Novel piperazine–triazole hybrids as PTP1B and alpha-amylase inhibitors: synthesis, biological evaluation and computational validation through network pharmacology, molecular docking, MD simulations, DFT and MMPBSA analysis

Abstract

The inhibition of α-amylase and PTP1B is essential for controlling hyperglycemia and improving insulin signaling. In this study, a series of piperazine conjugates were synthesized in 61–78% yields. All compounds were biologically screened against α-amylase to assess their anti-diabetic potential. Among these, compounds with Cl and OMe substitutions showed exceptional enzyme inhibition with IC₅₀ values of 2.39 ± 0.41 µM and 2.97 ± 0.31 µM, respectively, higher than standard acarbose (10.30 ± 0.20 µM). Further network pharmacology study was conducted, which spotted PTP1B as an important target to improve insulin signaling. Molecular docking analysis of potent compounds was performed to evaluate interactions of inhibitors with target receptors (PTP1B & α-amylase). Both compounds validated the in vitro protocols by showing excellent binding affinities −10.5 kcal mol⁻¹ and −9.7 kcal mol⁻¹ against α-amylase, surpassing standard acarbose (−8.1 kcal mol⁻¹). Likewise, both of them exhibited a similar binding pattern with binding affinities of −8.0 kcal mol⁻¹ & −7.8 kcal mol⁻¹ against PTP1B, even better than the reference acarbose (−7.4 kcal mol⁻¹). DFT studies were carried out to determine electronic properties, which were corroborated through NCI analysis. Furthermore, 500 ns MD simulation, binding free energy calculations, PCA, DCCM and FEL analysis were carried out to evaluate thermodynamic stability.

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1 Introduction

The WHO (World Health Organization) has identified noninfectious disorders such as obesity and diabetes mellitus as major causes of worldwide health crises.^1,2 Diabetes mellitus refers to persistent metabolic disease marked by elevated blood glucose (hyperglycemia) stemming from inadequate insulin secretion.^3–7 According to the IDF (International Diabetes Federation), this disease has affected more than 463 million people worldwide, which is predicted to rise to 578 million in the next five years.^8,9 This situation amplifies the threat of critical issues such as retinopathy,¹⁰ heart failure,¹¹ strokes¹² and nephropathy.¹³ Diabetes can be managed by controlling the blood glucose level through the suppression of carbohydrate-degrading enzymes such as α-amylase.^14–17 The α-amylase breaks down the starch into smaller subunits such as maltose and glucose. In diabetics, higher α-amylase activity can accelerate the breakdown of carbohydrates, resulting in a rise in blood glucose levels.^18–20

Similarly, PTP1B (Human Tyrosine Phosphatase-1B) is considered an important target against type II diabetes mellitus.^21–24 In the human body, PTP1B reduces leptin and insulin signaling and has demonstrated involvement in inflammation responses. PTPIB dephosphorylates IRS-1 (Insulin Receptor Substrate-1) and IR (Insulin Receptor), causing insulin resistance.^25–27 Therefore, suppression of this protein can improve insulin resistance in the body.²⁸ Enzymatic and in silico studies predict that synthesized inhibitors can interact with the active site to suppress the catalytic efficiency of α-amylase and PTPIB. The inhibition of α-amylase restricts the conversion of starch to glucose, resulting in a reduction of glucose absorption in the blood. Likewise, the inhibition of PTP1B results in the sustained insulin receptor phosphorylation and improved Akt/IRS-1/PI3K signaling (Fig. 1).^29–33

Fig. 1 Anti-diabetic dual action of synthesized compounds via enzyme inhibition and insulin signal amplification.

There are several drugs, such as acarbose 1 (containing valienamine fragment (with 1S,2S,3R,6S configuration) alongside three sugars with configurations 2R,3S,4S,5R,6R,2R,3S,4R,5R,6R, and 2R,3S,4R,6R^34–36), voglibose 2 ^37–39 (1S,2S,3S,4S,5S configuration) and miglitol 3 (2R,3R,4R,5S configuration),^40,41 that have been widely used for the inhibition of α-amylase, which are accountable for interrupting the hydrolysis of larger units (disaccharides and oligosaccharides) into smaller monosaccharide units. Despite their clinical usage, there are multiple adverse consequences, such as gastrointestinal distress, diarrhea, flatulence, and meteorism, resulting in discontinuation of treatment.⁴² Hence, to ensure effectiveness, they are frequently utilized together with other antidiabetic agents. Therefore, it is essential to identify new strategies for the management of diabetes with no or low potential for adverse effects.^43–45

Heterocycles containing nitrogen are an important class of organic compounds due to their high pharmacological and biological importance.^46–48 Notably, piperazine, an important six-membered heterocycle having two nitrogen atoms at the 1 and 4 positions, has a major impact in pharmaceutical research.^49–51 This moiety is found in a plenty of renowned medications with broad pharmacological effects, such as antidepressants, cardio-protectors, antianginals and antihistamines. Different piperazine derivatives 4 & 5 also displayed anti-diabetic potential. A piperazine moiety containing drug teneligliptin 6 has been approved as an oral DPP-4 inhibitor in some countries, for the treatment of type-II diabetes (Fig. 2).^52–54

Fig. 2 Previously reported anti-diabetic agents and current design strategy.

Additionally, 1,2,4-triazoles are globally acknowledged as important pharmacophores and have been widely investigated in antidiabetic drug design. Recent literature revealed that several triazole derivatives i.e., derivative 7 (IC₅₀ = 0.53 ± 0.14 nM) and derivative 8 (IC₅₀ = 0.97 ± 0.47 µM) exhibited anti-diabetic potential against α-amylase.^55,56 Moreover, 1,2,4-triazoles have also been explored against multiple anti-diabetic targets such as PTP1B (9, IC₅₀ = 6.45 µM) and DPP-4 (10, IC₅₀ = 1.50 nM), highlighting their wider scope in the treatment of diabetes.^57,58

The fragment-based drug design (FBDD) is considered an important strategy for the synthesis of biologically important hybrids. In this study, the FBDD approach was used to merge distinct pharmacophores such as piperazine, triazole and benzofuran into a single molecule to attain improved therapeutic effect.^59,60 The newly designed hybrids were biologically evaluated against α-amylase. Network pharmacology analysis was conducted, spotting PTP1B (Protein Tyrosine Phosphatase 1B) as an important target linked with insulin resistance. Additionally, molecular docking, MD simulation, MMPBSA, PCA, DCCM and FEL analyses were carried out to analyze the interaction and stability of the synthesized complexes. ADMET and DFT studies were also carried out to assess drug-likeness and electronic properties, respectively. These outcomes establish a firm basis for future improvements and the formation of more efficacious anti-diabetic molecules.

2 Results and discussion

2.1 Chemistry

The piperazine–tethered triazole hybrids 10a–10j were synthesized as per reported methodology, depicted in Scheme 1.⁶¹ According to it, tert-butyl 4-(2-bromoacetyl)piperazine-1-carboxylate 12 was allowed to react with substituted benzofuran triazoles 11a–11j using K₂CO₃, KI and DCM at room temperature. The desired products 13a–13j were obtained in good to excellent yields (61–78%) (Fig. 3).

Scheme 1 Synthetic protocol for the preparation of triazole–tethered piperazine conjugates 13a–13j.

Fig. 3 Substrate scope of piperazine–triazole conjugates 13a–13j.

2.2 Network pharmacology analysis

2.2.1 PPI network construction and hub gene identification. The PPI network was developed to analyze functional interactions using Cytoscape and String.⁶² A highly interconnected network was formed, indicating high cross-talk among protein targets. The hub gene analysis revealed that PTPN1 emerged as the most distinguished gene among multiple key genes interuupting such as NFKB1, NFE2L2, PTGS2, and HIF1A. The core position of PTPN1 in the PPI network signals its central role in various biological pathways connected to diabetes (Fig. 4).⁶³

Fig. 4 Identification of common targets, Hub gene and PPI interaction analysis.

PTPN1 represents protein tyrosine phosphatase non-receptor type 1 (PTP1B), a well-recognized inhibitor of insulin receptor (IR) and IR substrate signaling.⁶⁴ Excessive activation of PTPN1 results in disrupted insulin signaling that leads to insulin resistance.⁶⁵ Therefore, the suppression of PTPN1 (PTP1B) has been broadly acknowledged as encouraging a therapeutic approach to improve glucose homeostasis and insulin sensitivity. HIFIA plays a key role in glucose regulation, which is deregulated in hyperglycemic tissues.⁶⁶ The PPI interactions of PTPN1 indicated a strong connection with NFE2L2, which is considered to be a key regulator of oxidative stress and antioxidant defense.⁶⁷ Different research revealed that oxidative stress plays a significant role towards the progression of β-cell impairment along with insulin resistance.⁶⁸ These interactions regulate redox homeostasis and improve insulin signaling. Furthermore, PTPN1 also contributed toward PTGS2 and NFKB1 signaling, which emphasizes the engagement of PTPN1 in inflammation-induced insulin resistance.⁶⁹

2.2.2 KEGG pathway and GO enrichment analysis. The GO enrichment analysis uncovered that the target genes exhibited significant enrichment. In the biological process, enrichment analysis revealed that enrichment terms are predominantly allocated with process concerned with unfolded protein response (UPR) and endoplasmic reticulum.⁷⁰ Both of them are key players in insulin resistance. The long-term ER stress impairs insulin signaling and boosts inflammation. These enrichments suggest that PTPN1 might affect ER stress signaling, thus enhancing insulin sensitivity.⁷¹ The enriched terms in cellular components are mainly associated with the mitochondrial matrix, cytoplasmic side membrane, early endosome, and cytoplasmic side of the ER membrane.⁷² These associations are linked with functional property and intracellular localization of PTPN1, tied to the cytoplasmic ER membrane by making direct interactions with the insulin receptor. The analysis of molecular function revealed that there is a strong enrichment of terms associated with insulin receptor binding, phosphatase binding, protein tyrosine phosphatase activity, and kinase binding. These results explicitly endorse the function of PTPN1 as an insulin receptor regulator via the dephosphorylation of tyrosine residues (Fig. 5).⁷³

Fig. 5 GO Enrichment Analysis of target genes.

The KEGG pathway analysis validated the contribution of diabetes related signaling routes.⁷⁴ The most substantially enriched route involved the insulin signaling and resistance pathway alongwith other oxidative stress pathways (Fig. 6 and S21, SI).⁷⁵ Particularly, PTPN1 is explicitly associated with insulin regulation and resistance, during which it suppresses the insulin receptor substrate phosphorylation and insulin receptor.⁷⁶ The suppression of PTPN1 is thus anticipated to improve glucose uptake, GLUT4 translocation and PI3K-Akt signaling.⁷⁷ Moreover, oxidative stress also contributed to insulin resistance due to ROS (reactive oxygen species).⁶⁸ It was concluded from the KEGG and GO enrichment analysis that PTPN1 suppression might be beneficial in reducing oxidative stress, along with insulin signaling improvement.

Fig. 6 Representation of insulin signaling pathway via KEGG analysis.

2.3 Molecular docking analysis

All the synthesized compounds were docked against two primary targets (α-amylase and PTP1B) to evaluate their inhibitory potential. The docking protocols were validated through the determination of RMSD (Root Mean Square Deviation). For this purpose, the native ligand was redocked and upon superimposition, an RMSD value of 0.66 Å (<2.0 Å) was calculated. The low RMSD value generally verifies docking validity and conformational stability (Fig. 7).^78,79 The compounds 13a–13j gave docking scores in a range from −8.8 to −10.5 kcal mol⁻¹ against α-amylase (2QV4). Similarly, compounds 13a–13j exhibited binding affinities in a range from −7.2 to −8.0 kcal mol⁻¹ against PTP1B (1NNY). Among all, compounds 13c and 13e gave higher docking scores, even better than standard acarbose (Table 1).

Fig. 7 Validation of docking protocols by superimposition of redocked ligand (blue) with native ligand (red).

Table 1 Binding affinities of screened compounds 13a–13j

Compounds	α-Amylase (2QV4) kcal mol^−1	PTP1B (1NNY) kcal mol^−1
13a	−9.2	−7.6
13b	−8.7	−7.3
13c	−10.5	−8.0
13d	−8.8	−7.4
13e	−9.7	−7.8
13f	−8.5	−7.5
13g	−8.8	−7.3
13h	−9.4	−7.5
13i	−9.2	−7.2
13j	−9.5	−7.3
Acarbose	−8.1	−7.4

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The conformational analysis revealed that both compounds, 13c and 13e interacted with α-amylase by making different key interactions with active site residues such as GLU233, ASP197 (Fig. 8).⁸⁰ The compound 13c made significant electrostatic and aromatic contacts with the catalytic site of α-amylase. The strong interaction of 13c with the residue GLU233 is substantial due to its involvement in the acid-base chemistry of glycosidic bond breakage as reported by Neves et al.⁸¹ Likewise, the π-cationic contacts of ligand 13c with HIS201 can change the protonation state for further catalytic disruption.⁸² The interactions of the piperazine cage with TYR62 and TRP59 potentially inhibit the starch substrate binding,⁸³ which makes 13c a competitive inhibitor of α-amylase (Table 2). Similarly, LEU235 and LEU162 interacted with ligand 13c, thus destabilizing the catalytic environment.⁸⁴ Additionally, compound 13e broadly occupied the active site. It established hydrogen bonding interactions with LYS200 and GLN63 near the entrance of the catalytic site.^85,86 The compound 13e disrupts the electrostatic environment by interacting with ASP198 and GLU233 residues.⁸⁷ The residues TYR151 and TRP59 established hydrogen bonding and π-sigma interactions with the aromatic site and Boc functionality of compound 13e (Fig. 9) as explained by Gautam et al.⁸⁸ In comparison, the reference compound acarbose made strong conventional hydrogen bonding and carbon-hydrogen bonding interactions with GLN63, HIS101, ASN105, ALA106, HIS299, THR163, ASP300, TYR62, GLU23, ALA198 and GLU233 at distances of 2.59 Å, 2.59 Å, 2.73 Å, 2.08 Å, 2.21 Å, 2.18 Å, 2.59 Å, 2.78 Å, 2.77 Å, 3.74 Å and 3.31 Å respectively (Fig. 12).

Fig. 8 Binding analysis of compounds 13c (A) & 13e (B) with α-amylase (2QV4).

Table 2 Interactions of different residues of α-amylase with 13c & 13e

Compounds	Residue	Distance (Å)	Interactions	Type
13c	HIS201	3.59	Electrostatic	π-Cation
	GLU233	4.41	Electrostatic	Pi-anion
	TYR151	2.84	H-Bond	π-Donor hydrogen bond
	ILE235	3.86	Hydrophobic	π-Sigma
	TRP59	3.75	Hydrophobic	π-Sigma
	TRP59	3.67	Hydrophobic	Pi-sigma
	TYR151	4.04	Hydrophobic	π–π Stacked
	TYR151	4.68	Hydrophobic	π–π Stacked
	HIS201	4.6	Hydrophobic	π–π T-shaped
	TRP59	4.72	Hydrophobic	π-Alkyl
	TRP59	4.38	Hydrophobic	π-Alkyl
	TYR62	5.42	Hydrophobic	π-Alkyl
	ILE235	4.7	Hydrophobic	π-Alkyl
13e	GLN63	2.28	H-Bond	Conventional H-bond
	LYS200	2.53	H-Bond	Conventional H-bond
	ASP197	3.27	Other	Sulfur-X
	GLU233	3.00	Other	Sulfur-X
	HIS201	4.37	Electrostatic	π-Cation
	GLU233	4.14	Electrostatic	π-Anion
	TYR151	2.52	H-Bond	π-Donor hydrogen bond
	TRP59	3.66	Hydrophobic	π-Sigma
	TYR151	4.21	Hydrophobic	π-π Stacked
	TYR151	5.08	Hydrophobic	π-π Stacked
	TRP59	4.32	Hydrophobic	π-Alkyl
	TRP59	4.07	Hydrophobic	π-Alkyl
	ILE235	5.17	Hydrophobic	π-Alkyl
Acarbose	GLN63	2.59	H-Bond	Conventional H-bond
	HIS101	2.59	H-Bond	Conventional H-bond
	ASN105	2.73	H-Bond	Conventional H-bond
	ALA106	2.08	H-Bond	Conventional H-bond
	HIS299	2.21	H-Bond	Conventional H-bond
	THR163	2.18	H-Bond	Conventional H-bond
	ASP300	2.59	H-Bond	Conventional H-bond
	TYR62	2.78	H-Bond	Conventional H-bond
	GLU23	2.77	H-Bond	Conventional H-bond
	ALA198	3.74	H-Bond	Carbon H-bond
	GLU233	3.31	H-Bond	Carbon H-bond

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Fig. 9 (A & B) H-bonding and hydrophobic interactions of 13c with α-amylase (2QV4). (C &D) (B) H-bonding and hydrophobic interactions of 13e with α-amylase (2QV4).

The conformational analysis of compounds 13c and 13e demonstrated that they adopt a stable binding conformation within the active site of PTP1B (1NNY) (Fig. 10).⁸⁹ PTPIB facilitates phosphorylation of phosphotyrosine residues on insulin receptor via positive charge residues within the active site.⁷³ The residues LYS120 and LYS116 interacted with the aromatic region of 13c via conventional hydrogen bonding interactions (Fig. 11).⁹⁰ The interactions of these residues stabilize the positive residues, thus reducing the catalytic activity and improving insulin signaling.⁹⁰ Similarly, the other residues GLY220, GLY183, ALA217, and TYR46 interacted with compound 13c, promoting interference with substrate conformation, restricting conformational flexibility, and helping in inhibitor retention within the binding pocket. Similarly, compound 13e made π-cationic contact with LYS116 and hydrogen bonding interactions with LYS120 via the aromatic region. Similarly, other critical residues such as GLY183, TYR46, and ALA217 interacted via hydrophobic and hydrogen bonding interactions at distances of 2.25 Å, 5.1 Å, and 4.45 Å (Table 3).⁹¹ In comparison, the standard acarbose formed conventional hydrogen bonding interactions with ARG24, TRP179, GLY183, ARG221, ARG221 and GLY183 (Fig. 12).

Fig. 10 Binding analysis of compounds 13c (A) & 13e (B) with PTP1B (1NNY).

Fig. 11 (A & B) Hydrogen bonding and hydrophobic interactions of 13c with PTP1B (1NNY). (C &D) Hydrogen bonding and hydrophobic interactions of 13e with PTP1B (1NNY).

Table 3 Interactions of different residues of PTP1B (1NNY) with 13c & 13e

Compounds	Residue	Distance (Å)	Interactions	Type
13c	LYS116	2.71	H-Bond	Conventional H-bond
	LYS120	2.5	H-Bond	Conventional H-bond
	GLY183	2.28	H-Bond	Conventional H-bond
	GLY220	3.4	H-Bond	Carbon H-bond
	LYS116	3.48	Electrostatic	π-Cation
	LYS116	3.03	H-Bond; electrostatic	π-Cation; π-donor H-bond
	TYR46	5.13	Hydrophobic	π–π T-shaped
	LYS116	5.36	Hydrophobic	π-Alkyl
	LYS120	4.97	Hydrophobic	π-Alkyl
	ALA217	4.49	Hydrophobic	π-Alkyl
13e	LYS120	2.72	H-Bond	Conventional H-bond
	GLY183	2.25	H-Bond	Conventional H-bond
	LYS116	3.44	Electrostatic	π-Cation
	LYS116	3.02	H-Bond; electrostatic	π-Cation, π -donor H-bond
	TYR46	5.1	Hydrophobic	π–π T-shaped
	LYS120	5.11	Hydrophobic	π-Alkyl
	ALA217	4.45	Hydrophobic	π-Alkyl
Acarbose	ARG24	2.34	H-Bond	Conventional H-bond
	TRP179	2.48	H-Bond	Conventional H-bond
	GLY183	2.81	H-Bond	Conventional H-bond
	ARG221	2.25	H-Bond	Conventional H-bond
	ARG221	2.36	H-Bond	Conventional H-bond
	GLY183	2.59	H-Bond	Conventional H-bond

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Fig. 12 Interactions of compound acarbose with 2QV4 (A) and 1NNY (B).

2.4 Molecular dynamics simulation analysis

The potent compounds were selected to assess the overall stability of complexes during the 500 ns simulation time. The results were interpreted through measurement of RMSD, RMSF, contact analysis, and rGyr.

2.4.1 RMSD analysis of 13c and 13e. The structural stability of complex 13c_2QV4 and 13e_2QV4 was evaluated via RMSD. In the case of complex 13c_2QV4, the RMSD initially increased during the preliminary equilibration step. After the initial deviation period, the complex stabilized itself between 2.1 and 2.6 Å. Likewise, the ligand of complex 13c_2QV4 followed the same deviation pattern and stably bound within the active site. Similarly, the protein of complex 13e_2QV4 exhibited stable RMSD within a range of 2.0–2.3 Å. The ligand part of complex 13e_2QV4 displayed marked deviation due to conformational changes. Overall, both ligands maintained stable conformations within the active site of α-amylase (Fig. 13). Additionally, the RMSD of Cα atoms of PTP1B was determined to assess the stability of the backbone. The Cα atoms of PTP1B made stable conformations with an RMSD range from 2.5 to 3.0 Å. The ligand 13c firmly attached within the binding site with some minor deviations. Similarly, complex 13e_PTP1B displayed stable interaction around 2.7–3.1 Å after initial deviation. In both complexes, stable binding interactions were formed within the active site of PTP1B (Fig. 14).

Fig. 13 RMSD and RMSF calculations of complex 13c_2QV4 (A) and 13e_2QV4 (B).

Fig. 14 RMSD and RMSF calculations of complex 13c_PTP1B (A) and 13e_PTP1B (B).

2.4.2 RMSF analysis of 13c and 13e. The individual residual flexibility was determined through RMSF. It was observed from the complex 13c_2QV4 that most of the residues exhibited low RMSD (<1.5 Å) due to restricted fluctuations. Higher fluctuations were observed around the loop region that has inherent flexibility. Similarly, the same fluctuation profile was observed for 13e_2QV4 (Fig. 13). Both complexes displayed minimal fluctuations with the binding site residues of α-amylase. The RMSF analysis of 13c_PTP1B revealed that the majority of residues exhibited constrained backbone fluctuations (<1.5 Å). The loop and terminal residues exhibited higher fluctuations. Similarly, 13e_PTP1B demonstrated slightly higher fluctuations around the loop region than complex 13c_PTP1B (Fig. 14).

2.4.3 Interpretation of contacts between ligand and protein. The ligand–protein contacts analysis was performed to probe the type of interactions formed by the ligands with the active site of target proteins. Persistent key interactions were observed when complex 13c made strong interactions with active site residues GLN63, TRP59, TYR151, TRP58 and HIS201 of α-amylase. The binding profile was dominated by hydrophobic as well as hydrogen bonding interactions. It was observed from time-resolved interaction analysis that uniform interactions were formed throughout simulation time (Fig. 15). Similarly, the dynamic behavior of complex 13e, as it displayed irregular interactions, was compared with that of 13c (Fig. 16). The 2D interaction supported the evidence that both complexes maintained stable interactions within the catalytic site of α-amylase (Fig. 17). The complex 13c interacted with ARG254, ILE163 and ASP181. Hydrophobic and hydrogen bonding contacts surround these interactions, thereby playing significant role towards complex's stability. The timeline of interactions revealed that 13c made overall stable contacts despite several fluctuations of residues within the active site of PTP1B (Fig. 18). Similarly, 13e made strong interactions with residues ILE219, PRO31, VAL49, ASP48 and TYR46 via both hydrogen and hydrophobic interactions (Fig. 19). The 2D plot revealed that both compounds are involved in multiple interactions within the catalytic site (Fig. 20).

Fig. 15 Interaction analysis of complex 13c with α-amylase.

Fig. 16 Interaction analysis of complex 13e with α-amylase.

Fig. 17 2D protein–ligand contacts analysis of complex 13c_2QV4 (A) and 13e_2QV4 (B) against α-amylase.

Fig. 18 Time-resolved interaction analysis of complex 13c with PTP1B.

Fig. 19 Time-resolved interaction analysis of complex 13e with PTP1B.

Fig. 20 2D protein–ligand contacts analysis of complex 13c_PTP1B (A) and 13e_PTP1B (B).

2.4.4 Radius of gyration. The overall compactness of complexes 13c and 13e was determined through radius of gyration (rGyr). Stable rGyr was observed throughout the simulation time for compound 13c in complex with α-amylase (Fig. 21). The complex 13e exhibited marginally higher fluctuations. However, in both situations, the protein established well-folded and compact conformations within the binding pocket of α-amylase. Additionally, the 13c_PTP1B displayed observable fluctuations in rGyr due to conformational adjustments in the protein. In complex 13e_PTP1B, a bit higher fluctuations as compared to 13c_PTP1B complex were noticed due to higher flexibility. It was observed that the compound 13c binds with both α-amylase and PTP1B receptor, which exhibited higher compactness and stability (Fig. 21).

Fig. 21 (A & B) Measurement of rGyr of complex 13c_2QV4 (A) and 13e_2QV4 (B). (C & D) Measurement of rGyr of complex 13c_PTP1B (A) and 13e_PTP1B.

2.5 Estimation of binding free energies through MMPBSA

The binding free energies of the complexes were determined by using MMPBSA analysis. The complex 13c_2QV4 exhibited a higher binding energy of −43.71 kcal mol⁻¹. Similarly, complex 13e_2QV4 illustrated −26.41 kcal mol⁻¹ binding energy. Additionally, the complex 13c_PTP1B and 13e_PTP1B demonstrated binding energies of −20.69 kcal mol⁻¹ and −16.08 kcal mol⁻¹, correspondingly (Table 4). The individual component analysis revealed that van der Waals interactions are the major contributor among all individual binding components. This indicates the contribution of hydrophobic interactions in the stabilization of all complexes. WCA energy and electrostatic energies contributed generously to the stabilization of the complex; however, polar solvation energy contributed unfavorably to the stability of the complex. Overall, the complex 13c exhibited higher stability within the binding pocket of target proteins, thus indicating more substantial inhibitory potential (Fig. 22).

Table 4 Estimation of binding free energies of target compounds

MMPBSA	13c_2QV4	13e_2QV4	13c_PTP1B	13e_PTP1B
van der Waals energy	−59.12	−53.78	−33.43	−25.39
Electrostatic energy	−11.2	−17.56	−12.48	−1.83
Polar solvation energy	28.21	47.1	28.18	11.22
WCA energy	−1.6	−2.17	−2.96	−0.082
Binding energy	−43.71	−26.41	−20.69	−16.082

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Fig. 22 Representation of individual binding components along with total binding energies of complexes 13c_2QV4 (A), 13e_2QV4 (B), 13c_PTP1B (C) and 13e_PTP1B (D).

2.6 DCCM analysis

The correlated and anti-correlated movements with active site residues upon binding with ligands were assessed through DCCM (Dynamic Cross-Correlation Matrix). Both complexes i.e., 13c_2QV4 and 13e_2QV4 exhibited positive correlation across the diagonal, indicating the structural stability and synchronized motion surrounding residues. Similarly, the complexes 13c_PTP1B and 13e_PTP1B showed a noticeable correlation, as complex 13c_PTP1B exhibited marked correlated movements, suggesting enhanced internal stability. In contrast, higher anti-correlated regions were observed in the 13e complex due to conformational fluctuations. Generally, the complex 13c demonstrated favorably correlated movements with both receptors (Fig. 23).

Fig. 23 DCCM analysis of complex 13c_2QV4 (A), 13e_2QV4 (B), 13c_PTP1B (C) and 13e_PTP1B (D).

2.7 PCA and FEL analysis

The key collective movements observed throughout the simulation time were investigated through PCA (Principal Component Analysis). Among both 13c_2QV4 and 13e_2QV4 complexes, the complex 13c_2QV4 occupied a more distinct and compact motion along PC1 and PC2. Likewise, 13e_2QV4 showed higher conformational variability, suggesting more flexibility. Additionally, 13c_PTP1B exhibited constrained structural dynamics due to more compact clustering across PC1 and PC2. Similarly, 13e_PTP1B demonstrated scattered distribution along PC2 and PC1 (Fig. 24). Furthermore, the thermodynamic stability of individual conformation was evaluated through FEL (Free Energy Landscape). Among all, complex 13c_2QV4 and 13c_PTP1B exhibited distinct and intense global minima, suggesting thermodynamically stable conformations. The deeper basins indicated the stable binding with low-energy conformations. Likewise, complexes 13e_2QV4 and 13e_PTP1B showed a higher energy barrier between different states. Overall, it was observed from PCA, DCCM, and FEL analysis that complex 13c illustrated enhanced correlated dynamics, higher dynamic stability, and favorable energy minima (Fig. 25).

Fig. 24 PCA analysis of complex 13c_2QV4 (A), 13e_2QV4 (B), 13c_PTP1B (C) and 13e_PTP1B (D).

Fig. 25 FEL analysis of complex 13c_2QV4 (A), 13e_2QV4 (B), 13c_PTP1B (C) and 13e_PTP1B (D).

2.8 Density functional Theory analysis

The stability of synthesized compounds 13c and 13e was assessed through DFT studies. A Gaussian program was used to perform all the calculations. The optimization of structures was done by using the B3LYP functional with the 631G(d) basis set.

2.8.1 Geometry optimization of compounds 13c and 13e. The geometry-optimized structures of compounds 13c and 13e were depicted in Fig. 26. The geometry optimization gave stable structures, which were used to calculate the bond angles and bond lengths as mentioned in the Table 5. It was observed from the geometry optimization that both compounds 13c and 13e adopted a non-planar conformation because of the aromatic ring and heteroatoms, leading to electronic and steric effects. All of the observed bond lengths were noted to be in the expected range for heterocyclic compounds.

Fig. 26 Geometry optimized structures 13c (A) and 13e (B).

Table 5 Determination of bond lengths and bond angles of geometry optimized structures 13c & 13e

Bond lengths (Å)			Bond angles (°)
Geometry parameters	13c	13e	Geometry parameters	13c	13e
C1–C2	1.4	1.4	C2–C1–C6	122.7	121.71
C1–C6	1.38	1.39	C1–C2–C3	120.3	122.06
C2–C3	1.39	1.39	C2–C3–C4	116.76	115.69
C3–C4	1.38	1.4	C3–C4–C5	123.55	122.84
C4–C5	1.4	1.4	C4–C5–C6	119.26	120.24
C5–C6	1.4	1.4	C3–C4–O7	126.01	126.96
C4–O7	1.36	1.37	C4–O7–C8	106.41	106.57
O7–C8	1.37	1.37	O7–C8–C9	111.46	111.29
C8–C9	1.36	1.36	O7–C8–C10	114.63	114.32
C8–C10	1.44	1.44	C8–C10–N11	125.33	125.37
C10–N11	1.39	1.39	C8–C10–N14	124.68	124.68
C10–N14	1.31	1.31	C10–N11–C12	103.88	103.94
N11–C12	1.37	1.37	N11–C12–N13	110.55	110.57
C12–N13	1.31	1.31	N11–C12–S15	121.82	121.66
C12–S15	1.76	1.76	N13–C12–S15	127.62	127.76
S15–C16	1.85	1.85	C12–S15–C16	99.68	99.75
C16–C17	1.53	1.53	S15–C16–C17	111.05	111.06
C17–O24	1.22	1.22	C16–C17–O24	118.41	118.43
C31–C32	1.22	1.22	O24–C17–C18	122.67	122.66

---

2.8.2 Frontier molecular orbital analysis. The chemical reactivity and electronic properties of the synthesized compounds 13c and 13e were determined through FMO. The HOMO energy for compound 13c was noted to be −0.218 eV and the LUMO is found at −0.05 eV. Likewise, the HOMO energy gap in compound 13e is found at −0.205 eV, while LUMO is located at −0.047 eV. The energy gaps for compounds 13c and 13e were noted to be 0.168 eV and 0.158 eV, respectively (Fig. 27). Both compounds have comparable energy gaps, which is vital to estimate the chemical reactivity and polarizability. To get deep insights into the stability of 13c and 13e, multiple global descriptors such as softness, electron affinity, chemical potential, electronegativity, etc., were determined from HOMO–LUMO as mentioned in Table 6.

Fig. 27 FMO analysis of compounds 13c (A) and 13e (B).

Table 6 Estimation of global descriptors through HOMO–LUMO^a

Quantum descriptors	13c	13e
a ΔE = energy gap, I = ionization potential, A = electron affinity, µ = chemical potential, χ = electronegativity, η = chemical hardness, σ = softness and ω = electrophilicity index.
HOMO (eV)	−0.218	−0.205
LUMO (eV)	−0.05	−0.047
ΔE (eV)	0.168	0.158
I (eV)	0.218	0.205
A (eV)	0.05	0.047
µ (eV)	−0.134	−0.126
χ (eV)	0.134	0.126
η (eV)	0.084	0.079
σ (eV^−1)	5.95	6.33
ω (eV)	0.107	0.101

---

2.8.3 Molecular electrostatic potential analysis of compounds 13c and 13e. MEP analysis of hybrids 13c and 13e was conducted to estimate the distribution of charge on the molecular structures. This helps to determine the possible nucleophilic and electrophilic regions (Fig. 28). The yellow and red colors correspond to an electronically rich region, whereas the light blue color represents the electronically poor region. Both compounds 13c and 13e possess electronically rich regions around hetero atoms because of their high electronegativity. However, the other areas, such as carbons and hydrogen atoms, act as electrophilic sites due to poor electron density.

Fig. 28 MEP analysis of compounds 13c (A) and 13e (B).

2.8.4 NCI analysis of compounds 13c and 13e. The non-covalent interaction (NCI) analysis was carried out to probe weak intra- and intermolecular interactions in compounds 13c and 13e. NCI analysis stems from a reduced density gradient that permits grouping and visualization of non-covalent interactions via sign(λ₂)ρ vs. RDG plots. The negative and positive values of sign(λ₂)ρ represent hydrogen bonding and repulsive attractions, respectively, however, zero value of sign(λ₂)ρ represents weak interactions such as van der Waals. Both compounds 13c and 13e demonstrated weak hydrogen bonding (blue color) around hetero atoms. Weak van der Waals interactions (green color) have also been observed between aliphatic parts and aromatic rings. The red regions were also observed due to the presence of bulky groups; however, they did not affect the overall stability of the compounds (Fig. 29).

Fig. 29 NCI analysis of compounds 13c (A) and 13e (B).

2.9 α-Amylase inhibition studies

The synthesized piperazine–triazole conjugates 13a–13j were examined through in vitro studies to assess anti-diabetic potential against α-amylase. The results suggested that all compounds exhibited higher potential against α-amylase as compared to the reference compound acarbose (10.30 ± 0.20 µM). Their IC₅₀ values fell within the range of 2.39 ± 0.41 µM to 4.54 ± 0.39 µM. It was found that compound 13c exhibited higher potential among all compounds with an IC₅₀ of 2.39 ± 0.41 µM, showing higher potency than the reference compound. Similarly, compound 13e also displayed significant inhibition with an IC₅₀ of 2.97 ± 0.31 µM. In addition, compounds 13b and 13f exhibited slightly higher IC₅₀ of 4.03 ± 0.28 µM and 4.54 ± 0.39 µM, respectively, as compared to other compounds. Likewise, compounds 13i, 13j, 13h, 13g, 13a and 13d exhibited strong inhibition against α-amylase with IC₅₀ values i.e., 3.47 ± 0.15 µM, 3.03 ± 0.16 µM, 3.09 ± 0.30 µM, 3.76 ± 0.50 µM, 3.14 ± 0.43 µM and 3.55 ± 0.40 µM (Table 7).

Table 7 α-Amylase inhibition potential of piperazine–triazole conjugates 13a–13j

Compounds	IC50 (µM)
13a	3.14 ± 0.43
13b	4.03 ± 0.28
13c	2.39 ± 0.41
13d	3.55 ± 0.40
13e	2.97 ± 0.31
13f	4.54 ± 0.39
13g	3.76 ± 0.50
13h	3.09 ± 0.30
13i	3.47 ± 0.15
13j	3.03 ± 0.16
Acarbose	10.30 ± 0.20

---

2.10 Structure activity relationship

Based on in vitro and computational investigation, the SAR studies of piperazine–triazole conjugates were conducted. The synergistic role of all fragments of hybrids 13a–13j, comprising Boc piperazine, carbonyl, triazole, and substituted benzofurans, contributes to inhibitory activity against α-amylase. It was found that the inhibitory potential of synthesized conjugates appeared to be reliant on the substitution attached to the benzofuran ring. SAR studies revealed that the compound 13c with Cl substitution on the benzofuran ring has a strong influence on the enzyme inhibition of α-amylase. The presence of an electronegative group on the benzofuran ring gave a binding affinity of −10.5 kcal mol⁻¹ and IC₅₀ = 2.39 ± 0.41 µM. Likewise, the presence of the methoxy (7-OMe) group on the benzofuran ring of compound 13e also exerted a strong influence on the inhibition of alpha amylase. The in vitro assay revealed an IC₅₀ value of 2.97 ± 0.31 µM. Similarly, the in silico investigation has also demonstrated similar results, depicting binding affinity of −9.7 kcal mol⁻¹ (Fig. 30). It was observed that the compounds with no substitution (13b and 13f) on the benzofuran ring gave less inhibition (IC₅₀ = 4.03 ± 0.28 µM and 4.54 ± 0.39 µM) as compared with 13c and 13e. Likewise, the in silico docking affinities of compounds 13b and 13f (−8.7 kcal mol⁻¹ & −8.5 kcal mol⁻¹) were also found to be less than those of 13c and 13e. Additionally, the compounds with 13a, 13d, 13g, 13h, 13i and 13j with ethoxy, 5-Br, diethyl, 6-OMe, 6-Br and 7-Me gave moderate activity in a range from IC₅₀ = 3.03 ± 0.16–3.76 ± 0.50 µM and their docking scores were found within −8.8 kcal mol⁻¹ to −9.5 kcal mol⁻¹ range. However, all the synthesized hybrids exhibited higher activity against the reference compound acarbose (binding affinity = −8.1 kcal mol⁻¹ and IC₅₀ = 10.30 ± 0.20 µM).

Fig. 30 SAR studies of piperazine derivatives 13a–13j against α-amylase.

3 Experimental section

3.1 Chemicals and reagents

All solvents and reagents were purchased from Sigma-Aldrich which consumed after purification. WRS-1B melting point apparatus was utilized for observing melting points. ARX FT-NMR spectrometer was used for both ¹³C NMR spectra (150 MHz) and ¹H NMR (600 MHz). The MS spectra were obtained using Agilent 6400 series triple quadrupole mass spectrometer. FTIR specta were recorded using a Cary 630 FTIR spectrometer (Santa Clara, CA, USA).

3.2 Molecular docking and MD protocols

The crystal structures of both PTP1B (PDB: 1NNY) and α-amylase (PDB: 2QV4) was accessed from PDB (protein data bank) web server.⁸⁸ The protein structure was prepared in AutoDock tools before performing docking via AutoDockVina. The ligand–protein complexes were shifted to Desmond software for MD simulation analysis. The protein was prepared by using protein preparation module of Desmond. Further orthorhombic box, 0.15 NaCl and TIP3P solvent model was used. A 500 ns (2000 frames) simulation were performed using OPLS_2005 force field at 1 atm pressure.⁹² Furthermore, MMPBSA analysis was performed by converting trajectories to g_mmpbsa script.

3.3 Enzymatic assay

In order to perform an enzyme inhibition experiment, α-amylase (EC number: 232-588-1, MDL number: MFCD00081319; UNSPSC code: 12352204; eCl@ss: 32160401) was purchased commercially (Sigma-Aldrich) with a concentration of around 30 U/mg sourced out of Aspergillus oryzae. The analytical grade enzyme was used without any additional purification. The soluble starch with a concentration of 1% (w/v) was employed as substrate in enzymatic assay. Acarbose, an α-amylase inhibitor, was characterized well and taken as a reference standard at a concentration of 1 mg mL⁻¹. Inhibition test was performed using 0.1 M phosphate buffer (pH 6.9) according to the methodology described in the literature with slight changes.⁹³ In short, 100 µL of the test sample was incubated with immobilized enzyme beads (9 U mg) for 15 minutes at 37 °C. Next, 700 µL of 1% soluble starch solution was combined with the mixture and incubated over 5 min at 50 °C. This reaction was stopped by pouring 300 µL DNS reagent and then the mixture was boiled at 100 °C within 5 min. On cooling, 1000 µL of distilled water was added and the absorbance of 540 nm was taken. The rate of percentage inhibition was computed based on the following equation:

% Inhibition = (ΔA control − ΔA sample)/ΔA control × 100

3.4 Compound and disease targets identification through network pharmacology

The synthesized compounds' targets were identified through the Swiss target prediction web server, followed by the removal of duplicate targets. The visualization of the targets was achieved through Cytoscape software.⁹⁴ The therapeutic significance of hyperpigmentation genes was extracted from the GenCard web server using “diabetes” as a keyword. The 33 compound targets were identified through the Swiss target prediction server after taking out replica proteins. Furthermore, a total of 996 diabetes associated genes were extracted from the GeneCards website using “diabetes” as a keyword. Upon convergence, 43 targets were identified as core genes that might exert therapeutic potential.

3.5 General synthetic procedure

A solution of tert-butyl 4-(2-bromoacetyl)piperazine-1-carboxylate 12 (1.1 equivalent) in DCM (5 mL) was allowed to react with substituted benzofuran triazoles 11a–11j (1 equivalent) in the presence of K₂CO₃ (1.1 equivalent) and KI (0.16 equivalent) for 12–24 hours. The reaction progress was continuously observed via TLC. Upon reaction completion, addition of ice-cold water to reaction mixture resulted in the formation of precipitates, which were dried (in an oven) and column chromatography was carried out to obtain the pure products.

3.5.1 Characterization of 13a. Off-white powder; yield 63%; m.p. 139 °C; IR (KBr cm⁻¹): 2974 (C–H), 1669 (C=O, Boc barbonyl), 1636 (C=O, amide), 1559 (C=N), 1499 (C=C, Ar), 1450 (C–H bending), 1363 (CH₃ ending, ^tBu), 1226 (O–Et/C–O), 1161 (C–O), 1122 (C–N), 1073 (C–O), 690 (C–S); ¹H NMR (600 MHz, DMSO-d6) δ = 1.35 (t, J = 6 Hz, 3H, ethoxy–CH₃), 1.41 (s, 9H, Boc tert-butyl), 3.39–3.43 (m, 6H, piperazine–CH₂), 3.48–3.50 (m, 2H, piperazine–CH₂), 4.10 (q, J = 6 Hz, 2H, ethoxy–OCH₂–), 4.38 (s, 2H, –S–CH₂–), 6.50 (s, 1H, benzofuran–H), 6.95 (d, J = 6 Hz, 1H, benzofuran Ar–H), 7.13–7.15 (m, 2H, benzofuran Ar–H), 7.58 (d, J = 12 Hz, 2H, phenyl Ar–H), 7.65–7.69 (m, 3H, phenyl Ar–H); ¹³C NMR (150 MHz, DMSO-d6) δ = 15.2, 28.5, 28.5, 28.5, 37.1, 41.9, 41.9, 45.7, 45.7, 64.7, 79.7, 107.7, 110.1, 114.3, 128.2, 128.2, 129.2, 129.2, 129.2, 130.7, 131.2, 133.8, 143.2, 143.2, 144.6, 147.5, 152.8, 154.3, 165.8; MS m/z: 564.1 [M + 1]⁺; anal. elem. calc. for C₂₉H₃₃N₅O₅S: C, 61.79: H, 5.90: N, 12.42: found: C, 61.64: H, 5.89: N, 12.41.

3.5.2 Characterization of 13b. Yellow solid; yield 61%; m.p. 135 °C; IR (KBr cm⁻¹): 2980 (C–H), 1696 (C=O, Boc carbonyl), 1630 (C=O, amide), 1554 (C=N), 1494 (C=C, Ar), 1444 (C–H bending), 1365 (CH₃ bending, ^tBu), 1228 (C–O), 1162 (C–O), 1122 (C–N), 1072 (C–O), 694 (C–S); ¹H NMR (600 MHz, DMSO-d6) δ = 1.37 (s, 9H, Boc tert-butyl), 3.35–3.39 (m, 6H, piperazine CH₂), 3.44–3.46 (m, 2H, piperazine CH₂), 4.34 (s, 2H, –S–CH₂–), 6.40 (s, 1H, benzofuran H), 7.21 (t, J = 6 Hz, 1H, benzofuran Ar–H), 7.32 (t, J = 6 Hz, 1H, benzofuran Ar–H), 7.52–7.57 (m, 4H, Ar–H (phenyl + benzofuran)), 7.62–7.66 (m, 3H, phenyl Ar–H); ¹³C NMR (150 MHz, DMSO-d6) δ = 28.5, 28.5, 28.5, 37.1, 41.9, 41.9, 45.7, 45.7, 79.7, 107.4, 111.8, 122.6, 124.2, 126.7, 127.5, 128.2, 128.2, 128.2, 130.7, 131.3, 133.7, 143.2, 147.6, 152.9, 154.3, 154.4, 165.7; MS m/z: 520 [M + 1]⁺; anal. elem. calc. for C₂₇H₂₉N₅O₄S: C, 62.41: H, 5.63: N, 13.48: found: C, 62.39: H, 5.59: N, 13.49.

3.5.3 Characterization of 13c. Off-white solid; yield 75%; m.p. 148 °C; IR (KBr cm⁻¹): 2974 (C–H), 1700 (C=O, Boc carbonyl), 1633 (C=O, amide), 1560 (C=N), 1493 (C=C, Ar), 1448 (C–H bending), 1362 (CH₃ bending, ^tBu), 1227 (C–O), 1159 (C–O), 1121 (C–N), 1069 (C–O), 690 (C–S); ¹H NMR (600 MHz, DMSO-d6) δ = 1.37 (s, 9H, Boc tert-butyl), 3.25–3.27 (m, 2H, piperazine CH₂), 3.35–3.39 (m, 4H, piperazine CH₂), 3.44–3.46 (m, 2H, piperazine CH₂), 4.36 (s, 2H, –S–CH₂–), 6.39 (s, 1H, benzofuran H), 7.34–7.36 (m, 1H, benzofuran Ar–H), 7.55–7.59 (m, 3H, Ar–H (phenyl + benzofuran)), 7.63–7.69 (m, 4H, phenyl Ar–H); ¹³C NMR (150 MHz, DMSO-d6) δ = 28.5, 28.5, 28.5, 37.1, 41.9, 41.9, 45.7, 45.7, 79.7, 106.9, 113.5, 122.0, 126.6, 126.6, 128.1, 128.6, 128.6, 129.2, 130.8, 131.44, 133.6, 144.7, 147.2, 153.0, 153.2, 154.3, 165.7; MS m/z: 555.9 [M + 2]⁺; anal. elem. calc. for C₂₇H₂₈ClN₅O₄S: C, 58.53: H, 5.09: N, 12.64: found: C, 58.45: H, 5.10: N, 12.61.

3.5.4 Characterization of 13d. Light yellow powder; yield 73%; m.p. 118 °C; IR (KBr cm⁻¹): 2976 (C–H), 1682 (C=O, Boc carbonyl), 1639 (C=O, amide), 1556 (C=N), 1498 (C=C, Ar), 1437 (C–H bending), 1385 (CH₃ bending, ^tBu), 1221 (C–O), 1159 (C–O), 1119 (C–N), 1072 (C–O), 686 (C–S); ¹H NMR (600 MHz, DMSO-d6) δ = 1.36 (s, 9H, Boc tert-butyl), 3.34–3.38 (m, 6H, piperazine CH₂), 3.43–3.45 (m, 2H, piperazine CH₂), 4.35 (s, 2H, –S–CH₂–), 6.39 (s, 1H, benzofuran H), 7.46 (d, J = 12 Hz, 1H, benzofuran Ar–H), 7.50–7.55 (m, 4H, phenyl Ar–H), 7.59–7.67 (m, 2H, phenyl Ar–H), 7.82 (s, 1H, benzofuran Ar–H), ¹³C NMR (150 MHz, DMSO-d6) δ = 28.5, 28.5, 28.5, 37.1, 41.9, 41.9, 45.7, 45.7, 79.7, 106.8, 111.6, 113.9, 116.5, 121.2, 124.7, 125.1, 125.1, 128.1, 128.1, 128.1, 129.3, 130.7, 132.5, 148.8, 153.2, 153.2, 165.7; MS m/z: 599.9 [M + 2]⁺; anal. elem. calc. for C₂₇H₂₈BrN₅O₄S: C, 54.18: H, 4.72: N, 11.70: found: C, 54.16: H, 4.69: N, 11.69.

3.5.5 Characterization of 13e. Off-white solid; yield 78%; m.p. 151 °C; IR (KBr cm⁻¹): 2970 (C–H), 1685 (C=O, Boc carbonyl), 1647 (C=O, amide), 1585 (C=N), 1501 (C=C, Ar), 1415 (C–H bending), 1364 (CH₃ bending, ^tBu), 1271 (C–O), 1164 (C–O), 1129 (C–N), 1033 (C–O), 693 (C–S); ¹H NMR (600 MHz, DMSO-d6) δ = 1.46 (s, 9H, Boc tert-butyl), 3.40–3.42 (m, 2H, piperazine CH₂), 3.50–3.52 (m, 2H, piperazine CH₂), 3.58–3.61 (m, 4H, piperazine CH₂), 3.83 (s, 3H, Ar–OCH₃), 4.35 (s, 2H, –S–CH₂–), 6.56 (s, 1H, benzofuran H), 6.78 (d, J = 6 Hz, 1H, benzofuran H), 7.03–7.11 (m, 2H, benzofuran H), 7.37 (d, J = 12 Hz, 2H, phenyl H), 7.54–7.60 (m, 3H, phenyl H); ¹³C NMR (150 MHz, DMSO-d6) δ = 28.4, 28.4, 28.4, 36.3, 42.2, 42.2, 46.1, 46.1, 56.5, 80.5, 107.9, 109.1, 113.8, 124.3, 124.3, 127.5, 127.5, 129.2, 130.2, 130.7, 133.3, 142.9, 144.5, 145.6, 148.0, 153.1, 154.5, 165.8; MS m/z: 550 [M + 1]⁺; anal. elem. calc. for C₂₈H₃₁N₅O₅S: C, 61.19: H, 5.69: N, 12.74: found: C, 61.17: H, 5.63: N, 12.71.

3.5.6 Characterization of 13f. Off-white powder; yield 70%; m.p. 139 °C; IR (KBr cm⁻¹): 2969 (C–H), 1700 (C=O, Boc carbonyl), 1653 (C=O, amide), 1559 (C=N), 1494 (C=C, Ar), 1437 (C–H bending), 1365 (CH₃ bending, ^tBu), 1231 (C–O), 1159 (C–O), 1119 (C–N), 1072 (C–O), 693 (C–S); ¹H NMR (600 MHz, DMSO-d6) δ = 1.46 (s, 9H, Boc tert-butyl), 3.41–3.62 (m, 8H, piperazine CH₂), 4.37 (s, 2H, –S–CH₂–) 6.92 (s, 1H, benzofuran H), 7.42–7.72 (m, 9H, Ar–H), 7.85–7.89 (m, 2H, naphthyl Ar–H); ¹³C NMR (150 MHz, DMSO-d6) δ = 28.4, 28.4, 28.4, 38.9, 42.2, 42.2, 46.1, 46.1, 80.5, 106.4, 112.4, 123.0, 123.4, 125.2, 125.2, 126.8, 127.3, 127.3, 127.3, 127.6, 128.9, 128.9, 128.9, 130.3, 130.9, 133.4, 142.2, 148.3, 152.8, 154.5, 165.8; MS m/z: 570 [M + 1]⁺; anal. elem. calc. for C₃₁H₃₁N₅O₄S: C, 65.36: H, 5.49: N, 12.29: found: C, 65.33: H, 5.45: N, 12.31.

3.5.7 Characterization of 13g. Light yellow solid; yield 67%; m.p. 141 °C; IR (KBr cm⁻¹): 2971 (C–H), 1691 (C=O, Boc carbonyl), 1641 (C=O, amide), 1555 (C=N), 1493 (C=C), 1438 (C–H bending), 1393 (CH₃ bending) 1254 (C–O), 1213 (C–O), 1118 (C–N), 1028 (C–O), 694 (C–S); ¹H NMR (600 MHz, DMSO-d6) δ = 1.03 (t, J = 9 Hz, 6H, diethylamino CH₃), 1.37 (s, 9H, Boc tert-butyl), 3.25–3.27 (m, 2H, piperazine CH₂), 3.30–3.32 (m, 4H, –NCH₂CH₃), 3.37–3.40 (m, 4H, piperazine CH₂), 3.43–3.45 (m, 2H, piperazine CH₂), 4.29 (s, 2H, –S–CH₂–CO–), 6.05 (s, 1H, benzofuran H), 6.62 (d, J = 6 Hz, 1H, benzofuran Ar–H), 6.67 (s, 1H, benzofuran Ar–H), 7.25 (d, J = 12 Hz, 1H, benzofuran Ar–H), 7.51 (d, J = 6 Hz, 2H, phenyl Ar–H), 7.62–7.65 (m, 3H, phenyl Ar–H); ¹³C NMR (150 MHz, DMSO-d6) δ = 12.8, 12.8, 28.5, 28.5, 28.5, 37.1, 41.9, 41.9, 44.6, 44.6, 45.7, 45.7, 79.7, 93.1, 107.6, 110.6, 116.1, 122.6, 128.3, 128.3, 128.3, 130.7, 131.2, 133.9, 140.1, 147.7, 148.1, 151.9, 154.3, 157.2, 165.8; MS m/z: 590.9 [M]⁺; anal. elem. calc. for C₃₁H₃₈N₆O₄S: C, 63.03: H, 6.48: N, 14.23: found: C, 63.01: H, 6.42: N, 14.21.

3.5.8 Characterization of 13h. Off-white powder; yield 72%; m.p. 137 °C; IR (KBr cm⁻¹): 2968 (C–H), 1701 (C=O, Boc carbonyl), 1641 (C=O, amide), 1612 (C=N), 1496 (C=C, Ar), 1451 (C–H bending), 1365 (CH₃ bending, ^tBu), 1239 (C–O), 1155 (C–O), 1116 (C–N), 1081 (C–O), 692 (C–S); ¹H NMR (600 MHz, DMSO-d6) δ = 1.41 (s, 9H, Boc tert-butyl), 3.39–3.43 (m, 6H, piperazine CH₂), 3.47–3.49 (m, 2H, piperazine CH₂), 3.79 (s, 3H, Ar–OCH₃), 4.36 (s, 2H, –S–CH₂–CO–), 6.30 (s, 1H, benzofuran H), 6.87 (d, J = 12 Hz, 1H, benzofuran Ar–H), 7.18 (s, 1H, benzofuran Ar–H), 7.47 (d, J = 6 Hz, 1H, benzofuran Ar–H), 7.58 (d, J = 6 Hz, 2H, phenyl Ar–H), 7.66–7.71 (m, 3H, phenyl Ar–H); ¹³C NMR (150 MHz, DMSO-d6) δ = 28.5, 28.5, 28.5, 37.1, 41.9, 41.9, 45.7, 45.7, 56.2, 79.7, 96.1, 107.5, 120.6, 122.8, 122.8, 128.2, 128.2, 128.2, 130.8, 131.3, 133.8, 142.3, 147.7, 152.5, 154.3, 155.8, 159.5, 165.8; MS m/z: 550 [M + 1]⁺; anal. elem. calc. for C₂₈H₃₁N₅O₅S: C, 61.19: H, 5.69: N, 12.74: found: C, 61.15: H, 5.58: N, 12.73.

3.5.9 Characterization of 13i. Off-white solid; yield 68%; m.p. 128 °C; IR (KBr cm⁻¹): 2974 (C–H), 1691 (C=O, Boc carbonyl), 1638 (C=O, amide), 1558 (C=N), 1504 (C=C, Ar), 1446 (C–H bending), 1363 (CH₃ bending, ^tBu), 1234 (C–O), 1168 (C–O), 1119 (C–N), 1081 (C–O), 692 (C–S) ¹H NMR (600 MHz, DMSO-d6) δ = 1.41 (s, 9H, Boc tert-butyl), 3.39–3.43 (m, 4H, piperazine CH₂), 3.48–3.50 (m, 4H, piperazine CH₂), 4.39 (s, 2H, –S–CH₂–CO–), 6.41 (s, 1H, benzofuran H), 7.43 (d, J = 6 Hz, 1H, benzofuran Ar–H), 7.57–7.61 (m, 3H, phenyl Ar–H), 7.66–7.71 (m, 3H), 7.92 (s, 1H, phenyl Ar–H); ¹³C NMR (150 MHz, DMSO-d6) δ = 28.5, 28.5, 28.5, 37.1, 41.9, 41.9, 45.7, 45.7, 79.7, 107.3, 115.1, 119.2, 124.1, 126.9, 127.5, 128.1, 128.1, 128.3, 130.8, 131.4, 133.6, 143.9, 147.2, 153.2, 154.3, 154.8, 165.7; MS m/z: 599.9 [M + 2]⁺; anal. elem. calc. for C₂₇H₂₈BrN₅O₄S: C, 54.18: H, 4.72: N, 11.70: found: C, 54.13: H, 4.71: N, 11.71.

3.5.10 Characterization of 13j. White powder; yield 71%; m.p. 147 °C; IR (KBr cm⁻¹): 2981 (C–H), 1686 (C=O, Boc carbonyl), 1638 (C=O, amide), 1557 (C=N), 1502 (C=C, Ar), 1418 (C–H bending), 1364 (CH₃ bending, ^tBu), 1263 (C–O), 1167 (C–O), 1119 (C–N), 1026 (C–O), 693 (C–S). ¹H NMR (600 MHz, DMSO-d6) δ = 1.41 (s, 9H, Boc tert-butyl), 2.21 (s, 3H, benzofuran–CH₃), 3.39–3.43 (m, 6H, piperazine CH₂), 3.48–3.50 (m, 2H, piperazine CH₂), 4.36 (s, 2H, –S–CH₂–CO–), 6.83 (s, 1H, benzofuran H), 7.15 (d, J = 6 Hz, 2H, benzofuran Ar–H), 7.44 (t, J = 6 Hz, 1H, benzofuran Ar–H), 7.59 (d, J = 6 Hz, 2H, phenyl Ar–H), 7.63–7.69 (m, 3H, phenyl Ar–H); ¹³C NMR (150 MHz, DMSO-d6) δ = 14.5, 28.5, 28.5, 28.5, 37.0, 41.9, 41.9, 45.7, 45.7, 79.7, 107.8, 120.0, 121.5, 124.3, 127.0, 127.1, 128.3, 128.3, 128.3, 130.4, 131.0, 134.0, 143.2, 147.7, 152.6, 153.5, 154.3, 165.8; MS m/z: 534.0 [M + 1]⁺; anal. elem. calc. for C₂₈H₃₁N₅O₄S: C, 63.02: H, 5.86: N, 13.12: found: C, 63.05: H, 5.83: N, 13.13.

4 Conclusion

To summarize, a series of piperazine conjugates 13a–13j was synthesized in moderate to good yields (61–78%). The antidiabetic potential of all derivatives was determined via in silico and in vitro analysis. The in vitro studies identified 13c and 13e as potent compounds with lower IC₅₀ values i.e., 2.39 ± 0.41 µM and 2.97 ± 0.31 µM against standard acarbose (10.30 ± 0.20 µM). The molecular docking analysis revealed that both 13c and 13e were accurately aligned with in the binding pocket of both α-amylase and PTP1B. They were found to be involved in multiple hydrogen bonding and hydrophobic interactions, indicating strong linkage with important amino acids. Furthermore, FMO analysis disclosed that compounds 13c and 13e exhibited 0.168 eV and 0.158 eV energy gaps. NCI analysis indicated that both 13c and 13e illustrated both H-bonding and van der Waals interactions. The dynamic stability of compounds 13c and 13e was assessed via MD simulation (rGyr, RMSD, RMSF and protein–ligand contact analysis). Furthermore, binding free energy analysis revealed that both compounds 13c and 13e have higher stability within in the binding pocket of PTP1B and α-amylase. Free energy landscape, PCA, and DCCM analysis further validated that 13c and 13e have intense global minima and strong correlation within the active site. From these findings, 13c and 13e turn out to be effective candidates against diabetes. However, further confirmation via in vivo experiments is necessary to validate their effectiveness and safety. Ultimately, these results open a path for the synthesis of anti-diabetic drugs for the treatment of type II diabetes.

Conflicts of interest

The authors have no conflicts of interest for this research work.

Data availability

All data are contained in the manuscript and supplementary information (SI). Supplementary information: ¹³C NMR and ¹H NMR of synthesized derivatives, along with additonal figures supporting in silico studies. See DOI: https://doi.org/10.1039/d6ra00931j.

Acknowledgements

The authors gratefully acknowledge the facilities provided by the Government College University Faisalabad, Pakistan.

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