Comprehensive theoretical and experimental analyses of 5-Bromo-2-Hydroxybenzaldehyde: insights into molecular stability and drug design

Abstract

5-Bromo-2-Hydroxybenzaldehyde or 5-Bromosalicylaldehyde has been thoroughly scrutinized both experimentally and theoretically. To perform its theoretical calculations, we have used the DFT (Density Function Theory) approach and the most suitable basis set 6-311 + + G(d, p) was implied for its optimisation. As a result, the optimised structure and its output file served the basis for other calculations which includes the study of different vibrations in a molecule (O-H, C-H, C-C, C = O) that are responsible for its stabilization. The electron density maps (MEP, ELF) with the specialized colour gradients were plotted and examined to explore the distribution of electrons within the molecule. UV-Visible studies were carried out in different solvents to analyse its absorbance and the effect of solvent on its wavelength. Electron transfers associated with the band gaps of FMO’s were inspected for the evaluation of its Ionization Energy, Electron Gain Enthalpy, Electrophilicity index etc. The variation of its thermodynamic properties with the temperature was studied to find out the reaction feasibility and direction of equilibrium. The type and strength of bonding present in it (RDG) was also surveyed. The hybridisation and deviation in the hybridised orbitals and angles (NBO) were examined to analyse the chemical stability of the taken molecule. After that, we delved into exploring the nature of different type of attacking sites (Fukui Function) i.e., neutral, electrophilic and nucleophilic along with hyperconjugation (NBO) present in the molecule. To dig deep into other properties like its skin permeability, bioavailability etc., its derivatives were also examined for Drug Likeness. Molecular docking and molecular dynamics simulations were also executed for the study of interactions between the molecule and different proteins. All the above-mentioned studies have shown comparable results with the experimental calculations, making the molecule suitable for the implication in pharmaceutical drug synthesis.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13065-025-01562-7.

Introduction

5-Bromo-2-Hydroxybenzaldehyde is an organic compound with molecular formula C₇H₅BrO₂. It is also known as 5-bromo salicylaldehyde (5-BSA). It can be easily synthesized by the bromination of salicylaldehyde at low temperature [1]. The investigated molecule can be implied in the synthesis of an important compound called Schiff’s base by reacting it with Aniline [1]. A variety of techniques have been adopted to synthesize numerous Schiff’s bases [2–4] with different transition metals complexes [3]. It’s azomethine (N = C) group have shown remarkable antibacterial properties [1]. Among other transition metals, Copper (Cu) have been reported to be biologically relevant element due to its applications in nucleic acid chemistry [5]. It’s metal (Cu) complexes were reported to be applicable in dye industry, food industry, catalysis, anti-inflammable activity, fungicidal, agrochemical and other biological activities [1]. In the organotin (IV) complex, the derivative of the molecule 5-Bromo-2-Hydroxybenzaldehyde-4,4-dimethylthiosemicarbazone [6], it shows Anticancer property against HCT 116 (Human Colon cancer Line). When the investigated molecule, based Schiff’s base forms Ru (II) complex, it showed an increased catalytic activity at 0.01 mmol catalytic concentration and were able perform transfer hydrogenation (TH) to a broad range of ketones in presence of KOH [7]. Schiff’s base synthesized from the titled molecule when complexed with amino acids were reported to have antifungal, antiviral and antitubercular activity as well [8]. Recent studies also focused on the biological properties of Schiff’s base formed by 5-BSA for their antidiabetic [9], antioxidant [10], and antimicrobial [11] activities.

After the study of relevant literature regarding the titled molecule, it was observed that despite having a wide range of applications in varied fields mentioned above, theoretical investigation on 5-BSA is limited. In 2013, Reza Takjoo [12] and group carried out DFT calculations in which the molecules derivative (5-bromo-2-hydroxybenzaldehyde-S-ethylisothiosemicarbazone) formed Cu (II) complex with imidazole. Following this, in 2016, Osman Asheri [13] group carried out investigations on the kinetic and thermodynamic properties of dimethyl-6-bromo-2H-chromene-2,3-dicarboxylate, by computational and experimental studies. The reaction was performed between triphenylphosphine, dimethyl acetylene dicarboxylate and 5-Bromo-2-Hydroxybenzaldehyde acid in the presence of dichloromethane. After that, the sensitivity of the Schiff’s base (based on 5-BSA) and its complexes with Ni (II), Co (II), Zn (II) and Cu (II) was investigated and the structure of Schiff’s base for its electronegative coordinating areas, and they were proved by DFT calculations done by Sambamoorthy Santhi in 2023 [14]. The DFT calculations included the optimized structure, Mulliken charges, energy gap between HOMO-LUMO and Molecular electrostatic potential map. Understanding the connection between molecular structure and biological activity is essential to modern medicinal chemistry in order to progress the creation of novel medications. Simulations using Density Functional Theory (DFT) offer important information about the stable geometry, vibrational characteristics, interaction mechanisms, and reactive sites of molecules. We can learn more about the interactions of biologically active chemical systems and how they relate to material science by using local reactivity descriptors. This will help us find novel therapies and drug design applications. By providing crucial tools for comprehending the behavior of active physical systems, spectroscopic techniques significantly improve our capacity to assess their structure and interactions. A popular computational technique in theoretical chemistry, Density Functional Theory (DFT) is frequently used to explore the electronic structures and characteristics of molecules and materials. An examination of the literature found that there had not been many previous quantum chemistry studies of the target compound’s molecular properties, which served as the impetus for this study.

Based on the literature review, it was observed that most of the experimental as well as theoretical studies were done only on the Schiff’s base obtained from the titled molecule 5-BSA and its other derivatives. Specifically, our primary molecule 5-BSA is yet to be analysed thoroughly. The B3LYP/6–311 + + G(d, p) level of theory was used for the DFT calculations since it has a track record of correctness and computing efficiency. A hybrid method that combines Hartree-Fock exchange with DFT exchange-correlation, the B3LYP functional provides accurate predictions for a variety of molecular systems. Furthermore, both polarization and diffuse functions are included in the 6–311 + + G(d, p) basis set, which is necessary for precisely representing electron density in remote areas and for efficiently simulating anionic species and non-covalent interactions. In this work, we performed computational calculations on the molecule by applying Density Function Theory - DFT approach under the basis set 6-311 + + G(d, p), to unveil its structure, its vibrational and spectroscopic properties such as UV-Visible and IR spectra as done previously for other molecules [15–22]. To have a better idea of its charge distribution, MEP and ELF were obtained. The ease of the transfer of electron within the molecule was studied with the help of Frontal Molecular Orbitals FMO namely HOMO and LUMO [23–30]. Its chemical reactivity and toxicity were monitored using the TD-DFT approach. Variation in thermodynamic properties were analysed with respect to change in temperature [31–35]. In addition to this, the tendency of the titled molecule for binding with specific proteins for the development of biologically active and pharmaceutically useful drugs is incorporated herewith.

Since many important properties need to explore for this valuable and wonderful molecule for further development in the field of medicinal chemistry, drug discovery and other fields therefore we are further digging different unexplored properties in this paper.

Computational details

Computational studies provide insightful information about the molecule, describing its structure, interactions as well as its stability. The optimisation and frequency of 5-Bromo-2-Hydroxybenzaldehyde was done by “GAUSSIAN 09 [36] and GAUSS VIEW 5 [37]” software, for which Density Function Theory - DFT, B3LYP approach was applied under the basis set 6-311 + + G(d, p) [38]. Further calculations include TD-DFT, for which only 3 excited energy states were taken. The MEP i.e., Molecular Electrostatic Potential, IR, ELF, UV graphs were plotted by using the “Multiwfn 3.8 programme” [39]. For the PED assignments i.e., Potential Energy Distribution, DD2 file was used which is obtained by the “VEDA4 programme” [40]. Thermodynamic calculations and the graphs were performed and plotted with the help of “Shermo calculator”- an application of Atomistic Online [41]. The Reduced Density Gradient (RDG) required RDG-calculator [42] and VMD software [43]. The Drug Likeness parameters were analysed using the SWISS ADME [44] tool.

For a deeper understanding of the chosen molecule, the UV-Vis graphs under 4 different solvents, including Gas, Chloroform, Methanol and Water were plotted and merged using the “Origin 2019 (b) software” [45]. The molecule was docked with different proteins using “Chimera” [46] software followed by its 2D visualizatio6n with the help of “BIOVIA Discovery Studio” [47]. Finally, for Molecular Dynamic Simulation, GROMACS [48] software was used.

Experimental details

5-Bromo-2-Hydroxybenzaldehyde, with the molecular formula C7H5BrO2, was obtained in solid form from Sigma-Aldrich, India, and used without further modification. The Fourier Transform Infrared (FT-IR) spectra of 5-BSA was recorded in the range of 4000–450 cm^− 1 using a Perkin Elmer Spectrum, equipped with a KBr beam splitter, and bearing the serial number 105,627. Additionally, the UV-Visible spectra of 5-BSA in chloroform was measured using a UV-1280 multipurpose instrument (UV-Visible Spectrophotometer) covering an ultraviolet range of 200–1100 nm.

Result and discussion

Optimisation

Optimisation refers to the technique in which the most stable structure having minimum energy of the particular molecule was examined. It is used to analyse the reactivity and other chemical properties of the titled molecule, 5-BSA. It has a C1 point group. Its optimisation was performed using the Gaussian 09 software [36] by B3LYP method, taking 6-311 + + G(d, p) basis set. Gauss View 5 [37] software was used to perfectly label and number the optimised figure.

Table 1 includes comparative study between the calculated and experimental data of the bond angles and the bond lengths of 5-BSA and Fig. 1 represent the structure obtained by optimisation. The correlation coefficient (R) value is 0.963 for the bond lengths and 0.945 for the bond angles. Similarly, the Coefficient of Determination (R²) value is 0.928 and 0.892 for the bond lengths and bond angles respectively. The (C-C) bond length from the experimental data ranges between (1.3–1.4) A^° and its calculated value resides in between (1.3–1.4) A^°. The (C-H) bond length in the experimental data was reported to be (0.9301 or 0.9302) A^°, while the calculated data ranges from (1.0–1.1) A^°. The bond angle of (C1-C6-C5) is 0.1°and (C4-C5-H9) is 2.1°. The (C-C-C) bond angle varies from (118.5°– 121.1°) in experimental and (119.2°– 121.4°) in calculated data. The (C-C-H) bond angle stretches from (119.3°– 120.1°) and (117.9°– 121.7°) in the experimental and calculated data respectively.

Table 1.

Calculated and experimental bond distance and bond angle parameters of 5-Bromo-2-Hydroxybenzaldehyde

Atoms	Theoretical Bond length (Å)	Experimental Bond length (Å)	Atoms	Theoretical Bond angle (°)	Experimental Bond angle (°)
C1-C2	1.3895	1.3695	C2-C1-C6	119.6	119.9
C1-C6	1.3957	1.3895	C2-C1-H7	119.9	120.1
C1-H7	1.0824	0.9302	C6-C1-H7	120.3	119.9
C2-C3	1.3971	1.3877	C1-C2-C3	120.3	121.1
C2-H8	1.0858	0.9301	C1-C2-H8	119.6	119.3
C3-C4	1.4056	1.4080	C3-C2-H8	119.9	119.4
C3-O13	1.3635	1.3450	C2-C3-C4	119.8	118.5
C4-C5	1.4017	1.3964	C2-C3-O13	121.9	--
C4-C10	1.4837	1.4652	C4-C3-O13	118.1	--
C5-C6	1.3835	1.3676	C3-C4-C5	119.2	120.0
C5-H9	1.0828	0.9301	C3-C4-C10	121.4	120.7
C5-Br15	1.9163	1.7452	C5-C4-C10	119.2	119.1
C10-O11	1.2128	1.2171	C4-C5-C6	120.2	119.7
C10-H12	1.1030	0.9302	C4-C5-H9	117.9	120.1
O13-H14	0.9631	0.8207	C6-C5-H9	121.7	120.1
			C1-C6-C5	120.5	120.6
			C1-C6-Br15	119.3	--
			C5-C6-Br15	120.1	--
			C4-C10-O11	123.4	--
			C4-C10-H12	115.6	--
			O11-C10-H12	120.8	--
			C3-O13-H14	110.1	109.4

Fig. 1.

Optimized structure of 5-BSA

From the Table 1 and the above discussion it is clear that the calculated results differ slightly from the experimental values. This is because, experiment was done in solid phase and the computational calculations were performed in gas phase. Both the values were quite similar showing their accurate prediction due to the chosen basis set [49].

Vibrational frequency analysis

The molecule has 39 fundamental vibrational modes, obtained by using the formula (3 N-6); where N is the total number of atoms present in the molecule. Due to different phases in experimental and theoretical calculations, we needed to compare the experimental FT-IR data with the theoretical data of 5-BSA. Figure 2 (a) has the experimental IR and the theoretical data is present in Fig. 2 (b). Being geometrically stable with no imaginary frequencies, the optimised structure is present at the lowest position in the potential energy surface [49].

Fig. 2.

(a) Experimental IR Spectrum (b) Calculated IR Spectrum of 5-BSA

The comparison between the experimental (FT-IR) and calculated data with their respective PED values, IR frequencies as well as the intensities is listed in Table ST1 (Supporting information). It also consists different types of vibrations like stretching vibration, bending vibration, torsional vibration along with their intensity percentage.

Due to the approximate analysis of different vibrational frequencies, the calculated values are slightly higher than the experimental values (Unscaled), therefore they were multiplied with the normalisation factor 0.961 to get the calculated (Scaled) values. The normalization factor 0.961 for scaled vibrational frequencies with the B3LYP/6-311 + + G(d, p) method corrects systematic overestimation due to the harmonic approximation. This factor, derived from empirical fitting to experimental data, ensures better agreement with observed frequencies. Scaling accounts for anharmonicity and basis set limitations, improving accuracy in predicted IR spectra. It was also observed that the molecule contains 14 stretching (α), 13 bending (β), 9 torsional (τ) and 3 OUT modes.

O-H vibrations

The molecule has one OH group which appears at the 39th vibrational mode at the 3684 cm^− 1 regions with PED assignment is 100% for the stretching vibration. The experimental value for the O-H single bond stretching frequency is reported to be around (3700 − 3600) cm^− 1 [50] which is close to our calculated value obtained by the B3LYP method.

C-H and C-C vibrations

The C-H vibrational frequencies usually appear between 3110–3000 cm^− 1 and that of the C-C vibrational frequencies occur at 1300–800 cm^− 1 [51]. The stretching vibrations of C-H single bonds attached to the aromatic ring are in between (3078–2861) cm^− 1 regions at the mode no 38, 37, 36 and 35 with (22%, 27%, 31%), (38%, 21%, 27%), (32%, 34%) and (25%, 25%, 25%, 24%) PED assignments respectively. On the other hand, C-C bonds having partial double bond character lie in the region (1568–1048) cm^− 1 at 33, 32, 30, 25 and 22 mode no. with (53%), (23%), (21%, 22%), (27%, 24%) and (29%, 31%) PED assignment respectively, including both symmetric and asymmetric vibrations. As per our calculation, we can conclude that the calculated result of the vibrational frequencies is reported quite close to the experimental values.

C = O vibrations

The experimental value of the C = O double bond stretching frequency ranges from (1600–1700) cm^− 1 [52]. Its calculated value reports to be 1687 cm^− 1 with 89% PED assignment lying within the prescribed range.

Molecular electrostatic potential (MEP)

It is a 3-D surface map in which the molecule is shown graphically on the basis of the colour gradient. It is used to analyse the nature of bonding present inside the molecule, its 3-D interactions as well as charge distribution [49]. The colour variation tells us the positive, negative and neutral electrostatic potentials [53]. It is also responsible for the determination of size and structure of the chosen molecule. From MEP diagram, we can easily analyse the reactive sites of the molecule and can choose a proper reagent for a particular reaction.

The MEP graph of the molecule is shown in Fig. 3. The colour gradient includes red, blue, white, yellow, green and orange colours. The reddish yellow or reddish orange colour at the periphery of the aldehyde group represents the area having maximum electron density. It is the most negative region in the molecule and is highly reactive towards the attack of an electrophilic. It is near the oxygen atom (electronegative atom) of the aldehydic group having a lone pair. Similarly, the blue colour shows the most positive center having minimum electron density. It is prone to nucleophilic attack shown near the hydroxyl group and the hydrogen atoms of the ring [54].

Fig. 3.

Molecular Electrostatic Potential (MEP) of 5-BSA

Other colours including the green, blue, white and orange have their own respective meanings. Green colour represents the area of zero potential while that of the yellow and white are the symbol of neutrality in the molecule [55]. The plot range of the MEP graph of the molecule was observed to be from − 8.047 × 10^− 2 to 8.047 × 10^− 2 a.u. Its potential order is red < yellow < green < blue [55]. Notably, the molecule’s MEP has a larger white region than others, which influences its reactivity.

Electron localization function (ELF)

For the investigation of chemical properties of a molecule based on the distribution of electrons in it, Becky and Edgecombe [56] introduced a technique that serves as a direct connecting link between the molecule’s electron density and chemical structure. This technique is called ELF [57]. From the ELF, can easily determine the position and number of electrons corresponding to the reference electron with a specific spin. It helps us to better understand electron nature in a nuclear system qualitatively [49]. ELF of the 5-BSA is shown in Fig. 4 (a) Contour filled map and Fig. 4 (b) Shaded surface map with projection.

Fig. 4.

(a) Colour Filled Map (b) Shaded Surface Map with Projection of 5-BSA

ELF value ranges between 0 and 1. The highest probability region of finding an electron approaches to 1 and the lowest probability region approaches 0. In case of homogeneity, the ELF value tends to 0.5 [58]. The electrons in a molecule can be localized or delocalized. If the electrons are localized, they are referred to as chemical bonds, lone pairs or atomic shells and as a result they will experience maximum electron repulsion. Similarly, the delocalized electrons will be having the minimum repulsion [49]. ELF graph gives crucial information about the reactivity, bonding, electron density etc. hence it is used for the analysis of aromaticity, quantitatively [59].

High ELF value is shown by red colour followed by yellow, green and blue, going from 1 to 0. Region near the Hydrogen atoms (having a single electron) and that of Bromine atom has a red colour patch with the highest ELF value close to 1, as seen in Fig. 4 (a). In Fig. 4 (b), ELF values and electron densities were represented by different projections. A decent amount of the portion in the map is of blue colour showing the delocalization of the electrons in the molecule.

UV-Vis analysis

The titled molecule was studied both experimentally as well as theoretically in different solvents for its static and dynamic features. The experimental calculations were done in Chloroform, while that of the theoretical calculations were performed in 4 different solvents, Methanol, Gas, Water as well as Chloroform. For the chosen molecule only 3 excited energy states were calculated, as its UV falls into this range. Furthermore, we have calculated for 15 excited states separately as listed in Table 2 (b).

Table 2a.

Comparison of electronic properties of 5-Bromo-2-Hydroxybenzaldehyde attained experimentally and calculated by TD-DFT B3LYP method with 6-311 + + G(d, p)

	Experimental		TD-B3LYP/6–311 + + G(d, p)
	λmax (nm)	Band Gap (eV)	Gas phase λcal (nm)	Band Gap (eV)	Energy (cm− 1)	Oscillator Strength	Assignments
Gas			330.37	3.7529	30269.17	0.0001	H-1→L (86.18%)
			286.78	4.3232	34868.94	0.0804	H→L (90.34%)
			228.11	5.4352	43837.82	0.0	H→L + 4 (29.38%)
Chloroform	343	3.61	320.37	3.8700	31213.64	0.0002	H-1→L (87.05%)
			293.63	4.2225	34056.74	0.1180	H→L (92.59%)
			236.60	5.2403	42265.85	0.1616	H-2→L (81.09%)

Table 2 (a) manifests the experimental and theoretical analysis of the molecule. The calculated spectrum for Water, Gas, Methanol and Chloroform is present in Fig. 5 (a) and the spectrum of chloroform obtained experimentally, is present in Fig. 5 (b). Both the graphs were plotted and merged by using “Origin 2019 (b) programme” [45]. The experimental value observed of the maximum wavelength (λ_max) for Chloroform is 343 and 258 nm and the computed values are 303 and 251 nm. From Table 2 (a), λ_max for gas is 330.37, 286.78, 228.11 nm and for chloroform, it is 320.37, 293.63, 236.60 nm for first three excited states respectively, we have further calculated 15 excited states to see the proper transition and found that the highest oscillatory strength (0.2868) at 223 nm for the transition HL + 1. Other solvents including methanol and water have their respective values.

Table 2b.

Comparison of electronic properties of 5-Bromo-2-Hydroxybenzaldehyde attained experimentally and calculated by TD-DFT B3LYP method with 6-311 + + G(d, p)

	Experimental		TD-B3LYP/6–311 + + G(d, p)
	λmax (nm)	Band Gap (eV)	Gas phase λcal (nm)	Band Gap (eV)	Energy (cm− 1)	Oscillator Strength	Assignments
Chloroform	343	3.61	338.93	3.6581	29504.55	0.0001	H-1→L
			327.66	3.7839	30519.19	0.0856	H→L
			248.39	4.9915	40259.14	0.1773	H-2→L
			244.15	5.0782	40958.42	0.00	H→L + 2
			240.19	5.1618	41632.7	0.0003	H-3→L
			227.91	5.4401	43877.34	0.1811	H-4→L
			223.16	5.5559	44811.33	0.2868	H→L + 1
			217.71	5.6950	45933.25	0.0013	H→L + 3
			215.50	5.7534	46404.27	0.0001	H-1→L + 1
			204.69	6.0572	48854.59	0.1060	H→L + 5
			204.49	6.0630	48901.37	0.0092	H→L + 4
			198.77	6.2377	50310.42	0.0005	H-2→L + 2
			198.01	6.2614	50501.57	0.1198	H-1→L + 2
			192.49	6.4411	51950.95	0.00	H→L + 6
			192.46	6.4419	51957.4	0.0236	H-3→L + 1

Fig. 5.

(a) Computed UV of 5-BSA in Gas, Chloroform, Methanol and Water, (b) Experimental UV graph of 5-BSA in Chloroform

As per the above discussion, we can conclude that the experimental results are very much aligned with the computed values obtained by DFT method. Also, the values obtained from different solvents shows a meagre effect on the wavelength and other parameters. This indicates that the optical activity of 5-BSA was not much affected by the solvents.

Frontal molecular orbital (FMO)

FMO refers to the molecular orbitals HOMO and LUMO, that participate in bonding or interaction with a foreign entity. HOMO stands for the Highest Occupied Molecular Orbital and LUMO means Lowest Unoccupied Molecular Orbital. TD-DFT calculations results in different parameters listed in Table 3. It includes Chemical hardness, Softness, Electronegativity, Electrophilicity index etc. The diagrammatic representation of the HOMO -LUMO energy gap from + 3 to -3 is provided in Fig. 6. In the transitions from H→L; H-1→L + 1; H-2→L + 2 and H-3→L + 3 the Energy gap increased regularly as 7.1, 9.02, 9.61 and 10.35 eV respectively.

Table 3.

Calculated energy charge (eV) of 5-Bromo-2-Hydroxybenzaldehyde by B3LYP/6–311 + + G(d, p) method

Parameter (eV)	Charge
EHomo	-8.24
ELumo	-1.14
Ionization potential	8.24
Electron affinity	1.14
Energy gap	7.10
Electronegativity	4.69
Chemical potential	-4.69
Chemical hardness	3.54
Chemical softness	0.14
Electrophilicity index	3.09

Fig. 6.

Frontal molecular orbitals (FMO)

The band gap of HOMO (-8.24 eV) and LUMO (-1.14 eV) is 7.1 eV which indicates a good electron transfer, thereby increasing the stability of the titled molecule [60]. The values of Ionization energy (8.24 eV) and Electron Affinity (1.14 eV) are the required energies for the excitation of an electron from HOMO, and addition of an electron in LUMO, respectively. The calculated electronegativity is 4.69 eV, which tells us the extent to which an electron is attracted to a particular atom in a covalently bonded system. The parameter named Chemical Hardness is majorly responsible for the stability and reactivity of the molecule. It measures the disinclination of the molecule towards the change, taking place in the electron density and charge distribution among the molecule [58]. The value of Chemical Hardness is reported to be 3.54 eV and that of Chemical softness is 0.14 eV.

Among all the parameters, most important is Electrophilicity Index. It predicts the toxicological behaviour of the molecule. For any drug, if its value is near or less than 3, then it is assumed to have low toxicity [61]. As a result of our calculations, the reported value of the electrophilicity index is 3.09 eV for the titled molecule, due to which it is very less toxic and is suitable for medicinal use.

Thermodynamic properties

Laws of thermodynamics help us to examine the feasibility and direction of the reaction as well as the molecular interactions. Calculation for the thermodynamic properties such as Enthalpy (H), Internal Energy (U), Gibbs Free Energy (G), Entropy (S), Specific Heat Capacities Cv (at constant volume) and Cp (at constant pressure) was done using the above-mentioned method [60]. The graphs of U, G, H and S, Cv, Cp against temperature were plotted with the help of “Shermo calculator” - application of Atomistica online [41]. The temperature ranges from 0 K to 1000 K at an interval of 100 for the titled molecule for U, G and H; while that for the S, Cv and Cp, the temperature varies from 100 K to 1000 K. The variations in energies mentioned above are listed in Table ST2 (a) and (b), and the graphical representation is in Fig. 7 (a) and (b).

Fig. 7.

Thermodynamic Properties variation with temperature. (a) U, G, H (b) S, Cv, Cp

It can be seen from Fig. 7 (a) that the values of U, G and H is equal to -1.878 × 10⁶ kcal/mol at 0 K. On increasing the temperature, the value of the U and H was increased while that of G was decreased. In Fig. 7 (b), S, Cv and Cp have different values at 100 K but their behaviour is similar. All of them increased with increasing temperature.

High value of Entropy (S) leads to more randomness in the system and more negative value of Gibbs free Energy (G) leads to spontaneity. We can conclude that the trends observed from both the Fig. 7 (a) and 7 (b) favours the thermodynamic stability for the titled molecule 5-BSA with changing temperature.

Reduce density gradient (RDG) analysis

RDG helps us to identify the Non-Covalent Interactions (NCI) of a particular molecule in low density regions [62]. The RDG analysis is implemented on the titled molecule 5-BSA and can be seen in Fig. 8. To obtain the variation between RDG value and the product of sign(λ₂) and density ρ, we used an online RDG calculator [42] which is an application of Atomistica Online. Multiwfn 3.8 [39] programme followed by VMD [43] was used to run the file. To calculate the RDG value we used the following formula:

Fig. 8.

Reduced Density Gradient (RDG)

1

From the graph obtained, we can infer that the regions where λ₂ is negative, shows stabilized non covalent interactions, while the positive value indicates repulsive interactions. The stability of the molecule is determined by its binding strength which can be evaluated by the value of the product (sign(λ₂) x ρ).

In Fig. 8 it can be studied that for 5-BSA, the order of binding strength is H-bonds, greater than Van der waal interactions followed by steric effect present in ring/cage. The H-bonds and halogen bonds were present in the highest density region and have a blue colour gradient. The value of (sign(λ₂) x ρ) ranges from − 0.02 to -0.06 a.u. Van der waal interactions were represented by green colour in which λ₂ and ρ are similar and range from − 0.02 to 0.0 a.u. The red colour region is again a high-density region in which the steric repulsion including steric effect of ring/cage is the strongest and ranges from 0.01 to 0.05 a.u. approximately.

Molecular Docking

It refers to the technique and study of 3D structure obtained by the ligand-protein interactions [63]. It tells us the extent to which our chosen molecule (ligand), binds with the particular protein (receptor) at specific active sites. The site where the residues are located inside the protein and gets replaced by the ligand while docking are called Active Sites.

Swiss ADME Target prediction [64] was used to choose proteins and RCSB-PDB was used to download their PDB files [65], according to the type of ligand present inside their structure [66]. The software used for docking were UCSF Chimera [46] and Autodock Vina [67]; both of them are free and open sources to use. The titled molecule was docked with nine different proteins which are listed in Table 4 along with their respective binding scores. The binding score is proportional to the binding strength of the molecule with that protein.

Table 4.

Hydrogen bonding and molecular Docking with centromere associated protein inhibitor protein targets

S. No.	Protein PDB ID	Residues	H -Bond Distance (Å)	Binding Energy (kcal/mol)
1	4YVP	3	1.915, 2.243, 2.236	-6.0, -6.1, -6.2
2	4JQ2	3	2.347, 2.073	-6.0
3	4XO7	3	2.485, 2.296, 1.950	-6.0
4	3C3U	3	2.373, 2.506	-5.9
5	3NTY	3	2.497	-5.9
6	2HDJ	3	1.994, 2.201	-5.9
7	3LJ2	3	2.127	-5.7
8	3LJ1	3	1.835	-5.6
9	2RIO	3	1.835	-5.2

Ligand has been taken as optimized structure and further minimize with chimera with adding charges as gasteiger to the ligand. And protein first allow to dock prep where further gasteiger charges given to the protein and hydrogen were added to the system. The final step for docking was autodock vina where the final docking was done.

The protein docked with the highest (negative) binding score is of 4YVP. It was done thrice for better results and obtain a binding score of -6.1, -6.2 and -6.0 kcal/mol respectively. The coordinates of the marker for the highest score of -6.2 kcal/mol were with the center_X = -53.5039, center_Y = -0.629104, center_Z = 42.5389 and the grid box size (Å) 24.1802, 18.2186, 12.9322 with respect to X, Y and Z axis. 3D representation of top 8 proteins with the highest binding score were shown in Fig. 9 and their 2D visualization is present in Fig. 10, obtained by BIOVIA Discovery Studio [47]. The H-bond distance between the ligand and the protein were observed as 4YVP (2.326 A^°), 4JQ2 (2.347, 2.073 A^°), 4XO7 (2.485, 2.296, 1.950 A^°), 3C3U (2.373, 2.506 A^°), 3NTY (2.497 A^°), 2HDJ (1.994, 2.201 A^°) tabulated in Table 4.

Fig. 9.

3D interaction of 5-BSA with the proteins (a) 3C3U (b) 3NTY (c) 4YVP (d) 2HDJ (e) 4JQ2 (f) 4XO7 (g) 2RIO (h) 3LJ1

Fig. 10.

2D Visualization of 5-BSA for the proteins (a) 3C3U (b) 3NTY (c) 4YVP (d) 2HDJ (e) 4JQ2 (f) 4XO7 (g) 2RIO (h) 3LJ1

Drug-likeness

For the development of a pharmaceutical drug, the molecule5-BSA was studied thoroughly, based on the parameters listed in Table 5. It includes HBD, HBA, BBB-permeant, Bioavailability, TPSA, GI Absorption value, MR value, Lipinski Violation, log Kp value and CYP1A2 inhibitor value. The calculation for these parameters was governed by certain rules including Lipinski rule, MMRD rule, CMC 50 rule etc.

Table 5.

ADME properties of 4-bromosalicylaldehyde and other derivatives of salicylaldehyde

Derivatives	HBD	HBA	MR	TPSA A2	GI absorption	BBB permanent	CYP1A2 inhibitor	log Kp (cm/s)	Lipinski violations	Bioavailability Score
5-Bromo Salicylaldehyde	1	2	41.55	37.30	High	Yes	Yes	-6.40	0	0.55
5-Chloro Salicylaldehyde	1	2	38.86	37.30	High	Yes	No	-6.18	0	0.55
3,4-Dibromo-2-hydroxybenzaldehyde	1	2	49.25	37.30	High	Yes	Yes	-5.91	0	0.55
4-Bromo Salicylaldehyde	1	2	41.55	37.30	High	Yes	Yes	-6.40	0	0.55
3-Fluoro Salicylaldehyde	1	3	33.81	37.30	High	Yes	No	-5.96	0	0.55
3-Bromo Salicylaldehyde	1	2	41.55	37.30	High	Yes	Yes	-5.91	0	0.55

From Table 5, the values of HBD and HBA are 1 and 2 respectively, and are true according to the Lipinski rule (HBD < 5 and HBA < 10) having zero violation for the titled molecule. The value of TPSA should be less than 140 A² [68]. For the chosen derivatives of our molecule, it is 37.30 A². The MR value should ideally vary from 40 to 130 [68] and for the titled molecule, it is from 33.81 to 49.25. The BBB permeant is YES having a HIGH GI absorption value with skin permeability log Kp as − 6.40 (cm/s) with a bioavailability score of 0.55.

As per the conclusion, having a high value of skin permeability and bioavailability, the molecule and its derivative shows high likeness to be used as a useful pharmaceutical drug.

Natural bond orbital (NBO) analysis

It is a method used to assess the transfer of electron from Donor to Acceptor orbital i.e., from the filled (bonding) to empty (antibonding) orbital [69]. The interaction between the acceptor and the donor orbitals required a “Fock Matrix analysis by second order perturbation theory”. Table ST3 (a) lists the parameters evaluated by Fock matrix. It includes the perturbation energies and the donor-acceptor interactions. The given formula is used to calculate E2:

5

(F_ij)² - Fock matrix element.

Ɛ𝛔^*- Antibonding NBO.

Ɛ𝛔 - Bonding NBO.

n_𝛔 - S (donor) orbital population.

From Table ST3 (a), higher E2 values of the transitions from (C1-C2)𝝅→(C3-C4)𝝅^* and (C5-C6)𝝅^*; (C3-C4)𝝅→(C1-C2)𝝅^*, (C5-C6)𝝅^* and (C10-O11)𝝅^*; (C5-C6)𝝅→(C1-C2)𝝅^* and (C3-C4)𝝅^* are 23.22, 15.53, 16.38, 24.05, 18.32, 23.68 and 14.99 kcal/mol respectively results in a significant amount of conjugation within the titled molecule making it more stable [70]. Further study of the NBO provides us with the information of Natural Hybrid Orbitals (NHO), listed in Table ST3 (b) and ST3 (c). It includes the participation of Hybridisation and Polarisation in the orbitals and the deviation from symmetrically most stable structure.

From Table ST3 (b); the bond orbital 𝛔 C1-C2 in Hybrid (A) contains s (35.54%) p (64.41%) d (0.05%) characters with SP^1.81 and in Hybrid (B) it has SP^1.72 with s (36.70%) p (63.26%) d (0.04%) characters. The C_A and C_B for 𝛔 C1-C2 shows the electronegativities of C1 and C2 atoms respectively. Similarly, the same type atoms having different electronegativities are because of the neighbouring atoms attached with them in the optimized structure.

𝛔cc = 0.7058 (SP^1.81) + 0.7084 (SP^1.72).

For the lone pairs listed in Table ST3 (b), LP (1) O11 has s (59.18%) character and p (40.81%) with SP^0.69. LP (2) O11 with SP^1.00 has 99.93% p character. Since, LP (2) O11 has more p character than LP (1) O11, so it is more directional in nature.

From Table ST3 (c), the 𝛔 C1-C2 bond angle is slightly deviated from its Hybrid 1 by the factor of 1.4 and in case of 𝝅 C1-C2, it is deviated from its Hybrid 2 by a large factor of 90. The 𝛔 O11, 𝝅 O11, 𝛔 O13, 𝝅 O13, 𝝅 Br15 and 𝛅 Br15 are not at all deviated from their natural geometrical alignments in their Hybrid 1 and 2. From the above discussion we can conclude that the slight changes in geometrical parameters occurred due to the optimization of the titled molecule 5-BSA.

Fukui function and population analysis

It refers to the investigation of dipole moment, electronic charges, polarization of the molecule as well as its reactivity which is based on the atomic charges and their distribution within the molecule [55]. According to the electron density and radical formation, the molecular sites are considered as electrophilic, nucleophilic and neutral in nature. This can assist the type of reaction carried out by the molecule. Moreover, NMR shifts can also be determined by Fukui function analysis [57].

Above mentioned properties for the molecule(5-BSA) were investigated, for which we used the DFT approach under the above-mentioned basis set. Population analysis was done using Mulliken charges. Table ST4 includes the atomic charges, Fukui function and dual descriptors obtained by the NBO analysis in different charged systems of 5-BSA [54]. Three components of charges were calculated, including cationic (N + 1), anionic (N-1) and neutral (N) [70]. In case of neutral system, the multiplicity is singlet while that in cationic and anionic systems, it becomes doublet. For the calculation of the Fukui functions, we used the following formulas:

6

7

8

Fr⁺ is used to represent the Electrophilic attack, Fr⁻ denotes the Nucleophilic attack and Fr⁰ indicated the radical attack [55]. The change in the nucleophilic and electrophilic attack was calculated by (Δ Fr = Fr⁺ - Fr⁻). For electrophilic attack the Δ Fr should be less than 0, and for nucleophilic attack, Δ Fr should be greater than 0. In our molecule, C10 and C6 are the electrophilic centres with Fr⁺ (-0.1744 and − 0.0743 a.u.). This is because, C10 is attached to O11 of aldehydic group and C6 is attached to the Br15 (showing -I effect). Thus, we can conclude that the investigation of Mulliken atomic charges and Fukui functions of 5-BSA results in determining the nature of the molecule based on the electron density on different atoms.

Molecular dynamics simulation

After successfully conducting the molecular docking of the molecule with different proteins, we have finalised the protein 4YVP due to its best binding energy with 5-BSA. To further analyse its ligand-protein interaction, Molecular Dynamics Simulation (MD) was performed, in which we have used our docked ligand-protein complex. Gromacs [48] software was used to perform MD simulations and the CHARMM27 force field parameters were used to develop the protein topology file. Similarly, the ligand topology files were developed by Swiss Param online portal [71]. Force fields were employed for the modelling of both bonded and non-bonded interactions and to unveil the dynamics and stability of biomolecules [72]. To validate the complex stability, a 50 ns simulation was used for 2,50,000 steps in total. Water molecules were represented by the TIP3P water model and for further calculations, counter ions like Na⁺ and Cl⁻ were added to neutralize the complex and to balance the charge.

First step in MD simulation is to minimize the energy of the ligand, which was conducted in two phases. It was done by varying the time between 1 and 10 ns for 50,000 steps in each phase, with the steepest descent algorithm. In the first phase, minimization was done by taking the number of particles (N), Volume (V) and the temperature (T) as constants, i.e., the NVT step; and in the second phase, particle number (N), Pressure (P = 1 atm) and Temperature (T = 298 K) were taken as constants i.e., NPT step. Post completion of this step, the Gromacs in-built functions determined various parameters like RMSD– Root Mean Square Deviation, RMSF– Root Mean Square Fluctuation, Rg– Radius of Gyration as well as the ligand-protein H-bonding represented in Fig. 11.

Fig. 11.

: (a) RMSF (b) RMSD Protein (c) RMSD Ligand (d) Rg– Radius of Gyration (e) H-bonding between ligand and protein 4YVP (f) T.Protein Binding Energies (g) Solvent Accessible Surface Area

For 5-BSA, the binding energies for T. Protein-Ligand complex was found to be maximum around 300 K temperature and the solvent accessible surface area was average around 150 nm². The average radius of gyration for total and around axes (Rg) was 1.9 nm, and the Rgx, Rgy and Rgz was 1.5, 1.6 and 1.55 nm respectively. The calculated average value for RMS fluctuation was found to be at 0.1 nm and the RMSD value for ligand after the least square fit to protein and backbone was reported to be near 0.4 nm. Lower values of RMSF and RMSD favours stabilization for the ligand-protein complex [49]. On further analysis, it was observed that around the time frame of 5ns, 9 H-bonds were found and after the stabilization of the complex, 2 H-bonds were reported from 20 to 50 ns. It confirms the stability of our complex and is in agreement with the docking results [72]. For further verification, we have also conducted a 100 ns simulation of our molecule with the same target. The results complemented with that of 50 ns and are represented in Fig. 12. Hence, 5-BSA has proved to be a compatible molecule to be used in different pharmaceutical applications due to its astounding binding results with proteins.

Fig. 12.

(a) RMSF (b) RMSD Protein (c) RMSD Ligand (d) Rg– Radius of Gyration (e) H-bonding between ligand and protein 4YVP (f) T.Protein Binding Energies (g) Solvent Accessible Surface Area

Conclusion

As per the present study conducted on the molecule 5-BSA, it can be concluded that the theoretical calculations largely supported the experimental results. The DFT method used for the optimisation resulted in the most stable structure of the labelled molecule. The bond lengths and bond angles have a high similarity of around 90% with the experimental results. The presence of (OH) hydroxyl group and an aldehydic group (CHO) proved to enhance its stability by possessing Intra and Inter-molecular Hydrogen bonding. The Vibrational stretching frequencies and PED of different bonds (C-H, C-C, C = O) were evaluated using VEDA4 programme and were found to be in agreement with the experimental range. Multiwfn 3.8 programme was used to obtain the colour coded electron density maps i.e., MEP and ELF, that showed astounding results with precise charge distribution inside the molecule. The UV -Vis calculations result in having similar wavelengths and absorbance despite of being performed in different solvents (gas, methanol, water, chloroform). This concludes that the optical activity of the molecule remained unaffected by the solvents. The ground state energy band gap ( H ◊ L ) of 7.1 eV makes the molecule biologically active and enables a high charge transfer within itself. The chemical hardness and softness of 5-BSA were 3.54 and 0.14 respectively, which makes it more stable. The toxicity parameter i.e., Electrophilicity index, is 3.09 due to which it has proved to be less toxic and can be used for medicinal purpose. The trends in the variation of thermodynamic energies with temperature was also observed to favour the stability of our molecule. The type of non-covalent interactions and the order of their strengths within the molecule were evaluated using the reduced density gradient (RDG). H-bonds interactions were best observed in 5-BSA, followed by van der waal interactions and steric effect. Fukui functions were examined to find out the nature of attacking sites within the molecule. C10 and C6 were observed to be electrophilic centres having Fr⁺ -0.1744 and − 0.0743 a.u. respectively. The presence of an electron withdrawing group makes them more prone to the nucleophile attack. The electronic transition due to hyperconjugation, the hybridisation of the atomic orbitals and the deviation in hybrid orbitals and angles were well studied with the help of NBO analysis. The high energy transitions were reported to be from (C1-C2)𝝅→(C3-C4)𝝅^* and (C5-C6)𝝅^*; (C3-C4)𝝅→(C1-C2)𝝅^*, (C5-C6)𝝅^* and (C10-O11)𝝅^*; (C5-C6)𝝅→(C1-C2)𝝅^* and (C3-C4)𝝅^* with 23.22, 15.53, 16.38, 24.05, 18.32, 23.68 and 14.99 kcal/mol of energy respectively. This results in better conjugation and more stability of our molecule. The deviations observed in the bond angles were negligible and hence, the molecule was well optimized under the basis set 6-311 + + G(d, p). The comparable study of the chosen molecule with its derivates was done to evaluate its tendency to be used in drug synthesis. As a result, all of them found to have High GI absorption value, BBB permeant YES, 0.55 bioavailability score with no Lipinski violations. It was concluded that the molecule has a very good skin permeability which makes is suitable for the pharmaceutical use. Molecular docking and simulation results in the proven interaction of the molecule with the protein 4YVP having the best binding with a lowest binding energy of -6.1 kcal/mol. It has been the key factor in uplifting the applicability of the molecule in drug synthesis.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

Mohammad Shaheer: Writing– original draft, Naveen Garg: Writing– original draft, Ana Aftab: Validation, Investigation, Himanshu.: Visualization, Analysis, Iram Jan: Analysis, Shakeel Alvi: Analysis, Akhilesh Kumar: Proofreading, Visualization, Nazia Siddiqui: Writing– review & editing. Saleem Javed: Conceptualization, Software, Supervision. P. Divya: Analysis, Validation, Mudassar Shahid: Data curation, Analysis.

Funding

The authors acknowledge and extend their appreciation to the Ongoing Research Funding Program (ORF-2025-1005), King Saud University, Riyadh, Saudi Arabia for funding this study.

Data availability

All the data used present in the manuscript or supplementary material.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.