New cholesterol esterase inhibitors based on rhodanine and thiazolidinedione scaffolds

Article abstract of DOI:10.1016/j.bmc.2011.10.042

We present a new class of inhibitors of pancreatic cholesterol esterase (CEase) based on 'priviledged' 5-benzylidenerhodanine and 5-benzylidene-2,4- thiazolidinedione structural scaffolds. The lead structures (5- benzylidenerhodanine 4a and 5-benzylidene-

Full text of DOI:10.1016/j.bmc.2011.10.042

Bioorganic & Medicinal Chemistry 19 (2011) 7453–7463
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New cholesterol esterase inhibitors based on rhodanine
and thiazolidinedione scaffolds
Sabrina Heng a, William Tieu a, Stephanie Hautmann b, Kevin Kuan a, Daniel Sejer Pedersen c,
Markus Pietsch a,d, Michael Gütschow b, Andrew D. Abell a,
⇑
a Department of Chemistry and Physics, The University of Adelaide, North Terrace, SA 5005, Australia
b Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
c Department of Medicinal Chemistry, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark
d Department of Pharmacology, University of Cologne, Gleueler Strasse 24, D-50931 Cologne, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
We present a new class of inhibitors of pancreatic cholesterol esterase (CEase) based on ‘priviledged’
5-benzylidenerhodanine and 5-benzylidene-2,4-thiazolidinedione structural scaffolds. The lead structures
(5-benzylidenerhodanine 4a and 5-benzylidene-2,4-thiazolidinedione 4b) were identified in an in-house
screening and these inhibited CEase with some selectivity over another serine hydrolase, acetylcholines-
Received 31 August 2011
Revised 7 October 2011
Accepted 14 October 2011
Available online 20 October 2011
terase (AChE) (4a, CEase IC50 = 1.76
>100 M). A small library of analogs (5a–10a) containing a core amino acid in place of the glycerol group
of the lead structures, was prepared to explore other potential binding interaction with CEase. These ana-
logs inhibited CEase with IC50 values ranging from 1.44 to 85 M, with the majority exhibiting some
lM vs AChE IC50 = 5.14 lM and 4b, CEase IC50 = 5.89 lM vs AChE IC50
l
Keywords:
Cholesterol esterase
Acetylcholinesterase
Privileged scaffolds
Rhodanine
l
selectivity for CEase versus AChE. The most potent compound of the library (10a) had 17-fold selectivity
over AChE. We also report molecular docking (with CEase) and detailed kinetic analysis on the amino acid
analogs to further understand the associated structure–activity relationships.
Ó 2011 Elsevier Ltd. All rights reserved.
Thiazolidinedione
1. Introduction
butyrylcholinesterase (EC 3.1.1.8)17–20 reveal a high degree of sim-
ilarity in the N-terminal domain, especially around the active-site
Pancreatic cholesterol esterase (CEase, EC 3.1.1.13) is a member
of the /b hydrolase family of proteins that catalyzes the hydroly-
serine.21 It is interesting to note that an X-ray structure of pancre-
atic CEase has been solved at 1.6 Å by molecular replacement
based on a truncated model of Torpedo californica acetylcholines-
terase.16 This similarity of structure is consistent with reports that
inhibitors of CEase also inhibit AChE,22 with some inhibitors of
AChE approved as therapeutics. For example, rivastigmine (Exe-
lon), donepezil (Aricept) and galanthamine (Reminyl) are currently
used for the treatment of Alzheimer’s disease.23,24 Selectivity of
inhibition therefore presents a challenge and an important consid-
eration, if one is to develop pharmaceuticals based on the inhibi-
tion of CEase and/or AChE. Despite this need, there are few
a
sis of cholesterol esters into free cholesterol in the lumen of the
small intestine.1,2 It is also thought to play a role in the transport
of cholesterol from micelles to the enterocyte,1 although there
are conflicting reports on this.3 Given the combined roles of CEase
in the absorption and transport of cholesterol, its inhibition is of
much interest as a target for developing treatments of hypercho-
lesterolemia and associated diseases such as coronary heart
disease.4 Despite this need, existing inhibitors of CEase are some-
what limited to standard classes of mechanism-based inhibitors5
such as boronic acids, aryl haloketones, aryl phosphates and vari-
ous heterocycles.6–15 These inhibitors target the enzyme’s catalytic
triad (Ser-His-Asp)16 which is common to other members of the
studies6,7 that address this issue within the
a/b hydrolase family,
particularly with regards to CEase. There is a need for new classes
of inhibitors of CEase that are amenable to the preparation of a
range of derivatives that will provide an opportunity to explore
selectivity of inhibition.
a/b hydrolase family of proteins.
This family of proteins share a common a/b hydrolase fold that
consists of a mostly parallel 5 to 14-stranded b sheet surrounded
In this paper we present a new class of CEase inhibitors based
on ‘privileged’25,26 5-benzylidenerhodanine and 5-benzylidene-
2,4-thiazolidinedione structural scaffolds (see Fig. 1) that exhibit
some selectivity for CEase over AChE. The scaffolds were chosen
as a starting point since compounds containing them are known
to be pharmacologically active;27 for example as inhibitors of
protein tyrosine phosphatases.28 Certain representatives are of
by several
a
helices,16 and a high degree of overall structural
homology. For example, the reported structures of carboxyesteras-
es (EC 3.1.1.1), acetylcholinesterase (AChE, EC 3.1.1.7) and
⇑
Corresponding author.
E-mail address: andrew.abell@adelaide.edu.au (A.D. Abell).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.042

7454
S. Heng et al. / Bioorg. Med. Chem. 19 (2011) 7453–7463
X
S
OH
X
NH
O
O
S
5
NH
O
O
Figure 1. 5-Benzylidenerhodanine (X = S) and 5-benzylidene-2,4-thiazolidinedione
(X = O) core structures.
4a X = S
4b X = O
R
N
O
S
X
H
MeO
N
S
S
NH
H
O
NH
O
O
1
O
R
X
5a
5b
6a
6b
7a
7b
8a
8b
9a
10a
S
O
S
O
S
O
S
O
S
S
CH2Ph (Phe)
CH2Ph (Phe)
Me (Ala)
S
O
N
N
H
Me (Ala)
CH2OH (Ser)
CH2OH (Ser)
O
O
O
S
NH
CH2OC(Me)3 (SerOtBu)
CH2OC(Me)3 (SerOtBu)
CH2Ph-4-OH (Tyr)
O
F
CH2OCH2Ph (SerOBn)
F
2
CF3
Figure 3. 5-Benzylidenerhodanines and 5-benzylidene-2,4-thiazolidine-diones
prepared.
CF3
O
acids since the catalytic triad of CEase is known to be flanked by
hydrophobic domains, see later for a discussion.34 Specifically,
Phe (5a and 5b), Ala (6a and 6b), SerOtBu (8a and 8b) and SerOBn
(10a) were introduced into the base structures. More polar resi-
dues, Ser and Tyr, were also investigated (7a, 7b and 9a) to further
explore possible hydrogen bonding interactions. Derivatives 5–10
have the added advantage of ease of synthesis compared to 4a
and 4b.
Br
O
S
HN
O
3
Figure 2. Inhibitors of PDE429 (1), phospholipase A2 (2)30,31 and protein tyrosine
phosphatases (3)32 bearing the rhodanine and 2,4-thiazolidinedione scaffolds.
2.2. Chemistry
particular significance to our study as inhibitors of several ester-
ases and lipases, including PDE429 (1), phospholipase A2 (2)30,31
and protein tyrosine phosphatases (3)32 (Fig. 2).
The 5-benzylidene-2,4-thiazolidinedione (4b) was prepared by
Knoevenagel condensation of the aldehyde 1133 with 2,4-thiazolid-
inedione in ethanol, containing piperidine, as shown in Scheme 1.35
The novel 5-benzylidenerhodanine (4a) was prepared from rhoda-
nine under the same conditions as shown in Scheme 1. 1H NMR
analysis of both derivatives revealed a single olefinic resonance,
consistent with the formation of a single isomer, which was
2. Results and discussion
2.1. Inhibitor design
assigned to have the thermodynamically more stable
Z
Figure 3 shows the compounds that were prepared in the study.
The 5-benzylidenerhodanine (4a) and the corresponding 5-benzyl-
idene-2,4-thiazolidinedione (4b) were identified as our lead struc-
tures based on an in-house initial screening of a small library of
available rhodanines and thiazolidines against CEase and AChE.
The rhodanine 4a was shown to inhibit CEase with some selectivity
configuration.36,37
The amino acid-based examples 5–10 were prepared as out-
lined in Schemes 2 and 3. The amines 14a–e (prepared as shown
in Scheme 2) were separately coupled with 15a or 15b,30 in the
presence of O-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-
uronium hexafluorophosphate (HATU), 1-hydroxybenzotriazole
(HOBT) and diisopropylethylamine (DIPEA) to give 5, 6 and 8–10
(Scheme 3). The tert-butyl ether protecting groups of 8a and 8b
were then removed on treatment with 3 M HCl in dioxane to give
the serine derivatives 7a and 7b.
over AChE (CEase IC50 = 1.76 lM vs AChE IC50 = 5.14 lM, see Table
1). The oxo-analogue 4b, which has been reported to have antibac-
terial activity via an unknown mechanism,33 had slightly reduced
potency against CEase, but significantly improved selectivity over
AChE (CEase IC50 = 5.89 lM vs AChE IC50 >100 lM, see Table 1).
Given these preliminary results we proposed a small library of ana-
logs of 4a and 4b, where the glycerol group is replaced with an
amino acid core, in order to introduce structural diversity and
potential sites for interaction with CEase (see structures 5–10 in
Fig. 3). The 5-benzylidene and 2-propylphenyl groups of 4a/4b
were retained in all structures to allow direct comparison across
the series. We chose to incorporate mainly hydrophobic amino
2.3. Enzyme inhibition and structure–activity relationships
Derivatives 4–10 were assayed against both porcine pancreatic
CEase and Electrophorus electricus AChE with the results given in
Table 1. As discussed earlier, compound 4a inhibited CEase with
an IC50 of 1.76
lM, while compound 4b was slightly less potent
but more selective over AChE. All the amino acid-based examples

S. Heng et al. / Bioorg. Med. Chem. 19 (2011) 7453–7463
7455
Table 1
Inhibitory activity against CEase and AChE
Compound
CEase
AChEa
IC50
a
a
IC50
(
lM)
v[I]?1(%)b
(
l
M)
OH
OH
S
O
O
S
4a
1.76 0.17
5.89 0.42
40
18%
5.14 0.72c
NH
O
O
O
O
S
4b
17%
>100
NH
O
O
S
H
N
5a
5b
43
N
S
H
NH
O
O
O
O
H
N
40
14
N
H
S
NH
O
O
O
O
S
H
N
N
H
S
6a
6b
7a
7b
50
>100
>100
>100
>100
NH
O
O
O
H
N
N
H
S
54
NH
O
O
HO
O
S
H
N
N
H
S
31
NH
O
O
HO
O
O
H
N
N
H
S
S
5.70 0.43
12%
NH
O
O
O
O
O
S
H
N
8a
6.07 0.43
21.6 0.6c
N
H
NH
O
(continued on next page)

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S. Heng et al. / Bioorg. Med. Chem. 19 (2011) 7453–7463
Table 1 (continued)
Compound
CEase
AChEa
IC50
a
a
IC50
(l
M)
v[I]?1(%)b
(
l
M)
O
O
O
H
N
8b
85
66
N
H
S
NH
O
O
HO
O
S
H
N
9a
14.5 1.3
57
N
S
H
NH
O
O
O
O
O
S
H
N
10a
1.44 0.57
25%
25
N
H
S
NH
O
a
Limits without standard error were determined from duplicate experiments at a single inhibitor concentration. Values without standard error were from quadruplicate
experiments at a single inhibitor concentration. Values with standard error were from duplicate experiments with five different inhibitor concentrations.
b
Residual activity at infinite inhibitor concentration.
A three-parameter model was used for non-linear regression.
c
OH
OH
R
R
X
H
a
O
O
O
O
a
N
S
HO2C
NHBoc
NHBoc
NH
O
CHO
4a X = S
4b X = O
O
12a,b
13a,b
11
b
R
Scheme 1. a) piperidine, rhodanine or 2,4-thiazolidinedione, EtOH.
a
b
c
d
e
CH2Ph
CH2OCH2Ph
Me
CH2Ph-4-OH
CH2OC(Me)3
14a,b
R
H
N
were active against CEase, with compounds 7b, 8a and 10a being
the most potent. Some limited structure-activity relationships
can be drawn. A large hydrophobic amino acid side chain appears
to be well tolerated in 8a, 9a, 10a. Among the more hydrophilic
serine-based compounds, the thiazolidinedione 7b shows
enhanced potency compared to the rhodanine analogue 7a. The
activity of 7a (but not of 7b) was significantly improved on protect-
ing the hydroxyl group as the hydrophobic tert-butyl ether (8a). It
is noteworthy that we identified inhibitors (4b, 7b, 10a) that show
selectivity for CEase over AChE (greater than 15-fold). However,
these derivatives are significantly less potent than the known
phosphorylated flavones15, chloropyrones10 and chloroisocouma-
rins9 inhibitors.
NH2
O
14c-e
c
R
R
H
N
a
NHCbz
HO2C
NHCbz
O
12c-e
13c-e
Scheme 2. (a) EDCI, HOBT, DIPEA, 2-propylaniline, DMF; (b) 10% TFA, DCM; (c) 5%
Compounds 15–16 (Fig. 4) were also assayed against both CEase
Pd/C, cyclohexene, ethyl acetate.
and AChE, with all being inactive (IC50 values greater than 100 lM
for both enzymes). As such, the benzylidenerhodanine and ben-
zylidenethiazolidinedione groups alone are not sufficient for activ-
ity and a structural extension is required to inhibit these enzymes.
chosen since a structure of porcine CEase and a high-resolution
structure of E. electricus AChE (used in the assays) were not avail-
able. The two pdb files required minimal editing before application
of AutoDock40,41 and importantly, both contain ligands, taurocho-
late in the case of CEase and an active site bound oxime in the case
of AChE. An alignment of the two structures using PyMol42
revealed a strong similarity (see Fig. 5) and a calculated RMSD of
2.12 Å. This result is consistent with the observation that some
inhibitors of CEase also inhibit AChE.22
2.4. Docking studies
The inhibitors 4–10 were docked with bovine CEase (PDB entry:
1AQL38) and AChE from Mus musculus (PDB entry: 2JEY39) in order
to gain some insight into the inhibitory results shown in Table 1.
These X-ray crystallography structures (1AQL and 2JEY) were

S. Heng et al. / Bioorg. Med. Chem. 19 (2011) 7453–7463
7457
by the observation that both classes similarly inhibit CEase, see
Table 1. The 2-propylphenyl moieties of these inhibitors all bind
to a hydrophobic pocket defined by Ala108, Leu110, Leu272,
Leu285 and Phe324 (see box in Fig. 6, residues not shown), which
is distant from the catalytic triad. A similar pocket, found in the
structure of human CEase, is known to bind the hydrophobic fatty
acid portion of the native cholesterol ester substrates.43
R
O
X
H
a
N
14a-e
N
S
H
NH
O
O
R
X
Group 2 inhibitors (5a, 5b, 8a, 8b, 9a and 10a) all contain a rel-
atively large hydrophobic amino acid side chain (R in Fig. 3). For 5a
(R = CH2Ph) and 10a (R = CH2OCH2Ph), these groups occupy a
hydrophobic pocket defined by Leu282, Leu392 and Trp227, see
Figure 7. The 2-propylphenyl groups of each compound reside just
outside this pocket. Compounds 5b and 9a (not shown) bind to
CEase in a similar fashion. The docking of compound 8a and 8b
to CEase proved interesting, with its OtBu moiety orientated
towards Ala436 and the 2-propylphenyl functionality rotated
180° away from the hydrophobic pocket (Fig. 8).
5a
S
O
S
O
S
O
S
O
S
S
CH2Ph
CH2Ph
Me
X
5b
6a
6b
7a
7b
8a
8b
9a
10a
HO2C
S
NH
Me
CH2OH
CH2OH
CH2OC(Me)3
CH2OC(Me)3
CH2Ph-4-OH
CH2OCH2Ph
O
b
15a
X = S
15b X = O
b
The group 2 compounds are by and large more potent toward
AChE than are the group 1 compounds, see Table 1. This finding
might be helpful for further investigations directed towards selec-
tivity issues of esterase inhibitors.
Scheme 3. (a) 15a or 15b, HATU, HOBT, DIPEA, DMF; (b) 10% TFA, DCM.
X
R
S
2.5. Kinetic analysis of CEase inhibition
NH
O
The kinetics of this new class of CEase inhibitors were studied in
detail using compound 4a. Interestingly, this compound did not
completely inhibit CEase at high concentrations as plots of the rate
of the enzymatic reaction versus the inhibitor concentration, [I],
did not become asymptotic to the x-axis (see Fig. 9). Therefore, a
residual activity at infinite concentration of 4a (v[I]?1 = 18%) had
to be considered. Such a behavior was also observed for ben-
zylidenethiazolidinediones 4b and 7b as well as benzylidenerhoda-
nine 10a which showed values v[I]?1 of 12–25%. Occurrences of
such residual activities at an infinite inhibitor concentration has
recently been described for the inhibition of CEase44 and related
15a R = CO2H, X = S
15b R = CO2H, X = O
16a R = H, X = S
16b R = H, X = O
Figure 4. Benzylidenerhodanine and benzylidenethiazolidinedione analogs lacking
the amino acid or glycerol functionality.
Docking studies were then carried out using AutoDock4 by per-
forming 25 docking runs on each inhibitor using the Lamarckian
Genetic Algorithm40 and a maximum of 500,000 energy evalua-
tions. Meaningful results were obtained for CEase, with predicted
binding energies ranging from ꢀ10.2 to ꢀ7.0 kcal/mol. However,
despite the similarity of structure, AutoDock4 was unable to place
any of the inhibitors within the active site of AChE. Thus the fol-
lowing discussion on docking is limited to CEase, with some com-
parative analysis.
Based on the docking results, two groups of inhibitors of CEase
were apparent. Group 1 (4a, 4b, 6a, 6b, 7a and 7b) interact simi-
larly with CEase, with the thiazolidinedione and rhodanine moie-
ties binding in the vicinity of residues Ala113, Ala117, Leu124
and Tyr125 (see Fig. 6). This commonality of binding is reflected
a
/b hydrolases, such as AChE7 and butyrylcholinesterase.45 To
characterize the inhibition of CEase by 4a, a kinetic model that rep-
resents the general modifier mechanism46 was considered
(Scheme 4). In this model the substrate (S) and inhibitor (I) bind
to the enzyme (E) at different sites. The dissociation constant of
EI is Ki, and the dissociation constant of the ternary complex, ESI,
to form ES and I is
the catalytic constant kP. The complex ESI is still functional with
decreased rate of product formation governed by bkP. If
aKi. Product formation from ES is governed by
a
Figure 6. Docking of group 1 inhibitors (green) with CEase (PDB: 1AQL). The gray
(hydrophobic), blue (basic) and red (acidic) surface represents the active site of the
enzyme. The catalytic triad is shown (Ser194, Asp320 and His435). The rectangular
box highlights the hydrophobic pocket.
Figure 5. Overlay of bovine CEase (PDB entry: 1AQL, red) and Mus musculus AChE
(PDB entry: 2JEY, blue).

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S. Heng et al. / Bioorg. Med. Chem. 19 (2011) 7453–7463
Scheme 4. Kinetic model of the general modifier mechanism.
6
4
2
0
A
Figure 7. Docking of 5a and 10a within the active site of CEase. The catalytic triad is
shown. Trp227, Leu282, Leu392 define a key hydrophobic pocket.
0
0.2
0.4
0.6
0.8
1
/(1+ )
4
3
2
1
0
B
Figure 8. Docking of 8a within the active site of CEase, with Ala436 highlighted.
The catalytic triad is shown.
120
100
80
60
40
20
0
-0.1
0
0.1
1/[I]
0.2
0.3
Figure 10. (A) Specific velocity plot for the inhibition of CEase by compound 4a.
Rates measured at [I] = 4 M (open circles), 6 M (closed circles), 8 M (open
squares), and 10 M (closed squares) were used. The values /[1+ ], where = [S]/
Km, were calculated using the separately determined Km value of 178 M and
[S] = 100, 150, 200, 250, and 300 M. Linear regression according to Eq. 1 gave
l
l
l
l
r
r
r
l
l
values a and b. (B) Replots a/(aꢀ1) versus 1/[I] (closed circles) and b/(bꢀ1) versus 1/
[I] (open circles) for the inhibition of CEase by 4a. Linear regression according to Eq.
0
2
4
6
8
10
2
gave
regression according to Eq. 3 gave a slope
1/(1ꢀb) = 1.15. Values b = 0.13 (using the intercept of Eq. 3) and
Ki and Ki) were calculated. A value Ki = 1.22 M resulted from the quotient of slope
and intercept of the plot a/(aꢀ1) versus 1/[I].
a
slope
a
Ki/(
a
ꢀb) = 1.39
lM
and an intercept
a aꢀb) = 1.14; linear
/(
M and an intercept
= 5.5 (quotient of
[I] (µM)
a
Ki/(1ꢀb) = 7.68
l
a
Figure 9. Inhibition of CEase by compound 4a. The data are mean values of
quadruplicate experiments with substrate concentrations of [S] = 300 M (open
triangles), 250 (closed squares), 200 (open squares), 150 (closed
M (open circles). At a substrate concentration of 200 M, values
M and [I]?1 = 17.8 1.9% were obtained by nonlinear regres-
a
l
l
lM
lM
lM
circles), and 100
l
l
IC50 = 1.76 0.17
l
v
r
/[1+
r] = 0, as well as r/[1+r] = 1, that is, values a and b, were
used in replots according to Eqs. 2 and 3.
sion according to Eq. 5.
ꢀ
ꢁ
1
1
½Iꢁ
ꢀ
½Iꢁ
1 þ K
v0
vi
aKi
Ki
r
1 þ
0 < b < 1, the type of inhibition is referred to as ‘hyperbolic mixed-
type inhibition’.
i
¼
ꢂ
þ
r
ð1Þ
b½Iꢁ
b½Iꢁ
1 þ a
1 þ a
Ki
Ki
In order to obtain the kinetic parameters Ki,
bition by compound 4a was analyzed using the specific velocity
plot according to Eq. 147 where
0 and v are the rates in the absence
and presence of inhibitor and = [S]/Km. Values 0/v were plotted
versus /[1+ ], and the intercepts on the ordinate axis for
a, and b, CEase inhi-
a
a
Ki
1
a
¼
ꢂ
þ
ð2Þ
ð3Þ
a ꢀ 1
a
ꢀ b ½Iꢁ
a
ꢀ b
v
r
b
a
Ki
1
a
v
¼
ꢂ
þ
b ꢀ 1 1 ꢀ b ½Iꢁ 1 ꢀ b
r
r

S. Heng et al. / Bioorg. Med. Chem. 19 (2011) 7453–7463
7459
From these replots, the values Ki and
parameter was then calculated from Ki and
obtained from the intercept of Eq. 3.
a
Ki were obtained. The
and 39.55 ppm for 13C and CDCl3 were 7.26 ppm for 1H and
77.23 ppm for 13C. Structural assignment was confirmed with
COSY, ROESY, HMQC and HMBC. High-resolution mass spectra
(HRMS) were recorded on a Thermo Fisher Scientific LTQ orbitrap
a
aKi whereas b was
Analysis of CEase inhibition by 4a on the basis of Eqs. 1–3 re-
vealed hyperbolic mixed-type inhibition with a pronounced com-
FT MS equipment (
measurements were performed on a HP Series 1100 with a Phe-
nomenex Gemini C18 5
dissolved in dimethylformamide and eluted at a flow rate of 1 mL/
min with a linear gradient of 40–90% CH3CN in 0.01% TFA/H2O over
15 min, followed by 90% CH3CN in 0.01% TFA/H2O over 10 min.
Purity of tested samples was > 95% or as reported. Compounds
4b,33 11,33 16a54 and 16b55 were synthesized according to reported
procedures.
D <2 ppm). Analytical reversed-phased HPLC
petitive component
intersection point of the lines in the specific velocity plot at
/[1+ ] > 1 (Fig. 10A) and the intercepts of the replots being greater
than one (Fig. 10B).47 On the basis of these replots, the parameters
Ki = 1.22 M, = 5.5 and b = 0.13 were determined. The value
(a >1). This is shown by the common
lM (250 ꢂ 4.60 mm), tested samples were
r
r
l
a
a
indicates a 5- to 6-fold higher affinity of 4a to free CEase than to
the substrate-bound enzyme. However, the parameter b represents
a relative efficiency of ESI conversion to product (at saturating
[I]),48 which was found to be 13% compared to that of ES. This value
is in agreement with the value v[I]?1 = 18% calculated under the
assumption of an infinite inhibitor concentration. While the molec-
ular docking results (Fig. 6) suggest that 4a might interact with the
active site in the EI complex, the arrangement of the corresponding
ESI complex might be different to allow for a residual catalytic
activity.
The observed residual activity of CEase in the presence of high
concentrations of 4a (Fig. 9) might be explained with the so-called
‘side-door’, a secondary pore adjacent to the active site. This side
door in CEase and related carboxylesterases as found by crystallo-
graphic studies potentially acts as an alternative opening allowing
for the traffic of substrates and products.49–53
4.2. General procedures
4.2.1. General procedure A (13a–e)
The N-protected amino acid 12a-e (1 mmol), 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide (1.2 mmol), HOBt (1.2 mmol),
DIPEA (4.75 mmol) and 2-propylaniline (1.2 mmol) were sus-
pended in anhydrous DMF (5 mL). The reaction mixture was stirred
at rt overnight under nitrogen. 1 M HCl (50 mL) was added and the
mixture was extracted with ethyl acetate (3 ꢂ 50 mL). The com-
bined organic phases were washed with 1 M HCl (50 mL), brine
(50 mL), dried over anhydrous Na2SO4 and concentrated in vacuo.
The residue was purified by flash chromatography on silica gel to
yield 13a-e.
3. Conclusion
4.2.2. General procedure B (14a,b)
The N-Boc-amino acid 13a,b (0.3 mmol) was suspended in 10%
trifluoroacetic acid in anhydrous dichloromethane (5 mL). After
stirring overnight the reaction mixture was concentrated in vacuo.
The residue was diluted with methanol (5 mL). Water was added
and the precipitate was collected by filtration and dried in vacuo
to yield 14a and 14b.
CEase plays an important role in the absorption and transport of
cholesterol and is as such an interesting target for developing
treatments for diseases such as hypercholesterolemia and coronary
heart disease. The design of potent inhibitors of CEase, which show
selectivity against other members of the a/b hydrolase family, is a
significant challenge. The present study contributes one possible
solution to this problem. The ‘priviliged’ rhodanine and thiazolid-
inedione scaffolds were chosen, as substances bearing them are
known to inhibit structurally homologous proteins such as PDE4
and protein tyrosine phosphatases. A series of inhibitors with a
central glycerol or amino acid unit and an additional 2-propylphe-
nyl moiety was investigated and possible binding interactions with
CEase were explored using molecular docking and kinetic studies.
Most of the compounds displayed some selectivity for CEase over
the structurally homologous AChE. The optimum compound
4.2.3. General procedure C (14c–e)
The N-Cbz-amino acid 13c–e (0.4 mmol) and cyclohexene
(2.0 mmol) in ethyl acetate (2 mL) was stirred at rt under nitrogen.
10% Pd/C (0.16 mmol) was added and the reaction was refluxed for
2 h. After cooling to rt the mixture was filtered and washed with
ethyl acetate. The filtrate was dried over anhydrous Na2SO4 and
concentrated in vacuo to yield the desired amine to yield 14c–e.
4.2.4. General procedure D (5a, 5b, 6a, 6b, 8a, 8b, 9a, and 10a)
The carboxylic acids 15a,31 or 15b31 (0.22 mmol), HATU
(0.24 mmol), HOBT (0.24 mmol), DIPEA (0.95 mmol) and amino
acid anilide 14a–e (0.20 mmol) were suspended in anhydrous
DMF (2 mL). The reaction mixture was stirred at rt overnight under
nitrogen. 1 M HCl (10 mL) was added and the mixture was
extracted with ethyl acetate (3 ꢂ 25 mL). The combined organic
phases were washed with 1 M HCl (50 mL), brine (50 mL), dried
over anhydrous Na2SO4 and concentrated in vacuo. The residue
was purified by flash chromatography on silica gel to yield 5a,
5b, 6a, 6b, 8a, 8b, 9a, and 10a.
(10a) had an IC50 value of 1.44 lM against CEase, with 17-fold
selectivity for CEase versus AChE. The paper presents a new class
of CEase inhibitors, with significant scope for further optimization
of potency and selectivity against other members of the
lase family.
a/b hydro-
4. Experimental
4.1. General chemistry
All reagents and solvents were from standard commercial
sources and of reagent grade. Reactions were monitored by ascend-
ing TLC using precoated plates (silica gel 60 F254, 250 lm, Merck,
4.3. Spectral data
Darmstadt, Germany), spots were visualized with ultraviolet light
at 254 nm and sulfuric acid–vanillin spray. Column chromatogra-
phy was performed with silica gel (40–63 lm, 60 Å, Davisil, Grace,
Germany). Melting points were recorded uncorrected on a Reichert
Thermovar Kofler microscope. 1H and 13C NMR spectra were
recorded on a Varian Gemini 2000 (300 MHz) or a Varian Inova
600 MHz. Chemical shifts are given in ppm (d) relative to the resi-
due signals, which in the case of DMSO-d6 were 2.50 ppm for 1H
4.3.1. (Z)-5-[[4-[(2R,S)-2-Hydroxy-3-(2-propylphenoxy)
propoxy]phenyl]methylene]-2-thioxo-thiazolidin-4-one (4a)
To a solution of rhodanine (31 mg, 0.23 mmol) in anhydrous
ethanol (10 mL), aldehyde 11 (73 mg, 0.23 mmol) and piperidine
(4.6 lL, 0.046 mmol) were added and refluxed under nitrogen for
8 hrs. The reaction was cooled to rt, diluted and precipitated with
glacial acetic acid (3 mL). The mixture was filtered and washed
with cold water (2 ꢂ 15 mL) and ethanol (2 ꢂ 15 mL). The

7460
S. Heng et al. / Bioorg. Med. Chem. 19 (2011) 7453–7463
precipitate was dissolved in toluene and concentrated in vacuo to
give 4a Yield: 50 mg, 50%. mp: 161–163 °C. 1H NMR (300 MHz,
DMSO-d6): d 13.92 (br s, 1H) 7.57–7.54 (m, 3H), 7.17–7.09 (m,
4H), 7.00 (d, J = 8.4 Hz, 1H), 6.89 (t, J = 7.2 Hz, 1H), 5.43 (br d,
J = 4.8 Hz, 1H), 4.25–4.02 (m, 5H), 2.54–2.49 (m, 6H, signal overlap
with DMSO), 1.56 (tq, J = 7.5, 7.2 Hz, 2H), 0.86 (t, J = 7.5, 3H). 13C
NMR (75 MHz, DMSO-d6): d 196.2, 170.9, 156.2, 133.6, 131.1,
130.3, 129.6, 127.0, 125.8, 123.3, 121.4, 115.5, 112.5, 72.3, 69.7,
68.8, 31.4, 23.6, 14.9. HRMS calcd for C22H22NO4S2 m/z [M-H]-:
428.0990; found: 428.0984. HPLC tR = 16.94 min (96.94%).
(600 MHz, DMSO-d6): d 9.32 (s, 1H), 8.74 (d, J = 6.6 Hz, 1H), 8.02
(d, J = 8.4 Hz, 2H), 7.66 (d, J = 8.4 Hz, 2H), 7.64 (s, 1H), 7.37 (d,
J = 7.2 Hz, 1H), 7.19 (d, J = 7.2 Hz, 1H), 7.09–7.17 (m, 2H), 6.51 (br
s, 1H), 4.65–4.70 (m, 1H), 2.46–2.55 (m, 2H), 1.42–1.50 (m, 5H),
0.83 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, DMSO-d6): d 171.4,
170.4, 168.0, 165.7, 136.9, 136.4, 135.5, 134.0, 129.4, 129.2,
128.2, 127.0, 126.0, 125.8, 125.4, 49.6, 32.9, 22.9, 17.5, 13.8. HRMS
calcd for C23H24N3O4S m/z [M+H]+ 438.1482; found 438.1489.
HPLC tR = 11.02 min (98.43%).
4.3.6. N-[(1S)-1-(Hydroxymethyl)-2-oxo-2-(2-propylanilino)
ethyl]-4-[(Z)-(4-oxo-2-thioxo-thiazolidin-5-ylidene)methyl]
benzamide (7a)
4.3.2. N-[(1S)-1-Benzyl-2-oxo-2-(2-propylanilino)ethyl]-4-[(Z)-
(4-oxo-2-thioxo-thiazolidin-5-ylidene)methyl]benzamide (5a)
Reaction of 15a (58 mg) with 14a (73 mg) according to general
method D gave 5a (60 mg, 57%). mp: 208–211 °C. 1H NMR
(600 MHz, DMSO-d6): d 10.09 (s, 1H), 8.85 (d, J = 8.4 Hz, 1H), 7.92
(d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 7.2 Hz, 2H)
7.31–7.35 (m, 1H), 7.28–7.31 (m, 3H), 4.90–4.94 (m, 1H), 3.69 (br
s, 1H), 2.89 (dd, J = 4.8 Hz, 13.8 Hz, 1H), 2.81–2.86 (m, 1H), 2.13–
2.22 (m, 2H), 1.14 (tq, J = 7.2 Hz, 7.8 Hz, 2H), 0.51 (t, J = 7.2 Hz,
3H). 13C NMR (150 MHz, DMSO-d6): d 198.6, 170.1, 165.6, 163.2,
137.9, 137.0, 136.3, 135.2, 133.5, 129.2 (ꢂ2), 128.9, 128.3, 127.9,
126.1, 125.7, 125.6, 125.5, 125.3, 55.2, 36.7, 32.6, 22.6, 13.6. HRMS
calcd for C29H28N3O3S2 m/z [M+H]+ 530.1569; found 530.1567.
HPLC tR = 16.11 min (97.40%).
Compound 8a (47 mg, 0.1 mmol) was suspended in 10% trifluo-
roacetic acid in dichloromethane (5 mL) at rt. After stirring over-
night the reaction mixture was concentrated in vacuo and
dissolved in methanol (5 mL). Water (50 mL) was added and the
precipitate was filtered and air dried to give a off-white powder
(15 mg, 37%). mp: 165–168 °C. 1H NMR (600 MHz, DMSO-d6):
13.9 (s, 1H), 9.45 (s, 1H), 8.79 (d, J = 7.8 Hz, 1H), 8.03 (d,
J = 7.8 Hz, 2H), 7.66 (d, J = 7.8 Hz, 2H), 7.51 (s, 1H), 7.36 (d,
J = 7.8 Hz, 1H), 7.21 (d, J = 7.2 Hz, 1H), 7.11–7.20 (m, 2H), 4.76–
4.80 (m, 1H), 3.71–3.77 (m, 2H), 2.55–2.58 (m, 2H), 1.44 (qt,
J = 7.2 Hz, 6.6 Hz, 2H), 1.19 (s, 9H), 0.78 (t, J = 7.2 Hz, 3H). 13C
NMR (150 MHz, CDCl3): d 168.6, 165.9, 138.1, 136.6, 136.5, 135.3,
134.3, 129.8, 129.4, 128.7, 128.4, 128.2, 127.5, 127.4, 127.3,
126.0, 125.9, 125.6, 72.2, 69.4, 32.8, 22.8, 13.8. HRMS calcd for
4.3.3. N-[(1S)-1-Benzyl-2-oxo-2-(2-propylanilino)ethyl]-4-[(Z)-
(2,4-dioxothiazolidin-5-ylidene)methyl]benzamide (5b)
Reaction of 15b (54 mg) with 14a (73 mg) according to general
method D gave 5b (71 mg, 69%). mp: 175–178 °C. 1H NMR
(600 MHz, DMSO-d6): d 9.52 (br s, 1H), 8.84 (d, J = 7.8 Hz, 1H),
7.95 (d, J = 8.4 Hz, 2H), 7.69 (s, 1H), 7.65 (d, J = 8.4 Hz, 2H), 7. 42
(d, J = 7.8 Hz, 2H), 7.28–7.34 (m, 2H), 7.11–7.21 (m, 3H), 6.53 (br
s, 1H), 4.92–4.96 (m, 1H), 3.23 (dd, J = 4.8 Hz, 13.8 Hz, 1H), 3.10–
3.26 (m, 1H), 2.46–2.55 (m, 2H), 1.47 (tq, J = 7.2 Hz, 7.8 Hz, 2H),
0.84 (t, J = 7.2 Hz). 13C NMR (150 MHz, DMSO-d6): d 171.4, 170.5,
169.8, 165.8, 138.2, 136.7, 136.6, 135.4, 134.3, 129.5 (ꢂ2), 129.3,
128.2, 126.4, 126.1, 125.7, 49.8, 36.1, 32.9, 23.0, 13.9. HRMS calcd
for C29H28N3O4S m/z [M+H]+ 514.1795; found 514.1801. HPLC
tR = 14.85 min (98.16%).
C
23H24N3O4S2 m/z [M+H]+ 470.1203; found 470.1269. HPLC
tR = 11.03 min (95.30%).
4.3.7. 4-[(Z)-(2,4-Dioxothiazolidin-5-ylidene)methyl]-N-[(1S)-1-
(hydroxymethyl)-2-oxo-2-(2-propylanilino)ethyl]benzamide
(7b)
Compound 8b (45 mg, 0.1 mmol) was suspended in 10% trifluo-
roacetic acid in dichloromethane (5 mL) at rt. After stirring over-
night the reaction mixture was concentrated in vacuo and
dissolved in methanol (5 mL). Water (50 mL) was added and the
precipitate was filtered and air dried to give a white powder
(20 mg, 50% yield). mp: 116 °C. 1H NMR (600 MHz, DMSO-d6): d
8.54 (s, 1H), 7.94 (d, J = 8.2 Hz, 2H), 7.83 (d, J = 8.2 Hz, 1H), 7.78
(s, 1H), 7.55 (d, J = 7.8 Hz, 2H), 7.47 (d, J = 6.0 Hz, 1H), 7.24–7.08
(m, 3H), 4.87–4.75 (m, 1H), 4.09 (dd, J = 9.0, 1.8 Hz, 1H), 3.60 (t,
J = 9.0 Hz, 1H), 2.60 (t, J = 7.2 Hz, 2H), 1.69–1.59 (m, 2H), 0.99–
0.93 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, CDCl3): d 169.0, 166.6,
166.4, 166.3, 136.1, 135.0, 134.6, 133.8, 132.4, 130.2, 129.6,
128.1, 127.9, 126.6, 125.6, 124.7, 123.7, 75.3, 61.4, 33.2, 22.9,
13.9. HRMS calcd for C23H24N3O5S m/z [M+H]+ 454.1437; found
454.1452. HPLC tR = 14.52 min (95.00%).
4.3.4. N-[(1S)-1-Methyl-2-oxo-2-(2-propylanilino)ethyl]-4-[(Z)-
(4-oxo-2-thioxo-thiazolidin-5-ylidene)methyl]benzamide (6a)
Reaction of 13c (134 mg) according to general method C gave
14c (56 mg, 69%). mp: 124–125 °C. 1H (300 MHz, CD3OD): d 9.86
(br s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.02–7.23 (m, 3H), 3.63 (q,
J = 7.2 Hz, 1H), 2.58 (t, J = 7.8 Hz, 2H), 1.63 (qt, J = 7.8 Hz, 7.2 Hz,
2H), 1.42 (d, J = 7.2 Hz, 3H) 0.98 (t, J = 7.2 Hz, 3H); 13C NMR
(75 MHz, CD3OD): d 173.5, 135.3, 132.0, 129.5, 126.6, 124.2,
121.4, 51.3, 33.5, 22.8, 21.5, 13.9.
Reaction of 15a (58 mg) with 14c (41 mg) according to general
method D gave 6a (54 mg, 60%). mp: 144–146 °C. 1H NMR
(600 MHz, DMSO-d6): d 9.32 (s, 1H), 8.73 (d, J = 6.6 Hz, 1H), 8.01
(d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 7.8 Hz, 1H),
7.31 (s, 1H), 7.09–7.19 (m, 3H), 6.51 (s, 1H), 4.64–4.69 (m, 1H),
2.49–2.55 (m, 2H), 1.43–1.51 (m, 2H), 1.46 (d, J = 7.2 Hz, 3H),
0.83 (t, J = 7.8 Hz, 3H). 13C NMR (150 MHz, DMSO-d6): d 171.4,
165.7, 137.5, 136.4, 135.5, 133.5, 129.4, 129.3, 128.2, 126.0,
125.8, 125.4, 124.1, 49.7, 32.9, 22.9, 18.3, 13.8. HRMS calcd for
4.3.8. N-[(1S)-1-(tert-Butoxymethyl)-2-oxo-2-(2-propylanilino)
ethyl]-4-[(Z)-(4-oxo-2-thioxo-thiazolidin-5-ylidene)
methyl]benzamide (8a)
Reaction of 15a (58 mg) with 14e (55 mg) according to gen-
eral method D gave 8a (92 mg, 88%). mp: 156–160 °C. 1H NMR
(600 MHz, DMSO-d6): d 9.16 (s, 1H), 8.31 (d, J = 7.8 Hz, 1H),
8.02 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.53 (d,
J = 8.4 Hz, 1H), 7.51 (s, 1H), 7.10–7.20 (m, 3H), 4.85 (dd,
J = 5.7 Hz, 13.4 Hz, 1H), 3.76–3.86 (m, 2H), 2.55–2.60 (m, 2H)
1.50–1.63 (m, 2H), 1.24 (s, 9H), 0.9 (t, J = 7.2 Hz, 3H). 13C NMR
(150 MHz, CDCl3): d 196.7, 172.6, 167.5, 164.6, 135.3, 134.2,
134.2, 133.8, 132.9, 129.7, 128.3, 127.9, 126.8, 126.1, 124.5,
123.9, 123.6, 71.9, 60.2, 53.2, 31.5, 25.9, 21.4, 12.4. HRMS calcd
for C27H32N3O4S2 m/z [M+H]+ 526.1829; found 526.1840. HPLC
tR = 16.41 min (97.22%).
C
23H24N3O3S2 m/z [M+H]+ 454.1254; found 454.1258. HPLC
tR = 13.14 min (97.66%).
4.3.5. 4-[(Z)-(2,4-Dioxothiazolidin-5-ylidene)methyl]-N-[(1S)-1-
methyl-2-oxo-2-(2-propylanilino)ethyl]benzamide (6b)
Reaction of 15b (54 mg) with 14c (41 mg) according to general
method D gave 6b (65 mg, 74%). mp: 168–170 °C. 1H NMR

S. Heng et al. / Bioorg. Med. Chem. 19 (2011) 7453–7463
7461
4.3.9. N-[(1S)-1-(tert-Butoxymethyl)-2-oxo-2-(2-propylanilino)
ethyl]-4-[(Z)-(2,4-dioxothiazolidin-5-ylidene)
methyl]benzamide (8b)
1H NMR (600 MHz, CDCl3): d 7.95 (br s, 1H), 7.84 (d, J = 7.8 Hz, 1H),
7.30–7.36 (m, 5H), 7.20 (dd, J = 7.2, 7.8 Hz, 1H), 7.16 (d, J = 7.2 Hz,
1H), 7.10 (dd, J = 7.2, 7.2 Hz, 1H), 5.28 (br s, 1H), 5.17 (d,
J = 12.6 Hz, 1H), 5.15 (d, J = 12.6 Hz, 1H), 4.36–4.45 (m, 1H), 2.49–
2.52 (m, 2H), 1.53–1.63 (m, 2H), 1.49 (d, J = 7.2 Hz, 3H), 0.95 (t,
J = 7.2 Hz, 3H). 13C NMR (150 MHz, CDCl3): d 170.3, 156.4, 135.8,
134.8, 133.4, 129.7, 128.6, 128.4, 128.1, 126.7, 125.3, 123.2, 67.4,
51.2, 33.4, 23.1, 17.7, 13.9; HRMS calcd for C20H25N2O3 m/z
[M+H]+ 341.1864; found 341.1860.
Reaction of 15b (54 mg) with 14e (55 mg) according to general
method D gave 8b (87 mg, 85%). mp: 141–143 °C. 1H NMR
(600 MHz, DMSO-d6): d 12.59 (br, 1H), 9.40 (s, 1H), 8.57 (d,
J = 7.8 Hz, 1H), 8.01 (d, J = 7.8 Hz, 2H), 7.23 (s, 1H), 7.68 (d,
J = 7.8 Hz, 2H), 7.35 (d, J = 7.8 Hz, 1H), 7.20 (d, J = 7.2 Hz, 1H),
7.16 (t, J = 7.2 Hz, 1H), 7.11 (dd, J = 7.2 Hz, 7.8 Hz, 1H), 4.75–4.78
(m, 1H), 3.69–3.76 (m, 2H), 2.52–2.55 (m, 2H), 1.49 (tq,
J = 7.2 Hz, 6.6 Hz, 2H), 1.15 (s, 9H), 0.84 (t, J = 6.6 Hz, 3H). 13C
NMR (150 MHz, CDCl3): d 169.1, 169.0, 165.7, 136.5, 136.4, 135.5,
134.5, 129.5, 129.3, 128.7, 128.3, 125.9, 125.8, 125.6, 73.0, 61.6,
54.8, 31.3, 27.3, 22.7, 13.9. HRMS calcd for C27H32N3O5S m/z
[M+H]+ 510.2057; found 510.2068.
4.3.14. Benzyl N-[(1S)-1-[(4-hydroxyphenyl)methyl]-2-oxo-2-(2-
propylanilino)ethyl]carbamate (13d)
Reaction of 12d (314 mg) with 2-propyl-aniline (160 mg)
according to general method A gave 13d (198 mg, 46%). mp:
125–129 °C. 1H NMR (300 MHz, DMSO-d6): d 8.28 (s, 1H), 7.92 (d,
J = 8.1 Hz, 1H), 7.07–7.37 (m, 8H), 5.51 (br s, 1H), 4.64 (d,
J = 11.7 Hz, 1H), 4.57 (d, J = 11.7 Hz, 1H), 4.39–4.50 (m, 1H), 4.04
(dd, J = 3.6 Hz, 9.3 Hz, 1H), 3.69 (dd, J = 6.6 Hz, 9.3 Hz, 1H), 2.45
(t, J = 7.5 Hz, 2H), 1.40–1.51 (m, 2H), 0.88 (t, J = 6.9 Hz, 3H). 13C
NMR (75 MHz, DMSO-d6): d 168.2, 155.3, 136.8, 134.6, 132.6,
129.3, 128.2, 127.7, 127.6, 126.4, 124.7, 122.5, 80.2, 73.3, 69.4,
54.1, 32.9, 27.9, 22.5, 13.5. HRMS calcd for C26H29N2O4 m/z
[M+H]+ 433.2126; found 433.2122.
4.3.10. N-[(1S)-1-[(4-Hydroxyphenyl)methyl]-2-oxo-2-(2-
propylanilino)ethyl]-4-[(Z)-(4-oxo-2-thioxo-thiazolidin-5-
ylidene)methyl]benzamide (9a)
Reaction of 15a (58 mg) with 14d (60 mg) according to general
method D gave 9a (34 mg, 31%). mp: 262–265 °C. 1H NMR
(600 MHz, DMSO-d6): d 13.90 (s, 1H), 9.57 (s, 1H), 8.96 (d,
J = 7.8 Hz, 1H), 8.21 (d, J = 8.4 Hz, 2H), 7.98 (d, J = 8.4 Hz, 2H),
7.79 (d, J = 8.4 Hz, 2H), 7.68 (m, 3H), 7.31 (d, J = 7.8 Hz, 1H),
7.09–7.25 (m, 3H), 6.66 (d, J = 9.0 Hz, 1H), 4.95–4.99 (m, 1H),
3.25–3.31 (m, 1H, overlap with H2O), 3.15–3.20 (m, 1H),
2.51–2.56 (m, 2H), 1.44–1.52 (m, 2H), 0.85 (t, J = 7.8 Hz, 3H). 13C
NMR (150 MHz, DMSO-d6): d 195.6, 170.3, 165.7, 163.9, 149.1,
137.9, 136.7, 135.4, 135.1, 130.6, 130.5, 130.3, 130.2, 129.9,
129.4, 128.7, 127.3, 126.0, 125.7, 121.5, 55.4, 36.3, 32.9, 22.9,
13.9. HRMS calcd for C29H28N3O4S2 m/z [M+H]+ 546.1516; found
546.1524. HPLC tR = 14.77 min (95.38%).
4.3.15. Benzyl N-[(1S)-1-(tert-butoxymethyl)-2-oxo-2-(2-
propylanilino)ethyl]carbamate (13e)
Reaction of 12e (294 mg) with 2-propylaniline (160 mg) accord-
ing to general method A gave 13e (280 mg, 68%). mp: 114–116 °C.
1H NMR (600 MHz, CDCl3): d 8.29 (br s, 1H), 7.83 (d, J = 8.1 Hz, 1H),
7.37–7.32 (m, 5H), 7.21 (t, J = 7.2 Hz, 1H), 7.12 (d, J = 7.2 Hz, 1H),
7.11 (t, J = 7.2 Hz, 1H), 5.85 (br s, 1H), 5.19 (s, 2H), 4.50–4.32 (m,
1H), 4.04–3.87 (m, 1H), 3.56–3.50 (m, 1H), 2.55 (t, J = 7.8 Hz, 2H),
1.55–1.63 (m, 2H), 1.23 (br s, 9H) 0.96 (t, J = 7.8 Hz, 3H). 13C
NMR (600 MHz, CDCl3): d 168.7, 156.2, 156.2, 134.8, 133.5, 129.5,
128.6, 128.3, 128.2, 126.7, 125.3, 123.4, 67.2, 61.8, 55.5, 33.3,
4.3.11. N-[(1S)-1-(Benzyloxymethyl)-2-oxo-2-(2-
propylanilino)ethyl]-4-[(Z)-(4-oxo-2-thioxo-thiazolidin-5-
ylidene)methyl]benzamide (10a)
27.5, 22.9, 13.9. HRMS calcd for
413.2441; found 413.2435.
C
24H33N2O4 m/z [M+H]+
Reaction of 15a (58 mg) with 14b (85 mg) according to general
method D gave 10a (95 mg, 85%). mp 112–115 °C. 1H NMR
(600 MHz, DMSO-d6): d 9.48 (s, 1H), 8.79 (d, J = 7.2 Hz, 1H), 8.03
(d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.4, 2H), 7.48 (s, 1H), 7.27–7.36 (m,
5H), 7.10–7.19 (m, 3H), 6.51 (br, 1H), 4.97 (dd, J = 7.2 Hz, 13.2 Hz,
1H), 4.57–4.61 (m,2H), 3.84–3.90 (m, 2H), 2.45–254 (m, 2H),
1.41–1.46 (m, 2H), 0.78 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz,
DMSO-d6): d 196.0, 168.7, 165.9, 138.1, 136.7, 136.5, 135.4,
134.2, 129.8 (ꢂ2), 129.4, 129.3, 128.7, 128.5, 128.3, 128.2, 127.5,
127.4, 126.8, 126.0, 125.8, 125.6, 72.2, 55.8, 42.1, 32.8, 22.8, 13.9.
4.3.16. (2S)-2-Amino-3-phenyl-N-(2-propylphenyl)
propanamide trifluoroacetate salt (14a)
Reaction of 13a (114 mg) according to general method B gave
14a (67 mg, 57%). mp: 172–176 °C. 1H NMR (600 MHz, CD3OD): d
7.16–7.40 (m, 9H), 4.41 (t, J = 7.8, 1H), 3.34 (dd, J = 14.4 Hz,
7.2 Hz, 1H) 3.21 (dd, J = 13.9 Hz, 7.2 Hz, 1H), 2.37–2.47 (m, 2H),
1.52 (qt, J = 7.8 Hz, 7.2 Hz, 2H), 0.90 (t, J = 7.8 Hz, 3H). 13C NMR
(150 MHz, CD3OD): d 169.4, 139.1, 136.1, 135.7, 131.2, 131.1,
130.7, 129.5, 128.6, 127.9, 127.8, 56.5, 39.3, 34.4, 24.7, 14.7. HRMS
calcd for C18H23N2O m/z [M+H]+ 283.1803; found 283.1810.
HRMS calcd for
C
30H30N3O4S2 m/z [M+H]+ 560.1672; found
560.1680. HPLC: 16.63 min (94.22%).
4.3.12. tert-Butyl N-[(1S)-1-benzyl-2-oxo-2-(2-propylanilino)
ethyl]carbamate (13a)
4.3.17. (2S)-2-Amino-3-benzyloxy-N-(2-propylphenyl)
propanamide trifluoroacetate salt (14b)
Reaction of 12a (264 mg) with 2-propylaniline (160 mg)
according to general method A gave 13a (243 mg 59%). mp:
75–78 °C. 1H NMR (600 MHz, DMSO-d6): d 7.82 (d, J = 8.4 Hz, 1H),
7.65 (s, 1H), 7.24–7.33 (m, 5H), 7.19 (dd, J = 7.2, 7.8 Hz, 1H), 7.12
(d, J = 7.2 Hz), 7.08 (t, J = 7.2 Hz, 1H), 5.08 (br, 1H), 4.47–4.49 (m,
1H), 3.19 (d, J = 7.2 Hz, 2H), 2.27–2.38 (m, 2H), 1.43–1.50 (m,
12H) 0.89 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, DMSO-d6): d
169.5, 155.6, 136.6, 134.7, 133.1, 129.5, 129.3, 128.8, 127.1,
126.7, 125.2, 122.9, 80.7, 56.7, 38.0, 33.0, 28.3, 22.9, 13.9. HRMS
calcd for C23H31N2O3 m/z [M+H]+ 383.2330; found 383.2329.
Reaction of 12b (295 mg) with 2-propylaniline (160 mg)
according to general method A gave 13b (308 mg, 81%). mp: 94–
99 °C. 1H NMR (300 MHz, DMSO-d6): d 8.28 (s, 1H), 7.92 (d,
J = 8.1 Hz, 1H), 7.07–7.37 (m, 8H), 5.51 (br s, 1H), 4.64 (d,
J = 11.7 Hz, 1H), 4.57 (d, J = 11.7 Hz, 1H), 4.39–4.50 (m, 1H), 4.04
(dd, J = 3.6 Hz, 9.3 Hz, 1H), 3.69 (dd, J = 6.6 Hz, 9.3 Hz), 2.45 (t,
J = 7.5 Hz, 2H), 1.40–1.51 (m, 11H), 0.88 (t, J = 6.9 Hz, 3H). 13C
NMR (300 MHz, DMSO-d6): d 168.2, 155.3, 136.8, 134.6, 132.6,
129.3, 128.2, 127.7, 127.6, 126.4, 124.7, 122.5, 80.2, 73.3, 69.4,
32.9, 27.9, 22.5, 13.5.
Reaction of 13b (119 mg) according to general method B then
gave 14b (126 mg, 99%). 1H NMR (600 MHz, CD3OD): d 7.20–7.41
(m, 9H), 4.65–4.69 (m,, 2H), 4.37 (dd, J = 4.2 Hz, 5.4 Hz, 1H), 3.99
(dd, J = 4.2 Hz, 10.2 Hz, 1H), 3.96 (dd, J = 5.4 Hz, 10. 2 Hz, 1H),
2.55 (dt, J = 2.4 Hz, 7.8 Hz, 2H), 1.55 (tq, J = 7.2 Hz, 7.8 Hz, 2H)
4.3.13. Benzyl N-[(1S)-1-methyl-2-oxo-2-(2-propylanilino)
ethyl]carbamate (13c)
Reaction of 12c (222 mg) with 2-propylaniline (160 mg) accord-
ing to general method A gave 13c (282 mg, 83%). mp: 128–131 °C.

7462
S. Heng et al. / Bioorg. Med. Chem. 19 (2011) 7453–7463
0.88 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, CD3OD): d 167.2, 139.0,
138.5, 135.4, 131.5, 131.0, 129.6, 129.2, 128.2, 127.7, 127.6, 75.2,
69.8, 55.3, 34.9, 24.9, 14.8. HRMS calcd for C19H25N2O2 m/z
[M+H]+ 313.1910; found 313.1916.
Compounds 5a, 5b, 6b and 8b were analyzed in quadruplicate
experiments, 6a and 7a in sixtuplicate experiments, and compounds
15a–16b in duplicate experiments, each at a single inhibitor
concentration (50 lM). IC50 values were calculated using Eq. 4,
where v0 and
v
are the rates in the absence and the presence of
4.3.18. (2S)-2-Amino-3-(4-hydroxyphenyl)-N-(2-propylphenyl)
propanamide (14d)
inhibitor.
½Iꢁ
À
Á
IC50
¼
ð4Þ
Reaction of 13d (172 mg) according to general method C gave
14d (83 mg, 70%). mp: 120–122 °C. 1H NMR (300 MHz, CD3OD): d
7.50 (d, J = 7.2 Hz, 1H), 7.07–7.19 (m, 5H), 6.71–6.75 (m, 2H),
3.69 (dd, J = 7.2 Hz, 6.3 Hz, 1H), 3.03 (dd, J = 6.3 Hz, 13.5 Hz, 1H),
2.84 (dd, J = 7.2 Hz, 13.5 Hz, 1H), 2.42 (t, J = 7.8 Hz, 2H), 1.49 (qt,
J = 7.8 Hz, 7.5 Hz, 2H), 0.89 (t, J = 7.5 Hz, 3H). 13C NMR (75 MHz,
CD3OD): d 175.9, 157.5, 137.2, 136.2, 131.5, 130.7, 129.4, 127.4,
127.0, 125.9, 116.4, 58.2, 41.5, 34.3, 24.3, 14.3; HRMS calcd for
v0
v
ꢀ 1
For inhibitors 4a, 4b, 7b, 8a, 9a and 10a five different concentra-
tions were used in duplicate experiments at a single concentration
of pNPB (200 lM). IC50 values for 4a, 4b, 7b and 10a, resulted from
plots of rates versus [I] and non-linear regression according to Eq.
5, where v[I]?1 is the residual activity at infinite concentration of
the inhibitor. The IC50 value calculated by Eq. 5 corresponds to
the concentration of the inhibitor which reduces the rate of the en-
C
18H23N2O2 m/z [M+H]+ 299.1754; found 299.1754.
zyme-catalyzed reaction to a velocity (v0
the velocity half between 0 and v[I]?1. Data v[I]?1 are given as rel-
ative velocities with respect to v0 at a substrate concentration of
200 M.
– v[I]?1)/2 + v[I]?1, i.e.
4.3.19. (2R)-2-Amino-3-tert-butoxy-N-(2-propylphenyl)
propanamide (14e)
v
Reaction of 13e (163 mg) according to general method C gave
14e (95 mg, 86%). 1H NMR (600 MHz, CD3OD): d 9.68 (s, 1H),
8.08 (dd, J = 7.8 Hz, 1H), 7.21 (dd, J = 7.2, 6.6 Hz, 1H), 7.16 (d,
J = 7.8 Hz), 7.05 (dd, J = 7.2, 6.6 Hz, 1H), 3.74–3.59 (m, 3H), 2.62
(t, J = 7.8 Hz, 2H), 1.88 (br s, 2H), 1.75–1.57 (m, 2H), 1.20 (br s,
9H) 1.04 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, CD3OD): d 170.2,
134.4, 131.1, 128.5, 125.7, 123.3, 120.6, 72.4, 62.6, 55.1, 32.7,
l
ð
v0
ꢀ
v
½Iꢁ!1Þ
ꢀ
ꢁ
v
¼
þ
v
ð5Þ
½Iꢁ!1
½Iꢁ
1 þ IC
50
IC50 values for 8a and 9a resulted from plots of rates versus [I]
and non-linear regression according to Eq. (6).
26.5, 21.9, 13.0. HRMS calcd for
279.2065 found 279.2067.
C
16H27N2O2 m/z [M+H]+
V0
ꢀ
ꢁ
v
¼
ð6Þ
½Iꢁ
1 þ IC
50
4.4. Biological assays
Substrate dependency of CEase inhibition by 4a was analyzed in
quadruplicate experiments at five different inhibitor concentra-
tions (2–10 M) with five different substrate concentrations
(100–300 M). The rate of the uninhibited reaction in the presence
4.4.1. General methods and material
l
Enzymatic assays were done on a Varian Cary 50 Bio UV/VIS
spectrometer and a Varian Cary 100 Bio UV/VIS spectrometer,
both with a cell holder equipped with a constant temperature
water bath. Cholesterol esterase (CEase) from porcine pancreas,
acetylthiocholine iodide (ATCh), 5,50-dithiobis(2-nitrobenzoic
acid) (DTNB), sodium taurocholate (TC), and p-nitrophenyl buty-
rate (pNPB) were obtained from Sigma (Steinheim, Germany).
Acetylcholinesterase (AChE) from E. electricus, anhydrous DMSO,
and acetonitrile (HPLC grade) were purchased from Fluka
(Deisenhofen, Germany).
l
of a substrate concentration of 200
lM was set 100% in these
experiments.
4.4.3. AChE inhibition assay
Inhibition of acetylcholinesterase from E. electricus was assayed
spectrophotometrically at 412 nm at 25 °C as previously de-
scribed.7 Assay buffer was 100 mM sodium phosphate, 100 mM
NaCl, pH 7.3. The enzyme stock solution (ꢃ100 U/mL) in assay buf-
fer was kept at 0 °C. Appropriate dilutions were prepared immedi-
ately before starting the measurement. ATCh (10 mM) and DTNB
(7 mM) were dissolved in assay buffer and kept at 0 °C. The final
4.4.2. CEase inhibition assay
Porcine cholesterol esterase inhibition was assayed spectro-
photometrically at 405 nm at 25 °C as previously described.7
Assay buffer was 100 mM sodium phosphate, 100 mM NaCl, pH
concentration of acetonitrile was 6%, of ATCh 500
350 M. Assays were performed with a final concentration of
ꢃ30 mU/mL of AChE which corresponded to an initial rate of
ꢃ0.8 M/min. Into a cuvette containing 830 L assay buffer, 50
of the DTNB solution, 55 L acetonitrile, 5 L solution of the test
compound, and 10
L of an enzyme solution (ꢃ3 U/mL) were
added and thoroughly mixed. After incubation for 15 min at
25 °C, the reaction was initiated by adding 50 L of the ATCh solu-
lM, and of DTNB
l
7.0. A stock solution of CEase (122 lg/mL) was prepared in
l
l
ll
100 mM sodium phosphate buffer, pH 7.0 and kept at 0 °C. A
1:122 dilution was done with the same buffer immediately before
starting the measurement. TC (12 mM) was dissolved in assay
buffer and kept at 25 °C. A stock solution of pNPB (20 mM) was
prepared in acetonitrile. The final concentration of acetonitrile
l
l
l
l
tion. IC50 values were calculated from the linear steady-state turn-
over of the substrate.
Compounds 5a, 8b, 9a, and 10a were analyzed in quadruplicate
experiments, compound 5b in a sixtuplicate experiment, and com-
pounds 4b, 6a, 6b, 7a, 7b and 15a–16b in duplicate experiments,
was 3%, of DMSO 3%, of the substrate pNPB 200 lM, and of TC
6 mM, which corresponds to the duodenal bile salt concentra-
tion.56 Assays were performed with a final concentration of
10 ng/mL of CEase which corresponded to an initial rate of
5.0
500
DMSO, 10
l
l
M/min. Into
L of the TC solution, 10 or 20
L of the pNPB solution, and 10
a
cuvette containing 430
L acetonitrile, 30 or 20
L of a inhibitor solu-
lL assay buffer,
each at a single inhibitor concentration (5–25 lM) and IC50 values
l
lL
were calculated with Eq. (4). Inhibitors 4a and 8a were analyzed in
duplicate experiments at five different concentrations using a
three-parameter Eq. 7.
l
l
tion in either actonitrile or DMSO were added and thoroughly
mixed. After incubation for 5 min at 25 °C, the reaction was
v0
initiated by adding 10 lL of the enzyme solution (1 lg/mL) and
followed over 6 min. IC50 values were calculated from the linear
steady-state turnover of the substrate.
v
¼
ð7Þ
ꢀ
ꢁ
X
½Iꢁ
1 þ IC
50

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Acknowledgments
The authors acknowledge financial support from the Australian
Research Council (ARC). M.P. thanks the German Academic Ex-
change Service for a fellowship within the ‘‘Postdoc-Programm’’.
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