Synthesis and biological investigation of the β-thiolactone and β-lactam analogs of tetrahydrolipstatin

Article abstract of DOI:10.1039/c2ob06976h

The synthesis of β-thiolactone and β-lactam analogs of tetrahydrolipstatin is described from a common late-stage β-lactone derivative. These analogs, and a cis-disubstituted β-lactone analog of tetrahydrolipstatin, were screened for activity against porcine pancreatic lipase and for inhibition of cell growth of a panel of four human cancer lines. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob06976h

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PAPER
Synthesis and biological investigation of the β-thiolactone and β-lactam
analogs of tetrahydrolipstatin†
Sylvain Aubry,a Geneviève Aubert,a Thierry Cresteila and David Crich*a,b
Received 23rd November 2011, Accepted 16th January 2012
DOI: 10.1039/c2ob06976h
The synthesis of β-thiolactone and β-lactam analogs of tetrahydrolipstatin is described from a common
late-stage β-lactone derivative. These analogs, and a cis-disubstituted β-lactone analog of
tetrahydrolipstatin, were screened for activity against porcine pancreatic lipase and for inhibition of cell
growth of a panel of four human cancer lines.
Introduction
Over the past few decades naturally occurring β-lactones (2-
oxetanones) and their synthetic congeners have been recognized
by the scientific community as promising drug candidates for
several human disease states.1 Tetrahydrolipstatin (Orlistat), a
saturated analog of the natural lipstatin isolated from Strepto-
Fig. 1 Lipstatin and tetrahydrolipstatin.
myces toxytricini in 1987,2 is one such molecule that is currently
marketed as Xenical® for the treatment of obesity (Fig. 1).3 The
biological target for tetrahydrolipstatin is the active site serine of
the pancreatic and gastric lipases with which it forms an irrevers-
ible ester bond by ring opening of the trans-fused β-lactone,4
resulting in a decrease of the rate of triglyceride hydrolysis and
adsorption of dietary fat by the small intestine.5 More recently,
Orlistat and some of its analogs have been found to inhibit the
thioesterase domain of fatty acid synthase, an essential enzymatic
process involved in the growth and survival of tumor cells and
a validated drug target for the discovery of new anti-tumor
antibiotics.6
β-thiolactones arising from the longer C–S bond and the smaller
C–S–CvO angle as compared to their oxygen analogs, which
offer the potential of a useful compromise between stability and
reactivity.9
As a part of a program to uncover the potential of the
β-thiolactones in medicinal chemistry,10 we report here on the
synthesis of sulfur analogs (β-thiolactones) of tetrahydrolipstatin
and, for the purposes of comparison of the corresponding nitro-
gen analogs (β-lactams), with the two series of compounds
obtained efficiently from a common late-stage β-lactone inter-
mediate. The β-thiolactones and β-lactams prepared in this
manner were evaluated for activity against porcine pancreatic
lipase and for the inhibition of four human cancer cell lines.
Unlike the β-lactones1,7 and β-lactams,8 which have received
enormous attention from the synthetic and medicinal chemistry
communities, the β-thiolactones have been essentially ignored
and thus represent an untapped potential for drug discovery. The
β-thiolactones present a unique reactivity profile toward active
site nucleophiles, including both acylating and alkylating capa-
bilities, which differ from those of the corresponding β-lactones.
This is because of the different physicochemical properties of
Synthesis
Selective ring opening of (S)-(−)-epichlorohydrin with
C10H21MgBr in presence of CuI in THF at −45 °C gave the
chlorohydrin 1 in 87% yield (Scheme 1) that was converted to
the epoxide 2 by the action of KOH in Et2O in 81% yield.11
Subsequent ring opening with vinylmagnesium bromide, again
in the presence of CuI resulted in the formation of the homo-
allylic alcohol 3 in 92% yield, which was protected as PMB
ether 4 in 92% yield. Oxidative cleavage of the alkene by a one-
pot procedure12 consisting of dihydroxylation in the presence of
OsO4 and NMO followed by diol cleavage with PhI(OAc)2,
afforded the corresponding aldehyde 5 in 88% yield. Subsequent
reaction with the boron enolate derived from oxazolidinone
aCentre de Recherche de Gif, Institut de Chimie des Substances
Naturelles, CNRS, 1 Avenue de la Terrasse, 91190 Gif-sur-Yvette,
France. E-mail: Dcrich@icsn.cnrs-gif.fr; Fax: +33 1690 77752;
Tel: +33 1698 23089
bDepartment of Chemistry, Wayne State University, Detroit, MI 48202,
USA. E-mail: Dcrich@chem.wayne.edu; Fax: +1 313 577 8822;
Tel: +1 313 577 6203
†Electronic supplementary information (ESI) available: Full experimen-
1
tal details, characterization data and copies of H and 13C NMR spectra.
See DOI: 10.1039/c2ob06976h
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6 delivered the aldol 7 in 76% yield and a diastereomeric excess
of >99% (Scheme 1).13 Removal of the oxazolidinone group
from 7 was accomplished by nucleophilic displacement with the
lithium salt of BnSH at −10 °C in THF, furnishing the benzyl
thioester 8 in 73% yield. Subsequent methanolysis of 8 with
MeONa in MeOH then gave rise to the methyl ester 9 in 72%
yield.
Attempted conversion of the alcohol 9 to the corresponding
configurationally inverted thiol by Mitsunobu reaction with
thioacetic acid, or by displacement of the derived mesylate or
triflate esters was unsuccessful. Consequently, it was envisaged
that the desired substitution might be achieved via the β-lactone.
Accordingly, hydrolysis of methyl ester 9 was conducted to
afford the corresponding acid 10 in quantitative yield, which was
cyclized using PhSO2Cl in pyridine to give the β-lactone 11 in
67% yield (Scheme 2). With 11 in hand, PMB removal with
TFA in CH2Cl2 gave the secondary alcohol 12 in 94% yield,
which, on esterification with Boc-Leu-OH in the presence of
EDCI and DMAP, provided the N-protected amino ester 13 in
86% yield. Subsequent removal of the Boc group with TFA was
followed by formylation with formic acetic anhydride to afford
the cis-isomer of tetrahydrolipstatin 14 in 86% yield
(Scheme 2).14
Returning to the formation of the β-thiolactone, and taking
into consideration previous reports on the alkylation of soft
nucleophiles including thiols by oxetanones,15 β-lactone 11 was
subjected to BnSLi, but only O-acyl fission of β-lactone 11
affording benzyl thioester 8 was observed. The use of NaSH
as nucleophile also proved to be fruitless due to competitive
intermolecular reaction after the initial SN2 cleavage of 11.
Fortunately, the use of cesium thioacetate as nucleophile led to
opening of the β-lactone 11 in the desired manner and gave rise
to the corresponding thioacetate-substituted carboxylic acid
intermediate (Scheme 3). Cleavage of acetate group from this
intermediate was then accomplished by the action of hydrazine
hydrate, and this was followed by cyclization with EDCI and
C6F5OH in CH2Cl2, to cleanly afford the β-thiolactone 15 in
65% yield over three steps (Scheme 3). With this strategy for the
preparation of β-thiolactone 15 in hand, the use of β-lactone 11
as a synthon for the β-lactam analog was considered. To this
end, ring opening of 11 was achieved with NaN3 in DMF at
60 °C and furnished the desired azido carboxylic acid as
Scheme 1 Synthesis of methyl ester 9.
Scheme 3 Synthesis of the β-thiolactone and β-lactam precursors.
Scheme 2 Synthesis of the β-lactone analog 14 of tetrahydrolipstatin.
2630 | Org. Biomol. Chem., 2012, 10, 2629–2632
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Table 1 IC50 values of Orlistat, β-lactone 14, β-thiolactone 22, and
β-lactam 23 for the inhibition of porcine pancreatic lipase
Entry
Compound
IC50a (nM)
1
2
3
4
Orlistat
14
22
7.5
15.0
>100
>100
23
a Average of duplicate measures.
Table 2 IC50 (μM) values of Orlistat, β-lactone 14, β-thiolactone 22,
and β-lactam 23 for the inhibition of KB, HCT116, PC3 and
MDA231 human cancer cell line proliferation in vitro
Entry
Compound
KBa
PC3a
HCT116a
MDA231a
1
2
3
4
Orlistat
14
22
50.6
29.7
54.4
13.6
57.2
43.2
36.9
10.2
32.0
54.6
15.4
11.5
13.0
28.2
40.7
12.4
Scheme 4 Completion of the synthesis of the β-thiolactone and
β-lactam analogs of tetrahydrolipstatin.
23
demonstrated by mass spectrometric analysis using ESI in the
negative ion mode (Scheme 3).15c,16 In what might be dubbed an
intramolecular Staudinger–Vilarrasa β-lactamization,17 this inter-
mediate was then directly engaged in a one-pot procedure con-
sisting first of azide reduction and subsequent cyclization to the
β-lactam 16 through the aegis of triphenylphosphine and 2,2′-
dipyridyl disulfide in CH3CN–THF at 60 °C in 79% over two
steps. Related lactam-forming reactions from β-amino acids
using Ohno’s protocol have previously been reported by several
groups (Scheme 3).18
a Average of duplicate measures.
neither the β-thiolactone 22 nor the β-lactam 23 inhibited the
lipase to any significant extent (Table 1, entries 3 and 4), thereby
revealing the importance of the reactivity of the β-lactone for
lipase inhibition. In addition, none of the synthetic intermediates
(11–13, 15–16, 18–21) prepared, revealed any inhibition of the
pancreatic lipase.22
With respect to the inhibition of cancer cell proliferation, IC50
values were determined for compounds 14, 22, 23 and Orlistat
against a range of four human cancer cell lines (KB nasopharynx
human carcinoma, PC3 prostate carcinoma, MDA231 human
breast adenocarcinoma, and HCT116 colorectal carcinoma). For
both the KB and PC3 cell lines β-lactam 23 (Table 2, entry 4)
was found to be between two to four-fold more cytotoxic in the
10 μM range than Orlistat, 14 and 22 (Table 2). Concerning the
HCT116 cell line, β-thiolactone 22 and β-lactam 23 (Table 2,
entries 3 and 4) exhibited similar cytotoxicity and were more
potent than the β-lactone analogs (Table 2, entries 1 and 2). This
observation may be linked to the inhibition of reactive cysteine
residues, that might provide a different mechanism of action
towards β-thiolactone or β-lactam.23
Finally, Orlistat and β-lactam 23 showed similar inhibitory
activity against the MDA231 cancer cell line (Table 2, entries 1
and 4) and showed a similar cytotoxic profile, while the
β-thiolactone 22 was the least cytotoxic of the four compounds
surveyed (Table 2, entry 3). Overall, in this brief screen of cyto-
toxicity the β-lactam congener 23 of Orlistat was discovered to
be generally more cytotoxic (IC50 = 10.2–13.6 μM) than either
Orlistat itself, the cis-analog of Orlistat 14 and the β-thiolactone
analog 22, perhaps suggesting a parallel with earlier observations
on the inhibition of the thioesterase domain of fatty acid synthase
by β-lactam congeners of Orlistat.6,8d
Removal of the PMB group from 15 with TFA in CH2Cl2
proved to be more difficult than expected due the concomitant
formation of a six-membered ring δ-lactone 17, which was
accompanied by the migration of the PMB group to the sulfur
atom (Scheme 4). To avoid this issue, the reaction was conducted
at 5 °C and, critically, in the presence of Et3SiH as a scavenger
when 18 was obtained in 88% yield. In the case of β-lactam 16,
acidic cleavage of the PMB ether gave the secondary alcohol 19
in 86% yield. Subsequent derivatization of 18 and 19, by esterifi-
cation of the secondary alcohol function was accomplished as
described above for the lactone, and gave the corresponding
N-protected amino ester 20 and 21 in 76 and 87% yield, respect-
ively (Scheme 4). Finally, removal of the Boc group with TFA,
followed by formylation with formic acetic anhydride led to the
β-thiolactone 22 and β-lactam 23 in 80% and 92% yields,
respectively (Scheme 4).
Biology
With compounds 14, 22 and 23 in hand, their capacity to inhibit
porcine pancreatic lipase was first assessed with the aid of the 6′-
methylresorufin ester of 1,2-di-O-lauryl-rac-glycero-3-glutaric
acid as substrate,19 and Orlistat as control.19 Orlistat proved to be
the best inhibitor of porcine pancreatic lipase with an IC50 of 7.5
nM,20 but its cis-isomer 14, which retained the β-lactone func-
tionality, showed only a two-fold loss of activity (IC50 = 15 nM)
(Table 1, entries 1 and 2).14 This observation potentially opens
the way to more extensive studies of Orlistat analogs bearing a
cis- rather than a trans-disubstituted β-lactone as potential inhibi-
tors of fatty acid synthase.21 In contrast to Orlistat and 14,
Conclusions
To conclude, an efficient synthesis of the β-thiolactone and
β-lactam analogs of tetrahydrolipstatin has been developed that
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