(Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide as carbonic anhydrase inhibitor: exploration of its in vitro and in silico studies

Abstract

Human Carbonic Anhydrase inhibitors (CAIs) have been clinically used to treat a variety of disorders, such as cancer, obesity, haemolytic anaemia, glaucoma, retinopathy, and epilepsy. To develop a Carbonic Anhydrase inhibitor, Iminothiazoline analogue ((Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide) was synthesized and characterized. Single crystal X-Ray diffraction studies and Hirshfeld surface analysis (HSA) were conducted to find the exact molecular structure as well as intermolecular interactions. DFT Calculations indicated the soft and reactive nature of molecule. In-Vitro carbonic anhydrase inhibition studies showed the excellent inhibition potential of (Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide (IC₅₀ value of 0.147 ± 0.03 µM). Four hydrogen bonds and a multiple hydrophobic interactions were observed between synthesized molecule and the enzyme during Molecular docking studies. Molecular dynamic simulation studies showed that Protein–ligand complex generally remained stable throughout the time. ADMET studies suggested the need of structural modification for the drug like behavior of synthesized molecule.

Introduction

Carbonic Anhydrases (CA) belong to the family of metalloenzymes, categorized into six major classes i.e. α-, β-, γ- δ-, ζ- and η- class [1]. All human carbonic anhydrases (hCAs) lies within the α-class, which are characterized by the presence of a Zn(II) ion at the active site. Carbonic anhydrase (CA) isoforms display notable dissimilarities in terms of their molecular characteristics, subcellular localization, tissue and organ distribution, expression levels, and responsiveness to diverse classes of inhibitors. The α-class isoforms, namely h-CA I-III, h-CA VII, and h-CA XIII, primarily reside within the cytosolic compartment, contributing to intracellular pH regulation [2, 3]. The membrane-bound isoforms encompass h-CA IV, h-CA IX, h-CA XII, and h-CA XIV influence processes such as pH regulation, ion transport, and cell signaling across plasma membranes. Mitochondrial isoforms h-CA VA and h-CA VB are involved in particular metabolic pathways. This diverse array of CA isoforms, with the distinct cellular localizations and functionalities, collectively contributes to the intricate regulation of pH homeostasis and cellular processes in various organs and tissues [4].The remarkable ubiquity of this enzyme within the human body is evident through its presence in numerous tissues, including renal cortex, gastric mucosa, red blood cells, lungs, pancreas, central nervous system as well as various ocular tissues [5]. CA IX (and subsequently CA XII) have been demonstrated to play pivotal roles in the development of tumors, primarily due to their participation in the metabolic processes of hypoxic and acidic tumor environments. Their overexpression in tumor cells, resulting from the activation of the hypoxia-inducible factor-1(HIF-1) cascade, leads to the generation of H + and bicarbonate ions, the most basic metabolites derived from CO₂ as a substrate [6, 7]. Carbonic anhydrases are also responsible for the production of fatty acid in liver as well as adipocytes. Moreover, the role of carbonic anhydrase (CA3) expression in promoting the fat deposition during the process in which preadipocytes are differentiated to adipocytes is well studied [8]. In addition to this, CAs play role in the pathophysiology of obesity which is responsible for various comorbidities which includes nervous system disorders, cardiovascular diseases and metabolic complications. It has been established that carbonic anhydrase inhibiting drugs has an efficient potential of weight loss [9]. The side effect of significant weight loss in obese patients being treated for epilepsy with topiramate and zonisamide is interesting from a pharmacological perspective. It is believed that the strong anti-obesity effect of these drugs may be attributed to their potent inhibitory action on several carbonic anhydrase isozymes, such as h-CA II and h-CA VA/VB [10]. Thus, blockage of CAs is a significant issue for therapeutic drugs that can be used to treat cancer, glaucoma, edema, obesity, epilepsy, osteoporosis, and many other physiological complaints [11].

Among the various classes of compounds, heterocyclic compounds bearing thiazolidine structural core exhibit various pharmacological applications in the medicinal industries. These compounds displayed anti allergic [12], antimicrobial activity [13] and anti-inflammatory properties [14, 15], particularly, 3-methyl-2-thiazolidinimine significantly inhibited the indole ethylamine N-methyltransferase so can be employed for treatment of schizophrenia [16, 17]. Moreover, 2-imino-3-(benzoylmethyl)thiazolidine another thiazolidine, found to have an effective radioprotector activity towards γ-radiation [18, 19]. Previously, different amides and thiazolidine derivatives have been reported to have an excellent inhibitory activities against hCA isoforms [20] and among them 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamides showed excellent carbonic anhydrase Inhibition activity [21]. -23]. Different benzamide derivatives, which have been recognized as the most pharmacologically active class of CAIs and for the treatment of glaucoma, congestive cardiac failure, diuresis, epilepsy and altitude sickness, are given in Fig. 1 [22,23,24].

Fig. 1

Chemical Structure of biologically active heterocyclic compounds [22,23,24]

Based on the abovementioned data and as a continuation of our research interest in the field of synthesis and biological assessment of more potential inhibitor of carbonic anhydrase and medicinal importance of thiazolidine based benzamide derivatives, the current study was focused to synthesize new molecule with improved therapeutic potential. The synthesis of this compound was driven by a strategic approach to develop a potent and selective carbonic anhydrase (CA) inhibitor. The rationale involves the integration of diverse functional groups to enhance enzyme binding, specificity, and physicochemical properties.

The synthesized molecule (Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide was characterized by various chromatographic and spectroscopic techniques including NMR, FTIR and elemental analysis. Following the structural analysis, single crystal XRD was employed to get the detailed structural information. Hirshfeld surface analysis was carried out to characterize the intermolecular interactions. Density Functional Theory (DFT) studies were performed to get the optimized structure and quantum mechanical descriptors. After the estimation of geometrical properties, the molecular docking studies were performed where the compound was docked within the active pocket of various carbonic anhydrases to observe the binding interactions of the (Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide with targeted CAs. In order to support the in silico work, the in vitro studies were performed against CA-II where the results supported the in silico work. To study the ligand–protein interactions and ligand–protein complex stability molecular dynamics simulations were performed. At the end, ADMET Analysis was carried out for the assessment of drug like behavior of synthesized molecule.

Experimental

Materials and methods

Aluminum pre-coated silica gel plates Kiesel 60 F₂₅₄ was purchased from Merck. R_f value of the synthesized compound was determined by thin layer chromatography (TLC). Gallenkamp melting point apparatus was used for the melting point (M.P) determination by open capillary method. Bruker FT-IR Bio-Rad-Excalibur Series Mode No. FTS 300 MX spectrometer was used to record IR spectrum. The ¹H NMR and ¹³C NMR spectra were recorded on Bruker 300 MHz NMR spectrometer in deuterated chloroform, Acetone solvent with tetramethylsilane (TMS) as an internal standard. HPLC–MS analysis was performed by instrument LC system Agilent 1200 series. LECO-183 CHNS analyzer was employed for elemental analyses. Rigaku Oxford Diffraction Eos Gemini diffractometer was used to collect the single crystal XRD data (From QAU Islamabad) Rigaku Oxford Diffraction Eos Gemini diffractometer was used to collect the single crystal XRD data. ( Hacettepe University, Beytepe-Ankara, 06800, Turkey).

General procedure for the synthesis of (Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide

Thiourea (0.01 mol) was taken in the flask containing dry dichloromethane (30 mL), triethylamine (0.01 mol) was added to this solution. Dissolve 2,4-dibromoacetophenone (0.01 mol) in dry dichloromethane (7 mL) in a separate beaker, then added it dropwise through dropping funnel in the above reaction mixture under inert atmosphere. The reaction mixture was stirred at room temperature for 4–5 h. Reaction progress was monitored on TLC. Finally, the reaction mixture was filtered and filtrate was concentrated under reduced pressure. The crude product was recrystallized in ethanol to get the pure product.

(Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide 5

White solid, m.p = 172–174 °C, Yield = 74%, R_f = 0.46 (n-Hexane: Ethyl acetate 4:1); FTIR (cm⁻¹) 3375 (OH), 3098 (sp² CH), 2984 (sp³ CH),1682 (C = N),1464,1621 (C = C), 794 (C-S); ¹H-NMR (300 MHz, acetone-d₆): δ (ppm): 10.87 (s 1H OH), 7.89 (d, 2H, J = 7.2, 1.7 Hz, Ar–H), 7.87–7.51 (m, 8H, Ar–H), 7.37–7.35 (m, 3H), 3.25 (s, 2H, Thiazolidine ring-H), 2.92 (t, 2H, J = 9.5 Hz, aliphatic-H), 1.42 (m, 2H), 1.21–1.11 (m, 6H, aliphatic-H) 0.86 (t, 3H, J = 7.2 Hz, aliphatic-H); ¹³C-NMR (75 MHz, acetone-d₆):175.0 (C = O), 168.2 (C = N), 141.4, 139.8, 136.1, 136.0, 131.3, 131.1, 130.5, 128.9, 128.6, 128.2, 128.0, 127.5, 126.1, 107.5 (C = C, Aromatic), 39.8, 38.4, 31.1, 29.7, 23.1, 22.2, 13.4 (Aliphatic-C). Anal. Calcd.for C₂₉H₃₁BrN₂O₂S: C, 63.15; H, 5.67; N, 5.08; S, 5.81 found: C, 63.17; H, 5.69; N, 5.09; S, 5.82 HRMS (ESI): m/z Calcd for [C₂₉H₃₁BrN₂O₂S + H] ⁺ 550.1290. Found 550.1292.

X-ray crystallography

The data was collected on a Rigaku Oxford Diffraction Eos Gemini diffractometer using Cu K_α radiation (λ = 1.54184 Å). The data was solved and refined using SHELX [25].The position of Hydrogen atoms were optimized at distances of 0.84 Å, 0.95 Å, 0.99 Å and 0.98 Å for OH, CH, CH₂ and CH₃ respectively, riding model was applied for Hydrogen atoms by using the constraints of U_iso(H) = k X U_eq (C, O). Crystal structure was submitted in Cambridge crystallographic data center vide CCDC Deposition Number 2252779.Experimental details are given in Table 1 while Hydrogen bond symmetry are mentioned in Table 2.

Table 1 Experimental details

Table 2 Hydrogen-bond geometry (Å, º)

Symmetry codes: (i) − x + 2, − y + 1, − z + 1; (ii) x, y + 1, z. Cg2 is the centroid of ring B (C12-C17).

Computational studies

DFT studies

B3LYP is most commonly used functional now a days due to its accuracy and cost effectiveness. Because of their efficient energy convergence properties, the correlation consistent (cc) basis sets gained popularity. Moreover, these basis set provide a systematic route for calculation accuracy [27, 28]. cc-pVDZ is double zeta basis set developed by dunning and colleagues [29]. B3LYP/cc-pVDZ level of theory was used for DFT Studies due to better accuracy to cost ratio.

Geometry of the molecule was optimized using B3LYP/cc-pVDZ level of theory. DFT calculations were performed with the GAUSSIAN 09 software [30]. GAUSS VIEW 6 software [31] was used for visualization purpose. Based on quantum mechanical approach, electronic properties including Frontier Molecular orbital analysis and global chemical reactivity descriptors were calculated.

Molecular docking

The molecular docking technique is a useful tool for simulating the atomic level interactions between a protein and a small molecule. This approach is specifically useful in understanding the behavior of small molecules within the binding site of target proteins [32]. To perform the molecular docking, chemical structure of the molecule was sketched in ChemDraw 12.0 software in SDF format followed by energy minimization in Chem3D Pro 12.0 [33]. Autodock structure format (PDBQT) of ligand was obtained by using openbabel [34]. Protein structure intended to be used for docking was downloaded from protein data bank https://www.rcsb.org/ pdb id: 3HS4 accessed on March 15, 2023. Protein preparation was made using Auto Dock tool [35].Water molecules were removed and polar hydrogen were added subsequently. Gridbox dimensions of Co crystal ligand acetazolamide complexed with protein i.e., x = -5.406769, y = 3.078538, z = 15.029308 with size value 40 and exhaustiveness value of 20. Acetazolamide was docked along with AS-TU-BCC by using the Autodock Vina [36]. Binding energy and RMSD values were analyzed and visualization was made using discovery studio visualizer [37] and UCSF Chimera [38].

Carbonic anhydrase assay

In vitro carbonic anhydrase assay was performed with slight modification in already prescribed protocol [39, 40]. Reaction mixture consists of 120 µL of Tris-Sulfate buffer 50 mM, 20 µL of compound under study and 20 µL of bovine enzyme (50 U; from calzyme laboratories, 3443 Miguelito Ct, San Luis Obispo, CA 93401, United States) per well. Substrate (p-nitrophenyl acetate) after preparation was added in a way that final concentration of 0.6 mM per well could be achieved. Acetazolamide was taken as standard inhibitor. Absorbance was recorded at 348 nm using microplate reader. IC₅₀ values were calculated by applying nonlinear regression of GraphPad prism software version 5.0.

Molecular dynamic simulation studies

Crystallographic information provide compelling evidence about the impact of protein flexibility on ligand binding. Due to costly and laborious nature of conducting such studies, exploration of computational methodologies capable of predicting protein motions are becoming popular [41]. Molecular dynamics (MD) simulation is a computational technique used to analyze the temporal behavior of molecular interactions within docked complexes. The present study utilized the NAMD software version 1.9.3 to predict the dynamic behavior of a protein–ligand complex [42].

The complex exhibiting the highest binding energy, as determined by molecular docking analysis, was chosen for molecular dynamics (MD) simulations [43]. The topology files utilized for the simulations were created using CHARM-GUI 56. The molecular entities, including both ligands and proteins, were characterized utilizing the CHARMM36 force field parameters within the framework of periodic boundary conditions, as discussed earlier [44]. The molecule was solvated with water (TIP3P) at normal physiological conditions, and counter ions, specifically NaCl (0.15 M), were introduced to achieve neutralization of the simulation box. Before conducting the simulation, the system underwent equilibration in both the NVT (constant number of particles, volume, and temperature) and NPT (constant number of particles, pressure, and temperature) ensembles. This equilibration process involved the use of the Berenson thermostat and barostat, and was carried out for a duration of 2 femtoseconds at a temperature of 300 Kelvin. The root mean square deviation (RMSD) and root mean square fluctuation (RMSF) were computed using VMD 1.9.3, employing the Ewald summation technique 57. The molecular dynamics (MD) simulations were conducted for a duration of 100 ns.

ADMET studies

The process of drug development requires substantial investments of time, resources, and expertise to successfully bring a new drug from its initial concept to market availability while ensuring safety, efficacy, and adherence to regulatory guidelines. Assessing the pharmacokinetic profile of a drug at an early stage through the utilization of computational models is crucial in the lengthy and expensive drug development process. It allows for valuable insights into the drug's absorption, distribution, metabolism, and excretion properties, aiding in the optimization of drug candidates and the prediction of their behavior in the human body [45].In silico ADMET analysis was performed by widely used and reliable online webserver ADMET lab2.0 [46]. Smiles format was used for input structures.

Results and discussion

Chemistry

Iminothiazoline analogue was synthesized by reacting the potassium thiocyanate with octenal chloride 1 in dry acetone solvent followed by the addition of [1,1'-biphenyl]-2-amine to prepare the acyl thiourea 3. After purification, the acyl thiourea 3 was treated with p-bromophenacylbromide 4 to yield the (Z)-4-bromo-N-(4-butyl-3-(quinolin-3-yl)thiazol-2(3H)-ylidene)benzamide 5 as shown in Scheme 1. The reactions were carried out in dry solvents because isothiocyanate 2 undergo hydrolysis in the presence of moisture. The last step was carried out under nitrogen atmosphere to avoid side products, as discussed earlier [47].

Scheme 1

General scheme for the synthesis of (Z)-4-bromo-N-(4-butyl-3-(quinolin-3-yl)thiazol-2(3H)-ylidene)benzamide

Characterization of (Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide

The conformational analysis of these synthesized compounds was done by NMR spectroscopy and physical parameters. The n-Hexane: Ethyl acetate (20%) solvent system was used for the determination of R_f values. In ¹H NMR the characteristic signal for thiazoline ring proton appeared as a signlet at δ3.25 ppm. the aromatic protons appeared in the range of δ7.86–7.33 ppm and OH proton appeared as a signlet at 10.87. While aliphatic protons observed at δ2.86–0.89 ppm. In ¹³C NMR, the most deshielded signal for carbonyl carbon appeared at δ175.0 ppm.while the C = N (imine) group carbon signal observed at δ168.2 ppm. The characteristic signal for thiazoline ring carbon attached with hydrogen atom appeared at δ107.5 ppm.(¹H NMR, ¹³C NMR and IR spectra are given in supplementary information fig.S1 fig.S2 and fig.S3).

X-Ray structure

Crystal structure of the molecule consists of (Fig. 2) four rings A, B, C and D which are oriented at dihedral angles of B/C = 82.34(6)°, B/D = 87.49(7)° and C/D = 46.06(6)°.Ring A is in the envelope conformation with the puckering parameter of φ₂ = 316.2(6)°, where atom C3 is at the flap position at the distance of -0.2615(22)Å away from the best plane of the other four atoms. Bromine atom is at the distance of 0.0416(4)Å away from the least-square plane of ring B. The crystal structure of the molecule are linked into centrosymmetric dimers, enclosing R₂²(18) ring motifs.

Fig. 2

Thermal ellipsoids drawn at the 50% probability level

Hirshfeld surface analysis

Hirshfeld surface (HS) analysis as well as two dimensional fingerprint plots were carried out using the Crystal Explorer 17.5 [48]. Hirshfeld surfaces can be used to explore intermolecular interactions by using different colors and color intensity to indicate short and long contacts and the nature of these interactions (Fig. 3). The red circular depressions in the surface, which signify strong hydrogen bonding contacts, were created by mapping van der Waal's radii onto the Hirshfeld surface. The positive d_norm value, shown in blue on the Hirshfeld surface, denotes a distance greater than the sum of van der Waal's radii. The d_norm value is zero for intermolecular distances that are close to van der Waal radii. Negative d_norm values are shown in red signify a shorter distance than the van der Waal's radii [49].

Fig. 3

Three-dimensional Hirshfeld surface Visualization plotted over d_norm

The overall two-dimensional fingerprint plot are represented in Fig. 4a, [50] The major dominant interaction is H … H contributing 55.6% to the overall crystal packing, which is reflected as widely scattered points of high density due to the large hydrogen content of the molecule with the tip at d_e = d_i = 1.12 Å as shown in Fig. 4b.In the absence of C—H … π interactions, the pair of characteristic wings in the fingerprint plot delineated into H … C/C … H contacts (Fig. 4c, 17.1% contribution to the HS) has the tips at d_e + d_i = 2.77 Å. The pair of wings resulting in the fingerprint plot delineated into H … Br/Br … H, Fig. 4d, contacts with 9.5% contribution to the HS is viewed with the tips at d_e + d_i = 3.20 Å. The spikes of the H ··· O/O ··· H contacts (Fig. 4e, 5.0% contribution to the HS) have a symmetrical distribution of points with the tips at d_e + d_i = 1.75 Å. The H ··· S/S ··· H (Fig. 4f) and C ··· Br/Br ··· C (Fig. 4g) contacts have 4.2% and 2.4% contributions to the HS, and they are observed as the pairs of wings with the tips at d_e + d_i = 3.04 Å and d_e + d_i = 3.55 Å, respectively. Minor contribution to Hirshfeld surface includes C … S/S … C (Fig. 4h), C … C (Fig. 4i), N … S/S … N (Fig. 4j), H … N/N … H (Fig. 4k), O … S/S … O (Fig. 4l), Br … Br (Fig. 4m) and C … O/O … C (Fig. 4n) contacts with 0.9%, 0.9%, 0.6%, 0.5%, 0.4%, 0.2% and 0.2% contributions. These are represented as distributions of the scattered points of very low densities. The Hirshfeld surface analysis confirms the importance of H-atom contacts in establishing the packing. The large number of H … H, H … C/C … H and H … Br/Br … H interactions suggest that van der Waals interactions and hydrogen bonding play the major roles in the crystal packing (Fig. 5).

Fig. 4

Finger print plots of various interactions along with their percentage contribution to crystal packing

Fig. 5

The concentrations vs percentage Inhibition and IC₅₀ Curves (dose response) of a) (Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide and b) Acetazolamide by non-linear regression analysis using GraphPad Prism 5.0

DFT calculations

Density Functional Theory studies including structure optimization, calculations of quantum mechanical parameters and HOMO, LUMO orbital analysis were performed using the B3LYP/cc-pVDZ level of theory. Optimized Structure of molecule under study is given in Fig. 6 below.

Fig. 6

Optimized structure calculated at B3LYP/cc-pVDZ

Frontier molecular orbitals (FMO) analysis

FMO analysis is commonly employed to elucidate the optical and electronic properties of organic compounds. Understanding the characteristics of the Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO provides valuable insights into the chemical reactivity of molecules. During molecular interactions, the LUMO acts as an electron acceptor, with its energy corresponding to the electron affinity (EA), while the HOMO represents electron donors, and its energy is associated with the ionization potential (IP) [51]. Frontier molecular orbitals i.e. HOMO, LUMO analysis of studied compound revealed that highest occupied molecular orbitals has energy of -0.23307 a.u while the energy associated with LUMO is -0.05782 a.u.

It is evident from the Fig. 7 that maximum electron density is localized on bromine substituted phenyl ring and thiazolidine ring in LUMO. In case of HOMO, maximum electron density was observed on substituted thiazolidine ring and 1,1' -biphenyl ring directly attached with the hetrocyclic ring. Localization of electron density on thiazolidine ring specifies it importance in establishing the reactivity pattern of the molecule.

Fig. 7

A: LUMO B: HOMO visualization along with their energy gap

The energy gap between the Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO), E_LUMO-E_HOMO, plays a significant role in explaining the charge transfer interactions occurring within a molecule. Moreover, it serves as a critical factor in determining the molecular electrical transport properties. A molecule having large E_LUMO-E_HOMO energy gap requires significant energy input to facilitate the electron transfer process from lower energy level HOMO to higher energy level LUMO so exhibits low chemical reactivity and high kinetic stability and vice versa [52]. Calculated values of quantum mechanical parameters and Global reactivity descriptors calculated from HOMO and LUMO energy values are given in Table 4.

Table 3 Quantum mechanical parameters based electronic properties of the molecule

Lower value of energy gap (E_LUMO-E_HOMO) and hardness while higher softness value of studied compound indicates that the molecule under study is highly reactive and soft.

Molecular docking

Molecular docking, highly useful techniques for modern drug design, analyze the ligand conformations within the binding sites of receptors [53]. Information about the active pocket amino acid residues is important in structure based drug design. Active pocket amino acid residue of carbonic Anhydrase has been visualized in Fig. 8.

Fig. 8

3D visualization of Amino acid residue present in the active pocket of carbonic anhydrase at the distance of < 4 Å in the activation loop from the ligand molecule

Binding energies played considerable role in intermolecular recognition process can be estimated by molecule docking. Values of binding energies are given in Table 5.

Table 4 Binding energies calculated during molecular docking procedure

Molecular docking results of different type of carbonic anhydrases (CAs) i.e. h-CA-II, h-CA-IX, h-CA-XII and b-CA-II with (Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide suggested that the synthesized compound exhibited strong binding interactions with all the targeted proteins. Moreover, an interesting observation was made during analysis that this compound is the best potential candidate against carbonic anhydrase II rather than the other types of carbonic anhydrases. This comparison was observed in two different species i.e., human and bovine source. In all cases, the Ligand conformation having the minimal RMSD values were considered for binding interaction analysis.

Binding interactions of (Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide with h-CA-II

Within the active pocket of h-CAII, four hydrogen bonds were observed between CA and AS-TU-BCC. Briefly the sulphur atom of thiazolidine ring made one conventional hydrogen bond with amino acid ASN67 while the nitrogen atom of thiazolidine 2-imine formed second hydrogen bond with amino acid GLN92. The oxygen atom and hydrogen atom of hydroxyl group –OH attached directly with the thiazolidine ring was involved in hydrogen bonding with amino acid residues THR200 and PRO201 respectively. Hydrogen bonding involving the amino acid residues ASN67,GLN92,THR200 and PRO201 is crucial for binding stability of ligand–protein [54].

In addition to the hydrogen bonding, one π -Sigma interaction was found between phenyl ring and amino acid LEU198. Alkyl and π-alkyl interactions were observed between AS-TU-BCC and amino acid residues; HIS64, ILE91, VAL121, PRO202. It can be concluded that larger number of hydrophobic van der waals contacts played role in formation as well as stability of ligand–protein complex. In case of acetazolamide, hydrogen bond was formed involving the acetamide group of ligand and amino acid residue THR199.Carbonyl oxygen of acetazolamide was involved in the formation of hydrogen bond with amino acid residue THR200. Metal acceptor interaction was also observed between Zn atom of h-CA-II and nitrogen atom of acetamide group as shown in Fig. 9.Contrary to the reference inhibitor acetazolamide, no metal ion interaction was observed. This might be due to steric hindrance caused by bulkier thiazolidine ring.

Fig. 9

3D and 2D visualization of Protein–Ligand (synthesized derivative and reference standard) interaction

Binding interactions between protein and reference compound observed in molecular docking were similar as observed from the experimental XRD data retrieved from Protein data bank. This justifies reliability of docking procedure.

Binding interactions of (Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide with b-CA-II

Within the active pocket of b-CAII, hydrogen atom of hydroxyl group –OH attached directly with the thiazolidine ring was involved in hydrogen bonding with amino acid residues GLU234 while the oxygen atom of same group formed the corbon hydrogen bond with amino acid residue GLU232. Bromine atom made alkyl interaction with amino acid LYS166. In case of Acetazolamide, one hydrogen bond was formed between the acetamide group of ligand and amino acid residue GLY233. Two nitrogen atoms of thiadizole ring made two hydrogen bonds with amino acid residues ASN 228 and ASN230.Same ring was involved in the formation of π -anion interaction with amino acid GLU236.

Binding interactions of (Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide with h-CA-IX

Within the active pocket of h-CAIX, the oxygen atom of hydroxyl group –OH attached directly with the thiazolidine ring was involved in hydrogen bonding with amino acid residues THR201.Same amino acid residue was involved in the hydrogen bonding during the docking of reference ligand. THR 201 has been reported for its contribution in hydrogen bonding during the docking studies of CA-IX [55]. π-sulfur interaction was observed between sulfur atom of thiazolidine ring and amino acid HIS68. In addition to the hydrogen bonding, one π-Sigma interaction was found between phenyl ring and amino acid VAL121. Alkyl and π-Alkyl interactions were observed between AS-TU-BCC and amino acid residues; VAL130, LEU134, LEU140, LEU199, PRO203. The docking results are further supported by hydrophobic interactions revealed by CA-IX inhibitors involving the amino acid residues VAL121, VAL130,LEU134, LEU199 and PRO203 [56, 57].In case of Acetazolamide, four hydrogen bonds were formed involving the acetamide group of ligand and amino acid residues HIS94,HIS96,THR200 and THR201.Carbonyl oxygen of acetazolamide and nitrogen atom of thiadiazole ring was also involved in the formation of hydrogen bond with amino acid residue HIS68.

Binding interactions of (Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide with h-CA-XII

Within the active pocket of h-CAII, sulphur atom of thiazolidine ring made conventional hydrogen bond with amino acid ASP253 while the oxygen atom of hydroxyl group –OH attached directly with the thiazolidine ring was involved in hydrogen bonding with amino acid residue LEU26. Alkyl and π-Alkyl interactions were observed between AS-TU-BCC and amino acid residues; LEU25, LYS251, ARG255.In contrast to reference ligand, our synthesized derivative made week hydrophobic contacts with amino acid residues LYS251 and ARG255 anticipating its weaker activity against CA-XII.

In case of Acetazolamide, hydrogen bond was formed involving the acetamide group of ligand and amino acid residue LYS251.Carbonyl oxygen of acetazolamide was also involved in the formation of hydrogen bond with amino acid residue ARG255.

2D and 3D interactions of (Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide with b-CA-II, h-CA-IX and h-CA-XII are visualized in supplementary fig S4.

Carbonic anhydrase assay

The Table 3 represents a comparative analysis of the b-CA-II enzyme inhibitory activity exhibited by compound (Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide and the standard compound Acetazolamide. In vitro studies were conducted, and the IC₅₀ values were determined for both compounds, representing the concentrations required to inhibit 50% of the CA-II enzyme's activity. The synthesized compound exhibited more strong inhibition potential as compared to Acetazolamide with IC₅₀ value of 0.147 ± 0.03 µM, while acetazolamide exhibited an IC₅₀ value of 0.997 ± 0.13 µM. According to the stability studies the synthesized compound was found to have good properties that are required for the best drug like candidate. Further investigations, including in vivo experiments and clinical trials, are necessary to validate these findings and assess the therapeutic potential and safety profiles of these compounds.

Table 5 The comparative analysis of CA-II enzyme inhibitory activity of synthesized compound and the standard compound Acetazolamide

The graphical representation of results of in-vitro carbonic anhydrase inhibition studies are given in Fig. 5.

Molecular dynamic simulation

One of the key parameters for analyzing a protein–ligand complex is the measurement of the root mean square deviation (RMSD) of the protein backbone C-α atoms. RMSD values serve to characterize the overall conformational stability of the protein within a dynamic state during the simulation. The low RMSD levels with consistent fluctuations throughout the simulation indicate that the system has reached equilibrium and stability. Conversely, higher fluctuations are indicative of reduced stability. Pronounced deviations observed in the RMSD graphs suggest significant conformational transitions occurring in the protein to achieve a stable conformation with the bound ligand [58].

RMSD analysis

Upon the completion of simulation runs for 100 ns, RMSD Trajectories for protein, ligand and protein–ligand complex were analyzed. Data were manipulated in form of line plot which are given in Figs. 10 and 11. It is evident that after initial stability flipping occur at around 25 ns after that protein–ligand complex remained stable throughout. This depicts that ligand binding to protein structure does not induce any major conformational changes. RMSD analysis of protein shows that RMSD Value slightly deviates between 15 to 40 ns. After 40 ns it is equilibrated and showed a stable conformation. Ligand RMSD remained stable throughout the simulation time.

Fig. 10

RMSD in Angstrom vs Time in Nano seconds for protein and ligand–protein complex

Fig.11

RMSD in Angstrom vs Time in Nano seconds for ligand

RMSF analysis

RMSF analysis holds significant importance in identifying and characterizing the rigid and flexible regions within a protein structure. RMSF analysis enables the comprehensive assessment of flexibility in both the protein's backbone elements and the associated ligand, enhancing understanding of their dynamic behavior [59].

Line plot of RMSF Analysis for Residue No. vs RMSF is depicted in Fig. 12.

Fig. 12

RMSF analysis of protein back bone

Although higher RMSF fluctuations were observed from Residue No.34 to 41 at around 110 and 150 from the reference line, yet the overall RMSF results indicated the structural stability of protein.

ADMET studies

Drug-likeness parameters were selected and results were manipulated against the reference range for each selected parameter given on ADMETLAB 2.0 platform.

Although the range of molecular weight according to lipinski’s rule of 5 is upto 500 yet molecular weight lied within the acceptable range of 100–600.It is evident from Table 6 that the values for total polar surface area (TPSA), number of hydrogen bond donors (nHD), and number of hydrogen bond acceptors (nHA) lied with in the optimal range for drug-like molecule. However, range of aqueous solubility LogS, lipophilicity indicator LogP and typically octanol–water partition coefficient Log D deviated from the acceptable ranges. These solubility parameters which affects the absorption can be corrected by appropriate structural modifications.

Table 6 Physicochemical Properties

Human intestinal absorption HIA < 30% has been considered as HIA positive. Table 7 showed that the molecule has excellent human intestinal absorption. Volume of distribution lied within the reference range. However, molecule has plasma protein binding that is above the acceptable range. Proposed molecule can cross the blood brain barrier. Values for MDCK permeability and CACO-2 permeability lied with in the optimal ranges. (Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide behaved as an excellent P-glycoprotein inhibitor as is evident from Table 7.

Table 7 Absorption and Distribution

(Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide interacts with the cytochrome P450 (CYP) enzyme family up to different extent. It can act as an enzyme inhibitor of CYP2C9, CYP 2C19 and CYP3A4 while it does not inhibit the CYP1A2 and CYP2D6.Molecule under investigation showed a shorter half-life and low clearance rate as shown in the results given in Table 8.

Table 8 Metabolism and excretion

Computational toxicity studies (Table 9) showed that proposed molecule showed a good safety profile. AMES test which indicates the mutagenic potential of any compound is negative. Moreover, studied molecule showed moderate carcinogenic potential. Lipinski’s rule (RO5) was not followed by the synthesized molecule. A large number of drug molecules do not obey RO5 [60]. (Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4 bromobenzamide is extremely safe in terms of respiratory and ocular toxicity.

Table 9 Toxicity profile

Conclusion

(Z)-N-(3-([1,1'-biphenyl]-2-yl)-4-heptyl-4-hydroxythiazolidin-2-ylidene)-4-bromobenzamide was synthesized and characterized. Single crystal XRD Analysis including Hirshfeld surface analysis was carried out to explore the exact molecular structure as well as intermolecular interactions. Various physical and quantum mechanical parameters which play role in establishing the reactivity pattern of molecule were assessed by Density Functional Theory calculations. Efficient carbonic anhydrase inhibition was observed during in vitro enzyme inhibition assay. Molecular docking revealed the pattern of interactions of molecule with the enzyme. Results of Molecular Dynamic Simulation proved the stable protein–ligand complex. ADMET profiling suggested that synthesized molecule has an excellent safety profile and may act as drug like molecule by modifications to improve solubility parameters which may result in acceptable pharmacokinetic profile. It can be suggested that synthesized molecule can be used for the development of drug for the treatment of diseases linked with increased level of carbonic anhydrase i.e. glaucoma, diabetes mellitus, obesity etc.