BI-97C1, an optically pure apogossypol derivative as pan-active inhibitor of antiapoptotic B-cell lymphoma/Leukemia-2 (Bcl-2) family proteins

Article abstract of DOI:10.1021/jm1001265

In our continued attempts to identify novel and effective pan-Bcl-2 antagonists, we have recently reported a series of compound 2 (Apogossypol) derivatives, resulting in the chiral compound 4 (8r). We report here the synthesis and evaluation on its optically pure individual isomers. Compound 11 (BI-97C1), the most potent diastereoisomer of compound 4, inhibits the binding of BH3 peptides to Bcl-X_L, Bcl-2, Mcl-1, and Bfl-1 with IC ₅₀ values of 0.31, 0.32, 0.20, and 0.62 μM, respectively. The compound also potently inhibits cell growth of human prostate cancer, lung cancer, and lymphoma cell lines with EC₅₀ values of 0.13, 0.56, and 0.049 μM, respectively, and shows little cytotoxicity against bax ^-/-bak^-/- cells. Compound 11 displays in vivo efficacy in transgenic mice models and also demonstrated superior single-agent antitumor efficacy in a prostate cancer mouse xenograft model. Therefore, compound 11 represents a potential drug lead for the development of novel apoptosis-based therapies against cancer.

Full text of DOI:10.1021/jm1001265

4166 J. Med. Chem. 2010, 53, 4166–4176
DOI: 10.1021/jm1001265
BI-97C1, an Optically Pure Apogossypol Derivative as Pan-Active Inhibitor of Antiapoptotic
B-Cell Lymphoma/Leukemia-2 (Bcl-2) Family Proteins
Jun Wei,† John L. Stebbins,† Shinichi Kitada,† Rupesh Dash,‡ William Placzek,† Michele F. Rega,† Bainan Wu,†
Jason Cellitti,† Dayong Zhai,† Li Yang,† Russell Dahl,† Paul B. Fisher,‡ John C. Reed,† and Maurizio Pellecchia*,†
†Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, and ‡Department of Human and
Molecular Genetics, VCU Institute of Molecular Medicine, VCU Massey Cancer Center, Virginia Commonwealth University, School of Medicine,
Richmond, Virginia 23298-0678
Received January 29, 2010
In our continued attempts to identify novel and effective pan-Bcl-2 antagonists, we have recently
reported a series of compound 2 (Apogossypol) derivatives, resulting in the chiral compound 4 (8r).
We report here the synthesis and evaluation on its optically pure individual isomers. Compound 11
(BI-97C1), the most potent diastereoisomer of compound 4, inhibits the binding of BH3 peptides to Bcl-XL,
Bcl-2, Mcl-1, and Bfl-1 with IC50 values of 0.31, 0.32, 0.20, and 0.62 μM, respectively. The compound also
potently inhibits cell growth of human prostate cancer, lung cancer, and lymphoma cell lines with EC50
values of 0.13, 0.56, and 0.049 μM, respectively, and shows little cytotoxicity against bax-/-bak-/- cells.
Compound 11 displays in vivo efficacy in transgenic mice models and also demonstrated superior single-
agent antitumor efficacy in a prostate cancer mouse xenograft model. Therefore, compound 11 represents a
potential drug lead for the development of novel apoptosis-based therapies against cancer.
Introduction
pro-apoptotic members.5 Thus, molecules that mimic the
BH3 domain of pro-apoptotic proteins may be effective in
either inducing apoptosis and/or in abrogating the ability of
antiapoptotic Bcl-2 proteins to inhibit cancer cell death.
We and others have reported that the natural product 1
(Gossypol) (Figure 1A) is a potent inhibitor of Bcl-2, Bcl-XL,
and Mcl-1, functioning as a BH3 mimic.11-15 The (-) atropi-
somer of compound 1 is currently in phase II clinical trials
(AT101), displaying single-agent antitumor activity in patients
with advanced malignancies.13-15 Given that compound 1
may have off-target effects, likely due to two reactive aldehyde
groups, we designed compound 2 (Apogossypol) (Figure 1A),
a molecule that lacks these aldehydes but retains activity
against antiapoptotic Bcl-2 family proteins in vitro.16 Re-
cently, we further compared the efficacy and toxicity in mice
of compounds 1 and 2. Our preclinical in vivo data show that
compound 2 has superior efficacy and markedly decreased
toxicity compared to 1.17 We also evaluated the single-dose
pharmacokinetic characteristics of compound 2 in mice. Com-
pound 2 displayed superior blood concentrations over time
compared to compound 1 due to slower clearance.18 These
observations indicate that compound 2 is a promising lead
compound for further development of cancer therapies.
Recently, we reported the separation and characterization
of atropisomers of compound 2.19 These studies revealed that
the racemiccompound 2 isaseffectiveasitsindividual isomers
in vitro.19 We further reported the synthesis and evaluation of
5,50 amide and ketone substituted compound 2 derivatives.20,21
Among these derivatives, compounds 3 (BI-79D10) and 4
(8r)20,21 displayed improved in vitro and in vivo efficacy
compared to compound 2 (Figure 1A,B). However, compound
4 has three centers of chirality and is a mixture of diastere-
isomers (Figure 1B). Chirality has a significant effect on the
Programmed cell-death (apoptosis) plays critical roles in
the maintenance of normal tissue homeostasis, ensuring a
proper balance of cell production and cell loss.1,2 Defects in
the regulation of programmed cell death promote tumorgen-
esis and also contribute significantly to chemoresistance.3,4
B-cell lymphoma/leukemia-2 (Bcl-2a) family proteins are
central regulators of this process.5-7 To date, six antiapopto-
tic members of the Bcl-2 family have been identified and
characterized, including Bcl-2, Bcl-XL, Mcl-1, Bfl-1, Bcl-W,
and Bcl-B. Given that overexpression of antiapoptotic Bcl-2
family proteins occurs in many human cancers and leukemias,
these proteins are very attractive targets for the development
of novel anticancer agents.8-10 Members of the Bcl-2 family
proteinsalso include pro-apoptoticeffectorssuchasBak, Bax,
Bad, Bim, and Bidthatare antagonizedbyantiapoptotic Bcl-2
family proteins via heterodimerization3 involving a hydro-
phobic crevice on the surface of antiapoptotic Bcl-2 family
proteins and the R helix BH3 dimerization domain of
*To whom correspondence should be addressed. Phone: (858)
6463159. Fax: (858) 7955225. E-mail: mpellecchia@burnham.org.
a Abbreviations: Bcl-2, B-cell lymphoma/leukemia-2; EDCI, 1-ethyl-
3-(30-dimethylaminopropyl)carbodiimide; 1D-1H NMR, one-dimen-
sional titration calorimetry; WT, wild type; MEF, mouse embryonic
fibroblast cells; DKO, Bax/Bak double knockout; MEF/DKO, Bax/Bak
double knockout mouse embryonic fibroblast cells; ACN, acetonitrile;
LC-MS, liquid chromatography and tandem mass spectrometry; HPLC,
high-performance liquid chromatography; TROSY, transverse relaxa-
tion-optimized spectroscopy; ADME, absorption, distribution, meta-
bolism, and excretion; DMSO, dimethyl sulphoxide; PAMPA, parallel
artificial membrane permeation assay; FITC, fluorescein isothiocya-
nate; GST, glutathione-S-transferase; PBS, phosphate-buffered saline;
SE, standard error; PI, propidium iodide; NADPH, nicotinamide
adenine dinucleotide phosphate; rpm, rotations per min.
r
pubs.acs.org/jmc
Published on Web 05/05/2010
2010 American Chemical Society

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4167
Figure 1. (A) Structure of compounds 1, 2, and 3. (B) Structure of compound 4 (8r). (C) NMR binding studies. Aliphatic region of the 1H
NMR spectrum of Bcl-XL (15 μM, black) and Bcl-XL in the presence of compound 11 (150 μM, red), 12 (150 μM, green), 13 (150 μM, gray), and
14 (150 μM, blue). (D) Superpositionof2D[15N,1H]-TROSY spectra of free Bcl-XL (200μM; red) and after addition of compound 11 (200μM; blue).
behavior of compounds in vitro and in vivo, partially because
different enantiomers and diastereoisomers have different phy-
sical, chemical, and pharmacology properties. In principle,
different enantiomers or diastereoisomers should be treated
as different compounds. Indeed, the (-) atropisomer of com-
pound 1 displayed a markedly differential activity compared to
its natural racemic mixture. On the basis of these premises, in
this current work, we focus our attention on preparing four
optically pure isomers (11-14) of compound 4 (Scheme 1),
followed by further investigation of their in vitro and in vivo
activities.
isomers (R, -, R)-11 and (R, þ, R)-12 (Scheme 1). The atrop-
isomers (S, (, S)-10 were similarly resolved to afford the other
two optically pure isomers (S, -, S)-13 and (S, þ, S)-14
(Scheme 1). The optical configuration and purity of each
atropisomer was determined using a polorimeter and liquid
chiral column chromatography (Table 1 and Supporting In-
formation Figure 1). The optical rotation ([R]) generated by
atropisomer (axial chirality) and 2-phenyl propyl groups in
compound 4 was approximately (18.5° and 33.5°, respectively
(Table 1).
Four pure diastereoisomers, namely compounds 11, 12, 13,
and 14, were first tested by one-dimensional 1H nuclear mag-
netic resonance spectroscopy (1D-1H NMR) binding assays
against Bcl-XL, as we reported previously (Figure 1C).24 Com-
pounds 11-14 displayed significant binding to Bcl-XL in these
assays (Figure 1C). However, compared to other diastereo-
isomers, compound 11 induced larger chemical shift perturba-
tions in the active site methyl groups (region between -0.38
and 0.42 ppm) in the one-dimensional 1H NMR spectrum of
Bcl-XL (Figure 1C). To confirm the result from the one-
Results and Discussion
Wehad recently reported thatcompound 4 was a promising
inhibitor of Bcl-XL and Bcl-2 with improved in vitro and in
vivo efficacy compared to compound 2.20 However, com-
pound 4 was not optically pure, having two centers of chirality
generated from 2-phenyl propyl groups (Figure 1B). In addi-
tion, compound 4 displayed axial chirality due to restricted
rotation around the binaphthyl bond (Figure 1B). Therefore,
it was attractive to explore whether optically pure isomers of
compound 4 presented different in vitro and in vivo activities.
A synthetic route (Scheme 1) was developed to prepare
optically pure isomers. Synthesis of atropisomers (()-2, (()-
5, and (()-6 had been previously reported.20 The racemic
carboxylic acid (()-6 was then coupled with optically pure
chiral amines, (R)-β-methylphenethylamine and (S)-β-methyl-
phenethylamine, respectively, in the presence of 1-ethyl-3-(30-
dimethylaminopropyl)carbodiimide (EDCI) at room tempera-
ture to give atropoisomers (R, (, R)-7 and (S, (, S)-8,
respectively.22 Subsequent demethylation of the compound
(R, (, R)-7 and (S, (, S)-8 using boron tribromide afforded
atropisomers (R, (, R)-9 and (S, (, S)-10, respectively.23 The
atropisomers (R, (, R)-9 were readily resolved using a liquid
chiral column chromatography to afford two optically pure
1
dimensional H NMR binding assay, we also produced uni-
formly 15N-labeled Bcl-XL and measured 2D [15N,1H]-TROSY
correlation spectra in the absence and presence of compound 11
(Figure 1D). Compound 11 displayed low to submicromolar
binding affinity to Bcl-XL, as qualitatively evaluated by the
nature of the shifts at the ligand/protein ratio of 1:1. To further
confirm these results, we evaluated the binding affinity of
four pure isomers 11-14 using isothermal titration calorimetry
assay (ITC), competitive fluorescence polarization assays,
(FPA), and cell viability assays (Figure 2, Tables 2 and 3). In
agreement with NMR binding assays, compound 11 displayed
tight binding affinity to Bcl-XL, with a Kd value of 0.11 μM in
the ITC assay, which is 4-20 times more potent than other
isomers 12-14 in the same assay (Table 2). Compound 11
also displayed the best inhibitory properties against Bcl-XL
in the FP assay compared to the other pure isomers 12-14

4168 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Wei et al.
Scheme 1a
a Reagents and conditions: (a) NaOH, H2O, reflux; (b) H2SO4; (c) DMS, K2CO3; (d) TiCl4, Cl2CHOCH3, rt; (e) HCl, H2O; (f ) NaClO2, H2O2,
KH2PO4, CH3CN, rt; (g) (R)-(þ)-β-methylphenethylamine, EDCI, NH2R, HOBT, rt; (h) (S)-(-)-β-methylphenethylamine, EDCI, NH2R, HOBT, rt;
(i) BBr3, CH2Cl2; ( j) HCl, H2O; (k) chiral column chromatography separation.
and isomer mixture 4, with an IC50 value of 0.31 μM
(Figure 2A and Table 2). Compound 11 also displayed
superior cell membrane permeability compared to other com-
pounds (12-14) (Table 4). Consistent with NMR binding,
ITC, FPA, and cell permeability data, compound 11 was
more effective compared to other compounds (4, 12-14) in
inhibiting growth of PC3 cells, which expressed high levels
of Bcl-XL. The EC50 value of 11 in killing PC3 cells was

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4169
Table 1. Optical Activity and Chiral HPLC Purity of Four Diastereoisomers of Compound 4
a Optical activity [R]22/D þ 35.0°, C = 1 in ethanol, commercially available from Sigma-Aldrich. b Optical activity [R]22/D - 35.0°, C = 1 in ethanol,
commercially available from Sigma-Aldrich.
0.13 μM, hence 4-36-fold more potent than other com-
pounds (4 and 12-14) (Figure 2B and Table 3). Overall,
considering both in vitro binding and displacement assays
and cell killing properties, we concluded that anti-Bcl-XL
properties of optically pure isomers 11-14 were affected
largely by the atropisomers, with the (-) isomers being
consistently more potent than the corresponding (þ)
isomers. This was in agreement with previous observations
that lead to the selection of (-)-1 (Gossypol, AT101) for
clinical trials.13 In fact, atropisomers (-)-1 and (þ)-1 bound
to Bcl-XL with IC50 values of 0.48 and 0.54 μM, respec-
tively, in FPA assays, while their EC50 values in killing PC3
cells were 3.3 and 17.8 μM, respectively.13 Compound 11
was more potent compared to (-) 1 in both assays.
To further rationalize these observations, molecular dock-
ing studies of diastereoisomers 11-14 (Figure 3) into the BH3
binding groove in Bcl-XL were performed. These studies
suggested that although the left half components of (-) and
(þ) atropisomers bound similarly to Bcl-XL, their right
parts had remarkably different binding models (Figure 3).
(-) Atropisomers 11 and 13 not only fully occupied the right
hydrophobic pockets (P2) but also formed hydrogen bonding
involving their 10 hydroxyl groups on the right naphthalene
ring (Figure 3A). On the contrary, (þ) atropisomers 12 and 14
could not occupy the right hydrophobic pocket (P2) or form a
hydrogen bond with their 10 hydroxyl groups. The GOLD
score of (-) atropisomer 11 was 39.03, which was greater than
34.16 for (þ) atropisomer 12. In agreement with FPA and cell
data for Bcl-XL, molecular docking studies further suggested
that the two centers of chirality generated from 2-phenyl
propyl groups had little effect for isomers 11-14 to bind to
Bcl-XL. For example, compound 11 and 13 with reverse
configurations on 2-phenyl propyl groups had very similar
orientations in Bcl-XL (Figure 3A,C) and their GOLD score
were very similar, with values of 39.03 and 39.78, respectively.
This binding trend was also observed for compound 12 and 14
(Figure 3B,D), with similar gold score of 34.16 and 34.49,
respectively. This was not unexpected given that these isomers
only differ for the arrangement of a small hydrogen atom and
methyl group on the chiral carbon on 2-phenyl propyl group.
On the basis of these observations, we decided not to explore
the asymmetric synthesis of the (R, -, S) isomer but rather to
focus our effects in the characterization of the (R, -, R)
isomer, compound 11, as reported below.
In addition to Bcl-XL, other members of the Bcl-2 family
were known to play critical roles in cancer cell survival.25,26
Therefore, we further evaluated the binding properties and
specificity of isomers 11-14 against Bcl-2, Mcl-1, and Bfl-1
using FP assays (Table 2 and Figure 2C). All four isomers
(11-14) displayed significant displacement properties against
Bcl-2, Mcl-1, and Bfl-1 in FP assays with average IC50 values
of 0.32, 0.22, and 0.65 μM, respectively (Table 2). To confirm
the result from FP assays, we also produced uniformly 15N-
labeled Mcl-1 protein and measured 2D [15N,1H]-TROSY
correlation spectra in the absence and presence of compound
11 (Supporting Information Figure 2A). Compound 11 dis-
played a significant binding to Mcl-1, as qualitatively evalu-
ated by the nature of the shifts at the ligand/protein ratio of
2:1. Compound 11 showed inhibitory properties against Bcl-2
compared to other compounds (4, 12-14), with an IC50 value

4170 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Wei et al.
Figure 2. (A) Fluorescence polarization-based competitive binding curves of compound 11 for Bcl-XL (red squares), Bcl-2 (blue dots), Bfl-1
(black up triangle), and Mcl-1 (green up triangle). (B) Inhibition of cell growth by compound 4 (red dots), 11 (blue dots), 12 (green dots), 13
(yellow dots), and 14 (orange dots) in the PC-3 human prostate cancer cell line. Cells were treated for 3 days, and cell viability was evaluated
using ATP-LITE assay. (C) Inhibition of cell growth by compound 4 (deep-blue dots), 11 (red square), 12 (green down triangle), 13 (light-blue
diamonds), and 14 (gray up triangle) in the H460 human lung cancer cell line. Cells were treated for 3 days, and cell viability was evaluated using
ATP-LITE assay. (D) Inhibition of cell growth by compound 11 (black square) in the human BP3 cell line. Apoptosis was monitored by
Annexin V-FITC assays.
Table 2. Cross-Activity of Diastereoisomers of 4 against Bcl-XL, Bcl-2, Mcl-1, and Bfl-1 in Fluorescence Polarization Displacement Assays and Binding
to Bcl-xL as Measured via Isothermal Titration Calorimetrya
IC50 ( μM) FPA
Kd ( μM) ITC
compd
Bcl-XL, IC50
Bcl-2, IC50
Bfl-1, IC50
Mcl-1, IC50
Bcl-XL
11
12
13
14
4
0.31 ( 0.05
0.68 ( 0.09
0.44 ( 0.02
0.65 ( 0.06
0.39 ( 0.02
0.32 ( 0.05
1.14 ( 0.09
0.43 ( 0.01
0.43 ( 0.03
0.62 ( 0.04
0.59 ( 0.07
0.75 ( 0.07
0.77 ( 0.05
0.42 ( 0.09
0.20 ( 0.02
0.18 ( 0.02
0.24 ( 0.02
0.24 ( 0.02
0.33 ( 0.02
0.1
2.4
0.4
2.0
ND
0.66 ( 0.02
a ND = Not determined.
Table 3. Efficacy (EC50 Values in μM) of Diastereoisomers of Com-
pound 4 against Prostate Cancer Cells (PC3), Lung Cancer Cells (H460)
and Lymphoma Cell (BP3)
Table 4. Plasma Stability, Microsomal Stability, and Cell Membrane
Permeability of Diastereoisomers 11-14
plasma stability
(T = 1 h), %
microsomal stability
(T = 40 min), %
cell permeability
(LogPe)
compd
PC3aEC50, μM
H460 aEC50, μM
BP3 bEC50, μM
compd
11
12
13
14
4
0.13 ( 0.02
4.64 ( 1.07
0.47 ( 0.10
1.61 ( 0.45
2.45 ( 0.50
0.42 ( 0.09
0.78 ( 0.10
0.56 ( 0.08
0.80 ( 0.09
0.47 ( 0.18
0.049
0.072
0.31
11
12
13
14
41
28
42
41
89.6 ( 7.0
83.4 ( 8.1
86.3 ( 7.0
91.6 ( 1.6
-6.70 ( 0.30
-7.69 ( 0.14
-7.78 ( 0.06
-7.85 ( 0.13
0.12
0.61
a Determined using the ATP-LITE assay. b Determined using Annexin
V-FITC and propidium iodide assay.
Figure 2C). These observations were consistent with atrop-
isomers (-)-1 and (þ)-1, which bound to Bcl-2 with an IC50
value of 0.26 and 0.30 μM, respectively, in FPA assays and
their EC50 value in killing MDA-MB-231 breast cancer cells
was around 2.0 and 10.0 μM, respectively.13 Compound 11
had similar binding affinity as other isomers (12-14) for
Mcl-1 and Bfl-1 in FP assays, which could be due to structural
difference of their BH3 binding pockets (Supporting Informa-
tion Figure 2B,C)30,31
of 0.32 μM in FP assays. Consistent with FPA data, com-
pound 11 displayed best efficacies compared to other com-
pounds (4, 12-14) in inhibiting growth of H460 cells, which
expressed high level of Bcl-2.27-29 The IC50 values of 11 in
killing H460 cells was 0.42 μM, hence approximately 2-fold
more potent than compounds 12 and 14 (Tables 2, 3 and

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4171
Figure 3. Molecular docking studies. Stereo views of docked structures of (A) compound 11. (B) Compound 12. (C) Compound 13. (D)
Compound 14 into Bcl-XL (PDB ID: 2YXJ).
Figure 4. Mouse embryonic fibroblast cells with wild-type (MEF/WT; dark square) or bax-/-bak-/- double knockout (MEF/DKO, red
square) genotypes were treated with compounds 11, 14, and 4 at various concentrations and apoptosis was monitored by Annexin V-FITC
assays. (A-C) Compounds 11, 14, and 4. (D) Effects of compounds 11-14 on shrinkage of Bcl-2 mouse spleen at a single intraperitoneal
injection dose of 42 mg/kg. All shrinkage data are percentage of maximum reduction of mice spleen size.
We further evaluated the ability of compounds 11-14 to
induce apoptosis of the human BP3 cell line, which originated
from a human diffuse large B-cell lymphoma (DLBCL).20,26
For these assays, we used Annexin V-FITC and propidium
iodide (PI) double staining, followed by flow-cytometry ana-
lysis (Table 3). Compounds 11-14 effectively induced apoptosis
of the BP3 cell line in a dose-dependent manner (Figure 2D,
Table 3 and Supporting Information Figure 3A,B). In parti-
cular, compound 11 was most effective, with an IC50 value of
0.049 μM, which was approximately 2- to 6-fold more potent
than other diastereoisomers 12-14 (Figure 2D, Table 3 and
Supplementary Figure 3A,B). The mRNA ratio of Bfl-1,
Bcl-XL, and Mcl-1 was approximately 10:3:1 in BP3 cell lines.26
However, we determined by Western Blot that BP3 cells

4172 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Wei et al.
Figure 5. Characterization of compounds 4 and 11 in tumor xenografts model. Tumor xenografts from M2182 cells were established in
athymic nude mice on the left and right flanks. After establishing visible tumors of ∼75 mm3, DMSO or compounds (4 or 11) were given (ip)
every two days (total of nine injections). A minimum of five animals was used per experimental group. For in vivo imaging of tumors, the mice
were anesthetized and injected ip with 150 mg/kg luciferin and light emitted from each tumor was determined in a xenogen system with CCD
camera using an integration time of 1 min. Luminescence measurements were made using Living Image software (version 2.50.1; Xenogen). At
the end of the experiment, the animals were sacrificed, and the tumors were removed and photographed. (A) Left flank tumor treated with
compound 4 at dose of 10 mg/kg and 25 mg/kg, respectively. (B) Right flank tumor treated with compound 4 at dose of 10 mg/kg and 25 mg/kg,
respectively. (C) Left flank tumor treated with compound 11 at dose of 1, 3, and 5 mg/kg, respectively. (D) Right flank tumor treated with
compound 11 at doses of 1, 3, and 5 mg/kg, respectively.
expressed high levels of both Bfl-1 and Mcl-1.20 In agreement
with these observations, the potent dual Bcl-XL and Bcl-2
antagonist ABT-73732 displayed no cytotoxic activity against
BP3 cell lines, presumably because ABT737 was not effective
against Mcl-1 and Bfl-1.25,32,33
permeability compared to other diastereoisomers 12-14. The
LogPe value of compound 11 was -6.7, which indicates good
cell membrane permeability, while LogPe values of other
compounds (12-14) were around -7.8, which corresponded
to relative poor cell membrane permeability. Compound 11
also displayed relatively good microsomal stability (Table 4)
in which the compound degraded 10.4% after 40 min incuba-
tion in rat microsomal preparations. In contrast, compound
12 displayed decreased plasma and microsomal stability
compared to other diastereoisomers. Compound 11 also
displayed better chemical stability compared to other com-
pounds (12-14) at different temperatures (Supporting Infor-
mation Figure 5)
Hence, using a combination of NMR-based binding assays,
FP assays, ITC assays, cytotoxicity assays, and preliminary in
vitro ADME data, we selected pan-Bcl-2 antagonists to be
further tested in vivo models. Unlike currently available
antagonists,32,36 our compounds were effective in inhibiting
several of the antiapoptotic Bcl-2 proteins and hence were
expected to display in vivo efficacy against a variety of mice
models of cancer that relied on different Bcl-2 proteins for
growth and progression. To test this hypothesis, we selected
two different models: Bcl-2 transgenic mouse model and
prostate cancer xenograft model that relied on Mcl-1 over-
expression. B-cells of the B6 transgenic mice overexpressed
human Bcl-2 and accumulated in the spleen of mice.17,20
Because we had determined that the spleen weight was highly
consistent in age- and sex-matched Bcl-2-transgenic mice,
varying by only (2% among control Bcl-2 mice,17 the spleen
weight was used as an end-point for assessing in vivo activity.
We tested the in vivo activities of isomers 11-14 in two Bcl-2
We next explored whether pure diastereoisomers 11-14
and diastereoisomer mixture 4 had cytotoxic properties
against wild type mouse embryonic fibroblast cells (MEF/
WT) and transformed Bax/Bak double knockout MEF cells
(MEF/DKO) in which antiapoptotic Bcl-2 family proteins
lacked a cytoprotective phenotype.34,35 Compound 11 dis-
played slight toxicity in Bak/Bak double knockout mouse
embryonic fibroblast cells (MEF/DKO) at 30 μM, while it
killed almost 70% wild type mouse embryonic fibroblast cells
(MEF/WT) at the same concentration using FITC-Annexin
V/PI assays (Figure 4A), implying that the compound 11 only
displayed slight off-target effects. In contrast, compound 14
seemed almost equally effective in killing both MEF/WT and
MEF/DKO at 30 μM (Figure 4B), suggesting that other
possible killing mechanisms not related to Bcl-2 inhibition
were induced by this compound. Accordingly, the mixture of
isomers 4 displayed higher cytotoxicity in MEF/DKO cells at
3-30 μM comparedto the optically pure isomer 11, indicating
that the optically pure isomer was more selective (Figure 4C).
Next, we examined the pharmacological properties of the
isomers, as chirality could greatly affect such properties due to
stereoselective metabolism. To test the pharmacological prop-
erties of diastereoisomers 11-14, we determined their in vitro
rat plasma stability, rat microsomal stability, and cell mem-
brane permeability (Table 4). From these studies, we could
conclude that compound 11 displayed superior cell membrane

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4173
transgenic mice with a single intraperitoneal (ip) injection at
42 mg/kg. In agreement with in vitro data, compound 11
displayed superior in vivo activity compared to other isomers
(12-14) in this model. It induced more than 30% spleen
weight reduction compared to e20% induced by other dia-
stereoisomers. Because the maximum spleen shrinkage would
be no more than 50% in this experimental model,20 these
compounds induced near 65% maximal biological activity,
while other isomers 12-14 induced e40% of maximum
reduction in spleen weight at the same dose. In particular,
compound 12 displayed weak in vivo activity, which was
inconsistent with its relatively weak cell activity and poor
pharmacological properties. All mice tolerated the treatment
well, with only mild signs of GI toxicity.
against bax-/-bak-/- cells compared to compound 4, indicat-
ing that it kills cancers cells predominantly via the intended
mechanism. Compound 11 displays in vivo efficacy in trans-
genic mice in which Bcl-2 is overexpressed in splenic B-cells
and also demonstrates superior single-agent antitumor effi-
cacy compared to compound 4 in a prostate cancer mouse
xenograft model that depends on Mcl-1 for survival. Con-
sidering the critical roles of antiapoptotic Bcl-2 family pro-
teins in tumorgenesis, chemoresistance, and the potent
inhibitory activity of compound 11 against antiapoptotic
Bcl-2 family proteins, compound 11 represents a viable drug
candidate for the development of novel apoptosis-based
cancer therapies.
As mentioned, current available experimental treatments
targeting Bcl-2 proteins failed to address Mcl-1 as a critical
regulator of cancer survival. In fact, the potent Bcl-XL/Bcl-2
antagonist ABT-737 (Abbott Laboratories) and the Bcl-2
antisense Genasense (Genta) were not effective against cancer
cells that overexpress Mcl-1.32,36 Therefore, to further exam-
ine the therapeutic potential of compound 11 as a single agent
against tumors thatrelied on Mcl-1for survival, compound11
was evaluated side by side with compound 4 in a prostate
cancer xenograft using the M2182 cell line. M2182 was a
tumorigenic variant of normal prostate epithelial P69 cell and
highly overexpressed Mcl-1.37-40 A quantity of 1 ꢀ 106 M2182
cells were injected subcutaneously in the left and right flanks of
male athymic nude mice, and the tumors were allowed to grow
to an average size of ≈75 mm3 prior to initiation of therapy.
Compounds 4 and 11 were administrated (ip) every 2 days
(total of nine injections), and compound 4 was injected at two
doses, 10 and 25 mg/kg while compound 11 was administrated
at three lower doses, 1, 3, and 5 mg/kg due to its superior in
vitro properties compared to compound 4. Compounds 11 and
4 displayed a marked inhibitory effect of tumor size compared
with the control group (Figure 5 and Supporting Information
Figure 5A,B). In fact, compound 11 at the dose of 5 mg/kg
(Figure 5C,D, Supporting Information Figure 5A,B) and
compound 4 at the dose of 25 mg/kg (Figure 5A,B) induced
near complete inhibition of tumor growth in both flanks
compared with their control groups. As anticipated, com-
pound 11 displayed better tumor growth inhibitory effect
compared to compound 4. Even at the dose of 3 mg/kg,
compound 11 inhibited tumor growth to ∼60% of the tumor
volume in the control group (Figure 5C,D, Supporting In-
formation Figure 5A,B) while compound 4 displayed weak
inhibitory effect of tumor size at the dose of 10 mg/kg
(Figure 5A,B). All mice tolerated the treatment well with no
apparent signs of toxicity.
Experimental Section
General Synthetic Procedures. Unless otherwise indicated, all
reagents and anhydrous solvents (CH2Cl2, THF, diethyl ether,
etc.) were obtained from commercial sources and used without
purification. All reactions were performed in oven-dried glass-
ware. All reactions involving air or moisture sensitive reagents
were performed under a nitrogen atmosphere. Silica gel or
reverse phase chromatography was performed using prepacked
silica gel or C-18 cartridges (RediSep), respectively. All final
compounds were purified to >95% purity, as determined by a
HPLC Breeze from Waters Co. using an Atlantis T3 3 μM
4.6 mm ꢀ 150 mm reverse phase column. Compounds 11-14 were
isolated using a CHIRALCEL OD-RH 5 μM 250 mm ꢀ 10 mm
reverse phase chiral column, and the enantiomeric purity of
compounds 11-14 was analyzed using a CHIRALCEL OD-RH
5 μM 250 mm ꢀ 4.6 mm reverse phase chiral column on a HPLC
from Water Corp. The eluant was a linear gradient with a flow rate
of 5 mL/min for preparative and 1 mL/min for analytical column,
respectively, from 60% A and 40% B to 20% A and 80% B in 15
minfollowedby5minat100% B(solventA:H2O with 0.1% TFA;
solvent B: ACN with 0.1% TFA). Compounds were detected at
λ = 254 nm. 1H NMR spectra were recorded on Bruker 600 MHz
instruments. Chemical shifts are reported in ppm (δ) relative to 1H
(Me4Si at 0.00 ppm). Coupling constants (J ) are reported in Hz
throughout. Mass spectral data were acquired on Shimadzu
LCMS-2010EV for low resolution and on an Agilent ESI-TOF
for high resolution.
Compound 1 is commercially available from Yixin Pharma-
ceutical Co. HPLC purity 99.0%, tR = 12.50 min and synthesis
of compounds 2, 4, 5, and 6 have been previously reported.20
5
5
1,10,6,60,7,70-Hexamethoxy-3,30-dimethyl-N ,N -bis(2-phenyl-
propyl)-2,20-binaphthyl-5,50-dicarboxamide (7). Compound 6 (1.35 g,
2.45 mmol), EDCI (1.30 g, 6.76 mmol), and HOBT (910 mg,
6.76 mmol) were dissolved in 30 mL of dry CH2Cl2 and stirred at
room temperature for 15 min under nitrogen atmosphere. (R)-β-
Methylphenethylamine (0.81 mL, 5.63 mmol) and N,N-diisopro-
pylethylamine (1.7 mL, 9.8 mmol) were added, and the reaction
mixture was stirred at room temperature for 20 h. The mixture was
then poured onto 100 mL of water and the solution was extracted
with CH2Cl2 (3 ꢀ 100 mL). The ether extracts were washed with
water and brine, dried over magnesium sulfate, and filtered.
Evaporation of the solvent in vacuo and the residue was purified
by silica chromatography to give 1.46 g (76%) of compound 7 as a
yellow solid. 1H NMR (600 MHz, CD3OD) δ 7.36 (s, 2H), 7.33 (d,
J = 6.6 Hz, 4H), 7.32 (t, J1 = 7.2 Hz, J2 = 6.6 Hz, 4H), 7.21 (m,
4H), 4.59(s, 4H), 3.98 (s, 6H), 3.85 (s, 6H), 3.76 (m, 2H), 3.63 (m,
2H), 3.54 (s, 3H), 3.53 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H), 1.38 (d,
J1 = 6.0 Hz, 6H). HRMS calcd for C48H52N2O8 785.3796 (M þ H),
found 785.3790.
0
Conclusions
In summary, we synthesized and evaluated four diastereoi-
somers (11-14) of compound 4 in a variety of in vitro and in
vivo assays. The optically pure compound 11 inhibits the
binding of BH3 peptides to Bcl-XL, Bcl-2, Mcl-1, and Bfl-1
with IC50 values of 0.31, 0.32, 0.20, and 0.62 μM, respectively.
The compound 11 also potently inhibits cell growth of human
prostate cancer, lung cancer, and BP3 B-cell lymphoma cell
lines with EC50 values of 0.13, 0.56, and 0.049 μM, respec-
tively. Compound 11 displays approximately 20-fold and 12-
fold greater efficacy in inhibiting growth of PC-3 and BP3 cell,
respectively, compared to the compound 4, which was pre-
viously disclosed.20 Compound 11 also shows less cytotoxicity
50
1,10,6,60,7,70-Hexamethoxy-3,30-dimethyl-N5,N -bis(2-phenyl-
propyl)-2,20-binaphthyl-5,50-dicarboxamide (8). Compound 6 (1.0 g,
1.81 mmol), EDCI (960 mg, 5.0 mmol), and HOBT (181 mg,
1.34 mmol) were dissolved in 25 mL of dry CH2Cl2 and stirred
at room temperature for 10 min under nitrogen atmosphere.

4174 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Wei et al.
(S)-β-Methylphenethylamine (0.60 mL, 4.17 mmol) and N,N-
diisopropylethylamine (1.26 mL, 7.3 mmol) were added, and the
reaction mixture was stirred at room temperature for 24 h. The
mixture was then poured onto 50 mL of water and the solution was
extracted with CH2Cl2 (3 ꢀ 100 mL). The ether extracts were
washed with water and brine, dried over magnesium sulfate, and
filtered. Evaporation of the solvent in vacuo and the residue was
purified by silica chromatography to give 0.87 g (60%) of
compound 8 as a yellow solid. 1H NMR (600 MHz, CD3OD) δ
(600 MHz, CD3OD) δ 7.48 (s, 2H), 7.36 (d, J = 6.0 Hz, 4H), 7.32
(t, J1 = 7.2 Hz, J2 = 7.2 Hz, 4H), 7.22 (m, 4H), 4.59 (s, 4H), 3.98
(s, 6H), 3.84 (s, 6H), 3.76 (m, 2H), 3.63 (m, 2H), 3.54 (s, 3H), 3.53
(s, 3H), 2.01 (s, 3H), 1.99 (s, 3H), 1.38 (d, J1 = 6.6 Hz, 6H).
HRMS calcd for C48H52N2O8 785.3796 (M þ H), found
785.3788.
MgSO4. The solvent was concentrated in vacuo, and the residue
was purified using C-18 column chromatography (H2O/acet-
onitrile) to give 200 mg of compound 10 (72%) as white-yellow
solid. 1H NMR (600 MHz, CD3OD) δ 7.56 (d, J = 6.6 Hz, 2H),
7.39 (t, J1 = 6.0 Hz, J2 = 3.0 Hz, 4H), 7.35 (t, J1 = 6.0 Hz, J2 =
6.6 Hz, 4H), 7.23 (t, J1 = 6.0 Hz, J2 = 6.6 Hz, 2H), 7.06 (s, 1H),
6.99 (s, 1H), 3.76 (dd, J1 = 6.6 Hz, J2 = 11.4 Hz, 1H), 3.68 (m,
3H), 3.22 (m, 2H), 1.90 (s, 3H), 1.89 (s, 3H), 1.43 (m, 6H). HRMS
calcd for C42H40N2O8 701.2857 (M þ H), found 701.2853.
Compound 10 (150 mg) was further purified using a CHIRAL-
CEL OD-RH 5 μM 250 mm ꢀ 10 mm reverse phase chiral column
to give 50 mg of compound 13 and 58 mg of compound 14,
respectively.
(S)-1,10,6,60,7,70-Hexahydroxy-3,30-dimethyl-N5-((S)-2-phenyl-
50
propyl)-N -((S)-2-phenylpropyl)-2,20-binaphthyl-5,50-dicarboxamide
50
1,10,6,60,7,70-Hexahydroxy-3,30-dimethyl-N5,N -bis(2-phenyl-
(13). 1H NMR (600 MHz, CD3OD) δ 7.57 (s, 2H), 7.38 (d, J = 7.2
Hz, 4H), 7.33 (t, J1 = 7.2Hz, J2 = 7.8 Hz, 4H), 7.22 (t, J1 = 7.2Hz,
J2 = 7.8 Hz, 2H), 6.99 (s, 2H), 3.75 (dd, J1 = 7.8 Hz, J2 = 13.2 Hz,
2H), 3.62 (dd, J1 = 7.8 Hz, J2 = 13.2 Hz, 2H), 3.20 (m, 2H), 1.89 (s,
6H), 1.40 (d, J = 6.6 Hz, 6H). 13C NMR (600 MHz, CD3OD)
δ 169.66, 149.17, 144.62, 144.54, 143.92, 133.85, 128.13, 127.16,
126.96, 126.11, 118.36, 116.60, 115.15, 114.34, 105.55, 46.33, 39.66,
19.35, 18.67. HPLC purity 99.4%, tR = 9.31 min. Enantiomeric
purity 99.1%, tR = 12.38 min. HRMS calcd for C42H40N2O8-
701.2857 (M þ H), found 701.2849.
propyl)-2,20-binaphthyl-5,50-dicarboxamide (9). First, 0.65 mL of
BBr3 solution (1.72 g, 6.85 mmol) was added dropwise into a
solution of compound 7 (420 mg, 0.55 mmol) in 20 mL of
anhydrous CH2Cl2 at -78 °C. Stirring was continued at -78 °C
for 1 h, 0 °C for 1 h, and ambient temperature for 1 h. 50 g of ice
containing 10 mL of 6 M HCl was added to the mixture and stirred
for 30 min at room temperature. The aqueous layer was then
extracted with ethyl acetate (3 ꢀ 60 mL). The combined organic
layer was washed with water and brine and dried over MgSO4. The
solvent was concentrated in vacuo, and the residue was purified
using C-18 column chromatography followed by preparative
HPLC (H2O/acetonitrile) to give 150 mg of compound 9 (39%)
as white-yellow solid. 1H NMR (600 MHz, CD3OD) δ 7.39 (d, J =
4.2 Hz, 2H), 7.19 (m, 4H), 7.15 (t, J1 = 6.0 Hz, J2 = 6.0 Hz, 4H),
7.03 (t, J1 =6.0Hz, J2 = 6.0 Hz, 2H), 6.89 (s, 1H), 6.81 (s, 1H), 3.49
(m, 4H), 3.02(m, 2H), 1.72(s, 3H), 1.71(s, 3H), 1.25(s, 3H), 1.24(s,
3H). HRMS calcd for C42H40N2O8 701.2857 (M þ H), found
701.2865. Compound 9 (100 mg) was further purified using a
CHIRALCEL OD-RH 5 μM 250 mm ꢀ 10 mm reverse phase
chiral column to give 25 mg of compound 11 and 28 mg of
compound 12, respectively.
(R)-1,10,6,60,7,70-Hexahydroxy-3,30-dimethyl-N5-((S)-2-phenyl-
50
propyl)-N -((S)-2-phenylpropyl)-2,20-binaphthyl-5,50-dicarboxamide
(14). 1H NMR (600 MHz, CD3OD) δ 7.57 (s, 2H), 7.37 (d, J =
7.8 Hz, 3H), 7.33 (t, J1 = 7.2 Hz, J2 = 7.8 Hz, 4H), 7.22 (t, J1 =
J2 = 7.2 Hz, 2H), 7.06 (s, 2H), 3.66 (m, 4H), 3.22 (m, 2H), 1.90 (s,
6H), 1.40 (s, J = 6.6 Hz, 6H). 13C NMR (600 MHz, CD3OD)
δ169.71, 149.16, 14.64, 144.58, 144.03, 133.84, 128.15, 127.16,
126.95, 126.09, 118.41, 116.56, 115.23, 114.41, 105.57, 46.40,
39.56, 19.34, 18.60. HPLC purity 98.6%, tR = 9.22 min. Enantio-
meric purity 99.5%, tR = 10.57 min. HRMS calcd for C42H40N2O8
701.2857 (M þ H), found 701.2852.
Molecular Modeling. Molecular modeling studies were con-
ducted on a Linux workstation and a 64 3.2 GHz CPUs Linux
cluster. Docking studies were performed using the crystal structure
of Bcl-XL and Mcl-1 in complex with a BH3 mimetic ligand (Protein
Data Bank codes 2YXJ and 2NL9, respectively).30,32,41,42 The
ligand was extracted from the protein structure and was used to
define the binding site for small molecules. Compounds 11-14 were
docked into the Bcl-2 family protein by the GOLD43 docking
program using GoldScore44 as the scoring function. The active site
(S)-1,10,6,60,7,70-Hexahydroxy-3,30-dimethyl-N5-((R)-2-phenyl-
50
propyl)-N -((R)-2-phenylpropyl)-2,20-binaphthyl-5,50-dicarboxamide
(11). 1H NMR (600 MHz, CD3OD) δ 7.57 (s, 2H), 7.37 (d, J =
7.8 Hz, 4H), 7.33 (t, J1 = 7.2 Hz, J2 = 7.8 Hz, 4H), 7.22 (t, J1 =
J2 = 7.2 Hz, 2H), 7.07 (s, 2H), 3.67 (m, 4H), 3.23 (m, 2H), 1.90 (s,
6H), 1.43 (d, J = 7.2 Hz, 6H). 13C NMR (600 MHz, CD3OD) δ
169.73, 149.20, 144.64, 143.95, 133.86, 128.15, 127.17, 126.95,
126.10, 118.38, 116.60, 115.20, 114.37, 105.57, 46.40, 39.56, 19.33,
18.58. HPLC purity 99.0%, tR = 9.13 min. Enantiomeric purity
99.7%, tR = 12.35 min. HRMS calcd for C42H40N2O8 701.2857 (M
þ H), found 701.2854.
˚
radius was set at 10 A and 10 GA solutions were generated for each
molecule. The GA docking procedure in GOLD43 allowed the small
molecules to flexibly explore the best binding conformations,
whereas the protein structure was static. The protein surface was
prepared with the program MOLCAD45 as implemented in Sybyl
(Tripos, St. Louis, MO) and was used to analyze the binding poses
for studied small molecules.
(R)-1,10,6,60,7,70-Hexahydroxy-3,30-dimethyl-N5-((R)-2-phenyl-
50
propyl)-N -((R)-2-phenylpropyl)-2,20-binaphthyl-5,50-dicarboxamide
(12). 1H NMR (600 MHz, CD3OD) δ 7.57 (s, 2H), 7.38 (d, J = 7.8
Hz, 4H), 7.33 (t, J1 = 7.2 Hz, J2 = 7.8 Hz, 4H), 7.22 (t, J1 = J2 =
7.2 Hz, 2H), 6.99 (s, 2H), 3.75 (dd, J1 = 7.2 Hz, J2 = 13.2 Hz, 2H),
3.62 (dd, J1 = 7.2 Hz, J2 = 13.2 Hz, 2H), 3.20 (m, 2H), 1.89 (s, 6H),
1.42 (d, J = 6.6 Hz, 6H). 13C NMR (600 MHz, CD3OD) δ 169.67,
149.18, 144.62, 144.54, 143.92, 133.85, 128.14, 127.16, 126.97,
126.12, 118.36, 116.59, 115.14, 114.35, 105.56, 46.34, 39.66, 19.63,
18.78. HPLC purity 99.0%, tR = 9.30 min. Enantiomeric purity
99.5%, tR = 10.28 min. HRMS calcd for C42H40N2O8 701.2857
(M þ H), found 701.2848.
Fluorescence Polarization Assays (FPAs). A Bak BH3 peptide
(F-BakBH3) (GQVGRQLAIIGDDINR) was labeled at the N-ter-
minus with fluorescein isothiocyanate (FITC) (Molecular Probes)
and purified by HPLC. For competitive binding assays, 100 nM
GST-Bcl-XL ΔTM protein was preincubated with the tested com-
pound at varying concentrations in 47.5 μL PBS (pH = 7.4) in
96-well black plates at room temperature for 10 min, and then
2.5 μL of 100 nM FITC-labeled Bak BH3 peptide was added to
produce a final volume of 50 μL. The wild-type and mutant Bak
BH3 peptides were included in each assay plate as positive and
negative controls, respectively. After 30 min incubation at room
temperature, the polarization values in millipolarization units46
were measured at excitation/emission wavelengths of 480/535 nm
with a multilabel plate reader (PerkinElmer). IC50 was determined
by fitting the experimental data to a sigmoidal dose-response
nonlinear regression model (SigmaPlot 10.0.1, Systat Software,
Inc., San Jose, CA). Data reported are mean of three independent
experiments ( standard error (SE). Performance of Bcl-2 and
50
1,10,6,60,7,70-Hexahydroxy-3,30-dimethyl-N5,N -bis(2-phenyl-
propyl)-2,20-binaphthyl-5,50-dicarboxamide (10). First, 0.45 mL of
BBr3 solution (1.18 g, 4.73 mmol) was added dropwise into a
solution of compound 8 (310 mg, 0.40 mmol) in 20 mL of
anhydrous CH2Cl2 at -78 °C. Stirring was continued at -78 °C
for 1 h, 0 °C for 1 h, and ambient temperature for 1 h. Then 50 g of
ice containing 10 mL of 6 M HCl was added to the mixture and
stirred for 1 h at room temperature. The aqueous layer was
extracted with dichloromethane (3 ꢀ 50 mL). The combined
organic layer was washed with water and brine and dried over

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4175
Mcl-1 FPA are similar. Briefly, 50 nM of GST-Bcl-2 or -Mcl-1 were
incubated with various concentrations of compound (4 and 11-14)
for 2 min, and then 15 nM FITC-conjugated-Bim BH3 peptide47
was added in PBS buffer. Fluorescence polarization was measured
after 10 min.
activity as we determined that spleen weight is highly consistent
in age- and sex-matched Bcl-2-transgenic mice in preliminary
studies.17 Variability of spleen weight was within (2% among
control-treated age-matched, sex-matched B6Bcl2 mice.
M2182 Cell Lines and Stable Clones. M2182 progressed prostate
cancer cells were obtained from Dr. Joy Ware (Virginia Common-
wealth University, School of Medicine, Richmond, VA) and
cultured as described.37 M2182 is a tumorigenic but nonmetastatic
variant of normal prostate epithelial P69 cells. pGL3 basic plasmid
(Promega) was used to transfect M2182 cells using Lipofectamine
2000 (Invitrogen) according to the manufacturer’s protocol. Cells
were then selected for 2 weeks in 200 μg/mL of hygromycin, and
individual colonies were isolated, expanded, and maintained in
5 μg/mL hygromycin. The overexpression of luciferase in these
clones was confirmed by measuring luciferase activity.
Human Prostate Cancer Xenografts in Athymic Nude Mice.
M2182-Luc cells (1ꢀ 106) were injected sc in 100 μL of PBS in the
left and right flanks of male athymic nude mice (NCRnu/nu, 4
weeks old, 20 g body weight) as described previously.40 After
establishing visible tumors of ∼75-mm3, requiring ∼5-6 days,
compound dissolved in 500 μL of solvent (ethanol/Cremophor
EL/saline = 10:10:80) were injected intraperitoneally (ip). The
injections were given every 2 days for a total of nine injections.
Three treatment groups were established for the experiment of
compound 4, i.e., DMSO only, 10 mg/kg, and 25 mg/kg of
compound 4. Four treatment groups were established for the
experiment of compound 11, i.e., DMSO only, 1 mg/kg, 3 mg/kg,
and 5 mg/kg of compound 11. A minimum of five animals was
used per experimental condition. For in vivo imaging of tumors,
the mice were anesthetized and injected ipwith 150 mg/kg luciferin
and light emitted from each tumor was determined using a
Xenogen system with CCD camera with an integration time of
1 min. Luminescence measurements were made using Living Image
software (version 2.50.1; Xenogen). At the end of the experiment,
the animals were sacrificed and the tumors were removed and
photographed.
Cell Viability and Apoptosis Assays. The activity of the com-
pounds against human cancer cell lines (PC3, H460, H1299) were
assessed by using the ATP-LITE assay (PerkinElmer). All cells
were seeded in either 12F2 or RPMI1640 medium with 5 mM
L-glutamine supplemented with 5% fetal bovine serum (Mediatech
Inc.), penicillin, and streptomycin (Omega). For maintenance, cells
were cultured in 5% FBS. Cells plated into 96-well plates at varying
initial densities depending on doubling time. H460 and H1299
plated at 2000 cells/well and PC3 at 3000 cells/well. Compounds
were diluted to final concentrations with 0.1% DMSO. Prior to
dispensing compounds onto cells, fresh 5% media was placed into
wells. Administration of compounds occurred 24 h after seeding
into the fresh media. Cell viability was evaluated using ATP-LITE
reagent (PerkinElmer) after 72 h of treatment. Data were normal-
ized to the DMSO control-treated cells using Prism version 5.01
(Graphpad Software).
The apoptotic activity of the compounds against BP3 cells was
assessed by staining with Annexin V- and propidium iodide (PI).
BP3 cell line was cultured in RPMI 1640 medium (Mediatech Inc.,
Herndon, VA 20171) containing 10% fetal bovine serum
(Mediatech Inc., Herndon, VA) and penicillin/streptomycin
(Mediatech Inc., Herndon, VA). Cells were cultured with various
concentrations of compounds 4 and 11-14 for 1-2 days. The
percentage of viable cells was determined by FITC-Annexin V- and
propidium iodide (PI)-labeling, using an Apoptosis Detection kit
(BioVision Inc.) and analyzing stained cells by flow cytometry
(FACSort; Bectin-Dickinson, Inc.; Mountain View, CA). Cells that
were annexin-V-negative and PI-negative were considered viable.
The apoptotic activity of compounds 4 and 11-14 against
mouse embryonic fibroblast wild-type cells (MEF/WT) and
mouse embryonic fibroblast BAX/Bak double knockout cells
(DKO/MEF) was assessed by staining with Annexin V- and
propidium iodide (PI). Wild-type MEF and DKO/MEF were
seeded in 24-well plate at a seeding density of half a million per
well (in 1 mL of DMEM medium supplemented by 10% FCS).
Next day, compound was added to wild-type and DKO cells at
final concentration of 0, 2.5, 5.0, 7.5, 10, and 30 μM. On the
following day, floating cells were pooled with adherent cells
harvested after brief incubation with 0.25% Trypsin/EDTA
solution (Gibco/Invitrogen Inc.). Cells were centrifuged and
supernatant was discarded, and cell pellet was resuspended with
0.2 mL of Annexin-V binding buffer, followed by addition of
1 μL Annexin-FITC and 1 μL PI (propidium iodide). The per-
centage of viable cells was determined by a 3-color FACSort
instrument and data analyzed by Flow-Jo program, scoring
Annexin V-negative, PI-negative as viable cells.
Acknowledgment. We thank NIH (grants CA113318 and
CA149668 to M.P. and J.C.R.) and Coronado Biosciences
(CSRA no. 08-02) for financial support.
Supporting Information Available: Experimental section in-
cluding information on isothermal titration calorimetry assays
and in vitro ADME studies. This material is available free of
charge via the Internet at http://pubs.acs.org.
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