Solid-State Synthesis, Characterization, Biological Studies and Theoretical Evaluation of (E)-2-(2-(naphthalene-2-yloxy)-1-phenylethylidene) Hydrazine Thiocarboxamide

Introduction

Thiosemicarbazones are imine derivatives obtained by the condensation reaction between aldehydes or ketones and thiosemicarbazide. Different derivatives of thiosemicarbazone can be obtained by suitable substitution. These are versatile intermediates and can be synthesized using simple methods with high yields. Their biological activity is considered to stem from their ability to form chelates with metals. As a class these compounds are known to exhibit various biological activities like antitumour, antiviral, antibacterial, antiprotozoal and other activities [1-3]. Their tendency to form Schiff bases with metal cations has attracted the attention of researchers to consider thiosemicarbazones as interesting ligands for synthesizing metal complexes [4]. It is this chelating ability of thiosemicarbazones with endogenous metals that makes them biologically important molecules. Modifications of their structure with different aromatic substituents that can interact with biomolecules enhance their biological activity.

Thiosemicarbazones used to exist in conformational isomeric forms. The stability of the isomeric forms depends on the structural features of the thiosemicarbazone under consideration. Karabatsos et al. [5-9] have carried out NMR spectral analysis on aromatic ring substituted phenyl hydrazones, thiosemicarbazones and semicarbazones. The existence of two different isomers of semicarbazones has been reported by Sternberg et al. [10] using proton NMR spectra.

In the present work, the synthesis and characterization of unreported (E)-2-(2-(naphthalene-2-yloxy)-1-phenylethylidene) hydrazine thiocarboxamide (NAPTCAR) using ¹HNMR, ¹³CNMR and FTIR spectral analysis has been presented. The carbon nuclear magnetic resonance spectral data confirm that NAPTCAR exists as two different conformer structures.

Experimental

Solid-State Synthesis of (E)-2-(2-(naphthalene-2-yloxy)-1-phenylethylidene) hydrazine thiocarboxamide

A mixture of 2-(naphthalene-2-yloxy)-1-phenylethanone (0.1 mol), thiosemicarbazide(0.1 mol) was taken in a test tube. Then the reaction mixture was treated with 5 drops acetic acid and heated at 80^oC for about 30 minutes in water bath. Thin layer chromatography using petroleum ether and ethyl acetate (4:1) as an eluent was used to monitor the progress of the reaction. At the end of the reaction, the reaction mixture was allowed to coolto room temperature.  The product was separated, filtered, dried and recrystallized using ethanol. Yield: 86%, Melting range :158-160 °C

Scheme 1: Synthesis of (E)-2-(2-(naphthalene-2-yloxy)-1-phenylethylidene) hydrazine thiocarboxamide (2) Click here to View scheme

Physical Measurements

The uncorrected melting points were found in an open capillary tube. Infra-red spectra of the compounds were recorded using FTIR–Shimadzu model IR-Affinity-1 spectrophotometer using KBr discs in the range 400 – 4000 cm-1. ¹H NMR and ¹³C NMR spectra have been recorded at 400 and 100 MHz, respectively, using a Bruker Avance III HD Nanobay 400MHz FT-NMR spectrometerat a temperature of 25°C. CDCl₃ and TMS wereused as the solvent and internal standard, respectively.

Antimicrobial Examinations

The antifungal and antibacterial activity of thiocarboxamide were allowed to take place in vitro for the growth inhibiting nature against different bacterialand fungal strains utilizing agar disk diffusion method. The cultures were inoculated and incubated for18 hrs for the two microorganisms, the nutrient broth and fungi, in potato dextrose agar incline. For the readiness of cultivated agar plate, the liquid medium was poured in sterile petri dish to get a profundity of 5mm. The medium was allowed to solidify and the test organism was seeded. 5 ml sterile water was added to agar incline culture of organisms to get suspension. A sterile cotton swab was dipped in suspension, daintily spread over the solidified medium. The petri dish was left for a couple of moments. 5 mmwhatmann filter paper no.1 discs were dipped in the sample solution. The disc was then removed and placed in a sterile petri dish so that the solvent evaporates. After 10 minutes, the discs were moved to cultivated agar plates. 2 circles were kept on the cultivated agar plate. At last, dishes were incubated at 37°C for bacteria and fungi. Inhibition zones were detected around each disc.

Results and Discussion

Proton and Carbon NMR Studies

¹H NMR spectrum of (E)-2-(2-(naphthalene-2-yloxy)-1-phenylethylidene) hydrazine thiocarboxamide (NAPTCAR)

In ¹H NMR (400 MHz, CDCl₃), the singlet (Fig. 1) at 5.3 ppm is ascribed to methylene (-CH₂) group in the product. Another singlet appearing at 10.4 ppm is assigned to the -NH group. The resonance of the -NH₂ hydrogens gives rise to a broad signal at 6.4 ppm. The remaining phenyl and naphthyl ring protons resonances are given below: 7.20 -7.28 (d, 1H), 7.34-7.46 (m, 6H), 7.67-7.71 (t, 3H), 7.79-7.81 (t, 2H) ppm.

Figure 1:1H NMR spectrum of (E)-2-(2-(naphthalene-2-yloxy)-1-phenylethylidene) hydrazine thiocarboxamide in CDCl3 at 25°C Click here to View figure

Figure 2: 13C NMR spectrum of (E)-2-(2-(naphthalene-2-yloxy)-1-phenylethylidene)  hydrazine thiocarboxamide in CDCl3 at 25°C. Click here to View figure

Figure 3: Expanded 13CNMR Spectrum of (E)-2-(2-(naphthalene-2-yloxy)-1-phenylethylidene) hydrazine thiocarboxamide in the range of a) 180.0 to 130 ppm, b) 131 to 124 ppm and c) 120 to 64 ppm. Click here to View figure

¹³C NMR spectrum of (E)-2-(2-(naphthalene-2-yloxy)-1-phenylethylidene) hydrazine thiocarboxamide (NAPTCAR)

Thiosemicarbazones are well known to exhibit conformational isomerism. The ¹³C NMR spectrum (Fig. 2) of NAPTCAR displays two sets of carbon resonance peaks indicating the presence of two conformer structures(Fig. 4) which are given below.

Figure 4: [A] And [B] Are The Conformational Structures Of NAPTCAR. Click here to View figure

Conformer A

The singlet at 65.1 ppm is ascribed to methylene (-CH₂) group in the product.  The two singlet signals at 155.9 and 179.3 ppm are assigned to the -C=N-NH- and -CSNH₂ groups, respectively. The remaining phenyl and naphthyl ring carbon peaks are given below: 107.5, 118.3, 124.1, 126.5, 126.8, 127.0, 127.7, 128.9, 129.7, 129.9, 130.2, 134.1, 135.5, 148.7 ppm.

Conformer B

The singlet at 70.9 ppm is ascribed to methylene (-CH₂) group in the product.  The two singlet signals at 154.7 and 179.5 ppm are assigned to the -C=N-NH- and -CSNH₂ groups, respectively. The remaining phenyl and naphthyl ring carbon absorptions are: 107.9, 118.6, 124.6, 126.7, 126.9, 127.4, 127.8, 129.3, 129.8, 130.2, 130.7, 134.3, 144.2 ppm.

FTIR Spectral analysis

In this present work, the ν_s (C – H) has been assigned to the wavenumber higher than the ν_as (C – H) band position. This is the usual phenomenon that the substituted aromatic compounds [11-15] produce the  C – H  symmetric stretching peaks at higher wavenumbers than the asymmetric vibrational bands. The ν_as (CH₂) and ν_s (CH₂) bands absorb at 2922.2, and 2723.5 cm^-1, respectively. The ν_as (CH₂) and ν_s (CH₂) ν(C =) bands are noticed at 2854.7, and 2675.3 cm^-1, respectively.  All these bands have been shifted to higher wavenumber side in the spectrum (Fig. 5 and Table 1) of NAPTCAR whose formation has been confirmed by the emergence of additional bands due to the stretching vibrations of NH₂ , N – H, C = N  and C = S groups. The ν_as (NH₂) , ν_s (NH₂) , ν(N – H) , ν(C = N) and ν(C = S)  bands are observed at 3444.9, 3342.8, 3568.3, 1606.7 and 684.7 cm^-1, respectively. The molecules that have NH₂ group exist usually as the H – bonded networks which can be ascertained from the broad absorption in the region. But, the FTIR spectrum (Fig. 5 and Table 1) of NAPTCAR displays no such spectral feature and this indicates that NAPTCAR molecules do not involve in self-association among themselves. NH₂  ν_s (NH₂) ν(N – H).

Figure 5: Experimental vibrational spectrum of NAPTCAR. Click here to View figure

The theoretical infrared spectra of Conformer A and Conformer B of NAPTCAR are given in Fig. 6 and the assignment of vibrational bands are given in Table 1. The v_as(NH₂) , v_s(NH₂) , ν(N – H) , ν_s (C – H), ν_as (C – H),ν_as (CH₂) , ν_s (CH₂ ),ν(C = N) and ν(C = S) bands are observed at 3704.6, 3571.3, 3610.9, 3211.7, 3181.4, 3119.6, 3068.7, 1610.9 and 744.52 cm^-1 for Conformer A and 3685.15, 3553.51, 3506.78, 3202.06, 3179.79, 3105.38, 2995.57, 1649.65 and 772.23 cm^-1 for Conformer B, respectively. Theoretical FTIR spectra of the compound showed that the results are in agreement with experimental.

Figure 6: Theoretical vibrational spectrum of NAPTCAR [(a) Conformer A and (b) Conformer B]. Click here to View figure

Table 1: Experimental and theoretical vibrational bands (cm^-1) of NAPTCAR.

Vibrational bands	NAPTCAR
	Experimental	Theoretical Conformer A	Experimental	Theoretical Conformer B
νas (NH2)	3444.87	3704.55	3415.93	3685.15
νs (NH2)	3342.84	3571.34	3244.27	3553.51
ν(N – H)	3568.31	3610.94	3502.73	3506.78
νs (C – H)	3149.76	3211.73	3055.24	3202.06
νas (C – H)	—-	3181.40	—-	3179.79
νas (CH2)	2922.16	3119.56	2854.65	3105.38
νs (CH2)	2723.49	3068.73	2675.27	2995.57
ν(C = N)	1606.70	1610.85	1627.92	1649.65
ν(C = S)	684.73	749.314	744.52	772.23

Molecular geometry

The structures of the two conformers of NAPTCAR molecule have been optimizedat B3LYP [16-17] functional of Density Functional Theory (DFT) with the basis set 6-311++G(2d, 2p) using Gaussian 09W program [18] are shown in Fig. 7.

Figure 7: Ground state optimized structures of NAPTCAR a) Conformer A and b) Conformer B. Click here to View figure

Frontier molecular orbital (FMO) studies

The frontier molecular orbital examination affords clear ideas about the spatial distribution of the electron density throughout the molecular framework [19-20]. The energy difference between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of a molecule is known as the HOMO – LUMOgap. Using the HOMO – LUMOgap, electrical transport properties [21] and hence the reactive nature of the conjugated system [22] can be explored. The conjugated molecules with a smaller HOMO – LUMOgap are very good candidates for nonlinear optical applications [23-24].The HOMO and LUMO diagram of NAPTCAR conformers A and B are shown in Fig. 8. The HOMO which is the electron rich part of the moleculeis found to be spread over the carbothioamide group. The LUMO is mainly distributed in the phenyl ring. This is common for both the conformers of NATPCAR. But unlike in conformer A, the LUMO on carbothioamide is a bit more in conformer B. The HOMO – LUMOenergy for conformer A is (-0.2141-(-0.0788)) = 0.13 eV which is almost the same that for conformer B -0.2098-(-0.0691)) = 0.14 eV]. The lower energy gap means that the molecules are more reactive because of the possibility for the easy transition of the electrons from ground state to the excited state.

Figure 8: The HOMO (a and c) of Conformer A and Conformer B form of NAPTCAR and LUMO of Conformer A and Conformer B form (b and d). Click here to View figure

Molecular electrostatic potential

The molecular electrostatic potential (MEP) can be used to obtain information on the electrophilic and nucleophilic sites and hydrogen bonding interactions [25-28]. Fig. 9shows the MEP ofNAPTCAR conformer A and B from which it can be noticed that the electron density is rich over the region (red) around C = S of conformer A while the region around the C = S of conformer B loses the electron density much. At the same time, the electron deficient region (blue) in both the conformers is around the NH₂ hydrogens. Therefore, conformer A has a stronger nucleophilic site, the C = S , than conformer B. But both the conformers are equally good as far as the electrophilic site (blue region) is concerned.

Figure 9: Electrostatic potential (ESP) surface of (a and b) Conformer A and Conformer  B form of  NAPTCAR. Click here to View figure

Reduced density gradient

The RDG isosurface, which can display the presence of various interactions at different parts of the molecule, has been given in Fig. 10.The different types of interactions are indicated by various colours. The presence of strong steric interactions (strong red region) within the phenyl rings in both the conformers can be observed. Van der Waals interactions (green and brown) are found to be operative in conformer A (Fig. 10a) between i) C = N  nitrogen and one of the C – H  hydrogens of naphthyl ring adjacent to the C – O – C oxygen, ii) CH₂ carbon and one of the C – H hydrogens of the phenyl ring and iii) NH₂  hydrogen and C = N carbon. In conformer B (Fig. 10b), the second interaction continues to be present while new Van der Waals interactions between i) CH₂ carbon and one of the C – H hydrogens of the naphthyl ring, ii) C = N nitrogen and one of the hydrogens of the phenyl ring, iii) C = S sulphur and C = N nitrogen, and iv) notably, a very weak intramolecular H – bond (pale blue) between N – H hydrogen and C – O – C oxygen and steric interaction (pale red) between N – H nitrogen and C – O – C oxygen are operative. Therefore, it can be stated that conformer B is stabilized by more interaction forces than conformer A.

Figure 10: The reduced density gradient (RDG) isosurfaces of a) conformer A and b) conformer B of NAPTCAR. Click here to View figure

Antimicrobial activity

The antimicrobial properties of NAPTCAR were evaluated against Gram negative bacterial strains (Escherichia Coli, Klebsiella pneumonia, Pseudomonas aeruginosa,Serratia marcescens), Gram positive bacterial strains (Bacillus cereus, Bacillus subtilis, Enterococcus, Staphylococcus aureus) and Fungal Species (Candida albicans, Aspergillus niger). The antimicrobial activity of test compound are expressed as the area of zone of inhibition and summarized in Table 2. The results of antimicrobial screening, Compound semi exhibited good activity against Pseudomonas aeruginosa, Serratia marcescens, Bacillus cereus and Enterococcus. This compound was inactive against fungal species. The thio-semi compound was tested against tem pathogens and active against Klebsiella pneumonia, Serratia marcescens, Bacillus cereus, and Enterococcus and Candida albicans. The results of NAPTCAR against pathogenic bacteria and fungi indicate that these may be used for the development of new drugs that canbe used to treat microbial infections.

Table 2: Antimicrobial activities of  NAPTCAR.

			Zone of Inhibition in mm
Gram positive Bacteria			NAPTCAR	Control (Amikacin)
1		Bacillus Subtilis	13	26
2		Bacillus Cereus	11	26
3		Enterococcus	13	22
4		Staph Aureus	8	27
Gram Negative Bacteria
1	E.Coli		11	22
2	Klebsiella		12	20
3	Pseudomonas Aeruginosa		10	27
4	Serratia Marcescens		13	25
Fungai				Control (Nystatin)
1		Candida albicans	15	18
2		Aspergillus Niger	8	12

Conclusion

The synthesis of (E)-2-(2-(naphthalene-2-yloxy)-1-phenylethylidene) hydrazine thiocarboxamide by simple condensation method and its characterization using NMR (¹H and ¹³C), FTIR and DFT calculations. Two conformers of NAPTCAR have been identified using ¹³CNMR spectral data.  The HOMO – LUMO energy gap is found to be almost the same for both the conformers.The molecular electrostatic potential surface shows that Conformer A is relatively a stronger nucleophilic than conformer B. The structure of both the conformers are stabilized by mostly Van der Waals and the steric interactions. As suggested by the results of NAPTCAR against pathogenic bacteria and fungi indicate, both the conformers may be used for the development of new drugs that canbe used to treat microbial infections.

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