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

Article abstract of DOI:10.1021/acsmedchemlett.8b00050

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.

Full text of DOI:10.1021/acsmedchemlett.8b00050

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Letter
Stereochemical Structure Activity Relationship
Studies (S-SAR) of Tetrahydrolipstatin
Xiaofan Liu, Yanping Wang, Richard I Duclos, and George A. O'Doherty
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00050 • Publication Date (Web): 21 Feb 2018
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Stereochemical Structure Activity Relationship Studies (S-SAR) of
Tetrahydrolipstatin
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Xiaofan Liu,§ Yanping Wang,§ Richard I. Duclos, Jr.,* George A. O’Doherty*
* Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115
Supporting Information Placeholder
ABSTRACT: 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 activity
(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 three orders of magnitude of campothecin.
KEYWORDS: tetrahydrolipstatin, pancreatic lipase, diastereomers, inhibitory activity, cytotoxicity, structure activity relationship
,
Tetrahydrolipstatin (THL, 1)1 2 is an FDA approved oral anti-
shown to inhibit the in vitro growth of Giardia duodenalis, a
gastrointestinal parasite.25 THL may also have value as an an-
tifungal agent against fungi that lack lipase enzymes and rely
obesity drug that inhibits pancreatic lipase digestive en-
, ,
zyme.3 4 5 Inhibition of pancreatic lipase in the intestinal lu-
,
on free fatty acids for their nutrition and growth.26 27
men results in the inability to digest dietary triacylglycerides
(fat) and diacylglycerides to free fatty acids and monoacyl-
glycerides that can be absorbed into the bloodstream and lym-
phatic system. Dietary fat and diacylglycerides are then elim-
inated as bodily waste without the caloric consequences of
their taste and consumption. As part of an effort aimed at de-
veloping a novel treatment for pancreatitis, we became inter-
ested in structure activity space around (THL) 1. Pancreatitis
is believed to result from the excessive release of active pan-
creatic 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 re-
moving excess pancreatic lipase.
NHCHO
NHCHO
Regioselective
Carbonylation
O
O
O
O
O
O
O
C11H23
C11H23
C6H13
C6H13
1-4
or
5a/b
or
(ent)-5a/b
(ent)-1-4
Asymmetric
Synthesis
Diastereoselective
Epoxidation
O
OH
C11H23
C11H23
C7H15
C6H13
7
6
or
NHCHO
1: THL
2: 2'-epi-THL
3: 5-epi-THL
4: 5,2'-epi-THL
(ent)-1: ent-THL
(ent)-2: 2,3,5-epi-THL
(ent)-3: 2,3,2'-epi-THL
(ent)-4: 2,3-epi-THL
(ent)-6
2'
1'
O
O
O
O
Scheme 1. Retrosynthetic Analysis for THL
3
1
5
2
4
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 ac-
C5H11
THL (1)
Figure 1. Structures of THL (1) and seven diastereomers
THL (1) is known to be a potent inhibitor of porcine pancre-
atic 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 b-lactone by a lipase active
,
, , ,
tivities (e.g., 2, 3, and the 3-epi-THL).16 23 28 29 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-b-lactone
isomer (3-epi-THL)23 has been evaluated for lipase inhibitory
activity (IC50 = 15 nM). However, no systematic study of the
trans-b-lactone stereoisomers has been reported. Thus, we
identified seven trans-b-lactone stereoisomers of THL (2-4
and (ent)-1-4) as compounds as a convenient method to iden-
tify the structural space around the enzyme active site (Figure
1). Importantly, these type of comprehensive stereochemical-
, ,
site serine to form a stable ester.7 8 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 treat-
,
,
ment of pancreatitis by intravenous,10 intraperitoneal,11 12 13
and direct injection into pancreatic tissue.9,11,12,13,14 In addi-
tion, 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 16 17 18 19 20 21 22 23 24 THL has been
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SAR studies (S-SAR) of natural products and their deriva-
tives, require an asymmetric synthetic approach to the struc-
at all assay concentrations. Using this assay, the Km (Michae-
lis-Menten constant) for our PPL was determined to be 2.41
mM (see supporting information) which corresponds well
with a related assay.54
1
2
3
4
5
6
7
8
,
,
,
,
,
,
37
ture of interest.31 32 33 34 35 36
Over the years there have been numerous syntheses of THL
,
, ,
and related molecules.38 39 40 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 diastereose-
lective epoxidation of 6 and regioselective Coates carbonyla-
NO2
PPL
plus inhibitor
NO2
+
O
O
H7C3
OH
H7C3
O
(9:1) H2O/DMSO
pH 7.5, RT
HO
9
10
11
9
10
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Scheme 3. Hydrolysis of p-nitrophenyl butyrate (pNPB) to p-
nitrophenol (pNP) by porcine pancreatic lipase (PPL) in the pres-
ence of tetrahydrolipstatin and its diastereomers (1-4 and (ent)-
1-4).
,
, , , , ,
tion42 43 44 45 46 47 48 of 5 to afford either enantiomer of the four
diastereomers (1-4).49 Key to the success of this stereodiver-
gent approach was the recognition that all eight THL stereoi-
somers (1-4 and (ent)-1-4) could be diastereo- and regio-se-
lectively prepared from the homo-allylic epoxide 8 and its en-
antiomer in only three steps (Scheme 2).
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 sub-
NHCHO
2'
,
,
NHCHO
2'
strate.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% con-
fidence limits for run in triplicates on three separate occasions
(N = 9) and are consistent with slight variations in pH, en-
zyme concentration, and timing the start of the assays.
NHCHO
2'
O
O
O
2
O
5
3
C11H23
O
O
O
O
O
O
2
O
O
5
5
C6H13
2
3
3
1: THL
C11H23
C11H23
C6H13
C6H13
2: 2'-epi-THL
(ent)-4: 2,3-bis-epi-THL
NHCHO
2'
NHCHO
2'
OH
O
O
O
O
O
O
C11H23
(+) or (–)
O
2
O
O
5
8
5
2
C6H13
3
C11H23
3
C11H23
C6H13
C6H13
Table 1. IC50 of THL diastereomers against pancreactic lipase a
3: 5-epi-THL
(ent)-3: 2,3,2'-tris-epi-THL
Cpd
Epimerization
IC50 (nM)
NHCHO
NHCHO
2'
2'
NHCHO
1
2
THL
O
O5
O
O
4.0 (±1.3)
15.4 (±2.6)
17.4 (±3.1)
8.0 (±1.5)
O
O
2
O
O
2'
5
2
3
3
C11H23
C11H23
2'-epi-THL
5-epi-THL
O
O
O
2
O
C6H13
C6H13
5
3
3
C11H23
(ent)-2: 2,3,5-tris-epi-THL
4: 5,2'-epi-THL
C6H13
(ent)-1: ent-THL
4
5,2'-epi-THL
ent-THL
ent-1
ent-2
ent-3
76.9 (±17.6)
20 (±7.2)
Scheme 2. Asymmetric approach to THL and 7 stereoisomers
2,3,5-epi-THL
2,3,2'-epi-THL
930 (±340)
Herein we report the evaluation of the eight THL diastereoi-
somers (1-4 and (ent)-1-4). All the stereoisomers were eval-
uated for their lipase inhibitory properties using porcine pan-
creatic lipase (PPL). To ensure that the various stereochemi-
cal inversions on THL did not introduce any unwanted toxic-
ities, the cytotoxicity of all eight stereoisomers were deter-
mined against three cancer cell lines (H460, MCF-7 and
MDA-MB-231).
ent-4
2,3-epi-THL
14.7 (±2.9)
a The results are an average of three independent triplicate ex-
periments (N = 9). The 95% confidence limits are indicated in
parentheses.
100
,
Porcine pancreatic lipase is frequently used,7,50 51 since the ac-
ent-3
tive site of the porcine lipase is highly homologous to the hu-
man 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 en-
zyme concentration of 5.0 µg/200µL. This concentration was
consistent with values used for a related assay.51 The concen-
tration 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
80
ent-1
ent-2
3
60
ent-4
2
40
4
1 (THL)
20
0
-2
0
2
4
Inhibitor Concentration
(log nM)
Figure 2. IC50 curves for THL and seven stereoisomers against
pancreatic lipase using inhibitor concentrations between 0.01 nM
and 10 µM.
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The eight stereoisomers were assayed for cytotoxicity against
1
2
3
4
5
6
7
8
100
80
60
40
20
0
three human cancer cell lines: two human breast adenocarci-
noma 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 viabil-
1 (THL)
2
3
,
ity was used.55 56 The MDA-MB-231 was chosen as it has
4
ent-1
ent-2
ent-3
been previously used in several studies of THL cytotoxici-
ties.16, 23
ent-4
CPT
9
Table 2. IC50 of THL diastereomers against cancer cells (nM)a
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Compound
1 to (ent)-4
camptothecin
H460 MDA-MB-231 MCF-7
0
1
2
3
4
5
Inhibitor Concentration
(log nM)
>105
110
>105
540
>105
160
Figure 4. IC50 curves for THL and seven stereoisomers against
MCF-7 cell line using inhibitor concentration from 0.1 nM to
100 µM. CPT = camptothecin.
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 un-
treated wells as controls. The viability was measured by UV ab-
sorbance at 570 nm. The dose-dependent experiment was per-
formed in 2 replicate wells of each compound for a single con-
centration with 3 independent experiments (N = 6).
100
80
1 (THL)
2
3
60
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
4
ent-1
ent-2
40
ent-3
ent-4
CPT
20
0
,
,
and assay.57 58 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, demon-
strated no significant difference in cytotoxicity across the
three cell lines.
0
1
2
3
4
5
Inhibitor Concentration
(log nM)
Figure 5. IC50 curves for THL and seven stereoisomers against
H460 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 surpris-
ing PPL-inhibitory activity (20 nM), as it is enantiomeric at
the key b-lactone portion of the molecule. For comparison,
the enantiomer of THL, (ent)-1, was much less active (77
nM). Interestingly, this systematic inversion of stereochem-
istry around the THL structure had no deleterious effects on
the cytotoxicity of these molecules. These S-SAR findings
suggest that there is potential for future medicinal chemistry
around the b-lactone structural motif of THL. Our efforts
along these lines will be reported in due course.
100
80
1 (THL)
2
3
60
4
ent-1
ent-2
40
ent-3
ent-4
20
CPT
0
-1
0
1
2
3
4
5
Inhibitor Concentration
(log nM)
Figure 3. 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.
ASSOCIATED CONTENT
Supporting Information. Experimental details for synthetic
procedure and compound characterization. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
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* Email: G.ODoherty@neu.edu, r.duclosjr@northeastern.edu
Acknowledgment: We would like to thank Michael Mulzer and
Brandon J. Tiegs for help with the Coates carbonylation. This
work was supported NSF (CHE- 1565788 to G.A.O.)
1
2
3
4
5
6
Present Addresses
† Department of Chemistry and Chemical Biology, Northeastern
University, Boston, MA 02115
Author Contributions:
§ Co-first authors, the order is alphabetical.
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Tetrahydrolipstatin (THL) is a natural product derivative that has been approved by the FDA as an oral anti-obesity drug,
which acts by the inhibition of the pancreatic lipase enzyme in the digestive track. Synthetic access to THL and seven
diastereomers enabled a novel stereochemistry based structure activity relationship study of this class of biologically
active natural products. Several stereoisomers of THL were discovered with significant pancreatic lipase inhibitory ac-
tivity.
TOC:
NHCHO
2'
OH
1'
O
O
O
O
O
C11H23
(+) or (â“)
3
1
C6H13
5
2
4
C5H11
THL (1)
1: THL
(ent)-1: ent-THL
2: 2'-epi-THL
3: 5-epi-THL
4: 5,2'-epi-THL
(ent)-2: 2,3,5-epi-THL
(ent)-3: 2,3,2'-epi-THL
(ent)-4: 2,3-epi-THL
five stereoisomers retained potent activity
(IC50 = 8.0 to 20 nM)
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