A bluish-green emitting organic compound methyl 3-[(E)-(2-hydroxy-1-naphthyl)methylidene]carbazate: spectroscopic, thermal, fluorescence, antimicrobial and molecular docking studies

Abstract

The present paper describes the physicochemical properties and biological activities of an organic single crystal, methyl 3-[(E)-(2-hydroxy-1-naphthyl)methylidene]carbazate which was grown by the slow evaporation solution growth technique. A powder X-ray diffraction study of the compound was carried out and the (hkl) values were found to be comparable with the reported values. The vibrational properties of the compound were analysed on the basis of FT-IR and FT-RAMAN spectra. The proton NMR spectrum was consistent with the chemical structure of the compound. From UV-VIS-DRS analysis and fluorescence studies, the absorption range and the bluish-green emission property of the title compound were found. The melting point of the compound was found to be at 205 °C from the TG-DTA-DSC analysis. The antimicrobial activities of the title compound were screened using the resazurin reduction assay against human pathogenic bacteria such as Shigella dysenteriae, Vibrio cholerae, Streptococcus faecalis, and Bacillus cereus and fungi such as Candida krusei, Candida albicans, and Candida glabrata. Molecular docking studies demonstrated that the title compound could bind well with the active site of human estrogen receptor and act as a potential inhibitor of ERα and ERβ.

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Introduction

Nonlinear optical single crystals have their applications in high-energy lasers for inertial confinement fusion research, color displays, electro-optical switches, frequency generations, etc.¹ Organic second order nonlinear optical chromophores and polymers have been the subject of extensive research for their range of applications in optical communication, optical interconnects for computing and for the generation of THz radiation.² Fluorescent molecules have played a key role in the chemical, biological and medical sciences.³ Fluorescence in aldehydes and ketones arises from the occurrence of a low lying n–π* transition associated with the carbonyl group which favours high yields of singlets to triplet intersystem crossing. Naphthaldehyde has analytical interest because of the fluorogenic properties of the naphthyl group and the high reactivity of the carbonyl group which is useful in the fluorescent derivatives of non-fluorescent analytes.⁴ The naphthalene group as a fluorophore has been studied extensively due to its characteristic photophysical properties. Schiff base compounds can be classified according to their photo and thermochromic characteristics. 2-Hydroxyl Schiff base ligands are of interest due to the resistance (O–H⋯N & N–H⋯O) type hydrogen bonds and tautomerism between the enol–imine and keto–enamine forms.³ A vast array of Schiff bases having excellent activity in a broad spectral range forms an invaluable part of the present antimicrobial, antifungal, antitubercular, anticancer and anti-HIV toolbox of clinicians. Schiff bases exhibit a variety of biological therapeutic properties and are the fastest growing antibacterial class in terms of global revenue, increasingly being used in both the hospital and community sectors for a broad range of infections.⁵

Breast cancer is the second most widespread type of cancer after lung cancer with the rate of 10.4% and also in 2008, 458 503 deaths were caused due to breast cancer (World Health Organization international agency for research on cancer). Breast cancer is 100 times less common in men.^6,7 Both normal and breast cancer cells have receptors to bind estrogen and progesterone circulating in the blood.⁸

Estrogen interacts with the estrogen receptors ERα and ERβ, and it controls several functions in mammalian tissues. It also plays an important role in female reproduction, bone formation, and cardiovascular and Central Nervous System (CNS) health.⁹ Selective Estrogen Receptor Modulators (SERMs) on Schiff base compounds act as an alternative approach to Hormone Replacement Therapy (HRT).¹⁰ Breast cancer cells are estrogen receptor positive and more likely to respond to hormonal therapies with tamoxifen, raloxifene, and toremifene. These cells also have a better prognosis than cancers that are hormone receptor negative.⁸ Tamoxifen (Nolvadex R) is a drug, taken orally as a tablet, which interferes with the activity of estrogen. The serious side effects of tamoxifen include blood clots, stroke, uterine cancer, and cataracts. Side effects of raloxifene are serious blood clots in the legs, lungs, or eyes. Other reactions include leg swelling/pain, trouble breathing, chest pain, and vision changes. These side effects discourage the use of these drugs and therefore studies are required to find a better alternative.

Methyl 3-[E]-(2-hydroxy-1-napthyl)methylidene]carbazate (MNMC) is such a Schiff base compound with the molecular formula C₁₃H₁₂N₂O₃. Sheng et al.¹¹ have already reported structural studies of this compound. The molecule adopts an E or trans configuration with respect to the C=N bond. The crystal structure of MNMC is stabilized by intramolecular O–H⋯N interaction and intermolecular N–H⋯O interaction.¹¹ In the present investigation, elaborate studies on the spectral, thermal, electrochemical and fluorescence properties of the title compound were made. The antimicrobial activity of the Schiff base ligand was screened against selected kinds of bacteria and fungi and the minimum inhibitory concentrations (MICs) against human pathogenic bacteria, such as Shigella dysenteriae, were analysed by the resazurin reduction assay described by Sarker et al.¹²Molecular docking of the MNMC Schiff base was carried out with human estrogen receptor as the target protein.

Experimental

Materials and methods

The title compound was synthesized by following the procedure given in the literature by Sheng et al.¹¹ A solution of 1 mmol of methyl carbazate (Sigma-Aldrich) in 5 mL of ethanol was added slowly to a solution of 1 mmol of 2-hydroxy-1-naphthaldehyde (Sigma-Aldrich) in 15 mL of absolute ethanol, under heating and stirring. The mixture was refluxed for 3 h, then cooled to room temperature. Yellow block-shaped crystals were formed on slow evaporation of the solvent. The reaction scheme is shown in Scheme 1.

Scheme 1 Reaction scheme for MNMC.

The obtained methyl 3-[(E)-(2-hydroxy-1-napthyl)methylidene]carbazate (MNMC) crystals were further purified by a recrystallization process using ethanol and dimethylformamide as solvents. Yellow block-shaped MNMC single crystals (Fig. 1a and b) were obtained after a period of 3 months. The morphology of the MNMC single crystal (Fig. 1c) was predicted using WinXMorph.^13,14 The shape of a MNMC single crystal was analysed using Apex Bruker Software (Fig. 1d). All the characterization studies were performed on the MNMC single crystal grown from ethanol solvent.

Fig. 1 As grown single crystals of MNMC (a) from ethanol solvent (b) from dimethyl formamide solvent; (c) morphology of MNMC single crystal; (d) shape of MNMC single crystal in different orientations.

Characterization

Single crystal X-ray diffraction analysis was carried out on the grown MNMC single crystal using an Enraf (Bruker) Nonius CAD4 Single Crystal X-ray Diffractometer. A D2 PHASER powder X-ray diffractometer was used to analyse the cell parameter and the (hkl) planes. An Alpha Bruker FT-IR spectrometer in the region of 400–4000 cm⁻¹ and a Bruker 27 multiRAM stand-alone FT-RAMAN spectrometer with a scanning range of 50–4000 cm⁻¹ were used for analysing the spectral properties of the compound. Proton NMR study of the MNMC compound was performed using a Bruker AVANCE III 500 MHz multinuclei solution spectrometer. Shimadzu 2450 UV-VIS spectrophotometer was utilized for the study of the electronic absorption range of the compound in the region 200–800 nm. Cyclic voltammetry measurements were made for the title compound using a CHI 600D electrochemical analyzer (room temperature) with a 3 electrode cell in a solution of BuNClO₄ in dichloromethane at a scanning rate of 100 mV s⁻¹. A glassy carbon electrode was used as the working electrode, platinum foil was used as the counter electrode and the reference electrode Ag/AgCl was calibrated after each measurement using ferrocene (Fc). The fluorescence study was carried out using a Jobin Yvon fluorolog-3-11 spectrofluorometer having a xenon lamp with 450 W as the light source in the range 180–1550 nm with a resolution of 0.2 nm.

A NETZSCH STA 449 F3 Jupiter instrument was utilized for the TG-DSC analysis of the compound in the range 25–1400 °C with the initial sample weight of 4.28 mg in a nitrogen atmosphere with a nitrogen flow rate of 20 mL min⁻¹. The graph was plotted using exothermic behavior in the downward direction. A frequency-doubled, Q-switched Nd:YAG (Spectra-Physics, INDI 40) laser, delivering 6 ns laser pulses at 1064 nm at a repetition rate of 10 Hz, passed through the powdered form of the title material and the output intensity was measured for screening the second harmonic generation efficiency of the MNMC compound.

Preparation of resazurin dye solution

The resazurin dye solution was made by dissolving a 270 mg tablet in 40 mL of sterile distilled water. The vortex mixer was used to ensure the formation of a homogeneous resazurin solution.

Preparation of the activity plates

96 well plates were prepared under aseptic conditions. A volume of 200 μL of the compound (1 mg mL⁻¹) in 5% (v/v) dimethyl sulfoxide was pipetted into the first row of the sterile 96 well plate. 100 μL of nutrient broth was added to other wells for the bacteria cells and 100 μL of Sabouraud dextrose broth for the fungus cells. The serial dilutions were performed with sterile pipette tips such that each well had 100 μL of the test material in serially descending concentrations and then 10 μL of the resazurin dye solution was added to all of these wells. A 10 μL bacterial suspension (5 × 10⁶ cells per mL) was added to each well to achieve a concentration of 5 × 10⁵ cells per mL. The commercial antibiotic streptomycin (against bacteria) and amphotericin B (against fungus) were used as positive controls in the assay plate. The plates were placed in an incubator at 37 °C for 18–24 h. The color change was then observed visually. The color changes from purple to pink or colorless were recorded as a reduction of the dye by the viable bacteria. The lowest concentration at which no color change occurred was taken as the MIC value.

Protein, ligand preparation and induced fit docking

The protein human estrogen acceptor data were downloaded from the Protein Data Bank (PDB id: 2IOK). The water molecules were removed and side chains were fixed using the module protein preparation wizard and the energy was minimized using the Optimized Potentials for Liquid Simulations (OPLS) force field. All computational works were performed on a CentOS EL-5 workstation using the molecular modelling software Maestro (Schrodinger LLC 2009, USA). GLIDE-5.5 (Grid-based Ligand Docking with Energetics) performs flexible Induced Fit Docking (IFD) between the ligand molecule with a macromolecule, usually a protein. PyMOL software was used for graphical visualization, analyzing hydrogen bond interactions and producing quality images. The crystal structure of the compound was drawn using the software Chemsketch and the energy was minimized using the impact minimization. The ligand was prepared as a three dimensional structure of a drug-like molecule in maestro format. The impact module performed conversions, applied corrections to the structures, generated variations on the structures and optimized the structures. The structures were minimized using impact energy minimization with 1000 cycles of steepest descent and 5000 cycles of conjugate gradient.

An induced fit docking study was carried out for the MNMC compound and compared with the co-crystal ligand.

Results and discussion

FT-IR and FT-RAMAN spectral analysis

FT-IR and FT-RAMAN spectra gave information about the vibrational modes of the MNMC compound which aided in the identification of the functional groups. The FT-IR and FT-RAMAN spectra of the MNMC compound are shown in Fig. 2a and b. The assignment of the IR and Raman bands along with their respective vibrational modes are listed in Table S2.†

Fig. 2 (a) FT-IR spectrum of the MNMC compound (b) FT-RAMAN spectrum of the MNMC compound.

The weak band at 3749 cm⁻¹ in the IR spectrum and the medium peaks at 1239 cm⁻¹ and 1195 cm⁻¹ in the Raman spectrum are assigned to aromatic O–H stretching. The symmetric stretching of the N–H band in the IR spectrum is observed at 3362 cm⁻¹ and 3246 cm⁻¹. The weak peaks at 3068 cm⁻¹ and 3246 cm⁻¹ in the IR spectrum and the medium peak at 3077 cm⁻¹ in the Raman spectrum correspond to the aromatic C–H stretching of the molecule. The C=O symmetric stretching vibration is found as a very high intensity peak at 1701 cm⁻¹ in the IR spectrum and as a weak band at 1623 cm⁻¹ and 1669 cm⁻¹ in the Raman spectrum. The medium peak in the IR spectrum at 1669 cm⁻¹ is assigned to C=N symmetry stretching whereas in the Raman spectrum the stretching is represented by a medium peak and a very high intensity peak at 1604 cm⁻¹ and 1576 cm⁻¹, respectively.^15–17

¹H NMR studies

¹H NMR spectra give information about the different types of protons in a molecule and also provide the nature of the immediate environment to each of them in the molecule. The ¹H NMR spectral data for the MNMC compound was recorded using DMSO as the solvent (Fig. 3). The proton peaks of the methoxy group appeared at 3.7 ppm. With different multiplicity and coupling constants, doublet downfield peaks of the aromatic region occur between 7.2 and 8.2 ppm. The azomethine proton undergoes a significant shift at 11.4 ppm as a singlet indicating the coordination of the azomethine nitrogen. Moreover, the singlet at 9.0 ppm confirms the N–H proton of the MNMC molecule. The OH peak shifted towards 12.2 ppm due to its interaction with the azomethine nitrogen.

Fig. 3 ¹H NMR spectrum of the MNMC compound.

Ultraviolet-visible-diffuse reflectance spectroscopy (UV-Vis-DRS) studies

The absorption of UV light by a molecule depends on its electronic structure. Hence, the UV spectrum reveals the presence of specific bonding arrangements in the molecule. UV-Vis-DRS was employed for acquisition of the absorption spectra of MNMC (Fig. 4).

Fig. 4 The absorption spectrum of the MNMC compound.

There are three main absorption bands centered at 222, 360, 450 nm. The maximum absorption peak was found at 360 nm with a cut-off wavelength of 405 nm. The π–π* transition of the C=N chromophore and the highly conjugated naphthalene group caused the absorption at 222 and 360 nm, respectively, in the near UV region. The absorption band in the visible region arose at 450 nm as a result of the n–π* transitions of the C=N chromophore.^18,19 As there is no absorption observed in the Nd:YAG laser second harmonic generation wavelength (532 nm), the material can be utilized in optical applications above 500 nm. Study of the absorption edge is essential in connection with the theory of electronic structure, which leads to the prediction of whether the band structure is affected near the band extreme. The most direct way of extracting the optical band gap is to simply determine the photon energy at which there is a sudden increase in the absorption. Using the relation E_g = hc/λ, the optical band gap value of MNMC was calculated from the maximum absorption edge as 3.0 eV.

Cyclic voltammetry

A cyclic voltammetry measurement was carried out to measure the HOMO–LUMO energy levels and electrochemical band gap value. The HOMO–LUMO energy levels of the MNMC compound can be calculated from the electrochemical data by utilizing eqn (1) & (2).²⁰ The onset oxidation potential [E_Ox(onset)] 0.529 V and the onset reduction potential [E_Red(onset)] – 0.687 V were analysed from the cyclic voltammogram (Fig. 5).

HOMO = −[4.65 V − E_Ox(onset)] = −4.121 eV (1)

LUMO = −[4.65 V − E_Red(onset)] = −5.337 eV (2)

The electrochemical band gap value was calculated from the HOMO and LUMO energy levels using the formula E_g = LUMO − HOMO as 1.2 eV. We arrived at a lesser energy band gap value from the cyclic voltammogram when compared with the optical band gap value calculated from the UV-Vis-DRS spectrum.

Fig. 5 Cyclic voltammogram of the MNMC compound.

Fluorescence studies

The fluorescence spectrum of MNMC was recorded at room temperature. The MNMC compound was excited at a wavelength of 365 nm. A broad emission peak at 495 nm (2.5 eV) indicates bluish-green fluorescence emission in the MNMC crystal (Fig. 6). The fluorescence peak was obtained at a photon energy which was less than the effective band observed by absorption and the peak was shifted towards a lower energy. The emission peak exhibits inhomogeneous broadening (Gaussian distribution) which indicates the formation of defect levels in the material.²¹

Fig. 6 The fluorescence emission spectrum of MNMC (inset: deconvoluted plot).

Antimicrobial studies

The MIC of the compound was determined by the resazurin dye reduction assay method described by Sarker et al.¹² The color change of the dye from blue or purple to pink indicates that the cells are viable. The enzyme oxido-reductase present inside the bacterial cells or the unicellular fungus convert the resazurin to resorufin which is pink in color. If the color of the dye remains blue then it indicates that there is no activity of viable cells. The compound destroys the bacterial and fungal cells after the incubation period. This was inferred from the color change in the respective wells. The pink color change in the wells even after treating with the test compound or commercial drugs indicates the presence of viable cells. Thus, the least dilution at which the color remained blue was taken as the MIC value of the particular compound.

The test compound was active against all tested human bacterial and fungal pathogens. The compound shows MIC values between 0.78 μg mL⁻¹ and 25 μg mL⁻¹. The compound was more effective against the Gram positive bacteria Streptococcus faecalis and Bacillus cereus than Gram negative bacteria Shigella dysenteriae and Vibrio cholera (Fig. 7a). The MIC value of the title compound was equipotent to the reference antibiotic streptomycin with the MIC value of 1.56 μg mL⁻¹ against the Gram positive bacteria Streptococcus faecalis. In the antifungal study (Fig. 7b), the test compound was more active against Candida glabrata than the other two fungi Candida krusei and Candida albicans. The test compound was less active than the positive control amphotericin B (reference drug) for all the tested fungi. The results are tabulated in Table 1. Thus, the given test compound has very good antimicrobial activity. Further development of this compound may lead to biological applications.

Fig. 7 (a) MIC of the compound against bacteria by the resazurin reduction assay. X1 – control − compound + reagent + without bacteria, X2 – control − reagent + Shigella dysenteriae + without compound, X3 – control − reagent + Vibrio cholerae + without compound, X4 – control − reagent + Streptococcus faecalis + without compound, X5 – control − reagent + Bacillus cereus + without compound, Y1 – compound + reagent + Shigella dysenteriae, Y2 – compound + reagent + Vibrio cholerae, Y3 – compound + reagent + Streptococcus faecalis, Y4 – compound + reagent + Bacillus cereus, Z1 – streptomycin + reagent + Shigella dysenteriae, Z2 – streptomycin + reagent + Vibrio cholerae, Z3 – streptomycin + reagent + Streptococcus faecalis, Z4 – streptomycin + reagent + Bacillus cereus. (b) MIC of the compound against fungi by the resazurin reduction assay, C1 – control − compound + reagent + without fungus, C2 – control − reagent + Candida krusei + without compound, C3 – control − reagent + Candida albicans + without compound, C4 – control − reagent + Candida glabrata + without compound, T1 – compound + reagent + Candida krusei, T2 – compound + reagent + Candida albicans, T3 – compound + reagent + Candida glabrata, P1 – amphotericin B + reagent + Candida krusei, P2 – amphotericin B + reagent + Candida albicans, P3 – amphotericin B + reagent + Candida glabrata.

Table 1 Minimum inhibitory concentration of test compound against microbial pathogens by resazurin reduction assay

Microbial pathogens	MIC μg mL^−1
	Compound	Streptomycin
Shigella dysenteriae	6.25	1.56
Vibrio cholerae	25.0	6.25
Streptococcus faecalis	1.56	1.56
Bacillus cereus	6.25	1.56
Candida krusei	12.5	1.56 (amphotericin B)
Candida albicans	25	1.56 (amphotericin B)
Candida glabrata	6.25	0.78 (amphotericin B)

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Molecular docking

Molecular docking is an effective tool to get an insight into ligand–receptor interactions and to screen molecules for binding affinities against a particular receptor.²² The molecular docking study showed that the compound bound well at the active site of the human estrogen receptor. Table 2 represents the induced fit docking studies of the compound with the human estrogen receptor. The oxygen atom of the co-crystal ligand interacts with the oxygen atom of the residue THR347 and the oxygen atom of GLU353 at a distance of 3.0 Å and 2.7 Å, respectively with the glide score of −9.33 and glide energy of −59.95 kcal mol⁻¹ (Fig. 8a).

Table 2 Induced fit docking studies of the compound with the human estrogen receptor

Compound	H-bond interaction D–H⋯A	Distance (Å)	Glide score	Glide energy (kcal mol^−1)
IOK (cocrystal)	N–H⋯O(THR 347)	3.0	−9.33	−59.95
	O–H⋯O(GLU 353)	2.7
Compound	O–H⋯O(GLU 353)	3.1	−6.05	−38.90
	N–H⋯O(GLU 353)	2.8
	(LYS449)N–H⋯O	3.0
	(LYS449)N–H⋯O	3.2

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Fig. 8 (a) Interactions of the co-crystal ligand (IOK) at the active site residues. (b) Interactions of the compound Schiff base at the active site residues.

In the MNMC compound under study, the oxygen interacts with the oxygen atom of the GLU353 and nitrogen interacts with another oxygen atom of the residue GLU353 at a distance of 3.1 Å and 2.8 Å, respectively. The two oxygen atoms of the compound interact with the residue LYS449 at a distance of 3.0 Å and 3.2 Å, respectively with the glide score of −6.05 and glide energy of −38.90 kcal mol⁻¹ (Fig. 8b).

Conclusion

A methyl 3-(E)-(2-hydroxy-1-naphthyl)methylidene]carbazate single crystal was grown by employing the slow evaporation solution growth technique. The cell parameters were analysed by single crystal X-ray diffraction analysis and powder X-ray diffraction analysis of the title compound which were correlated well with the already reported results. Spectral analyses of the compound were performed using FT-IR, FT-RAMAN and proton NMR and confirmed the formation of the title compound. The TG-DTA-DSC study revealed the purity of the compound with the sharp melting of the compound at 205 °C and it exhibit two stages of weight loss left with 12.27% residue of mass. From the UV-Vis-DRS studies, the band gap value was found to be 3.0 eV corresponding to the UV absorption edge of 405 nm with the maximum absorption peak at 360 nm. The electrochemical band gap value was calculated as 1.2 eV from cyclic voltammetry measurements which is less than the optical band gap value. MNMC showed a bluish-green emission with a broad peak emission at 495 nm under 365 nm of excitation. Hence, the title compound can be utilised for fluorescence applications. The title compound has very good antimicrobial activity against human pathogenic bacteria and fungi. The title compound forms a stable complex with human estrogen receptor which is evident from the ligand–receptor interactions and it may be an effective inhibitor of human estrogen receptor if further biological studies are carried out on the compound in consideration.

Acknowledgements

One of the authors, G. Gomathi, acknowledges the Centre for Research, Anna University, Chennai-25 for providing the Anna Centenary Research Fellowship (Proceeding no. CR/ACRF/JAN.2011/33). We acknowledge SAIF, IITM, Chennai – 36 for using Single crystal X-ray diffraction, FT-IR, FT-RAMAN, TG-DTA-DSC and fluorescence measurements. We also acknowledge Prof. D. Narayana Rao, Department of Physics, University of Hyderabad for extending his lab facility for SHG studies.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra04964d

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