Structure-activity relationship studies of phenanthridine-based Bcl-X L inhibitors

Article abstract of DOI:10.1021/jm8005433

Despite their structural similarities, the natural products chelerythrine (5) and sanguinarine (6) target different binding sites on the pro-survival Bcl-X_L protein. This paper details the synthesis of phenanthridine-based analogues of the natu

Full text of DOI:10.1021/jm8005433

J. Med. Chem. 2008, 51, 6699–6710
6699
Structure-Activity Relationship Studies of Phenanthridine-Based Bcl-XL Inhibitors
Paul H. Bernardo,† Kah-Fei Wan,‡ Thirunavukkarasu Sivaraman,§ Jin Xu,† Felicity K. Moore,† Alvin W. Hung,†
Henry Y. K. Mok,§ Victor C. Yu,‡ and Christina L. L. Chai*,†
Institute of Chemical & Engineering Sciences, Agency for Science Technology and Research (A*STAR), 1 Pesek Road,
Jurong Island, Singapore 627833, Institute of Molecular & Cell Biology, Agency for Science Technology and Research (A*STAR),
61 Biopolis DriVe, Singapore 138673, Departmental of Biological Sciences, National UniVersity of Singapore, Singapore 117543
ReceiVed May 10, 2008
Despite their structural similarities, the natural products chelerythrine (5) and sanguinarine (6) target different
binding sites on the pro-survival Bcl-XL protein. This paper details the synthesis of phenanthridine-based
analogues of the natural products that were used to probe this difference in binding profiles. The inhibitory
constants for these compounds were then measured in a fluorescence polarization assay against Bcl-XL and
the tagged Bak-BH3 peptide. The results led to structure-activity relationship studies, which identified the
structural motifs required for binding-site specificity as well as inhibitory activity. We also identified synthetic
analogues of the natural products that display similar binding modes but with more potent IC50 values.
Introduction
Apoptosis, or programmed cell death, is a process by which
unwanted or damaged cells undergo a series of regulated cellular
events resulting in the safe destruction of the targeted cells.
Many cancer cells, however, can prevent apoptosis from
occurring by overproducing antiapoptotic proteins such as Bcl-2
and Bcl-XL.1 One strategy for the development of therapeutics
for cancer chemotherapy is to inhibit these antiapoptotic proteins
and thus restore the potential to undergo apoptosis.2 Several
small molecule inhibitors of Bcl-XL have already been reported
in the literature (Figure 1) including 1 (ABT-737),3 2 (BH3I-1),4,5
gossypol (3),6,7 and its analogue 4 (TW-37).8,9 These compounds
display good (nM to µM) binding affinities toward Bcl-XL, and
in addition they are potent inducers of apoptosis in various
cancer cell lines. Recently, Chan et al. identified the natural
product chelerythrine (5) as an inhibitor of Bcl-XL through high-
throughput screening of natural products.10 Further investiga-
tions using NMR spectroscopy and computational methods show
that despite their structural similarity, chelerythrine and its
homologue sanguinarine (6) bind at different sites on the Bcl-
XL protein.11 To understand this difference in binding as well
as to develop a structure-activity relationship study, a number
of phenanthridine-based compounds that are structurally related
to chelerythrine and sanguinarine were synthesized using
complementary synthetic methods. These compounds were
prepared in order to assess the structural motifs required for
efficient binding of chelerythrine and sanguinarine analogues
to Bcl-XL. The results can be used to optimize the structure of
the chelerythrine and sanguinarine fragments to further enhance
binding to the target protein.
Synthesis
The compounds chosen for the study were phenanthridine-
based analogues of chelerythrine and sanguinarine that contain
the ABC-ring systems. The chelerythrine analogues are defined
Figure 1. Structures of some small molecule inhibitors of Bcl-XL.
as the compounds whose structures contain freely rotating
methoxy substituents at the 7- and 8-positions. The sanguinarine
analogues are defined as the compounds with a fused dioxolo
(-OCH2O-) group at the 7,8-positions. Previous studies have
shown that chelerythrine binds to Bcl-XL through H-bonding
interactions of the methoxy groups at an allosteric site known
as the BH groove. Similarly, sanguinarine has been shown to
* To whom correspondence should be addressed. Phone: +65 6796 3902.
Fax: +65 6316 6184. E-mail: christina_chai@ices.a-star.edu.sg.
†
Institute of Chemical & Engineering Sciences, Agency for Science
Technology and Research.
‡
Institute of Molecular & Cell Biology, Agency for Science Technology
and Research.
§
Departmental of Biological Sciences, National University of Singapore.
10.1021/jm8005433 CCC: $40.75
2008 American Chemical Society
Published on Web 10/17/2008

6700 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Bernardo et al.
Figure 2. Structures of compounds synthesized for study.
Scheme 1
bind to a site on the BH1 region adjacent to the BH3 binding
site, which can accommodate the constrained dioxolo group.
We therefore prepared a number of 7,8-dimethoxy compounds,
dioxolo compounds, and selected 7,8-dihydroxy compounds to
probe their binding to the Bcl-XL protein (Figure 2). These
structural analogues of chelerythrine and sanguinarine are then
tested against Bcl-XL and the Bak-peptide to enable the
measurement of the inhibition constants, and the most potent
inhibitors will be studied by NMR methods to determine the
binding sites on the protein.
Several A-ring substituted phenanthridines were synthesized
using a one-pot Suzuki-Miyaura coupling between pinacol
N-mesyl-2-aminophenylboronate (7) and the appropriate 2-bro-
mobenzaldehydes 8-10 using a modification of the methodol-
ogy reported by Snieckus.12 This synthetic methodology is
outlined in Scheme 1. Under the conditions utilized, the
formation of a carbon-carbon bond between the bromoben-
zaldehyde and aminophenyl boronic acid occurred, followed by
an in situ intramolecular Schiff base formation to yield the
neutral phenanthridines 11-13 in good yields. To obtain the
N-substituted species, these phenanthridines were treated with
iodomethane to afford the desired N-methylphenanthridinium
bromide salts 14-16 in 84-99% yields. Alternatively, the
corresponding N-oxides were prepared by treating the phenan-
thridines 11-13 with mCPBAa to obtain the zwitterionic
compounds 17-19 in high yields.
a Abbreviations: DDQ, 2,3-Dichloro-5,6-dicyano-p-benzoquinone; DIBAL,
diisobutylaluminium hydride; FPA, fluorescence polarization assay; mCPBA,
meta-chloroperoxybenzoic acid; MOMBr, methoxymethyl bromide; TBAF,
tert-butylammonium fluoride.

Phenanthridine-Based Bcl-XL Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6701
Scheme 2
Scheme 3
The general synthetic methodology employed for the synthesis
of 2-substituted phenanthridines is shown in Scheme 2. The
appropriately substituted bromobenzaldehydes 8 and 9 were
oxidized with AgNO3 to obtain the bromobenzoic acids 20 and
21. The bromobenzoic acids were then converted to the acid
chlorides in situ using SOCl2, and these acid chlorides were
then reacted with the anilines 22 and 23 to obtain the benzamides
24-26 in high yields.
To effect the intramolecular cyclization of the benzamides
to the corresponding phenanthridinones, it is necessary to
convert the secondary amides to the tertiary amides.13-16 This
transformation was accomplished by treating the benzamides
with NaH and MOMBr in dry THF to give the corresponding
N-MOM benzamides 27-29. The N-MOM benzamides were
then cyclized to the phenanthridinones 30-32 via an intramo-
lecular Heck reaction using catalytic Pd(OAc)2 and PPh3 in good
yields.
genated with DDQ to obtain the N-methylphenanthridinium
methyl ethers 40-41 in quantitative yields.
To access the 7,8-dihydroxy compounds 42-44, the com-
pounds 14, 40, and 41 were treated with BBr3 followed by
workup with aqueous HCl to obtain the desired products in good
yields (Scheme 3).
Fluorescence Polarization Assay (FPA)
Previous reports have shown that the reduction of the
N-methoxymethylphenanthridinones such as 30-32 can be
achieved using LiAlH4 to obtain the corresponding neutral
phenanthridines.13-16 Although the reduction to obtain the
neutral phenanthridines was successful, we found that the use
of DIBAL as the reducing agent directly furnished the N-methyl
5,6-dihydrophenanthridines in one step. Subsequent dehydro-
genation using DDQ and treatment with dilute HCl gave the
desired phenanthridinium salts 33-35 in high yields. The
structure of compound 35 was confirmed by X-ray analysis.
The phenanthridinones 31 and 32 were also used to prepare
2-substituted methyl ethers using a series of transformations.
In the first step, the removal of the TBDMS protecting group
was achieved by treating the phenanthridinones with TBAF to
obtain the hydroxy compounds 36-37. In the second step, these
hydroxy compounds were methylated using dimethyl sulfate to
obtain the methyl ethers 38-39. Finally, the methyl ethers
38-39 were reduced with DIBAL and subsequently dehydro-
Several Bcl-XL/Bcl-2 inhibitors have been identified from
high throughput screening based on fluorescence polarization.2,17
To assess the binding activity of each synthesized compounds
in Figure 2, a competitive binding assay based on fluorescence
polarization was performed to test the ability of these compounds
in disrupting the binding of the Bak-BH3 peptide to Bcl-XL.10,18,19
In the fluorescence polarization assay, the Bcl-XL protein is
incubated with a fluorescein-tagged Bak-BH3 peptide. The Bcl-
XL:Bak-BH3 peptide complex is then titrated with the test
compounds, and the displacement of the Bak-BH3 peptide is
measured as a function of decreasing polarized fluorescence.
In our assay, the concentrations of the test compounds ranged
from 125 to 0.5 µM. The binding affinities are reported as the
inhibitory concentration of the titrant required to displace 50%
of the Bak-BH3 peptide (IC50). The results are summarized in
Table 1. The results show that the benzophenanthridine 6 (IC50
) 19 µM) has a much stronger binding affinity to the Bcl-XL
protein than 5 (IC50 ) 100 µM). The chelerythrine analogue

6702 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Bernardo et al.
Table 1. IC50 Values From for the Displacement of Fluorescein-Tagged
Bak Peptide From Bcl-XL as Measured By Fluorescence Polarization
Assaya
and N-oxide) shows that the N-methyl compound 15 shows
favorable binding to Bcl-XL. The neutral compound 12 and its
N-oxide 18 however show poor binding activity toward Bcl-
XL. These results indicate that the presence of the cationic
N-methyl group is essential for efficient binding to the Bcl-XL
protein. This increase in binding may be due to a charge-charge
interaction of the cationic nitrogen with a negatively charged
carboxylate residue on the Bcl-XL protein.
IC50
(µM)
IC50
(µM)
IC50
(µM)
IC50
(µM)
compd
compd
compd
compd
5
6
11
12
13
100
19
>100
>100
>100
14
15
16
17
18
>100
12
>100
>100
>100
19
33
34
35
40
>100
>100
11
3
11
41
42
43
44
6
18
20
22
With respect to the substituents on the A-ring for the
sanguinarine analogues, it is interesting to note that the
dimethyldioxolo compound 16 is inactive. This compound
differs from the highly active compound 15 due to the presence
of an acetonide group (OC(CH3)2O) rather than a dioxolo group
(OCH2O). It is possible that the additional steric bulk provided
by the methyl groups, as opposed to the smaller hydrogen
groups, is sufficient to prevent the compound from binding. This
suggests that these compounds may bind at a site with a small
pocket, which can accommodate the constrained dioxolo group,
and bulkier groups such as the acetonide group, which is present
in compound 16, or the dimethoxy groups of the chelerythrine
analogues may prevent these compounds from binding effectively.
In summary, the SAR study shows that the 7,8-dimethoxy
chelerythrine analogues including the parent compound display
very poor binding activity. The 7,8-dihydroxy compounds, on
the other hand, are more potent than chelerythrine and equipotent
with sanguinarine toward Bcl-XL. With respect to the sangui-
narine analogues, several compounds show better promise as
inhibitors of Bcl-XL. This study also shows that the presence
of a quaternary nitrogen is also essential for the binding activity
of these phenanthridines. The constrained dioxolo ring system
also enhances the binding activity of these compounds. Fur-
thermore, the DE rings are not required for good binding
activity. Elaboration of the C-ring using ethylhydroxy and its
methyl ether (i.e., compounds 35 and 41) also significantly
improve the binding activity of these compounds to Bcl-XL.
To understand and study the binding of selected active
compounds from the SAR studies obtained from the FPA, we
turned our attention to NMR studies and docking simulations.
a Note: IC50 values >100 µM Indicates Inactive.
14, which lacks the DE-rings, fails to displace the Bak-BH3
peptide at the concentrations tested. Interestingly, none of the
7,8-dimethoxyphenanthridines (chelerythrine analogues 11, 14,
17, 33) displaced the Bak peptide at the concentrations tested.
For the sanguinarine analogues, five compounds (15, 34-35,
40-41) display more potent binding activity than the parent
compound with IC50 values ranging from as low as 3 to 11 µM.
All three 7,8-dihydroxy phenanthridines (42-44) show im-
proved inhibitory activity as compared to chelerythrine and equal
potency to sanguinarine with IC50 values ranging from 18-22
µM. It is also noteworthy that none of the neutral phenan-
thridines nor the N-oxides displayed significant activity in the
concentration range tested.
Structure-Activity Relationship Study
Our studies show that none of the 7,8-dimethoxyphenan-
thridines (chelerythrine analogues) had IC50 values below 100
µM. However, it should also be noted that the parent compound
chelerythrine itself shows poor binding activity against Bcl-XL
and the Bak-peptide in FPA (IC50)100 µM).11 However, several
7,8-dihydroxy phenanthridines (42-44) showed lower µM
inhibitory activity against Bcl-XL. The 7,8-dihydroxyphenan-
thridinium chloride (42) showed the greatest activity against
Bcl-XL (IC50) 18 µM). The 2-substituted compounds 43 and
44 showed similar but slightly weaker activity than 42.
Interestingly, these compounds are equipotent to sanguinarine
but more active than chelerythrine, suggesting that hydroxy
groups improved the inhibitory toward Bcl-XL as compared to
methoxy groups. It is also interesting to note that, despite the
presence of C2 groups on compounds 43-44, these compounds
show similar binding activity as the simpler compound 42. This
suggests that C7 and C8 substituents have a greater impact on
the inhibitory activity against Bcl-XL as compared to the C2
substituents.
With respect to the sanguinarine analogues, several com-
pounds (15, 34-35, 40-41) exhibit better binding activity
toward Bcl-XL than the parent compound 6 (IC50 ) 19 µM).
These results suggest that the DE-rings are not necessary for
the binding of sanguinarine to Bcl-XL. For instance, the simplest
sanguinarine analogue 15 has an IC50 of 12 µM. Furthermore,
the compounds 34-35 have IC50 values of 11 and 3 µM
respectively, and the methyl ethers 40-41 have IC50 values of
11 and 6 µM. Compounds 34 and 40 are the 2-substituted
analogues that have a methylhydroxy and methylmethoxy group,
respectively. These two compounds are only moderately more
active than the analogue 15, which suggests that the additional
binding conferred by the short tail is weak. However, the longer
tail of the ethylhydroxy and ethylmethoxy compounds 35 and
41 are 2-4 times more potent than the analogue 15. This may
be explained by an additional hydrogen bonding interaction
between the oxygen atom on the 2-ethyloxy group and a protein
residue that enhances binding.
NMR Studies and Docking Simulations
NMR titration studies were carried out to determine the
binding site of the ligands 35 and 42 to the Bcl-XL proteins.
This was carried out by first preparing 15N-labeled human Bcl-
XL as described previously.11 The labeled proteins were then
titrated with the test compounds, and the chemical shift
perturbations of the amide cross-peaks in the ligand:protein
complex relative to the unbound protein were determined using
1H-15N heteronuclear single quantum correlation (HSQC)
experiments. The results of the NMR titration studies with
human Bcl-XL are shown in Figure 3. The results show that
compound 42 strongly perturbs the 11 different residues on the
protein by more than 0.075 ppm. Similarly, compound 35 causes
significant chemical shifts perturbations for 13 protein residues
over the same threshold. One striking observation is that
chemical shift perturbation for each ligand-protein complex is
very different in terms of the residues affected as well as the
magnitude of the shifts. The results show that the two complexes
have only three shifted residues in common: L99, R132, and
L178. However, both complexes share a common perturbed
region at the BH1 domain where residues are disturbed by
compound 42 (Q125, R132, A142, F146), while six residues
are disturbed by compound 35 (S122, V126, E129, R132, D133,
G134). These results indicate that the binding sites of the two
compounds are not identical, but they are likely to share a
A study of the sanguinarine-based analogues (12, 13, and
18) with variations at the 5-position (neutral nitrogen, N-methyl

Phenanthridine-Based Bcl-XL Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6703
Figure 3. Combined amide proton and nitrogen chemical shift perturbations of Bcl-XL derived from the 1H-15N HSQC spectra after titration with
compounds 35 (A) and 42 (B) are plotted against the residual number of the protein. The molar ratio of ligand to protein was 10:1 in both titrations.
Residues that are perturbed by more than an arbitrary threshold value of 0.075 ppm are labeled.
Figure 4. Docking models of compounds 35 and 42 bound to Bcl-XL. The protein is represented as a surface diagram (A) and ribbon diagram (B),
and the docked ligands on the protein are shown as sticks. Compounds 35 and 42 bind at the BH1 domain and BH groove of the protein respectively.
Residues such as E129, D133, V135, A142, Y173, and E184 that play an essential role for binding of compound 35 or 42 are colored in yellow.
common region on the protein that is likely to be the BH1
domain. This BH1 domain is comprised of a portion of helices
4-5 and its connecting loop and is located between the BH
groove (chelerythrine binding site) and the BH3 binding groove
(sanguinarine binding site).
hybrid GA-LS runs were carried out. A total of 100 possible
binding conformations were generated and grouped into clusters
based on a 1.0 Å cluster tolerance.
The docking results for the compound 42 show 98 out of
100 possible conformers in a single cluster when the cluster
tolerance is set to 1.0 Å. The cluster is located at the top portion
of the BH groove (Figure 4 A). It was found that the hydrophilic
sidechains from residues H177 and Y173 are in close proximity
to the hydroxy groups of compound 42. The sidechains of
residues V135, L178, and W181 are also shown to interact with
compound 42 via hydrophobic interactions. The docking model
suggests that compound 42 binds selectively at the BH groove,
the region where chelerythrine has been shown to bind.
The docking results for compound 35 in Figure 4 yielded
two clusters with a cluster tolerance of 1.0 Å. The major cluster
contains 72 conformers docked at the BH1 region (Figure 4A).
Furthermore, a single cluster is obtained when the tolerance is
adjusted to 1.1 Å. The docking results show that the side chain
of the residues L108, L130, F131, A142, and F143 interact with
the sanguinarine analogue by hydrophobic interactions, while
E129 is in close contact to the cationic nitrogen atom of
It should be noted however that the chemical shift perturba-
tions can be caused by direct protein-ligand interactions or by
indirect protein conformational changes induced by the ligand.
Docking studies using AutoDockSuite 4.0.1 were thus employed
using a rigid protein structure to determine if the observed
chemical shift perturbations are likely to arise from direct
ligand-protein interactions.
20
The NMR structure of the human Bcl-XL was used as the
rigid protein for docking studies, and the ligand structures of
35 and 42 were generated using Sybyl. The Autodock 4.0.1
program equipped with ADT was used for generating docking
models by employing a Genetic Algorithm-Local Search (GA-
LS) method for the docking of the flexible ligands to the rigid
protein.21 A grid box was selected to cover the residues in the
regions of interest (BH groove, BH3 binding groove, and BH1
region) for AutoDock calculations. For each docking job, 100

6704 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Bernardo et al.
Table 2. Degree of Overall Chemical Shift Perturbation Caused By
Compounds 42 and 35 on the Backbone NHs of Various Human Bcl-XL
Mutants as Examined by 1H-15N HSQC Spectra
mutant
structural context
compd 35
compd 42
E129A
L130A
D133K
V135A
R139A
A142G
Y173F
E184A
BH1
BH3 groove
BH1
BH groove
BH3 groove
BH1
BH groove
BH groove
+ + +
+ + +
+ + +
+ + + +
+ + + +
+
+ + + +
+ + +
+ + + +
+ +
+ + + +
+ +
+ + + +
+ + + +
+
+ + +
compound 35 (Figure 4B). These results suggest that compound
35 binds at the same region where sanguinarine binds.
To verify the binding sites predicted by docking studies,
further NMR studies were conducted in which mutants of the
Bcl-XL protein were employed. The point mutations were based
on the residues predicted by the model to be essential for binding
(E129A, L130A, D133K, V135A, R139A, A142G, Y173F, and
E184A). These mutations correspond to residues from three
different regions of the protein (R139A and L130A from the
BH3 binding groove, E129A, D133K, and A142G from the BH1
domain, and V135A, Y173F, and E184A from the BH groove).
The mutants were then titrated with compounds 35 or 42 and
analyzed by HSQC. The qualitative results are shown in
Table 2.
Figure 5. Effect of mutations at different sites of human Bcl-XL on
the binding of the analogue 35 and the analogue 42 based on the extent
of chemical shift perturbation. (A) The 1H-15N HSQC spectra of
R139A and A142G mutant before and after titration with 35. (B) The
1H-15N HSQC spectra of R139A and Y173F mutant before and after
titration with 42. In all spectra, the cross peaks are colored black for
free Bcl-XL mutant protein and red for protein in the presence of 42 or
35.
The overall chemical shift perturbation showed that A142G
mutation, which is in the BH1 region, drastically inhibited the
binding of compound 35 to Bcl-XL (Figure 5). The binding of
35 to Bcl-XL was moderately inhibited by mutations such as
E129A and D133K, which are also in the BH1 domain.
However, mutation on the BH groove such as V135A, Y173F,
and E184A showed little effect on the binding of 35 to Bcl-XL
(Table 2).
analogues together to see determine if a more potent bidentate
compound can be obtained.
In contrast, mutations on the BH groove such as V135A,
Y173F, and E184A inhibited significantly the binding of
compound 42 to Bcl-XL (Figure 5). It was also noted that the
mutations on the BH1 region such as E129A and D133K showed
little effect on the binding of 42 (Table 2). Taken together, the
extent of chemical shift perturbation obtained for human Bcl-
XL mutants upon titration as well as the docking models for
these compounds on the protein demonstrates that compounds
35 and 42 bind at different binding sites, the BH1 domain, and
the BH groove of human Bcl-XL, respectively. Furthermore,
these experiments provide strong evidence that the analogue
42 binds to the same site as chelerythrine and, similarly, the
analogue 35 binds to the same site as sanguinarine.
Experimental Section
Chemistry. General Experimental Section. 1H NMR and 13
C
NMR spectra were acquired on a Bruker 400 UltraShield spec-
trometer operating at 400 and 100 MHz, respectively. All the proton
spectra were referenced to the respective residual solvent peaks
(MeOH-d4: 3.20; DMSO-d6: 2.50; D2O: 4.79; CD2Cl2: 5.31 ppm),
except for those recorded in CDCl3, were referenced to TMS.
Carbon spectra were referenced to the central peak of the respective
residual solvents (MeOH-d4: 49.0; DMSO-d6: 39.5; CDCl3: 77.0;
CD2Cl2: 53.7 ppm). Low-resolution and high-resolution electron
impact mass spectra (EIMS) were measured using a Finnigan
MAT95XP double-focusing mass spectrometer. Low-resolution
electrospray ionization (ESI) mass spectra were recorded using
Waters Quattro Micro API, and high resolution mass spectra were
obtained using the Agilent 6210 time-of-flight LC/MS. Infrared (IR)
spectra were measured on a BioRad FTIR spectrophotometer with
samples analyzed as KBr discs or thin films on a KBr plate.
Elemental analysis was performed using a EuroEA3000 series
CHNS Analyzer. X-ray analysis data was obtained using a Rigaku
single crystal X-ray diffraction system with Saturn-70 CCD detector.
Preparative HPLC was carried out using either on a Waters 600
controller and 2996 detector with an Xterra C18 19 mm × 50 mm
column, or a Waters 2545 binary gradient module and 2996 detector
with a Luna C18 21.2 mm × 250 mm column.
Conclusion
We have thus synthesized a number of chelerythrine and
sanguinarine analogues and tested them in FPA assays for their
inhibitory activity against Bcl-XL and the Bak-peptide. We have
shown that three dihydroxy compounds show greater potency
against Bcl-XL than the natural product chelerythrine. We have
also identified five sanguinarine analogues that are more potent
than the corresponding natural product. A combination of NMR
titration studies and in silico docking studies have shown that
our most potent compounds, the sanguinarine analogue 35 and
compound 42, bind at the BH1 domain and BH groove
respectively, similar to the binding preference of their natural
parent compounds. Further work is underway to link these
Experimental Details. Preparation of 2-Bromo-5,6-dimethoxy-
benzaldehyde (8). A mixture of 2-bromo-5-methoxy-6-hydroxy-
benzaldehyde (4.62 g, 20 mmol), dimethyl sulfate (3 mL, 32 mmol),
and potassium carbonate (8.28 g, 60 mmol) in acetone (300 mL)
was refluxed for 5 h and then cooled down. The solvent was
removed in vacuo, and the residue was partitioned between water

Phenanthridine-Based Bcl-XL Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6705
and ether, then further extracted with ether (3 × 200 mL). The
ether extract was washed successively with saturated NaHCO3,
water, and brine and then dried with MgSO4. Concentration of the
organic layer left a solid, which was then recrystallized from ether
as pale-yellow needles (4.4 g, 90% yield). Rf (1:4 EtOAc/hexanes):
0.34; mp: 75-77 °C (lit.22 76-77 °C). 1H NMR (CDCl3): δ 10.34
(s, 1H), 7.34 (d, J ) 9 Hz, 1H), 6.96 (d, J ) 9 Hz, 1H), 3.93 (s,
3H, OMe), 3.89 (s, 3H, OMe). HRMS (EI+): calcd 243.9769 for
C9H979BrO3 [M]+and 245.9715 for C9H981BrO3 [M]+; found,
243.9730 and 245.9709, respectively.
0.88 (s, 9H, 3 × CH3), 0.00 (s, 6H, 2 × CH3). HRMS (ESI+):
calcd 252.1778 for C14H25NOSi [M + H]+; found, 252.1795.
Synthesis of Phenanthridines via Suzuki Coupling. 7,8-Dimethox-
yphenanthridine (11). To a solution of 2-bromo-5,6-dimethoxy-
benzaldehyde (500 mg, 2 mmol) and pinacol 2-aminophenylbor-
onate (608 mg, 2 mmol) in DMF (15 mL) under nitrogen was added
anhydrous Na2CO3 (0.4 g, 3.9 mmol), Pd(OAc)2 (43 mg, 0.20
mmol), and PPh3 (0.2 g, 0.78 mmol). The mixture was thrice
degassed and flushed with nitrogen, then heated in a sealed
tube for 2h at 160 degrees under microwave conditions (Biotage
initiator). The reaction mixture was filtered through Celite, and the
DMF solvent was removed under reduced pressure. The crude
product was extracted with CHCl3 (3 × 150 mL), and the organic
layer was washed with brine, dried with MgSO4, and then filtered.
The solvent was removed under reduced pressure, and the resulting
crude mixture was subjected to column chromatography on silica
to give the desired phenanthridine as yellow crystals in 76% yield.
Pale-yellow solid; mp: 111-113 °C from EtOAc/hexane (lit.
121-122 °C, petroleum ether).25 1H NMR (CDCl3): δ 9.64 (s, 1H),
8.49 (br d, J ) 8 Hz, 1H), 8.33 (d, J ) 9 Hz, 1H), 8.20 (br d, J )
8 Hz, 1H), 7.68 (m, 2H), 7.59 (d, J ) 9 Hz, 1H), 4.11(s, 3H, OMe),
5-Bromobenzo[d][1,3]dioxole-4-carbaldehyde (9). This com-
pound was purchased from Sigma-Aldrich (product no. 597376).
Preparation of 5-Bromo-2,2-dimethylbenzo[d][1,3]dioxole-4-car-
baldehyde (10). To a solution of 5-bromo-2,2-dimethylbenzo[d][1,3]-
dioxole (1.2 g, 5.24 mmol) in dry THF (15 mL) was added N,N-
diisopropylamine (0.812 mL, 5.76 mmol) and n-BuLi (1.3 M in
hexanes, 4.44 mL, 5.76 mmol). The mixture was stirred at -78 °C
for 15 min and then DMF (0.51 mL, 6.55 mmol) was added and
the resulting mixture was stirred at -78 °C for a further 15 min.
The resulting mixture was then warmed to rt and stirred for a further
4 h. The reaction was quenched by the addition of saturated Na2CO3.
The crude product was extracted with ethyl acetate (3 × 150 mL),
and the organic layer was washed with brine, dried with MgSO4,
and then filtered. The solvent was removed under reduced pressure,
and the resulting crude mixture was subjected to column chroma-
tography on silica to give the product as pale-yellow crystals in
74% yield. Rf (1:10, EtOAc/hexane): 0.2; mp: 92 °C (lit. 91-92
°C).23 1H NMR (CDCl3): δ 10.22 (s, 1H), 6.99 (d, J ) 8 Hz, 1H),
6.69 (d, J ) 8 Hz, 1H), 1.68 (s, 6H).
2-Bromo-5,6-dimethoxybenzoic Acid (20). To 2-bromo-5,6-
dimethoxybenzaldehyde (8) (300 mg, 1.22 mmol) in MeCN (5 mL)
was added AgNO3 solution (0.1 M, 24 mL) and 10% NaOH solution
(6 mL). The mixture was stirred at room temperature for 16 h and
then filtered through Celite 545. MeCN was removed in vacuo.
The aqueous phase was acidified with 4 M HCl until the pH ) 5,
and then the organic acid was extracted with EtOAc (20 mL). The
organic layer was washed with 1 M HCl, dried with MgSO4, and
concentrated in vacuo to afford the product (274 mg, 86% yield)
as a colorless oil. Rf (1:9 MeOH/CH2Cl2): 0.18. 1H NMR (CDCl3):
δ 7.27 (d, J ) 9 Hz, 1H), 6.86 (d, J ) 9 Hz, 1H), 3.92 (s, 3H,
OMe), 3.88 (s, 3H, OMe). MS (CI-) (m/z, %): 259 (95), 261 [M
- H]- 100).
+
4.05 (s, 3H, OMe). HRMS (ESI+): calcd 240.1019 for C15H14NO2
[M + H]+; found, 240.1014.
[1,3]Dioxolo[4,5-i]phenanthridine (12). Pale-yellow solid; mp:
148-149 °C. 1H NMR (CDCl3): δ 9.34 (s, 1H), 8.44 (br d, J ) 8
Hz, 1H), 8.14 (br d, J ) 8 Hz, 1H), 8.11 (d, J ) 9 Hz, 1H), 7.66
(m, 2H), 7.42 (d, J ) 9 Hz, 1H), 6.27 (s, 2H, OCH2O). HRMS
(ES+): calcd 223.0633 for C14H9NO2 [M]+; found, 223.0637.
Preparation of 2,2-Dimethyl-[1,3]dioxolo[4,5-i]phenanthridine
(13). Rf (1:5 EtOAc/hexanes): 0.4; mp: 118-120 °C. 1H NMR
(CDCl3): δ 9.25 (s, 1H), 8.34 (d, J ) 8 Hz, 1H), 8.07 (d, J ) 8
Hz, 1H), 7.98 (d, J ) 9 Hz, 1H), 7.56 (m, 2H), 7.27 (d, J ) 9 Hz,
1H), 1.75 (s, 6H). 13C NMR (CDCl3): δ 146.8, 145.9, 143.9, 142.5,
129.9, 127.8, 127.6, 126.8, 124.5, 122.0, 120.7, 114.6, 113.7, 112.0,
26.05. IR (KBr): 3469, 2933, 1641, 1469, 1379, 1290, 1255, 1236,
1222, 1109, 1022, 951, 916, 826, 760 cm-1. HRMS (EI+): calcd
251.0946 for C16H13NO2 [M]+; found: 251.0940.
General Procedure for the N-Methylation of the Phenan-
thridines. To a suspension of the phenanthridine (typically 50 mg)
in dry toluene was added iodomethane (5 eq). The reaction mixture
was heated at 80 °C for 16 h in a sealed tube. The resulting
precipitate was then filtered and washed with cold toluene (5 mL)
and diethyl ether (5 mL) and then dried in vacuo.
5-Bromobenzo[d][1,3]dioxole-4-carboxylic Acid (21). The title
compound was obtained in 98% yield as a white solid from
commercially available 5-bromobenzo[d][1,3]dioxole-4-carbalde-
hyde (9) following the same procedure for the preparation of 20;
mp: 167-168 °C (lit. 166-168 °C).24 1H NMR (MeOH-d4): δ 7.09
(d, J ) 8 Hz, 1H), 6.81 (d, J ) 8 Hz, 1H), 6.06 (s, 2H). HRMS
(ESI+): calcd for 266.9263 for C8H579BrO4Na [M + Na]+and
268.9248 for C8H581BrO4Na [M + Na]+; found, 266.9269 and
268.9250.
7,8-Dimethoxy-5-methylphenanthridinium Iodide (14). Obtained
as an orange solid from 11 (79 mg, 99% yield); mp: 202-208 °C
1
(dec). H NMR (DMSO-d6): δ 10.18 (s, 1H), 9.07 (m, 1H), 8.82
(d, J ) 9 Hz, 1H), 8.47 (m, 1H), 8.25 (d, J ) 9 Hz, 1H), 8.06 (m,
2H), 4.71 (s, 3H, NCH3), 4.15 (s, 3H, OMe), 4.10 (s, 3H, OMe).
13C NMR (DMSO-d6): δ 151.3, 151.0, 146.7, 133.1, 130.8, 130.3,
127.4, 125.5, 125.3, 124.3, 119.9, 119.1, 119.0, 62.3, 57.0, 45.8.
HRMS (ESI+): calcd 254.1181 for C16H16NO2 [M]+; found,
254.1176.
(4-Aminophenyl)methyl-tert-butyldimethylsilyl Ether (22). To a
solution of (4-aminophenyl)methanol (3.69 g, 30 mmol) in anhy-
drous DMF (60 mL) under argon was added imidazole (4.08 g, 60
mmol). The mixture was stirred at room temperature for 30 min,
and then TBDMSCl (4.6 g, 30.5 mmol) in DMF (40 mL) was
slowly added via syringe. The reaction mixture was stirred for 3 h.
The solvent was removed under reduced pressure, and the residue
was purified by column chromatography on silica (15% ethyl acetate
in hexane) to give the desired product (4.32 g, 61% yield) as a
[1,3]Dioxolo-5-methyl-[4,5-i]phenanthridinium Iodide (15). Pre-
pared from 12. The product was further purified by preparative
HPLC (Luna C18, 21.2 × 250 mm, flow rate: 20 mL/min, mobile
phase: acetonitrile in water with 0.1% formic acid, gradient:
10-70% over 15 min, retention time (Rt) 9.1 min) to afford the
pure product as an orange solid (68 mg, 84% yield). mp: 125-127
1
°C. H NMR (DMSO-d6): δ 10.17 (s, 1H), 8.95 (m, 1H), 8.56 (d,
J ) 9 Hz, 1H), 8.42 (m, 1H), 8.04 (d, J ) 9 Hz, 1H), 8.01 (m,
2H), 6.58 (s, 2H, OCH2O), 4.63 (s, 3H, NCH3). 13C NMR (DMSO-
d6): δ 150.3, 147.8, 147.6, 133.0, 130.7, 130.6, 126.5, 125.7, 124.4,
120.2, 119.3, 116.9, 109.0, 104.9, 45.5. HRMS (ESI+): calcd
238.0868 for C15H12NO2 [M]+; found, 238.0861.
1
colorless oil. Rf (1:4 EtOAc/hexane): 0.25. H NMR (CDCl3): δ
7.10 (d, J ) 8 Hz, 2H), 6.65 (d, J ) 8 Hz, 2H), 4.62 (s, 2H), 0.91
(s, 9H, 3 × CH3), 0.08 (s, 6H, 2 × CH3). HRMS (CI+): calcd
238.1622 for C13H24NOSi [M + H]+; found, 238.1684.
Preparation of 2,2-Dimethyl-[1,3]dioxolo-5-methyl-[4,5-i]phenan-
thridinium Iodide (16). Prepared from 13. Recrystallization from
CHCl3/hexane yielded the desired product as orange crystals in
(4-Aminophenyl)ethyl-tert-butyldimethylsilyl Ether (23). (4-
Aminophenyl)ethyl-tert-butyldimethylsilyl ether was obtained as
a colorless oil from 2-(4-aminophenyl)ethanol in 95% yield
following the general procedure as above. Rf (1:4 EtOAc/hexane):
0.27. 1H NMR (CDCl3): δ 6.99 (d, J ) 8 Hz, 2H), 6.62 (d, J ) 8
Hz, 2H), 3.74 (app. t, J ) 7 Hz, 2H), 2.71 (app. t, J ) 7 Hz, 2H),
1
quantitative yield; mp: 192-195 °C (dec). H NMR (CDCl3): δ
10.55 (s, 1H), 8.65 (m, 1H), 8.25 (m, 1H), 8.20 (d, J ) 9 Hz, 1H),
7.93 (m, 2H), 7.68 (d, J ) 9 Hz, 1H), 4.90 (s, 3H), 1.93 (s, 6H).
13C NMR (CDCl3): δ 150.2, 148.5, 148.0, 133.0, 130.8, 130.7,

6706 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Bernardo et al.
126.6, 126.4, 124.7, 124.1, 119.41, 119.38, 115.3, 109.3, 47.2,
26.28. IR (KBr, cm-1): 3450, 3050, 2991, 1641, 1617, 1489, 1347,
1297, 1251, 1213, 1156, 762. HRMS (EI+): calcd 266.1177 for
C17H16NO2 [M]+; found, 266.1179.
General Procedure for the N-Oxidation of Phenanthridines. To
a solution of the phenanthridine (typically 90 mg) in DCM (1 mL)
was added mCPBA (177 mg). The mixture was stirred at rt for
16 h, and then saturated NaHCO3 was added, followed by addition
of more DCM (15 mL). The DCM layer was washed with water
and then brine and dried over Na2SO4. After removal of DCM in
vacuo, the crude product was purified by column chromatography
on silica (1:9 i-PrOH/hexanes) to give desired product.
(ESI+): calcd 486.0707 for C21H2679BrNO4SiNa [M + Na]+; found,
486.0699. Anal. (C21H26BrNO4Si) C, H, N.
5-Bromo-N-(4-(2-tert-butyldimethylsilyloxyethyl)phenyl)benzo[d]-
[1,3]dioxole-4-carboxamide (26). The product was obtained as a
white solid in 93% yield from 21 and 23 using the same reaction
conditions for the preparation of 24. Rf (1:4 EtOAc/hexanes): 0.24;
1
mp: 103-104 °C. H NMR (CDCl3): δ 7.68 (br s, 1H, NH), 7.55
(d, J ) 8 Hz, 2H), 7.20 (d, J ) 8 Hz, 2H), 7.10 (d, J ) 8 Hz, 1H),
6.75 (d, J ) 8 Hz, 1H), 6.07 (s, 2H, OCH2O), 3.79 (app. t, J ) 7
Hz, 2H), 2.81 (app. t, J ) 7 Hz, 2H), 0.88 (s, 9H, 3 × CH3), 0.00
(s, 6H, 2 × CH3). 13C NMR (CDCl3): δ 161.0, 147.5, 146.5, 136.0,
135.5, 129.7, 126.5, 120.0, 119.5, 111.0, 110.8, 102.5, 64.5, 39.1,
25.9, 18.3, -5.4. FTIR (KBr, cm-1): 3289, 2929, 2857, 1657, 1605,
1542, 1411, 1325, 1247, 1110, 1046, 834, 775. HRMS (ESI+): calcd
478.1044 for C
7,8-Dimethoxy-5-methylphenanthridine-5-oxide (17). Yellow
1
solid; mp: 144-162 °C (dec). H NMR (CDCl3): δ 9.17 (s, 1H),
22H2979BrNO4Si [M + H]+ and 480.1029 for
8.87 (m, 1H), 8.45 (m, 1H), 8.20 (d, J ) 9 Hz, 1H), 7.76 (m, 2H),
7.44 (d, J ) 9 Hz, 1H), 4.05 (s, 3H, OCH3), 4.04 (s, 3H, OCH3).
13C NMR (CDCl3): δ 151.0, 142.5, 138.2, 130.6, 129.3, 128.7,
126.6, 122.4, 122.1, 121.5, 120.7, 118.1, 116.3, 61.7, 56.5. HRMS
(ESI+): calcd 256.0968 for C15H14NO3 [M + H]+; found, 256.0967.
[1,3]Dioxolo[4,5-i]phenanthridine-5-oxide (18). Yellow solid; mp:
178-197 °C (dec). 1H NMR (CDCl3 + MeOH-d4): δ 8.87 (s, 1H),
8.79 (d, J ) 3 Hz, 1H), 8.42 (d, J ) 5 Hz, 1H), 8.01 (d, J ) 8 Hz,
1H), 7.76 (m, 2H), 7.31 (d, J ) 5 Hz, 1H), 6.25 (s, 2H, OCH2O).
13C NMR (CDCl3 + MeOH-d4): δ 146.9, 142.1, 138.0, 129.9,
129.4, 129.0, 127.0, 122.8, 121.9, 120.6, 116.0, 112.2, 111.7, 103.1.
HRMS (ESI+): calcd 240.0655 for C14H10NO3 [M + H]+; found,
240.0648.
C22H2981BrNO4Si [M + H]+; found, 478.1041 and 480.1024,
respectively. Anal. (C22H28BrNO4Si) C, H, N.
General Procedure for the N-MOM Protection of Benza-
mides. 2-Bromo-N-(4-(tert-butyldimethylsilyloxymethyl)phenyl)-
N-(methoxymethyl)-5,6-dimethoxybenzamide (27). To the benza-
mide 24 (100 mg, 0.208 mmol) in THF (5 mL) under argon was
added portionwise NaH (50 mg, 1.25 mmol). The mixture was
stirred at room temperature for 20 min, and then MOMBr (37.8
µL, 0.42 mmol) was added via syringe to the reaction mixture.
The mixture was warmed to 40 °C and stirred for a further
3 h. The reaction was quenched by the addition of saturated
NaHCO3. The crude product was extracted with diethyl ether (3 ×
50 mL), and the organic layer was washed with brine. The organic
layer was separated and dried with MgSO4 and then filtered. The
solvent was removed under reduced pressure, and the residue was
subjected to column chromatography on silica to afford 27 as a
colorless wax (98 mg, 90% yield). Rf (1:4 EtOAc/hexanes): 0.22;
mp: 77-79 °C. 1H NMR (CDCl3): δ 7.33 (d, J ) 8 Hz, 2H), 7.13
(d, J ) 8 Hz, 2H), 7.00 (d, J ) 8 Hz, 1H), 6.59 (d, J ) 8 Hz, 1H),
5.29 (d, J ) 10 Hz, 1H, OCH2N), 5.21 (d, J ) 10 Hz, 1H, OCH2N),
4.62 (s, 2H, CH2O), 3.90 (s, 3H, OMe), 3.76 (s, 3H, OMe), 3.62
(s, 3H, OMe), 0.88 (s, 9H, 3 × CH3), 0.01 (s, 6H, 2 × CH3). 13C
NMR (CDCl3): δ 167.4, 151.7, 145.7, 141.3, 139.1, 134.1, 127.8,
127.3, 126.2, 113.7, 108.9, 78.8, 64.4, 61.8, 56.8, 55.9, 25.9, 18.3,
-5.3. FTIR (KBr, cm-1): 2932, 2857, 1660, 1473, 1301, 1266,
1063, 1009, 837, 778. HRMS (EI+): calcd 523.1384 for
C24H3479BrNO5Si [M]+ and 525.1364 for C24H3481BrNO5Si [M]+;
found, 523.1395 and 525.1383 respectively.
2,2-Dimethyl-[1,3]dioxolo[4,5-i]phenanthridine-5-oxide (19). Pale-
yellow solid (75 mg, 78% yield); mp: 156-158 °C. Rf (1:9, IPA/
hexane) 0.21. 1H NMR (CDCl3): δ 8.85 (s, 1H), 8.83 (m, 1H),
8.38 (m, 1H), 7.94 (d, J ) 8 Hz, 1H), 7.71 (m, 2H), 7.19 (d, J )
8 Hz, 1H), 1.81 (s, 6H, 2 × CH3). 13C NMR (CDCl3): δ 146.3,
141.4, 138.2, 129.5, 128.8, 128.4, 127.1, 122.5, 121.0, 120.8, 115.1,
111.9, 111.6, 26.0. ESI-MS (m/z, %): 268 ([M + H]+, 100), 290
([M + Na]+, 20).
General Procedure for the Synthesis of the Benzamides.
2-Bromo-N-(4-(tert-butyldimethylsilyloxymethyl)phenyl)-5,6-
dimethoxybenzamide (24). The benzoic acid 20 (1.51 g, 5.79 mmol)
was refluxed in neat thionyl chloride (2 mL) for 2 h then allowed
to cool to rt. The excess thionyl chloride was removed under
reduced pressure to yield the crude acid chloride. A solution of
(4-aminophenyl)methyl-tert-butyldimethylsilyl ether (22) in DCM
(5 mL) was added to a solution of the crude acid chloride in DCM
(15 mL), followed by triethylamine (1.62 mL, 11.65 mmol). The
mixture was stirred at room temperature under argon for 3 h, after
which the solvent was removed. The residue was purified by column
chromatography on silica to afford the product (2.24 g, 90% yield)
as a pale-yellow solid. Rf (1:4 EtOAc/hexane): 0.15; mp: 121-122
5-Bromo-N-(4-(tert-butyldimethylsilyloxymethyl)phenyl)-N-
(methoxymethyl)benzo[d][1,3]dioxole-4-carboxamide (28). The prod-
uct was obtained as a colorless gum in 84% yield from 25 following
a procedure similar to that used for the preparation of 27 above. Rf
1
(3:7 EtOAc/hexanes): 0.43. H NMR (CDCl3): δ 7.26 (d, J ) 8
Hz, 2H), 7.16 (d, J ) 8 Hz, 2H), 6.85 (d, J ) 8 Hz, 1H), 6.47 (d,
J ) 8 Hz, 1H), 5.93 (d, J ) 1 Hz, 1H), 5.72 (d, J ) 1 Hz, 1H), s,
2H, CH2O), 3. 5.25 (d, J ) 10 Hz, 1H, OCHaHbO), 5.22 (d, J )
10 Hz, 1H, OCHaHbO), 4.64 (58 (s, 3H, OMe), 0.89 (s, 9H, 3 ×
CH3), 0.02 (d, J ) 1 Hz, 6H, 2 × CH3). 13C NMR (CDCl3): δ
165.7, 146.7, 144.2, 141.6, 139.0, 127.2, 126.2, 125.3, 120.8, 110.3,
109.7, 101.9, 78.7, 64.4, 56.9, 25.9, 18.3, -5.3. FTIR (KBr, cm-1):
2932, 1665, 1513, 1450, 1302, 1241, 1042, 924, 873, 801. HRMS
(ESI+): calcd 530.0969 for C23H3079BrNO5SiNa [M + Na]+; found,
530.0951.
1
°C. H NMR (CDCl3): δ 7.61 (br s, 1H, NH), 7.59 (d, J ) 8 Hz,
2H), 7.32 (d, J ) 8 Hz, 2H), 7.25 (d, J ) 8 Hz, 1H), 6.79 (d, J )
8 Hz, 1H), 4.72 (s, 2H, CH2O), 3.87 (s, 3H, OMe), 3.86 (s, 3H,
OMe), 0.94 (s, 9H, 3 × CH3), 0.10 (s, 6H, 2 × CH3). 13C NMR
(CDCl3): δ 163.5, 152.2, 146.9, 138.0, 136.4, 133.8, 128.3, 126.8,
120.1, 114.3, 109.9, 64.6, 62.2, 56.1, 26.0, 18.4, -5.2. FTIR (KBr,
cm-1): 3262, 2931, 2857, 1659, 1604, 1542, 1414, 1280, 1092,
1060, 1006, 838, 776. HRMS (EI+): calcd 479.1122 for
C22H3079BrNO4Si [M]+ and 481.1102 for C22H3081BrNO4Si [M]+;
found, 479.1120 and 481.1106, respectively. Anal. (C22H30BrNO4Si)
C, H, N.
5-Bromo-N-(4-(2-tert-butyldimethylsilyloxyethyl)phenyl)-N-
(methoxymethyl)benzo[d][1,3]dioxole-4-carboxamide (29). The com-
pound 29 was obtained as a colorless gum in 80% yield from 26
using the same procedure as that used for the preparation of 27.
The 1H NMR spectrum reveals two rotamers as evidenced by two
distinct singlet resonances for the dioxolane ring at δ 5.96 and 5.76
5-Bromo-N-(4-(tert-butyldimethylsilyloxymethyl)phenyl)benzo[d]-
[1,3]dioxole-4-carboxamide (25). The product was obtained as white
needles in 86% yield following the procedure for compound 24
using benzoic acid 21. Rf (3:7 EtOAc/hexanes): 0.4; mp: 125-126
1
1
°C. H NMR (CDCl3): δ 7.78 (br s, NH, 1H), 7.60 (d, J ) 8 Hz,
ppm. Rf (1:1:8 EtOAc/acetone/hexanes): 0.40. H NMR (CDCl3):
2H), 7.31 (d, J ) 8 Hz, 2H), 7.09 (d, J ) 8 Hz, 1H), 6.73 (d, J )
8 Hz, 1H), 6.06 (s, 2H, OCH2O), 4.72 (s, 2H, CH2O), 0.94 (s, 9H,
3 × CH3), 0.10 (s, 6H, 2 × CH3). 13C NMR (CDCl3): δ 161.1,
147.5, 146.5, 138.2, 136.2, 126.8, 126.5, 120.0, 119.5, 110.9, 110.8,
102.5, 64.6, 26.0, 18.4, -5.2. FTIR (KBr, cm-1): 3300, 2955, 2855,
1662, 1611, 1548, 1452, 1248, 1092, 1043, 838, 774. HRMS
δ 7.22 (d, J ) 8 Hz, 2H), 7.06 (d, J ) 8 Hz, 2H), 6.86 (d, J ) 8
Hz, 1H), 6.48 (d, J ) 8 Hz, 1H), 5.96 (s, 1H, OCH2O), 5.76 (s,
1H, OCH2O), 5.22 (d, J ) 12 Hz, 1H, NCHaHbO), 5.21 (d, J ) 12
Hz, 1H, NCHaHbO), 3.73 (app. t, J ) 6 Hz, 2H), 3.58 (s, 3H, OMe),
2.71 (app. t, J ) 6 Hz, 2H), 0.82 (s, 9H, 3 × CH3), -0.13 (s, 3H,
CH3), -0.14 (s, 3H, CH3). 13C NMR (CDCl3): δ 165.6, 146.7,

Phenanthridine-Based Bcl-XL Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6707
144.2, 139.7, 138.5, 129.6, 127.1, 125.3, 120.9, 110.3, 109.7, 101.9,
78.9, 63.9, 56.9, 39.0, 25.9, 18.2, -5.50, -5.53. FTIR (KBr, cm-1):
2930, 2857, 1670, 1451, 1301, 1241, 1110, 1043, 836, 778. HRMS
(ESI+): calcd 544.1131 for C24H3279BrNO5SiNa [M + Na]+ and
546.1110 for C24H3281BrNO4SiNa [M + Na]+; found, 544.1088
and 546.1112, respectively.
thridin-4(5H)-one (36). White solid (yield 99%). Rf (1:1 EtOAc/
hexanes): 0.07; mp: 202-204 °C. H NMR (CDCl3): δ 8.08 (s, J
1
) 9 Hz, 1H), 7.77 (d, J ) 9 Hz, 1H), 7.48 (d, J ) 9 Hz, 1H), 7.40
(dd, J ) 9 and 2 Hz, 1H), 7.22 (d, J ) 9 Hz, 1H), 6.25 (s, 2H,
NCH2O), 5.74 (s, 2H, OCH2O), 4.74 (s, 2H, CH2OH), 3.46 (s, 3H,
OMe). 13C NMR (CDCl3): δ 160.4, 148.3, 148.0, 136.2, 135.1,
128.3, 127.5, 121.3, 119.3, 116.2, 115.4, 113.5, 110.4, 102.9, 73.3,
64.1, 56.6. FTIR (KBr, cm-1): 3445, 3318, 2939, 1656, 1456, 1316,
1263, 1086, 1032, 951, 804. HRMS (ESI+): calcd 336.0842 for
C17H15NO5Na [M + Na]+; found, 336.0836. Anal. C17H15NO5 C,
H, N.
Procedure for the Palladium Catalyzed Aryl-Aryl Coupling.
Synthesis of 2-(tert-butyldimethylsilyloxymethyl)-7,8-dimethoxy-
5-(methoxymethyl) Phenanthridin-6(5H)-one (30). To a solution
of the compound 27 (4 g, 7.63 mmol) in dry DMF (200 mL) was
added anhydrous Na2CO3 (1.62 g, 15.28 mmol), Pd(OAc)2 (171
mg, 0.76 mmol), and PPh3 (0.5 g, 1.91 mmol) under argon. The
mixture was thrice degassed under reduced pressure and then filled
with argon and then refluxed under argon for 7 h. The reaction
mixture was cooled to rt and then diluted with diethyl ether and
filtered through Celite 545 to remove the precipitated Pd. Concen-
tration of the solution under reduced pressure gave a residue, which
was subjected to purification via column chromatography on silica
(EtOAc/hexanes, gradient 20-100%) to afford the title compound
as a white amorphous solid (2.37 g, 70% yield). Rf (1:1 EtOAc/
hexanes): 0.51; mp: 107-108 °C. 1H NMR (CDCl3): δ 8.12 (br s,
1H), 8.03 (d, J ) 9 Hz, 1H), 7.50 (d, J ) 9 Hz, 1H), 7.41 (d, J )
9 Hz, 1H), 7.40 (dd, J ) 9 and 2 Hz, 1H), 5.79 (s, 2H, NCH2O),
4.84 (s, 2H, CH2O), 4.00 (s, 3H, OMe), 3.98 (s, 3H, OMe), 3.49
(s, 3H, OMe), 0.98 (s, 9H, 3 × CH3), 0.14 (s, 6H, 2 × CH3). 13C
NMR (CDCl3): δ 160.4, 153.2, 150.7, 135.9, 135.4, 128.9, 126.8,
120.2, 119.9, 118.8, 118.1, 118.0, 115.8, 73.7, 64.7, 61.4, 56.6,
56.6, 26.0, 18.5, -5.1. FTIR (KBr, cm-1): 2927, 2854, 1655, 1499,
1458, 1318, 1278, 1103, 1080, 836, 774. HRMS (EI+.): calcd
443.2123 for C24H33NO5Si [M]+·; found, 443.2144.
8-(2-Hydroxyethyl)-5-(methoxymethyl)-[1,3]dioxolo[4,5-i]phenan-
thridin-4(5H)-one (37). Compound 37 was synthesized from 32
following the general procedure. As the product is polar and poorly
soluble in most organic solvents, the crude product was obtained
by vacuum filtration. The precipitate was washed with small
portions of DCM, followed by acetone and then water. The
precipitate was dried in vacuo to yield the desired product as white
amorphous solid (685 mg, 85% yield). Rf (100% EtOAc): 0.36;
mp: 216-217 °C. 1H NMR (CDCl3): δ 7.97 (br s, 1H), 7.79 (d, J
) 8 Hz, 1H), 7.52 (d, J ) 9 Hz, 1H), 7.33 (d, J ) 8 Hz, 1H), 7.40
(d, J ) 9 Hz, 1H), 6.26 (s, 2H, OCH2O), 5.77 (s, 2H, OCH2N),
3.94 (app. t, J ) 6 Hz, 2H, CH2O), 2.97 (app. t, J ) 6 Hz, 2H,
ArCH2). 13C NMR (CDCl3): δ 160.3, 148.4, 148.1, 134.9, 133.5,
129.6, 128.3, 123.4, 119.5, 116.5, 115.4, 113.4, 110.7, 102.9, 73.4,
63.6, 56.8, 38.7. FTIR (KBr, cm-1): 3420, 2919, 2863, 1654, 1458,
1307, 1266, 1060, 809. MS-ESI+ (m/z, %): 350 ([M + Na]+, 100).
HRMS (ESI+): calcd 328.1180 for C18H18NO5 [M + H]+, and
350.0999 for C18H17NO5Na [M + Na]+; found, 328.1175 and
350.0983, respectively.
O-Methylation of 2-Substituted Phenanthridinones. 5,8-Bis-
(methoxymethyl)-[1,3]dioxolo[4,5-i]phenanthridin-4(5H)-one (38).
To a suspension of compound 36 (30 mg, 0.0958 mmol) in THF
(5 mL) was added NaH (60% in mineral oil, 19 mg, 0.475 mmol).
The mixture was stirred for 20 min at rt, and then dimethyl sulfate
(27 µL, 0.285 mmol) was added slowly by syringe. The reaction
mixture was refluxed for 6 h, after which the mixture was cooled
to rt and water was added slowly to quench the excess NaH. The
mixture was diluted with EtOAc (20 mL) and water (20 mL), and
the EtOAc layer was washed with brine and dried over Na2SO4.
Removal of solvent in vacuo gave a white residue, which was
subjected to silica gel chromatography (70% EtOAc in hexanes).
The product was obtained as a white solid in 96% yield. Rf (7:3
EtOAc/hexane): 0.29; mp: 193-194 °C. 1H NMR (CDCl3): δ 8.08
(d, J ) 2 Hz, 1H), 7.81 (d, J ) 9 Hz, 1H), 7.55 (d, J ) 8 Hz, 1H),
7.40 (dd, J ) 8 and 2 Hz, 1H), 7.24 (d, J ) 9 Hz, 1H), 6.26 (s,
2H, OCH2O), 5.78 (s, 2H, OCH2N), 4.54 (s, 2H, CH2OH), 3.48 (s,
3H, OMe), 3.44 (s, 3H, OMe). 13C NMR (CDCl3): δ 160.4, 148.4,
148.1, 135.7, 133.1, 128.4, 122.2, 119.5, 116.3, 115.5, 113.5, 110.7,
102.9, 74.2, 73.4, 58.2, 56.8. MS-ESI+ (m/z, %): 350 ([M + Na]+,
100). HRMS (ESI+): calcd 328.1180 for C18H18NO5 [M + H]+,
and 350.0999 for C18H18NO5Na [M + Na]+; found, 328.1179 and
350.0997 respectively.
8-(2-Methoxyethyl)-5-(methoxymethyl)-[1,3]dioxolo[4,5-i]phenan-
thridin-4(5H)-one (39). Compound 39 was prepared from 37
following the general procedure for the preparation of 38. The
product was a white solid (quantitative yield). Rf (1:1 EtOAc/
hexanes): 0.23. 1H NMR (CDCl3): δ 7.94 (d, J ) 2 Hz, 1H), 7.77
(d, J ) 9 Hz, 1H), 7.50 (d, J ) 8 Hz, 1H), 7.31 (dd, J ) 9 and 2
Hz, 1H), 7.23 (d, J ) 8 Hz, 1H), 6.25 (s, 2H), 5.77 (s, 2H), 3.66
(app. t, J ) 7 Hz, 2H), 3.47 (s, 3H, OMe), 3.38 (s, 3H, OCH3),
2.97 (app. t, J ) 7 Hz, 2H). HRMS (ESI+): calcd 364.1155 for
C19H19NO5Na [M + Na]+; found, 364.1144.
8-(tert-Butyldimethylsilyloxymethyl)-5-(methoxymethyl)-[1,3]di-
oxolo[4,5-i]phenanthridin-4(5H)-one (31). The product was obtained
as a white solid in 71% yield from 28 following the procedure
outlined for compound 30. Rf (1:1EtOAc/hexanes): 0.39; mp:
128-129 °C. 1H NMR (CDCl3): δ 7.98 (s, 1H), 7.66 (d, J ) 8 Hz,
1H), 7.46 (d, J ) 8 Hz, 1H), 7.33 (d, J ) 8 Hz, 1H), 7.15 (d, J )
8 Hz, 1H), 6.19 (s, 2H, OCH2O), 5.70 (s, 2H, NCH2O), 4.78 (s,
2H, CH2O), 3.43 (s, 3H, OMe), 0.95 (s, 9H, 3 × CH3), 0.12 (s,
6H, 2 × CH3). 13C NMR (CDCl3): δ 160.2, 148.3, 147.9, 136.2,
135.1, 128.5, 126.8, 120.3, 119.1, 116.1, 115.2, 113.3, 110.6, 102.9,
73.3, 64.5, 56.7, 26.0, 18.4, -5.2. FTIR (KBr, cm-1): 2929, 2856,
1659, 1459, 1369, 1306, 1253, 1064, 1912, 838, 775. HRMS
(ESI+): calcd 428.1888 for C23H30NO5Si [M + H]+; found,
428.1878. Anal. (C23H29NO5Si) C, H, N.
8-(2-tert-Butyldimethylsilyloxyethyl)-5-(methoxymethyl)-[1,3]di-
oxolo[4,5-i]phenanthridin-4(5H)-one (32). Compound 32 was syn-
thesized from 29 using the procedure for the preparation of 30.
The desired product was obtained as a white solid in 80% yield. Rf
1
(1:1 EtOAc/hexanes): 0.45; mp: 128-129 °C. H NMR (CDCl3):
δ 7.94 (s, 1H), 7.75 (d, J ) 8 Hz, 1H), 7.48 (d, J ) 9 Hz, 1H),
7.29 (d, J ) 9 Hz, 1H), 7.22 (d, J ) 8 Hz, 1H), 6.24 (s, 2H,
OCH2O), 5.77 (s, 2H, NCH2O), 3.86 (app. t, J ) 6 Hz, 2H), 3.47
(s, 3H, OMe), 2.90 (app. t, J ) 6 Hz, 2H), 0.88 (s, 9H, 3 × CH3),
0.00 (s, 6H, 2 × CH3). 13C NMR (CDCl3): δ 160.3, 148.4, 147.9,
134.6, 134.2, 129.8, 128.6, 123.5, 119.2, 116.1, 115.3, 113.3, 110.8,
102.9, 73.3, 64.4, 56.7, 39.1, 25.9, 18.3, -5.4. FTIR (KBr, cm-1):
2954, 2857, 1657, 1461, 1307, 1256, 1057, 912, 833, 775. HRMS
(ESI+): calcd 442.2044 for C24H32NO5Si [M + H]+; found,
442.2056. Anal. (C24H31BrNO5Si) C, H, N.
General Procedure for the Deprotection of the TBDMS Group.
To a stirred solution of TBS-protected ethers 31 or 32 (typically
500 mg) in dry THF (50 mL) was added tetra-n-butylammonium
fluoride (1 M in THF, 2.84 mL) slowly under argon. The mixture
was stirred for 1 h at rt and quenched by adding saturated NH4Cl.
The reaction mixture was partitioned between water (50 mL) and
EtOAc (50 mL) and then further extracted with EtOAc (2 × 50
mL). The combined organic layer was washed with brine, dried
with MgSO4, and concentrated in vacuo to yield a crude product,
which was then purified by column chromatography to give the
desired product as a white solid.
Procedure for the Synthesis of N-Methyl Phenanthridinium
Chlorides. 2-(Hydroxymethyl)-7,8-dimethoxy-5-methylphenanthri-
dinium Chloride (33). To a solution of compound 30 (300 mg, 0.68
mmol) in dry THF (20 mL) was slowly added a solution of
diisobutylaluminium hydride (DIBAL, 1 M in toluene, 2.71 mL)
at -10 °C under argon. The mixture was stirred at -10 °C for 1 h
and then warmed to rt. Saturated MgSO4 was added dropwise until
a colorless precipitate was formed, and the mixture was stirred
8-(Hydroxymethyl)-5-(methoxymethyl)-[1,3]dioxolo[4,5-i]phenan-

6708 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Bernardo et al.
vigorously for an additional 30 min. The mixture was diluted with
diether ether and filtered through Celite. Removal of solvents gave
a colorless residue. The residue was dissolved in toluene (10 mL),
and a solution of 2,3-dicyano-5,6-dichloro-parabenzoquinone (DDQ,
227 mg in 10 mL toluene, 1 mmol) was then added slowly. The
reaction mixture was stirred for 2 h prior to acidification with 2 M
HCl (15 mL). The acidic solution was stirred for an additional 15
min, during which a yellow aqueous layer was formed. This aqueous
layer was separated then washed with ether (15 mL) and then
chloroform (2 × 15 mL), and the organic layers were discarded.
Removal of water under reduced pressure gave an orange residue,
which was recrystallized from MeOH-ether to afford 33 as yellow
J ) 9 Hz, 1H), 7.72 (d, J ) 9 Hz, 1H), 7.69 (dd, J ) 9 and 1 Hz,
1H), 7.48 (d, J ) 9 Hz, 1H), 6.28 (s, 2H, OCH2O), 4.24 (s, 3H,
NMe), 3.80 (app. t, J ) 7 Hz, 2H), 3.36 (s, 3H, OCH3), 3.02 (app.
t, J ) 7 Hz, 2H). 13C NMR (D2O): δ 147.5, 147.3, 147.2, 143.1,
131.8, 130.5, 125.4, 124.8, 123.0, 119.2, 119.1, 115.8, 107.9, 105.2,
72.0, 57.9, 45.4, 34.8. FTIR (KBr, cm-1): 3400, 2948, 1615, 1498,
1475, 1301, 1248, 1104, 1067, 892, 839. HRMS (ESI+): calcd
296.1281 for C18H18NO3 [M]+; found, 296.1279.
Procedure for Cleavage of Methyl Ethers. 7,8-Dihydroxy-5-
methylphenanthridinium Chloride (42). To the suspension of 14
(20 mg, 0.052 mmol) in dry DCM (5 mL) under argon was slowly
added an excess amount of BBr3 (1 M in DCM, 0.2 mL) at 0 °C.
The mixture was warmed to rt and stirred for 1 h. The reaction
mixture was cooled to 0 °C and quenched with dry MeOH. The
solvent was removed in vacuo to yield an orange crude product,
which was acidified with dilute HCl and purified by reverse phase
HPLC (Luna C18, 21.2 mm × 250 mm, flow rate: 20 mL/min;
mobile phase: acetonitrile in water with 0.1% formic acid; gradient:
10-70%, 15 min, Rt 6.6 min) to afford pure product 42 as a beige
solid in 86% yield; mp: 263-266 °C. 1H NMR (DMSO-d6): δ 10.00
(s, 1H), 8.83 (br s, 1H), 8.30 (br s, 1H), 8.20 (br s, 1H), 7.92 (m,
3H), 4.57 (s, 3H, NMe). 13C NMR (DMSO-d6): δ 150.6, 147.6,
145.3, 132.7, 129.9, 129.7, 126.8, 125.9, 125.7, 123.9, 119.6, 115.1,
112.5, 45.1. FTIR (KBr): 3421 (br), 3093 (br), 1613, 1543, 1491,
1458, 1359, 1339, 1240, 1175, 1005, 838, 765, 710 cm-1. HRMS
1
needles (200 mg, 93% yield). mp: 187-189 °C (dec). H NMR
(DMSO-d6): δ 10.15 (s, 1H), 8.93 (s, 1H), 8.77 (d, J ) 9 Hz, 1H),
8.43 (d, J ) 8 Hz, 1H), 8.24 (d, J ) 9 Hz, 1H), 8.00 ((d, J ) 8 Hz,
1H), 4.85 (s, 2H, CH2OH), 4.70 (s, 3H, NMe), 4.14 (s, 3H, OMe),
4.10 (s, 3H, OMe). 13C NMR (DMSO-d6): δ 151.5, 150.9, 147.1,
146.0, 132.5, 129.7, 127.8, 125.8, 125.6, 121.3, 120.2, 119.6, 119.5,
62.7, 62.6, 57.4, 46.3. FTIR (KBr, cm-1): 3371, 3214, 3022, 2957,
1596, 1509, 1447, 1335, 1292, 1252, 1067, 957, 809, 781. MS-
ESI+ (m/z, %): 284 ([M]+, 100), 269 ([M-Me]+, 37). HRMS
(ESI+): calcd 284.1287 for C17H18NO3 [M]+; found, 284.1283.
8-(Hydroxymethyl)-5-methyl-[1,3]dioxolo[4,5-i]phenanthridin-
ium Chloride (34). Compound 34 was prepared from 31 following
the procedure used for the preparation of compound 33. Orange
1
+
(ESI+): calcd 226.0863 for C14H12NO2 [M]+; found, 226.0860.
solid, 90% yield.); mp: 230-236 °C (dec). H NMR (CDCl3):
δ 9.77 (s, 1H), 8.67 (br s, 1H), 8.33 (d, J ) 10 Hz, 1H), 8.25
(d, J ) 10 Hz, 1H), 7.93 (dd, J ) 9 and 2 Hz, 1H), 7.83 (d, J
) 9 Hz, 1H), 6.44 (s, 2H), 4.94 (s, 2H), 4.58 (s, 3H, N-Me).
13C NMR (CDCl3): δ 148.0, 147.6, 147.4, 143.8, 131.3, 129.2,
125.9, 125.2, 120.6, 119.3, 119.2, 115.9, 108.3, 105.1, 62.6, 45.4.
FTIR (KBr, cm-1): 3333, 2977, 1615, 1500, 1472, 1293, 1062,
1030, 903, 832. HRMS (ESI+): calcd 268.0968 for C16H14NO3
[M]+; found, 268.0975.
8-(2-Hydroxyethyl)-5-methyl-[1,3]dioxolo[4,5-i]phenanthridin-
ium Chloride (35). Compound 35 was prepared from 32 following
the general procedure. The crude product was recrystallized from
methanol to afford yellow prism crystals (45 mg, 92% yield); mp:
243-246 °C (dec). 1H NMR (MeOH-d4): δ 9.94 (s, 1H, N ) CH),
8.77 (s, 1H), 8.55 (d, J ) 8 Hz, 1H), 8.34 (d, J ) 8 Hz, 1H), 7.94
(d, J ) 8 Hz, 1H), 7.92 (d, J ) 8 Hz, 1H), 4.67 (s, 3H, NMe), 3.96
(app. t, J ) 6 Hz, 2H, CH2O), 3.17 (app. t, J ) 6 Hz, 2H, ArCH2).
13C NMR (MeOH-d4): δ 150.4, 149.8, 149.5, 145.0, 133.4, 128.6,
127.8, 125.4, 120.7, 120.6, 118.0, 110.9, 106.5, 63.4, 46.5, 40.0.
FTIR (KBr, cm-1): 3384, 3232, 3070, 2920, 1614, 1502, 1472,
1293, 1275, 1064, 1029, 841, 812. HRMS (ESI+): calcd 282.1125
for C17H16NO3 [M]+; found, 282.1124.
7,8-Dihydroxy-2-(hydroxymethyl)-5-methylphenanthridinium
Chloride (43). The title compound was prepared following the
previous procedure for compound 42. The crude product was stirred
at 80 °C for 5 h in the presence of a few drops of 1 M HCl to
convert any 2-methylbromo-5-methyl-7,8-dihydroxyphenanthri-
dinium byproduct to the title compound 43. The solvent was then
removed in vacuo, and the crude product was recrystallized from
MeOH to afford the desired product as an orange solid (67% yield).
1H NMR (D2O): δ 9.19 (s, 1H), 7.99 (s, 1H), 7.88 (d, J ) 9 Hz,
1H), 7.72 (d, J ) 9 Hz, 1H), 7.40 (d, J ) 9 Hz, 1H), 7.22 (d, J )
9 Hz, 1H), 4.79 (s, 2H, CH2OH), 4.27 (s, 3H, NMe). 13C NMR
(DMSO-d6): δ 150.1, 145.4, 144.9, 144.7, 131.7, 128.6, 127.5,
126.2, 125.5, 120.6, 119.7, 115.3, 113.7, 62.3, 45.5. ESI-MS (m/z,
%): 256 ([M]+, 100). HRMS (ESI+): calcd 256.0968 for
+
C15H14NO3 [M]+; found, 256.0960.
7,8-Dihydroxy-2-(2-hydroxyethyl)-5-methylphenanthridinium (44).
The reaction of compound 41 with BBr3 for 16 h afforded the
desired product, which was purified by preparative HPLC. HPLC
condition: Luna C18 21.2 mm × 250 mm, flow rate: 20 mL/min;
mobile phase: acetonitrile in water; gradient: 20% (5 min), 20-80%
1
(20 min), 80% (5 min), Rt 3.0 min. Yellow solid, 84% yield. H
8-(Methoxymethyl)-5-methyl-[1,3]dioxolo[4,5-i]phenanthridin-
ium Chloride (40). Compound 40 was prepared from 38 following
the procedure for compound 33. Purification via reverse phase
HPLC (Luna 21.2 mm × 250 mm; flow rate: 20 mL/min; mobile
phase: acetonitrile in water with 0.1% formic acid; gradient: 10%
(10 min), 10-70% (10 min), 70% (5 min), Rt 7.9 min) afforded
the product, which was obtained as an orange solid in quantitative
yield; mp: 199-202 °C (dec). 1H NMR (D2O): δ 9.53 (s, 1H),
8.14 (s, 1H), 8.01 (d, J ) 9 Hz, 1H), 7.82 (d, J ) 8 Hz, 1H), 7.77
(dd, J ) 8 Hz, 1H), 7.60 (d, J ) 9 Hz, 1H), 6.38 (s, 2H, OCH2O),
4.66 (s, 2H, CH2O), 4.40 (s, 3H, NMe), 3.54 (s, 3H, OMe). 13C
NMR (D2O): δ 148.3, 147.7, 147.5, 140.9, 131.5, 129.9, 125.8,
125.1, 121.6, 119.4, 119.3, 115.9, 108.3, 105.2, 72.8, 58.3, 45.4.
FTIR (KBr, cm-1): 3442, 3045, 1619, 1471, 1445, 1295, 1274,
1105, 1061, 900, 831. HRMS (ESI+): calcd 282.1125 for
C17H16NO3 [M]+; found, 282.1122.
8-(2-Methoxyethyl)-5-methyl-[1,3]dioxolo[4,5-i]phenanthridin-
ium Chloride (41). Compound 41 was prepared from 39 following
the general procedure for preparing 33. Reverse phase HPLC (Luna
21.2 mm × 250 mm, flow rate: 20 mL/min; mobile phase:
acetonitrile in water with 0.1% formic acid; gradient: 10% (10 min),
10-70% (10 min), 70% (5 min), Rt 10.6 min) yielded the product
as an orange solid in quantitative yields; mp: 146-147 °C (dec).
1H NMR (D2O): δ 9.28 (s, 1H), 7.99 (d, J ) 1 Hz, 1H), 7.85 (d,
NMR (MeOH-d4): δ 9.98 (s, 1H), 8.72 (s, 1H), 8.30 (d, J ) 9 Hz,
1H), 8.29 (d, J ) 9 Hz, 1H), 7.89 (d, J ) 9 Hz, 1H), 7.82 (d, J )
9 Hz, 1H), 4.65 (s, 3H, NMe), 3.95 (app. t, J ) 6 Hz), 3.16 (app.
t, J ) 6 Hz). ESI-MS (m/z, %): 270 ([M]+, 95). 13C NMR (DMSO-
d6): δ 149.8, 145.3, 144.6, 142.4, 131.4, 131.3, 127.4, 126.1, 125.5,
123.7, 119.4, 115.2, 113.8, 61.7, 45.2, 38.6. ESI-MS (m/z, %): 270
([M]+, 95). HRMS (ESI+): calcd 270.1125 for C16H16NO3+ [M]+;
found, 270.1143.
Biology. Fluorescence Polarization Assay. The Bak-BH3 pep-
tide labeled with fluorescein at the N terminus was synthesized
by Mimotopes (Clayton, Victoria, Australia) and purified by
HPLC. The peptide was dissolved in DMSO at 1 mM, and stock
solutions of the test compounds (4 mM in DMSO) were used
for serial dilutions (250-0.65 µM final concentrations). The
reaction was carried out in a total volume of 100 µL/well
containing 3 µg glutathione S-transferase (GST)-BclXL∆C19 and
60 nM labeled peptide in assay buffer (50 mM Tris, pH 8, 150
mM NaCl and 0.1% bovine serum albumin). To each well was
added 10 µL of the test compounds, and the reaction mixture
was incubated at rt for 1 h. The fluorescence polarization values
were determined using a Tecan GeniosPro plate reader using
the excitation/emission wavelengths 485/535 nm.
Protein Expression, Purification and NMR Titration. A vector
modified from pET-32a containing the construct for human Bcl-

Phenanthridine-Based Bcl-XL Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6709
Nature 2005, 435 (7042), 677–681.
XL (from residues 1-218 with the flexible loop from residues
45-84 removed) as described by Zhang et al.11 was used for
expression of wild-type Bcl-XL and its mutants (E129A, L130A,
D133K, V135A, R139A, A142G, Y173F, and E184A). The DNA
sequences of all the constructs were confirmed by Big Dye
sequencing. The expression, purification, and thrombin cleavage
of His-tagged Bcl-XL protein were performed as described
previously.11 The purified proteins were concentrated to around
0.25 mM and buffer exchanged to 20 mM sodium phosphate
buffer (pH 7.0) using Amicon Centriprep-10 (Millipore) for
NMR experiments. For NMR titrations, both compounds 35 and
42 (stock solutions of 50 mM in DMSO) were titrated into wild-
type and mutant Bcl-XL at inhibitor to protein molar ratio of
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1
10:1. H-15N HSQC spectrum of each titrated sample (∼0.25
mM protein in 20 mM phosphate buffer containing 10% D2O
v/v) was recorded on a 500 MHz Bruker DRX spectrometer
equipped with cryoprobe at 298 K. All spectra were acquired
with 1024 complex data points in t2 and 128 points in t1 with
spectral widths of 26.4 ppm and 16 ppm in the 15N and 1H
dimensions, respectively, using 16 scans. All NMR data were
processed using NMR Pipe/NMRDraw suite26 and analyzed by
Sparky.27 The combined amide proton and nitrogen chemical
shift perturbation from the 1H-15N HSQC spectra of Bcl-XL
after titration with the test compounds were calculated using
the following relation:
2
1/2
2
)
{
[
]}
(
chemical shift perturbation ) ∆H + (∆N) × 0.2
where ∆H and ∆N are change in chemical shift in 1H and 15N
dimensions, respectively.
Molecular Docking. The NMR structure of human Bcl-XL (Bcl-
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The 3D structures of the inhibitors were generated using Sybyl7.3
followed by Powell energy minimization. Autodock 4.0.1 program
equipped with ADT was used for generating docking models.
Autogrids covering residues that were perturbed more than the
threshold value of 0.075 ppm in the BH groove, BH3 binding
groove, and BH1 region were defined for both compounds 35 and
42 in the AutoDock calculations using a grid spacing of 0.375 Å.
The GA-LS algorithm was adopted using default settings except
the number of individuals in the population was changed to 500
and maximum number of energy evolutions was increased to
15000000. For each docking job, 100 hybrid GA-LS runs were
carried out. A total of 100 possible binding conformations were
generated and grouped into clusters based on a 1.0 Å cluster
tolerance.
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Acknowledgment. We thank the Agency for Science,
Technology, and Research (A*STAR), Singapore, for the
research funding for this project. The authors also thank Mr.
Janarthanan Krishnamoorthy (NUS) for providing assistance on
aspects of this project.
Supporting Information Available: 1H and 13C NMR spectra,
elemental analysis, and HPLC traces. This material is available free
of charge via the Internet at http://pubs.acs.org.
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