water bath at about 50°C and then add one drop of phenolphthalein. Prepare a saturated solution of KOH in another beaker and add it drop by drop into the first beaker until the light pink color appears. Meanwhile, in another beaker prepare a saturated solution of CuSO₄ (approx 3-4 g in 5 ml H₂O) and mix it into the above solution with constant stirring till the blue coloured soap is formed. Filtered it and washed with warm water and 10% ethyl alcohol, then dried and recrystallized with hot benzene (Scheme 3).

Scheme 3 Synthesis of copper palmitate soap.

Complexation of purified copper palmitate obtained from palmitic acid and substituted phenylthiourea was done by adding 0.001-mole copper palmitate with 0.002-mole phenylthiourea in 25-30 ml ethyl alcohol and the mixture was refluxed for about two hours with constant stirring (Schemes 4 and 5). After cooling, the solid separated out was filtered, dried and recrystallized with hot benzene.

Scheme 4 Synthesis of complex from copper palmitate soap and 4-nitro phenyl thiourea.

Scheme 5 Synthesis of complex from copper palmitate soap and 4-methoxy phenyl thiourea.

TLC is probably the most versatile technique for the analysis of complexes and their mixtures. TLC is quite an effective technique for separation and identification of synthesized soap, ligand and their complexes in sub-microgram amounts using silica gel plates in various non-aqueous solvent systems. Rf of copper soap, ligands and the synthesized complexes in non-aqueous solvent systems has been determined. A thin layer chromatographic applicator was used for coating sorbent material on 20×3 cm glass plates to obtain an absorbent layer of 0.25 mm thickness [15Sharma, A.K.; Sharma, R.; Saxena, M. Synthesis, spectroscopic and fungicidal studies of Cu (II) soaps derived from groundnut and sesame oils and their urea complexes. Bulletin Pure Appl. Sci., 2017, 36(2), 26-37.
[http://dx.doi.org/10.5958/2320-320X.2017.00004.8] ]. The coated plates were developed in glass jars of 24×6 cm. A glass sprayer was used to spray chromogenic reagent on the plate to detect the spot. Silica gel G acted as stationary phase whereas the following solvent systems were used as mobile phase.

1. Acetone: Carbon tetrachloride
2. Acetone: Petroleum ether
3. Benzene: Acetone: Petroleum ether

Silica gel G and double distilled water are mixed in 1:3 (w/v) ratio with constant mechanical shaking until the homogenous slurry was obtained to prepare the plates. Approx. 10μl of the test solution was applied on thin layer plates by using micropipette. The solvent ascent was fixed to 10-12 cm in all cases. When the development was completed, the plates are withdrawn from the jar and dried at room temperature. The spots were visible in daylight.

The spectral measurements (IR and NMR) and elemental analysis were carried out at the (RSIC, CDRI) Lucknow U.P. India. The IR spectra of the complexes were obtained as KBr discs in the range 400-4000 cm–1 on Perkin Elmer spectrophotometer. 1H NMR spectra were recorded at CDRI, Lucknow using CDCl3 as a reference. ESR spectra of the complexes were recorded at SAIF, IIT, Mumbai in the X-band region at microwave frequency 9.1 GHz and microwave power 5 mW under the magnetic field of strength 2000G at room temperature. (TCNE) was used as the standard. Magnetic susceptibility measurements were conducted at S.P. University, Vallabh Vidyanagar, Gujrat.

Step 1: Processing of the Sample.

The unknown compound is suspended in DMSO in mass concentration (w/v).

Step 2: Antimicrobial Susceptibility Testing (Kirby-Bauer and Stokes' methods.) [16Mathur, N.; Bargotya, S. Advances in the Research of Novel N/S Containing Metal Complexes as Potent Antioxidants. J. Appl. Chem., 2017, 10(8), 18-23.].

I. Hinton Susceptibility Test Agar

Sabouraud Dextrose Agar is the only susceptibility test medium that has been validated by NCCLS. Mueller-Hinton agar should always be used for disk diffusion susceptibility testing, according to Fig. (1)

II. McFarland turbidity standard

A McFarland 0.5 standard should be prepared and quality controlled prior to beginning susceptibility testing. If tightly sealed to prevent evaporation and stored in the dark, the standard can be stored for up to 6 months. The McFarland standard is used to adjust the turbidity of the inoculum for the susceptibility test

III. Preparation of inoculum

Each culture to be tested should be streaked onto a no inhibitory agar medium to obtain isolated colonies. After incubation at 35°C overnight, select 4 or 5 well-isolated colonies with an inoculating needle or loop and transfer the growth to a tube of sterile saline or nonselective broth (Mueller-Hinton broth, Peptone water) and vortex thoroughly.

IV. Inoculation procedure

a. Within 15 minutes after adjusting the turbidity of the inoculum suspension, dip a sterile cotton swab into the suspension. Pressing firmly against the inside wall of the tube just above the fluid level, rotate the swab to remove excess liquid. Streak the swab over the entire surface of the medium three times, rotating the plate approximately 60 degrees after each application to ensure an even distribution of the inoculum. Finally, swab all around the edge of the agar surface.

b. The Mueller-Hinton plate should be swabbed over the entire surface of the medium three times, rotating the plate 60 degrees after each application.

V. Loading the plate with Positive, Negative Control and Sample

a. The working supply of antibiotic (streptomycin, positive control) should be stored in the refrigerator (4°C). 50 µl of the antibiotic suspension was dispensed in the well labeled with C (control) to the plates as soon as possible, but no longer than 15 minutes after inoculation. Diffusion of the drug in the well begins immediately.

b. 50 µl of the sample (S) and 50 µl of the reference (R, negative control) were dispensed in the well labeled with C (control) to the plates as soon as possible, but no longer than 15 minutes after inoculation.

VI. Recording and interpreting results

After the Loading of C, S, R on the plate, invert the plate and incubate at 35°C for 16 to 18 hours. After incubation, measure the diameter of the zones of complete inhibition (including the diameter of the well) and record it in millimeters. The measurements can be made with a ruler on the undersurface of the plate without opening the lid. The zones of growth inhibition should be compared and recorded as susceptible, intermediate, or resistant to each drug tested. Colonies growing within the clear zone of inhibition may represent resistant variants or a mixed inoculum. The distance from the colonies closest to the well to the center of the well should be measured and then doubled to obtain a diameter. The diameter of the outer clear zone should be recorded as well and an interpretation recorded for each diameter. The colonies inside the zone should be picked, re-isolated, re-identified, and retested in the well diffusion test to confirm the previous results. The presence of colonies within a zone of inhibition may predict eventual resistance to that agent.

Note:

• Dimethyl sulfoxide (negative control) does not show any activity against the test organism.

• Antifungal Sensitivity (Itraconozole 5 mg/well) serves as a positive control.

• Sample (unknown chemical compound) (5mg/ well (w/v))

Fig. (1) Flow diagram for antimicrobial study.

Table 1
Analytical and physical data of ligands, copper soap and complexes.

The results were reproducible with in ±0.02 Rf unit values for various substituted compounds using the procedure for three solvent systems – the main part of the chromatographic studies was an attempt to obtain the standard conditions for their resolution and identification. The best shaped spots given by the solvents were considered to be suitable, and are A = Acetone = carbon tetrachloride, B = Acetone = Petroleum ether, C = Benzene = Acetone = Petroleum ether. In pure acetone spots run along with the solvent front hence could not be used [17Sharma, A. K.; Sharma, R.; Saxena, M. Biomedical and antifungal application of Cu(II) soaps and its urea complexes derived from various oils. Open Access J. Trans. Med. Res., 2018, 2(2), 40-43.]. The single spot was given by all compounds, uniform in shape in the present investigation, so all of them are chromatographically pure. The results of copper palmitate, substituted phenylthiourea and complexes are given in Table 2 [18Bargotya, S.; Mathur, N.; Sharma, A.K. Physico-chemical analysis of environmentally safe complexes of copper “ISBN978-613-8-34672-2”., 2018, ].

Table 2
Rf Values of ligands, copper soap and complexes.

Table 3
IR Spectral Data for Cu (II) Complexes.

Table 4
NMR Spectral Data for Cu (II) Complexes.

Table 5
ESR Spectral Data for Cu (II) Complexes.

Table 6
Antifungal activity of the synthesized complexes against test fungi.

Table 7
ANOVA and descriptive statics results for antifungal activities of synthesized complexes

As is reported earlier, the presence of copper ion leads to a magnetic moment of 1.73 BM. Various scientists have reported subnormal values for the magnetic moment of copper soap and fatty acid, inferring that the soap exists as a binuclear molecule. This has been attributed to the formation of δ-bonds arising from inter-molecular exchange demagnetization through the lateral overlapping of 3dx2-y2 orbital on each copper ion. The studies also reveal that these values of magnetic moments in the range 1.92-2.08 BM is due to the sub-dispersion unit of soap/complex which retains its binuclear configuration even in solution. It may be suggested that sub-dispersion units are bound together by weak polar forces resulting from the interaction between 3dxy and 3dxz orbital of two copper atoms. The lower values of magnetic moments of copper ions in copper complex in benzene, as compared to those in solid state, reflect the terminals of binuclear molecules as [Cu₂(RCOO)₄L₂] Where L represents the solvent molecule in solution but is replaced by the ligand on complexation [27Mathur, N.; bargotya, S.; Mathur, R. Thermogravimetric analysis of microwave assisted novel macromolecular complexes of metal surfactants. J. App. Chem., 2014, 3(2), 712-719.].

The nature of the complexes is found to be non-electrolytic as the molar conductance values in benzene, lies in the range of 3.9-6.14 mhos cm2mol^-1 [28Tank, P.; Sharma, R.; Sharma, A.K. A Pharmaceutical approach & Antifungal activities of Copper Soaps with their N & S donor complexes derived from Mustard and Soyabean oils. Glob. J. Pharmaceu Sci., 2017, 3(4), 1-6.]

The study aims to evaluate the bioactivity of synthesized complexes against test organisms (Fig. 3). Antimicrobial sensitivity was performed for synthesized complexes (Compound S1, S2) on Muller Hinton Agar against Candida abicans, Trichoderma harzianum by Kirby-Bauer Stokes' method [29Mathur, N.; Manna, B. TGA analysis of transition metal complexes derived from phenothiazine ligands. Int. J. Sci. Eng. Res., 2018, 6(3), 47-57.]. Results obtained using Itraconazole (5 mg (w/v)) as antifungal agent control (C) and for negative reference, Ethanol/Methanol (R) are mentioned in Table 6 and Fig. (4)

Fig. (3) Test disks presenting sensitivity of Candida albican fungi against complexes. A- Inhibition zone by fungi Candida albicans s against complex CP[PTU]A B- Inhibition zone by fungi Candida albicans against complex CP[PTU]NA

Fig. (4) Plots presenting the comparative study of sensitivity of fungi against complexes.

Activity index = zone of inhibition of sample[S]/zone of inhibition of reference [R] and the activity can be found out by

Activity = Activity index x 10

If the Zone of Inhibition is [30Sharma, A.K.; Sharma, R.; Gangawal, A. Biomedical and fungicidal application of copper surfactants derived from pure fatty acid. Org. Med. Chem. I J., 2018, 5(5), 1-3 .]

< 13 = it means that the extract is inactive,

13 – 18 = it means that the extract is bioactive,

> 18 = it means that the extract is highly active

The results of ANOVA [31Mathur, N.; Bargotya, S. A facile synthesis and biological evaluation of some macro cyclic copper complexes. Int. J. Pharm. Sci. Res., 2015, 6(6), 2538-2345.] for the antifungal activities for all complexes are shown in Table 7. The predicted R2 are in reasonable agreement and closer to1.0. This confirms that the experimental data are well satisfied. The descriptive statics results of all complexes also confirm satisfactory results in triplet [32Mathur, N.; Bargotya, S. Synthesis characterization and plant growth regulatory activity of metal complexes with some bioactive ligands. World J. Pharm. Pharm. Sci., 2016, 5(11), 945-955.]. This work also coincided with Sharma et al. [33Sharma, A.K.; Saxena, M.; Sharma, R. Fungicidal activities and characterization of novel biodegradable Cu (II) surfactants derived from lauric acid. Open Chem. J., 2018, 5, 89-101.
[http://dx.doi.org/10.2174/1874842201805010089] ], Mathur et al. [34Mathur, N.; Jain, N.; Sharma, A.K. Biocidal activities of substituted benzothiazole of copper surfactants over Candida albicans & Trichoderma harzianumon Muller Hinton Agar. Open Pharm. Sci. J., 2018, 5, 24-35.
[http://dx.doi.org/10.2174/1874844901805010024] ], Bhutra el al. [35Bhutra, R.; Sharma, R.; Sharma, A.K. Antimicrobial studies and characterization of copper surfactants derived from various oils treated at high temperatures by P.D.A. technique. Open Phar Sci. J., 2018, 5, 36-40.
[http://dx.doi.org/10.2174/1874844901805010036] ] on fungicidal activities of copper surfactants derived from lauric acid, substituted complex of benzothiazole of copper palmitic acid and copper surfactants derived from fried oils treated and untreated at higher temperatures, respectively.