Identification of Novel Piperidine and Pyrrolidine Derivatives as Potent Inhibitors of Pancreatic Lipase-Based Molecular Docking and In Vitro Testing

Abstract

Obesity is a major global health concern associated with increased risks of chronic diseases and mortality. Inhibiting pancreatic lipase, a key enzyme in dietary fat absorption, presents a promising therapeutic approach. This study aimed to evaluate the inhibitory potential of piperidine derivatives (1 and 2) and pyrrolidine derivatives (3–13) against pancreatic lipase (PL) through both enzymatic assays and molecular docking simulations. Among the tested compounds, compound 12 demonstrated the highest PL inhibitory activity with IC₅₀ 0.143 ± 0.001 mg/mL and the strongest binding energy (−8.24 kcal/mol), attributed to extensive hydrogen bonding with Gly76, Phe77, Asp79, and His151. Compounds 10 and 13 also exhibited notable inhibitory activity, attributed to their extensive hydrogen bond network with residues Gly76, Phe77, Asp79, and His151. Particularly the presence and position of hydroxy and carbonyl groups and the length of alkyl side chains critically influenced binding stability and specificity. These findings demonstrate that specific structural modifications in pyrrolidine derivatives significantly affect pancreatic lipase inhibition. Compound 12, with its optimal molecular architecture and interaction profile, stands out as the most promising candidate for further development as an anti-obesity agent, with compounds 10 and 13 offering additional scaffolds for future optimization.

1. Introduction

Obesity is a major health concern and is defined as abnormal or excessive fat accumulation, which can cause the risk of chronic diseases, both illness and mortality. The prevalence of overweight and obesity increased steadily across the globe, in every region and country, from 1990 to 2021. Based on these ongoing trends, it’s predicted that by 2050, the total number of adults living with overweight or obesity will surpass 3.80 billion, representing more than half of the projected global adult population at that time [1]. Treatment typically begins with lifestyle adjustments like diet and exercise. Currently, several anti-obesity drugs like orlistat and sibutramine are available as a next step for patients who are unresponsive to initial conservative management [2]. The effectiveness and side effect profiles of these medications vary. Orlistat, a lipase inhibitor, is primarily linked to gastrointestinal issues and has no effects on the nervous system (CNS), whereas sibutramine’s side effects include CNS effects, nausea, vomiting, heart palpitations, and sweating [3]. The development of novel pharmacological agents with minimal or no side effects is of particular interest, especially as part of long-term strategies targeting both prevention and treatment of obesity. Understanding how derivatives interact with enzyme active sites enables the optimization of their inhibitory potency and specificity. This optimization process involves modifying the chemical structure of potential compounds to enhance their binding affinity and selectivity towards the target enzymes while minimizing off-target effects [4,5].

Pyrrolidine and piperidine, as saturated nitrogen-containing heterocyclic scaffolds, hold substantial importance in medicinal chemistry [6,7]. Their widespread utilization stems from their inherent structural flexibility and frequent occurrence in both natural products and diverse bioactive compounds [6,7]. These five-membered (pyrrolidine) and six-membered (piperidine) rings are recognized as privileged frameworks, adept at engaging various biological targets through hydrogen bonding and hydrophobic bonding [4,5]. Both scaffolds are associated with a diverse range of pharmacological actions, including anti-inflammatory, antimicrobial, and antidiabetic effects [7,8]. Their integration into potential drug molecules has been shown to enhance bioavailability, target specificity, and pharmacokinetic properties [6]. Pyrrolidine and piperidine derivatives represent promising candidates for developing novel pancreatic lipase inhibitors. Strategic structural modifications, such as functional group substitution or aromatic fusion, can further optimize their lipophilicity and binding affinity, thereby supporting their significant role in anti-obesity drug discovery.

Preussin (originally named asperidine B) was isolated from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178. Notably, its structure was initially proposed as a piperidine but was later revised to that of a known pyrrolidin-3-ol alkaloid. It displays the promising and potent cholesterol-lowering effects [9]. Furthermore, the synthetic 2,6-disubstituted piperidin-3-ol derivatives (1 and 2) exhibited a lipid-lowering effect [10]. Interestingly, modifying the chemical structure of potential or synthetic compounds offers a promising strategy to enhance inhibitory activity while minimizing side effects. By exploring structural variations in piperidine and pyrrolidine derivatives. This study employed molecular docking simulations to evaluate two piperidine derivatives (1 and 2) and eleven pyrrolidine derivatives (3–13) for their potential to bind and inhibit pancreatic lipase. Our primary objective was to identify potent inhibitors that could serve as foundational structures for developing next-generation anti-obesity therapeutics.

2. Materials and Methods

Porcine pancreatic lipase (type II), propanesulfonic acid (MOPS), orlistat, and p-nitrophenyl butyrate (p-NPB) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Piperidine derivatives (1 and 2) and pyrrolidine derivatives (3–13) were synthesized using our reported procedure [10,11]. The purity of all tested compounds was confirmed by NMR spectroscopy. All other chemical reagents used in this study were of analytical grade.

2.1. Molecular Docking Studies

The interaction between compounds 1–13 and pancreatic lipase was conducted utilizing the AutoDock 4.2 software developed by The Scripps Research Institute, USA. The X-ray crystallographic structure of pancreatic lipase was procured from the Protein Data Bank (www.rcsb.org (accessed on 17 October 2024), accessed on LAPTOP-0753RSFT, PDB ID: 1LPS). The ligands examined in this investigation include two piperidine derivatives (1 and 2) and 11 pyrrolidine analogs (3–13). These compounds were represented in three-dimensional structure through the use of ChemDraw Ultra (version 12.0.2) software. The Lamarckian Genetic Algorithm was employed to ascertain optimal ligand binding conformations, facilitating the accommodation of flexible ligands within rigid protein binding sites. The parameters for AutoDock were configured as follows: the total number of genetic algorithm runs was established at 100, the population size was designated as 150, and the maximum number of energy evaluations was augmented to 2,500,000 per run. All other parameters for the run were maintained at their default configurations. The final docked conformations underwent clustering utilizing a tolerance of 2 Å RMSD. The molecular docking outcomes were evaluated based on the criteria of binding structure, binding energy, and potential interactions between the ligand and the pivotal residues of the protein.

2.2. Pancreatic Lipase Activity Assay

The inhibitory activity of pancreatic lipase was evaluated utilizing p-nitrophenyl butyrate (p-NPB) as the substrate, in accordance with the previously established methodology with slight modifications [12]. Reaction mixtures comprising compounds (at various concentrations) or orlistat and lipase (dissolved in 10 mM MOPS and 1 mM EDTA (pH 6.8) to 100 mM Tris-HCl and 5 mM CaCl₂ (pH 7.0)) were subjected to incubation at 37 °C for a duration of 15 min. Following this incubation period, the reaction was initiated by the addition of the substrate (10 mM p-NPB in dimethylformamide). After a subsequent incubation at 37 °C for 30 min, the absorbance values were recorded at 405 nm using a microplate reader. All samples underwent analysis in triplicate. The percentage of inhibition was determined in accordance with the following specified formula:

$${\displaystyle \frac{\mathrm{\%}\mathrm{ }\mathrm{p}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{ }\mathrm{l}\mathrm{i}\mathrm{p}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{ }\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=[{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}}_{\left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)}-{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}}_{\left(\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\right)}]}{{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}}_{\left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)}}}$$

Then, the values were represented in IC₅₀ value.

2.3. Statistical Analyses

Data are expressed as Mean  ±  SEM. IC₅₀ values, defined as the concentration inducing 50% inhibition, were estimated using GraphPad Prism (version 5) through nonlinear regression (inhibitor vs. normalized response-variable slope model). Analysis of Variance (ANOVA) determined significant differences among replicate means, with p < 0.05 indicating statistical significance.

3. Results

3.1. Chemical Structures

The chemical structures of two piperidine derivatives (1 and 2) and 11 pyrrolidine analogs (3–13) are depicted in Figure 1. Compound 2 was an N-methyl derivative of the piperidine 1. The pyrrolidine analogs include the C3-(R)-hydroxy (3), the C3-(S)-methoxy (4), the NH (5), the n-nonyl side chain with hydroxy and carboxyl groups (6–9), the n-heptyl side chain (10) with hydroxy and carboxyl groups (11 and 12) and the n-pentyl side chain (13). Additionally, compounds 5 and 7–11 are previously reported natural products.

3.2. Pancreatic Lipase Activity

The effect of these compounds on the activity of pancreatic lipase was monitored at saturation conditions, using p-NPB as a substrate. The pancreatic lipase activities of piperidine derivatives (1 and 2), pyrrolidine derivatives (3–13), and orlistat are depicted in

Table 1. Inhibitory effects (IC₅₀ values) of piperidine derivatives (1 and 2) and pyrrolidine derivatives (3–13) on pancreatic lipase.

Compounds	Pancreatic Lipase Inhibition (IC50; mg/mL)	Compounds	Pancreatic Lipase Inhibition (IC50; mg/mL)
Orlistat	0.0005 ± 0.0005	7	0.329 ± 0.001
1	>10	8	0.655 ± 0.001
2	>10	9	0.591 ± 0.001
3	4.045 ± 0.003	10	0.362 ± 0.001
4	0.769 ± 0.001	11	1.837 ± 0.001
5	1.314 ± 0.001	12	0.143 ± 0.001
6	0.846 ± 0.001	13	0.226 ± 0.001

Mean ± SEM.

. Among the tested compounds, compound 12 with an n-heptyl alcohol side chain showed the highest pancreatic lipase inhibitory activity with an IC₅₀ value of 0.143 ± 0.001 mg/mL, whereas compounds 7, 10, and 13 with an n-nonanoic acid, n-heptyl, and n-pentyl side chains, respectively, displayed significant potential with the IC₅₀ values of 0.329 ± 0.001, 0.362 ± 0.001, and 0.226 ± 0.001 mg/mL, respectively. Orlistat, the standard drug, showed the IC₅₀ value of 0.0005 ± 0.0005 mg/mL (1.06 ± 1.053 µM).

3.3. Molecular Docking

The redocking of the crystallized ligand into the binding pocket of the target protein yielded an RMSD value of 1.88 Å between the experimental and predicted poses. The protein template and docking parameters used in this study are considered robust and appropriate for subsequent docking analyses. The binding affinity, % member in cluster, and H-bond interaction in a protein-binding context of all compounds are demonstrated in

Table 2. Molecular interaction of piperidine derivatives (1 and 2), pyrrolidine derivatives (3–13), and orlistat with pancreatic lipase.

Compounds	Binding Energy (kcal/mol)	% Member in Cluster	H-Bond Interaction
Orlistat	−5.72	8	-
1	−6.59	32	O-H---O=C (Phe77)
2	−6.27	33	-
3	−7.42	57	-
4	−7.20	28	-
5	−6.87	21	O-H---O=C (Phe77)
6	−6.19	16	-
7	−6.79	20	C=O---H-N (His263)
8	−7.43	15	H-O---H-N (Gly76) O-H---O=C (Asp79) H-O---H-N (His151)
9	−7.20	12	H-O---H-N (Gly76) O-H---O=C (Asp79) H-O---H-N (His151)
10	−7.39	50	H-O---H-N (Phe77) H-O---H-O (Ser152)
11	−6.78	27	C=O---H-N (Gly76) C=O---H-N (His151)
12	−8.24	32	H-O---H-N (Gly76) O-H---O=C (Phe77) O-H---O=C (Asp79) H-O---H-N (His151)
13	−7.24	83	H-O---H-N (Phe77) H-O---H-O (Ser152)

Mean ± SEM.

. Compound 12 shows the highest pancreatic lipase inhibitory activity stands out with the highest binding energy of −8.24 kcal/mol, indicating the strongest binding compared to other compounds. It has a 32% member presence in the cluster. It forms hydrogen bond interactions, such as H-O---H-N and O-H---O=C, with specific amino acid residues, including Gly76, Phe77, Asp79, and His151, marked by green dotted lines (Figure 2). The hydrogen bond with Gly76 secures an initial positioning of compound 12, while additional bonds with Phe77 and Asp79, involving their carbonyl groups, enhance stability by creating multiple attachment points. Finally, the interaction with His151 provides further reinforcement, suggesting a highly stable and energetically favorable binding. Together, these hydrogen bonds demonstrate a strong affinity of compound 12 for this protein site, ensuring it is well-stabilized within the binding environment, making this compound a potentially effective compound for binding to this protein target compared to other compounds. Compounds 3, 4, 8–10, and 13 display similar binding energy in the range of –7.20 to −7.43 kcal/mol. However, only compounds 3, 10, and 13 had 50–83% of their predicted binding poses grouped within the same molecular docking cluster, indicating a relatively consistent binding orientation. Additionally, molecular docking simulations revealed that both compounds 10 and 13, but not compound 3, bind favorably to the active site of pancreatic lipase, interacting with the catalytic triad and nearby residues through hydrogen bonding and hydrophobic contacts (Figure 3). Compound 10 exhibited a high binding energy at –7.39 kcal/mol, suggesting a stronger binding affinity. Hydrogen bonding interactions were observed with Phe77 and Ser152, with 50% docking conformations falling into the dominant binding cluster, indicating moderately consistent binding. Compound 13 with similar binding energy (–7.24 kcal/mol) had a higher percentage of cluster members (83%), implying greater conformational consistency and stability in binding. It also formed hydrogen bonds with the same amino acid residues (Phe77 and Ser152) as those of compound 10. These findings demonstrate that compounds 10, 12, and 13 possess favorable molecular interactions with stabilizing interactions and the potential to inhibit pancreatic lipase activity.

4. Discussion

The study emphasizes that variations in the molecular architecture of piperidine and pyrrolidine compounds are critical for their pancreatic lipase inhibitory activity. A key distinction appears to be the size of the ring: six-membered piperidine rings (1 and 2) versus five-membered pyrrolidine rings (3–13). In drug design, piperidines can interact directly with enzymes, or they can serve as a flexible structural unit to optimize the drug’s conformation and physical properties [13]. Pyrrolidine-triazole-aurone hybrids have emerged as potential anti-obesity agents [14]. Hybrid aurones are more potent lipase inhibitors when their phenyl ring is either unsubstituted or substituted with electron-releasing groups such as -CH₃ or -OCH₃, compared to those with electron-withdrawing halogen substituents [14]. Amino acid residues, SER105 and SER140, form hydrogen bonds in lipase’s active site [14]. Differences in ring size can influence their orientation, steric bulk, and affinity for the enzyme’s active site [15]. The presence of specific functional groups, such as hydroxy (-OH) and carbonyl (-CO-), on these rings is likely critical in either enhancing or hindering these interactions. When the compounds bound to other active amino acids, they triggered a conformational change in the enzymes. This change then affected the enzyme–substrate interaction, ultimately resulting in either a boost or a reduction in the enzyme’s activity [16]. Pancreatic lipase (PL) is composed of two functional regions. The N-terminal domain adopts an α/β hydrolase fold and contains the enzyme’s active site, which includes a catalytic triad much like those seen in serine proteases [17]. At its core, the enzyme’s function in digesting lipids relies on an active site with a catalytic triad of Ser152, Asp176, and His263, mirroring those found in serine proteases. This setup is key to breaking down triglycerides. A unique surface loop, referred to as the lid, plays a vital role in controlling substrate entry to the active site [17]. The active site of pancreatic lipase is designed with a hydrophobic groove that binds to the acyl chain of triglyceride substrates [18]. The presence of inhibitors, for instance, C11 alkyl phosphonate, triggers changes in the shape of surface loops, which in turn improves access to the catalytic triad [18]. The inhibitor forms a strong bond with Ser152 at the active site, and His263 likely plays a role in breaking down the acyl-lipase intermediate. This structural setup is key to the enzyme’s catalytic activity and substrate binding [18]. Orlistat is a powerful drug that specifically targets and blocks gastric and pancreatic lipases, enzymes essential for digesting dietary fat. It binds covalently to the serine residue of the active site of these lipases [19].

Pyrrolidine derivatives demonstrated stronger inhibition of pancreatic lipase than piperidine derivatives. This difference can be attributed to the smaller and more conformationally flexible pyrrolidine ring, which facilitates a better fit into the active site pocket of pancreatic lipase. In addition, the orientation of functional groups such as hydroxy (–OH) and carbonyl (–CO–) in pyrrolidine derivatives may enhance hydrogen bonding and hydrophobic interactions, thereby improving binding affinity and leading to lower IC₅₀ values compared with the corresponding piperidine derivatives. This study identified compound 12 as the most potent inhibitor of pancreatic lipase among the tested compounds. In comparison, the reference drug orlistat exhibited significantly higher potency. However, despite the greater inhibitory activity of orlistat, it is important to note that compound 12 still demonstrated promising activity, particularly considering it as a novel synthetic compound. This effect is likely attributed to its distinct molecular architecture, specifically, the combined influence of its pyrrolidine ring and its associated functional groups facilitating a more optimal fit and interaction within the enzyme’s active site. The high inhibitory potential of compound 12 is underscored by its strong binding affinity and the inherent stability of its complex with the lipase enzyme, reflected by a high binding energy. At the molecular level, compound 12 engages in critical hydrogen bonding interactions with key amino acid residues: Gly76, Phe77, Asp79, and His151. These interactions establish a robust and stable network that effectively anchors compound 12 in an optimal orientation. Each hydrogen bond contributes significantly to the overall stability and precise positioning of compound 12, thereby enhancing its binding efficacy. For instance, the interaction with Gly76 likely provides the initial stabilization point, while additional bonds with Phe77 and Asp79 are instrumental in securing compound 12 in a conformation highly conducive to lipase inhibition. The interaction involving His151 further strengthens this multi-point attachment, culminating in a highly energetically favorable and stable enzyme-inhibitor complex. This pattern of multi-point hydrogen bond formation suggests that the remarkable potency of compound 12 directly correlates with its ability to establish these reinforcing interactions, which are critical for its sustained positioning and effective blockade of lipase enzymatic activity. The strongest performance of compound 12 suggests that adding or repositioning groups like hydroxyl and carbonyl to boost hydrogen bonding could further optimize how they interact. Compounds 10 and 13 also demonstrate notable inhibitory properties, suggesting moderate to high potential. The observed variability in binding energies and inhibitory activities among the compounds highlights the significance of binding flexibility. Compounds exhibiting strong binding energies can still be effective for inhibitors if they maintain essential hydrogen bonds and a favorable orientation within the active site. The IC₅₀ of compound 12 is weaker than that of orlistat, reflecting differences in potency. This discrepancy can be attributed to both structural and mechanistic factors. In addition, the gastrointestinal side effects commonly associated with orlistat are largely attributed to its covalent and irreversible inhibition of pancreatic lipase. In contrast, our synthetic compounds interact with the enzyme through non-covalent binding modes, which are typically reversible. This type of interaction may reduce the risk of prolonged enzyme inactivation. This suggests a potential advantage over orlistat in minimizing gastrointestinal side effects. The inhibitory performance cannot be fully captured by conventional affinity metrics alone but requires consideration of the broader molecular context. This insight suggests that beyond sheer binding strength, structural adaptability and specific molecular interactions are also crucial determinants of inhibitory efficacy. Moreover, although the present study primarily focused on the structural optimization and biological evaluation of the lead compounds, consideration of their pharmacokinetic and safety profiles (ADMET properties) is crucial for further translational development. Recent reports have highlighted that poor solubility and limited bioavailability often restrict the therapeutic potential of small molecules [20]. Nanoparticle-based drug delivery systems represent promising approaches to overcome these challenges by enhancing stability, controlled release, and targeted delivery [20]. Incorporating these perspectives into future work could facilitate the progression of compound 12 and related analogs toward preclinical validation.

5. Conclusions

In conclusion, this study establishes a valuable understanding of how specific structural characteristics of piperidine (six-membered rings) and pyrrolidine derivatives (five-membered rings), as saturated nitrogen-containing heterocyclic scaffolds, influence their capacity to inhibit pancreatic lipase. In particular, hydrogen bonding interactions, involving functional groups such as hydroxy (–OH) and carbonyl (–CO–), play an important role in this activity. Taken together, the results suggest that compound 12 is the most promising candidate, exhibiting both the highest inhibitory potency and the strongest enzyme-ligand interactions. However, compounds 10 and 13 also possess desirable pharmacological profiles, particularly in terms of binding stability and interaction specificity, and may serve as viable leads for further optimization in the development of novel pancreatic lipase inhibitors.