Discovery of a potent inhibitor of the antiapoptotic protein Bcl-X L from NMR and parallel synthesis

Article abstract of DOI:10.1021/jm0507532

The antiapoptotic proteins Bcl-x_L and Bcl-2 play key roles in the maintenance of normal cellular homeostasis. However, their overexpression can lead to oncogenic transformation and is responsible for drug resistance in certain types of cancer. This makes Bcl-x_L, and Bcl-2 attractive targets for the development of potential anticancer agents, Here we describe the structure-based discovery of a potent Bcl-x_L inhibitor directed at a hydrophobic groove on the surface of the protein. This groove represents the binding site for BH3 peptides from proapoptotic Bcl-2 family members such as Bak and Bad. Application of NMR-based screening yielded an initial biaryl acid with an affinity (K_d) of ～300 μM for the protein. Following the classical "SAR by NMR" approach, a second-site ligand was identified that bound proximal to the first-site ligand in the hydrophobic groove. From NMR-based structural studies and parallel synthesis, a potent ligand was obtained, which binds to Bcl-x_L with an inhibition constant (K _i) of 36 ± 2 nM.

Full text of DOI:10.1021/jm0507532

6
56
J. Med. Chem. 2006, 49, 656-663
L
Discovery of a Potent Inhibitor of the Antiapoptotic Protein Bcl-x from NMR and Parallel
Synthesis
†
†
Andrew M. Petros, Jurgen Dinges, David J. Augeri, Steven A. Baumeister, David A. Betebenner, Mark G. Bures,
Steven W. Elmore, Philip J. Hajduk, Mary K. Joseph, Shelley K. Landis, David G. Nettesheim, Saul H. Rosenberg, Wang Shen,
Sheela Thomas, Xilu Wang, Irini Zanze, Haichao Zhang, and Stephen W. Fesik*
Global Pharmaceutical Research and DeVelopment, Abbott Laboratories, Abbott Park, Illinois 60064
ReceiVed August 1, 2005
The antiapoptotic proteins Bcl-x
However, their overexpression can lead to oncogenic transformation and is responsible for drug resistance
in certain types of cancer. This makes Bcl-x and Bcl-2 attractive targets for the development of potential
anticancer agents. Here we describe the structure-based discovery of a potent Bcl-x inhibitor directed at a
L
and Bcl-2 play key roles in the maintenance of normal cellular homeostasis.
L
L
hydrophobic groove on the surface of the protein. This groove represents the binding site for BH3 peptides
from proapoptotic Bcl-2 family members such as Bak and Bad. Application of NMR-based screening yielded
an initial biaryl acid with an affinity (K
NMR” approach, a second-site ligand was identified that bound proximal to the first-site ligand in the
hydrophobic groove. From NMR-based structural studies and parallel synthesis, a potent ligand was obtained,
d
) of ∼300 µM for the protein. Following the classical “SAR by
which binds to Bcl-x
L
with an inhibition constant (K
i
) of 36 ( 2 nM.
Introduction
of protein-protein interactions is that these interactions typically
involve extensive surface contact between the two partners,
Apoptosis (programmed cell death) plays a vital role in
2
typically 750-1500 Å for each side of the interface. Protein-
normal development, tissue remodeling, immune response, and
overall maintenance for all multicellular organisms.1,2 Disruption
protein binding sites also tend to be shallow and featureless
compared to small molecule binding sites. The binding site in
Bcl-2 and Bcl-xL is very different from the typical case. For
of this process is implicated in a number of disease states,
including Alzheimers disease, autoimmune disorders, and
cancer. The B-cell lymphoma (Bcl) family of proteins, which
includes both antiapoptotic members, such as Bcl-2, Bcl-xL, and
Bcl-w, and proapoptotic members, such as Bak, Bax, and Bad,
2
Bak peptide binding to Bcl-xL, only approximately 500 Å of
the protein surface is involved. Furthermore, the binding site
on Bcl-xL is a deep, hydrophobic groove. This groove accom-
3
modates an amphipathic helix upon binding of Bcl-x to its
play key roles in both normal and abnormal apoptotic processes.
L
proapoptotic partners. Here we describe the use of fragment-
In general, homeostasis is maintained by the highly regulated
expression of these pro- and antiapoptotic proteins coupled with
their tissue-specific patterns of distribution. Under normal
circumstances, apoptotic stimuli such as DNA damage or
hypoxia induce expression and/or activation of proapoptotic
family members and subsequent apoptosis of the aberrant cell.
For some cancers, however, this apoptotic process is circum-
vented by overexpression of the antiapoptotic proteins Bcl-2
based methods, specifically “SAR by NMR”, and parallel
synthesis to obtain a potent inhibitor that binds in the hydro-
phobic groove of Bcl-xL.
Results and Discussion
Discovery of a First-Site Ligand. An NMR-based screen
was conducted using a form of Bcl-xL that lacked the putative
transmembrane helix and the long unstructured loop connecting
4
,5
and/or Bcl-xL. Therefore, Bcl-2 and Bcl-xL are attractive
8
targets for the development of anticancer agents.6
helix 1 to helix 2. A 10 000 compound fragment library with
an average compound molecular weight of 210 Da was screened
The three-dimensional structures of several Bcl-2 family
proteins have been determined and all display the same overall
and yielded the fluoro biaryl acid 1 (Table 1) as a ligand for
1
5
7
Bcl-xL. Figure 1 shows a section from a N HSQC spectrum
of Bcl-xL in the presence (red) and absence (black) of compound
fold. This fold consists of two predominately hydrophobic, cen-
tral R-helices surrounded by five to seven amphipathic helices
of varying length. The antiapoptotic proteins Bcl-xL and Bcl-2
have a hydrophobic groove on their surface, which serves as
the binding site for the proapoptotic proteins such as Bak, Bax,
and Bad. The three-dimensional-structure of BH3 peptides from
Bak and Bad bound to Bcl-xL have been determined by NMR.
In both cases, the peptide binds as an amphipathic helix and
makes key hydrophobic interactions in the groove of the protein.
1
. The chemical shift changes (e.g., G94 and G138) observed
in this region of the spectrum along with those observed in other
regions are consistent with the compound binding in the
hydrophobic groove of Bcl-xL. An NMR-based titration of this
compound yielded a dissociation constant (Kd) of ∼300 µM.
8
,9
To explore the structure-activity relationships for binding
to this site, several biaryl analogues from our corporate
compound repository were tested for binding to Bcl-xL. Some
of these are shown in Table 1. From these studies it was clear
that the carboxylate was critical for binding, as analogues
containing a phenol (3) or methyl ester (4) exhibited weaker
affinities compared to the corresponding acids. In addition, the
position of the carboxylate proved to be important, as a shift of
the acid moiety from para to meta decreased affinity (compare
A challenging aspect of targeting Bcl-xL is that its activity is
manifested through interactions with other proteins. Historically,
such interactions have been difficult to disrupt using small
molecules, resulting in a strong bias against targeting protein-
protein interactions.10 A primary hurdle in designing inhibitors
*
To whom correspondence should be addressed. Tel: (847) 937-1201.
5
with 2), as did homologation of the carboxylate (compounds
Fax: (847) 938-2478. E-mail: stephen.fesik@abbott.com.
†
These authors contributed equally to this work.
6 and 7). The fluorine atom could be replaced with either a
1
0.1021/jm0507532 CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/20/2005

Inhibitor of the Antiapoptotic Protein Bcl-x
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Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 657
Table 2. Affinities of Selected Second-Site Bcl-xL Binders
Table 1. Affinities of Selected Biaryl Compounds for Bcl-xL
methyl group (8) or a chlorine atom (9) and the fluorine-bearing
ring could be replaced with a naphthalene (10).
of compound 1 using a 3500 compound library with an average
molecular weight of 125 Da. From this screen, several naphthol
analogues and a biaryl phenol (Table 2) were identified that
bound to Bcl-xL in the presence of the fluoro biaryl acid. These
second-site ligands bound to Bcl-xL more weakly then the first-
site ligands with dissociation constants in the low millimolar
NMR-based structural studies on Bcl-xL complexed to
compound 1 showed that this compound binds at the center of
11
the hydrophobic groove and occupies approximately the same
8
position as the key leucine residue of the bound Bak peptide.
Furthermore, compound 1 is positioned to make an electrostatic
1
1
interaction, through its carboxylate, with R139 of the protein.
15
range. In the section of the N HSQC spectrum shown in Figure
, the cross-peaks from the complex of Bcl-xL with both
For the Bak peptide, an analogous interaction was observed
between an aspartic acid residue of the peptide (D83) and R139.
Discovery of a Second-Site Ligand. The comparison of the
structure of Bcl-xL complexed to 1 with the structure of the
Bcl-xL/Bak peptide complex indicates the existence of a
proximal second site. In the structure of the Bak peptide
complex, this site is filled by the side chain from I85 of the
peptide. To identify ligands for this proximal site, a second-
site screen was performed in the presence of an excess (2 mM)
1
compounds 1 and 11 are shown in green. In addition to minor
shift changes of the G94 and G138 amides, the amide of G196
shifts upon the addition of this second-site ligand. The shift
changes are consistent with binding of the ligand in the
hydrophobic groove of Bcl-xL.
Linking Strategy. To propose an appropriate linking strategy
for these two fragments, the structure of the ternary complex
of Bcl-xL with compounds 1 and 11 was obtained using NMR.
Figure 2. Superposition of seven low-energy structures calculated for
Bcl-x complexed to 1 and 11. For clarity, the average-minimized
L
structure of the protein is shown as a solvent-accessible surface, color
coded as follows: oxygen and oxygen-bound protons are red, and
nitrogen and nitrogen-bound protons are blue, while all other atoms
are gray. The positions of compounds 1 and 11 in the average-
minimized structure are shown in orange.
Figure 1. Selected region of 15N HSQC spectra recorded on uniformly
15
N-labeled Bcl-x
(red), and in the presence of 2 mM biaryl acid 1 and 5 mM naphthol
derivative 11 (green).
L
alone (black), in the presence of 2 mM biaryl acid
1

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58 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Petros et al.
L
Figure 3. Fragment linking strategy used for Bcl-x . The best
compound incorporated the second-site biaryl and a trans-olefin linker.
Scheme 1a
Figure 4. NMR-derived model of trans-olefin linked compound 25
bound to Bcl-x . The side chain of F97 divides the first site from the
L
second site. Protein is shown as a solvent-accessible surface color-
coded as in Figure 2.
Table 3. Affinities of Selected Acylsulfonamides for Bcl-xL.
a
Reagents and conditions: (a) 4-fluorophenylboronic acid, PdCl2(PCy3)2,
CsF, NMP; 90 °C 15 h; (b) BBr3 DCM, -78 °C to rt, 16 h; (c) Tf2NPh,
(i-Pr)2NEt, DCM, 0 °C to rt; (d) vinyldibutylboronate, Pd(PPh3)4, CsF, 2:1
DME:MeOH, reflux, 15 h; (e) 3-bromobiphenyl, Pd2(dba)3, NEt3, P(o-tol)3,
:1 CH3CN:DMF, 85 °C, 10 h; (f) LiOH‚H2O, THF, MeOH, rt, 15 h.
2
A superposition of seven low-energy structures for the Bcl-xL
ternary complex is shown in Figure 2. The ortho position of
the biaryl acid seemed to provide the most direct trajectory to
the second site. On this basis, various linkers were used to
connect the ortho position of the fluoro biaryl acid to either a
naphthol moiety or a biaryl moiety (Figure 3). Most of the
resulting compounds bound with an inhibition constant (Ki)
greater than 10 µM, as measured in a flourescence polarization
assay.12 However, compound 25 (Scheme 1), which incorporates
a trans-olefin linker and a biaryl second-site fragment, binds
to Bcl-xL with an inhibition constant (Ki) of 1.4 ( 0.4 µM.
This represents an appoximately 200-fold improvement in
potency over the original biaryl acid 1.
Structural Characterization of Linked Compound. The
binding of 25 to Bcl-xL was characterized structurally by NMR
in order to uncover areas for further gains in affinity. As the
solubility of this compound was limited, it was not possible to
obtain NMR data of sufficient quality to do a full structure deter-
mination. However, nine unambiguous intermolecular NOEs
were obtained for this ligand when bound to Bcl-xL, from which
a model was generated of the compound bound to the protein
a
Compound in slow exchange.
trans olefin may not be the best linker for maintaining the opti-
mal position of the first and second-site fragments and that a
different trajectory from the first-site fragment might be
necessary.
Discovery of Acylsulfonamide Linker. Acylsulfonamides
are common isosteres for carboxylates, as they possess pKa’s
in the range of 3-5. To overcome the shortcomings of the trans-
olefin linker, it was proposed that the p-carboxylate of the biaryl
acid be replaced with an acylsulfonamide, which could then
function as a linker. The advantage of this moiety is that it can
bridge the wall between the two sites while the key electrostatic
interaction that the first-site ligand makes with R139 of the
protein is maintained. To test whether the acylsulfonamide could
serve as a suitable replacement for the carboxylic acid, methyl
biaryl acylsulfonamide 26 was synthesized and tested for binding
to Bcl-xL. The methyl biaryl acylsulfonamide displayed virtually
the same affinity as the biaryl acid (Table 3). On the basis of
13
(Figure 4). The model shows that compound 25 interacts with
this observation, a structurally diverse set of sulfonamides and
sulfonyl chlorides was selected from commercial sources and
our corporate compound repository to prepare a small library
both site 1 and site 2 in the hydrophobic groove of the protein.
However, from this NMR-based model, we concluded that the

Inhibitor of the Antiapoptotic Protein Bcl-x
L
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 659
Scheme 3a
Scheme 2a
a
Reagents and conditions: (a) Pd(PPh3)4, DME, 4-fluorophenylboronic
acid in EtOH, aq Na2CO3, reflux, 16 h; (b) 2 N NaOH, 2:1 THF:MeOH, rt,
1
6 h; (c) RSO2NH2, P-EDC, DMAP, ClCH2CH2Cl/t-BuOH, rt, 24 h, then
MP-TsOH, EtOAc, rt, 4 h.
of acylsulfonamides. The sulfonyl chlorides were converted to
their sulfonamide analogues through reaction with ammonium
hydroxide in diethyl ether at room temperature. The biphenyl
acid 1 was prepared through Suzuki coupling of ethyl 4-bro-
mobenzoate 27 with 4-fluorophenylboronic acid and followed
by ester cleavage (Scheme 2). A total of 120 acylsulfonamides
were then synthesized in parallel using the method of Sturino
1
4
et al., which was modified to include the use of resin-bound
1
5
tosic acid scavenging reagent. Of these, compound 28 (Table
) exhibited the greatest affinity for Bcl-xL, with an inhibition
a
Reagents and conditions: (a) NH4OH, diethyl ether, 0 °C to rt, 30 min;
b) 1, EDC, DMAP, CH2Cl2, rt, 16 h; (c) HNRR′, DMF, 100 °C, 16 h.
3
(
constant (Ki) of 0.245 ( 0.013 µM.
Structural Characterization of Compound 28. To guide
subsequent rounds of synthesis, an NMR-based structure of 28
bound to Bcl-xL was determined. The averaged-minimized
structure is shown in Figure 5. The biaryl portion of this
compound makes essentially the same contacts as the original
biaryl acid, with the exception that it is tilted slightly toward
the second site with respect to the free acid. The nitrophenyl
sits on top of F97 and below Y194, forming a hydrophobic
π-stacked arrangement. This type of interaction was not
observed for either the bound Bak or Bad peptide.
Second Parallel Synthesis. As the acylsulfonamide, nitro-
phenyl linker appeared to provide an energetically favorable
way of connecting a first-site fragment to a second-site fragment,
another round of parallel synthesis was carried out in order to
optimize second-site binding. Toward this end, an acylsulfona-
mide core was prepared that contained a 4-chloro-3-nitrophenyl
moiety. This core allowed the SAR to be rapidly established
through the nucleophilic aromatic substitution of its 4-chloro
substituent with selected amines (Scheme 3). The commercially
Figure 5. NMR-derived structure of acylsulfonamide compound 28
L
Figure 6. NMR-derived structure of compound 31 bound to Bcl-x .
bound to Bcl-x
with F97 and Y194 of the protein. (B) Bcl-x
accessible surface. Atoms are color-coded as in Figure 2.
L
. (A) The nitrophenyl portion of 28 forms a π-stack
The nitrophenyl portion of 31 forms a π-stack with F97 and Y194 of
the protein. In addition, the compound adopts an unexpected intramo-
lecular π-stack arrangement.
L
is shown as a solvent-

6
60 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Petros et al.
the highest binding affinity for Bcl-xL, with an inhibition
constant of 36 ( 2 nM.
Structural Characterization of Compound 31. To under-
stand why 31 binds so potently to Bcl-xL compared to other
compounds in the library, an NMR-based structure of 31 bound
to Bcl-xL was determined (Figure 6). A total of 103 protein/
ligand NOEs was used to dock this compound into the Bcl-xL
binding groove. The positions of the biaryl, acylsulfonamide,
and nitrophenyl portions of this compound superimpose well
with analogous portions of compound 28 bound to Bcl-xL.
Surprisingly, the S-phenyl ring is bent back underneath the
nitrophenyl. This is very different from what was observed for
the isothiochroman moiety of 28. To accommodate this ligand
conformation, the side chain of F97 rotates downward about
ø1 and its phenyl ring is stacks against the S-phenyl ring of the
ligand. The S-phenyl ring of the ligand, in turn, forms an
intramolecular stack with the nitrophenyl ring, while the
nitrophenyl ring stacks with Y194 of the protein. It is presum-
ably this extensive π-stacking arrangement that accounts for
the greater affinity of this ligand compared to others from the
library.
Analysis of the Fragment-Based Approach. Several groups
have reported on the identification of inhibitors of Bcl-xL or
Bcl-2, including antisense oligonucleotides, peptide inhibitors,
2
3
and small molecule antagonists. The organic small-molecule
inhibitors include natural products (e.g., antimycin A and
gossypol) and other compounds with liabilities that limit their
therapeutic potential, such as large size, low potency, poor
solubility, or synthetic intractability. In contrast, we have
successfully developed a highly potent inhibitor of Bcl-xL by
independently identifying and optimizing the multiple hot spots
on the protein surface and capitalizing on the combinatorial
advantage of fragment-based screening (see Figure 7A). For the
application to Bcl-xL described here, the first and second-site
libraries consisted of 10 000 and 3500 compounds, respectively,
and the set of linkers contained 21 moieties. The product of
these three libraries represents ∼700 million virtual compoundss
more than the total of all commercial screening libraries
combined. Thus, SAR by NMR accesses significantly larger
portions of chemical diversity space than do conventional lead
generation methods. In addition, the structure-guided iterative
library approach allowed for a highly focused and rapid
optimization of the initial leads to low nanomolar potency. An
8300-fold gain in potency was realized through library optimi-
zation (see Figure 7B).
Figure 7. (A) A summary of the SAR by NMR approach as applied
to Bcl-xL, in which two weakly binding ligands (1 and 11) that bound
to proximal sites on the protein were identified and linked to produce
single-digit micromolar inhibitors (22) of the protein. This lead was
further optimized using structure-based iterative library synthesis to
produce nanomolar leads (31). (B) A summary of the potency increases
realized through the iterative library approach, in which a 1200-fold
gain was obtained in the initial library and a 7-fold gain was achieved
in the second iteration, resulting in a 8300-fold gain over the initial
fragment leads.
Conclusions
We have discovered a high-affinity ligand for the antiapop-
totic protein Bcl-xL through a combination of fragment-based
screening (SAR by NMR) and parallel synthesis. The affinity
of our initial biaryl ligand (Kd ∼ 300 µM) for Bcl-xL was
increased by almost 4 orders of magnitude to obtain a lead
compound with an affinity (Ki) of 36 ( 2 nM. While potent,
the utility of compound 31 as a therapeutic agent was limited
by its poor aqueous solubility and tight binding to serum albumin
(unpublished observations). Through subsequent rounds of
structure-guided synthesis these limitations were overcome and,
as previously discussed, a compound that causes the regression
of established tumors in a mouse xenograft model was obtained.
NMR-based structure determination played an important role
in the discovery process, providing structural information for
key intermediates and thus guiding the parallel synthesis. Finally,
this study provides a fragment-based paradigm for the discovery
of molecules aimed at disrupting protein-protein interactions.
available 4-chloro-3-nitrophenylsulfonyl chloride 29 was con-
verted to the sulfonamide as described above. Acid 1 was then
coupled with 29 using EDC and DMAP to yield the desired
acylsulfonamide core 30. Substitution of the chlorine in 30 with
amines was easily achieved by heating the core in the presence
of a 5-fold excess of the nucleophile at 100 °C in DMF
overnight. A library of 125 compounds was prepared from a
group of amines selected on the basis of traditional medicinal
11
chemistry principles,1
6-20
diversity calculations,
13,21,22
and mo-
lecular modeling. Of these, compound 31 (Figure 6) exhibited

Inhibitor of the Antiapoptotic Protein Bcl-x
L
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 661
Experimental Section
fluorescein-labeled BH3 peptide derived from the Bad protein
(
NLWAAQRYGRELRRMSDK(FITC)FVD) (prepared in-house) as
the probe. The dissociation constant (K ) for the binding of this
peptide to Bcl-x is 20 nM. Test compounds were diluted in DMSO
Protein Preparation. A previously described,8 loop-deleted
version of Bcl-x that lacked the putative transmembrane helix was
employed in these studies. Uniformly N-labeled protein was
prepared by growing bacteria containing the Bcl-x plasmid on
minimal media containing 15N-labeled ammonium chloride as the
d
L
L
1
5
to concentrations between 100 µM and 1 pM and introduced into
each well of the plate. A mixture totaling 125 µL per well of assay
buffer (20 mM sodium phosphate, pH 7.4, 1 mM EDTA, 50 mM
L
1
5
13
sole nitrogen source while uniformly N-, C-labeled protein was
prepared by growing bacteria on minimal media containing 1 N-
labeled ammonium chloride as the sole nitrogen source and 13C-
labeled glucose as the sole carbon source. The hexahistidine-tagged
protein was purified by affinity chromatography on a nickel-
ProBond column (Invitrogen), concentrated, and exchanged into
L
NaCl, 0.05% PF-68), 6 nM BcL-x protein, 1 nM fluorescein-
5
labeled BAD peptide, and the DMSO solution of the compound
was shaken for 2 min and placed in a LJL Analyst (LJL Bio
Systems, CA). Polarization was measured at room temperature using
a continuous fluorescein lamp (excitation 485 nm, emission 530
nm). Inhibition constants (K ) were calculated using an in-house
i
written program in Microsoft Excel, and the reported standard
deviations are based on three measurements.
40 mM sodium phosphate buffer, pH 7.0, containing either 10%
or 100% D O plus 5 mM deuterated dithiothreitol. Protein samples
2
for NMR were 0.5-1.0 mM in microcells.
NMR-Based Screening. NMR-based screening was conducted
Chemistry. General Methods. All reactions were carried out
under inert atmosphere (N ) and at room temperature unless
2
1
5
1
by acquiring sensitivity-enhanced N/ H HSQC spectra on 500 µL
of uniformly 15N-labeled protein (100 µM) in the presence and
absence of added compound(s). For the initial screen, 9373
compounds (〈MW〉 ∼ 210) were added as mixtures of 10 at 1 mM
each. Sixty-six of the mixtures caused significant chemical shift
changes upon addition to Bcl-x
identified by individually testing the 660 compounds that comprised
these active mixtures, yielding a total of 49 ligands exhibiting K
values less than 5 mM. The second-site screen was performed in
the presence of 2 mM 4-fluoro-biphenyl-4-carboxylic acid (1). In
this case, 3472 compounds (〈MW〉 ∼ 150) were added as mixtures
of five at 5 mM each. The second-site ligands were identified by
individually testing the 60 compounds that comprised the active
otherwise noted. Solvents and reagents were obtained commercially
and were used without further purification. All reported yields are
of isolated products and are not optimized. P-EDC was purchased
from Polymer Laboratories and MP-TsOH was purchased from
1
Argonaut Technologies. H NMR spectra were obtained on a Varian
L
. The first-site ligands were
UNITY or Inova (500 MHz), Varian UNITY (400 MHz), or Varian
UNITY plus or Mercury (300 MHz) instrument. Chemical shifts
are reported as δ values (ppm) downfield relative to TMS as an
internal standard, with multiplicities reported in the usual manner.
Mass spectra determinations were performed by the Analytical
Research Department, Abbott Laboratories; DCI indicates chemical
ionization in the presence of ammonia, ESI indicates electron spray
ionization, APCI indicates atmospheric pressure chemical ionization
with ammonia. Elemental analyses were performed by Quantitative
Technologies, Inc., Whitehouse, NJ, or by Robertson Microlit
Laboratories, Inc., Madison, NJ. Column chromatography was
carried out in flash mode on silica gel (Merck Kieselgel 60, 230-
D
d
mixtures, yielding a total of 24 compounds exhibiting K values
less than 5 mM. Dissociation constants were obtained by monitoring
the chemical shift changes as a function of ligand concentration
using a single binding site model. A least squares grid search was
performed by varying the values of K
the fully saturated protein. Standard deviations were derived for
each K by monitoring three different cross-peaks in the HSQC
spectrum.
NMR-Based Structural Studies. NMR spectra for structural
studies were recorded on Bruker DRX600 and DRX800 spectrom-
eters at 303 K. Resonance assignments for liganded Bcl-x were
extrapolated from those of the apo protein by comparing two-
d
and the chemical shift of
4
00 mesh). Unless otherwise noted, preparative HPLC samples were
purified on a Waters Symmetry C8 column (25 × 100 mm, 7 µm
particle size) using a gradient of 10-100% CH CN:0.1% TFA over
min (10-min run time) at a flow rate of 40 mL/min. Analytical
HPLC spectra were acquired on an Agilent Zorbax SB-C8 column
d
3
8
(
4.6 × 75 mm, 3.5-µm particle size) using a gradient of 30-100%
L
CH
CN:water over 8 min and an Agilent Zorbax SB-C18 column
3
(
4.6 × 75 mm, 3.5 µm particle size) using a gradient of 10-100%
1
3
15
13
dimensional C and N HSQC spectra and three-dimensional C-
CH
3
CN:water over 8 min. UV detection at 254 nm.
and 15N-edited NOESY spectra of the liganded and the unliganded
4′-Fluoro-3-methoxybiphenyl-4-carboxylic Acid Methyl Ester
protein. Intraprotein NOEs for those residues of Bcl-x
L
that line
(22). A 250-mL round-bottom flask was charged with methyl
1
3
the binding groove were extracted from three-dimensional C- and
4-chloro-2-methoxybenzoate (21) (6.5 g, 32.5 mmol), 4-fluorophe-
15
N-edited NOESY spectra recorded with a mixing time of 80 ms.
nylboronic acid (4.56 g, 32.5 mmol), and CsF (14.8 g, 97.5 mmol)
followed by NMP (65 mL) and trans-dichlorobis(tricyclohexy-
lphosphine)palladium(II) (0.72 g, 0.975 mmol) and was heated to
13
Protein-ligand NOEs were extracted from three-dimensional C-
edited, 12C-filtered NOESY spectra recorded with mixing times
ranging from 150 to 250 ms.
90 °C for 12 h. The resulting mixture was diluted with ethyl acetate
Structure Calculations. All structure calculations were carried
(300 mL), washed with water and brine, dried over Na SO , and
2
4
out with the program CNX.24 Initially, Bcl-x
L
ligands were
concentrated under reduced pressure. Purification by silica gel
chromatography eluting with 5% ethyl acetate in hexanes yielded
randomly positioned near the binding groove of the apo protein
and the observed intermolecular NOEs were used to dock the
ligands into the groove. This docking was followed by energy
minimization and a standard simulated annealing protocol.25 During
the simulated annealing, coordinates of the protein were held fixed
with the exception of those residues that line the binding groove
1
6.50 g (77%) of the desired product: H NMR (300 MHz, CDCl3)
δ 7.87 (d, J ) 8.1 Hz, 1H), 7.59-7.53 (m, 2H), 7.18-7.10 (m,
+
4H), 3.97 (s, 3H), 3.91 (s, 3H); MS (DCI) m/z 261 (M + H) .
Anal. (C H FO ) C, H.
15
13
3
4′-Fluoro-3-trifluoromethanesulfonyloxybiphenyl-4-carboxy-
(
residues 96-112, 127-142, 191-194). This protocol was based
lic Acid Methyl Ester (23). A solution of 22 (6.5 g, 25.0 mmol)
in DCM (100 mL) was cooled to -78 °C, treated dropwise with
BBr (40 mL of a 1M solution in DCM, 40.0 mmol), and stirred
3
on our structural studies of Bak peptide binding to Bcl-x , for which
L
we observed structural rearrangements only for residues lining the
hydrophobic groove upon peptide binding. The conformation of
these residues was governed by the observed intraprotein NOEs
and intermolecular NOEs.
For the initial biaryl acid, compound 1, a total of 75 intermo-
lecular NOEs were employed to dock the ligand to the protein.
For the ternary complex of Bcl-x
intermolecular NOEs were used for compound 1 with 14 NOEs
for compound 13.
for 2.5 h. Methanol (30 mL) was added slowly, and the mixture
was allowed to warm to room temperature and concentrated under
reduced pressure. Purification by silica gel chromatography eluting
with 5% ethyl acetate in hexanes yielded 5.30 g (86%) of the
intermediate phenol: 1H NMR (400 MHz, CDCl ) δ 7.87 (d, J )
3
L
with compounds 1 and 13, 75
8.3 Hz, 1H), 7.59-7.53 (m, 2H), 7.16-7.10 (m, 3H), 7.06 (dd, J
) 8.3 Hz, J ) 1.9 Hz, 1H), 3.96 (s, 3H); MS (DCI) m/z 247 (M +
+
H) . Anal. (C H FO ) C, H.
1
4
11
3
Fluorescence Polarization Assay. Assays for the inhibition of
A solution of the above phenol (5.0 g, 20.7 mmol) and
triethylamine (6.0 mL, 42.0 mmol) in DCM (100 mL) was cooled
L
Bcl-x were performed in 96-well microtiter plates using a

6
62 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Petros et al.
to 0 °C and treated dropwise with trifluromethanesulfonic anhydride
4.53 mL, 26.9 mmol). The mixture was allowed to warm to room
temperature and to stir overnight. The resulting mixture was diluted
with DCM (300 mL); washed with 1.5% aqueous HCl, saturated
aqueous NaHCO , water, brine; dried over Na SO ; and concen-
3 2 4
trated under vacuum. Purification by silica gel chromatography
eluting with 10% ethyl acetate in hexanes yielded 7.0 g (90%) of
ethyl ester: 1H NMR (500 MHz, CDCl
7.62-7.57 (m, 4H), 7.17-7.14 (m, 2H), 4.40 (q, J ) 7.3 Hz, 2H),
1.42 (t, J ) 7.0 Hz, 3H); MS (DCI) m/z 245 (M + H) .
3
) δ 8.11-8.10 (m, 2H),
(
+
A solution of the above ethyl ester (7.1 g, 29.09 mmol) in THF
(80 mL)/MeOH (80 mL) was treated with a 2 N aqueous solution
of NaOH (50 mL) at room temperature overnight. The reaction
mixture was concentrated down to the water layer under reduced
pressure and was acidified by addition of a 2 N aqueous solution
of HCl. The mixture was extracted with ethyl acetate, the combined
the desired product: 1H NMR (400 MHz, CDCl
) δ 8.15 (d, J )
.3 Hz, 1H), 7.63 (dd, J ) 8.3, 1.9 Hz, 1H), 7.59-7.53 (m, 2H),
.44 (m, 1H), 7.21-7.16 (m, 2H), 3.99 (s, 3H); MS (DCI) m/z 396
3
8
7
4
organic extracts were dried over MgSO , and the solvents were
+
(
M + NH
′-Fluoro-3-vinylbiphenyl-4-carboxylic Acid Methyl Ester
24). A solution of 23 (1.51 g, 4.0 mmol) in DME (20 mL) and
4 10 4 3
) . Anal. (C15H F O S) C, H.
removed under reduced pressure to afford 5.94 g (94%) of the
1
4
desired product: H NMR (300 MHz, DMSO-d ) δ 12.96 (bs, 1H),
6
(
8.04-8.01 (m, 2H), 7.82-7.76 (m, 4H), 7.37-7.29 (m, 2H); MS
+
+
methanol (10 mL) was treated with vinyldibutylboronate (0.96 g,
.2 mmol), Pd(PPh (0.23 g, 0.20 mmol), and CsF (1.82 g, 12.0
(DCI) m/z 216 (M ), 234 (M + NH ) .
4
5
3 4
)
Parallel Synthesis A: Acylsulfonamide Formation. The reac-
tions were carried out in 12-mL glass vials fitted with Teflon-lined
screw caps. Each vial was charged with the appropriate sulfonamide
mmol). The resulting mixture was brought to reflux and stirred
overnight. After allowing to cool to room temperature, the solvent
was removed under vacuum and the residue partitioned between
diethyl ether (300 mL) and water (60 mL). The organic layer was
washed with water and brine, dried over Na SO , and concentrated
2 4
under reduced pressure. Purification by silica gel chromatography
eluting with 4% ethyl acetate in hexanes yielded 0.86 g (84%) of
(0.31 mmol) in a mixture of ClCH
2 2
CH Cl (2.8 mL) and t-BuOH
(2.8 mL). DMAP (56 mg, 0.46 mmol), 1 (100 mg, 0.46 mmol),
and P-EDC (443 mg, 0.62 mmol, loading ) 1.4 mmol/g) were
added and the mixtures were agitated at room temperature for 24
h. Each reaction mixture was then diluted with 3 mL of ethyl
acetate, MP-TsOH (561 mg, 2.3 mmol, loading ) 4.1 mmol/g) was
added, and agitation was continued for another 4 h. The mixtures
were filtered and the solvents were evaporated under reduced
pressure. The crude products were purified by preparative HPLC.
the desired product: 1H NMR (400 MHz, CDCl
8
(
) δ 7.96 (d, J )
.0 Hz, 1H), 7.72 (d, J ) 1.6 Hz, 1H), 7.60-7.51 (m, 3H), 7.48
dd, J ) 8.3, 1.6 Hz, 1H), 7.15 (t, J ) 8.6 Hz, 2H), 5.70 (d, J )
7.2 Hz, 1H), 5.40 (d, J ) 10.7 Hz, 1H), 3.92 (s, 3H); MS (DCI)
3
1
+
m/z 257 (M + H) . Anal. (C16
2
H13FO ) C; H.
N-(4′-Fluorobiphenyl-4-ylcarbonyl)methanesulfonamide (26).
Compound 26 (57 mg, 63%) was obtained according to the
procedure given for parallel synthesis A from 1 and methane-
3
-(2-Biphenyl-3-ylvinyl)-4′-fluorobiphenyl-4-carboxylic Acid
(25). A solution of 24 (0.10 g, 0.40 mmol) and 3-bromobiphenyl
(0.12 g, 0.50 mmol) in DMF (2 mL) and acetonitrile (4 mL) was
sulfonamide: 1H NMR (300 MHz, CDCl
3
) δ 8.62 (bs, 1H), 7.93-
treated with Pd
2
(dba)
3
(0.037 g, 0.04 mmol), tri-o-tolylphosphine
N (111 µL, 0.8 mmol) and heated to 85
C overnight. After allowing to cool to room temperature, the
7
.90 (m, 2H), 7.70-7.67 (m, 2H), 7.62-7.57 (m, 2H), 7.21-7.15
(0.025 g, 0.08 mmol), Et
3
+
(
m, 2H), 3.47 (s, 3H); MS (DCI) m/z 311 (M + NH
N-(4′-Fluorobiphenyl-4-ylcarbonyl)-4-(N′-isothiochroman-4-
ylidenehydrazino)-3-nitrobenzenesulfonamide (28). Compound
8 (82 mg, 46%) was obtained according to the procedure given
for parallel synthesis A from 1 and 4-(N′-isothiochroman-4-
4
) .
°
mixture was concentrated under vacuum, dissolved in ethyl acetate
100 mL), washed with water and brine, dried over Na SO , and
(
2
4
2
concentrated under reduced pressure. Purification by silica gel
chromatography eluting with 4% ethyl acetate in hexanes yielded
1
1
ylidenehydrazino)-3-nitrobenzenesulfonamide (Maybridge):
NMR (400 MHz, DMSO-d ) δ 11.04 (s, 1H), 8.73 (m, 1H), 8.18-
.16 (m, 3H), 7.98-7.95 (m, 2H), 7.81-7.77 (m, 5H), 7.37-7.28
H
0
.11 g (67%) of the intermediate methyl ester: H NMR (400 MHz,
6
CDCl
.87 (d, J ) 1.8 Hz, 1H), 7.75 (m, 1H), 7.64-7.61 (m, 3H), 7.58
m, 1H), 7.52-7.42 (m, 5H), 7.38-7.34 (m, 2H), 7.17 (t, J ) 8.8
Hz, 2H), 7.12 (d, J ) 16.3 Hz, 1H), 3.94 (s, 3H); MS (DCI) m/z
3
) δ 8.12 (d, J ) 16.3 Hz, 1H), 8.02 (d, J ) 8.3 Hz, 1H),
8
7
+
(
m, 5H), 4.03 (s, 2H), 3.88 (s, 2H); MS (ESI) m/z 577 (M + H) ;
HRMS (ESI) m/z calcd (C28 ) 577.1010, found 577.1012
4.627 min (C8), t 5.673 min (C18).
-Chloro-3-nitrobenzenesulfonamide (29). To an ice-cooled
solution of 4-chloro-3-nitrobenzenesulfonyl chloride (9.0 g, 35.0
mmol) in diethyl ether (150 mL) was added dropwise NH OH (66
mL, 28-30% NH ). After the addition was complete, the ice bath
was removed and the reaction mixture was stirred for an additional
0 min at room temperature. Water (100 mL) was added and the
mixture was extracted with ethyl acetate. The combined organic
extracts were dried over MgSO and the solvents were removed
under reduced pressure to give 8.0 g (97%) of the desired product:
(
H
4 5 2
21FN O S
+
+
(M + H) ; HPLC t
R
R
4
09 (M + H) . Anal. (C28
The above methyl ester (0.050 g, 0.122 mmol) was dissolved in
THF (1 mL)/MeOH (0.5 mL)/H O (0.25 mL), treated with LiOH‚
O (0.025 g, 0.595 mmol), and stirred overnight. The mixture
2
H21FO ) C, H.
4
2
4
H
2
3
was treated with 1 N HCl (2 mL) and extracted with ethyl acetate.
The organic layer was washed with water and brine, dried over
Na SO , and concentrated under vacuum. Purification by silica gel
2 4
3
chromatography eluting with 3% methanol in 1:1 ethyl acetate:
hexanes yielded 0.045 g (94%) of the desired product: 1H NMR
4
(
400 MHz, CDCl
Hz, 1H), 7.95 (d, J ) 8.3 Hz, 1H), 7.90-7.85 (m, 4H), 7.70 (d, J
8.0 Hz, 1H), 7.67 (dd, J ) 8.0, 1.6 Hz, 1H), 7.64-7.58 (m,
H), 7.52-7.46 (m, 4H), 7.42-7.32 (m, 3H); MS (APCI) m/z 395
3
) δ 8.09 (d, J ) 6.4 Hz, 1H), 8.08 (d, J ) 8.0
1
H NMR (300 MHz, CDCl ) δ 8.42 (d, J ) 2.3 Hz, 1H), 8.05 (dd,
3
J ) 8.5, 2.0 Hz, 1H), 7.73 (d, J ) 8.5 Hz, 1H), 4.94 (bs, 2H); MS
(
)
2
+
DCI) m/z 235, 237 (M ).
+
-
(
M + H) , 393 (M - H) . Anal. (C27
′-Fluorobiphenyl-4-carboxylic Acid (1). To a solution of Pd-
PPh (1.0 g, 0.86 mmol) in DME (250 mL) was added ethyl
-bromobenzoate (27) (5.0 mL, 30.62 mmol), and the mixture was
H19FO
2
) C, H.
4-Chloro-N-(4′-fluorobiphenyl-4-ylcarbonyl)-3-nitrobenzene-
sulfonamide (30). A mixture of 1 (5.9 g, 27.5 mmol), 29 (7.2 g,
0.3 mmol), EDC (6.3 g, 33.0 mmol), and DMAP (1.7 g, 13.8
mmol) in CH Cl (100.0 mL) was stirred at ambient temperature
4
3
(
4
3 4
)
2
2
for 24 h. The solvent was removed under reduced pressure and the
residue was purified by silica gel chromatography eluting with 5%
MeOH in DCM + 0.5% HOAc to give 6.8 g (57%) of desired
stirred at ambient temperature for 10 min. A solution of 4-fluo-
rophenylboronic acid (5.0 g, 35.73 mmol) in EtOH (40 mL) was
added and stirring was continued for 15 min. A 2 M aqueous
product: 1H NMR (300 MHz, DMSO-d
) δ 8.63 (d, J ) 2.2 Hz,
H), 8.27 (dd, J ) 8.5, 2.2 Hz, 1H), 8.07 (d, J ) 8.5 Hz, 1H),
.99-7.97 (m, 2H), 7.83-7.78 (m, 4H), 7.36-7.30 (m, 2H): MS
2 3
solution of Na CO (69 mL) was added and the reaction mixture
6
1
7
was heated to reflux overnight. After cooling, the organic solvents
were evaporated under reduced pressure, water (100 mL) and diethyl
ether (200 mL) were added, and the mixture was filtered through
Celite. The layers were separated, and the aqueous layer was
extracted with diethyl ether. The combined organic extracts were
dried over MgSO and evaporated under reduced pressure, and the
4
residue was purified by silica gel chromatography eluting with 33%
ethyl acetate in hexanes to give 7.1 g (95%) of the intermediate
+
(DCI) m/z 452 (M + NH
4 2 5
) . Anal. (C19H12ClFN O S) C; H; N.
Parallel Synthesis B: Nucleophilic Substitution. The reactions
were carried out in 4-mL glass vials with Teflon-lined screw caps.
Each vial was charged with the appropriate amine (0.58 mmol), a
stock solution of 30 (50 mg, 0.12 mmol) in DMF (2.0 mL) was
added, and the mixtures were agitated and heated to 100 °C

Inhibitor of the Antiapoptotic Protein Bcl-x
L
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 663
overnight. The organic solvent was evaporated under reduced
pressure and the residue was purified by preparative HPLC.
N-(4′-Fluorobiphenyl-4-ylcarbonyl)-3-nitro-4-(2-phenylsulfa-
nylethylamino)benzenesulfonamide (31). Compound 31 (14 mg,
(10) Arkin, M. R.; Wells, J. A. Small-molecule inhibitors of protein-
protein interactions: Progressing towards the dream. Nat. ReV. Drug
DiscoVery 2004, 3, 301-317.
(
11) Oltersdorf, T.; Elmore, S. W.; Shoemaker, A. R.; Armstrong, R. C.;
Augeri, D. J.; et al. An inhibitor of Bcl-2 family proteins induces
regression of solid tumors. Nature 2005, 435, 677-681.
12) Zhang, H.; Nimmer, P.; Rosenberg, S. H.; Ng, S. C.; Joseph, M.
Development of a high-throughput fluorescence polarization assay
for Bcl-xL. Anal. Biochem. 2002, 307, 70-75.
2
2%) was obtained according to the procedure given for parallel
1
synthesis B from 30 and 2-phenylthioethylamine: H NMR (300
MHz, DMSO-d ) δ 12.50 (bs, 1H), 8.82-8.78 (m, 1H), 8.63 (d, J
2.0 Hz, 1H), 7.97-7.92 (m, 3H), 7.82-7.77 (m, 4H), 7.39-
.15 (m, 8H), 3.71-3.65 (m, 2H), 3.33-3.27 (m, 2H); MS m/z
(
6
)
(
(
(
13) Martin, Y. C. Molecular diversity: How we measure it? Has it lived
7
5
+
up to its promise? Farmaco 2001, 56, 137-139.
52 (M + H) . Anal. (C27
3 5 2
H22FN O S ) C; H; N.
14) Sturino, F. S.; Labelle, M. A convenient method for the preparation
of acylsulfonamide libraries. Tetrahedron Lett. 1998, 39, 5891-5894.
15) Lobb, K. L.; Hipskind, P. A.; Aikins, J. A.; Alvarez, E.; Cheung, Y.
Y.; et al. Acyl sulfonamide anti-proliferatives: Benzene substituent
structure-activity relationships for a novel class of antitumor agents.
J. Med. Chem. 2004, 47, 5367-5380.
Supporting Information Available: Elemental analysis and
HPLC data. This material is available free of charge via the Internet
at http://pubs.acs.org.
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