Structure-Based Optimization and Biological Evaluation of Pancreatic Lipase Inhibitors as Novel Potential Antiobesity Agents

The unusual fused β-lactone vibralactone was isolated from cultures of the basidiomycete Boreostereum vibrans and has been shown to significantly inhibit pancreatic lipase. In this study, a structure-based lead optimization of vibralactone resulted in three series of 104 analogs, among which compound C1 exhibited the most potent inhibition of pancreatic lipase, with an IC50 value of 14 nM. This activity is more than 3000-fold higher than that of vibralactone. The effect of compound C1 on obesity was investigated using high-fat diet (HFD)-induced C57BL/6 J obese mice. Treatment with compound C1 at a dose of 100 mg/kg significantly decreased HFD-induced obesity, primarily through the improvement of metabolic parameters, such as triglyceride levels. Electronic supplementary material The online version of this article (doi:10.1007/s13659-015-0062-6) contains supplementary material, which is available to authorized users.

market for antiobesity drugs is potentially large, accounting for 2-6 % of the total health care costs in several developed countries, and the obesity market has been predicted to grow continuously [5].
Energy intake begins from fat absorption through the digestion of fat into monoglycerides and fatty acids. Lipase is a key enzyme for lipid absorption. Among lipases, pancreatic lipase is responsible for the hydrolysis of 50-70 % of total dietary fats [5]. The reduction of fat absorption through pancreatic lipase inhibition is known to benefit the regulation of obesity [6][7][8][9].
Two drugs, orlistat (a lipase inhibitor) and sibutramine (an appetite suppressant), were used for the antiobesity [10]. These drugs are limited in their use due to their severe side effects, sibutramine has been withdrawn in 2010 duo to increased cardiovascular events from the market in countries including Australia, Canada, China, the United Kingdom, and the United States [11][12][13]. Therefore, reliable and effective antiobesity drugs are urgently required.
Orlistat forms a covalent but reversible bond with the active site serine residue of pancreatic lipase, rendering it unable to hydrolyze dietary fat into free fatty acids, therefore reducing the absorption of dietary fat [14]. We have previously reported the isolation of the unusual fused b-lactone vibralactone from cultures of the basidiomycete Boreostereum vibrans, which exhibited significant potency as a pancreatic lipase inhibitor [15]. Our ongoing investigations on the chemical constituents of the cultures of B. vibrans have led to a series of reports on bioactive vibralactone derivatives [16][17][18][19][20][21][22]. Zhou and Snide developed an elegant 10-step chemical route for the total synthesis of (±)-vibralactone and (-)-vibralactone C [23,24]. The Sieber group established that the unusual fused b-lactone bicyclic system of vibralactone may account for the binding of both types of caseinolytic peptidases that are vital for bacterial virulence [25,26]. Our recent investigation elucidated the biosynthetic pathway, which includes several interesting reactions that may involve unusual enzymes [27]. As shown in Fig. 1, the structure of vibralactone is interesting because it bears similarities to orlistat, which is a natural b-lactone-type lipase inhibitor. The pancreatic lipase inhibitory activity of vibralactone is most likely due to the b-lactone pharmacophore.
To further explore the potential of this unique molecule, a large-scale fermentation of the fungus B. vibrans was performed, and a large amount of vibralactone was isolated. Using the isolated vibralactone as the starting material, molecular modeling, chemical synthesis and biological evaluation were used to optimize the structure of vibralactone against pancreatic lipase. A study was performed to investigate the interactions between vibralactone and human pancreatic lipase. Three key subsites from the crystal structure of human pancreatic lipase were identified in the catalytic site, with which vibralactone interacts. In this study, three series of 104 analogs of vibralactone derivatives were designed and synthesized. All of the synthesized compounds were evaluated for their inhibitory activities against pancreatic lipase in vitro. Compound C1 exhibited the most potent inhibitory activity against pancreatic lipase, with an IC 50 value of 14 nM. This activity is more than 3000-fold higher than that of vibralactone. Compound C1 was selected for further in vivo evaluation. The effect of compound C1 on obesity was investigated in high-fat diet (HFD)-induced C57BL/6 J obese mice. Compound C1 was administered at a dose of 100 mg/kg for 33 days. The antiobesity activity was evaluated by measuring the body weight, epididymal white adipose tissue and metabolic plasma parameters. On day 33, the body weight of the compound C1-treated group was significantly lower compared with that of the HFD-treated group (model group). The metabolic parameters that increased in the HFD group were reduced following administration of compound C1. Particularly, the increased triglyceride levels were significantly reduced in the compound C1treated group. These results indicate that treatment with compound C1 significantly decreased HFD-induced obesity, primarily through the improvement of metabolic parameters, such as triglycerides. Therefore, compound C1, as a potent pancreatic lipase inhibitor, demonstrates potential benefits in the regulation of obesity.

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Structure-Based Optimization and Biological Evaluation of Pancreatic Lipase Inhibitors as… 131

Chemistry
Compounds A1-A54 were prepared as described in Scheme 1. A single long carbon chain was incorporated into the structure of vibralactone. A series of derivatives were synthesized using commercially available carboxyl acids with thionyl chloride in anhydrous dichloromethane. The corresponding acyl chlorides were generated and subsequently treated with vibralactone in the presence of triethylamine to yield compounds A1-A54 (Scheme 1). Compounds A1-A54 were then evaluated for their bioactivity to inhibit pancreatic lipase. Each test was performed in triplicate, and the IC 50 values were calculated based on the amount of inhibitor required to produce 50 % inhibition compared with the DMSO vehicle control [28]. The results are summarized in Table 1.
Compounds B1-B37 were prepared as described in Scheme 2. Vibralactone underwent oxidation to the corresponding aldehyde with pyridinium chlorochromate (PCC), followed by treatment with Grignard reagent to yield the secondary alcohol as a mixture of two diastereoisomers. Preparative HPLC separation was performed, and the two pure diastereoisomers were isolated. The absolute stereochemistry of the products was assigned using the Mosher method [29,30]. A series of vibralactone derivatives containing two long carbon chains was synthesized using commercially available carboxyl acids under standard conditions to generate the corresponding acyl chlorides, which were subsequently treated with the corresponding secondary alcohol in the presence of triethylamine to yield compounds B1-B37. We further designed the third series of vibralactone derivatives (C1-C13), which contained one amide bond. The structures of these derivatives and their synthetic route are shown in Scheme 3. Vibralactone underwent oxidation to the corresponding carboxyl acid with the Jones reagent, followed by treatment with (COCl 2 ) to generate the corresponding acyl chloride, which was subsequently treated with commercially available secondary amines in the presence of triethylamine to yield the N,N-dialkyl amide derivatives [31].

Biological Evaluation
The first series of compounds (A1-A54) was less potent than the other two series but exhibited significant pancreatic lipase inhibitory activities (Table 1). Compound A1 exhibited the highest inhibitory activity of this series, with an IC 50 value of 0.083 lM, whereas the IC 50 value of vibralactone is 47.26 lM. Vibralactone derivatives are active site-directed inhibitors that form stoichiometric long-lived acyl-enzyme complexes with pancreatic lipase following nucleophilic attack by the catalytic serine residue on the b-lactone group. An appropriately long carbon chain that enhances a compound's solubility in oil causes it to partition between the oil core and the lipid-water interface. Increasing the interfacial area through an emulsification process promotes a stronger diffusion of the compound from the oil core toward the interface.
As shown in Table 2, the second series of vibralactone derivatives was substantially more potent than the first series. Compound B1 exhibited the highest inhibitory activity of the second series, with an IC 50 value of 0.030 lM against pancreatic lipase. Several other compounds (compounds B1-B9) in this series exhibited higher pancreatic lipase inhibitory activities than that of compound A1, which was the best inhibitor of the first series. The rationale for the design of this second series of derivatives was to include two appropriately positioned long carbon chains to effectively occupy the hydrophobic pocket. This molecular design strategy may lead to molecules with enhanced pharmacological properties. The pancreatic lipase inhibitory activities of the third series of vibralactone derivatives are summarized in Table 3. Compound C1 exhibited the highest inhibitory activity among the three series, with an IC 50 value of 0.014 lM. Notably, two other compounds in this series, compounds C2 and C3, exhibited even higher inhibitory activities than compound A1.

Effects of Compound C1 on High-Fat Diet-Induced
Obese Mice The most active compound, C1, was selected for further study in vivo to evaluate its ability to cause body weight loss and to analyze possible side effects in mice [32]. After one month of continuous administration of either compound C1 (100 mg/kg) or orlistat (50 mg/kg), the total body weight (Fig. 2a) and the fat weight (epididymal white adipose tissue, Fig. 2b) of treated mice gradually decreased during this period, indicating that compound C1 can reduce the body weight of HFD-induced obese mice. The levels of triacylglyceride (TG) and cholesterol (CHO) are typically higher in obese animals. Lower levels of these two markers are considered important indicators for weight loss. Therefore, we further determined these levels in mice treated with compound C1 or orlistat and observed that these levels significantly decreased (Fig. 2c, d, respectively), further supporting that compound C1 reduced the body weight along with the TG and CHO levels.
Consistent with these in vitro results, compound C1, despite having a lower activity than that of orlistat in vivo, demonstrated efficacy in this model, with positive effects on weight loss throughout the duration of the studies without toxicity or adverse behavioral effects (up to 400 mg/kg, data not shown).

Molecular Modeling Studies
To gain further insight into the binding mode of the reported compounds, a series of docking experiments was  [36] in which pancreatic lipase adopts an active conformation with the b5 loop positioned away from the catalytic site. Hydrogen atoms were added, and water molecules that cocrystallized with the protein were removed from the original structure. This modified crystal structure of pancreatic lipase was used as the target for docking simulations using GOLD 5.2.2 software (CCDC, Cambridge, U.K.). The active site radius is 15 Å from OG atom 2376 of Ser153, which is one of the key residues in this serine protease. Nucleophilic attack on the b-lactone ring, which is the pharmacophore for this type of inhibitor, by the lipase active site serine residue is thought to form the longlived acyl-enzyme complex. Therefore, a restraint was used to limit the distance between the carbonyl carbon and the oxygen in Ser153. The ''Library screening'' parameters and 30 GA runs were used for each ligand.
To understand the interactions between the inhibitors and pancreatic lipase, the first round of docking simulations was performed using orlistat and vibralactone versus the crystal structure of pancreatic lipase. Figure 3 shows the binding conformation of orlistat (left) and vibralactone (right) in the active site. Three subsites in the catalytic site (indicated by three red circles) were found to be important for the interaction with the inhibitors. Subsites 1 and 3 comprise hydrophobic residues, whereas subsite 2 consists of hydrophobic residues and the charged residues Asp79, Glu83 and Arg256 (blue surface). As observed in the binding mode of orlistat, a good inhibitor should extensively interact with all three subsites. The binding mode obtained from the docking simulation was further used for structure optimization of vibralactone. Based on the docking results, the structure of vibralactone has space to expand in the three directions to optimize inhibitor interactions with pancreatic lipase. Therefore, the structure of vibralactone was accordingly optimized.
The vibralactone derivatives A1-A54 containing one long carbon chain toward subsite 2 were designed and synthesized, and the corresponding inhibitory activities against pancreatic lipase validated our strategy. As shown in Fig. 4b, the long chain of compound A1 occupies subsite 2 very well, which presented an inhibitory activity of 0.083 lM. Subsequently, derivatives containing two long carbon chains that could simultaneously interact with subsites 2 and 3 were synthesized. The docking conformation of derivative B1 in the catalytic site of pancreatic lipase indicated that the two chains could properly interact with subsites 2 and 3 ( Fig. 4c), with an IC 50 value of 0.030 lM. Considering that subsite 2 is partially charged by the residues Asp79, Glu83 and Arg256, two long chain N,Ndialkyl amide derivatives were designed and synthesized to hydrophilically interact with the lipase. Finally, we obtained thirteen amide derivatives, and the most active derivative is shown in Fig. 4d, exhibiting an IC 50 value of

Conclusion
In summary, three series of 104 vibralactone-based analogs were designed by altering the length and functionality of the chain linking the 3-position of the vibralactone moiety. All of the synthesized compounds were evaluated for their inhibitory activities against pancreatic lipase in vitro. Compound C1 appeared to be the most potent inhibitor of pancreatic lipase activity, with an IC 50 value of 14 nM, which is more than 3000-fold higher than that of vibralactone. Compound C1 was selected for further evaluation in vivo. The effect of compound C1 on obesity was investigated using HFD-induced C57BL/6 J obese mice. Compound C1 was administered at a dose of 100 mg/kg for 33 days. The antiobesity activity was evaluated by measuring the body weight, epididymal white adipose tissue and metabolic plasma parameters. From day 6 to day 33, the body weight of the compound C1-treated group was significantly low compared with the HFD-treated group. The metabolic parameters that increased in the HFD group decreased in the compound C1-treated group. Particularly, the increased triglyceride levels were significantly reduced in the compound C1-treated group. These results indicate that treatment with compound C1 decreased HFD-induced obesity, primarily through the improvement of metabolic parameters, such as triglyceride and cholesterol levels. Therefore, compound C1, as a potent pancreatic lipase inhibitor, demonstrates potential benefits in the regulation of obesity. Although the investigated compounds were less potent than orlistat and no data was reported showing that they are safer than orlistat at moment, but this provides a fact that this type of compounds can be optimized and the possibility for further optimization to find better antiobesity agents.

Materials Used for Chemical and Biological Experiments
All of the chemicals and reagents that were commercially available were purchased from Sigma-Aldrich and Acros and were used without further purification. All of the solvents were purified and dried using standard techniques and were distilled prior to use. All of the reactions were performed under a nitrogen atmosphere using oven-baked glassware unless otherwise noted. Flash chromatography was performed using mesh silica gel (200-300 mesh).

Vibralactone
The culture broth was filtered to remove the mycelium. The filtrate (500 L) was then successively extracted twice with ethyl acetate. The crude extract (150 g) was chromatographed on silica gel (200-300 mesh) and eluted with a gradient of petroleum ether/acetone to yield the vibralactone (10.5 g).

General Procedure to Synthesize Compounds A1-A54
To a stirring solution of the appropriate carboxyl acids (0.12 mmol) in CH 2 Cl 2 (2 mL) at 0°C, thionyl chloride (0.6 mmol) was added dropwise. The reaction was monitored by TLC, and following the complete reaction of the starting material, the reaction mixture was concentrated to yield a brown-yellow oil. To a solution of vibralactone (0.1 mmol) in dichloromethane (2 mL) at 0°C was added Et 3 N (0.2 mmol) and the corresponding acyl chloride dissolved in dichloromethane (2 mL). The reaction mixture was stirred at room temperature overnight. A saturated NH 4 Cl solution was added to quench the reaction, and the mixture was extracted with CH 2 Cl 2 (3 9 10 mL). The combined organic layers were dried over MgSO 4 and concentrated in vacuo. The products were purified using flash chromatography on silica gel.            SO 4 , filtered and concentrated in vacuo. The resultant oil was purified by chromatography on silica gel using petroleum ether/ethyl acetate (10:1) as the eluent, yielding the diastereoisomers. Preparative HPLC separation yielded the two pure diastereoisomers, and the absolute stereochemistry of the hydroxyl group in the products was assigned using the Mosher method. The synthesis of the Mosher ester derivatives was achieved with one of the diastereoisomers using 3. To a stirring solution of the appropriate carboxyl acids (0.12 mmol) in CH 2 Cl 2 (2 mL) at 0°C was added thionyl chloride (0.6 mmol) dropwise. The reaction was monitored Structure-Based Optimization and Biological Evaluation of Pancreatic Lipase Inhibitors as… 147 by TLC, and following the complete reaction of the starting material, the reaction mixture was concentrated to yield a brown-yellow oil. To a solution of (1R,5S)-3-(1-hydroxyalkyl)-1-(3-methylbut-2-en-1-yl)-6-oxabicyclo[3.2.0]hept-2-en-7-one (0.1 mmol) in dichloromethane (2 mL) at 0°C was added Et 3 N (0.2 mmol) and the corresponding acyl chloride dissolved in dichloromethane (2 mL). The reaction mixture was stirred at room temperature overnight. A saturated NH 4 Cl solution was added to quench the reaction, and the mixture was extracted with CH 2 Cl 2 (3 9 10 mL). The combined organic layers were dried over MgSO 4 and concentrated in vacuo. The products were purified using flash chromatography on silica gel.  , housed in a temperature-and humidity-controlled room with a 12-hour light/dark cycle, and allowed free access to solid food and tap water for one week prior to the experiments. All of the procedures were performed in accordance with the Institute Ethical Committee for Experimental Animal Use of the Yunnan Province and the Kunming Institute of Botany.

Reagents
The kits used to determine the triglyceride (TG) and cholesterol (CHO) levels were obtained from the Zhongsheng Beikong Bioengineering Institute.

Diet-Induced Obesity in Mice
Three-week-old C57BL/6 J male mice were fed a normal diet for 1 week for acclimatization. Randomly selected mice were fed a normal diet or a 45 % fat diet. After 4 months, the body weights of the mice in the obese group were 20 % higher than those in the control group. These obese mice were randomly divided into the following three groups: the model (45 % fat diet), orlistat (45 % fat diet ? orlistat) and Compound groups (45 % fat diet ? compound C1). The drugs (50 mg/kg orlistat or 100 mg/kg compound C1) were intragastrically administered for 1 month. During this period, the mice were weighed once every 3 days. Following administration of the final dose, blood samples were collected via the orbital vein after ethyl ether exposure, and epididymal fat was isolated. TG and CHO levels were measured using kits. The ratio of epididymal fat to body weight represents the amount of white adipose tissue (WAT) of each mouse (normalized to the body weight).

Statistical Analysis
Student's t test was used for comparisons between groups, as indicated in Fig. 2. #p \ 0.05, ## or **p \ 0.01.