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. 2018 Feb 21;9(3):274–278. doi: 10.1021/acsmedchemlett.8b00050

Stereochemical Structure Activity Relationship Studies (S-SAR) of Tetrahydrolipstatin

Xiaofan Liu 1, Yanping Wang 1, Richard I Duclos Jr 1,*, George A O’Doherty 1,*
PMCID: PMC5846039  PMID: 29541373

Abstract

graphic file with name ml-2018-000509_0009.jpg

Tetrahydrolipstatin (THL), its enantiomer, and an additional six diastereomers were evaluated as inhibitors of the hydrolysis of p-nitrophenyl butyrate by porcine pancreatic lipase. IC50s were found for all eight stereoisomers ranging from a low of 4.0 nM for THL to a high of 930 nM for the diastereomer with the inverted stereocenters at the 2,3,2′-positions. While the enantiomer of THL was also significantly less active (77 nM) the remaining five stereoisomers retained significant inhibitory activities (IC50s = 8.0 to 20 nM). All eight compounds were also evaluated against three human cancer cell lines (human breast cancers MCF-7 and MDA-MB-231, human large-cell lung carcinoma H460). No appreciable cytotoxicity was observed for THL and its seven diastereomers, as their IC50s in a MTT cytotoxicity assay were all greater than 3 orders of magnitude of camptothecin.

Keywords: tetrahydrolipstatin, pancreatic lipase, diastereomers, inhibitory activity, cytotoxicity, structure activity relationship

Tetrahydrolipstatin (THL, 1)1,2 is an FDA approved oral antiobesity drug that inhibits pancreatic lipase digestive enzyme.3−5 Inhibition of pancreatic lipase in the intestinal lumen results in the inability to digest dietary triacylglycerides (fat) and diacylglycerides to free fatty acids and monoacylglycerides that can be absorbed into the bloodstream and lymphatic system. Dietary fat and diacylglycerides are then eliminated as bodily waste without the caloric consequences of their taste and consumption. As part of an effort aimed at developing a novel treatment for pancreatitis, we became interested in structure activity space around THL (1). Pancreatitis is believed to result from the excessive release of active pancreatic digestive enzymes.6 As THL (1) is known to inhibit pancreatic lipase by covalent modification, it was identified as lead structure in our program aimed at capturing and removing excess pancreatic lipase.

THL (1) is known to be a potent inhibitor of porcine pancreatic lipase hydrolysis of triolein (fat) emulsion (IC50 of 360 nM).1 The mechanism of activity for THL (1) has been shown to occur via ring opening of the β-lactone by a lipase active site serine to form a stable ester.7−9 When the hydrolysis of the esterified serine is slow, this covalent modification of the lipase enzyme leads to essentially irreversible inactivation.

Previously, THL has been investigated in vivo for the treatment of pancreatitis by intravenous,10 intraperitoneal,11−13 and direct injection into pancreatic tissue.10−14 In addition, THL has been found to inhibit the thioesterase domain of fatty acid synthase (FAS)9,15,16 and has been investigated as anticancer chemotherapy.15−24 THL has been shown to inhibit the in vitro growth of Giardia duodenalis, a gastrointestinal parasite.25 THL may also have value as an antifungal agent against fungi that lack lipase enzymes and rely on free fatty acids for their nutrition and growth.26,27

In an effort to find novel structures that covalently modified pancreatic lipase, we decided to explore the structural space around THL (1). While there had been some structure activity relationship studies (SAR) of the lipstatins, there have been fewer studies of its stereoisomers. The syntheses of some THL diastereomers have been reported (e.g., 2, 3, (ent)-2, (ent)-4, and the 3-epi-THL), along with a smattering of lipase activities (e.g., 2, 3, and the 3-epi-THL).16,23,28−30 For instance, the C2′ epimer 2, was reported to be less potent than THL (1) in a diacylglycerol lipase assay.29 Similarly, the cis-β-lactone isomer (3-epi-THL)23 has been evaluated for lipase inhibitory activity (IC50 = 15 nM). However, no systematic study of the trans-β-lactone stereoisomers has been reported. Thus, we identified seven trans-β-lactone stereoisomers of THL (2-4 and (ent)-1–4) as compounds for a convenient method to identify the structural space around the enzyme active site (Figure 1). Importantly, these types of comprehensive stereochemical-SAR studies (S-SAR) of natural products and their derivatives, require an asymmetric synthetic approach to the structure of interest.31−37

Figure 1.

Figure 1

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Structures of THL (1) and seven diastereomers.

Over the years there have been numerous syntheses of THL and related molecules.38−41 Our own successful synthesis of THL was of the de novo asymmetric variety and as a result was stereodivergent in its approach. Specifically, it used a catalytic asymmetric reduction of achiral ynone 7 to install the initial stereocenter (Scheme 1), followed by a diastereoselective epoxidation of 6 and regioselective Coates carbonylation42−48 of 5 to afford either enantiomer of the four diastereomers (1–4).49 Key to the success of this stereodivergent approach was the recognition that all eight THL stereoisomers (1–4 and (ent)-1–4) could be diastereo- and regioselectively prepared from the homoallylic epoxide 8 and its enantiomer in only three steps (Scheme 2).

Scheme 1. Retrosynthetic Analysis for THL.

Scheme 1

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Scheme 2. Asymmetric Approach to THL and Seven Stereoisomers.

Scheme 2

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Herein we report the evaluation of the eight THL diastereoisomers (1–4 and (ent)-1–4). All the stereoisomers were evaluated for their lipase inhibitory properties using porcine pancreatic lipase (PPL). To ensure that the various stereochemical inversions on THL did not introduce any unwanted toxicities, the cytotoxicity of all eight stereoisomers were determined against three cancer cell lines (H460, MCF-7, and MDA-MB-231).

Porcine pancreatic lipase is frequently used,7,50,51 since the active site of the porcine lipase is highly homologous to the human lipase (Scheme 3).52 Porcine pancreatic lipase enzyme concentrations were varied between the range of 0.50 to 10.0 μg/200 μL in buffer solution to determine the optimal enzyme concentration of 5.0 μg/200 μL. This concentration was consistent with values used for a related assay.51 The concentration of the lipase substrate, p-nitrophenyl butyrate (p-NPB),53 in DMSO/buffer solutions were varied from 0.25 mM to 1.0 mM without any deleterious effects on IC50 values. Solubility issues became apparent at higher concentration, as such, all assays were run with a concentration of 0.25 mM p-NPB. A 10% DMSO concentration was used, which was found to be sufficient for maintaining homogeneous solutions at all assay concentrations. Using this assay, the Km (Michaelis–Menten constant) for our PPL was determined to be 2.41 mM (see Supporting Information) which corresponds well with a related assay.54

Scheme 3. Hydrolysis of p-Nitrophenyl Butyrate (pNPB) to p-Nitrophenol (pNP) by Porcine Pancreatic Lipase (PPL) in the Presence of Tetrahydrolipstatin and Its Diastereomers (1–4 and (ent)-1–4).

Scheme 3

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THL was found to be a very potent inhibitor of PPL with an IC50 of 4.0 nM, which correlates well with known IC50 from related assays with 1,2-diacyl-sn-glycerol as the substrate.23,29,51 None of the other seven diastereomers were more potent PPL inhibitors, with IC50 values ranging from 8.0 to 77 nM (cf, Figure 2, Table 1). One diastereomer, (ent)-3, did not show any significant inhibition of lipase activity below 100 nM, with an IC50 > 900. The error bars reflect 95% confidence limits for triplicate runs on three separate occasions (N = 9) and are consistent with slight variations in pH, enzyme concentration, and timing the start of the assays.

Figure 2.

Figure 2

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IC50 curves for THL and seven stereoisomers against pancreatic lipase using inhibitor concentrations between 0.01 nM and 10 μM.

Table 1. IC50 of THL Diastereomers against Pancreactic Lipasea.

Cpd Epimerization IC50 (nM)
1 THL 4.0 (±1.3)
2 2′-epi-THL 15.4 (±2.6)
3 5-epi-THL 17.4 (±3.1)
4 5,2′-epi-THL 8.0 (±1.5)
(ent)-1 ent-THL 76.9 (±17.6)
(ent)-2 2,3,5-epi-THL 20.0 (±7.2)
(ent)-3 2,3,2′-epi-THL 930 (±340)
(ent)-4 2,3-epi-THL 14.7 (±2.9)
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a

The results are an average of three independent triplicate experiments (N = 9). The 95% confidence limits are indicated in parentheses.

The eight stereoisomers were assayed for cytotoxicity against three human cancer cell lines: two human breast adenocarcinoma cancer cell lines MCF-7 and MDA-MB-231 and one human large-cell lung carcinoma cell line H460 (Table 2 and Figures 3–5). A standard 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay of cell viability was used.55,56 The MDA-MB-231 was chosen as it has been previously used in several studies of THL cytotoxicities.16,23

Table 2. IC50 of THL Diastereomers against Cancer Cells (nM)a.

compound H460 MDA-MB-231 MCF-7
1 to (ent)-4 >105 >105 >105
camptothecin 110 540 160
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a

The cell line was grown in 96-well plates at a density of 5 × 103 cell/well for 12 to 16 h. IC50 values were determined via MTT assay from a 72 h drug treatment (0.1 nM to 100 μM), with untreated wells as controls. The viability was measured by UV absorbance at 570 nm. The dose-dependent experiment was performed in 2 replicate wells of each compound for a single concentration with three independent experiments (N = 6).

Figure 3.

Figure 3

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IC50 curves for THL and seven stereoisomers against MDA-MB-231 cell line using inhibitor concentration from 0.1 nM to 100 μM. CPT = camptothecin.

Figure 5.

Figure 5

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IC50 curves for THL and seven stereoisomers against H460 cell line using inhibitor concentration from 0.1 nM to 100 μM. CPT = camptothecin.

Of the three cell lines we tested, MDA-MB-231 was most sensitive to THL compounds. The IC50 value we observed for THL against the MDA-MB-231 was ∼100 μM, which was significantly higher than previously reported (IC50 13 μM).23 This difference may be due to variations in the culture method and assay.57−59 For the MCF-7 cell line, the IC50’s of all of the THL diastereomers were at or above 100 μM, with the lowest being for THL 2 (see Figure 4). In general, varying the stereochemistry of THL had little effect on the cytotoxicity. Interestingly, (ent)-3, the least potent PPL inhibitor, demonstrated no significant difference in cytotoxicity across the three cell lines.

Figure 4.

Figure 4

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IC50 curves for THL and seven stereoisomers against MCF-7 cell line using inhibitor concentration from 0.1 nM to 100 μM. CPT = camptothecin.

In conclusion, synthetic THL and seven of its stereoisomers were evaluated in vitro as porcine pancreatic lipase inhibitors. This study found that the stereochemistry of THL plays a modulating role on inhibitory activity, with the IC50’s of THL and its diastereomers ranging from 4.0 to 930 nM. While THL proved to be the most potent inhibitor (4.0 nM), in this assay, several diastereomers retained significant inhibitory activity. Of these, diastereomers, (ent)-2 had the most surprising PPL-inhibitory activity (20 nM), as it is enantiomeric at the key β-lactone portion of the molecule. For comparison, the enantiomer of THL, (ent)-1, was much less active (77 nM). Interestingly, this systematic inversion of stereochemistry around the THL structure had no deleterious effects on the cytotoxicities of these molecules. These S-SAR findings suggest that there is potential for future medicinal chemistry around the β-lactone structural motif of THL. Our efforts along these lines will be reported in due course.

Acknowledgments

We would like to thank Michael Mulzer and Brandon J. Tiegs for help with the Coates carbonylation. This work was supported by the NSF (Grant CHE-1565788 to G.A.O.)

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.8b00050.

  • Experimental details for synthetic procedure and compound characterization (PDF)

Author Contributions

§ Co-first authors, the order is alphabetical.

The authors declare no competing financial interest.

Supplementary Material

ml8b00050_si_001.pdf (1.6MB, pdf)

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