Efficient synthesis of N-(ethylcarbamothioyl)-1-naphthamide: X-ray structure, Hirshfeld surface analysis, DFTs, and molecular modelling investigations as selective inhibitor of alkaline phosphatase

Abstract

Naphthyl Thiourea based derivative N-(ethylcaramothbioyl)-1-naphthamide (NA-MT) was synthesized by freshly prepared 1-naphthoyl isothiocyanate with ethyl amine to afford the products (NA-MT) high purity and characterized via spectroscopic techniques including FTIR, ¹H-NMR, ¹³C-NMR, elemental and HRMS analysis and single crystal X-ray diffraction. In-vitro analysis showed that the compound (NA-MT) possesses potent inhibitory effect with IC₅₀ = 9.875 ± 0.05 surpassing its reference inhibitor L-phenyl alanine (IC₅₀ = 80.2 ± 1.1) against cIAP. Additionally, the synthesized derivative (NA-MT) underwent an in-depth analysis of its electronic properties and reactivity using Density Functional Theory (DFT) calculations. Evaluations using SwissADME revealed the compound (NA-MT) possess acceptable physicochemical attributes, such as solubility and drug-likeness. Molecular docking studies demonstrated the compound (NA-MT) exhibit strong binding affinities to cIAP, which were further validated via Molecular Dynamics (MD) simulations. These integrated experimental and computational tools highlight the potential therapeutic uses of the synthesized compound, and pave the way for the development of novel pharmacologically active Alkaline phosphatase inhibitors with diverse applications.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s13065-025-01525-y.

Introduction

Alkaline phosphatase (ALP) refers to a family of isoenzymes responsible for catalyzing the hydrolysis of phosphate monoesters in alkaline pH environments [1], located on the exterior surface of the cell membrane and is distributed across various human tissues, including the liver, bone, placenta, kidneys, and intestine [2]. ALP participates in vital biological processes such as bone matrix mineralization, cellular signal transduction, and phospholipid metabolism. ALP measurements are frequently performed to assess liver and bone health [3]. In humans, they are classified into two basic types specifically based on localization i.e. tissue-specific AP (TSAP) and tissue nonspecific AP (TNAP) [1]. TSAP further divides into intestinal, placental, and germ cell APs, while TNAP exhibits high expression in bone, liver, and kidney tissues [4].

The current study primarily focused on intestinal type alkaline phosphatase. Intestinal alkaline phosphatase (IAP) is a brush-border enzyme found along the entire gastrointestinal tract [5], pivotal in mucosal defense of gut, maintaining its homeostasis, and regulating inflammatory mediators. IAP is Secreted by enterocytes, resides on the apical surface and detoxifies bacterial lipopolysaccharides (LPS), mitigating inflammation and tissue damage [6]. Reduced IAP levels are observed in gut-affecting conditions like inflammatory bowel disease (IBD) and type-2 diabetes [6]. IAP supplementation shows promise in treating these conditions by curbing inflammation and fortifying the gut barrier. Additionally, role of IAP in mineral metabolism regulation warrants further investigation, as optimal IAP levels are linked to normal bone functioning and cellular pathways [7].

Clinically, elevated level of intestinal alkaline phosphatase (IAP) is associated with a range of pathological conditions, including inflammatory bowel diseases [8], liver cholestasis, cirrhosis [1], various cancers such as cholangiocarcinoma and pancreatic adenocarcinoma [9]. Individuals infected with AIDS may experience elevated AP levels due to cholangiopathy resulting from infections like cytomegalovirus or cryptosporidiosis, or liver involvement in tuberculosis [10].

Previous research has highlighted the potential of naphthalene-based heterocycles, which hold considerable clinical significance. Naphthalene a bicyclic aromatic moiety is a component of various FDA accepted marketed drugs for instance, Tolnaftate, naphyrone, naftifine, nafcillin, propranolol, terbinafine, nafimidone, nabumetone, naproxen, lasofoxifene, duloxetine, bedaquiline [11]. The structure and function of some marketed drugs based on naphthalene is shown in Fig. 1. In addition, naphthalene ring is present in several bioactive molecules such as cardioprotective on the ischemia/reperfusion injury antimicrobial, anticancer, Inhibitors of SARS-CoV PLpro for the treatment of COVID-19 and KEAP1-NRF2 inhibitors [5–7]. Figure 1 shows examples of literature reported bioactive compounds containing naphthalene moiety.

Fig. 1.

Some marketed naphthalene-based drugs [5–7]

On the other hand, various acyl thioureas have attracted considerable attention in the past 30 years due to their distinctive clinical and pharmacological characters including analgesic, anti-asthmatic, antihypertensive, diuretic, anti-inflammatory, and anticholinergic [12–14].

Naphthoyl thioureas play a significant role in coordination chemistry. These derivatives feature two effective chelating motifs (C = S and C = O), which enhance their ability to encapsulate a variety of metal ions within their coordinating structures [14].

The extensive literature highlighting the medicinal importance of the naphthalene moiety, coupled with the expanding biological applications of thioureas, inspired us to create compounds that incorporate both pharmacophoric units into a single molecule. By utilizing a hybrid pharmacophore approach, we aimed to investigate their biological potential. The study aims to synthesize new naphthalene-based hybrid pharmacophore, targeting the IAP. Various computational approaches like Molecular docking, Ad-MET analysis, Density functional theory (DFT) analysis, and Molecular dynamic (MD) simulations were utilized to accurately assess the inhibitory potential of naphthalene-based hybrid pharmacophore against IAP and understand their interaction with its active site. Additionally, the ADMET profile was also analyzed for safety evaluation. Schematic representation of the suggestions behind the design of the N-(ethylcarbamothioyl)-1-naphthamide is given in Fig. 2.

Fig. 2.

Schematic Representation of the Design Concepts for N-(ethylcarbamothioyl)-1-naphthamide

Keeping in mind the extensive literature regarding the design of novel N-((substituted phenyl) carbamothioyl)-1-naphthamides analogs, a variety of substituent can be installed. We therefore synthesized a N-(ethylcarbamothioyl)-1-naphthamide and tested for on Alkaline Phosphatase inhibition assay.

Experimental

Materials and methods

Melting points were determined in open capillary using a digital Gallenkamp (SANYO) model MPD BM 3.5 apparatus and are uncorrected. Infrared spectra were recorded using a NICOLET 6700 purchased from thermoscientific and spectra were recorded using attenuated total reflectance (ATR) technique. ¹H NMR and ¹³C NMR spectra were determined at 400 MHz and 75 MHz using a Bruker AM-300 spectrophotometer using DMSO-d6 as solvent.

Synthesis of N-(ethylcarbamothioyl)-1-naphthamide (NA-MT)

Synthesis of 1-naphthoyl chloride

A solution of 1-naphthoic acid 1.14 g (5 mmol) and DMF (0.05 mL) in thionyl chloride 0.50 ml (7 mmol) was stirred under reflux for 3 h to afford corresponding acid chloride [15].

Synthesis of 1-naphthoyl isothiocyanate

A solution of potassium thiocyanate (5 mmol) in dry acetone (10 ml) freshly prepared suitably substituted acid chlorides (5.0 mmol) (2) was added drop wise using under inert environment for half an hours. Milky color of solution indicated the formation of naphthyl isothiocyanate intermediate (3). Reaction mixture was cooled at room temperature [16].

Synthesis of N-(ethylcarbamothioyl)-1-naphthamide

In final step a solution of ethyl amine (5.0 mmol) in dry acetone was added drop wise in the reaction mixture (3) and stirred for 8–9 h at 50 ^oC. Reaction progress was monitored by thin layer chromatography. After the reaction complete, the mixture was poured into crushed ice. The desire products (NA-MT).) Were precipitated as solids which was then filtered, washed with cold water, dried, and recrystallized from ethanol [16].

Characterization data of 1-naphthoyl chloride (2)

92%; R_f*: 0.87; IR (cm^-1): v = 3026 (Sp² CH Stretching Aromatic), 1780 (C = O), 1566 (C = C), 750 (C-Cl). Anal. Calcd. for C₁₁H₇ClO: C, 69.31; H, 3.70; found; C, 69.34; H, 3.72; HRMS (ESI): m/z Calcd for [C₁₁H₇ClO + H] ⁺ 190.0185, Found 190.0188.

Characterization data of yield synthesis of 1-naphthoyl isothiocyanate (3)

Yield: 82%; R_f*: 0.76; IR (cm^-1): v = 3052 (Sp² CH Stretching Aromatic), 1671 (C = O Amide), 1573 (C = C), 1246 (C = S). Anal. Calcd. for C₁₂H₇NOS: C, 67.58; H, 3.31; N, 6.57; S, 15.04 found; C, 67.56; H, 3.30; N, 6.56; S, 15.02 HRMS (ESI): m/z Calcd for [C₁₂H₇NOS + H] ⁺ 213.0248, Found 213.0251.

Characterization data of N-(ethylcaramothbioyl)-1-naphthamide (NA-MT)

Yield: 72%; M.P: 140 °C; R_f*: 0.57; IR (cm^-1): v = 3326 (N-H), 3046 (Sp² CH Stretching Aromatic), 2876 (Sp³ CH Stretching Aliphatic), 1671 − 1650 (C = O), 1563–1485 (C = C), 1244 (C = S). ¹H-NMR ((CD₃)₂SO, 300 MHz): δ 11.87 (s, 1H, NH), 11.47 (s, 1H, NH), 8.22–8.12 (m, 3H, Ar-H), 8.06–8.03 (m, 1H, Ar-H), 7.86–7.78 (m, 2H, Ar-H), 7.69–7.59 (m, 3H, Ar-H), 7.54–7.50 (m, 1H, Ar-H); 4.05 (q 2H CH₂), 1.93 (t 3H CH₃). ¹³C-NMR ((CD₃)₂SO, 75.5 MHz): δ 180.6 (C = S), 170.9 (C = O), 135.2, 133.4, 132.2, 131.9, 131.4, 130.0, 129.9, 129.6, 129.4, 27.4, 22.6. Anal. Calcd. for C₁₄H₁₄N₂OS: C, 65.09; H, 5.46; N, 10.84;; S, 12.41 found; C, 65.07; H, 5.42; N, 10.86;; S, 12.40 HRMS (ESI): m/z Calcd for [C₁₄H₁₄N₂OS + H] ⁺ 258.0827, Found 258.0830 Characterization spectra i.e. FTIR, ¹H-NMR and ¹³C-NMR data are given in Figures S1-S3 (supplementary file).

Alkaline phosphatase Inhibition assay

The inhibitory potential of the compound (NA-MT) against cIAP was assessed through a spectrophotometric method described in previous studies [17]. A reaction mixture was prepared with 50 mM Tris-HCl buffer at pH 9.5. cIAP of commercial scale was purchased from Calzyme laboratories (Catalog No:181A0300). A 10 µL sample of the compound was pre-incubated with 5 µL of CIAP (0.025 U/mL) for 10 min. To initiate the enzymatic reaction, 10 µL of 0.5 mM p-NPP (para-nitrophenyl phosphate disodium salt) was added, and the mixture was incubated at 37 °C for 30 min. The absorbance of the released p-nitrophenolate was measured at 405 nm using a Thermo Scientific Multiskan GO 96-well microplate reader. Each experiment was conducted in triplicate, and the inhibition activity and IC₅₀ values were determined through non-linear regression analysis using GraphPad Prism 5. L-phenylalanine was employed as a reference inhibitor for cIAP [18].

Density functional theory

Density Functional Theory (DFT) analysis is employed by the B3LYP method and 6-31G basis set to access the electronic properties of the molecule. The 6-31G basis set is justified in computational studies when a balance of accuracy and efficiency is desired, particularly for organic molecules and standard DFT calculations [19]. Comprehensive geometry optimization of the compounds was carried out using Gaussian 9w software [20]. For visualization purposes, GaussView 6 was employed to view the generated files as reported in our previous studies [21]. DFT studies provide thermochemical parameters derived from frontier molecular orbitals (FMO), including chemical hardness (η = (I - A) / 2), electron affinity (A = -ELUMO), chemical potential (µ = -(I + A) / 2), and chemical softness (σ = 1 / 2η). These parameters help in understanding the molecule’s stability and reactivity, with chemical hardness indicating resistance to changes in electron density, electron affinity reflecting the molecule’s ability to accept electrons, chemical potential representing the tendency to gain or lose electrons, and chemical softness quantifying the ease with which the molecule can gain or lose electrons [22].

Homology modeling

The homology modeling of calf intestinal alkaline phosphatase (cIAP) was conducted using a sequence database search, Uniprot. The protein sequence of cIAP was obtained from UniprotKB (https://www.uniprot.org/help/uniprotkb) with Swiss-Prot code P19111 and directly uploaded into the SwissModel server (https://swissmodel.expasy.org/). The server identified 2ef0.1.A as a suitable template with a high sequence identity of 99.34%. The selected sequence template was aligned with the target sequence and used for model building by SwissModel. Subsequently, the resulting model underwent assessment of its stereochemical properties through Procheck-Ramachandran plot analysis [23]. Following confirmation of its structural integrity, the model was deemed suitable for docking studies.

Molecular docking analysis

Molecular docking studies were performed to scrutinize the binding affinity between the synthesized derivatives and the targeted protein [24]. The three dimentional homology model structure of IAP was prepared as described in previous study [23]. The protein structure was organization using MGL tools. The structure of the synthesized derivative was drawn using ChemDraw 3D. MMFF94x force field was employed to minimize the energy of the synthesized compound [25]. The docking procedure was validated by initially separating the co-crystal ligand from the active site of the complex. Subsequently, re-docking was performed to assess and confirm the accuracy of the protocol [26]. The grid box dimensions were set for the active pocket were standardized as (x; 14.965582, y; 18.022452, z; 39.349934) for cIAP homology model. Docking analysis was accomplished on the cIAP model utilizing AutoDock, with the default genetic algorithm employed as the scoring function. Subsequently, the 100 different binding configurations of the compound (NA-MT) were tested. The configuration with the lowest binding energy, indicating the most stable arrangement, was chosen for further analysis in both 2D and 3D orientations. This approach aimed to elucidate the interactions between the synthesized compound and the targeted protein [27].

ADMET analysis

SwissADME (http://www.swissadme.ch/) online tool was utilized to predict the ADMET properties of synthesized compound, to assess the pharmacokinetic and toxicological profile [4]. Various physicochemical and pharmacological parameters were accessed. Using the radar charts and BOILED-Egg model, key ADME properties were particularly identified [23].

Molecular dynamic simulations

In Current study, the Nanoscale Molecular Dynamics (NAMD) software version 1.9.3 was utilized to predict the dynamic behavior of a protein-ligand complex [28]. The complex was prepared with the protein preparation wizard and optimized at pH 7.0. An orthorhombic box was created around the complex, and hydration was performed using the TIP3P water model [29]. The system was equilibrated at 300 K with 20,000 iterations using NPT and NVT ensembles [30]. Neutralization was achieved with 0.15 M counter ions. After equilibration, production was run for 100 nanoseconds under periodic boundary conditions. The final complex was analyzed for RMSF, and RMSD evaluation [31].

X-ray crystallography

The crystallographic data were collected using a Bruker AXS SMART APEX CCD diffractometer with Mo Kα radiation (λ = 0.71073 Å) at 130 K. A multi-scan absorption correction was applied using X-AREA software [32]. The structure was solved with direct methods and refined with full-matrix least squares against F² [33], using SHELXTL-Plus for analysis [34]. Non-hydrogen atoms were modeled with anisotropic displacement parameters, while hydrogen atoms were found in a difference Fourier map and refined with specific limits. O—H and C—H bond lengths were set geometrically, and hydrogen atoms were refined with specific Uiso values. Crystal structure drawings were generated using ORTEP-3 [35] and PLATON [36]. The crystallographic data for the structure described in this study have been deposited with the Cambridge Crystallographic Data Centre and are available as Supporting Information, CCDC No. 2,360,552. Copies of the data can be obtained through application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK. (fax: +44 1223 336033 or e-mail: deposit@ccdc.cam.ac.uk or at http://www.ccdc.cam.ac.uk).

Hirshfeld surface (HS) analysis

Hirshfeld surface (HS) analysis was carried out using Crystal Explorer 17.5 [37]. The intermolecular interactions of the crystal structure of the compound NA-MT were visulized. On the Hirshfeld surface, the distance to the closest nucleus inside is denoted as ‘di’, while the distance to the outermost nucleus is labeled ‘de’. The standard contact distance is represented by ‘d_norm’, which is color-coded in white, red, and blue described previously [38].

Interaction energy calculations

Intermolecular interaction energies were calculated using the CE–B3LYP/6–31G(d, p) model in Crystal Explorer 17.5 [39]. A cluster was generated within a 3.8 Å radius around a central molecule [40]. Total intermolecular energy (E_tot) is the sum of electrostatic (E_ele), polarization (E_pol), dispersion (E_dis), and exchange-repulsion (E_rep) energies with scale factors of 1.057, f.740, 0.871, and 0.618, respectively [41].

Results and discussion

Chemistry

The synthesis of 1-naphthoyl chloride (2), 1-naphthoyl isothiocyanate (3) and (NA-MT) are presented in (Scheme 1).

Scheme 1.

Synthesis scheme of N-((substituted phenyl)carbamothioyl)-1-naphthamide (NA-MT)

The FT-IR spectrum of 1-naphthoyl chloride (2) shows the disappearance of the OH band in the range 3450–3220 cm-1 and the appearance of carbonyl group bands at 1780 cm-1 (C = O) and 1244 cm-1 (C-Cl at 750 cm-1 is likely a separate peak). Elemental analysis of compound 2 revealed C, 69.31; H, 3.70, indicating the formation of 1-naphthoyl chloride. The HRMS spectrum showed a peak at 213.0251, further confirming the formation of compound (2).

The FT-IR spectrum of compound (3) displays typical peaks of particular carbonyl functional groups in the region 1671 cm-1 (amide) and 1244 cm-1 (C = S), confirming the synthesis of compound (3).

Characterization data

The FT-IR spectra of N-(ethylcaramothbioyl)-1-naphthamide (NA-MT) displayed a distinctive band for N-H stretching appear as broad absorptions in the range of 3326 cm -1. This feature is in agreement with previous reports on vibrational prop- erties of 1-acyl-3-monosubtituted thioureas, with the N-H stretching modes of the thioamide group being shifted to lower frequencies due to the formation of intramolecular N-H •••O = C hydrogen bond [16]. This interaction also affects the C = O stretching mode, which is observed as intense and well-defined band in the range 1671–1650 cm^− 1 [42]. The Napthyol group is characterized by the presence of aromatic C = C stretching frequencies are observed at 1563–1485 cm^− 1 and the C = S group absorbs at 1244–1230 cm -1. In 1 H NMR spectra, the two characteristic N-H protons signals appeared in the range of δ = 11.87 and δ = 11.47-ppm. The N-H proton connected to the electrophilic functional groups (carbonyl and thiocarbonyl) is more deshielded as compare to the N-H proton is connected to the thiocarbonyl moiety [43]. The low- f ield resonance observed for the thioamide proton is a signature for the occurrence of intramolecular N-H •••O = C hydrogen inter- action, as discussed below. The aromatic region protons were observed from δ = 8.06–7.50 ppm and the aliphatic region protons signals appeared in the range of δ = 4.05–1.93 ppm, as expected. In 13 C NMR, two differentiating signals of thiocarbonyl and carbonyl carbons appeared at δ = 180.6 and δ = 170.9 ppm, respectively. The difference in shift values between C = O and C = S groups is due to the presence of the NH groups that exert electron withdrawing effect. The aromatic carbon nucleus of the napthyol group resonates in the range ca. 135 − 129 ppm and the alkyl chains are observed at higher fields, as expected.

Alkaline phosphatase Inhibition assay

The cIAP inhibition bioassay was used to estimate the inhibitory potential of the synthesized compound (NA-MT). The results revealed that the compound NA-MT was found to be more potent than reference inhibitor i.e. L-phenylalanine with IC₅₀ 9.875 ± 0.05 µM and 80.2 ± 1.1 µM, respectively. The results shows that the compound NA-MT may prove as a potential inhibitor of Intestinal alkaline phosphatase as shown in Table 1; Fig. 3 and may serve as a good pharmacophore in future.

Table 1.

Enzyme inhibition potential of the compound NA-MT

Compound	CIAP IC50 ± SEM
NA-MT	9.875 ± 0.05
L–Phenylalaninea	80.2 ± 1.1

Reference. L–Phenylalanine^a,

Fig. 3.

IC₅₀ curves for the compound (NA-MT) were generated by plotting log concentrations against inhibition percentage

Density functional theory (DFT) calculations

In current study, we conducted lowest unoccupied molecular orbital (LUMO) and highest occupied molecular orbital (HOMO) analysis on N-(Ethylcarbamothioyl)-2-naphthamide (NA-MT) using DFT/B3LYP calculations with 6-31G basis set, as detailed in Table 2. This analysis is crucial for understanding various molecular properties such as chemical reactivity, molecular stability, and electrical and optical characteristics.

Table 2.

Global chemical reactivity descriptors in gas phase of NA-MT

Codes	NA-MT
Chemical Potential µ (eV)	-0.134
Electronegativity X (eV)	0.134
Hardness ƞ (eV)	0.070
softness S (eV-1)	7.162
Electrophilicity index ω (eV)	0.130
Optimization Energy	-1124.654
Dipole moment	4.986
Polarizability (α)	180.705
HOMO (Hartree)	-0.204
LUMO (Hartree)	-0.065
LUMO - HOMO (eV) *	3.78

* ΔE = 0.139Hartree×27.2114 eV/Hartree = 3.78 eV

The LUMO-HOMO band gap provides insights into a molecule’s reactivity. A smaller gap indicates lesser kinetic stability. Such molecules normally show HOMO–LUMO gaps between 2 and 6 eV depending on the level of conjugation and electronic distribution domination [44, 45]. Additionally, this energy gap also reflects the extent of intermolecular charge transfer along conjugated paths, from electron donor to electron acceptor groups. The HOMO energy signifies susceptibility to electrophilic attack, while the LUMO energy indicates susceptibility to nucleophilic attack. Electrophilic and nucleophilic regions were further heighted by calculating electrostatic potential (ESP) of the compound (NA-MT) as given in Fig. 4.

Fig. 4.

(A) Optimized structure of NA-MT, (B) Electrostatic potential of NA-MT, (C) LUMO and HOMO orbitals of NA-MT

Furthermore, the molecule (NA-MT) consists of a conjugated naphthalene ring attached to an amide group and a carbamothioyl group. The naphthalene ring, being aromatic and conjugated, played a crucial role in the electronic properties of the molecule. The HOMO is most likely localized on the π-system of the naphthalene ring, as the highest-energy electrons are typically found in the conjugated system. The ethylcarbamothioyl group, being an electron-donating group, may slightly influence the electron density on the aromatic ring, enhancing the HOMO’s localization in this region. Additionally, the lone pair of electrons on the sulfur atom in the carbamothioyl group could also contribute to the electron density distribution, making this region potentially involved in the HOMO.

On the other hand, the LUMO is likely to be localized on the carbonyl group (C = O) of the amide group and the sulfur atom in the carbamothioyl group, as these areas are electron-deficient and more prone to accepting electrons. The carbonyl group in particular, due to its electron-withdrawing nature, typically represents the site of lowest electron density and thus the primary region for LUMO localization. While the naphthalene ring could also contribute to the LUMO due to its conjugated system, the amide carbonyl and sulfur in the carbamothioyl group are more likely to dominate the LUMO characteristics as given in Fig. 4.

Homology modeling

Figure 5 illustrates a sequence alignment comparison between the target protein (cIAP) and a selected sequence. To assess the accuracy of the cIAP model, a Ramachandran plot was generated using PROCHECK, depicted in Fig. 6(A). This plot serves to validate and verify the homology model (cIAP), as displayed in Fig. 6(B). Notably, the suitable region of the model primarily occupies the first and third quadrants of the plot, affirming the reliability of the model. Additionally, the QMEAN server was utilized for further verification of the protein model, as depicted in Fig. 7 [23].

Fig. 5.

The sequence identity of template (2ef0.1) with cIAP model

Fig. 6.

(A) Ramachandran plot of cIAP (B) The homology model of cIAP generated using Swissmodel tool (https://swissmodel.expasy.org/)

Fig. 7.

Presents the absolute quality model for calf intestinal alkaline phosphatase. The dark zone indicates high-quality models (Z-score > 1), with the red marker representing a good model positioned within this zone

Molecular docking studies

Synthesized compound (NA-MT) was further evaluated for interactions within the active site of cIAP. The key amino acids involving in bonding interaction with their predicted binding scores are given in Table 3. The best configuration of complex was selected and analyzed further for 2D and 3D model building.

Table 3.

Docking profile of NA-MT within the active pocket of CIAP

Ligand Codes	Protein	Binding energy Kcal/mol	Hydrogen bond forming residues	Hydrophobic interactions residues
NA-MT	CIAP	-6.0	ALA285, LEU289	VAL65
L-phenylalaninea	CIAP	-6.0	VAL82, GLN79, ARG87	GLU53, VAL93

L–Phenylalanine^a, Reference

The detailed 3D and 2D binding interactions of the compound NA-MT within active pocket of cIAP is shown in Fig. 8. The amino acid residues involved in bonding interactions with NA-MT were ALA285, LEU289 and VAL65. Briefly substituted thiol moiety of parent compound was making strong hydrogen bond with bond length of 2.4 Å. Amino acid residue LEU289 was also involve in hydrogen bond interaction with the naphthalene ring and carbonyl oxygen of the compound NA-MT. Furthermore, it was observed that naphthalene ring of NA-MT was interacting with amino acid residue VAL65 via pi-alkyl linkage.

Fig. 8.

Tentative 3D (A) and 2D (B) binding mode of the compound NA-MT within the active site of CIAP model

In the docked conformation of the reference compound L-phenylalanine, several favorable interactions were observed. The stable complex formed with several amino acids, including VAL82, GLN79, ARG87, GLU53, and VAL93. L-phenylalanine exhibited a remarkable binding affinity of -6.0 kcal/mol and established three hydrogen bonds with cIAP. The terminal nitro groups and carboxylic acid group of L-phenylalanine contributed to the formation of conventional hydrogen bonds with VAL82, GLN79, and ARG87 given in Fig. 9. Additionally, interactions with GLU53 and VAL93 involved π-anion and π-alkyl, respectively. These findings were further validated through molecular dynamic simulations and in-vitro analysis.

Fig. 9.

Predicted 3D (A) and 2D (B) binding mode of compound L-phenylalanine and CIAP model

While both NA-MT and L-phenylalanine bind with the same affinity of -6.0 kcal/mol, their interactions within the active site of cIAP differ due to the unique set of amino acid residues and the distinct NA-MT and L-phenylalanine structures. As a small, naturally occurring amino acid, L-Phenylalanine also undergoes hydrophobic interactions with GLU53 and VAL93 which are commensurate with its polar head and aromatic side chain, forming hydrogen bonds with VAL82, GLN79, and ARG87. As a synthetic compound, NA-MT interacts through ALA285 and LEU289 using hydrogen bonding, while exhibiting hydrophobic interaction with VAL65. NA-MT differing set of interacting residues suggest that it adopts a different spatial orientation than L-phenylalanine and penetrates the binding pocket more deeply, likely due to the synthetic compound’s extended aromatic system and thiourea moiety, which are hypothesized to facilitate deeper binding. These structural differences are the reason behind distinct residue profiles, despite having the same binding energies, while also underscoring the lack of shifts on binding energies in terms of structure interactions.

ADMET analysis

The synthesized compound, NA-MT, was subjected to an assessment of its physicochemical properties using the SwissADME tool. Notably, the compound exhibited considerable oral bioavailability owing to its robust lipophilic characteristics. Table 4 provides a detailed overview of the compound’s comprehensive physicochemical and pharmacokinetic profiles.

Table 4.

Detailed ADMET profile of the compound NA-MT predicted via SWISSADME tool

Molecule	NA-MT
Formula	C14H14N2OS
MW	258.34
#Heavy atoms	18
#Aromatic heavy atoms	10
#Rotatable bonds	5
GI absorption	High
BBB permeant	Yes
Pgp substrate	No
CYP1A2 inhibitor	Yes
CYP2C19 inhibitor	Yes
CYP2C9 inhibitor	Yes
log Kp (cm/s)	-5.05
Lipinski #violations	0
Ghose #violations	0
Veber #violations	0
Egan #violations	0
Muegge #violations	0
Bioavailability Score	0.55
Brenk #alerts	1
Leadlikeness #violations	1
Synthetic Accessibility	1.71

Figure 10(A) illustrates a radar chart depicting the solubility of the compound, indicating a range from medium to moderate dissolution. Additionally, Fig. 10(B) presents the predicted targets for the compound NA-MT.

Fig. 10.

(A) Radar chart presentation of NA-MT, (B) Predicted Targets for the compound (NA-MT)

Additionally, the compound was also screened for its permeability studies by estimating boiled egg presentation as shown in Fig. 11. The analysis showed that the compound exhibited high lipophilic index and is not a substrate for p-glycoproteins (p-gp) resulting in good membrane permeability and enhanced bioavailability.

Fig. 11.

Predicted boiled egg presentation of the compound NA-MT

Molecular dynamic simulations

To determine the dynamic stability of the NA-MT and cIAP complex during the MD simulation, various parameters, including Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF), were accessed. From the RMSD plot (Fig. 12(a)), it is obvious that the NA-MT-cIAP complex demonstrates greater stability compared to the isolated apo protein and ligand, maintaining consistent conformational stability throughout the simulation with an RMSD of 1.87 Å. Notably, the ligand (NA-MT) exhibited the highest structural stability, with average RMSD values of 0.38 Å for the ligand and 2.26 Å for the apo protein.

Fig. 12.

(a) Predicted RMSD of ligand, apo-protein and ligand protein complex. (b) RMSF of apo-protein (cIAP)

The RMSF analysis presented in Fig. 12(b) indicates an average fluctuation of 0.98 Å, highlighting the dynamic flexibility of amino acid residues, which is essential for effective protein-ligand interactions. Importantly, the ligand (NA-MT) remained firmly bound within the protein’s active site throughout the simulation. The prominent peaks in Fig. 12(b) signify residues that experience significant vibrational oscillations, while the troughs represent residues that contribute to strong hydrophobic and hydrophilic interactions with the ligand. This data provides valuable insights into the dynamic behavior of the protein-ligand complex, highlighting key residues critical for stabilizing the interaction.

X-Ray structure

The experimental details are given in Table S1 (Supplementary file). In the molecule of the NA-MT (Fig. 13a), the planar, A (C1—C6) and B (C1/C6—C10), rings are oriented at a dihedral angle of A/B = 2.76(4)°. So, they are almost coplanar. Atom C11 is -0.1178(14) Å away from the best least-squares plane of ring B. Intramolecular N–H···O hydrogen bond (Table 5) forms an S(6) ring motif. In the crystal structure, intermolecular N–H···S hydrogen bonds (Table 5) link the molecules into centrosymmetric dimers, enclosing R₂²(8) ring motifs. Further, these dimers are linked through the bifurcated N–H···O hydrogen bonds into infinite double chains along the b-axis direction as given in Fig. 13b. Neither π···π nor C—H··· π interactions are observed.

Fig. 13.

(a) NA-MT compound with atom numbering; thermal ellipsoids at 50% probability. (b) Partial packing diagram along the a-axis, with N—H···O and N—H···S hydrogen bonds as dashed lines

Table 5.

Hydrogen-bond geometry of the compound NA-MT (Å)

D—H···A	D—H	H···A	D···A	D—H···A
N1—H1A···S1i	0.88	2.56	3.4243 (12)	166
N2—H2B···O1	0.88	1.98	2.6601 (15)	133
N2—H2B···O1iii	0.88	2.29	2.9980 (15)	137

Symmetry codes: (i) − x + 1, −y, −z + 1; (iii) − x + 1, −y + 1, −z + 1

The crystal structure of compound NA-MT, as shown in Table 6, confirms the stable geometry of the molecule through the specified interatomic distances.

Table 6.

Selected interatomic distances of the compound NA-MT (Å)

S1···N1i	3.4243 (12)	O1···H2B	1.98
S1···C9i	3.303 (2)	O1···H2Biii	2.29
S1···H13A	2.66	N1···H9A	2.71
S1···H1Ai	2.57	C2···C7iv	3.340 (4)
S1···H8Aii	2.95	C2···H13Biii	2.74
O1···N2	2.6601 (15)	C9···H1A	2.66
O1···C2	3.0069 (18)	C11···H2A	2.64
O1···N2iii	2.998 (3)	C11···H2B	2.47
O1···O1iii	2.985 (3)	H1A···H9A	2.36
O1···H2A	2.46	H2A···H14Av	2.37

Symmetry codes: (i) − x + 1, −y, −z + 1; (ii) − x, −y, −z + 1; (iii) − x + 1, −y + 1, −z + 1; (iv) x + 1, y, z; (v) − x + 2, −y + 1, −z + 1

Hirshfeld surface analysis

In Fig. 14a, the bright red spots on the HS plotted over d_norm indicate donor and/or acceptor roles. These spots correspond to blue and red regions on the HS mapped over electrostatic potential in Fig. 14b, representing positive and negative potentials, respectively. Figure 14c shows no π… π interactions in (NA-MT).

Fig. 14.

(a) 3D Hirshfeld surface of NA-MT over d_norm (-0.3188 to 1.5985 a.u.). (b) 3D Hirshfeld surface over electrostatic potential (-0.0500 to 0.0500 a.u., STO-3G, Hartree–Fock), with donors and acceptors in blue and red. (c) Hirshfeld surface over shape-index

The overall two-dimensional fingerprint plot of the compound is shown in Fig. 15a, and those delineated into H… H, H… C/C… H, H… S/S… H, H… O/O… H, H… N/N… H, C… S/S… C, C… C and C… N/N… C are illustrated in Fig. 15(b-i), respectively, together with their relative contributions to the Hirshfeld surface. The detailed description of 2D fingerprint plots is given in supplementary file. The geometrical parameters of molecule NA-MT obtained from X-ray diffraction (XRD) and Density Functional Theory (DFT) calculations showed good agreement, highlighting the stability and structure of the molecule. XRD results revealed nearly coplanar aromatic rings with a dihedral angle of 2.76° between rings A (C₁–C₆) and B (C₁/C₆–C₁₀), and atom C₁₁ is 0.1178 (14) Å from the best least-squares plane of ring B. Key hydrogen bond distances, such as N₁–H₁A···S₁ 3.4243(12) Å and N₂–H₂B···O₁ 2.6601(15) Å, confirm the molecular interactions. DFT calculations, using the B3LYP/6-31G method, produced optimized bond lengths and angles that align with the XRD data, further validating the structural stability. Additionally, the HOMO–LUMO gap calculated at 0.140 eV reflects the molecule’s chemical reactivity. Overall, the results from both XRD and DFT provide complementary insights into the molecular structure, with XRD offering physical bond lengths and DFT providing electronic reactivity details, demonstrating the robustness of the NA-MT structure.

Fig. 15.

a-i exhibiting the full two-dimensional fingerprint plots for the compound NA-MT

The nearest neighbor coordination environment of a molecule is determined by the color patches on the HS, indicating proximity to other molecules. Figure 16 (a-c) shows Hirshfeld surfaces with d_norm plotted for H… H, H… C/C… H, and H… S/S… H interactions, respectively.

Fig. 16.

(a-c) shows Hirshfeld surfaces with d_norm plotted for H… H, H… C/C… H, and H… S/S… H interactions, respectively

Hirshfeld surface analysis confirms the significance of H-atom contacts in packing. The numerous H… H, H… C/C… H, and H… S/S… H interactions indicate that van der Waals forces and hydrogen bonding are key factors in the crystal packing.

Crystal voids

The strength of the crystal packing is crucial for its response to applied mechanical force, depending on how robust the packing is. The detailed description of crystal voids is given in supplementary file. The volume of the crystal voids shown in Fig. 17 (a-c) and the percentage of free space in the unit cell are calculated as 80.39 ų and 12.31%, respectively, indicating no large cavities in the crystal packing.

Fig. 17.

Graphical views of voids in the crystal packing of the title compound. (a) along a-axis, (b) along b-axis and (c) along c-axis directions

Energy frameworks

Energy frameworks integrate the computation of intermolecular interaction energies with a visual depiction of their magnitude [46]. Interactions between pairs of molecules are depicted as cylinders connecting their centroids, with the cylinder’s radius reflecting the relative strength of the corresponding interaction energy. Three types of energy frameworks are generated: E_ele (represented by red cylinders), E_dis (green cylinders), and E_tot (blue cylinders) as given in Fig. 18 (a-c), respectively. Hydrogen bond energies for N1—H1A···S1: E_ele = -33.7, E_pol = -7.0, E_dis = -44.1, E_rep = 46.3, E_tot = -50.6 kJ/mol and for N2—H2B···O1: E_ele = -60.7, E_pol = -11.1, E_dis = -26.8, E_rep = 84.6, E_tot = -43.4 kJ/mol. Analysis of these frameworks suggests that electrostatic energy predominantly drives stabilization.

Fig. 18.

The energy frameworks of the compound (NA-MT) along the a-axis: (a) electrostatic, (b) dispersion, and (c) total energy. Cylindrical radii scaled equally at 80x with a 5 kJ mol⁻¹ cut-off within 2 × 2 × 2 unit cells

Conclusions

In this work, the new molecule N-((substituted phenyl)carbamothioyl)-1-naphthamide pharmacophore was designed and has been investigated for its inhibitory potential against calf intestinal alkaline phosphatase (CIAP). The structure of synthesized compound was screened through FTIR, ¹HNMR, and ¹³CNMR. In-vitro analysis showed that the compound NA-MT possess potent inhibitory effect with IC₅₀ = 9.875 ± 0.05 surpassing its reference inhibitor L-phenyl alanine (IC₅₀ = 80.2 ± 1.1). The DFT analysis shows that the reduced LUMO/HOMO gap and increased the dipole moment was the responsible of higher binding potency of compound NA-MT. Moreover, the docking study depicts their strong molecular interactions with binding pocket homology model of cIAP. The best binding mode was further analyzed via molecular dynamics simulation. These outcomes further indorse the ability of naphthalene-based conjugates to bind simultaneously to the active site of cIAP paving the way towards the rational design of diverse compounds. Additionally, the ADMET analysis predicted favorable pharmacokinetic properties such as high oral bioavailability, good membrane permeability, and the absence of P-glycoprotein interaction, suggesting potential for effective systemic absorption. The compound also demonstrated good human intestinal permeability. These findings collectively highlight the therapeutic potential of NA-MT as a promising lead compound for the development of selective inhibitors targeting alkaline phosphatase. The study, combining experimental and computational approaches, provides a solid foundation for designing more potent and effective inhibitors for clinical applications, especially in the treatment of conditions associated with altered alkaline phosphatase levels.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

A.S.; Methodology, and Investigations. P.A.C.; and S.A.E.; Supervision, Methodology, experimental material design. H.M.A.; Methodology, experimental material design, Investigations, Writing review & editing. S.A.C.; Investigation, Formal analysis. S.Q.; Methodology, Formal analysis, Q.A.; and U.F.; and T.H.; Investigation, experimental, Writing-review & editing.

Funding

This research was funded by University Higher Education Fund number CL/CO/A/8, King Khalid University, Saudi Arabia.

Data availability

All the associated data will be available on request from corresponding author.

Declarations

Ethics approval and consent to participate

Not Applicable.

Consent to publish

Not applicable.

Competing interests

The authors declare no competing interests.