Apogossypol derivatives as pan-active inhibitors of antiapoptotic B-cell lymphoma/leukemia-2 (Bcl-2) family proteins

Article abstract of DOI:10.1021/jm900472s

Guided by nuclear magnetic resonance (NMR) binding assays and computational docking studies, a series of 5,5′ substituted apogossypol derivatives was synthesized that resulted in potent pan-active inhibitors of antiapoptotic Bcl-2 family proteins. Compoun

Full text of DOI:10.1021/jm900472s

J. Med. Chem. 2009, 52, 4511–4523 4511
DOI: 10.1021/jm900472s
Apogossypol Derivatives as Pan-Active Inhibitors of Antiapoptotic B-Cell Lymphoma/Leukemia-2
(Bcl-2) Family Proteins
Jun Wei, Shinichi Kitada, Michele F. Rega, John L. Stebbins, Dayong Zhai, Jason Cellitti, Hongbin Yuan, Aras Emdadi,
Russell Dahl, Ziming Zhang, Li Yang, John C. Reed, and Maurizio Pellecchia*
Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, California 92037
Received April 13, 2009
Guided by nuclear magnetic resonance (NMR) binding assays and computational docking studies, a
series of 5,50 substituted apogossypol derivatives was synthesized that resulted in potent pan-active
inhibitors of antiapoptotic Bcl-2 family proteins. Compound 8r inhibits the binding of BH3 peptides to
Bcl-XL, Bcl-2, Mcl-1, and Bfl-1 with IC50 values of 0.76, 0.32, 0.28, and 0.73 μM, respectively. The
compound also potently inhibits cell growth of human lung cancer and BP3 human B-cell lymphoma cell
lines with EC50 values of 0.33 and 0.66 μM, respectively. Compound 8r shows little cytotoxicity against
bax-/-bak-/- cells, indicating that it kills cancers cells via the intended mechanism. The compound also
displays in vivo efficacy in transgenic mice in which Bcl-2 is overexpressed in splenic B-cells. Together
with its improved chemical, plasma, and microsomal stability relative to compound 2 (apogossypol),
compound 8r represents a promising drug lead for the development of novel apoptosis-based therapies
for cancer.
Introduction
and relatively large hydrophobic crevice on the surface of
antiapoptotic Bcl-2 family proteins that binds the BH3 dimer-
ization domain (an R-helical region) of proapoptotic family
members.10 Thus, molecules that mimic the BH3 domain of
proapoptotic proteins induce apoptosis and/or abrogate the
ability of antiapoptotic Bcl-2 proteins to inhibit cancer cell
death.
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 tumorgenesis
and also contribute significantly to chemoresistance.3,4 B-Cell
lymphoma/leukemia-2 (Bcl-2a) family proteins are central
regulators of apoptosis.5-7 In humans, six antiapoptotic
members of the Bcl-2 family have been identified and
characterized thus far, including Bcl-2, Bcl-XL, Mcl-1, Bfl-1,
Bcl-W, and Bcl-B. Overexpression of antiapoptotic Bcl-2
family proteins occurs in many human cancers and leukemias,
and therefore, these proteins are very attractive targets for the
development of novel anticancer agents.8-11 Members of the
Bcl-2 family proteins also include proapoptotic effectors such
as Bak, Bax, Bad, Bim, and Bid. Antiapoptotic and proapop-
totic Bcl-2 family proteins dimerize and negate each other’s
functions.3 Structural studies revealed the presence of a deep
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 mimetic.12-17 Com-
pound 1 is currently in phase II clinical trials, displaying
single-agent antitumor activity in patients with advanced
malignancies.14,17,18 In mice studies, compound 1 displays
some toxicity and off target effects likely due to two reactive
aldehyde groups, which are important for targeting other
cellular proteins such as dehydrogenases, for example. Our
previous molecular docking studies, however, suggested
that these two reactive groups are not essential for the
compound to bind to Bcl-2 proteins; hence, we designed
compound 2 (apogossypol) (Figure 1A) that lacks the
aldehydes. In agreement with our predicted docked struc-
ture, compound 2 retains activity against antiapoptotic Bcl-
2 family proteins in vitro and in cells.19 Recently, 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 reduced toxicity com-
pared to 1.20 We also evaluated the single-dose pharmaco-
kinetic characteristics of compound 2 in mice. Compound 2
displayed superior blood concentrations over time com-
pared to compound 1 because of slower clearance.21 These
observations indicate that compound 2 is a promising lead
compound for cancer therapy.
*To whom correspondence should be addressed. Phone: (858) 646-
3159. Fax: (858) 795-5225. E-mail: mpellecchia@burnham.org.
aAbbreviations: Bcl-2, B-cell lymphoma/leukemia-2; EDCI, 1-ethyl-
3-(30-dimethylaminopropyl)carbodiimide; 1D 1H NMR, one-dimen-
sional proton nuclear magnetic resonance spectroscopy; SAR, struc-
ture-activity relationship; FPA, fluorescence polarization assays; ITC,
isothermal titration calorimetry; WT, wild type; MEFs, mouse embryo-
nic fibroblast cells; DKO, Bax/Bak double knockout; DKO/MEFs,
Bax/Bak double knockout mouse embryonic fibroblast cells; ACN,
acetonitrile; LC-MS, liquid chromatography and tandem mass spectro-
metry; HPLC, high-performance liquid chromatography; TROSY,
transverse relaxation-optimized spectroscopy; ADME, absorption, dis-
tribution, metabolism, and excretion; DMSO, dimethyl sulfoxide;
PAMPA, parallel artificial membrane permeation assay; FITC, fluor-
escein isothiocyanate; GST, glutathione-S-transferase; PBS, phosphate-
buffered saline; SE, standard error; PI, propidium iodide; NADPH,
nicotinamide adenine dinucleotide phosphate; rpm, rotations per min-
ute; AUC, area under the curve.
Recently, we reported the separation and characterization
of atropoisomers of compound 2.22 These studies revealed
r
2009 American Chemical Society
Published on Web 06/25/2009
pubs.acs.org/jmc

4512 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Wei et al.
Figure 1. (A) Structure of compound 1, 2, and 3. (B) Structure of 5,50 substituted compound 2 derivatives. (C, D) Molecular docking studies.
Docked structures of (C) compound 2 and (D) compound 8r into Bcl-2 (PDB code 1YSW).
that the racemic compound 2 is as effective as its individual
isomers.22 We further reported the synthesis and evaluation of
5,50-ketone substituted compound 2 derivatives. Among these
derivatives, compound 3 (BI79D10)23 displayed improved in
vitroandin vivoefficacy comparedtocompound2(Figure 1A
andFigure 1B). However, contrary to what we observed with
compound 2, compound 3 also displayed mild GI toxicity in
mice. The observed toxicity in compound 3 may be attribu-
table to relatively active ketone groups.23 On the basis of these
premises, in this current work, we focused our attention on
preparing and evaluating activities of novel 5,50 substituted
compound 2 derivatives which further replace the reactive
ketone groups with more druggable amide and alkyl groups
(Figure 1B).
A synthetic route (Scheme 1) was developed to introduce a
variety of amide derivatives at the 5,50 positions. Compound 1
was treated with NaOH solution at 90 °C to provide com-
pound 2, which was readily methylated by dimethyl sulfate in
the presence of potassium carbonate to afford compound 4.26
Reaction of compound 4 with TiCl4 followed by dichloro-
methyl methyl ether at room temperature resulted in loss of
the isopropyl groups and simultaneous bisformylation to give
the aldehyde compound 5.26 The aldehyde groups of com-
pound 5 were converted to carboxylic acid 6 by mild oxidation
with sodium hypochlorite.27 The carboxylic acid 6 was then
coupled with a variety of commercially available amines in the
presence of 1-ethyl-3-(30-dimethylaminopropyl)carbodiimide
(EDCI) at room temperature to give compound 7.28 Subse-
quent demethylation of the compound 7 using boron tribro-
mide afforded compound 8.27 The synthesis of 5,50 alkyl
substituted compound 2 derivatives was outlined in Scheme 2.
Compound 5 was treated with different Grignard or lithium
reagents to afford a secondary alcohol 9, which was oxidized
to give the phenone 10 by pyridinium chlorochromate.
Triethylsilane reduced phenone 10 to alkyl compound 1129
followed by subsequent demethylation using boron tribro-
mide to afford compound 12 (Scheme 2). Compounds 13 and
14 (Scheme 3), with only hydrogen atom or carboxylic acid at
5,50 positions, weresynthesizedtoexploreif substitution atthe
5,50 position is important for enhancing biological activities.
Compound 13 was synthesized by treating compound 4 with
concentrated sulfuric acid to lose the isopropyl group.26 The
resulting product and compound 6 were then treated indivi-
dually with boron tribromide to give compounds 13 and 14,
respectively (Scheme 3).
Results and Discussion
We have recently reported that compound 2 is a promising
inhibitor of Bcl-XL and Bcl-2 with improved in vivo efficacy
and reduced toxicity compared to compound 1.12,19,20 Molec-
ular docking studies of compound 2 into the BH3 binding
groove in Bcl-224,25 (Figure 1C) suggest that 2 forms two
hydrogen bonds with residues Arg 143 and Tyr 105 in Bcl-2
through the 1 and 10 hydroxyl groups, respectively. Com-
pound 2 is also involved in hydrogen bonding interactions
with Trp 141 and Tyr 199 in Bcl-2 through the 60 hydroxyl
group on naphthalene ring. According to this model, the
isopropyl group on the left naphthalene ring inserts into the
first hydrophobic pocket (P1) in Bcl-2 (Figure 1C), while
the isopropyl group on the right naphthalene ring inserts into
the second hydrophobic pocket (P2) (Figure 1C). Hence, the
analysis of the predicted binding models indicates that while
the overall core structure of compound 2 fits rather well into
the BH3 binding groove of Bcl-2, the two isopropyl groups do
not apparently fully occupy the hydrophobic pockets P1 and
P2. Therefore, a library of 5,50 substituted compound 2
derivatives (Figure 1B) that replace the isopropyl groups with
suitable substituents was designed with the aim of deriving
novel molecules that could occupy the hydrophobic pockets
on Bcl-2 more efficiently.
The synthesized 5,50 substituted compound 2 derivatives
were first screened by one-dimensional 1H nuclear magnetic
resonance spectroscopy (1D 1H NMR) binding assays against
Bcl-XL, as we reported previously (Tables 1 and 2).30 Active
compounds in 1D 1H NMR binding assays were then selected
and evaluated in isothermal titration calorimetry assays
(ITC), competitive fluorescence polarization assays (FPA),
and cell viability assays (Tables 1-3). A group of compounds
(8r, 8q, 8m) displayed high binding affinity to Bcl-XL in these

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4513
Scheme 1a
a Reagents and conditions: (a) NaOH, H2O, reflux; (b) H2SO4; (c) DMS, K2CO3; (d) TiCl4, Cl2CHOCH3, room temp; (e) HCl, H2O; (f) NaClO2,
H2O2, KH2PO4, CH3CN, room temp; (g) HCl, H2O; (h) EDCI, NH2R, HOBT, room temp; (i) BBr3, CH2Cl2; (j) HCl, H2O.
Scheme 2a
a Reagents and conditions: (a) RMgBr or RLi, room temp; (b) NH4Cl, H2O; (c) pyridinium chlorochromate, CH2Cl2, room temp; (d) Et3SiH, TFA or
Pd/C, H2; (e) BBr3; (f) HCl, H2O.
Scheme 3a
has an IC50 value of 0.76 μM in the FP displacement assays,
which is 5 times more effective than 2 (Table 3 and Supporting
Information Figure 2A). To further confirm the results ob-
tained by the NMR binding data and the FP assays, we
further evaluated the binding affinity of compound 8r for
Bcl-XL using ITC assay (Table 3 and Supporting Information
Figure 1). In agreement with NMR binding and FPA data,
compound 8r displayed potent binding affinity to Bcl-XL with
a Kd value of 0.11 μM, which is 15 times more potent than
compound 2 (Kd=1.7 μM) in the same assay. Consistent with
NMR binding, FPA, and ITC data, compound 8r displayed
efficacy in inhibiting growth of PC3ML cells, which express
highlevelsof Bcl-XL. TheEC50 value of 8ris 1.7 μM and hence
6-fold more potent than 2 (EC50=10.4 μM). Compounds (8j-
8k and 8p-8s) displayed similar binding affinity as 8r for Bcl-
XL in these assays with average IC50 value of 2.8 μM (Table 1).
In addition to Bcl-XL, other members of the Bcl-2 family
are known to play critical roles in tumor cell survival.31,32
Therefore, we further evaluated the binding properties and
specificity of selected 5,50 substituted compound 2 derivatives
a Reagents and conditions: (a) H2SO4, room temp; (b) H2O; (c) BBr3,
CH2Cl2; (d) HCl, H2O.
assays (Table 1, Figure 2A). The most potent compound 8r
induced significant chemical shift changes in active site methyl
groups (region between -0.38 and 0.42 ppm) in the one-
dimensional 1H NMR spectra of Bcl-XL (Figure 2A) and also

4514 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Wei et al.
Table 1. Evaluation of 5,50 Substituted Compound 2 Derivatives Using a Combination of 1D 1H NMR Binding Assays and Cell Viability Assays
a*Four-point-rating scale: þþþ, very active; þþ, active; þ, mild; -, weak. b*Compounds against cell line using ATP-LITE assay. c*Compounds
against cell line using annexin V-FITC and propidium iodide assay. d*ND: not determined.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4515
Table 2. Evaluation of 5,50 Substituted Compound 2 Derivatives Using a Combination of 1D 1H NMR Binding Assays and Cell Viability Assays
a*Four-point-rating scale: þþþ, very active; þþ, active; þ, mild; -, weak. b*Compounds against cell line using ATP-LITE assay. c*Compounds
against cell line using annexin V-FITC and propidium iodide assay. d*ND: not determined.
Table 3. Cross-Activity of Selected 5,50 Substituted Compound 2
compound 2 against the same cell lines (Table 1, Figure 2C,
Derivatives against Bcl-XL, Bcl-2, Mcl-1, and Bfl-1
and Supporting Information Figure 3A). Molecular docking
IC50 (μM) FPA
studies with compound 8r and Bcl-2 (Figure 1D) suggest that
2-phenylpropyl groups at 5,50 positions could insert deeper
into hydrophobic pockets (P1 and P2) in Bcl-2, hence occupy-
ing these regions more efficiently compared to the isopropyl
groups of compound 2 (Figure 1B). In addition, the carbonyl
group on the right naphthalene ring also formed an additional
hydrogen bond with residue Tyr199. Other 5,50 substituted
derivatives, such as compounds 12e, 8n, 8p, 8q, 8k, also
displayed strong pan-active inhibitory properties against
Bcl-2, Mcl-1, and Bfl-1. The most potent derivative, com-
pound 8q, inhibits Bcl-2, Mcl-1, and Bfl-1 with IC50 values of
0.67, 0.59, and 1.3 μM (Figure2Band Table 3), respectively, in
FP assays. In agreement with these data, the compound
showed potent cell growth inhibitory activity against H460,
H1299, and BP3 cell lines, with IC50 values of 0.40, 0.36, and
0.20 μM, respectively (Table 1, Figure 2C, and Supporting
Information Figure 3A).
As anticipated by our initial docking studies with com-
pound 2, the synthesized derivatives reveal that substitution at
the 5,50 position with larger hydrophobic groups results in
improved binding affinity to the antiapoptotic Bcl-2 family
proteins tested. Consistent with these observations, com-
pounds 13 and 14 (Tables 1 and 2), with only hydrogen atoms
or carboxylic acid groups on 5,50 positions, displayed weak or
no inhibition in all reported in vitro and cellular assays
(Tables 1 and 2). A closer look at the emerging SAR for
the synthesized 5,50-amide substituted derivatives further
Kd (μM) ITC,
Bcl-XL
compd
Bcl-XL
Bcl-2
Mcl-1
Bfl-1
2
3.7
3.5
4.3
2.6
>10
5.0
1.7
0.41
0.85
ND
ND
0.12
NDa
0.11
0.11
NDa
12e
8m
8n
8p
8q
8j
0.48
0.71
0.15
4.4
0.83
0.78
0.30
3.2
1.1
2.0
0.80
6.3
0.55
NDa
1.3
NDa
0.40
0.73
0.67
0.93
0.8
0.67
0.70
0.49
0.32
0.70
0.59
1.1
8k
8r
0.27
0.76
0.85
0.23
0.28
0.35
8s
a ND: not determined.
against Bcl-2, Mcl-1, and Bfl-1 using FP assays (Table 3 and
Figure 2B). In these assays, compound 8r inhibited Bcl-2,
Mcl-1, and Bfl-1 with IC50 values of 0.32, 0.28, and 0.73 μM,
respectively, values thatcorrespondtoapproximately10 times
higher potency than what was observed with compound 2 in
similar FP assays (Figure 2B, Table 3, and Supporting
Information Figure 2A). Compound 8r was further evaluated
against H460, H1299, and BP3 cell lines, which express high
levels of Bcl-2, Mcl-1, and Bfl-1, respectively (Table 1).32-34
Consistent with FPA data, compound 8r displayed significant
efficacy in inhibiting growth of H460 and BP3 cells, with IC50
values of 0.33 and 0.66 μM, repectively, which are approxi-
mately 7-10 times more potent than what was observed with

4516 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Wei et al.
1
Figure 2. (A) NMR binding studies. Aliphatic region of the H NMR spectrum of Bcl-XL (25 μM, black) and Bcl-XL in the presence of
compound 8m (200 μM, gray), 8q (200 μM, blue), and 8r (200 μM, red). (B) Fluorescence polarization-based competitive binding curves
of compound 8m (solid squares), 8q (solid up triangle), 8r (solid down triangle), and 2 (solid dots) using Bcl-2. (C) Inhibition of cell growth by
compound 8m (red square), 8q (green triangle), 8r (blue diamond), 8p (dark triangle), and 2 (dark dots) in the H460 human lung cell line. Cells
were treated for 3 days, and cell viability was evaluated using ATP-LITE assay. (D) Mouse embryonic fibroblast cells with wild-type (MEF/
WT; blue bars) or bax-/-bak-/- double knockout (red bars) genotypes were treated with various 5,50 substituted compound 2 derivatives at
10 μM, and apoptosis was monitored by annexin V-FITC assays.
indicates that longer and flexible hydrophobic groups display
higher potency than small, short, and rigid hydrophobic
groups. Replacement of the methylcyclopropane (8l) or the
cyclopentyl (8b) groups by the longer methylcyclohexyl group
(8m) significantly increased cell inhibition potency. Also,
compounds (8n-s) having phenethyl groups at 5,50 positions
displayed potent cell activity in the H460 and PC3ML cell
lines with average EC50 values of 0.64 and 2.6 μM, respec-
tively, while related compounds (8a-e) having only phenyl
groups displayed relatively weaker cell activity with average
EC50 values of 8.6 and 11.3 μM, respectively (Table 1), against
the same cell lines.
These observations are corroborated by our computational
docking studies suggesting that longer and flexible groups
may insert deeper into the P1 and P2 pockets (Figure 1C and
Figure 1D).
We further explored the SAR of the 5,50-alkyl substituted
derivatives. Overall, we observed a similar trend that suggests
longer, hydrophobic groups to improve potency. Compounds
12a and 12b with isobutyl and isopentyl groups displayed
improved activity compared to 2 with isopropyl groups.
Similarly, compound 12e withphenethylgroupsismore active
than compound 12d with benzyl groups (Table 2).
Bcl-XL, and Mcl-1 is approximately 10:3:1.32 However, we
determined that BP3 cells express high levels of both Bfl-1 and
Mcl-1 by Western blot analysis (Supporting Information
Table 1). In agreement with these observations, the potent
dualBcl-XL and Bcl-2 antagonist, compound 15 (ABT-737),24
displayed no cytotoxic activity against BP3 cell lines presum-
ably because this molecule is not effective against Mcl-1 and
Bfl-1 (Supporting Information Figure 2B).24,31,36 By compar-
ison, our most active compounds 8q and 8r, targeting Bfl-1
and Mcl-1, showed submicromolar cell growth inhibitory
activity against BP3 cells with IC50 values of 0.20 and
0.66 μM, respectively (Table 1). We also evaluated the ability
of 5,50 derivatives to induce apoptosis of the human lympho-
ma RS11846 cell line, which expresses high levels of Bcl-2 and
Bcl-XL. For these assays, we used annexin V-FITC and
propidium iodide (PI) double staining, followed by flow-
cytometry analysis (Table 1). Most of synthesized derivatives
effectively induced apoptosis of the RS11846 cells in a dose-
dependent manner (Table 1 and Supporting Information
Figure 3B). In particular, compounds 8q, 8r, and 8n are
effective with EC50 values ranging from 3.0 to 5.8 μM, which
is consistent with previous results obtained for human
PC3ML and H460 cancer cell lines. Again, the negative
control compounds 13 and 14 induced little or no apoptosis
of the RS11846 cell line.
Next, we profiled the activity of the compounds in cells and
compared their cell killing ability with their reported levels of
Bcl-2 proteins. For example, the lung cancer H460 cell line has
been studied by several groups with respect to sensitivity to
Bcl-2 antagonists.33-35 In our studies, however, we also intro-
duce the BP3 cell line to profile compounds’ activity. The BP3
cell line originates from a human diffuse large B-cell lympho-
ma (DLBCL) overexpressing Bfl-1. The mRNA ratio of Bfl-1,
We next explored whether 5,50 substituted compound 2
derivatives had cytotoxicity against wild type mouse embryo-
nic fibroblast cells (wt-MEFs) and transformed Bax/Bak
double knockout MEF cells (DKO/MEFs) in which antia-
poptotic Bcl-2 family proteins lack a cytoprotective pheno-
type.37,38 The most potent pan-active Bcl-2 compound 2

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4517
Figure 3. Characterization of compounds in vitro and in vivo. (A) Chemical stability of 5,50-substituted compound 2 derivatives when left at
room temperature in powder form: 8m (red dot), 8p (green square), 8q (purple dot), 8r (blue triangle), 8k (pink dot), 12e (dark dot), 2*
(compound 2 with ascorbic acid, dark square), and 2 (pure 2, dark triangle). Chemical stability was evaluated in the air for 60 days at room
temperature. The stability was monitored using a combination of HPLC and LCMS. (B) Effects of 5,50 substituted compound 2 derivatives on
shrinkage of Bcl-2 mouse spleen at a single intraperitoneal injection dose of 0.072 mmol/kg. All shrinkage data are a percentage of maximum
reduction of mice spleen size. (C) Percent weight loss in mice induced by single ip injection of various amount of compound 8r. (D) Effects of
compound 8r at 42 mg/kg (0.06 mmol/kg) on reduction of spleen weight of six Bcl-2 mice treatment with a single intraperitoneal injection. Data
shown as the mean ( SE (n = 6). P < 0.0001.
derivatives, namely, compounds 8m, 8q, 8r, 8k, 8p, induced
nearly complete cell death (80-90%) of wt-MEFs at 10 μM
(Figure 2D). However, at the same concentration they are
ineffective in killing DKO/MEFs measured by the same
FITC-annexin V/PI assay (Figure 2D). These data strongly
suggest that cytotoxic activities of those compound 2 deriva-
tives are largely dependent on the Bcl-2 pathway. In contrast,
compound 1 seems equally effective in killing both wt MEFs
and MEF/DKOs at 10 μM (Figure 2D), suggesting that other
possible killing mechanisms not related to Bcl-2 inhibition are
induced by this compound. Because of the lack of the reactive
aldehydes of compound 1, compound 2 showed improved
selectivity against Bcl-2 proteins and hence reduced cytotoxi-
city against MEF/DKOs (Figure 2D). Recently, Vogler et
al.,38 on the basis of extensive sets of validation studies on
a panel of reported Bcl-2 antagonists, including compounds
1 and 2, concluded that only the Abbott compound 15 shows
remarkable selectivity for Bcl-2 and Bcl-xL. However, among
the compounds examined, Vogler et al. also concluded that it
is possible that analogues of compounds 1 and 2 may have the
potential for becoming selective Bcl-2 antagonists.38 This
conclusion is now corroborated by our studies that resulted
in more potent pan-active Bcl-2 antagonists.
powders at room temperature. Compound stability was mon-
itored using a combination of HPLC and LC-MS. Overall,
5,50-amide substituted compound 2 derivatives show superior
chemical stability compared to 2 (Figure 3A). In particular, 8r
and 8q were only 10% degraded after 60 days at room
temperature while 2 is almost 80% decomposed under the
same condition in the absence of ascorbic acid. Compound
12e, having phenethyl groups at the 5,50 positions, is also less
stablethanamide compounds, presumably because ofthe lack
of electron withdrawing groups.
To test the pharmacological properties of 5,50 substituted
compound 2 derivatives, we determined their in vitro plasma
stability, microsomal stability, and cell membrane permeabil-
ity (Table 4). From these studies, we could conclude that our
synthesized compounds displayed superior plasma and mi-
crosomal stability compared to 2 (Table 4). Compounds 8r
and 8m only degraded 4% and 11%, respectively, after 1 h of
incubation in rat plasma while 2 degraded 47% under the
same conditions. In addition, compounds 8r and 8m degraded
by 24% and 10%, respectively, after 1 h of incubation in rat
microsomal preparations, while 2 degraded by 36% under the
same conditions (Table 4). Compounds 8r and 8m also
showed similar or improved cell membrane permeability
compared to 2.
Compound 2 is a polyphenol scaffold with six hydroxyl
groups on the naphthalene ring (Figure 1A) which can be
oxidized to quinones. We previously stabilized 2 by cocrys-
tallizing it with ascorbic acid.22 Compound 2 can also be
stabilized by introducing electron withdrawing groups, such
as carbonyl groups on the naphthalene rings, which slow
oxidation and other side reactions. The chemical stability of
our compounds (8m, 8q, 8r, 8k, 8p, 12e) was evaluated as solid
Hence, using a combination of NMR-based binding assays,
FP assays, ITC assays, cytotoxicity assays, and preliminary in
vitro ADME data, we selected compounds to be tested in
subsequent in vivo studies using a Bcl-2 transgenic mouse
model. B-Cells of the B6 transgenic mice overexpress human
Bcl-2 and accumulate in the spleen of mice. The spleen weight
isusedas an end-point for assessing in vivoactivity, aswehave

4518 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Wei et al.
Table 4. Plasma Stability, Microsomal Stability, and Cell Permeability
of Selected 5,50 Substituted Compound 2 Derivatives
vitro and in vivo assays. The most potent compound, 8r, was
found to bind to Bcl-xL, Bcl-2, Mcl-1, and Bfl-1 with IC50
values of 760, 320, 280, and 730 nM, respectively. The com-
pound also potently inhibited growth in cultures of the
PC3ML, H460, H1299, and BP3 cancer cell lines, which
express Bcl-XL, Bcl-2, Mcl-1, and Bfl-1, respectively, with
EC50 values in the submicromolar to nanomolar range. Com-
pound 8r effectively induced apoptosis of the RS11846 human
lymphoma cell line in a dose-dependent manner and show
little cytotoxicity against Bax/Bak double knockout mouse
embryonic fibroblast cells in which antiapoptotic Bcl-2 family
proteins lack a cytoprotective phenotype. Hence, compound
8r is much more effective against Bcl-2 proteins in in vitro
assays, resulting in increased cytotoxicity against cancer cells
compared to compounds 1 and 2 and in descreased cytotoxi-
city against Bax/Bak MEF/DKO cells, suggesting that be-
cause of its increased affinity for the Bc-2 proteins, off-target
effects that characterize cell death induced by compounds
1 and 2, respectively, are much less prominent in this com-
pound. Finally, compound 8r showed favorable chemical
stability, in vitro ADME properties, and superior in vivo
efficacy compared to 2 in Bcl-2 transgenic mice in which Bcl-2
is overexpressed in B-cells. Considering the critical roles of
antiapoptotic Bcl-2 family proteins in tumorgenesis and
chemoresistance and the potent inhibitory activity of 8r
against antiapoptotic Bcl-2 family proteins, we speculate that
this compound represents a viable drug candidate for the
development of novel apoptosis-based cancer therapies.
T = 1 h
plasma
stability (%)
microsomal
stability (%)
cell
permeability
compd
2
53
80
64
89
75
60
90
90
87
76
71
-7.16
-6.61
-6.27
-6.49
-6.67
-7.71
-8.15
-7.51
-7.92
12e
12c
8n
8m
8p
8q
8r
81
63
89
NDa
94
96
94
8k
determined that the spleen weight is highly consistent in age-
and sex-matched Bcl-2-transgenic mice, varying by only (2%
among control Bcl2 mice.20 We first screened the in vivo
activities of compounds such as 8r and 8q side by side with 1
and 2 in Bcl-2 transgenic mice with a single intraperitoneal (ip)
injection at 72 μmol/kg. In agreement with the in vitro data,
tested 5,50-amide substituted compound 2 derivatives dis-
played superior in vivo activity compared to 1 and 2
(Figure 3B). In particular, compounds 8r, 8k, and 8p induced
more than 40% spleen weight reduction compared to only
e20% by 1 and 2. Since the maximum spleen shrinkage would
be no more than 50% in this experimental model,20 these
compounds induced near maximal (85-95%) biological ac-
tivity, while 1 and 2 induced e40% of maximum reduction in
spleen weight at the same dose. The negative control, com-
pound 13, displayed no activity in transgenic mice, as ex-
pected. Overall, the 5,50-alkyl substituted compound 2
derivatives tested(12cand 12e) displayedlowerinvivoactivity
compared to 5,50 amide substituted compound 2 derivatives.
However, the 5,50-alkyl substituted compound 2 derivatives
show no significant signs of toxicity at 72 μmol/kg and even at
120 μmol/kg while 5,50-amide substituted compound 2 deri-
vatives show evidence of toxicity at the 72 μmol/kg (Support-
ing Information Table 2). In particular, mice treated with
compound 8r had more apparent signs of GI toxicity at the 72
mol/kg dose (50 mg/kg).
To balance the toxicity versus efficacy of compound 8r, we
next explored the maximum tolerated dose (MTD) of 8r using
a group of five mice. Mice were treated with a single dose
of 100, 75, 50, and 25 mg/kg (ip) and observed for a period of
14 days monitoring morbidity (body weight loss) and mortal-
ity. All mice were alive after 14 days. The maximum weight
loss was observed at the fifth day, with 80-100% recovery
after 14 days (Figure 3C). The mice dosed at 25 mg/kg showed
slight weight loss, while the mice dosed at 50 mg/kg displayed
<15% weight loss. Therefore, the MTD of compound 8r is
likely between 25 mg to 50 mg/kg. We next evaluated the in
vivo activity and toxicity of the compound 8r in groups of six
mice each at a dose of 42 mg/kg (60 μmol/kg). Consistent with
the single mouse experiment, compound 8r treatment resulted
in a significant (∼70%) reduction of spleen weight (P <
0.0001) compared to the control group of six mice
(Figure 3D). All mice tolerated the treatment well, with only
mild signs of GI toxicity. The average weight loss of mice was
7.8% during the course of this study of compound 8r.
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), repectively. 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 for in vivo
studies were purified to g98% purity using preparative HPLC.
The eluant was a linear gradient with a flow rate of 1 mL/min
from 50% A and 50% B to 5% A and 95% B in 15 min followed
by 5 min at 100% B (Ssolvent A, H2O with 0.1% TFA; solvent
B, ACN with 0.1% TFA). Compounds were detected at λ=254
1
nm. H NMR spectra were recorded on Varian 300 or Bruker
600 MHz instruments. Chemical shifts are reported in ppm (δ)
relative to H (Me4Si at 0.00 ppm). Coupling constant (J) are
1
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.
1,10,6,60,7,70-Hexahydroxy-5,50-diisopropyl-3,30-dimethyl-
2,20-binaphthyl-8,80-dicarboxaldehyde (1). Compound 1 is com-
mercially available from Yixin Pharmaceutical Co. HPLC
purity 99.0%, tR=12.50 min.
5,50-Diisopropyl-1,10,6,60,7,70-hexahydroxy-3,30-dimethyl-2,20-bi-
naphthalene (2). Compound 1 (5 g, 8.65 mmol) in 50 mL of 40%
NaOH was heated under nitrogen at 90 °C for 3.5 h in the dark. The
reaction mixture was cooled and poured slowly onto ice (300 mL)
and concentrated H2SO4 (35 mL) mixture to form a white pre-
cipitation. The precipitation was filtered, washed with water and
1
dried to afford 3.8 g of compound 2 (95%) as a white solid. H
NMR (300 MHz, CDCl3) δ 7.61 (s, 2H), 7.50 (s, 2H), 5.93 (s, 2H),
5.27 (s, 2H), 5.13 (s, 2H), 3.88 (m, 2H), 2.12 (s, 6H), 1.55 (d, J=
5.5 Hz, 12H). HPLC purity 99.2%, tR=13.12 min. HRMS calcd
for C28H30O6 463.2115 (MþH), found 463.2108.
Conclusions
In summary, a library of 5,50 substituted compound 2
derivatives was synthesized and evaluated in a variety of in

Article
5,50-Diisopropyl-1,10,6,60,7,70-hexamethoxy-3,30-dimethyl-2,20-bi-
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4519
3.76-3.60 (m, 4H), 3.19 (m, 2H), 1.90 (d, J=3.0 Hz, 3H), 1.88 (d, J=
3.0 Hz, 3H), 1.41 (m, 6H). HPLC purity 99.4%, tR = 8.71 min.
HRMS calcd for C42H40N2O8 701.2857 (M þ H), found 701.2864.
Following the above-mentioned procedure and with use of
the appropriate starting materials and reagents, compounds
8a-t were synthesized.
naphthalene (4). Compound 2 (3.8 g, 8.21 mmol) was dissolved in
acetone (200 mL). K2CO3 (23.9 g, 206.7 mmol) and dimethyl sulfate
(16.3 mL, 206.7 mmol) were added, and the reaction mixture was
refluxed under nitrogen for 24 h. The solid was collected by
filtration and washed using acetone and water and dried to yield
4.2 g of compound 4 as a white solid (93%). 1H NMR (300 MHz,
CDCl3) 7.83 (s, 2H), 7.43 (s, 2H), 3.98 (m, 8H), 3.94 (s, 6H), 3.57
(s, 6H), 2.20 (s, 6H), 1.56 (s, 12H).
1,10,6,60,7,70-Hexamethoxy-3,30-dimethyl-2,20-binaphthalene-5,50-
dicarboxaldehyde (5). To a solution of compound 4 (1.6 g, 2.93
mmol) in dry methylene chloride (40 mL) at 0 °C was added tita-
nium tetrachloride (14.3 g, 75.5 mmol). After addition was com-
pleted, the dark-red solution was stirred an additional 15 min at
0 °C. Dichloromethyl methyl ether (2.93 g, 25.5 mmol) was added
dropwise over 15 min, and the reaction mixture was stirred at
ambient temperature under nitrogen for 12 h. The reaction mixture
was poured onto ice, and the resulting aqueous layer was extracted
twice with methylene chloride. The combined organic fractions were
washed with water and brine, dried over MgSO4, and concentrated
to give dark-red oil. The oil was chromatographed (acetonitrile/
methylene chloride) followed by trituration of crude product with
diethyl ether to afford compound 5 (0.60 g, 40%) as a yellow solid.
1H NMR (300 MHz, CDCl3) 10.84 (s, 2H), 8.93 (s, 2H), 7.82 (s, 2H),
4.10 (s, 6H), 4.03 (s, 6H), 3.48 (s, 6H), 2.22 (s, 6H).
50
1,10,6,60,7,70-Hexahydroxy-3,30-dimethyl-N5,N -diphenyl-2,20-bi-
naphthyl-5,50-dicarboxamide (8a). Yield, 75%; 1H NMR (600 MHz,
CD3OD) δ 7.77 (d, J=7.8 Hz, 4H), 7.63 (s, 2H), 7.38 (t, J1=7.8 Hz,
J2=7.2 Hz, 4H), 7.28 (s, 2H), 7.16 (t, J1=7.8 Hz, J2=7.2 Hz, 2H),
2.01 (s, 6H). HPLC purity 98.0%, tR = 5.95 min. HRMS calcd for
C36H28N2O8 617.1918 (MþH), found 617.1912.
5
5
0
N ,N -Dicyclopentyl-1,10,6,60,7,70-hexahydroxy-3,30-dimeth-
yl-2,20-binaphthyl-5,50-dicarboxamide (8b). Yield, 76%; 1H
NMR (600 MHz, CD3OD) δ 7.57 (s, 2H), 7.19 (s, 2H), 4.46
(m, 2H), 2.09 (m, 4H), 1.97 (s, 6H), 1.80 (m, 4H), 1.69 (m, 8H).
HPLC purity 98.1%, tR=4.23 min. HRMS calcd for C34H36-
N2O8 601.2544 (M þ H), found 601.2531.
50
1,10,6,60,7,70-Hexahydroxy-3,30-dimethyl-N5,N -bis(4-phenoxy-
phenyl)-2,20-binaphthyl-5,50-dicarboxamide (8c). Yield, 65%; 1H
NMR (600 MHz, CD3OD) δ 7.78 (d, J=8.4 Hz, 4H), 7.64 (s,
2H), 7.35 (t, J1=7.8 Hz, J2=7.8 Hz, 4H), 7.28 (s, 2H), 7.08 (m, 2H),
7.02 (m, 8H), 2.01 (s, 6H). HPLC purity 99.0%, tR=12.09 min.
HRMS calcd for C48H36N2O10 801.2443 (MþH), found 801.2425.
5
5
0
N ,N -Bis(3-ethylphenyl)-1,10,6,60,7,70-hexahydroxy-3,30-di-
methyl-2,20-binaphthyl-5,50-dicarboxamide (8d). Yield, 69%; 1H
NMR (600 MHz, CD3OD) δ7.63 (s, 4H), 7.60 (d, J=7.8 Hz,
2H), 7.28 (m, 4H), 7.02 (d, J=7.8 Hz, 2H), 2.68 (q, J1=J2 = 8.4
Hz, 4H), 2.01 (s, 6H), 1.28 (t, J1=J2=8.4 Hz, 6H). HPLC purity
99.5%, tR=10.27 min. HRMS calcd for C40H36N2O8 673.2544
(M þ H), found 673.2537.
1,10,6,60,7,70-Hexamethoxy-3,30-dimethyl-2,20-binaphthalene-5,50-
dicarboxylic Acid (6). Compound 5 (6.6 g, 12.7 mmol) was dissolved
in 40 mL of acetonitrile and 40 mL of THF in an ice bath. Sodium
dihydrogen phosphate (876 mg, 6.35 mmol) and 30% hydrogen
peroxide (2.6 mL, 25.4 mmol) were added. Sodium chlorite (4.14 g,
45.8 mmol) dissolved in 20 mL of water was added. The reaction
mixture was stirred overnight at room temperature and then poured
onto 100 g of ice with 30 mL of 6 M HCl. The solution was extracted
with ether (3 ꢀ 100 mL). The ether extracts were washed with brine,
dried over magnesium sulfate, and filtered. Evaporation of the
solvent in vacuo and the residue was purified by C-18 column
chromatography (H2O/acetonitrile) to give 5.9 g (85%) of com-
pound 6 as a red solid. 1H NMR (600 MHz, CD3OD) δ 8.0 (s, 2H),
7.68 (s, 2H), 4.1 (s, 6H), 4.06 (s, 6H), 3.54 (s, 6H), 2.21 (s, 6H).
50
1,10,6,60,7,70-Hexahydroxy-3,30-dimethyl-N5,N -bis(3-(triflu-
oromethyl)phenyl)-2,20-binaphthyl-5,50-dicarboxamide (8e). Yield,
69%; 1H NMR (600 MHz, CD3OD) δ 8.26 (s, 2H), 7.96 (d, J =
7.8 Hz, 2H), 7.65 (s, 2H), 7.57 (t, J1 = J2 = 6.6 Hz, 2H), 7.44 (d,
J=7.2 Hz, 2H), 7.27 (s, 2H), 2.01 (s, 6H). HPLC purity 99.0%,
tR=13.52 min. HRMS calcd for C38H26F6N2O8 751.1511 (Mþ
H), found 751.1506.
50
1,10,6,60,7,70-Hexahydroxy-3,30-dimethyl-N5,N -bis(1-phenylpro-
50
1,10,6,60,7,70-Hexamethoxy-3,30-dimethyl-N5,N -bis(2-phenylpro-
pyl)-2,20-binaphthyl-5,50-dicarboxamide (8f). Yield, 70%; 1H NMR
(600 MHz, CD3OD) δ 7.50 (m, 4H), 7.31 (m, 6H), 7.09 (s, 2H), 6.95
(s, 2H), 5.09 (m, 2H), 1.88 (m, 6H), 1.09 (m, 6H). HPLC purity
99.3%, tR=8.09 min. HRMS calcd for C40H40N2O8 701.2857 (Mþ
H), found 701.2867.
pyl)-2,20-binaphthyl-5,50-dicarboxamide (7r). Compound 6 (500 mg,
0.907 mmol), EDCI (522 mg, 2.72 mmol), and HOBT (244 mg, 1.81
mmol) were dissolved in 15 mL of dry CH2Cl2 and stirred at room
temperature for 10 min under nitrogen atmosphere. 2-Phenyl-1-
propanamine (0.30 mL, 2.09 mmol) and Et3N (0.51 mL, 3.7 mmol)
were added, and the reaction mixture was stirred at room tempera-
ture 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 was done in vacuo
and the residue was purified by silica chromatography to give
5
5
0
N ,N -Dibenzyl-1,10,6,60,7,70-hexahydroxy-3,30-dimethyl-2,20-bi-
naphthyl-5,50-dicarboxamide (8g). Yield, 78%; 1H NMR (600 MHz,
CD3OD) δ7.58 (s, 2H), 7.54 (d, J=7.2 Hz, 4H), 7.36 (t, J1=J2 =7.2
Hz, 4H), 7.27(t, J1=J2 =7.2 Hz, 2H), 7.16 (s, 2H), 4.70 (s, 4H), 1.91
(s, 6H). HPLC purity 99.0%, tR=4.92 min. HRMS calcd for C38-
H32N2O8 645.2231 (M þ H), found 645.2237.
5
5
0
1,10,6,60,7,70-Hexahydroxy-3,30-dimethyl-N ,N -bis(3-methylben-
zyl)-2,20-binaphthyl-5,50-dicarboxamide (8h). Yield, 75%; 1H NMR
(600 MHz, CD3OD) δ 7.58 (s, 1H), 7.36 (s, 2H), 7.30 (d, J=7.8 Hz,
2H), 7.23 (t, J1=7.8 Hz, J2=7.2 Hz, 2H), 7.16 (s, 2H), 7.08 (d, J =
7.2 Hz, 2H), 4.65 (t, J1=J2 =15.0Hz, 4H), 2.36(s, 6H), 1.91(s, 6H).
HPLC purity 99.0%, tR=6.22 min. HRMS calcd for C40H36N2O8
673.2544 (M þ H), found 673.2536.
1
320 mg (45%) of compound 7r as a yellow solid. H NMR (600
MHz, CD3OD) δ 7.56 (s, 2H), 7.37 (m, 8H), 7.22 (m, 4H), 3.98
(s, 6H), 3.85 (s, 6H), 3.77 (m, 2H), 3.62 (m, 2H), 3.55 (s, 3H), 3.53 (s,
3H), 3.20 (m, 2H), 2.01 (s, 3H), 2.00 (s, 3H), 1.38 (s, 3H), 1.39 (s, 3H).
50
1,10,6,60,7,70-Hexahydroxy-3,30-dimethyl-N5,N -bis(2-phenylpro-
pyl)-2,20-binaphthyl-5,50-dicarboxamide (8r). An amount of 0.45 mL
of BBr3 solution (1.18 g, 4.73 mmol) was added dropwise into a
solution of compound 7 (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 (3ꢀ50 mL). The combined orga-
nic layer was washed with water, brine and dried over MgSO4. The
solvent was concentrated in vacuo and the residue was purified using
C-18 column chromatography (H2O/acetonitrile) to give 200 mg of
5
5
0
N ,N -Bis(3-chlorobenzyl)-1,10,6,60,7,70-hexahydroxy-3,30-
dimethyl-2,20-binaphthyl-5,50-dicarboxamide (8i). Yield, 70%;
1H NMR (600 MHz, CD3OD) δ 7.59 (d, J=4.2 Hz, 4H), 7.46
(d, J=7.2 Hz, 2H), 7.35 (t, J1=J2=7.2 Hz, 2H), 7.28 (d, J=7.2
Hz, 2H), 7.15 (s, 2H), 4.68 (s, 4H), 1.93 (s, 6H). HPLC purity
98.0%, tR=6.71 min. HRMS calcd for C38H30N2O8 713.1452
(MþH), found 713.1426.
5
5
0
1,10,6,60,7,70-Hexahydroxy-3,30-dimethyl-N ,N -bis(2,4,6-tri-
methylbenzyl)-2,20-binaphthyl-5,50-dicarboxamide (8j). Yield,
70%; 1H NMR (600 MHz, CD3OD) δ 7.54 (s, 2H), 7.18 (s, 2H),
6.87 (s, 4H), 4.70 (s, 4H), 2.46 (s, 12H), 2.22 (s, 6H), 1.91 (s, 6H).
HPLC purity 98.8%, tR = 10.45 min. HRMS calcd for C44-
H44N2O8 729.3170 (M þ H), found 729.3167.
1
compound 8r (72%) as white-yellow solid. H NMR (600 MHz,
CD3OD) δ 7.56 (s, 2H), 7.37 (d, J=6.0 Hz, 4H), 7.32 (t, J1=J2 = 7.2
Hz, 4H), 7.20 (t, J1=J2=7.2 Hz, 2H), 7.07 (s, 1H), 6.99 (s, 1H),

4520 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Wei et al.
50
N5,N -Bis(1-(4-chlorophenyl)ethyl)-1,10,6,60,7,70-hexahydroxy-
followed by evaporation of the ether gave a yellow oil. The solution
of yellow oil in dry methylene chloride (10 mL) was added into
pyridinium chlorochromate (2.6 g, 12.1 mmol) in dry methylene
chloride (12 mL). The reaction mixture was stirred at ambient
temperature for 4 h and was filtered through Celite. The filtrate
was chromatographed to afford 0.3 g of 10e (22%). 1H NMR (600
MHz, CDCl3) δ 7.54 (s, 2H), 7.32 (m, 10H), 7.14 (s, 2H), 4.29 (s,
4H), 4.02 (s, 6H), 3.96 (s, 6H), 3.49 (s, 6H), 2.02 (s, 6H). To a
solution of compound 10e (170 mg, 0.29 mmol) in 10 mL of TFA
was added 0.6 mL of triethylsilane dropwise. The solution was
stirred overnight at room temperature and concentrated in vacuo
followed by silica gel column chromatography to give compound
11e as a colorless oil (140 mg, 90%). 1H NMR (600 MHz, CDCl3)
δ 7.67 (s, 2H), 7.44 (s, 2H), 7.35 (s, 8H), 7.25 (s, 2H), 4.04 (s, 6H),
3.95 (s, 6H), 3.60 (s, 6H), 3.41 (m, 4H), 3.02 (m, 4H), 2.18 (s, 6H).
3,30-Dimethyl-5,50-diphenethyl-2,20-binaphthyl-1,10,6,60,7,70-
hexaol (12e). An amount of 0.27 mL of BBr3 solution (0.72 g,
2.88 mmol) was added dropwise to a solution of 11e (200 mg,
0.30 mmol) in 8 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, respectively. Then 100 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, brine and dried over MgSO4. The solvent
was concentrated in vacuo and the residue was purified by C-18
column chromatography (H2O/acetonitrile) to give 128 mg of
compound 12e (75%) as an orange solid. 1H NMR (600 MHz,
CDCl3) δ 7.52 (s, 2H), 7.44 (s, 2H), 7.30 (m, 10H), 5.35 (s, OH,
4H), 5.17 (s, OH, 2H), 3.37 (t, J1=J2=6.6 Hz, 4H), 3.03 (t, J1=
J2=6.6 Hz, 4H), 2.13 (s, 6H). HPLC purity 99.5%, tR = 14.93
min. HRMS calcd for C38H34O6 587.2428 (M þ H), found
587.2425.
3,30-dimethyl-2,20-binaphthyl-5,50-dicarboxamide (8k). Yield,
73%; H NMR (600 MHz, CD3OD) δ 7.53 (m, 6H), 7.35 (m,
1
4H), 7.09 (s, 2H), 6.95 (s, 2H), 5.33 (m, 2H), 1.91 (s, 3H), 1.86 (s,
3H), 1.56 (m, 3H), 1.54 (m, 3H). HPLC purity 99.5%, tR=8.73
min. HRMS calcd for C40H34N2O8 741.1765 (M þ H), found
741.1763.
50
N5,N -Bis(cyclopropylmethyl)-1,10,6,60,7,70-hexahydroxy-3,30-
dimethyl-2,20-binaphthyl-5,50-dicarboxamide (8l). Yield, 70%; 1H
NMR (600 MHz, CD3OD) δ 7.58 (s, 2H), 7.26 (s, 2H), 3.36 (m,
4H), 1.97 (s, 6H), 1.18 (m, 2H), 0.57 (d, J=8.1 Hz, 4H), 0.37 (m,
4H). HPLC purity 98.5%, tR = 3.95 min. HRMS calcd for
C32H32N2O8 573.2231 (M þ H), found 573.2214.
0
N5,N5 -Bis(cyclohexylmethyl)-1,10,6,60,7,70-hexahydroxy-3,30-di-
methyl-2,20-binaphthyl-5,50-dicarboxamide (8m). Yield, 80%; 1H
NMR (600 MHz, CD3OD) δ 7.58 (s, 2H), 7.22 (s, 2H), 3.32 (m,
4H), 1.96 (s, 6H), 1.79 (d, J=7.2 Hz, 4H), 1.71 (d, J=8.4 Hz, 4H),
1.39-1.08 (m, 14H). HPLC purity 99.5%, tR=9.24 min. HRMS
calcd for C38H44N2O8 657.3170 (M þ H), found 657.3169.
5
5
1,10,6,60,7,70-Hexahydroxy-3,30-dimethyl-N ,N -diphenethyl-
0
1
2,20-binaphthyl-5,50-dicarboxamide (8n). Yield, 80%; H NMR
(600 MHz, CD3OD) δ 7.58 (s, 2H), 7.36 (d, J=7.2 Hz, 4H), 7.31
(t, J1=J2=7.2 Hz, 4H), 7.21 (t, J1=J2 = 7.2 Hz, 2H), 7.09 (s,
2H), 3.74 (m, 4H), 3.01 (t, J1=J2=7.2 Hz, 4H), 1.92 (s, 6H).
HPLC purity 99.2%, tR=5.89 min. HRMS calcd for C40H36-
N2O8 673.2544 (M þ H), found 673.2536.
5
5
0
1,10,6,60,7,70-Hexahydroxy-3,30-dimethyl-N ,N -bis(3-methylphe-
nethyl)-2,20-binaphthyl-5,50-dicarboxamide (8o). Yield, 76%; 1H
NMR (600 MHz, CD3OD) δ 7.57 (s, 2H), 7.23 (d, J=7.8 Hz,
4H), 7.11 (d, J=7.8 Hz, 4H), 7.06 (s, 2H), 3.80 (m, 4H), 2.96 (t, J1=
J2=7.2Hz, 4H), 2.29(s, 6H), 1.90(s, 6H), 1.40(s, 4H). HPLCpurity
98.0%, tR=7.83 min. HRMS calcd for C40H40N2O8 701.2857 (Mþ
H), found 701.2859.
50
N5,N -Bis(3-chlorophenethyl)-1,10,6,60,7,70-hexahydroxy-3,30-di-
Following the above-mentioned procedure and with use of
the appropriate starting materials and reagents, compounds
12a-e were synthesized.
methyl-2,20-binaphthyl-5,50-dicarboxamide (8p). Yield, 70%; 1H
NMR (600 MHz, CD3OD) δ 7.57 (s, 2H), 7.39 (s, 2H), 7.30 (t, J1
= 7.2 Hz, J2=6.6 Hz, 4H), 7.21 (d, J=6.6 Hz, 2H), 7.03 (s, 2H),
3.79 (m, 2H), 3.70 (m, 2H), 3.00 (m, 4H), 1.91(s, 6H). HPLC purity
98.5%, tR=7.72 min. HRMS calcd for C40H34N2O8 741.1765 (Mþ
H), found 741.1769.
5,50-Diisobutyl-3,30-dimethyl-2,20-binaphthyl-1,10,6,60,7,70-
1
hexaol (12a). Yield, 80%; H NMR (600 MHz, CD3OD) δ
7.44 (s, 2H), 7.34 (s, 2H), 2.95 (d, J=7.2 Hz, 4H), 2.14 (m,
2H), 2.05 (s, 6H), 1.02 (d, J=6.0 Hz, 6H), 1.00 (d, J=6.0 Hz,
6H). HPLC purity 98.5%, tR=13.19 min. HRMS calcd for
50
N5,N -Bis(4-ethylphenethyl)-1,10,6,60,7,70-hexahydroxy-3,30-
dimethyl-2,20-binaphthyl-5,50-dicarboxamide (8q). Yield, 75%;
1H NMR (600 MHz, CD3OD) δ 7.58 (s, 2H), 7.26 (d, J=7.8
Hz, 4H), 7.15 (d, J=7.8 Hz, 4H), 7.10 (s, 2H), 3.75 (m, 2H), 3.70
(m, 2H), 2.98 (t, J1=J2 = 7.2 Hz, 4H), 2.60 (q, J1=7.8 Hz, J2 =
7.2 Hz, 4H), 1.91 (s, 6H), 1.20 (t, J1=7.8 Hz, J2=7.2 Hz, 6H).
HPLC purity 99.7%, tR=11.44 min. HRMS calcd for C44H44-
N2O8 729.3170 (M þ H), found 729.3175.
C
30H34O6 491.2428 (M þ H), found 491.2429.
5,50-Diisopentyl-3,30-dimethyl-2,20-binaphthyl-1,10,6,60,7,70-
hexaol (12b). Yield, 79%; 1H NMR (600 MHz, CD3OD) δ 7.42
(s, 2H), 7.34 (s, 2H), 3.04 (t, J1=J2 = 5.4 Hz, 4H), 2.05 (s, 6H),
1.74 (m, 2H), 1.55 (m, 4H), 1.05 (d, J=3.6 Hz, 6H), 1.04 (d, J=
3.6 Hz, 6H). HPLC purity 99.5%, tR=15.56 min. HRMS calcd
for C32H38O6 519.2741 (M þ H), found 519.2739.
50
N5,N -Bis(2,3-dihydro-1H-inden-2-yl)-1,10,6,60,7,70-hexahy-
5,50-Bis(cyclopentylmethyl)-3,30-dimethyl-2,20-binaphthyl-
1,10,6,60,7,70-hexaol (12c). Yield, 78%; 1H NMR (600 MHz,
CD3OD) δ 7.41 (s, 2H), 7.37 (s, 2H), 3.06 (d, J=7.2 Hz, 4H),
2.36 (m, 2H), 2.03 (s, 6H), 1.72 (m, 8H), 1.50 (m, 8H). HPLC
purity 99.5%, tR = 16.93 min. HRMS calcd for C34H38O6
543.2741 (M þ H), found 543.2739.
droxy-3,30-dimethyl-2,20-binaphthyl-5,50-dicarboxamide (8s).
Yield, 72%; 1H NMR (600 MHz, CD3OD) δ 7.57 (s, 2H), 7.24
(s, 4H), 7.19 (s, 2H), 7.14 (s, 4H), 4.94 (m, 2H), 3.42 (m, 4H), 3.07
(m, 4H), 1.94 (s, 6H). HPLC purity 98.5%, tR = 6.66 min.
HRMS calcd for C42H36N2O8 697.2544 (M þ H), found
697.2541.
5,50-Dibenzyl-3,30-dimethyl-2,20-binaphthyl-1,10,6,60,7,70-hex-
aol (12d). Yield, 72%; 1H NMR (600 MHz, (CD3)2SO) δ 9.81 (s,
2H), 8.64 (s, 2H), 7.76 (s, 2H), 7.39 (s, 2H), 7.24 (m, 10H), 7.10
(m, 2H), 4.28 (dd, J1=15.0 Hz, J2=19.8 Hz, 4H), 1.94 (s, 6H).
HPLC purity 97.5%, tR=10.64 min. HRMS calcd for C36H30O6
559.2115 (M þ H), found 559.2112.
50
N5,N -Bis(4-chlorophenethyl)-1,10,6,60,7,70-hexahydroxy-3,30-di-
methyl-2,20-binaphthyl-5,50-dicarboxamide (8t). Yield, 75%; 1H
NMR (600 MHz, CD3OD) δ 7.57 (s, 2H), 7.35 (d, J=7.8 Hz,
4H), 7.30 (d, J=7.8Hz,4H),7.02(s,2H),3.76(m,2H),3.71(m,2H),
2.99 (t, J1=J2 = 6.6 Hz, 4H), 1.93 (s, 6H). HPLC purity 98.7%, tR
= 8.12 min. HRMS calcd for C40H34N2O8 741.1765 (M þ H),
found 741.1765.
3,30-Dimethyl-2,20-binaphthyl-1,10,6,60,7,70-hexaol (13). Com-
pound 4 (2.8 g, 5.1 mmol) was added in portions to 36 mL of
concentrated sulfuric acid. The solution was vigorous stirred for
50 min at room temperature. The reaction mixture was poured
onto ice and water mixture. The solution was extracted with
chloroform, and the organic layer was washed with water, dilute
ammonium hydroxide, and brine, dried, and concentrated
under vacuum. Purification by flash chromatography (10%
acetonitrile in CH2Cl2) followed by recrystallization from ben-
zene/methanol afforded 1.2 g of white solid as methylated
1,10,6,60,7,70-Hexamethoxy-3,30-dimethyl-5,50-diphenethyl-2,20-
binaphthalene (11e). To a fresh benzylmagnesium chloride (5.4
mmol) solution at room temperature was added a solution of 5
(1.0 g, 1.93 mmol) in anhydrous tetrahydrofuran (15 mL), and the
reaction mixture was stirred at this temperature for 12 h. The
reaction mixture was poured onto saturated ammonium chloride
solution, and the aqueous layer was extracted twice with diethyl
ether, washed with brine, and dried over MgSO4. Filtration

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4521
compound 13. Then 0.27 mL of BBr3 solution (0.72 g, 2.88 mmol)
was added dropwise into a solution of the above white solid
(87 mg, 0.19 mmol) in 8 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, respectively. An amount of 100 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 (3ꢀ50 mL). The combined organic
layer was washed with water, brine and dried over MgSO4. The
solvent was concentrated in vacuo and the residue was purified
by C-18 column chromatography (H2O/acetonitrile) to give 57 mg
Cells plated into 96-well plates at varying initial densities
depending on doubling time. H460 and H1299 plated at 2000
cells/well, A549 and PC3 at 3000 cells/well, and RS118456S at
10 000 cells/well. Compounds were diluted to final concentra-
tions with 0.1% DMSO. Prior to dispensing compounds onto
cells, fresh 5% medium was placed into wells. Administration of
compounds occurred 24 h after seeding into the fresh medium.
Cell viability was evaluated using ATP-LITE reagent (Perki-
nElmer) after 72 h of treatment. Data were normalized to the
DMSO control-treated cells using Prism, version 5.01 (Graph-
pad Software).
The apoptotic activity of the compounds against RS11846
cells was assessed by staining with annexin V and propidium
iodide (PI). Lymphoma cell line, RS11846, was cultured in
RPMI 1640 medium (Mediatech Inc., Herndon, VA 20171)
containing 10% fetal bovine serum (Mediatech Inc., Herndon,
VA 20171) and penicillin/streptomycin (Mediatech Inc., Herndon,
VA 20171). Cells were cultured with various concentrations of
5,50 substituted compound 2 derivatives for 1-2 days. The percen-
tage 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 the compounds, such as 8r and 8q
against mouse embryonic fibroblast wild-type cells (wt-MEFs)
and mouse embryonic fibroblast BAX/Bak double knockout
cells (DKO/MEFs), was assessed by staining with annexin V
and propidium iodide (PI). Wild-type MEFs and DKO/MEFs
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 concentrations of 0, 2.5, 5.0, 7.5, and 10 μM. On the
following day, floating cells were pooled with adherent cells
harvested after brief incubation with 0.25% trypsin/EDTA solu-
tion (Gibco/In-Vitrogen Inc.). Cells were centrifuged, and the
supernatant was discarded. The cell pellet was resuspended with
0.2 mL of annexin-V binding buffer, followed by addi-
tion of 1 μL of annexin-FITC and 1 μL of PI (propidium iodide).
The percentage of viable cells was determined by a three-color
FACSort instrument, and data were analyzed by Flow-Jo pro-
gram, scoring annexin V-negative, PI-negative as viable cells.
Bcl-2 Transgenic Mice Studies. Transgenic mice expressing
Bcl-2 have been previously described.44 The BCL-2 transgene
represents a minigene version of a t(14;18) translocation in
which the human BCL-2 gene is fused with the immunoglobulin
heavy-chain (IgH) locus and associated IgH enhancer. The
transgene was propagated on the Balb/c background. These
mice develop polyclonal B-cell hyperplasia with asynchronous
transformation to monoclonal aggressive lymphomas beginn-
ing at approximately 6 months of age, with approximately 90%
of mice undergoing transformation by the age of 12-24 months.
All animals used here had not yet developed aggressive lymphoma.
Mouse Experiments. Compounds dissolved in 500 μL of
solution (ethanol/Cremophor EL/saline = 10:10:80) were in-
jected intraperitoneally into age- and sex-matched B6Bcl2
mouse, while control mice were injected intraperitoneally with
500 μL of the same formulation without compound. After 24 h,
B6Bcl2 mice were sacrificed by intraperitoneal injection of a
lethal dose of Avertin. Spleen was removed and weighed. The
spleen weight of mice is used as an end-point for assessing
activity, as we determined that spleen weight is highly consistent
in age- and sex-matched Bcl-2-transgenic mice in preliminary
studies.20 Variability of spleen weight was within (2% among
control-treated age-matched, sex-matched B6Bcl2 mice.
1
of compound 13 (80%) as a white solid. H NMR (600 MHz,
CD3OD) δ 7.46 (s, 2H), 7.11 (s, 2H), 7.02 (s, 2H), 1.97 (s, 6H).
HPLC purity 99.0%, tR=3.46 min. HRMS calcd for C22H18O6
379.1176 (M þ H), found 379.1168.
Following above demethylation procedure, compound 14
was synthesized.
1,10,6,60,7,70-Hexahydroxy-3,30-dimethyl-2,20-binaphthyl-5,50-di-
carboxylic Acid (14). Yield, 70%; 1H NMR (600 MHz, CD3OD) δ
8.29 (s, 2H), 7.83 (s, 2H), 2.04 (s, 6H). HPLC purity 99.4%, tR=
2.56 min. HRMS calcd for C24H18O10 467.0973 (M þ H), found
467.0964.
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-2 in complex with a benzothiazole BH3 mimetic
ligand (Protein Data Bank code 1YSW).24,25 The ligand was
extracted from the protein structure and was used to define the
binding site for small molecules. Compound 2 and its derivatives
were docked into the Bcl-2 protein by the GOLD39 docking
program using ChemScore40 as the scoring function. The active
˚
site radius was set at 10 A, and 10 GA solutions were generated
for each molecule. The GA docking procedure in GOLD39
allowed the small molecules to flexibly explore the best binding
conformations, whereas the protein structure was kept static.
The protein surface was prepared with the program MOL-
CAD41 as implemented in Sybyl (Tripos, St. Louis) and
was used to analyze the binding poses for the studied small
molecules.
Fluorescence Polarization Assays (FPAs). A Bak BH3 peptide
(F-BakBH3) (GQVGRQLAIIGDDINR) was labeled at the
N-terminus 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 compound at varying concentrations in 47.5 μL of PBS
(pH 7.4) in 96-well black plates at room temperature for 10 min.
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 of
incubation at room temperature, the polarization values in
millipolarization units42 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 the mean of three independent experiments (
standard error (SE). Performance of Bcl-2 and Mcl-1 FPA is
similar. Briefly, 50 nM GST-Bcl-2 or -Mcl-1 was incubated with
various concentrations of compound 2 or its 5,50 substituted
derivatives for 2 min, then 15 nM FITC-conjugated-Bim BH3
peptide43 was added in PBS buffer. Fluorescence polarization
was measured after 10 min.
Cell Viability and Apoptosis Assays. The activity of the com-
pounds against human cancer cell lines (PC3 ML, H460, H1299,
RS11846) was assessed by using the ATP-LITE assay (Perkin-
Elmer). 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.
Acknowledgment. We thank the NIH (Grant CA113318
to M.P. and J.C.R.) and Coronado Biosciences (CSRA No.
08-02) for financial support.

4522 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Wei et al.
Supporting Information Available: Experimental details, che-
mical data for compounds 7a-7t and 11a-11c, NMR experi-
ments, isothermal titration calorimetry assays, in vitro ADME
studies, and in vivo MTD mice studies. This material is available
free of charge via the Internet at http://pubs.acs.org.
(22) Wei, J.; Rega, M. F.; Kitada, S.; Yuan, H.; Zhai, D.; Risbood, P.;
Seltzman, H. H.; Twine, C. E.; Reed, J. C.; Pellecchia, M. Synthesis
and evaluation of apogossypol atropisomers as potential Bcl-xL
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D. G.; Ng, S.; Nimmer, P. M.; O’Connor, J. M.; Oleksijew, A.;
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