Design, synthesis, and structure-activity relationship exploration of 1-substituted 4-aroyl-3-hydroxy-5-phenyl-1 H -pyrrol-2(5 H)-one analogues as inhibitors of the annexin A2′S100A10 protein interaction

Article abstract of DOI:10.1021/jm101212e

S100 proteins are small adaptors that regulate the activity of partner proteins by virtue of direct protein interactions. Here, we describe the first small molecule blockers of the interaction between S100A10 and annexin A2. Molecular docking yielded cand

Full text of DOI:10.1021/jm101212e

ARTICLE
pubs.acs.org/jmc
Design, Synthesis, and Structure-Activity Relationship Exploration
of 1-Substituted 4-Aroyl-3-hydroxy-5-phenyl-1H-pyrrol-2(5H)-one
Analogues as Inhibitors of the Annexin A2-S100A10 Protein
Interaction
Tummala R. K. Reddy, Chan Li, Xiaoxia Guo, Helene K. Myrvang, Peter M. Fischer, and Lodewijk V. Dekker*
School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, United Kingdom
S Supporting Information
b
ABSTRACT: S100 proteins are small adaptors that regulate the activity of partner
proteins by virtue of direct protein interactions. Here, we describe the first small
molecule blockers of the interaction between S100A10 and annexin A2. Molecular
docking yielded candidate blockers that were screened for competition of the
binding of an annexin A2 peptide to S100A10. Several inhibitory clusters were
identified with some containing compounds with potency in the lower micro-
molar range. We chose 3-hydroxy-1-(2-hydroxypropyl)-5-(4-isopropylphenyl)-
4-(4-methylbenzoyl)-1H-pyrrol-2(5H)-one (1a) as a starting point for struc-
ture-activity studies. These confirmed the hypothetical binding mode from the
virtual screen for this series of molecules. Selected compounds disrupted the
physiological complex of annexin A2 and S100A10, both in a broken cell
preparation and inside MDA-MB-231 breast cancer cells. Thus, this class of
compounds has promising properties as inhibitors of the interaction between
annexin A2 and S100A10 and may help to elucidate the cellular function of this
protein interaction.
’ INTRODUCTION
interacts with S100A10 as well as S100A4.2,13 It is highly
expressed in endothelial cells and is overexpressed in a number
of cancers, including breast and colon cancer, in which it has been
implicated in invasive behavior.14-16 Annexin A2 function is
crucial for blood vessel formation since its removal by genetic
deletion compromises the capacity of endothelial cells to digest
the extracellular matrix and to form new networks in the
extracellular matrix in the mouse in vivo.14 Similarly, blocking
the cell surface annexin A2 using antibodies reduces the capacity
of breast cancer cells to digest and migrate through a collagen
matrix.15
S100 proteins were first isolated from brain by virtue of their
solubility in saturated aqueous ammonium sulfate solution at neutral
pH. Since then, over 20 family members have been identified, each of
them unique in sequence and tissue distribution. S100 proteins belong
to the larger EF hand family of Ca2þ-binding proteins and primarily
exist as homodimers or heterodimers.1-5 Each S100 protein contains
two nonidentical EF hand motifs connected by a linker region. EF
hand motifs consist of two R-helices linked by a 12 amino acid long
Ca2þ binding loop.4 Most S100 proteins (dimers) are responsive to
Ca2þ by virtue of these EF hand motifs.6 The exception to this is
S100A10, which is permanently locked in a “Ca2þ on” configuration.
The principle function of S100 proteins is the regulation of the
localization and activity of other proteins by direct protein-
protein interactions. Some of these interactions potentially have
therapeutic relevance. For example, the interaction of S100A4
and the coiled coil region of the myosin-IIA heavy chain has been
implicated in cell movement in cancer.7 As such, S100 protein
interactions are attracting interest as drug targets.8 Indeed,
progress has been made in the identification of small molecule
blockers of S100B and S100A4.9-11
At the molecular level, the interaction between S100A10 and
annexin A2 is very well characterized both by mutagenesis and by
crystallography.2,17 Two S100A10 molecules form a dimeric
structure that yields two binding pockets, each of which accom-
modates the 14 amino acid N-terminal region of annexin A2.
Biophysical studies suggest that one of the two sites of the
S100A10 homodimer must be occupied by the annexin A2 N
terminus before the second annexin A2 protein can bind.18
A
synthetic peptide encompassing the N-terminal annexin A2
Members of the annexin family of Ca2þ/membrane binding
proteins are partners of several S100 proteins.12 Annexin A2
Received: September 16, 2010
Published: March 04, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
ARTICLE
region can disrupt preformed annexin A2-S100A10 complexes in
vitro, suggesting that it has a dominant negative effect on annexin
A2-S100A10 complex formation.19 This same N-terminal pep-
tide inhibited neoangiogenesis into matrigel plugs in a mouse
model system,14 and it also inhibited metastasis of prostate
cancer cells into the bone marrow in mice.20 Thus, complex
formation with S100A10 may be the key to regulating several of
the processes in which annexin A2 has been implicated.
analysis. The suggested binding poses were subjected to visual
inspection and ranked based on hit compounds apparently being
able (i) to occupy the hydrophobic regions H1, H2, and the
hydrophilic pocket H3 (see below); (ii) to form productive
hydrogen bonds with the receptor; and (iii) to form electrostatic
complementarity with the binding pocket.29 Furthermore, com-
pounds that required high entropic energy (for instance torsional
energy) to fit in the binding pocket were excluded.
On the basis of these criteria, a subset of 394 compounds was
selected, and a clustering analysis was performed using a hier-
archical clustering algorithm in which 2D fingerprints and atom
pairs were taken as metrics to quantify the differences in chemical
structures.30 A total of 27 different clusters were identified in this
way as well as three singletons. One distinct cluster contained 79
compounds, and the remaining 26 clusters contained on average
12 compounds.
To evaluate the virtual hit library, the above-prioritized 394
compounds were acquired and screened using a competitive
binding assay based on fluorescence resonance energy transfer
(FRET) between appropriately labeled S100A10 and an N-term-
inal annexin A2 peptide.31 A primary screen was performed in
which each compound was tested at a single concentration of 10
μM in quadruplicate. Compounds for which individual replicates
showed an inhibitory activity that differed by more than three
standard deviations from negative controls were rescreened and
half-maximal inhibition concentration (IC50) values determined.
This revealed that a total of 29 compounds possessed measurable
inhibition potency. These compounds belonged to 10 different
clusters (12 compounds from cluster 1, four each from clusters 9
and 19, two each from clusters 5 and 23, and one compound each
from clusters 3, 6, 17, 22, and 26). No active compounds were
obtained from the remaining clusters. Example compounds
representing six clusters are shown in Chart 1.
Detailed inspection of the binding site reveals that the
annexin-A2 N terminus buries ca. 660 Å2 of solvent accessible
surface area (SAS) in a compact lipophilic pocket of S100A10.2
Most of the binding energy derives from hydrophobic interac-
tions in the innermost portion of the pocket as well as charge-
enhanced H-bonds with the carboxyl groups of E5 and E9 of
S100A10.2,17 The main interacting residues at the N terminus (V3,
I6, L7, and L10) alone displace ca. 430 Å2 of SAS. The size of the
binding pocket is thus within reach of what are commonly
considered druglike molecules. Here, we describe the first small
molecule blockers of this binding interaction. We used molecular
docking to preselect candidate inhibitory molecules and tested these
for inhibition of the interaction between S100A10 and a fluorescent
annexin A2(1-14) ligand peptide. A pyrrol-2-one hit compound
thus identified was explored chemically, and structure-activity
relationship (SAR) patterns were observed to be compatible with
occupation of the target binding site. Inhibition of the cellular
protein interaction was then confirmed using immunoprecipita-
tion. Thus, we identified a novel class of chemical probes suitable
for functional exploration of the interaction between S100A10
and annexin A2.
’ RESULTS AND DISCUSSION
Structure-Based Virtual Screening. The N-terminal annexin
A2 peptide binding pocket of the S100A10 protein2 was used as
the target to perform a virtual screen of a total of 704511
structurally diverse small molecules. These were selected based
on a Mr range between 150 and 600, since modulators of
protein-protein interactions are frequently larger than inhibi-
tors of conventional drug targets.21,22 For similar reasons, the
number of hydrogen bond donors (up to 7), hydrogen bond
acceptors (up to 14), and nonterminal rotatable bonds (up to 12)
was allowed to exceed the limits of generally accepted druglike-
ness rules,23,24 although the calculated physicochemical proper-
ties of the majority of compounds (75%) in the screening
database conformed to such rules. The predicted partition
coefficient of compounds was kept comparatively low (XlogP
< 5)25 to avoid the selection of excessively lipophilic molecules.
Compounds were docked using the genetic optimization for
ligand docking (GOLD)26,27 program in virtual screening mode,
in which the genetic algorithm (GA) setting was fixed with 10000
operators; hence, search efficiency was limited, to permit faster
calculations. Docked ligands were ranked based on the fitness
scores (relating to predicted binding affinity)28 generated from
the virtual screening mode, and the top-scoring 1% of hit
compounds, whose fitness scores ranged from 40-71, were
chosen for further analysis.
Analysis of the Predicted Binding Poses. Analysis of the
molecular interaction between the annexin A2 N terminus and
the S100A10 protein shows that the annexin recognition site of
S100A10 is a comparatively compact, hydrophobic, and concave
binding pocket (Figure 1a), composed of residues from both
S100A10 molecules in the S100A10 dimer.2 The annexin A2 N
terminus forms an amphipathic helix, the hydrophobic face of
which interacts with this binding site. Mutagenesis studies
indicate that the hydrophobic annexin A2 residues Val3, Ile6,
Leu7, and Leu10 are the main contributors to the protein
interaction with S100A10.17 The N terminus of annexin A2 is
posttranslationally modified, and the Ser1 acetyl group is also
important for S100A10 binding.32 The crystal model of the
interaction is consistent with these suggestions. Hydrogen bonds
are observed from the backbone NH groups of Ser1 and Thr2 to
the side chain carboxyl of S100A10 B chain Glu9. Furthermore,
the imidazole NH group of annexin A2 His4 hydrogen bonds
with the B chain Glu5 carboxylate. Figure 1b shows that the
annexin A2 binding site can be divided into three distinct regions:
two predominantly hydrophobic pockets (H1 and H2) and a
somewhat hydrophilic subsite (H3), which in the native annexin
A2-S100A10 complex are occupied mainly by the annexin A2
residue side chains Thr2, Leu7, and AcSer1, respectively, whereas
the Val3 side chain is found at the intersection of the three
subsites (Figure 1a,b). H1 is composed of Tyr85, Phe86, and
Met90 of chain A and Glu9, Phe13, and Met12 of chain B. H2 is
formed by Phe38, Pro39, Gly40, Phe41, and Leu78 of chain A
and Glu5 and Met8 of chain B. Finally, the S100A10 residues that
constitute H3 are Pro1, Ser2, His6, and Glu9 of chain B.
The top-ranked 1% of compounds were then rescored using
GOLD in standard parameter mode. In this mode, the GA setting
was fixed with 100000 operators. As a result, the conformational
search efficiency of the ligands was increased. The resulting
fitness scores ranged from 41 to 77. On the basis of these scores,
775 top-ranked compounds were selected for binding mode
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Chart 1. Clusters of Blockers of the Annexin A2-S100A10 Protein Interaction Identified by GOLD Docking and Biochemical
Assay
The binding modes predicted by the docking studies for all
active compounds were analyzed, and the poses of five structu-
rally diverse compounds that showed significant inhibitory
activity are illustrated in Figure 1c-g. Compounds 2a (cluster
19) and 4 (cluster 17) interact mainly with the hydrophobic
regions H1 and H2 of the binding pocket, whereas compounds 3
(cluster 3), 1a (cluster 9), and 5 (cluster 26) additionally extend
toward the hydrophilic pocket H3.
The symmetrical compound 4 mainly occupies the hydro-
phobic regions of the binding pocket and is involved in good van
der Waals and hydrophobic interactions with the surrounding
amino acids. In addition, the p-aminophenyl moiety of one-half
of the molecule undergoes a π-π stacking interaction with the
phenyl ring of Tyr85. Compound 2a hydrogen bonds through
one of the protonated piperazine amine groups with a carbox-
ylate oxygen of Glu5. Additional close contacts result from van
der Waals interactions with residues Met12 and Phe86.
The amide moiety of 3 extends into the entrance of the
hydrophilic H3 site, whereas in the cases of 1a and 5, this site is
more fully occupied by aromatic ring substituents. Compound 3
forms hydrophobic interactions with both the H1 and the H2
sites, and its pose is stabilized by a hydrogen bond network
involving the ligand primary amide and sulfonyl groups with
Glu5 and Glu9 of the receptor. The 4-isopropylphenyl moiety of
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Journal of Medicinal Chemistry
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Figure 1. Structure-based design of annexin A2-S100A10 inhibitors. (a) The interaction between the helical N-terminal annexin A2 peptide (gray
CPK coloring) and a binding pocket of S100A10 (green CPK surface); the side chains, including the N-terminal acetyl group of the annexin peptide
involved in intimate contact with the receptor through either hydrophobic or hydrogen-bonding (dashed lines) interactions, are shown as solid sticks
and are labeled. (b) The hydrophobic (H1 and H2) and hydrophilic (H3) subsites of the S100A10 binding pocket are colored in cyan, yellow, and
magenta, respectively. Predicted binding poses of verified virtual screening hits (gray CPK stick models): (c) 1a, (d) 2a, (e) 3, (f) 4, and (g) 5 (refer
Chart 1).
Scheme 1. Synthesis of 1-Substituted 4-Aroyl-3-hydroxy-5-phenyl-1H-pyrrol-2(5H)-onesa
a Reagents and conditions: (a) 1,4-Dioxane, 15 min. (b) 1,4-Dioxane, overnight reaction. (c) (i) Microwave irradiation, 250 W, 30 °C, 5 min, 2 M
NaOMe/MeOH; (ii) Hþ (pH 3-4).
1a occupies the H1 site, whereas the 2-hydroxypropyl group
binds into the H2 site. The methyl group of the 2-hydroxypropyl
substituent undergoes a CH-π stacking interaction with the
phenyl ring of Phe41. The hydroxyl moiety donates a strong
hydrogen bond to the carboxylate oxygen of Glu5. In addition,
the 4-methylbenzoyl group is positioned in the H3 site.
Interestingly, the predicted binding mode of 5 is similar to that
of 1a, although the compounds are not similar in structure. The
3,4-dihydropyridine scaffold of 5 is located at the center of the
binding pocket, whereas the pyrazole side chain is buried deep
within the H2 pocket, making good van der Waals and hydro-
phobic interactions. Both the methyl and the ethyl ester side
chains are located in the hydrophobic region H1. The thiophene
side chain extends slightly into H3.
receptor residues with the inhibitors (1a, 2a, and 3) appear to
mimic the hydrogen bond interaction of Glu5 and Glu9 residues
with the annexin A2 N-terminal peptide in the crystal structure.
Overall, in view of the chemical nature of the inhibitor compounds
analyzed here and the nature of the interactions with the binding
pocket, it appears that the hydrogen bond acceptor sites of Glu5
and Glu9 and the two hydrophobic regions (H1 and H2) of the
receptor can be considered as the key pharmacophoric features.
Considering cluster SAR analysis, inhibitory potency, druglike
properties, and synthetic amenability, we chose cluster 9, and in
particular compound 1a, as a medicinal chemistry starting point for the
design and development of candidates that target the annexin A2-
S100A10 protein-protein interaction. In the following, we describe
our initial exploratory hit-to-lead conversion based on the 1-substi-
tuted 4-aroyl-3-hydroxy-5-phenyl-1H-pyrrol-2(5H)-one template.
From the above analysis of the binding poses, it appears that the
two hydrophobic regions (H1 and H2) play a dominant role in the
binding affinity of all of the inhibitors. The proposed hydrogen
bond formation of either or both of the Glu5 and the Glu9
1-Substituted 4-Aroyl-3-hydroxy-5-phenyl-1H-pyrrol-2(5H)-
one Analogue Synthesis. The required 1H-pyrrol-2(5H)-one
system was prepared using a simple two-step procedure
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Table 1. Chemical Structures and the Keto-Enol Form of
Methyl Phenylpyruvates 12a-k
Table 2. Inhibition of the S100A10 Interaction with Annexin
A2 by 5-Substituted 3-Hydroxy-1-(2-hydroxypropyl)-4-(4-
methylbenzoyl)-1H-pyrrol-2(5H)-ones and Activity in
Counterscreen Assay
no. R6
R7
R8
X
Y
keto-enol/diketone tautomer ratioa
12a
12b
12c
12d
12e
H
H
H
H
H
H
H
C
C
C
C
C
C
C
C
N
N
C
C
C
C
C
C
C
C
C
C
C
N
92:8
H
Me
H
92:8
Me
H
93:7
annexin-S100A10
OMe
H
93:7
interaction
counterscreen
% controlb
101 ( 7
OMe
H
90:10
91:9
IC50
(μM)
a
12f OEt
H
no. type
P
R2 R3
R4 R5
pIC50
12g
12h
12i
H
H
H
H
H
H
Cl
89:11
88:12
88:12
90:10
93:7
5.99 ( 0.10
4.85 ( 0.09
1
14
H
CN
Me
H
1a
I
H
H
iPr
H
13 II
14 III
<3
>1000
3.89 ( 0.15* 128
3.60 ( 0.33* 251
12j
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
H
H
H
H
H
H
H
H
H
H
H
CF3
H
H
H
H
H
H
iPr
H
H
H
H
H
H
H
H
CF3
H
CF3
H
Cl
Cl
H
H
Me
Et
nPr
tBu
NMe2
OH
OCF3
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
12k
H
4.94 ( 0.14
4.07 ( 0.10*
4.80 ( 0.25
4.74 ( 0.09
4.55 ( 0.05**
11
85
16
18
28
100 ( 16
a Tautomer ratios were calculated from the 1H NMR spectra based on
the CH signal of the keto-enol form against the CH2 signal of the
diketone form.
97 ( 9
123 ( 7
3.56 ( 0.55* 275
(Scheme 1). In the first step, substituted methyl pyruvates 12
were obtained by Claisen condensation of aryl methyl ketones 10
with dimethyl oxalate 11.33-35 Pyruvates 12 were then subjected
in a second step to a three-component coupling reaction with the
appropriate amines 7 and aldehydes 8.36-40
For the preparation of methyl pyruvates 12, we developed a rapid
protocol for the condensation of acetophenones 10 with dimethyl
oxalate 11 using 2 M MeONa in MeOH solution under microwave
irradiation (250 W) for 5 min at 30 °C. With most substrates, this
reaction gave rise to a precipitate of the sodium enolate Claisen
adduct, which upon acidification to pH 3-4 provided the methyl
pyruvate products 12 in maximally 52% isolated yields. Alterations
to neither base strength nor reaction time were found to result in
improved yields. Using this procedure, compounds 12a-k, which
exist predominantly in the keto-enol rather than the diketone form,
were prepared (Table 1).
<3
>1000
5.02 ( 0.11
5.20 ( 0.36
5.08 ( 0.09
5.12 ( 0.09
9
6
8
8
4
110 ( 8
102 ( 1
110 ( 6
102 ( 6
109 ( 3
CF3
H
CF3 5.41 ( 0.12
Cl
H
H
H
H
Cl
H
3.93 ( 0.21* 117
3.98 ( 0.15* 105
5.16 ( 0.15
5.12 ( 0.06
7
8
119 ( 4
121 ( 11
CF3 Cl
a Data were expressed as % of the specific signal of the nontreated control
and analyzed by nonlinear regression using Graphpad Prism. pIC50
(
standard error of the fit (n = 4-8 data points per concentration, 0.4-100
μM). The midpoint of the dose-response curve is given based on a floating
fit unless indicated as follows: *, bottom fixed at zero; **, bottom fixed at
zero, top fixed at 100%. b Percent remaining signal at 50 μM compound
relative to diluent only (mean ( standard error mean of >3
determinations). P = nonlabeled synthetic annexin N terminus peptide.
The three-component reaction of amines 7, aldehydes 8, and
methyl phenylpyruvates 12 occurs through initial formation of imines
9, which then undergo cyclocondensation with 12. When 4-isopropyl
benzaldehyde was added to 2-hydroxypropylamine in 1,4-dioxane,
mass spectromeric analysis after 15 min showed the presence of an
ion of m/z [M þ H]þ = 206.1537, corresponding to the imine.
Pyruvate ester 12b in 1,4-dioxane was then added to the reaction
mixture, and precipitation was observed within 1 h of the reaction.
Stirring of the mixture was continued overnight to afford authentic
compound 1a in 43% isolated yield. This procedure was adopted for
the preparation of all of the analogues described here (Tables 2-4).
SARs. Initially, a number of analogues were investigated in
which the substituent at C5 of the 1H-pyrrol-2(5H)-one system
present in hits 1a-d was varied (Table 2). Replacement of this
substituent with isopropyl (13) abolished the activity completely
and with unsubstituted phenyl (15) showed 18-fold decrease in
activity, suggesting that the substitution pattern of the phenyl
ring was important. We therefore prepared and tested a small set
of appropriate analogues 16-31 (Table 2). The facts that the 3-
and 4-isopropylphenyl derivatives 1a and 16 were equipotent
and that the 2-naphthyl analogue 14 retained some activity show
that steric bulk at either the meta- or the para position of the
phenyl group, or both, was productive in terms of binding.
Alteration of the phenyl para substituent (isopropyl) in 1a from
methyl (17) to ethyl (18), n-propyl (19), and t-butyl (20)
showed distinct SARs, suggesting that the alkyl chain length
and extent of branching in the isopropyl group are optimal.
Reference to the modeled binding pose of 1a (Figure 1c), where
the 4-isopropylphenyl group fits neatly into the H1 subsite,
supports this notion.
Replacement of the lipophilic isopropyl group with the roughly
isosteric but more polar dimethylamino group in analogue 21
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Journal of Medicinal Chemistry
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Table 3. Inhibition of the S100A10 Interaction with Annexin A2 by 1-Substituted 4-Aroyl-3-hydroxy-5-(4-isopropylphenyl)-1H-
pyrrol-2(5H)-ones and Activity in Counterscreen Assay
annexin-S100A10 interaction
counterscreen
% controlb
101 ( 7
a
no.
R1
R6
R7
R8
X
pIC50
IC50 (μM)
1a
32
33
34
35
36
37
38
39
40
41
42
43
44
CH2CHMeOH
iBu
H
H
Me
Me
Me
Me
Me
Me
H
C
C
C
C
C
C
C
C
C
C
C
N
C
C
4.85 ( 0.09
14
>1000
12
8
H
H
H
H
H
H
H
H
H
OEt
H
H
H
H
<3
CH2CH2OH
H
4.92 ( 0.16
5.12 ( 0.16
5.54 ( 0.27
5.19 ( 0.05**
4.84 ( 0.14
4.10 ( 0.10*
3.21 ( 0.35*
5.16 ( 0.05
5.15 ( 0.11
3.10 ( 0.01*
4.25 ( 0.31
5.57 ( 0.86
101 ( 6
CH2CH2OMe
CH2(CH2)2OMe
CH2CHdCH2
CH2CHMeOH
CH2CHMeOH
CH2CHMeOH
CH2CHMeOH
CH2CHMeOH
CH2CHMeOH
CH2CHMeOH
CH2CHMeOH
H
120 ( 11
111 ( 19
105 ( 14
H
3
H
6
H
14
79
616
7
Me
H
H
OMe
H
OMe
H
110 ( 3
H
7
112 ( 11
-
Me
CN
Cl
794
56
3
H
H
126 ( 5
a Data were expressed as % of the specific signal of the nontreated control and analyzed by nonlinear regression using Graphpad Prism. pIC50 ( standard
error of the fit (n = 4-8 data points per concentration, 0.4-100 μM). The midpoint of the dose-response curve is given based on a floating fit unless
indicated as follows: *, bottom fixed at zero; **, bottom fixed at zero, top fixed at 100%. b Percent remaining signal at 50 μM compound relative to diluent
only (mean ( standard error mean of >3 determinations).
resulted in loss of activity over 20-fold. Again, this is consistent with
the highly hydrophobic nature of the H1 subsite. Similarly, the even
more polar 4-phenol derivative 22 was inactive. Whereas the
dimethylamino group in 21 is somewhat hydrophobic and elec-
tron-donating, the trifluoromethoxy and trifluoromethyl groups, of
which the latter is isosteric with dimethylamino and isopropyl in the
comparatively more potent analogues 23 and 24, respectively, are
also hydrophobic but electron-withdrawing. As with the positional
isopropyl isomers 1a and 16, the 3- and 4-trifluoromethylphenyl
isomers 24 and 25 displayed similar potency. Interestingly, 3,5-di-
CF3 compound 27 was somewhat more active than the correspond-
ing 2,4-isomer 26 and was the most potent compound thus far. A
decrease in potency was observed with a chloro substituent at the
meta- (29) or para- (28) position of the phenyl ring. However, the
3,5-dichlorophenyl derivative 30 was 15-fold more potent than 28
and 29. Surprisingly, addition of a trifluoromethyl group to the meta
position of the phenyl ring in the context of the para-chloro
substituent resulted in a comparatively potent compound (31).
We next turned our attention to the 1H-pyrrol-2(5H)-one N1
substituent (32-36, Table 3). In 1a, this is a simple 2-hydro-
xypropyl group whose alcohol function donates a hydrogen bond
to the side chain carboxyl of Glu5 according to our binding
hypothesis (Figure 1c). As expected, therefore, replacement of
the hydroxyl group with a methyl group (32) resulted in
complete loss of activity. Replacement of the 2-hydroxypropyl
substituent of 1a with a 2-hydroxyethyl group (33) was well
tolerated. However, an unexpected increase in activity was
observed with compound 34, where a 2-methoxyethyl substitu-
ent, incapable of donating a hydrogen bond, was present. A
further gain in potency was observed upon lengthening the
substituent to 3-methoxypropyl (35). Introduction of a hydro-
phobic allyl side chain (36), incapable of participating in hydro-
gen bonds, also retained activity.
We finally probed the SARs of the 1H-pyrrol-2(5H)-one
4-aroyl group (37-44, Table 3). Inspection of the predicted
binding pose of 1a shows that the 4-methylbenzoyl group
occupies the comparatively shallow H3 subsite (Figure 1c) and
that substitution of the aromatic ring at the meta or ortho
position may result in better contacts. This might be achieved
with lipophilic or hydrophilic substituents since the H3 subsite is
weakly hydrophilic. Interestingly, switching of the methyl group
from the para- (1a) to the meta- (38) position resulted in 5-fold
lower activity, whereas removal of the methyl group (37) did not.
Surprisingly, replacement of the benzene ring with a more
hydrophilic 3-pyridyl ring (42) was poorly tolerated. When the
terminal methyl group was replaced with the larger and more
polar methoxy group either para (39) or meta (40), this resulted
in higher activity in the latter case. As expected from modeling,
the ortho-substituted ethoxyphenyl analogue 41 showed slightly
better activity than 1a. Replacement of the electron-donating
nonpolar 4-methyl group in 1a with the electron-withdrawing
polar 4-cyano group (43) resulted in loss of activity, whereas the
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Table 4. Inhibition of the S100A10 Interaction with Annexin A2 by 1,5-Substituted 4-Aroyl-3-hydroxy-1H-pyrrol-2(5H)-ones and
Activity in the Counterscreen Assay
annexin-S100A10 interaction
counterscreen
% controlb
a
no.
R1
R3
R6
R7
H
R8
X
pIC50
IC50 (μM)
45
46
47
48
49
50
51
52
53
CH2CHdCH2
H
H
H
H
H
F
H
H
N
N
C
N
N
N
C
C
C
<3
>1000
28
CH2CHdCH2
H
Me
Cl
H
4.55 ( 0.17
5.20 ( 0.06
102 ( 9
120 ( 6
CH2CHdCH2
H
6
CH2(CH2)2OMe
CH2(CH2)2OMe
CH2(CH2)2OMe
CH2(CH2)2OMe
CH2(CH2)2OMe
CH2(CH2)2OMe
H
<3
>1000
269
182
186
11
H
Me
Me
Cl
H
3.57 ( 0.01*
3.74 ( 0.01*
3.73 ( 0.42*
4.97 ( 0.09
5.31 ( 0.11
H
F
H
H
H
H
H
OEt
117 ( 9
F
OEt
H
5
127 ( 19
a Data were expressed as % of the specific signal of the nontreated control and analyzed by nonlinear regression using Graphpad Prism. pIC50 ( standard
error of the fit (n = 4-8 data points per concentration, 0.4-100 μM). The midpoint of the dose-response curve is given based on a floating fit unless
indicated as follows: *, bottom fixed at zero; **, bottom fixed at zero, top fixed at 100%. b Percent remaining signal at 50 μM compound relative to diluent
only (mean ( standard error mean of >3 determinations).
Figure 2. Alternative binding poses for N1-methoxyalkyl and allyl 1H-pyrrol-2(5H)-ones. Highly scoring S100A10-docked poses of compounds 35 (a),
36, (b), and 47 (c) are shown.
4-chlorophenyl derivative 44 retained comparatively better
potency.
the 1H-pyrrol-2(5H)-one C5 phenyl substituent. This difference
was not recapitulated in the context of the C5 2-ethoxybenzoyl
derivatives 52 and 53, which were both comparatively potent.
Overall, the divergence of the SARs depending on whether the
1H-pyrrol-2(5H)-one N1 substituent is the protic hydroxypropyl
or an aprotic (methoxyalkyl or allyl) group suggests that these
compounds may have different binding modes. Our modeling
studies show that an alternative pose for the methoxyalkyl and
allyl compounds (e.g., 35 and 36; Figure 2a,b) may be relevant,
where the 4-aroyl group binds in the H2 rather than the H3 site
and where the C5-isopropylphenyl occupies the region between
H1 and H2, with one of the methyl groups reaching into a small
pocket at the base of the binding site in a similar fashion to that
observed with the Val3 side chain of the annexin A2 peptide
(Figure 1a). A hydrogen bond with the B chain Glu5 carboxylate
Because of the somewhat unexpected potency of the 1H-pyrrol-
2(5H)-one 3-methoxypropyl (35) and allyl (36) N-substituted
analogues, the last phase of our exploratory SAR studies concerned
the combination of some of the individual modifications discussed
above in the context of these groups (Table 4). Like compound 42,
which also contains the 4-methyl-3-pyridyl group, the allyl (46) and
3-methoxypropyl (49 and 50) derivatives were also either less active
or inactive, and the corresponding 3-pyridyl analogues (45 and 48)
were completely inactive. However, the 4-chlorophenyl derivative
47 with the allyl N-substitution was one of the most potent
compound in our series, while a N-(3-methoxypropyl) derivative
(51) was comparatively inactive, although the latter compound
differs from 47 insofar as it contains an additional 3-fluoro group in
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is maintained but now involves the 1H-pyrrol-2(5H)-one C3-
hydroxyl rather than the N1 substituent. This alternative binding
pose does not appear to be favored when the C5-phenyl group
contains a para-trifluoromethyl group, however, since highly scoring
docking poses for, for example, the comparatively potent analogue
47 (Figure 2c) are similar to that of 1a (Figure 1c), except that the
alkene function of the N1-allyl group is stacked between the phenyl
rings of Phe38 and Phe41 within the H2 subsite.
the inhibition observed in the biochemical assay is not an artifact of
poor solubility.
The binding assay used for SAR exploration only reports the
interaction of the annexin N terminus with S100A10. While there is
ample evidence to suggest that this region of annexin A2 represents
the sole binding region on S100A10, we sought to confirm the
inhibitory activity of this class of compounds on the physiological
relevant complex of these two proteins. For this purpose, the
physiological complex was obtained by lysing MDA-MB-231 breast
cancer cells. The lysate was treated with compound after which
S100A10 was immunoprecipitated, and the immunoprecipitates were
analyzed by SDS-PAGE and Western blotting for the presence of
annexin A2. As can be seen in Figure 4a, annexin A2 is present in the
S100A10 immunoprecipitates, indicating that a complex between
these proteins exists in the cell lysate. After treatment with 1a or 27,
the amount of complex was reduced. Quantification of the coimmu-
noprecipitated protein showed that at inhibitor concentrations of 50
μM, around 70% of the complex had been lost, which was significantly
different from the noninhibitor control (Figure 4b). As expected, the
N-terminal annexin A2 peptide also disrupted the S100A10-annexin
A2 physiological complex. Thus, these compounds are able to disrupt
the complex of S100A10 and full length, intact annexin A2. Interest-
ingly, analogue 22, which is highly soluble (Table 5) but does not
inhibit the binding of the annexin N-terminal peptide in the in vitro
FRET binding assay (Table 2 and Figure 3b), does not inhibit the
complex between the native proteins, suggesting that the SAR patterns
observed in the FRET assay translate to the native proteins.
We also investigated the ability of selected analogues from this
series of compounds to inhibit the physiological complex inside the
cell. Intact MDA-MB-231 cells were pretreated with different con-
centrations of 1a or 27 after which the cells were lysed and
immunoprecipitation assays were performed. It can be seen in
Figure 4b that pretreatment of the cells with 27 leads to a statistically
significant disruption of the interaction between S100A10 and cellular
annexin A2. The extent of inhibition at 100 μM is similar to that
observed at 10 μM in the broken cell immunoprecipitation assay,
suggesting a 10-fold drop-off in potency, possibly due to limited cell
penetration of the compound. Compound 1a apparently inhibits the
cellular interaction, but this inhibition was not statistically significant
from the control treatment. Solubility may affect the assayability of 1a
at these higher concentrations (Table 5). Importantly, in contrast to
peptide blockers, 27 can penetrate the cell and can therefore be used
to evaluate the cellular function of this protein interaction.
Comparison of Biological Activity of Inhibitors. We com-
pared the dose response of inhibition of the unlabeled N-terminal
annexin A2 peptide with that of our 1H-pyrrol-2(5H)-one com-
pounds (examples are shown in Figure 3). From this analysis, an
IC50 value of 1 μM was calculated for the cognate annexin A2
peptide (which compares well with that measured by equilibrium
dialysis31 and isothermal titration calorimetry18). Thehitcompound
1a was about 14-fold less potent (IC50 = 14 μM), and our more
potent analogues (e.g., 47) were about 4-fold less potent (IC50 = 6
μM). The apparently steep Hill slopes for both peptide and
compounds may be a reflection of the complexity of the underlying
binding events as proposed.18 It is important to note that the
addition scheme in the binding assay involves preforming a complex
of the annexin A2 N-terminal peptide tracer and the S100A10 dimer
prior to exposure to compound; therefore, these compounds are
able to disrupt a preformed complex. Importantly, none of the
compounds inhibited a nonrelated protein interaction31 assayed in a
comparable fashion (Tables 2-4), suggesting that their inhibitory
potential is not due to nonspecific interference with the fluorescence
readout or to promiscuous inhibition of protein interactions. The
compounds (including inactive compounds) were soluble in aqu-
eous solution at the concentrations tested (Table 5), indicating that
Figure 3. Biological activity of peptide and small-molecule annexin
A2-S100A10 inhibitors. (A) Dose-response curves for the 14-residue
N-terminal annexin A2 peptide (() and compounds 1a (2), 47 (9), and
50 (1) in the FRET-based competitive annexin A2-S100A10 binding
assay are shown. Curve fit was performed in Graphpad Prism as
described in Table 1. Error bars represent the standard error of the
mean of four (compounds) or eight (peptide) observations. (B) As
under A except that compound 27 (b; n = 28 ( standard error of the
mean) or 22 (O; n = 4 ( standard error of the mean) was analyzed.
’ CONCLUSION
In this study, we have employed a structure-based virtual
screening approach to identify nonpeptide inhibitors of the
annexin A2-S100A10 protein interaction. Systematic alteration
of the 3-hydroxy-1H-pyrrol-2(5H)-one hit compound 1a
Table 5. Aqueous Solubility of Selected Analoguesa
no.
aqueous solubility (mM)
no.
aqueous solubility (mM)
no.
aqueous solubility (mM)
1a
13
15
18
21
22
0.3
5.3
4.1
0.8
2.7
5.8
24
25
26
27
31
33
1.5
4.1
3.5
2.3
2.0
1.7
40
42
45
47
48
53
1.1
4.9
2.3
0.3
3.4
3.1
a Numbers represent averages of two determinations, which differed by less than 5% from the average.
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Figure 4. Analysis of the native complex of S100A10 and annexin A2. (A) MDA-MB-231 cells were lysed and incubated with 50 μM compound as
indicated or with the annexin A2 N terminus peptide (P). S100A10 was immunoprecipitated, and the amount of annexin A2 in the immunoprecipitates
was analyzed by SDS-PAGE followed by Western blotting using an annexin A2 antibody. Annexin A2 was identified based upon comigration with an
annexin A2 reference on the blot. The section of the blot with the annexin A2 signal is shown. (B) MDA-MB-231 cells were lysed and incubated with
compound at concentrations as indicated or with the annexin A2 N terminus peptide (P). Western blots were obtained and scanned, and scans were
quantified using Scion Image software. Quantified data were expressed as % of nontreated control. Error bars represent the standard error of the mean
(n = 3 from three separate lysates). (C) As in B, except that MDA-MB-231 cells were treated with compound for 6 h after which the cells were lysed
and washed, and immunoprecipitations were performed as above. Error bars represent the standard error of the mean (n = 3 from three separate lysates).
*p < 0.05 (nonpaired t test).
resulted in distinct SARs that are in fairly good agreement with
our hypothetical binding mode for the series. Importantly,
the SAR translates to the native proteins, and the compounds
are potentially useful for exploration of the physiological
importance of the protein interaction. So far, peptides have
been used to compete with the interaction between these
proteins. While these peptides have generated some under-
standing of the function of the complex at the cell surface,
their use is limited. The blockers described here provide a
template to develop inhibitors for wider exploration of the
function of this complex. The current inhibitors may them-
selves be useful as probes, and the fact that we have identified
active as well as inactive analogues in this series indicates this
may be an option. However, further potency gains are
desirable, and a better understanding of the solubility vs
membrane penetration is required to allow this class of
compounds to become useful as probes of the intracellular
complex.
maximum iterations, 5000. The annexin A2 binding site of S100A10 was
defined as a cavity by a set of residues within 7 Å of the annexin A2 ligand
peptide from the energy-minimized complex. A centroid for the binding
site was calculated using a shell of all atoms within 5 Å of the ligand
peptide involving 283 atoms, and the peptide was then extracted from
the complex. There were five water molecules within the binding site,
and these were also deleted.
Compound Database Preparation. Compounds were selected
using the ZINC database (release 6, 2006)41 entries from the vendors
Asinex and Chembridge. A web-based query tool in ZINC was used to
specify molecular property constraints, and 3D structures of the selected
compounds were generated using the program Concord (Tripos).
Structures with inappropriate isomeric specifications and those contain-
ing disallowed atoms in terms of valence or with errors in ring definitions
were excluded. A database was generated from the remaining 704511
3D-encoded compounds.
Virtual Screening. Docking was performed using GOLD V3.0.1.
Ten docking runs were performed for each molecule in this database,
allowing early termination of the docking runs if the top three solutions
were within 1.5 Å rmsd of each other. A binding site radius of 12 Å was
found to be optimal. Flipping was not allowed for those ligands that have
ring-NHR and ring-NR1R2 groups to avoid the addition of huge
torsional energy penalties to the total fitness scores.
To optimize the fitness score, GOLD uses a GA, a search technique
that mimics the process of natural evolution. An initial population of
ligand conformations is transformed via GA operations (mutations,
crossovers, and migrations) into a final population. Individual ligand
conformations are known as chromosomes. Genes represent the flexible
torsion angles of the ligand. Each chromosome is assigned a fitness score
based on its predicted binding affinity, and the chromosomes within the
population are ranked according to the fitness score. The selection of
parent chromosomes is biased toward fitter members of the population,
’ EXPERIMENTAL SECTION
Preparation of the Annexin A2 N-Terminal Peptide-
S100A10 Complex Coordinates. The complex between the
S100A10 protein and the annexin A2 N-terminal peptide (PDB ID:
1BT6) was modified as follows: The chain termini were charged, and a
total of 1664 hydrogen atoms were added, including water molecules.
AMBER7 FF99 charges were assigned to the complex. Side chain amides
of Gln residues were reoriented to maximize hydrogen bonding. Then,
the complex was subjected to energy minimization in three steps using
AMBER7 FF99 parameters. Minimization method, Powell; initial
optimization, none; termination criteria, gradient 0.5 kcal/mol; and
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that is, chromosomes corresponding to ligand dockings with good
fitness scores. Each population in GA is called an island. Chromosomes
can migrate between adjacent islands using the migration operator.
Mutation is an intrachromosomal operator that introduces new genetic
material into the population. Crossover selects the genes from the parent
chromosomes and creates new offspring. The number of GA operations
applied over the course of a GA run determines the search efficiency of
the GOLD run.
During the run in virtual screening mode, 10000 GA operations were
performed on a single population (single island) of 100 individuals
(default value); hence, there cannot be any migration. Operator weights
for the crossover and mutation were set at default (100, 100, re-
spectively). A cutoff value of 2.0 Å was applied for the hydrogen bonds
to avoid the selection of poor hydrogen bonds during the GA run.
Similarly, a cutoff value of 10 Å was applied for the van der Waals
interactions, tolerating a few bad bumps during the GA run. Rescoring
was then performed using standard GA settings with the number of
operators increased to 100000. Five different populations (islands) each
consisting of 100 chromosomes were maintained during the entire GA
run. Ten migrations were allowed between the islands. For every
crossover, one mutation was applied. Cut-off values of 2.5 Å for
hydrogen bonds and 4 Å for van der Waals interactions were applied.
The parameters active site radius, early termination criteria, and ligand
flexibility remained unchanged during the rescoring of selected
compounds.
onto a Cy5-labeled goat IgG (3 μg/mL) was used in parallel to assess
nonspecific interference with the fluorescence readout. Compound
binding was calculated as percentage of nontreated control, and data
were analyzed by nonlinear regression (dose response-variable slope)
using Graphpad Prism Software to determine the IC50 of binding
(primary assay) or expressed as a percentage of nontreated controls
measured at the 50 μM concentration (counterscreen assay).
Immunoprecipitation Assay. Human MDA-MB-231 breast can-
cer cells were extracted in 50 mM Tris-HCl (pH 8.0) buffer containing
150 mM sodium chloride and 1% (v/v) NP-40. Lysates were centrifuged at
17000gand incubated at 4 °Cfor16hwith10μL of S100A10 antibody (BD
transduction Laboratories) and 50 μL of protein A/G agarose (Alpha
Diagnostic International Inc.). Protein A/G agarose was then recovered by
centrifugation at 2400g, washed three times with 10 mM phosphate buffer
(pH 7.4) containing 2.7 mM potassium chloride and 137 mM sodium
chloride, resuspended in 50 μL of 160 mM Tris-HCl (pH 6.8) buffer
containing 4% (w/v) sodium lauryl sulfate, 20% (v/v) glycerol, 0.04% (w/
v) bromophenol blue, and 10% (v/v) 2-mercaptoethanol, boiled at 99 °C
for 10 min, and centrifuged at 17000g for 10 min. The supernatant was then
analyzed by sodium lauryl sulfate polyacrylamide gel electrophoresis after
which the gel was transferred to a nitrocellulose filter. The filter was
incubated with an annexin A2 monoclonal antibody (1:3000; BD Trans-
duction Laboratories) followed by incubation with an antimouse horse-
radish peroxidase IgG conjugate (1:5000; GE Healthcare) and then
developed using the ECL detection reagent (GE Healthcare). Where
indicated, test compounds were incubated for 1 h with cell lysate prior to
immunoprecipitation. To determine inhibition of the intracellular complex,
MDA-MB-231 cells were pretreated with compound for 6 h after which the
cells were washed three times with 10 mM phosphate buffer (pH 7.4)
containing 2.7 mM potassium chloride and 137 mM sodium chloride, prior
to extraction and immunoprecipitation as described above.
Synthesis. All reagents were purchased directly from commercial
sources and used as supplied unless otherwise stated. Melting points
were measured using a Gallenkamp melting point apparatus and are
uncorrected. Accurate mass and nominal mass measurements were
performed using a Waters 2795-Micromass LCT electrospray mass
spectrometer. Infrared spectra were recorded using Avatar 360 FT-IR
system. Samples were prepared as KBr discs and scanned from 4000 to
500 cm-1. All NMR spectra were recorded in deutero-DMSO in 5 mm
tubes, with tetramethylsilane as an internal standard, using a Bruker ACS-
120 instrument at 400 (1H NMR) and 100.6 MHz (13C NMR). Thin-
layer chromatography was performed using aluminum-backed silica gel
60 plates (0.20 mm layer), and the ascending technique was used with a
variety of solvents. Visualization was by UV light at either 254 or 365 nm.
HPLC was carried out using two different methods. Method 1: Kromasil
(250 mm ꢀ 4.6 mm, 5 μM particle size) column with MeCN/H2O (5-
95% organic over 20 min at 1 mL/min). Method 2: Onyx Monolith C18
(100 mm ꢀ 4.6 mm, 5 μM particle size) column with MeOH/H2O (5-
95% organic over 10 min at 3 mL/min). In both of the methods,
detection was at 260 nm. All compounds were isolated in >95% purity
unless otherwise stated (refer to the Supporting Information).
Cluster Analysis. For cluster analysis, the Selector module of
SYBYL was used, and analysis was performed as described.30 Two-
dimensional fingerprints and atom pairs were selected as diverse metrics.
All of the compounds were processed using the hierarchical clustering
method with a weight metric of 1.00. A total of 27 clustering classes were
generated, and the diversity of these clusters was analyzed based on the
dendrogram obtained, in which nodes of the dendrogram correspond to
the common core structure.
Solubility Assay. Compounds were dissolved at 10 mM in
phosphate buffer (pH 7.4) containing 2.7 mM potassium chloride and
137 mM sodium chloride, by stirring for 27 h at room temperature. The
resulting solution was filtered using GHP Acrodisc 25 mm syringe filters
with 0.2 μm GHP membrane. Compound concentrations were mea-
sured by absorption on a Perkin-Elmer Envision plate reader, at the
wavelength showing maximum absorption in a scan between 250 and
700 nm, determined separately for each compound. The absorbance of
each filtrate was measured, and the concentration was determined by
comparison with a standard curve of compound in 5% aqueous DMSO.
Fluorescence Screening Assay. Routine assessment of com-
pound activity was performed as described in Li et al.31 Briefly, a Cy5-
labeled S100A10 tracer was developed, and binding of a Cy3-labeled
annexin A2(1-14) peptide ligand was assessed using a FRET readout.
Assays were carried out in Nunc black nontreated 384-well plates at
20 °C in 50 μL of buffer C. All incubations were performed in
quadruplicate. Compounds, peptide, and buffer controls were added
to the wells in a 10 μL volume in 5% DMSO. Cy5-labeled S100A10
tracer (407 nM) and Cy3-labeled annexin A2(1-14) peptide ligand
(1.33 μM) were preincubated for 5 min at 20 °C, and 40 μL of the
preformed complex was then added to the wells and mixed for 10 s to
yield a final DMSO concentration of 1%. After 5 min of incubation at
20 °C, readings were taken on a Perkin-Elmer Envision plate reader by
excitation at 488 nm and emission at 695 nm. Each screening plate
contained as controls the individual labeled proteins, as well as the two
protein partners with and without a 12.8 μM concentration of the
nonlabeled AA2(1-14) competitor (all in quadruplicate). FRET was
calculated by subtracting the sum of the fluorescence emission of
S100A10-Cy5 and AA2(1-14)-Cy3 individually from that measured
for the coincubated partners. A counterscreen assay31 that measured the
FRET signal from a Cy3-conjugated donkey antigoat IgG (4 μg/mL)
Step 1: Synthesis of Methyl Phenylpyruvates. Sodium (184
mg, 8 mmol) was dissolved in dry methanol (4 mL) at 0 °Ctoproducea2.00
M solution of sodium methoxide. Dimethyl oxalate (590 mg, 5.0 mmol, 1.0
equiv) was added, and the solution was stirred for 15 min. The appropriate
aromatic ketone (5.0 mmol, 1.0 equiv) was then added. A microwave-assisted
reaction was carried out for 5 min at 250 W, 250 P, and 30 °C.
Method A. After 5 min of the microwave reaction, for some products,
precipitation was observed immediately (12a,d,e,g-k). For other
products (12b,c), precipitation occurred after standing the solution
for 15 min. The precipitate was collected by filtration and was washed
with Et2O. The resulting solid was dissolved in H2O (volume depending
on amount of precipitate obtained) and stirred for 1 h at room
temperature. The solution was acidified by addition of glacial AcOH
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to pH 3-4 and kept at 0 °C for 1 h. The precipitate was filtered, washed
with distilled H2O, and dried in a freeze dryer.
251 °C. m/z (ES): found 394.2076 (C24H28NO4 [M þ H]þ) requires
394.1940.
3-Hydroxy-1-(2-hydroxy-propyl)-5-isopropyl-4-(4-methyl-benzoyl)-1,5-di-
hydro-pyrrol-2-one (13). The procedure was similar to the procedure
for 1a except that isobutyraldehyde (227 μL, 2.5 mmol, 1.0 equiv) was
used and the reaction mixture was allowed to stir at room temperature
for 24 h. The precipitate obtained was washed only with Et2O and gave
the desired product 13 as a white powder (14% yield); mp 214-216 °C.
m/z (ES): found 318.1652 (C18H24NO4 [M þ H]þ) requires 318.1627.
3-Hydroxy-1-(2-hydroxy-propyl)-4-(4-methyl-benzoyl)-5-naphtha-
len-2-yl-1,5-dihydro-pyrrol-2-one (14). The procedure was similar to
the procedure for 1a except that 2-naphthaldehyde (398 mg, 2.5 mmol,
1.0 equiv) was used. Product 14 was isolated as a white powder (54%
yield); mp 247-249 °C. m/z (ES): found 402.1804 (C25H24NO4 [M þ
H]þ) requires 402.1627.
3-Hydroxy-1-(2-hydroxy-propyl)-4-(4-methyl-benzoyl)-5-phenyl-1,5-dihy-
dro-pyrrol-2-one (15). The procedure was similar to the procedure for 1a
except that benzaldehyde (305 μL, 3.0 mmol, 1.0 equiv) was used, and the
reaction mixture was allowed to stir at room temperature for only 3 h. Product
15 was isolated as a white powder (80% yield); mp 251-253 °C. m/z (ES):
found 352.1584 (C21H22NO4 [M þ H]þ) requires 352.1471.
Method B. No precipitate was observed at the end of 5 min of the
microwave reaction (product 12f). The reaction mixture was poured
into 50 mL of ice-cold H2O and acidified with glacial AcOH to pH 3-4.
Precipitation was observed, and the reaction mixture was kept at 0 °C for
1 h. The precipitate was filtered, washed with distilled H2O, and dried in
a freeze dryer.
2-Hydroxy-4-oxo-4-phenyl-but-2-enoic Acid Methyl Ester (12a).
The product was isolated as a white powder (52% yield); mp 60-
62 °C. m/z (ES): found 205.0300 (C11H9O4 [M - H]-) requires
205.0579.
2-Hydroxy-4-oxo-4-p-tolyl-but-2-enoic Acid Methyl Ester (12b).
The product was isolated as a white powder (49% yield); mp 84-
86 °C. m/z (ES): found 221.0838 (C12H13O4 [M þ H]þ) requires
221.0736.
2-Hydroxy-4-oxo-4-m-tolyl-but-2-enoic Acid Methyl Ester (12c).
The product was isolated as a white powder (37% yield); mp 69-
71 °C. m/z (ES): found 219.0389 (C12H11O4 [M - H]-) requires
219.0736.
2-Hydroxy-4-(4-methoxy-phenyl)-4-oxo-but-2-enoic Acid Methyl
Ester (12d). The product was isolated as a pale white powder (47%
yield); mp 94-96 °C. m/z (ES): found 235.0806 (C12H11O5 [M -
H]-) requires 235.0685.
2-Hydroxy-4-(3-methoxy-phenyl)-4-oxo-but-2-enoic Acid Methyl
Ester (12e). The product was isolated as a white powder (26% yield);
mp 90-92 °C. m/z (ES): found 235.0647 (C12H11O5 [M - H]-)
requires 235.0685.
4-(2-Ethoxy-phenyl)-2-hydroxy-4-oxo-but-2-enoic Acid Methyl Es-
ter (12f). The product was isolated as a yellow powder (46% yield); mp
51-53 °C. m/z (ES): found 249.0685 (C13H13O5 [M - H]-) requires
249.0841.
4-(4-Chloro-phenyl)-2-hydroxy-4-oxo-but-2-enoic Acid Methyl Es-
ter (12g). The product was isolated as a white powder (48% yield); mp
109-111 °C. m/z (ES): found 239.0037 (C11H8ClO4 [M - H]-)
requires 239.0189.
3-Hydroxy-1-(2-hydroxy-propyl)-5-(3-isopropyl-phenyl)-4-(4-methyl-benz-
oyl)-1,5-dihydro-pyrrol-2-one (16). The procedure was similar to the
procedure for 1a except that 3-isopropyl benzaldehyde (371 μL, 2.5
mmol, 1.0 equiv) was used. No precipitation was observed after the
overnight reaction, and the reaction mixture was poured into cold H2O
(25 mL). The resulting solid was filtered and washed with EtOH/
petroleum ether 1:1 to afford 16 as a white powder (62% yield); mp
176-178 °C. m/z (ES): found 394.2066 (C24H28NO4 [M þ H]þ)
requires 394.1940.
3-Hydroxy-1-(2-hydroxy-propyl)-4-(4-methyl-benzoyl)-5-p-tolyl-1,5-dihy-
dro-pyrrol-2-one (17). The procedure was similar to the procedure for 1a
except that p-tolualdehyde (370 μL, 3.0 mmol, 1.0 equiv) was used and
the reaction mixture was allowed to stir at room temperature for only 3 h.
The precipitate obtained was washed with EtOH gave product 17 as a
white powder (4% yield); mp 233-235 °C. m/z (ES): found 366.2106
(C22H24NO4 [M þ H]þ) requires 366.1627.
4-(4-Cyano-phenyl)-2-hydroxy-4-oxo-but-2-enoic Acid Methyl Es-
ter (12h). The product was isolated as a pale white powder (30% yield);
mp 161-163 °C. m/z (ES): found 230.0474 (C12H8NO4 [M - H]-)
requires 230.0532.
2-Hydroxy-4-(6-methyl-pyridin-3-yl)-4-oxo-but-2-enoic Acid Meth-
yl Ester (12i). The product was isolated as a pale yellow powder (16%
yield); mp 100-102 °C. m/z (ES): found 222.0764 (C11H12NO4 [M þ
H]þ) requires 222.0688.
5-(4-Ethyl-phenyl)-3-hydroxy-1-(2-hydroxy-propyl)-4-(4-methyl-benzoyl)-
1,5-dihydro-pyrrol-2-one (18). The procedure was similar to the proce-
dure for 1a except that 4-ethyl benzaldehyde (420 μL, 3.0 mmol, 1.0
equiv) was used and the reaction mixture was allowed to stir at room
temperature for only 3 h. The precipitate obtained was washed with
EtOH and gave product 18 as a white powder (5% yield); mp 243-
245 °C. m/z (ES): found 380.1794 (C23H26NO4 [M þ H]þ) requires
380.1784.
2-Hydroxy-4-oxo-4-pyridin-3-yl-but-2-enoic Acid Methyl Ester
(12j). The product was isolated as a pale yellow powder (40% yield);
mp 121-123 °C. m/z (ES): found 208.0641 (C10H10NO4 [M þ H]þ)
requires 208.0532.
2-Hydroxy-4-oxo-4-pyridin-4-yl-but-2-enoic Acid Methyl Ester
(12k). The product was isolated as a yellow powder (12% yield); mp
99-101 °C. m/z (ES): found 206.0398 (C10H8NO4 [M - H]-)
requires 206.0532.
3-Hydroxy-1-(2-hydroxy-propyl)-4-(4-methyl-benzoyl)-5-(4-propyl-phen-
yl)-1,5-dihydro-pyrrol-2-one (19). The procedure was similar to the
procedure for 1a except that 4-propyl benzaldehyde (380 μL, 2.5 mmol,
1.0 equiv) was used and the reaction mixture was allowed to stir at room
temperature for only 3 h. The precipitate obtained was washed with
EtOH and gave product 19 as a white powder (52% yield); mp 248-
250 °C. m/z (ES): found 394.2096 (C24H28NO4 [M þ H]þ) requires
394.1940.
5-(4-tert-Butyl-phenyl)-3-hydroxy-1-(2-hydroxy-propyl)-4-(4-methyl-ben-
zoyl)-1,5-dihydro-pyrrol-2-one (20). The procedure was similar to the
procedure for 1a except that 4-tert-butyl benzaldehyde (431 μL, 2.5
mmol, 1.0 equiv) was used and the reaction mixture was allowed to stir at
room temperature for only 3 h. Product 20 was isolated as a white
powder (62% yield); mp 263-265 °C. m/z (ES): found 408.2273
(C25H30NO4 [M þ H]þ) requires 408.2097.
Step 2: Synthesis of 1,5-Disubstituted-4-substituted Ar-
oyl-3-hydroxy-5-phenyl-1H-pyrrol-2(5H)-ones. 3-Hydroxy-1-(2-hy-
droxy-propyl)-5-(4-isopropyl-phenyl)-4-(4-methyl-benzoyl)-1,5-dihydro-pyr-
rol-2-one (1a). 4-Isopropylbenzaldehyde (308 μL, 2.0 mmol, 1.0 equiv) and
1-amino-2-propanol (166 μL, 2.0 mmol, 1.0 equiv) were stirred in 1,4-dioxane
(5 mL) for 15 min. To this was added a solution of 2-hydroxy-4-oxo-4-p-tolyl-
but-2-enoic acid methyl ester (12b) (440 mg in 5.0 mL of 1,4-dioxane, 2.0
mmol, 1.0 equiv), and the reaction mixture was allowed to stir at room
temperature overnight. The yellow precipitate formed was filtered, washed
with Et2O, followed by EtOH, and was then dried in vacuo. The desired
product 1a was obtained as a white powder (335 mg, 43% yield); mp 249-
5-(4-Dimethylamino-phenyl)-3-hydroxy-1-(2-hydroxy-propyl)-4-(4-meth-
yl-benzoyl)-1,5-dihydro-pyrrol-2-one (21). The procedure was similar to
the procedure for 1a except that 4-dimethyl amino benzaldehyde
(448 mg, 3.0 mmol, 1.0 equiv) was used and the reaction mixture was
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allowed to stir at room temperature for only 3 h. Product 21 was isolated
as a yellow powder (32% yield); mp 242-244 °C. m/z (ES): found
395.2034 (C23H27N2O4 [M þ H]þ) requires 395.1893.
5-(3-Chloro-phenyl)-3-hydroxy-1-(2-hydroxy-propyl)-4-(4-methyl-
benzoyl)-1,5-dihydro-pyrrol-2-one (29). The procedure was similar to
the procedure for 1a except that 3-chloro benzaldehyde (293 μL, 2.5
mmol, 1.0 equiv) was used. Product 29 was isolated as a white powder
(47% yield); mp 235-237 °C. m/z (ES): found 386.1232
(C21H21ClNO4 [M þ H]þ) requires 386.1081.
3-Hydroxy-5-(4-hydroxy-phenyl)-1-(2-hydroxy-propyl)-4-(4-methyl-benz-
oyl)-1,5-dihydro-pyrrol-2-one (22). The procedure was similar to the
procedure for 1a except that 4-hydroxy benzaldehyde (249 mg, 2.0
mmol, 1.0 equiv) was used. Product 22 was isolated as a pale yellow
powder (15% yield); mp 225-227 °C. m/z (ES): found 368.1541
(C21H22NO5 [M þ H]þ) requires 368.1420.
5-(3,5-Dichloro-phenyl)-3-hydroxy-1-(2-hydroxy-propyl)-4-(4-methyl-
benzoyl)-1,5-dihydro-pyrrol-2-one (30). The procedure was similar to
the procedure for 1a except that 3,5-dichloro benzaldehyde (451 mg, 2.5
mmol, 1.0 equiv) was used. No precipitate was observed after the
overnight reaction, and the reaction mixture was heated under reflux for
10 h to complete the reaction. As precipitation was not observed on
standing, the reaction mixture was poured onto crushed ice (50 mL). A
brown precipitate formed that was filtered and recrystallized from
MeOH. Product 30 was isolated as a white powder (18% yield); mp
245-247 °C. m/z (ES): found 420.0893 (C21H20Cl2NO4 [M þ H]þ)
requires 420.0691.
3-Hydroxy-1-(2-hydroxy-propyl)-4-(4-methyl-benzoyl)-5-(4-trifluorom-
ethoxy-phenyl)-1,5-dihydro-pyrrol-2-one (23). The procedure was
similar to the procedure for 1a except that 4-(trifluoromethoxy)-
benzaldehyde (372 μL, 2.5 mmol, 1.0 equiv) was used and the reaction
mixture was allowed to stir at room temperature for only 3 h. The
precipitate obtained was washed with EtOH and gave product 23 as a
white powder (32% yield); mp 246-248 °C. m/z (ES): found 436.1570
(C22H21F3NO5 [M þ H]þ) requires 436.1294.
5-(4-Chloro-3-trifluoromethyl-phenyl)-3-hydroxy-1-(2-hydroxy-propyl)-
4-(4-methyl-benzoyl)-1,5-dihydro-pyrrol-2-one (31). The procedure
was similar to the procedure for 1a except that 4-chloro-3-trifluoro-
methyl benzaldehyde (532 μL, 2.5 mmol, 1.0 equiv) was used and the
reaction mixture was allowed to stir at room temperature for only 3 h.
The precipitate obtained was washed with EtOH and gave product 31 as
a white powder (45% yield); mp 225-227 °C. m/z (ES): found
454.1246 (C22H20ClF3NO4 [M þ H]þ) requires 454.0955.
3-Hydroxy-1-(2-hydroxy-propyl)-4-(4-methyl-benzoyl)-5-(4-trifluorometh-
yl-phenyl)-1,5-dihydro-pyrrol-2-one (24). The procedure was similar to
the procedure for 1a except that trifluoro-p-tolualdehyde (409 μL, 3.0
mmol, 1.0 equiv) was used and the reaction mixture was allowed to stir at
room temperature for only 3 h. The precipitate obtained was washed
with EtOH and gave product 24 as a white powder (19% yield); mp
256-258 °C. m/z (ES): found 420.1533 (C22H21F3NO4 [M þ H]þ)
requires 420.1344.
3-Hydroxy-1-isobutyl-5-(4-isopropyl-phenyl)-4-(4-methyl-benzoyl)-1,5-
dihydro-pyrrol-2-one (32). The procedure was similar to the procedure
for 1a except that isobutyl amine (201 μL, 2.0 mmol, 1.0 equiv) was used
and the reaction mixture was allowed to stir at room temperature for
only 3 h. The precipitate obtained was washed with Et2O followed by
MeOH and gave product 32 as a white powder (57% yield); mp 262-
264 °C. m/z (ES): found 392.2286 (C25H30NO3 [M þ H]þ) requires
392.2147.
3-Hydroxy-1-(2-hydroxy-ethyl)-5-(4-isopropyl-phenyl)-4-(4-methyl-
benzoyl)-1,5-dihydro-pyrrol-2-one (33). The procedure was similar to
the procedure for 1a except that ethanolamine (151 μL, 2.5 mmol, 1.0
equiv) was used and the reaction mixture was allowed to stir at room
temperature for only 3 h. The precipitate obtained was washed with
Et2O followed by MeOH and gave product 33 as a white powder (74%
yield); mp 236-238 °C. m/z (ES): found 380.1957 (C23H26NO4 [M þ
H]þ) requires 380.1784.
3-Hydroxy-1-(2-hydroxy-propyl)-4-(4-methyl-benzoyl)-5-(3-trifluorometh-
yl-phenyl)-1,5-dihydro-pyrrol-2-one (25). The procedure was similar to
the procedure for 1a except that 3-trifluoromethyl benzaldehyde
(276 μL, 2.0 mmol, 1.0 equiv) was used. No precipitation was observed
after the overnight reaction, and the reaction mixture was poured into
cold H2O (25 mL). The resulting solid was filtered and washed with
distilled H2O followed by recrystallization from MeOH. Product 25 was
isolated as a white powder (9% yield); mp 205-207 °C. m/z (ES):
found 420.1573 (C22H21F3NO4 [M þ H]þ) requires 420.1344.
5-(2,4-Bis-trifluoromethyl-phenyl)-3-hydroxy-1-(2-hydroxy-propyl)-4-(4-
methyl-benzoyl)-1,5-dihydro-pyrrol-2-one (26). The procedure was
similar to the procedure for 1a except that 2,4-bis-(trifluoromethyl)
benzaldehyde (417 μL, 2.5 mmol, 1.0 equiv) was used. The precipitate
obtained was washed only with Et2O and gave the desired product 26 as
a white powder (21% yield); mp 204-206 °C. m/z (ES): found
488.1429 (C23H20F6NO4 [M þ H]þ) requires 488.1218.
3-Hydroxy-5-(4-isopropyl-phenyl)-1-(2-methoxy-ethyl)-4-(4-meth-
yl-benzoyl)-1,5-dihydro-pyrrol-2-one (34). The procedure was similar
to the procedure for 1a except that 2-methoxy ethylamine (218 μL, 2.5
mmol, 1.0 equiv) was used and the reaction mixture was allowed to stir at
room temperature for only 3 h. The precipitate obtained was washed
with Et2O followed by MeOH and gave product 34 as a white powder
(68% yield); mp 247-249 °C. m/z (ES): found 394.2099 (C24H28NO4
[M þ H]þ) requires 394.1940.
5-(3,5-Bis-trifluoromethyl-phenyl)-3-hydroxy-1-(2-hydroxy-propyl)-4-(4-
methyl-benzoyl)-1,5-dihydro-pyrrol-2-one (27). 1-Amino-2-propanol
(416 μL, 5.00 mmol) and 3,5-bis-(trifluoromethyl) benzaldehyde
(850 μL, 5.00 mmol) were stirred in 1,4-dioxane (10 mL) for 15 min.
To this was added 12b (1.101 g in 10 mL of 1,4-dioxane, 5.00 mmol),
and the reaction mixture was stirred at room temperature for 22 h. The
precipitate that formed was filtered, washed with Et2O, and then dried in
vacuo to give 27. To recover more of the compound 27, the filtrate was
then poured into cold H2O (50 mL) and allowed to stand for 6 h. The
resulting yellow precipitate was filtered, washed with water, and dried in
vacuo. To the dried yellow precipitate was added Et2O (120 mL), and
the mixture was stirred for 4 h. The white precipitate that formed was
filtered and dried in vacuo to give 27. The total amount of 27 obtained
from both steps was 1.021 g (42% yield); mp 234-236 °C. m/z (ES):
found 488.1485 (C23H20F6NO4 [M þ H]þ) requires 488.1218.
3-Hydroxy-5-(4-isopropyl-phenyl)-1-(3-methoxy-propyl)-4-(4-methyl-
benzoyl)-1,5-dihydro-pyrrol-2-one (35). The procedure was similar to
the procedure for 1a except that 3-methoxy propylamine (256 μL, 2.5
mmol, 1.0 equiv) was used and the reaction mixture was allowed to stir at
room temperature for only 3 h. The precipitate obtained was washed
with Et2O followed by MeOH and gave product 35 as a white powder
(47% yield); mp 252-254 °C. m/z (ES): found 406.2099 (C25H28NO4
[M - H]-) requires 406. 2097.
5-(4-Chloro-phenyl)-3-hydroxy-1-(2-hydroxy-propyl)-4-(4-methyl-
benzoyl)-1,5-dihydro-pyrrol-2-one (28). The procedure was similar to
the procedure for 1a except that 4-chloro benzaldehyde (362 mg, 2.5
mmol, 1.0 equiv) was used and the reaction mixture was allowed to stir
at room temperature for only 3 h. Product 28 was isolated as a white
powder (28% yield); mp 238-240 °C. m/z (ES): found 386.1226
(C21H21ClNO4 [M þ H]þ) requires 386.1081.
1-Allyl-3-hydroxy-5-(4-isopropyl-phenyl)-4-(4-methyl-benzoyl)-1,5-di-
hydro-pyrrol-2-one (36). The procedure was similar to the procedure
for 1a except that allyl amine (189 μL, 2.5 mmol, 1.0 equiv) was used and
the reaction mixture was allowed to stir at room temperature for only 3 h.
The precipitate obtained was washed with Et2O followed by MeOH and
gave product 36 as a white powder (52% yield); mp 249-251 °C. m/z
(ES): found 376.1973 (C24H26NO3 [M þ H]þ) requires 376.1834.
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4-Benzoyl-3-hydroxy-1-(2-hydroxy-propyl)-5-(4-isopropyl-phenyl)-1,5-
dihydro-pyrrol-2-one (37). The procedure was similar to the procedure
for 1a except that 12a (618 mg, 3.0 mmol, 1.0 equiv) was used and the
reaction mixture was allowed to stir at room temperature for only 3 h.
The precipitate obtained was washed with EtOH and gave product 37 as
a white powder (50% yield); mp 257-259 °C. m/z (ES): found
380.1749 (C23H26NO4 [M þ H]þ) requires 380.1784.
temperature overnight. The yellow precipitate formed was filtered,
washed with diethyl ether followed by ethanol, and then dried in vacuo.
Product 45 was isolated as a white powder (84% yield); mp 265-
267 °C. m/z (ES): found 389.1143 (C20H16F3N2O3 [M þ H]þ)
requires 389.1035.
1-Allyl-3-hydroxy-4-(6-methyl-pyridine-3-carbonyl)-5-(4-trifluoromethyl-
phenyl)-1,5-dihydro-pyrrol-2-one (46). 4-Trifluoromethyl benzaldehyde
(273 μL, 2.0 mmol, 1.0 equiv) and allyl amine (152 μL, 2.0 mmol, 1.0
equiv) were stirred in 1,4-dioxane (5 mL) for 15 min. To this was added
a solution of 12i (442 mg in 5.0 mL of 1,4-dioxane, 2.0 mmol, 1.0 equiv),
and the reaction mixture was allowed to stir at room temperature for 2.30
h. The yellow precipitate obtained was washed only with Et2O and gave
the desired product 46 as a pale yellow powder (18% yield); mp 237-
239 °C decomp. m/z (ES): found 403.1359 (C21H18F3N2O3 [M þ
H]þ) requires 403.1191.
3-Hydroxy-1-(2-hydroxy-propyl)-5-(4-isopropyl-phenyl)-4-(3-methyl-
benzoyl)-1,5-dihydro-pyrrol-2-one (38). The procedure was similar to
the procedure for 1a except that 12c (661 mg, 3.0 mmol, 1.0 equiv) was
used and the reaction mixture was allowed to stir at room temperature
for only 3 h. Product 38 was isolated as a pale white powder (17% yield);
mp 221-223 °C. m/z (ES): found 394.2122 (C24H28NO4 [M þ H]þ)
requires 394.1940.
3-Hydroxy-1-(2-hydroxy-propyl)-5-(4-isopropyl-phenyl)-4-(4-methoxy-
benzoyl)-1,5-dihydro-pyrrol-2-one (39). The procedure was similar to
the procedure for 1a except that 12d (591 mg, 2.5 mmol, 1.0 equiv) was
used. Product 39 was isolated as a white powder (29% yield); mp
238-240 °C. m/z (ES): found 410.2090 (C24H28NO5 [M þ H]þ)
requires 410.1889.
3-Hydroxy-1-(2-hydroxy-propyl)-5-(4-isopropyl-phenyl)-4-(3-methoxy-
benzoyl)-1,5-dihydro-pyrrol-2-one (40). The procedure was similar to
the procedure for 1a except that 12e (709 mg, 3.0 mmol, 1.0 equiv) was
used. Product 40 was isolated as a white powder (12% yield); mp 227-
229 °C. m/z (ES): found 410.2022 (C24H28NO5 [M þ H]þ) requires
410.1889.
4-(2-Ethoxy-benzoyl)-3-hydroxy-1-(2-hydroxy-propyl)-5-(4-isopro-
pyl-phenyl)-1,5-dihydro-pyrrol-2-one (41). The procedure was similar
to the procedure for 1a except that 12f (500.5 mg, 2.0 mmol, 1.0 equiv)
was used. No precipitation was observed after the overnight reaction,
and the reaction mixture was poured into cold H2O (25 mL). The
resulting solid was filtered and washed with distilled H2O to afford
product 41 as a whitish brown powder (44% yield); mp 128-130 °C.
m/z (ES): found 424.2231 (C25H30NO5 [M þ H]þ) requires 424.2046.
The purity was obtained 91% (see the Supporting Information).
3-Hydroxy-1-(2-hydroxy-propyl)-5-(4-isopropyl-phenyl)-4-(6-methyl-pyr-
idine-3-carbonyl)-1,5-dihydro-pyrrol-2-one (42). The procedure was
similar to the procedure for 1a except that 12i (331.8 mg, 1.5 mmol, 1.0
equiv) was used. The precipitate obtained was washed only with Et2O
and gave the desired product 42 as a pale yellow powder (46% yield); mp
234-236 °C. m/z (ES): found 395.1867 (C23H27N2O4 [M þ H]þ)
requires 395.1893.
4-[4-Hydroxy-1-(2-hydroxy-propyl)-2-(4-isopropyl-phenyl)-5-oxo-2,5-di-
hydro-1H-pyrrole-3-carbonyl]-benzonitrile (43). The procedure was
similar to the procedure for 1a except that 12h (462.4 mg, 2.0 mmol,
1.0 equiv) was used. The reaction was carried out overnight at 70 °C. As
precipitation was not observed on standing, the reaction mixture was
poured onto crushed ice (40 mL). The precipitate formed was filtered
and washed with Et2O, followed by recrystallization from MeOH.
Product 43 was isolated as a white powder (12% yield); mp 234-
236 °C. m/z (ES): found 405.1912 (C24H25N2O4 [M þ H]þ) requires
405.1736.
1-Allyl-4-(4-chloro-benzoyl)-3-hydroxy-5-(4-trifluoromethyl-phenyl)-1,5-
dihydro-pyrrol-2-one (47). 4-Trifluoromethyl benzaldehyde (273 μL, 2.0
mmol, 1.0 equiv) and allyl amine (152 μL, 2.0 mmol, 1.0 equiv) were
stirred in 1,4-dioxane (5 mL) for 15 min. To this was added a solution of
12g (481.3 mg in 5.0 mL of 1,4-dioxane, 2.0 mmol, 1.0 equiv), and the
reaction mixture was allowed to stir at room temperature overnight. The
yellow precipitate obtained was washed only with Et2O and gave the
desired product 47 as a white powder (59% yield); mp 264-266 °C. m/z
(ES): found 422.0850 (C21H16ClF3NO3 [M þ H]þ) requires 422.0693.
3-Hydroxy-1-(3-methoxy-propyl)-4-(pyridine-3-carbonyl)-5-(4-tri-
fluoromethyl-phenyl)-1,5-dihydro-pyrrol-2-one (48). 4-Trifluoro-
methyl benzaldehyde (273 μL, 2.0 mmol, 1.0 equiv) and 3-methoxy
propylamine (205 μL, 2.0 mmol, 1.0 equiv) were stirred in 1,4-dioxane
(5 mL) for 15 min. To this was added a solution of 12j (414 mg in
5.0 mL of 1,4-dioxane, 2.0 mmol, 1.0 equiv), and the reaction mixture
was allowed to stir at room temperature overnight. The yellow pre-
cipitate obtained was washed only with Et2O and gave the desired
product 48 as a white powder (59% yield); mp 253-255 °C. m/z (ES):
found 421.1439 (C21H20F3N2O4 [M þ H]þ) requires 421.1297.
3-Hydroxy-1-(3-methoxy-propyl)-4-(6-methyl-pyridine-3-carbonyl)-5-(4-
trifluoromethyl-phenyl)-1,5-dihydro-pyrrol-2-one (49). 4-Trifluoro-
methyl benzaldehyde (273 μL, 2.0 mmol, 1.0 equiv) and 3-methoxy
propylamine (205 μL, 2.0 mmol, 1.0 equiv) were stirred in 1,4-dioxane
(5 mL) for 15 min. To this was added a solution of 12i (442.4 mg in
5.0 mL of 1,4-dioxane, 2.0 mmol, 1.0 equiv), and the reaction mixture
was allowed to stir at room temperature for 3 h. The yellow precipitate
obtained was washed only with Et2O and gave the desired product 49 as
a white powder (57% yield); mp 261-263 °C. m/z (ES): found
435.1588 (C22H22F3N2O4 [M þ H]þ) requires 435.1453.
5-(3-Fluoro-4-trifluoromethyl-phenyl)-3-hydroxy- 1-(3-methoxy-propyl)-
4-(6-methyl-pyridine-3-carbonyl)-1,5-dihydro-pyrrol-2-one (50). 3-
Fluoro-4-trifluoromethyl benzaldehyde (205 μL, 1.5 mmol, 1.0 equiv)
and 3-methoxy propylamine (154 μL, 1.5 mmol, 1.0 equiv) were stirred
in 1,4-dioxane (5 mL) for 15 min. To this was added a solution of 12i
(332 mg in 5.0 mL of 1,4-dioxane, 1.5 mmol, 1.0 equiv), and the reaction
mixture was allowed to stir at room temperature overnight. The yellow
precipitate obtained was washed only with Et2O and gave the desired
product 50 as a white powder (65% yield); mp 257-259 °C. m/z (ES):
found 453.1475 (C22H21F4N2O4 [M þ H]þ) requires 453.1359.
4-(4-Chloro-benzoyl)-3-hydroxy-1-(2-hydroxy-propyl)-5-(4-isopropyl-
phenyl)-1,5-dihydro-pyrrol-2-one (44). The procedure was similar to
the procedure for 1a except that 12g (481.3 mg, 2.0 mmol, 1.0 equiv).
The precipitate obtained was washed only with Et2O and gave thedesired
product 44 as a white powder (56% yield); mp 256-258 °C. m/z (ES):
found 414.1546 (C23H25ClNO4 [M þ H]þ) requires 414.1394.
1-Allyl-3-hydroxy-4-(pyridine-3-carbonyl)-5-(4-trifluoromethyl-
phenyl)-1,5-dihydro-pyrrol-2-one (45). 4-Trifluoromethyl benzalde-
hyde (273 μL, 2.0 mmol, 1.0 equiv) and allyl amine (152 μL, 2.0 mmol,
1.0 equiv) were stirred in 1,4-dioxane (5 mL) for 15 min. To this was
added a warm solution of 12j (414 mg in 5.0 mL of 1,4-dioxane, 2.0
mmol, 1.0 equiv), and the reaction mixture was allowed to stir at room
4-(4-Chloro-benzoyl)-5-(3-fluoro-4-trifluoromethyl-phenyl)-3-hydroxy-
1-(3-methoxy-propyl)-1,5-dihydro-pyrrol-2-one (51). 3-Fluoro-4-triflu-
oromethyl benzaldehyde (273 μL, 2.0 mmol, 1.0 equiv) and 3-methoxy
propylamine (205 μL, 2.0 mmol, 1.0 equiv) were stirred in 1,4-dioxane
(5 mL) for 15 min. To this was added a solution of 12g (481 mg in
5.0 mL of 1,4-dioxane, 2.0 mmol, 1.0 equiv), and the reaction mixture
was allowed to stir at room temperature overnight. The yellow pre-
cipitate formed was filtered, washed with diethyl ether followed by
ethanol, and then dried in vacuo. Product 51 was isolated as a white
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powder (76% yield); mp 252-254 °C. m/z (ES): found 470.0819
(C22H17ClF4NO4 [M - H]-) requires 470.0860.
(6) Pathuri, P.; Vogeley, L.; Luecke, H. Crystal Structure of Metas-
tasis-Associated Protein S100A4 in the Active Calcium-Bound Form. J.
Mol. Biol. 2008, 383, 62–77.
4-(2-Ethoxy-benzoyl)-3-hydroxy-1-(3-methoxy-propyl)-5-(4-trifluorometh-
yl-phenyl)-1,5-dihydro-pyrrol-2-one (52). 4-Trifluoromethyl benzalde-
hyde (279 μL, 2.0 mmol, 1.0 equiv) and 3-methoxy propylamine (205
μL, 2.0 mmol, 1.0 equiv) were stirred in 1,4-dioxane (5 mL) for 15 min.
To this was added a solution of 12f (501 mg in 5.0 mL of 1,4-dioxane, 2.0
mmol, 1.0 equiv), and the reaction mixture was allowed to stir at room
temperature overnight. No precipitation was observed, and the reaction
mixture was poured onto cold water (25 mL). The resulting solid was
filtered and washed with distilled H2O followed by Et2O to afford
product 52 as a white powder (6% yield); mp 138-140 °C. m/z (ES):
found 462.1536 (C24H23F3NO5 [M - H]-) requires 462.1607.
4-(2-Ethoxy-benzoyl)-5-(3-fluoro-4-trifluoromethyl-phenyl)-3-hydroxy-1-
(3-methoxy-propyl)-1,5-dihydro-pyrrol-2-one (53). The procedure was
similar as for 52 except that 3-fluoro-4-trifluoromethyl benzaldehyde
(273 μL, 2.0 mmol, 1.0 equiv) was used. Product 53 was isolated as a
white powder (9% yield); mp 140-142 °C. m/z (ES): found 480.1387
(C24H22F4NO5 [M - H]-) requires 480.1512.
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’ ASSOCIATED CONTENT
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signalling to membrane dynamics. Nat. Rev. Mol. Cell. Biol. 2005, 6, 449–461.
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Supporting Information. IR, 1H and 13C NMR spectro-
S
b
scopic data, and assessment of compound purity by HPLC. This
material is available free of charge via the Internet at http://pubs.
acs.org.
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Silverstein, R. L.; Hempstead, B.; Mark, W. H.; Hajjar, K. A. Annexin II
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’ AUTHOR INFORMATION
Corresponding Author
*Fax: þ44 115 95 13412. E-mail: lodewijk.dekker@nottingham.
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ac.uk.
’ ACKNOWLEDGMENT
We thank Wen Yuen Lim and Catherine Pereira for experi-
mental help at various stages of this research and Dr. Cristina de
Matteis for virtual screening software. This research was sup-
ported by grants from Cancer Research UK. H.K.M. was funded
by a Biotechnology and Biological Sciences Research Council
studentship.
’ ABBREVIATIONS USED
FRET, fluorescence resonance energy transfer; GA, genetic
algorithm; GOLD, genetic optimization for ligand docking; SAS,
solvent accessible surface area
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