Development of selective inhibitors for anti-apoptotic Bcl-2 proteins from BHI-1

Article abstract of DOI:10.1016/j.bmc.2006.12.020

A series of inhibitors for anti-apoptotic Bcl-2 proteins based on BHI-1 were synthesized and their binding interactions with Bcl-2, Bcl-X_L, and Bcl-w were evaluated. It was found that modification of BHI-1 resulted in varied binding profiles am

Full text of DOI:10.1016/j.bmc.2006.12.020

Bioorganic & Medicinal Chemistry 15 (2007) 2167–2176
Development of selective inhibitors for anti-apoptotic
Bcl-2 proteins from BHI-1
Chengguo Xing,* Liangyou Wang, XiaoHu Tang and Yuk Y. Sham
Department of Medicinal Chemistry, University of Minnesota, 8-101 WDH 308 Harvard ST SE, Minneapolis, MN 55455, USA
Received 9 August 2005; revised 4 December 2006; accepted 11 December 2006
Available online 14 December 2006
Abstract—A series of inhibitors for anti-apoptotic Bcl-2 proteins based on BHI-1 were synthesized and their binding interactions
with Bcl-2, Bcl-XL, and Bcl-w were evaluated. It was found that modification of BHI-1 resulted in varied binding profiles among
Bcl-2, Bcl-XL, and Bcl-w, and a set of inhibitors with varied selectivity to Bcl-2, Bcl-XL, and Bcl-w proteins have been identified.
Molecular modeling of the interaction of the BHI-1 based analogues with the anti-apoptotic Bcl-2 proteins suggested that the bind-
ing site for the BHI-1 based inhibitor was the least conserved section among Bcl-2, Bcl-XL, and Bcl-w: targeting the non-conserved
section may account for the observed selectivity of the BHI-1 based inhibitors among the anti-apoptotic Bcl-2 proteins. The validity
of the model was supported by a strong correlation between the model-calculated binding energy and the experimental binding
affinity. In summary, our studies suggest that most of the reported inhibitors for anti-apoptotic Bcl-2 proteins are nonselective
and BHI-1 is a promising template to distinguish among Bcl-2, Bcl-XL, and Bcl-w by targeting the non-conserved domain among
the anti-apoptotic Bcl-2 proteins. Molecular-modeling-aided rational development of BHI-1 based selective inhibitor for anti-
apoptotic Bcl-2 proteins is underway.
Published by Elsevier Ltd.
1. Introduction
tein.11–15 For instance, in a study of head and neck
carcinoma by Thompson et al., 52% of the patients
had up-regulated Bcl-XL and 17% of the patients had
up-regulated Bcl-2. There was no overlap between these
two groups.12 In Kaposi’s sarcoma, the expression level
of Bcl-XL protein was elevated, while Bcl-2 protein
remained at the basal level.13 In mastocytosis, the
expression of Bcl-2 significantly increased while no
up-regulation of Bcl-XL was detected.16 In gastric and
colorectal adenocarcinomas, only Bcl-w was up-regulat-
ed.14,15 Besides their selective over-expression in cancer
tissues, anti-apoptotic Bcl-2 proteins are expressed in
healthy tissues in a tissue-selective manner17–19 and
function to protect healthy cells.19 The cancer/tissue-
selective expression of anti-apoptotic Bcl-2 proteins
suggests that as therapeutic agents, member-selective
inhibitors for anti-apoptotic Bcl-2 proteins could be saf-
er than the non-selective counterparts in those tumors
with the selective elevation of one single anti-apoptotic
Bcl-2 protein. However, it is still debatable whether
the selective inhibitor will be as effective as the non-se-
lective ones in overcoming drug resistance. To address
these questions, selective inhibitors and non-selective
inhibitors of the same class need to be developed. In
addition, the selective inhibitors for the anti-apoptotic
Bcl-2 proteins can be useful chemical probes to help
Drug resistance is one major barrier in the fight against
cancer.1 At the molecular level, drug resistance can be
acquired through the over-expression of anti-apoptotic
Bcl-2 proteins which protect cancer cells from apoptosis
induced by cancer therapy.2–4 Inhibiting anti-apoptotic
Bcl-2 proteins, therefore, holds promise as a way to
overcome drug resistance for cancer treatment. This
concept has been proved valid through anti-sense
approach.5–7
Anti-apoptotic Bcl-2 proteins are a subgroup of the Bcl-
2 protein family, including Bcl-2, Bcl-XL, Bcl-w, Mcl-1,
and A1. In some cancers, more than one anti-apoptotic
Bcl-2 protein is co-over-expressed.8–10 Under such cir-
cumstance, a non-selective inhibitor for multiple anti-
apoptotic Bcl-2 proteins may be needed to antagonize
these proteins simultaneously to overcome the drug
resistance. Some cancers, however, exhibit exclusive
over-expression of one specific anti-apoptotic Bcl-2 pro-
Keywords: Apoptosis; Bcl-2; Inhibitor; Selectivity.
*
Corresponding author. Tel.: +1 612 626 5675; fax: +1 612 624
0139; e-mail: xingx009@umn.edu
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2006.12.020

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C. Xing et al. / Bioorg. Med. Chem. 15 (2007) 2167–2176
define the biological functions of the individual anti-
apoptotic Bcl-2 proteins.
With initial NMR and molecular modeling study sug-
gesting that BHI-1 targets the less conserved section of
the hydrophobic cleft,25 BHI-1 was selected for study
to investigate its potential as lead templates for develop-
ing member-selective inhibitors for anti-apoptotic Bcl-2
proteins.
Mechanistically, anti-apoptotic Bcl-2 proteins protect
cells from apoptosis by antagonizing pro-apoptotic
Bcl-2 proteins through dimerization.20,21 Structural
studies of a complex of Bcl-XL protein and a pro-apop-
totic peptide (Bak BH3) have revealed a hydrophobic
cleft on Bcl-XL protein as the binding pocket for the
pro-apoptotic peptide22 and molecules binding to that
hydrophobic cleft may overcome the protective effect
of the Bcl-XL protein.23 These observations have stimu-
lated the recent discovery of several small molecules tar-
geting the hydrophobic cleft of Bcl-XL or Bcl-2 proteins
with a partial list shown in Figure 1.24–32 As the hydro-
phobic cleft is very conserved among the anti-apoptotic
Bcl-2, Bcl-XL, Bcl-w, and Mcl-1 proteins,22,33–36 these
reported inhibitors could have cross activities among
these anti-apoptotic Bcl-2 proteins as has been estab-
lished for ABT-737.30 However, it is possible to develop
selective inhibitors among the anti-apoptotic Bcl-2 pro-
teins as the anti-apoptotic Bcl-2 proteins can differenti-
ate the peptides derived from the BH3 domain of the
pro-apoptotic Bcl-2 proteins with selectivity up to 10
fold.37,38 In addition, a small-molecule inhibitor (YC-
137) was recently discovered by Wang et al. to selective-
ly target Bcl-2 protein over Bcl-XL protein.29 Biological
evaluation of YC-137 demonstrated that YC-137 can
selectively eliminate the tumors with the over-expression
of Bcl-2 protein over those with the over-expression of
Bcl-XL protein. More excitingly, YC-137 showed mini-
mal toxicity to the normal healthy cells with no eleva-
tion of Bcl-2 protein, further underscoring the need to
develop selective inhibitors for anti-apoptotic Bcl-2
proteins.
2. Results and discussion
2.1. Chemistry
A group of BHI-1 analogues (compounds 3a–o) possess-
ing variation at the amino acid and alkylidene sections
were synthesized with a 20–40% overall yield by first
cyclizing respective amino acids with CS2 and a-chloro-
acetate to form rhodanines by using a procedure similar
to that previously reported,39 followed by Knoevenagel
condensation of rhodanines with varied aldehydes or ke-
tones to form the final analogues (Scheme 1). The con-
densation generated two isomers (E and Z) and the
major product was the thermodynamically stable Z iso-
mer as characterized by the down-field shift of the meth-
ylene proton of the Z isomer compared to that of the E
isomer. No racemerization was detected in the synthesis
of the BHI-1 analogues as demonstrated in the Supple-
menting Materials.
2.2. Biology
The binding affinities of the synthesized small molecules
and several reported inhibitors to three anti-apoptotic
Bcl-2 proteins, Bcl-2, Bcl-XL, and Bcl-w, were deter-
mined by using a fluorescence polarization (FP) based
competition assay against an Oregon Green fluoresce-
in-labeled Bak BH3 domain peptide (Flu-Bak).25 First,
the binding affinities of the Flu-Bak peptide to the three
Bcl-2 proteins were determined by using a Saturation
binding model with a constant concentration of Flu-
Bak peptide (45 nM) and varied concentrations of the
proteins. The binding affinity of Flu-Bak peptide to
the three Bcl-2 proteins is summarized in Table 1. The
binding affinity of Flu-Bak peptide to Bcl-XL protein
Since there is no obvious structural similarity among the
reported inhibitors, we hypothesized that their respec-
tive interactions with anti-apoptotic Bcl-2 proteins in-
volve distinct sections along the long hydrophobic
cleft. Inhibitors targeting the less conserved portion of
the hydrophobic cleft would be more promising tem-
plates in the search for member-selective inhibitors.
HO2C
I
O
Cl
O
O
N
N
O
C
O
S
H
O
O
N
S
O
S
Cl
Br
O
N
O
MeO
Me
Cl
O
NH2
OMe
Br
BHI-2
HA14-1
Chelerythrine
BHI-1
H3C
O
O
H
OH
OH
O
H
OH
H
N
O
O
HO
HO
N
O
CH3
CH3
H
OH
O
OH
O
CH3
H
O
O
Antimycin A3
Figure 1. Reported inhibitors for Bcl-2 or Bcl-XL proteins.
Gossypol

C. Xing et al. / Bioorg. Med. Chem. 15 (2007) 2167–2176
2169
O
R1
R4O
O
N
O
S
O
a, b, c
d
R1
R1
NH2
R4O
S
S
R4O
R2
N
O
S
R3
1
2
3
Scheme 1. Synthesis of BHl-1 based BCl-2 inhibitors 3a–o. Reagents and conditions: (a) NaOH, CS2, 23 ꢀC, overnight; (b) ClCH2CO2Na, 23 ꢀC, 3 h;
(c) HCl, reflux, 12 h; (d) benzaldehyde/ketone, NH4OAc, toluene, reflux, 1–12 h.
Table 1. The binding affinities of Flu-Bak peptide to Bcl-2, Bcl-XL,
and Bcl-w proteins
in this study (Kd = 6200 1500 nM) is quite different
from the one determined by Gemperli et al.
(Kd = 900 100 nM),37 it is possible that the difference
is because of the different forms of recombinant Bcl-2
proteins used in these two studies. The recombinant
Bcl-2 protein used in this study is transmembrane-do-
main deleted. With the binding affinities of Flu-Bak
peptide to the proteins determined, the interactions of
BHI-1 based small molecules to the Bcl-2 proteins were
indirectly evaluated by assaying their capabilities of
competing against Flu-Bak peptide binding to the
Bcl-2 proteins. The capabilities of the small molecules
to compete against Flu-Bak peptide binding to Bcl-2
proteins (Ki) are summarized in Table 2 for reported
inhibitors and Table 3 for BHI-1 based inhibitors.
Compound
Flu-Bak
Kd SD (nM)
Bcl-XL
Bcl-2
Bcl-w
990 100
362 36
555 41
(Kd = 362 36 nM) is comparable to the binding affinity
determined by Sattler et al., Kd = 340 nM.22 While the
binding affinity of Flu-Bak peptide to Bcl-2 determined
Table 2. The inhibitory capability of reported inhibitors against
Flu-Bak binding to Bcl-2, Bcl-XL, and Bcl-w proteins
Compound
Ki SD (lM)
Bcl-2
Bcl-XL
Bcl-w
As we have hypothesized, the reported inhibitors bound
to all the three anti-apoptotic Bcl-2 proteins tested with-
out much selectivity. In our effort to identify an optimal
template for the search of selective inhibitors, a series
of analogues of BHI-1 were synthesized. Of note, modifi-
cations resulted in a wider range of binding affinities
Antimycin A3
Chelerythrine
Gossypol
HA14-1
124 10.6
171 24.3
10.1 0.9
254 17.2
43.4 3.9
127 16.3
96 8.9
127 11.9
106 6.3
17.7 1.3
210 18
24.7 1.8
86.2 7.9
133 7.6
BHI-1
124 12.5
Table 3. The inhibitory capability of BHI-1 based candidates against Flu-Bak binding to Bcl-2, Bcl-XL, and Bcl-w proteins
R4
O
S
N
O
S
R1
R3
R2
Compound
R1
R2
R3
R4
Ki SD (lM) (selectivity)
Bcl-2
Bcl-XL
Bcl-w
124 12.5
BHI-1
3a
3b
3c
2-Propyl
Me
H
Br
Br
Br
Br
Br
Br
H
OH
OH
43.4 3.9 (2.85)
115 8.4 (2.11)
69.6 4.9 (1.26)
2.85 0.21 (11.51)
320 21.7 (1.91)
14.2 1.2
133 7.6
374 16.3
134 20.3
32.8 2.7
H
243 14.7
88 14.2
CH3S(CH2)2
3-Indolyl-CH2
C(O)OH(CH2)2
PhCH2
H
OH
OH
H
54.2 5.5
610 31.4
21.5 1.8
41.1 3.7
31.2 3.8
24.1 1.7
58.3 4.2
61.9 6.9
6.98 0.38
>1000
3d
3e
H
OH
OH
848 39.0
5.35 0.39 (2.65)
27.9 2.7
H
n-Propyl
3f
PhCH2
OH
OH
12 0.8 (2.33)
7.43 0.65
3g
3h
3i
(CH3)2CHCH2
(CH3)2CHCH2
(CH3)2CHCH2
(CH3)2CHCH2
(CH3)2CHCH2
Me
H
n-Propyl
Br
H
2.99 0.18 (2.48)
41.3 4.2
OH
OH
13.5 0.6 (1.79)
21.2 1.6 (2.74)
57.8 4.6 (1.07)
1.95 0.13 (3.58)
192 12.4 (1.40)
70.0 4.4 (2.01)
>1000
Me
Ph
Ph
H
H
128.2 5.9
68.8 5.5
8.7 0.46
3j
H
OH
OH
3k
3l
Ph
Br
Br
Br
Ar
O-2-Propenyl
N-Morpholinyl
O-2-Propenyl
O-2-Propenyl
268 18.4
228 19.7
244 20.1
176 14.6
3m
3n
3o
Me
PhCH2
PhCH2
H
146 17.6
56.8 2.9 (4.29)
116 11.8 (1.52)
H
H
260 19.7

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C. Xing et al. / Bioorg. Med. Chem. 15 (2007) 2167–2176
Table 4. Non-conserved amino acids in the hydrophobic cleft of Bcl-2,
Bcl-XL, and Bcl-w
(1.95–848 lM) and modification also changed the bind-
ing profiles of these inhibitors for Bcl-2, Bcl-XL, and
Bcl-w proteins. For instance, 3e and 3g are more selective
for Bcl-XL, L-Phe esters (3n and 3o) are more selective for
Bcl-w, while 3c and the ketone analogues (3f, 3h, 3i, 3j,
and 3k) are selective for Bcl-2 with 3c > 10-fold selective
for Bcl-2 over both Bcl-XL and Bcl-w. The distinct bind-
ing profiles of BHI-1 based inhibitors among Bcl-2, Bcl-
XL, and Bcl-w suggest that BHI-1 is a promising template
for the development of selective inhibitors.
104
108
122
Bcl-XL
Bcl-2
Bcl-w
Ala
Asp
Thr
Leu
Met
Leu
Ser
Arg
Arg
Figure 3A shows the modeled three-dimensional struc-
ture of one BHI-1 candidate (3e) bound to Bcl-XL pro-
tein. Based upon sequence alignment of Bcl-2, Bcl-XL,
and Bcl-w, the major distinctions among these three
proteins in the hydrophobic cleft are amino acids 104,
108, and 122 (Table 4, Amino acids numbered based
on Bcl-XL sequence).40 Amino acids 104 and 108 are
located at the modeled binding site for BHI-1 based
2.3. Molecular modeling
To understand the observed binding selectivity of BHI-1
based inhibitors for Bcl-2, Bcl-XL, and Bcl-w, molecular
modeling of the binding interaction by InsightII was
performed to define the binding section in the hydro-
phobic cleft on the proteins for these small molecules.
The validity of the model first was evaluated by correlat-
ing (1) the model-estimated binding energies of the
inhibitors to the protein with (2) experimentally deter-
mined binding affinities; the estimated binding energy
correlates highly with the experimental binding affinity
(Fig. 2 shows the correlation for seven BHI-1 based
inhibitors for Bcl-XL protein, R2 = 0.88; similar correla-
tions were obtained when Bcl-2 and Bcl-w homology
models were used for docking as well). The high correla-
tion between modeled and experimental results suggest-
ed that the binding model likely reflects the actual
binding interactions.
˚
inhibitors, while amino acid 122 is ꢀ2 to 6 A from 3k
inhibitor (Fig. 1B). The modeling studies suggest that
BHI-1 based inhibitors bind to the least conserved sec-
tion of the hydrophobic cleft. The distinct D104 in
Bcl-2 protein indicates that incorporating basic func-
tional groups that will be protonated at physiological
pH at the amino acid section of BHI-1 template will
introduce binding preference to Bcl-2 and may account
for the observed selective binding of 3c to Bcl-2 protein.
A104 and L108 on Bcl-XL may be responsible for the
moderate selectivity of 3e and 3g for Bcl-XL because
of steric effects and hydrophobic interactions. Interest-
ingly, all of the analogues with a R2 substituent prefer
Bcl-2 and we hypothesize that this selectivity trend is
due to the relative wideness of the cleft among Bcl-2,
Bcl-XL, and Bcl-w; because Bcl-XL has the narrowest
cleft followed by Bcl-2, with Bcl-w the widest,40 the ana-
logues with a R2 substituent may fit well into the hydro-
phobic cleft of Bcl-2 while they are too bulky for Bcl-XL
and not big enough for Bcl-w. Incorporation of larger
substituents is expected to result in analogues selective
for Bcl-w. This spatial difference of the hydrophobic
cleft also explains the selectivity of 3n and 3o for Bcl-
w, while 3l and 3m do not bind Bcl-w selectively.
0
-5
R2 = 0.8816
-10
-15
-20
-25
-30
-35
3. Conclusion
-8.00
-7.50
-7.00
-6.50
-6.00 -5.50
-5.00
-4.50
-4.00
logKd
In this study, we evaluated the binding interactions of
several reported inhibitors for anti-apoptotic Bcl-2 pro-
teins with Bcl-2, Bcl-XL, and Bcl-w proteins. None of
Figure 2. Correlation between the calculated binding energy and
experimental binding affinity of BHl-1 based BCl-2 inhibitors.
Figure 3. (A) Modeled interaction of BHI-1 based inhibitor 3e with Bcl-XL protein. (B) The relative position of amino acid 104, 108, and 122 (side
˚
chains are colored purple and distance in A) to 3e.

C. Xing et al. / Bioorg. Med. Chem. 15 (2007) 2167–2176
2171
these inhibitors showed selectivity among the three anti-
apoptotic Bcl-2 proteins. We also synthesized and eval-
uated a series of analogues based upon BHI-1 to search
for selective inhibitors for the anti-apoptotic Bcl-2 pro-
teins, as BHI-1 targets the less conserved section of the
hydrophobic cleft. Our results suggest that modifica-
tions of BHI-1 can introduce dramatic binding affinity
changes and varied binding profile among Bcl-2,
Bcl-XL, and Bcl-w proteins. Modeling of the binding
interaction of this series of analogues with the three pro-
teins supports that the binding site for BHI-1 is the least
conserved section in the long hydrophobic cleft. The
model also explains the observed binding selectivity of
BHI-1 based inhibitors for Bcl-2, Bcl-XL, and Bcl-w
proteins. Molecular-modeling-aided rational design of
member-selective inhibitors based on the BHI-1 tem-
plate is in progress.
dilutions of the anti-apoptotic Bcl-2 protein was pre-
pared in a PBS solution, pH 7.0, with 45 nM Flu-Bak
peptide and 1 mM DTT, and incubated at 23 ꢀC for
one hour (a time-course study of binding process of
Flu-Bak peptide to all the anti-apoptotic Bcl-2 proteins
demonstrated that binding interaction reaches equilibri-
um within 5 min). To each well in a 96-well half-area
black plate (Corning, NY) the solution (50 ll) was add-
ed and fluorescence polarization (FP, in mP unit) was
measured. The binding affinity was determined by fitting
the FP values to the concentrations of protein with a sin-
gle-binding site saturation model, by using Prism soft-
ware package (GraphPad, CA).41
To determine the binding affinity of small molecules for
an anti-apoptotic Bcl-2 protein, a series of 3-fold dilu-
tions of small molecules were prepared in dimethylsulf-
oxide, that is, 10, 3.33, 1.11, 0.37, 0.123, 0.041, 0.014,
and 0 mM. To each well in a 96-well half-area black
plate, 5 ll of the small molecule stock solution was add-
ed. A solution containing 50 nM Flu-Bak peptide and
the anti-apoptotic Bcl-2 protein to be tested in PBS buff-
er, pH 7.0, 1 mM DTT was prepared and incubated at
23 ꢀC for 1 h. The concentration of the anti-apoptotic
Bcl-2 protein used corresponds to the one that resulted
in 60% of Flu-Bak peptide complex with the anti-apop-
totic Bcl-2 protein and such a concentration was deter-
mined in the Flu-Bak and Bcl-2 protein saturation
binding experiment. There are two reasons that we
chose the condition with 60% complex formation for
the competing assay. First, with 60% complex forma-
tion, there will be a FP increase of >100 mP over the
background. Such a FP increase would generate a con-
siderable FP change in the presence of effective compe-
tition over background variation. Second, with 60%
complex formation, relatively less competing ligand
needs to be added to detect a considerable FP change
compared to the competition with higher percentage of
complex formation. The corresponding concentrations
of Bcl-2, Bcl-XL, and Bcl-w needed to achieve 60% com-
plex formation under the assay conditions are 1000, 880,
and 1200 nM, respectively. To each well the Flu-Bak
peptide and anti-apoptotic Bcl-2 protein solution
(45 ll) was added by auto-injection at a rate of 200 ll/s.
The sample was incubated at 23 ꢀC for 1 h and shaken
for two seconds before fluorescence polarization (in
mP unit) was measured. Controls included dose–re-
sponse measurements in the absence of proteins to assess
for any interactions between the compounds and the
Flu-Bak peptide. Eventual effects were taken into ac-
count by subtraction. The inhibitory constant (Ki) was
determined by fitting the FP changes to the concentra-
tion of the small molecule competing ligand by using
an equation developed by Shaomeng Wang et al. with
GraphPad Eq. 1.42
4. Experimental
4.1. Preparation of recombinant Bcl-2, Bcl-XL, and
Bcl-w proteins
The DNA sequence encoding Bcl-2DC21 inserted into a
His6 tag fused pET-25b(+) vector was a generous gift
from Stanley Korsmeyer at Harvard University; the
DNA sequence encoding Bcl-wDC22 and the DNA se-
quence encoding Bcl-XLDloopDC40 inserted into a
His6 tag fused pET-29b(+) vector were generous gifts
from Kalle Gehring at McGill University. The plasmids
were transformed into the Escherichia coli strain
ER2566 (New England Biolab., MA). The expression
of the fusion proteins was induced by 1 mM isopropyl-
b-D-thiogalactopyranoside (IPTG) and the fusion pro-
teins were purified by Ni–NTA resin following a native
protein purification protocol provided by manufacturer
(Qiagen, CA). Recombinant proteins were concentrated
with Centrifugal Filter Devices (Millipore, MA) and
dialyzed against phosphate-buffered saline (PBS) con-
taining 15% glycerol, 1 mM dithiothreitol (DTT). The
concentration of the recombinant protein was deter-
mined by the Bradford method with bovine serum albu-
min (BSA) as a standard, and stored at ꢁ20 ꢀC.
4.2. Fluorescence polarization assays (FPA)
The Bak BH3 (GQVGRQLAIGDDINR) peptide was
synthesized at The Oligonucleotide & Peptide Synthesis
Facility at the University of Minnesota, purified by
HPLC. The purified peptide was labeled with Oregon
Green 488ꢁ fluorescein at the N terminus following
the manufacturer’s protocol (Promega, CA), purified
by HPLC, analyzed by mass spectrometry (Calculated:
2119 Da; Observed: 2119.7 Da), and named as Flu-
Bak. Flu-Bak was dissolved in double distilled water
and stored at ꢁ20 ꢀC as aliquot. FPA were conducted
with the Flu-Bak peptide by using a GENios Pro plate
reader (Tecan US, NC) with all assays performed in
triplicate and each assay performed twice.
FPmax
FP ¼
ð1Þ
1 þ X ꢂ Kd=ð2 ꢂ Ki ꢂ L0 þ Ki ꢂ P0 þ Ki ꢂ KdÞ
FPmax, the fluorescence polarization value when 60% of
Flu-Bak binds to the protein; FP, the fluorescence polar-
ization value with the addition of the small molecule;
Kd, dissociation constant of Flu-Bak to the protein;
To determine the binding affinity of the Flu-Bak peptide
for an anti-apoptotic Bcl-2 protein, a series of 3-fold

2172
C. Xing et al. / Bioorg. Med. Chem. 15 (2007) 2167–2176
L0, the total concentration of Flu-Bak peptide; P0, the
total concentration of protein; Ki, the inhibitory con-
stant of the small molecule to the binding of Flu-Bak
to protein.
(22–29 min), 80%–0B1 (29–30 min); MA3 was 100% A
(0–5 min), 0–70% B1 (5–7 min), 70–80% B1
(7–22 min), 80–85% B1 (22–29 min), 85%–0B1
(29–30 min); MB1 was 100% A (0–5 min), 0–95% B2
(5–7 min), 95% B2 (7–29 min), 95%–0B2 (29–30 min);
MB2 was 100% A (0–5 min), 0–85% B2 (5–7 min),
85% B2 (7–29 min), 85%–0B2 (29–30 min); MB3 was
100% A (0–5 min), 0–80% B2 (5–7 min), 80–90% B2
(7–22 min), 90–95% B2 (22–29 min), 95%–0B2 (29–
30 min).
4.3. Molecular modeling
Molecular modeling studies were conducted on a SGI
Octane2 workstation using InsightII (Accelrys Inc.,
CA). The NMR energy-minimized average structure
of Bcl-XL was extracted from the complex of Bcl-XL
and Bak peptide from the protein data bank (PDB
code: 1BXL). The structures of Bcl-2 and Bcl-w used
for modeling studies were generated by homology-
mimicking the structure of Bcl-XL from the complex
of Bcl-XL and Bak peptide based on sequence align-
ments (PDB code: 1G5M, 1BXL, and 1MK3, respec-
tively, for Bcl-2, Bcl-XL, and Bcl-w) with side chains
energy-minimized. The structures of all the inhibitors
were created and energy-minimized within InsightII,
followed by docking of the inhibitors into the Bak
peptide binding site.22 The energy minimization of
the ligand–protein complex was carried out by keeping
the binding site rigid using a dielectric constant of 80.
The binding energy of the ligand to the protein was
calculated by using e = 6.
4.5. General procedure for synthesis of BHI-1 based
inhibitors
In a round-bottomed flask equipped with a magnetic
stirrer, the amino acid or ester (1 mmol) was dissolved
with sodium hydroxide (80 mg, 2 mmol) in water
(10 ml). Then, carbon disulfide (60 ll, 1 mmol) was add-
ed to the reaction mixture, which was stirred vigorously
overnight. An aqueous solution of ClCH2CO2Na (1 ml,
1 M, 1 mmol) was added and stirring was continued at
23 ꢀC for 3 h. Then hydrochloric acid solution (3 ml,
5.5 N, 16.5 mmol) was added and the reaction mixture
was refluxed overnight. The reaction mixture was neu-
tralized with saturated NaHCO3 solution. The solvent
was removed under vacuum and the cyclized product
was purified by flash chromatography. The cyclized
intermediates are reported before and no characteriza-
tion data are included herein.39
4.4. Chemistry
All commercial reagents and anhydrous solvents were
purchased from vendors and were used without further
purification or distillation, unless otherwise stated. Ana-
lytical thin-layer chromatography (TLC) was performed
on EM Science silica gel 60 F254 (0.25 mm). Compounds
were visualized by UV light and/or stained with either
p-anisaldehyde, potassium permanganate, or cerium
molybdate solutions followed by heating. Flash column
chromatography was performed on Fischer Scientific sil-
ica gel (230–400 mesh). Melting points were determined
by using a Thomas Hoover capillary melting point
apparatus. IR spectra were recorded on a Nicolet Por-
tege 460 FT-IR instrument. NMR (1H) spectra were
recorded on a Varian 300 MHz spectrometer and cali-
brated using an internal reference. High-resolution mass
spectra (HRMS) were recorded on a BrukerBio TOF II
mass spectrometer. Reversed-phase high-performance li-
quid chromatography (RP-HPLC) was performed on a
Beckman–Coulter System Gold 126 solvent module
and 168 detector. Results were analyzed using the 32
Karat software package. Method MA1, MA2, MA3,
and MB3 were used on a Celius C18 column,
4.6 · 150 mm. Method MB1 and MB2 were used on a
Phenomenex Polar-RP column, 4.6 · 250 mm. Mobile
phase A was 0.1%TFA in water, B1 was 0.1%TFA in
acetonitrile, and B2 was 0.1%TFA in methanol. Flow
rate was 1.0 mL/min. The run duration was 30 min.
MA1 and MB1 were detected at 375 nm; MA2 and
MB2 were detected at 355 nm; MA1 and MB3 were
detected at 395 nm. The time program for MA1
was 100% A (0–5 min), 0–35% B1 (5–7 min), 35–95%
B1 (7–22 min), 95% B1 (22–29 min), 95%–0B1
(29–30 min); MA2 was 100% A (0–5 min), 0–65% B1
(5–7 min), 65–75% B1 (7–22 min), 75–80% B1
4.6. General procedure for condensation with
aldehydes/ketones
To a round-bottomed flask equipped with a reflux con-
denser and a magnetic stirrer, cyclized rhodanine inter-
mediate (1 mmol) in toluene (20 ml) was added. To
this, the aldehyde/ketone (3 mmol) was added along
with ammonium acetate (3 mmol). The mixture was re-
fluxed overnight and the solvent was removed under
vacuum. The final rhodanine candidate was purified by
flash chromatography.
4.7. 2-[5-(4-Bromobenzylidene)-4-oxo-2-thioxothiazoli-
din-3-yl]-3-methylbutanoic acid (BHI-1)
Yield: 85%. TLC (CH2Cl2/MeOH = 5:1), Rf: 0.40. IR
(KBr): 3700–2200, 2967, 2897, 1727, 1710, 1603, 1582,
1486, 1382, 1327, 1242, 1202, 1128, 1072, 1005,
815 cmꢁ1. 1H NMR (CDCl3): d 7.62 (1H, s, 50-dene pro-
ton), 7.57 (2H, d, J = 8.5 Hz, 200 and 600Ph protons), 7.30
(2H, d, J = 8.5 Hz, 300 and 500Ph protons), 5.34 (1H, m,
2-methine proton), 2.84 (1H, m, 3-methine proton),
1.25 (6H, d, J = 7.4 Hz, 3, 3-dimethyl protons);
ESI-MS (negative): m/z 398, 400 (MꢁꢁH).
4.8. 2-[5-(4-Bromobenzylidene)-4-oxo-2-thioxothiazoli-
din-3-yl]propanoic acid (3a)
Yield: 81%. Yellow powder. TLC (CH2Cl2/
MeOH = 10:1), Rf 0.11. Mp: 223–224 Cꢀ. IR (KBr):
3700–2200, 2922, 1714, 1602, 1583, 1486, 1400, 1342,
1
1249, 1125, 1071, 1006, 829 cmꢁ1. H NMR (CDCl3):
d 7.64 (1H, s, 50-dene proton), 7.62 (2H, d, J = 8.7 Hz,

C. Xing et al. / Bioorg. Med. Chem. 15 (2007) 2167–2176
2173
200 and 600 Ph protons), 7.35 (2H, d, J = 8.7 Hz, 300 and 500
Ph protons), 5.79 (1H, m, 2-methine proton), 1.68 (3H,
d, J = 7.6 Hz, 3-methyl protons); ESI-MS (negative):
m/z 370, 372 (MꢁꢁH). HRMS (C13H9BrNO3S2)
[MꢁH]ꢁ: found m/z 369.9191, calcd m/z 369.9205;
RP-HPLC method A1, minor isomer tR = 19.150 min
(2.19%) and major isomer tR = 20.300 min (97.81%);
method B1, minor isomer tR = 15.083 min (2.12%) and
major isomer tR = 15.500 min (87.93%).
(MꢁꢁH). HRMS (C15H11BrNO5S2) [MꢁH]ꢁ: found
m/z 427.9268, calcd m/z 427.9260; RP-HPLC method
MA1, minor isomer tR = 16.417 min (15.06%) and ma-
jor isomer tR = 17.333 min (84.94%); method MB1, min-
or isomer tR = 13.900 min (11.14%) and major isomer
tR = 14.433 min (92.53%).
4.12. 2-[5-(4-Bromobenzylidene)-4-oxo-2-thioxothiazoli-
din-3-yl]-3- phenylpropanoic acid (3e)
4.9. 2-[5-(4-Bromobenzylidene)-4-oxo-2-thioxothiaz-
olidin-3-yl]-4-methylthiobutanoic acid (3b)
Yield: 89%. Yellow powder. TLC (CH2Cl2/MeOH =
10:1), Rf 0.13. Mp: 242–243 Cꢀ. IR (KBr): 3700–2200,
2923, 2361, 1700, 1600, 1487, 1400, 1342, 1239, 1174,
1
Yield: 81%. TLC (CH2Cl2/MeOH = 5:1), Rf 0.29. Mp:
165–166 Cꢀ. IR (KBr): 3700–2200, 2922, 1714, 1602,
1583, 1486, 1400, 1342, 1249, 1125, 1071, 1006,
829 cmꢁ1. 1H NMR (CDCl3): d 7.62–7.45 (3H, m, 50-de-
ne proton and 200 and 600 Ph protons), 7.28–7.24 (2H, d,
J = 8.4 Hz, 300 and 500 Ph protons), 5.76 (1H, m,
2-methine proton), 2.60–2.26 (4H, m, 3- and 4-methy-
lene protons), 2.01 (3H, s, 4-methylthio methyl protons);
ESI-MS (negative): m/z 430, 432 (MꢁꢁH); 386, 388
(MꢁꢁH–CO2). HRMS (C15H13BrNO5) [MꢁH]ꢁ: found
m/z 429.9250, calcd m/z 429.9239; RP-HPLC method
MA1, minor isomer tR = 20.367 min (12.07%) and ma-
jor isomer tR = 21.483 min (87.93%); method MB1, min-
or isomer tR = 15.767 min (12.67%) and major isomer
tR = 16.300 min (87.33%).
1073, 1008, 829, 700, 546 cmꢁ1. H NMR (CDCl3): d
7.64–7.32 (3H, m, 50-dene proton and 200 and 600 Ph pro-
tons), 7.26–7.00 (7H, m, 300 and 500 Ph and 3-Ph protons),
5.93 (1H, m, 2-methine proton), 3.55 (2H, m, 3-methylene
protons); ESI-MS (negative): m/z 446, 448 (MꢁꢁH); 402,
404 (MꢁꢁH–CO2). HRMS (C19H13BrNO3S2) [MꢁH]ꢁ:
found m/z 425.9534, calcd m/z 445.9518; RP-HPLC meth-
od MA1, minor isomer tR = 21.050 min (5.42%) and ma-
jor isomer tR = 22.067 min (94.58%); method MB1, minor
isomer tR = 15.583 min (6.80%) and major isomer tR =
16.167 min (93.20%).
4.13. 2-[5-(1-Phenylbutylidene)-4-oxo-2-thioxothiaz-
olidin-3-yl]-3-phenylpropanoic acid (3f)
Yield: 43%. Orange oil. TLC (hexane/ethyl acetate =
1:1), Rf 0.38. IR (KBr): 3700–2200, 3027, 2959, 2929,
2870, 1711, 1589, 1569, 1455, 1441, 1382, 1332, 1230,
1175, 1029, 970, 920, 751, 698 cmꢁ1. 1H NMR (CDCl3):
d 7.43–7.18 (10H, m, 3-Ph and Ph00 protons), 6.05
(1H, m, 2-methine proton), 3.60 (2H, d, J = 7.2 Hz,
3-methylene protons), 3.17 (2H, m, (CH3CH2CH2)00 pro-
tons), 1.41 (2H, m, (CH3CH2CH2)00 protons), 0.94 (3H,
t, J = 7.2 Hz, (CH3CH2CH2)00 protons). ESI-MS (nega-
tive): m/z 410 (MꢁꢁH). HRMS (C22H21NO3S2)
[MꢁH]ꢁ: found m/z 410.0892, calcd m/z 410.0883; RP-
HPLC method MA2, minor isomer tR = 23.233 min
(35.97%) and major isomer tR = 23.983 min (64.03%);
method MB2, minor isomer tR = 22.717 min (34.23%)
and major isomer tR = 23.833 min (65.77%).
4.10. 2-[5-(4-Bromobenzylidene)-4-oxo-2-thioxothiazoli-
din-3-yl]-3-(3-indolyl)-propanoic acid (3c)
Yield: 78%. Yellow powder. TLC (CH2Cl2/
MeOH = 5:1), Rf 0.24. Mp: 158–159 Cꢀ. IR (KBr):
3700–2200, 2924, 1691, 1601, 1351, 1138 cmꢁ1 1H
.
NMR (CDCl3): d 7.73 (2H, d, J = 8.4 Hz, 200 and 600
Ph protons), 7.62 (1H, d, J = 8.1 Hz, indole-7 proton),
7.60 (1H, s, 50-dene proton), 7.48 (2H, d, J = 8.4 Hz,
300 and 500 Ph protons), 7.28 (1H, d, J = 8.1 Hz, indole-
4 proton), 7.14–6.99 (2H, m, indole-5,6 protons), 7.00
(1H, s, indole-2 proton), 5.90 (1H, m, 2-methine pro-
ton), 4.04 (1H, dd, J1 = 13.2 Hz, J2 = 3.9 Hz, 3-methy-
lene proton), 3.80 (1H, dd, J1 = 13.2 Hz, J2 = 3.9 Hz,
3-methylene proton); ESI-MS (negative): m/z 485, 487
(MꢁꢁH);
441,
443
(MꢁꢁH–CO2);
HRMS
4.14. 2-[5-(4-Bromobenzylidene)-4-oxo-2-thioxothiazoli-
din-3-yl]-4-methylpentanoic acid (3g)
(C21H14BrNO3S2) [MꢁH]ꢁ: found m/z 484.9634, calcd
m/z 484.9627; RP-HPLC method MA1, minor isomer
isomer
tR = 19.467 min
(1.23%)
and
major
Yield: 79%. Yellow powder. TLC (CH2Cl2/MeOH =
10:1), Rf 0.22. Mp: 155–156 Cꢀ. IR (KBr): 3700–2200,
2956, 1716, 1700, 1601, 1558, 1341, 1273, 1239,
tR = 20.250 min (98.73%); method MB1, minor isomer
tR = 14.700 min (5.44%) and major isomer tR = 15.100
min (94.56%).
1
1207 cmꢁ1. H NMR (CDCl3): d 7.64 (1H, s, 50-dene
proton), 7.53 (2H, d, J = 8.1 Hz, 200 and 600 Ph protons),
7.23 (2H, d, J = 8.1 Hz, 300 and 500 Ph protons), 5.53 (1H,
m, 2-methine proton), 2.00 (2H, m, 3-methylene pro-
tons), 1.23 (1H, m, 4-methine proton), 0.86 (6H, m,
4,4-dimethyl protons); ESI-MS (negative): m/z 412,
414 (MꢁꢁH); 358, 360 (MꢁꢁH–CO2). HRMS
(C16H16NO3S2) [MꢁH]ꢁ: found m/z 411.9702, calcd
m/z 411.9675; RP-HPLC method MA1, minor isomer
tR = 22.167 min (9.20%) and major isomer tR =
23.433 min (90.80%); method MB1, minor isomer
tR = 15.317 min (13.02%) and major isomer tR =
15.767 min (86.98%).
4.11. 2-[5-(4-Bromobenzylidene)-4-oxo-2-thioxothiazoli-
din-3-yl]-glutaric acid (3d)
Yield: 76%. Yellow powder. TLC (CH2Cl2/MeOH =
1:1), Rf 0.28. Mp: 227–228 Cꢀ. IR (KBr): 3700–2200,
1700, 1602, 1487, 1401, 1345, 1252, 1174, 1073, 1008,
819 cmꢁ1
. d 7.77 (2H, d,
1H NMR (CD3OD):
J = 8.4 Hz, 200 and 600 Ph protons), 7.70 (1H, s, 50-dene
proton), 7.57 (2H, d, J = 8.4 Hz, 300 and 500 Ph protons),
5.64 (1H, m, 2-methine proton), 2.30–2.04 (4H, m, 3 and
4-methylene protons); ESI-MS (negative): m/z 428, 430

2174
C. Xing et al. / Bioorg. Med. Chem. 15 (2007) 2167–2176
4.15. 2-[5-(1-Phenylbutylidene)-4-oxo-2-thioxothiaz-
olidin-3-yl]-4- methylpentanoic acid (3h)
(MꢁꢁH). HRMS (C28H24NO3S2) [MꢁH]ꢁ: found
m/z 486.1196, calcd m/z 486.1196; RP-HPLC method
MA3, minor isomer tR = 22.783 min (39.89%) and ma-
jor isomer tR = 23.750 min (60.11%); method MB3,
minor isomer tR = 23.317 min (42.08%) and major iso-
mer tR = 24.317 min (57.92%).
Yield: 46%. Orange oil. TLC (hexane/ethyl ace-
tate = 1:1), Rf 0.17. IR (KBr): 3700–2200, 2959, 2930,
2870, 1709, 1590, 1332, 1232, 1213 cmꢁ1 1H NMR
.
(CDCl3): d 7.51–7.20 (5H, m, Ph00 protons), 5.79 (1H,
m, 2-methine proton), 3.21 (2H, m, (CH3CH2CH2)00 pro-
tons), 2.20 (2H, m, (CH3CH2CH2)00 protons), 2.01 (2H,
m, 3-methylene protons), 1.60 (1H, m, 4-methine
proton), 1.10–0.80 (9H, m, 4,4-dimethyl and
(CH3CH2CH2)00 protons). ESI-MS (negative): m/z 376
(MꢁꢁH). HRMS (C19H22NO3S2) [MꢁH]ꢁ: found m/z
376.1050, calcd m/z 376.1039; RP-HPLC method
MA1, minor isomer tR = 22.617 min (37.27%) and
major isomer tR = 22.083 min (62.73%); method MB2,
minor isomer tR = 22.733 min (34.89%) and major
isomer tR = 15.750 min (63.11%).
4.19. 2-Propenyl 2-[5-(4-bromobenzylidene)-4-oxo-2-thi-
oxothiazolidin-3-yl]-3-propanoate (3l)
Yield: 66%. Yellow powder. TLC (Hexane/CH2Cl2 =
1:1), Rf 0.50. Mp: 105–106 Cꢀ. 1H NMR (CDCl3): d
7.63 (1H, s, 50-dene proton), 7.62 (2H, d, J = 8.4 Hz,
200 and 600 Ph protons), 7.35 (2H, d, J = 8.4 Hz, 300 and
500 Ph protons), 5.87 (1H, m, 2-methine proton), 5.73
(1H, m, CH2CH@CH2 proton), 5.30 and 5.23 (2H, d
and d, J = 17.4 Hz and J = 9.6 Hz, CH2CH@CH2 pro-
tons), 4.66 (2H, m, CH2CH@CH2 protons), 1.69 (3H,
d, J = 9.6 Hz, 3-methyl protons); ESI-MS (positive):
m/z 412, 414 (M++H); 368, 370 (M++H–CO2). HRMS
(C16H14BrNO3S2Na) [M+Na]+: found m/z 433.9504,
calcd m/z 433.9495; RP-HPLC method MA1, minor iso-
mer tR = 23.083 min (3.45%) and major isomer
tR = 24.267 min (96.55%); method MB1, minor isomer
tR = 16.917 min (0.95%) and major isomer tR =
17.70 min (99.05%).
4.16. 2-[5-(1-Phenylethylidene)-4-oxo-2-thioxothiaz-
olidin-3-yl]-4- methylpentanoic acid (3i)
Yield: 38%. Orange oil. TLC (hexane/ethyl acetate =
1:1), Rf 0.17. IR (KBr): 3700–2200, 2957, 2869, 1708,
1591, 1570, 1490, 1442, 1331, 1213, 1141, 1028, 990,
1
760, 698 cmꢁ1. H NMR (CDCl3): d 7.59–7.20 (5H, m,
Ph00 protons), 5.73 (1H, m, 2-methine proton), 2.72
(3H, s, 5-methyl00 protons), 2.14 (2H, m, 3-methylene
protons), 1.55 (1H, m, 4-methine proton), 0.95 (6H, m,
4,4-dimethyl protons). ESI-MS (negative): m/z 348
(MꢁꢁH). HRMS (C17H18NO3S2) [MꢁH]ꢁ: found m/z
348.0756, calcd m/z 348.0726; RP-HPLC method
MA2, minor isomer tR = 17.800 min (23.19%) and ma-
jor isomer tR = 18.983 min (76.81%); method MB2, min-
or isomer tR = 19.183 min (27.46%) and major isomer
tR = 20.133 min (72.54%).
4.20. 5-(4-Bromobenzylidene)-3-(1-morpholino-1-oxo-3-
phenylpropan-2-yl)-4-oxo-2-thioxothiazolidine (3m)
Yield: 97%. Yellow oil. TLC (methanol/CH2Cl2 = 1:99),
1
Rf 0.40. H NMR (CDCl3): d 7.62 (1H, s, 50-dene pro-
ton), 7.57 (2H, d, J = 8.4 Hz, 200 and 600 Ph protons),
7.35 (2H, d, J = 8.4 Hz, 300 and 500 Ph protons), 5.56
(1H, q, J = 7.5 Hz, 2-methine proton), 3.83 (4H, t,
J = 5.4 Hz, N(CH2)2(CH2)2O protons), 3.07 (4H, t,
J = 5.4 Hz, N(CH2)2(CH2)2O protons), 1.63 (3H, d,
J = 7.2 Hz, 3-methyl protons); ESI-MS (positive): m/z
441, 443 (M++H); 397, 399 (M++H–CO2). HRMS
(C17H17BrN2O3S2Na) [M+Na]+: found m/z 462.9758,
calcd m/z 462.9761; RP-HPLC method MA1, minor iso-
mer tR = 20.050 min (8.66%) and major isomer
tR = 21.150 min (91.34%); method MB1, minor isomer
tR = 16.600 min (8.71%) and major isomer tR =
17.300 min (91.29%).
4.17. 2-[5-(Diphenylmethylidene)-4-oxo-2-thioxothiazoli-
din-3-yl]-4- methylpentanoic acid (3j)
Yield: 30%. Orange oil. TLC (hexane/ethyl ace-
tate = 1:1), Rf 0.26. IR (KBr): 3700–2200, 3056, 2956,
2869, 1712, 1616, 1579, 1558, 1444, 1330, 1212, 1133,
1093, 752, 697 cmꢁ1 1H NMR (CDCl3): d 7.51–7.20
.
(10H, m, Ph00, Ph00 protons), 5.76 (1H, m, 2-methine pro-
ton), 2.16 (2H, m, 3-methylene protons), 1.60 (1H, m,
4-methine proton), 1.10–0.93 (6H, m, 4,4-dimethyl pro-
tons). ESI-MS (negative): m/z 410 (MꢁꢁH). HRMS
(C22H20NO3S2) [MꢁH]ꢁ: found m/z 410.0896, calcd m/
z 410.0883; RP-HPLC method MA1, tR = 22.700 min;
method MB1, tR = 16.083 min.
4.21. 2-Propenyl 2-[5-(4-bromobenzylidene)-4-oxo-2-thi-
oxothiazolidin-3-yl]-3-phenylpropanoate (3n)
Yield: 82%. Yellow powder. TLC (CH2Cl2), Rf 0.54. Mp:
146–147 Cꢀ. 1H NMR (CDCl3):
d 7.61 (2H, d,
J = 8.1 Hz, 200 and 600 Ph protons), 7.60 (1H, s, 50-dene
proton), 7.31 (2H, d, J = 8.1 Hz, 300 and 500 Ph protons),
7.20 (5H, m, 3-Ph protons), 5.92 (1H, m, CH2CH@CH2
proton), 5.88 (1H, m, 2-methine proton), 5.30 and
5.25 (2H, d and d, J = 18.5 Hz and J = 10.5 Hz,
CH2CH@CH2 protons), 4.69 (2H, d, J = 4.8 Hz,
CH2CH@CH2 protons), 3.63 (2H, d, J = 8.4 Hz, 3-meth-
ylene protons); ESI-MS (positive): m/z 488, 490 (M++H);
444, 446 (M++H–CO2). HRMS (C22H18BrNO3S2Na)
[M+Na]+: found m/z 509.9834, calcd m/z 509.9808; RP-
HPLC method MA1, minor isomer tR = 24.700 min
(12.28%) and major isomer tR = 25.600 min (87.72%);
4.18. 2-[5-(Phenyl(4-biphenyl)methylidene)-4-oxo-2-thi-
oxothiazolidin-3-yl]-4-methylpentanoic acid (3k)
Yield: 23%. Orange oil. TLC (hexane/ethyl ace-
tate = 1:1), Rf 0.19. IR (KBr): 3700–2200, 3029,
2956, 2869, 1712, 1352, 1330, 1213, 1179, 752,
697 cmꢁ1 1H NMR (CDCl3): d 7.65-7.26 (14H, m,
.
Ph00 and biphenyl00 protons), 5.77 (1H, m, 2-methine
proton), 2.17 (2H, m, 3-methylene protons), 1.60
(1H, m, 4-methine proton), 1.00–0.90 (6H, m, 4,4-di-
methyl protons). ESI-MS (negative): m/z 486

C. Xing et al. / Bioorg. Med. Chem. 15 (2007) 2167–2176
2175
14. Lee, H. W.; Lee, S.-S.; Lee, S. J.; Um, H.-D. Cancer Res.
2003, 63, 1093.
method MB1, minor isomer tR = 17.983 min (9.07%) and
major isomer tR = 19.017 min (90.93%).
15. Wilson, J. W.; Nostro, M. C.; Balzi, M.; Faraoni, P.;
Cianchi, F.; Becciolini, A.; Potten, C. S. Br. J. Cancer
2000, 82, 178.
16. Hartmann, K.; Artuc, M.; Baldus, S. E.; Zirbes, T. K.;
Hermes, B.; Thiele, J.; Mekori, Y. A.; Henz, B. M. Am.
J. Pathol. 2003, 163, 819.
4.22. 2-Propenyl 2-[5-biphenylmethylidene-4-oxo-2-thioxo-
thiazolidin-3-yl]-3- phenylpropanoate (3o)
Yield: 83%. Yellow oil. TLC (hexane/CH2Cl2 = 6:4), Rf
1
0.20. H NMR (CDCl3): d 7.64 and 7.60 (4H, d and d,
´
17. Hamner, S.; Skoglo¨sa, Y.; Lindholm, D. Neuroscience
1999, 91, 673.
J = 8.1 Hz and J = 8.1 Hz, (C6H4C6H5)00 protons), 7.62
(1H, s, 50-dene proton),7.45 (5H, m, (C6H4C6H5)00 pro-
tons), 7.30 (5H, m, 3-Ph protons), 5.93 (1H, m,
CH2CH@CH2 proton), 5.89 (1H, m, 2-methine proton),
5.30 and 5.25 (2H, d and d, J = 17.8 Hz and J = 10.5 Hz,
CH2CH@CH2 protons), 4.69 (2H, d, J = 5.4 Hz,
CH2CH@CH2 protons), 3.66 (2H, d, J = 7.8 Hz, 3-
methylene protons). ESI-MS (positive): m/z 486
(M++H); 442 (M++H–CO2). HRMS (C28H23NO3S2Na)
[M + Na]+: found m/z 508.1028, calcd m/z 508.1067; RP-
HPLC method MA1, minor isomer tR = 26.233 min
(15.50%) and major isomer tR = 27.083 min (84.50%);
method MB1, minor isomer tR = 19.167 min (13.53%)
and major isomer tR = 19.950 min (86.47%).
18. Sorenson, C. M. Biochim. Biophys. Acta 2004, 1644, 169.
19. Gibson, L.; Holmgreen, S. p.; Huang, D. C. S.; Bernard,
O.; Copeland, N. G.; Jekins, N. A.; Sutherland, G. R.;
Baker, E.; Adams, J. M.; Cory, S. Oncogene 1996, 13, 665.
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