Structure-Guided Development of Potent Benzoylurea Inhibitors of BCL-XLand BCL-2

Article abstract of DOI:10.1021/acs.jmedchem.0c01771

The BCL-2 family of proteins (including the prosurvival proteins BCL-2, BCL-XL, and MCL-1) is an important target for the development of novel anticancer therapeutics. Despite the challenges of targeting protein-protein interaction (PPI) interfaces with small molecules, a number of inhibitors (called BH3 mimetics) have entered the clinic and the BCL-2 inhibitor, ABT-199/venetoclax, is already proving transformative. For BCL-XL, new validated chemical series are desirable. Here, we outline the crystallography-guided development of a structurally distinct series of BCL-XL/BCL-2 inhibitors based on a benzoylurea scaffold, originally proposed as α-helix mimetics. We describe structure-guided exploration of a cryptic "p5"pocket identified in BCL-XL. This work yields novel inhibitors with submicromolar binding, with marked selectivity toward BCL-XL. Extension into the hydrophobic p2 pocket yielded the most potent inhibitor in the series, binding strongly to BCL-XL and BCL-2 (nanomolar-range half-maximal inhibitory concentration (IC50)) and displaying mechanism-based killing in cells engineered to depend on BCL-XL for survival.

Full text of DOI:10.1021/acs.jmedchem.0c01771

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Article
Structure-Guided Development of Potent Benzoylurea Inhibitors of
BCL‑XL and BCL‑2
Michael J. Roy, Amelia Vom, Toru Okamoto, Brian J. Smith, Richard W. Birkinshaw, Hong Yang,
Houda Abdo, Christine. A. White, David Segal, David C. S. Huang, Jonathan B. Baell, Peter M. Colman,
Peter E. Czabotar,* and Guillaume Lessene*
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ABSTRACT: The BCL-2 family of proteins (including the prosurvival proteins BCL-2, BCL-XL, and MCL-1) is an important target
for the development of novel anticancer therapeutics. Despite the challenges of targeting protein−protein interaction (PPI)
interfaces with small molecules, a number of inhibitors (called BH3 mimetics) have entered the clinic and the BCL-2 inhibitor, ABT-
199/venetoclax, is already proving transformative. For BCL-XL, new validated chemical series are desirable. Here, we outline the
crystallography-guided development of a structurally distinct series of BCL-XL/BCL-2 inhibitors based on a benzoylurea scaffold,
originally proposed as α-helix mimetics. We describe structure-guided exploration of a cryptic “p5” pocket identified in BCL-XL. This
work yields novel inhibitors with submicromolar binding, with marked selectivity toward BCL-XL. Extension into the hydrophobic
p2 pocket yielded the most potent inhibitor in the series, binding strongly to BCL-XL and BCL-2 (nanomolar-range half-maximal
inhibitory concentration (IC50)) and displaying mechanism-based killing in cells engineered to depend on BCL-XL for survival.
The PPI interfaces underpinning the interactions between
BCL-2 family proteins are typified by large, shallow, and
mainly hydrophobic interaction surfaces that represent
challenging targets for medicinal chemistry. Foremost among
these are the binding interfaces formed by amphipathic BH3
domains and the flexible surface grooves formed on their
binding partners (reviewed extensively elsewhere13). In
addition, many studies have overlooked the complexity of
the BCL-2 family biology, leading to questionable conclusions
regarding the mechanism of action.4,14 Despite many putative
BCL-2 inhibitors being published, the number of mechanis-
tically well-validated and structurally distinct series of selective
inhibitor molecules remains relatively limited (e.g., ABT-737
(1), Figure 1a,15 and clinical relatives ABT-263 and
venetoclax;7,16 molecules based on the AbbVie scaffold;17−20
WEHI-539 and related derivatives;21−24 S638458). In the case
of BCL-XL, the recent development of targeted protein
INTRODUCTION
■
The BCL-2 family of proteins is widely recognized as an
attractive set of cancer therapeutic targets. The balance
between pro- and antiapoptotic members of this family
mediates intra- and extracellular stress signals through a
cascade of protein−protein interactions (PPIs), leading to a
key mitochondrial permeabilization event and subsequent
caspase activation that ultimately results in cell demolition.
Decreased apoptotic signaling, due to overexpression of
prosurvival proteins such as BCL-2, BCL-XL, and MCL-1, is
a key driver in tumorigenesis and in resistance to chemo-
therapy, as most anticancer drugs rely on a functional BCL-2
family-mediated apoptotic cascade.1−3
Targeting prosurvival proteins to reactivate a blunted
apoptotic response in cancer cells has been a longstanding
and ambitious goal of a number of pharmaceutical and
academic research programs.4,5 Just as the expression levels of
individual BCL-2 proteins in different cell types vary,6 so do
their individual contributions to different cancers. Recent
developments also support the therapeutic potential of
selectively targeting individual BCL-2 family proteins: a first-
in-class selective BCL-2 inhibitor, venetoclax, is now approved
for patients with chronic lymphocytic leukemias7 and three
selective MCL-1 inhibitors have entered the clinic.5,8−12
Received: October 8, 2020
Published: April 27, 2021
https://doi.org/10.1021/acs.jmedchem.0c01771
© 2021 American Chemical Society
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Figure 1. (a) Structures of ABT-737 (1)15 and novel benzoylurea hit 2. (b) Crystal structure of 2 in complex with BCL-XL, determined to 2.15 Å
(PDB code: 6UVD, carbon: light blue, BCL-XL in surface representation in gray), showing canonical hydrophobic pockets p2 and p4 and a cryptic
pocket p5 along the BH3-binding groove. (c) Overlaid crystal structures of 2 (from the BCL-XL:2 complex) with the BIM BH3 peptide (from the
BCL-XL:BIMBH3 complex, PDB code: 3FDL, magenta),33 highlighting key conserved residues of the BH3 domain including hydrophobic amino
acids h1−h4 and Asp99 (that form an important salt bridge interaction with Arg139 of BCL-XL). (d) Compounds 3−6 were synthesized to explore
the negative structure−activity relationship (SAR) and validate the observed binding mode of 2 with BCL-XL.
degraders (also known as proteolysis-targeting chimeras,
PROTACs) that aim to target BCL-XL for proteasomal
degradation,25 including in a tissue-specific manner, offers
new potential to ameliorate the known on-target platelet
toxicity of BCL-XL inhibition.26−28 BCL-XL targeting pro-
drugs29 and antibody−drug conjugates (ADCs)30 are also
being developed with this aim. To date, these molecules all
derive from similar chemotypes (either ABT-263 or structural
relatives of WEHI-539). New structural knowledge and novel
chemical series are thus desirable to support the next
generation of selective BCL-2 family inhibitors and degraders,
particularly in light of recently published structural insights to
guide PROTAC development targeting BCL-XL.31
We had previously outlined a structure-guided de novo
design strategy, using a benzoylurea scaffold to spatially
reproduce the projections of α-helical peptide pharmaco-
phores, such as those of BH3 domains within BH3:groove
interactions characteristic of the BCL-2 family (Figure S1).32
That initial work led to benzoylurea inhibitors of BCL-XL in
the low micromolar range.
Herein, we report another series initially developed toward
targeting BCL-XL, based on a different benzoylurea hit,
compound 2 (Figure 1a). Using the crystal structure of 2
bound to BCL-XL, including an unexpected interaction in a
binding site we have termed the “cryptic p5” pocket (Figure
1b), we outline the rational elaboration of 2 to target the p2
and cryptic p5 pockets of BCL-XL, leading to potent
benzoylurea molecules with half-maximal inhibitory concen-
tration (IC50) values in the nanomolar range for both BCL-XL
and BCL-2. We show that the most potent molecule in the
series can elicit mechanism-based cell killing of cells dependent
on BCL-XL for survival, proceeding via release of cytochrome c
from mitochondria.
RESULTS AND DISCUSSION
■
As part of our previous work, a modular synthesis strategy was
used to prepare a library of benzoylurea molecules (∼130
compounds) encompassing multiple orientations of various
side chains to explore a relatively wide structure−activity
relationship (SAR) space (Figure S1, Supporting Information).
This library was subsequently screened using a bead-based
AlphaSCREEN (AS) assay for displacement of the BCL-XL/
BIMBH3 or MCL-1/BAKBH3 interactions (Supporting Informa-
tion).32,34 In this screen, we identified benzoylurea hit 2
(Figure 1a) with a promising IC50 value for BCL-XL in the
submicromolar range (Table 1). The comparatively weaker
IC50 value of 2 for MCL-1 also hinted at selectivity across the
family of proteins.
We next obtained a crystal structure of 2 in complex with
BCL-XL (Figures 1b and 2), which confirmed binding to the
hydrophobic groove of BCL-XL responsible for interacting with
BH3 domains (Figure 1c). Two binding orientations of 2 were
observed in this structure. One of these we considered to be a
“secondary” mode, as the binding appeared to be influenced by
its location at a crystal packing interface (Figure S2). The other
binding mode we have termed the “primary” mode (Figure 2),
as subsequent data suggest that it is more likely to reflect the
predominant bound conformation in solution. This mode
exhibited some key features of the original benzoylurea scaffold
“peptidomimetic” design, including formation of an intra-
molecular hydrogen bond (pseudo-six-membered ring) to
partially preorganize the molecule.35
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form similar interactions as those of Leu94 and Phe101 of the
BIMBH3 peptide with BCL-XL. Interestingly, the carboxylic acid
moiety of 2 also lies proximal to Asp99 of BIMBH3 (Figure 1c),
a conserved acidic residue in all BH3 domains that mediates a
key salt bridge interaction, with residue Arg139 in the case of
BCL-XL. However, it does not precisely align with Asp99 of
BIMBH3, nor does it directly engage Arg139 of BCL-XL (Figure
S2b), but rather aligns to a position one helix-turn further
along the BIMBH3 peptide (Figure 1c). Notably, the S-benzyl
moiety of 2 bound unexpectedly in a relatively unexplored
pocket (adjacent to p4, Figure 1b) that is created by the
displacement of Tyr195 (Figure 2a). We refer to this binding
site as the cryptic p5 pocket (Figure 2a). This pocket is not
present in unliganded BCL-XL (Figure 2b,c), nor is it observed
with BH3 peptides (Figure 2d) or the BCL-XL inhibitor ABT-
737 (1) (Figure S3a). Interestingly, the triflone moiety of
ABT-263 does occupy a similar pocket when bound to BCL-
XL and also BCL-2 (Figure S3b), as does the methoxy
substituent of A-1342192 when bound to BCL-XL, which was
first described while this manuscript was under review (Figure
S3c).36 A-1342192 (referred to as compound 14 in the
relevant publication) is a BCL-XL inhibitor, structurally related
to WEHI-539 and A-1155463.
Table 1. IC50 Values for Compounds 2−8 Determined by
AlphaSCREEN (AS) Assay
a
IC50 (μM, AS)
compounds
BCL-XL
MCL-1
b
b
ABT-737 (1)
0.004 0.005
0.51 0.07
13.1 0.3
>100
>100
40 60
2
3
4
5
6
7
8
7.7 1.6
b
b
44
9
>100
>100
28
31
5
5
0.24 0.02
0.21 0.05
1.5 0.1
2.0 0.1
a
IC50 values are reported in micromolar units. Unless noted, all data
are reported as mean standard error of mean (SEM) from N = 3
b
independent experiments. Data are mean standard deviation (SD)
from N = 2 independent experiments.
Surprisingly, the orientation of the bound compound in the
primary mode was in the opposite direction along the groove
to that intended in our original design and to that observed in
our previously reported series of BCL-XL inhibitors (Figure
S1).32 Nonetheless, many of the interactions of 2 with BCL-XL
in this binding mode also resembled those observed for BH3
domains bound to BCL-XL (e.g., BCL-XL:BIMBH3 complex,
Figure 1c). In particular, the biphenyl and tolyl moieties of 2
(that engage the p2 and p4 pockets of BCL-XL, respectively)
To help validate the binding mode of 2 observed in the
crystal structure, we next generated a series of analogues to
ascertain the role of the acid moiety of 2 and the relative
importance of interactions in the p4 and cryptic p5 pockets.
Figure 2. Displacement of the side chain of Tyr195 of BCL-XL creates the cryptic p5 binding pocket. (a) The side chain of Tyr195 is displaced by
the S-benzyl moiety of 2 in the BCL-XL:2 complex (PDB code: 6UVD, carbon: light blue), forming a cryptic p5 pocket that is lined by the C-
terminus of the α8 helix and side chains of Trp137, Phe191, Leu194, and Tyr195 of BCL-XL. (b, c) Crystal structure of apo BCL-XL (PDB code:
1MAZ), (b) surface representation and (c) colored by B-factor (dark blue, low B-factor; red, high B-factor),40 the side chain of Tyr195 lines the
hydrophobic groove adjacent to the p2, p3, and p4 ligand binding pockets. B-factor analysis suggests that the region of BCL-XL in which Tyr195
lies (extending from the C-terminal end of the α8 helix), in addition to the α3 helix and α2−3 hinge regions, all exhibit some structural flexibility.
(d) The cryptic p5 pocket is not present in any structure of BCL-XL bound to native ligands such as in the BCL-XL:BAXBH3 structure (PDB code:
3PL7; overlaid with 2 from the BCL-XL:2 complex).41 In many of these structures, a pocket that could be termed the “canonical p5” pocket exists,
into which residues turn up from the h4 residue inset, in this case Met74 of the BAXBH3. The cryptic p5 pocket is adjacent to this as demonstrated
by the position of the S-phenyl moiety of 2 in this overlay.
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Thus, compounds 3−6 (Figure 1d) were evaluated in an
AlphaSCREEN (AS) assay (Table 1) and revealed the acidic
moiety of 2 to be essential, as its removal or methylation
(compounds 4 and 5) completely abrogated binding to both
BCL-XL and MCL-1, similar to the importance of the
conserved acidic residue on BH3 domains. The carboxylate
of 2 does not appear to directly engage Arg139 of BCL-XL in
the bound structure, instead interacting with Asn136 (Figure
3a). A similar result was also observed in the ABT-737 series
benzyl moiety of 2 to an S-methyl weakened the IC50 for BCL-
XL by 25-fold (compound 3, Figure 1d), while replacing the p4
tolyl moiety of 2 for an n-propyl had a more significant effect
on BCL-XL binding (60-fold weaker IC50 value of 6 for BCL-
XL relative to 2) than MCL-1 (only 4−5-fold weaker IC50 of 6
for MCL-1 relative to 2). This result was also consistent with
mutagenesis studies, which showed that mutation of the
corresponding “h4” Phe101 residue of BIM to Ala diminishes
the affinity of human BIM for BCL-XL by 90-fold but is less
important for MCL-1 binding.42 Combined, these results
validated that the crystal structure of the BCL-XL:2 complex
does indeed reflect the binding of 2 to BCL-XL in solution. We
therefore next looked to utilize this structure for the rational
optimization of 2 to improve the affinity of the series for BCL-
XL.
Previous work by our team as well as by researchers at
AbbVie has described the significant gains in potency toward
BCL-XL/BCL-2 achievable via interactions targeted to the p2
pocket.43 Using an overlay of the BCL-XL:2 X-ray crystal
structure with that of the BCL-XL:ABT-737 (1) complex
(Figure 4a), we noticed two suitable positions of 2 to redesign
the scaffold and grow the molecule toward a cryptic pocket of
BCL-XL/BCL-2 (in the region of the the p2 pocket) that is
engaged by the chlorobiphenyl moiety of ABT-737 (1). Bis-
biphenyl compound 7 and tetrahydroisoquinoline (THIQ) 8
were the outcomes of this scaffold redesign (Figure 4b). These
two target compounds were prepared, and their binding modes
in complex with BCL-XL were elucidated through X-ray crystal
structures (Figure 4c,d). Overlays of each of these structures
with those of 2 or 1 in complex with BCL-XL highlight the
success of our molecular grafting strategy in achieving the
designed binding mode (Figure 4e). However, while 7 and 8
displayed improved binding toward BCL-XL (based on IC50
determined by the AlphaSCREEN assay), they were both still
significantly weaker than ABT-737 (1) (Table 1).
We next turned our attention to the cryptic p5 pocket to
explore whether the interactions of 2 with BCL-XL in this
region could be further optimized either by modifying the
thioether linker of 2 (bridging the p4 and cryptic p5 pockets)
or by conferring greater three-dimensionality to the hydro-
phobic S-benzyl moiety to better fill the cryptic p5 pocket. The
X-ray crystal structure of BCL-XL:ABT-737 (1) suggested that
a suitable hydrogen-bond acceptor introduced into the
thioether linker region of 2 might form an additional
interaction with the backbone NH of Gly138 of BCL-XL
(Figure 3b,c).
An analogue of 2 with an all-carbon linker was found to
retain equivalent BCL-XL binding (compound 9, Table 2);
similarly, additional hydrogen-bond acceptors could also be
accommodated into the bridging linker (compounds 10, 11),
albeit with little overall improvement in binding to BCL-XL,
based on AlphaSCREEN-determined IC50 (Table 2). This may
be understood partially due to the linker location, at the rim of
the BH3 groove, being highly solvent-exposed (often
rationalized in terms of entropy/enthalpy compensation);44
in addition to this, sulfones are generally regarded as weak
hydrogen-bond acceptors.45 To explore the effect of size/shape
in the cryptic p5 pocket, cyclohexyl and adamantyl analogues
12 and 13 were generated; however, for these direct analogues
of 2, any improvement in BCL-XL binding was also modest at
best (Table 2).
Figure 3. Key interactions between acidic moieties of 2 and ABT-737
(1) with BCL-XL. (a) Key interactions of compound 2 with Asn136 of
BCL-XL. (b, c) Side-by-side comparison of the crystal structures of
ABT-737 (1) with BCL-XL (magenta, shown in stick conformation,
PDB: 2YXJ)37 and the primary conformation of 2 with BCL-XL (light
blue). A hydrogen-bond interaction is apparent between the backbone
−NH atom of Gly138 of BCL-XL and the carbonyl oxygen atom of
the acylsulfonamide moiety of ABT-737 (1). Notably, the thioether
of the S-benzyl moiety of 2 (primary conformation, light blue)
occupies a similar position, which suggested possibilities for further
elaboration.
where the acid isostere acylsulfonamide is key for binding but
lies away from Arg139 in the bound state, instead interacting
with the backbone amide −NH of Gly138 (Figure 3b).37
Protein/ligand interactions are not static; in addition, charge−
charge interactions can operate over relatively long distances in
molecular interactions (e.g., up to 5−10 Å) including to
enhance the kinetics favoring complex formation.38,39 Thus,
the effect of the acidic moiety on binding in this context may
not be particularly sensitive to the charge spacing.
The negative SAR study also revealed the relative
importance of interactions in the cryptic p5 and the p4
pockets for the binding of 2 to BCL-XL: pruning back the p5 S-
We also made similar changes for the tetrahydroisoquinoline
8 to see whether modifications in the cryptic p5 pocket and the
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Figure 4. (a) Overlaid X-ray crystal structures of compound 2 (PDB code: 6UVD, carbon: light blue) and ABT-737 (1) (PDB code: 2YXJ, carbon:
magenta)37 both bound to BCL-XL, indicating sites for scaffold grafting (BCL-XL from the BCL-XL:ABT-737 (1) complex is shown in surface
representation in gray). (b) Bis-biphenyl compound 7 and tetrahydroisoquinoline 8 designed with a chlorobiphenyl extension to access the cryptic
p2 pocket. (c) X-ray crystal structure of compound 7 bound to BCL-XL (PDB code: 6UVE, carbon: dark blue). (d) X-ray crystal structure of
compound 8 bound to BCL-XL (PDB code: 6UVC, carbon: light orange). (e) Overlays of compounds 7 with 2 and 8 with ABT-737 (1), all in
their BCL-XL bound poise.
p2 extension would have any combined effect. In this case,
while replacing the thioether linker of 8 with a sulfonyl moiety
again did not, on its own, improve binding to BCL-XL
(compound 11), additional replacement of the benzyl group
of 11 with a cyclohexyl (compound 15) or adamantyl
(compound 16) led to >4-fold improvement in IC50 for
BCL-XL (and left the binding to MCL-1 relatively unaffected).
Together, these changes resulted in the most potent analogues
in the series (15 and 16), which showed nanomolar IC50 for
BCL-XL in the AlphaSCREEN assay.
We next sought to validate these binding results using
another complementary biophysical assay technique and to
assess binding to other BCL-2 family members, including BCL-
2. We had previously described a surface plasmon resonance
(SPR)-based competition experiment to detect groove-specific
binding to various BCL-2 family proteins, including BCL-XL,
BCL-2, BCL-W, MCL-1, and A1.21,23,34,46 Using this assay, we
found that for the benzoylurea series, the trends for BCL-XL
binding observed in the primary AlphaSCREEN screening
assay were maintained (Table 2 and Figure S4, Supporting
Information). For the positive control ABT-737 (1), the IC50
for BCL-XL was tighter in the SPR competition assay (SPR
IC50 < 0.25 nM) than in the AlphaSCREEN assay (Table 2,
but in line with the reported Ki of ABT-737 (1) for BCL-XL, Ki
< 1 nM).16 In this case, the AlphaSCREEN may perhaps
underestimate the “true” IC50, falling outside the lower
measurable limit of the assay. For the benzoylurea analogues,
consistent with the AlphaSCREEN results, compounds 15 and
16 showed the tightest binding to BCL-XL in the SPR
competition assay, with IC50 values for BCL-XL of 41 and 44
nM, respectively. Strikingly, SPR competition experiments also
revealed that a number of analogues also showed marked
binding to BCL-2, in particular 15, which binds BCL-2 with an
IC50 value of 18 nM. This reflected a broader trend, whereby
analogues based on the simpler biphenyl scaffold (11, 12, 13)
showed negligible binding to BCL-2, whereas incorporation of
the chlorobiphenyl extension designed to interact with the
cryptic p2 pocket of BCL-XL/BCL-2 substantially enhanced
BCL-2 binding (extended THIQ scaffold analogues 8, 14−16).
Comparing analogues 11 and 14, or 12 and 15, being matched
pairs on the simple biphenyl and extended THIQ scaffolds, this
additional p2 interaction led to >189-fold and >690-fold
enhancements in BCL-2 binding, respectively. This highlights
the critical importance of engaging the cryptic p2 pocket for
development of inhibitors targeting BCL-2. For BCL-XL, this
requirement appears less marked, although it remains to be
seen whether high-affinity BCL-XL inhibitors could be
generated that do not make use of the p2 pocket. As was
observed for the AlphaSCREEN assay, binding of all
compounds to MCL-1 was relatively weaker and via SPR no
measurable binding was observed over the concentration range
tested (in all cases IC50 > 12.5 μM). The differences in the
observed binding affinities for MCL-1 across technology likely
reflect the use of a BAK competitor BH3 peptide in the
AlphaSCREEN assay, as compared to a BIM BH3 peptide used
in the SPR assay. All compounds showed weak or undetectable
binding to BCL-W and A1 in the SPR competition assay over
the concentration range tested (in all cases IC50 > 4.5 μM).
These results reveal 15 and 16 to be bona fide binders of both
BCL-XL and BCL-2 (nanomolar-range IC50) and the most
potent described to date utilizing the modular benzoylurea
scaffold.
X-ray crystal structures were also solved for each of
compounds 12, 13, and 15 in complex with BCL-XL in an
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Table 2. IC50 Values for Compounds Determined by the AlphaSCREEN (AS) Assay and the Surface Plasmon Resonance
a
(SPR) Competition Assay
a
b
See Figure S4, Supporting Information. IC50 values are reported in micromolar units. Unless noted, all data are reported as mean standard
c
error of mean (SEM) from N = 3 independent experiments. For each independent experiment, duplicate technical replicates were performed. Data
are mean standard deviation (SD) from N = 2 independent experiments.
Figure 5. (a) X-ray crystal structure of 12 in complex with BCL-XL (PDB code: 6UVF, carbon: light green). (b) X-ray crystal structure of 13 in
complex with BCL-XL (PDB code: 6UVG, carbon: dark green). (c) X-ray crystal structure of tetrahydroisoquinoline 15 in complex with BCL-XL
(PDB code: 6UVH, carbon: orange).
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Figure 6. MEFs (wild-type, WT; MCL-1−/−; or MCL-1−/−/BAX−/−/BAK−/−) were cultured for 24 h in the presence of varying concentrations of
(a) ABT-737 (1), (b) 6, or (c) 15 in an FMA medium containing 1% fetal calf serum (FCS) and then harvested, stained using propidium iodide
(PI), and analyzed by flow cytometry. Viability plots for each titration series depict the percentage of live cells (PI negative, normalized to vehicle
control, mean SEM of three independent experiments), which were fitted using nonlinear regression to determine the corresponding EC50 values.
(d) Plot showing the percentage of cells with released cytochrome c from mitochondria. Data are shown as mean SEM and are representative of
four independent experiments ***p < 0.001 (multiple unpaired t test, Figure S7).
attempt to better understand the basis of the improvement in
binding (Figure 5a−c). In all cases, these structures confirmed
that one of the sulfonyl oxygens within the linker moiety did in
fact form an additional hydrogen bond to the backbone NH of
Gly138 of BCL-XL (Figures 5 and S5d; compare to Figure
3b,c), in spite of being a relatively weak hydrogen-bond
acceptor. In addition, as anticipated, the structures confirmed
that the modified p5 substituents (cyclohexyl or adamantyl)
could be accommodated in the hydrophobic cryptic p5 pocket
(Figure S5a−c,e). This study suggests that sp3-rich groups with
the necessary flexibility, rather than aromatic, are preferred for
optimal binding in this pocket of both BCL-XL and BCL-2
with complementary close-packing hydrophobic interactions.
Combined with subtle conformational effects in the case of the
THIQ scaffold of 15 and 16, this effect results in the improved
binding (IC50) observed for these two compounds for BCL-
XL/BCL-2. The weaker binding of 16 for BCL-2 relative to 15
may point toward a more restricted cryptic p5 pocket in BCL-
2.
Using the most potent compound 15, we next sought to
evaluate whether this compound could demonstrate mecha-
nism-based cellular killing via inhibition of one or more BCL-2
family proteins. We first decided to utilize cells engineered to
be reliant on BCL-XL for survival. MCL-1-deficient mouse
embryo fibroblast cells have been shown to be sensitive to
BCL-XL inhibition and undergo apoptosis in a BAX-/BAK-
dependent manner when BCL-XL is neutralized, and so this
has become a robust and widely used cellular model to
evaluate on-target inhibition of BCL-XL.14,21,24,47,48 Thus,
ABT-737 (1) induced potent killing of MCL-1−/− mouse
embryonic fibroblasts (MEFs) (EC50 = 13 nM) but not wild-
type (WT) or MCL-1−/−/BAX−/−/BAK−/− MEFs (Figure 6a).
Similarly, compound 15 showed on-target activity, albeit at
significantly higher concentrations, killing MCL-1−/− MEFs
(EC50 = 19 μM) but not WT or MCL-1−/−/BAX−/−/BAK−/−
MEFs (Figure 6a). These results strongly suggest that killing of
MCL-1−/− MEFs following treatment with 15, as for ABT-737
(1), results primarily from on-target inhibition of BCL-XL, as it
requires BAX/BAK (i.e., proceeds via the intrinsic apoptotic
pathway) and sensitivity to both molecules is lost in WT MEFs
in which a nontargeted prosurvival protein (MCL-1) is
present. In comparison, benzoylurea compound 6, which
only binds weakly to BCL-XL, did not induce killing of MCL-
1−/− MEFs over the concentration range tested. The
significantly weaker cellular activity of 15 relative to ABT-
737 (1) reflects the weaker binding of 15 to BCL-XL/BCL-2
(SPR-derived IC50 for BCL-XL and BCL-2 suggest >100-fold
difference in binding), modest solubility of 15, as well as the
likelihood that 15 is highly serum bound. We observed that 15
lost all activity in this MEF viability assay if 10% fetal calf
serum (FCS) is used rather than 1% FCS (Figure S6a,
Supporting Information). Relevantly, extensive optimization
was undertaken during the development of ABT-737 (1) to
reduce the extent of serum binding (which improved BCL-XL
binding of this series by up to 70-fold in the presence of 1%
serum and decreased binding to domain III of human
albumin).43,49 In comparison, compound 15, unlike ABT-
737 (1), has not yet been optimized in this manner to reduce
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Scheme 1
a
Reagents and conditions: (a) thionyl chloride, MeOH, 0 °C to room temperature (r.t.); (b) HCl (4 M) in dioxane; (c)
(bromomethyl)cyclohexane, tetrabutylammonium iodide (TBAI) (3 mol %), NaOH (2 N, aq)/EtOH (1:1); (d) meta-chloroperoxybenzoic
acid (m-CPBA), CH2Cl2, 0 °C; (e) triflic anhydride, pyridine, CH2Cl2, 0 °C to r.t.; (f) trimethylphosphine, tetrahydrofuran (THF); (g) 25, K2CO3,
18-crown-6, CH3CN; (h) trifluoroacetic acid (TFA).
serum binding or to optimize solubility. We also evaluated 15
in the BCL-XL-dependent cancer cell line COLO205 (Figure
S6b,c, Supporting Information); however, no effect on viability
was observed over the concentration range tested. As the
threshold affinity required to show effects on the viability of
cancer cell lines would typically be higher than in MCL-1−/−
MEFs,21 lack of activity in this context is not surprising; more
potent molecules will be required.
To verify mechanistically whether the BAX-/BAK-depend-
ent activity on the viability of MCL-1−/− MEFs correlated with
the release of cytochrome c from mitochondria, we adopted an
assay to measure mitochondrial retention of cytochrome c by
intracellular staining. Briefly, cells were treated for 18 h with
compound ABT-737 (1), 6, 15, or vehicle; then, the outer
membrane was permeabilized with digitonin (leaving mito-
chondria intact), washed, fixed, and stained for cytochrome c
using an anticytochrome c primary and phycoerythrin (PE)-
conjugated secondary antibody, and then analyzed using
fluorescence-assisted cell sorting (FACS, Figure 6b). While
treatment of MCL-1−/− MEFs with the less potent benzoylurea
analogue 6 (25 μM), or with a vehicle control, did not show
evidence of cytochrome c loss from mitochondria, treatment
with ABT-737 (1) (2.5 μM) as expected caused virtually all
cells to lose cytochrome c (>90% of MCL-1−/− MEFs).
Similarly, treatment with compound 15 (25 μM, Figure 6c)
caused a statistically significant proportion of cells to have lost
cytochrome c from mitochondria (approximately 30% of MCL-
1−/− MEFs with released cytochrome c). No loss of
cytochrome c from mitochondria was observed for wild-type
or MCL-1−/−/BAX−/−/BAK−/− MEFs treated with either 6 or
15. This suggests that 15 selectively induces cytochrome c
release in MCL-1−/− MEFs relative to either WT or MCL-
1−/−/BAX−/−/BAK−/− MEFs (Figures 6c and S7). Thus, the
BCL-XL-dependent cell killing exhibited by 15 appears to be
on-target, both requiring BAX/BAK and proceeding mecha-
nistically via loss of cytochrome c from mitochondria.
CHEMISTRY
■
Some analogues in this study necessitated the preparation of
amino acids not commercially available (Scheme 1). We
prepared the methyl-protected version of S-benzyl-cysteine
(19) through a simple sequence involving a thionyl chloride-
mediated conversion of the carboxylic acid of compound 17
into the methyl ester 18 followed by deprotection of the t-Boc
group.
The cyclohexylmethylsulfonyl cysteine derivative 23 was
obtained first by alkylation of t-Boc-protected cysteine with
(bromomethyl)cyclohexane in the presence of the phase-
transfer reagent, subsequent oxidation with m-CPBA, and t-
Boc deprotection to obtain the HCl salt 23.
The final unnatural amino acid required, bearing an
adamantylmethylsulfonyl side chain (30), was a more
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Scheme 2
a
Reagents and conditions: (a) 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), N,N-diisopropylethylamine
(DIEA) and then 4-methylbenzylamine and dimethylformamide (DMF); (b) (i) trimethylsilyl trifluoromethanesulfonate (TMSOTf), NEt3, Et2O;
(ii) COCl2 20% in toluene, 0 °C to r.t.; (c) 2-(benzylthio)ethanaminium chloride, NEt3, CH2Cl2; (d) 19, NEt3, propylene oxide, CH2Cl2; (e)
NEt3, CH3CN, or CH2Cl2; (f) N,O-bistrimethylsilylacetamide, propylene oxide, CH3CN, or CH2Cl2; (g) 39, 40, 41, 42, 43, 44, CH3CN, or
CH2Cl2, 0 °C to r.t.; (h) m-CPBA, CH2Cl2, 0 °C; (i) 4-bromo-N-propylbenzamide, phenylboronic acid, Pd(PPh3)4, sat Na2CO3 EtOH. (j)
CH3CN or CH2Cl2, 0 °C to r.t.
challenging target to prepare. After a number of attempts, the
successful approach entailed first the preparation of triflated
adamantylmethanol 25 (Scheme 1) using triflic anhydride in
the presence of pyridine. Our strategy also required a version
of cysteine protected on both the amino and carboxylic acid
groups by acid-labile protecting groups (t-Boc and t-Bu,
respectively). We obtained compound 27 (Scheme 1) by
reducing the disulfide bond from the suitably protected cystine.
The thiol group was then alkylated using triflate 25, using
K2CO3 as a base. This alkylation required the addition of 18-
crown-6 to achieve reasonable yields (Scheme 1). The
resulting compound was then doubly deprotected, to obtain
amino acid 30.
The synthesis of the compounds described in this work
relies on the formation of the core benzoylurea, which we have
reported previously.32,35,50 This two-step multicomponent
reaction involves first the preparation of a carbamoyl chloride
from a corresponding amide and the reaction of this reactive
intermediate onto a transiently protected amino acid.
Specifically, for this work, preparation of compounds 2−6
and 9−13 followed this general principle (Scheme 2), using
either amide 32 or 46 and the required amino acid (either
commercially available or prepared in the lab, Scheme 1).
For the preparation of the extended scaffold compounds 7,
8, and 14, we chose to have the corresponding amino acid
starting materials on hand before initiating the synthesis. This
was particularly important for the sulfonyl version of S-
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Scheme 3
a
Reagents and conditions: conditions: (a) m-CPBA, CH2Cl2, 0 °C; (b) HCl (4 M) in dioxane; (c) 4-chloro-phenylboronic acid, Pd(OAc)2 (5 mol
%), tetra-n-butylammonium bromide (1 mol %), K2CO3, acetone/water (1:1), 40 °C; (d) 1,2,3,4-tetrahydroisoquinoline-6-carboxylate
hydrochloride, NEt3, sodium triacetoxyborohydride, CH2Cl2, r.t.; (e) LiOH, MeOH/water/THF (1:1:3), r.t.; (f) HBTU, DIEA and then 4-
methylbenzylamine, DMF; (g) (1) TMSOTf, NEt3, Et2O; (2) COCl2 20% in toluene, 0 °C to r.t.; (h) NEt3, CH3CN, or CH2Cl2; (i) N,O-
bistrimethylsilylacetamide, propylene oxide, CH3CN, or CH2Cl2; (j) 56 or 57, CH3CN or CH2Cl2, 0 °C to r.t.; (k) CH2Cl2, 0 °C to r.t.
benzylcysteine (compound 49, Scheme 3) as late-stage
oxidation could potentially impact the stability of this set of
more complex target compounds. Thus, compound 49 was
easily obtained using an oxidation/deprotection sequence used
above (Scheme 3). Compounds 7, 8, and 14 also required
more complex amide starting intermediates. We successfully
obtained the THIQ analogue by first generating the chloro-
biphenylaldehyde 51 with a Suzuki coupling from 50, followed
by a reductive amination onto 1,2,3,4-tetrahydroisoquinoline-
6-carboxylate hydrochloride. This THIQ intermediate was
then easily converted into tolylmehtyl amide compound 54 in
two steps (Scheme 3).
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describes a mixture of hexanes in the bp range 40−60 °C. Analytical
thin-layer chromatography was performed on Merck silica gel 60F254
aluminum-backed plates and were visualized by fluorescence
quenching under UV light or by ninhydrin or KMnO4 staining.
Flash chromatography was performed with silica gel 60 (particle size
0.040−0.063 mm) either manually or using a Combiflash platform.
Some of the final compounds described herein were purified using
mass-directed preparative high-performance liquid chromatography
(HPLC) using a mixture of 0.1% formic acid in water and acetonitrile.
1H and 13C NMR spectra were recorded on a Bruker Avance DRX
300 (1H NMR at 300 MHz, 13C NMR at 75 MHz), Varian 600 (1H
NMR at 600 MHz), or a Bruker AVANCE III 600 MHz (1H NMR at
600 MHz, 13C NMR at 151 MHz) using the solvents indicated.
Chemical shifts (δ) for all 1H are reported in parts per million (ppm)
and were referenced to proteo-solvent residual peaks, such as δ 7.26
ppm for deuterated chloroform (CDCl3), δ 2.50 ppm for d6-dimethyl
sulfoxide (d6-DMSO), or δ 3.31 for d4-methanol (CD3OD). Each
proton resonance was assigned according to the following convention:
chemical shift (δ), multiplicity, coupling constant(s) (J Hz), and
number of protons. Multiplicities are denoted as s, singlet; d, doublet;
t, triplet; q, quartet; p, pentet; sext, sextet; m, multiplet; br, broad; and
app, apparent.
Liquid chromatography−mass spectrometry (LCMS) was recorded
on either a Waters ZQ 3100 using a 2996 diode array detector
(Waters, Milford, MA) or an Agilent G6120B coupled to an Agilent
1260 Infinity G4212B diode array detector (Agilent, Palo Alto, CA).
LCMS conditions used to assess purity of compounds were as follows:
for the Waters system, column: XBridge TM C18 5 μm 4.6 × 100
mm; injection volume: 10 μL; gradient: 10−100% B over 10 min
(solvent A: water 0.1% formic acid; solvent B: acetonitrile 0.1%
formic acid); flow rate: 1.5 mL/min; detection: 100−600 nm; for the
Agilent system, column: Poroshell 120 EC-C18, 2.7 μm 3.0 × 50 mm;
injection volume: 5 μL; gradient: 5−100% B over 3 min (solvent A:
water 0.1% formic acid; solvent B: acetonitrile 0.1% formic acid); flow
rate: 0.8 mL/min; detection: 254 nm. All final compounds were
analyzed using ultrahigh-performance liquid chromatography/ultra-
violet/evaporative light-scattering detection coupled to mass spec-
trometry. Unless otherwise noted, all compounds were found to be
>95% pure by this method.
In parallel, the bis-biphenyl structure 55 was sourced
externally and used directly in the construction of the
benzoylurea core. The final compounds 7, 8, and 14 were
prepared using the standard protocol using amino acids 34 or
58. Yields for the synthesis of the THIQ derivatives 8 and 14
were more variable as we observed some debenzylation of the
tertiary amine, an issue that was partially resolved by carefully
controlling the amount of phosgene added to the reaction.
Our most optimized compounds, 15 and 16, were prepared
according to the same protocol reacting the carbamoyl chloride
57 with the trimethylsilyl (TMS)-protected amino acids 41 or
44 (Scheme 3).
CONCLUSIONS
■
In summary, this work describes a novel series of inhibitors of
BCL-XL as well as potent dual inhibitors of BCL-XL and BCL-2
that exhibit a number of structurally distinct features from
existing series of potent inhibitors published to date, including
use of the benzoylurea scaffold and the specific targeting of
interactions in a cryptic p5 pocket of BCL-XL/BCL-2. To our
knowledge, this pocket is only present in two other published
BCL-XL structures, that of the BCL-XL:ABT-263 complex
(Figure S3b, Supporting Information) and the BCL-XL:A-
1342192 complex (Figure S3c, Supporting Information),36
recently published while this manuscript was under review. In
the case of ABT-263, pocket formation likely occurs
unintentionally through the presence of the triflone electron-
withdrawing group specific to ABT-263, and for A-1342192,
the methoxy-adamantane only addresses part of this pocket
and variations of the methoxy are not explored. As such, this
pocket has not previously been specifically targeted with
hydrophobic moieties in this manner.
These new benzoylurea molecules have binding IC50 values
from the micromolar to nanomolar range for BCL-XL and
BCL-2. Structure-guided modifications generated new inter-
actions within the p2 and cryptic p5 pockets of BCL-XL/BCL-
2, leading to the most potent analogue in the series, 15, with an
SPR-determined IC50 value of around 40 nM for BCL-XL and
20 nM for BCL-2, and significantly weaker binding to other
prosurvival proteins, including BCL-W, MCL-1, and A1.
Consistent with its potent inhibition of both BCL-XL and
BCL-2 in the SPR competitive binding assay, we showed that
15 displays a window of mechanism-based cytochrome c
release and killing activity in MEF cells that are engineered to
depend on BCL-XL for survival. This work also illustrates the
versatility of our benzoylurea library to target challenging
protein−protein interfaces. It adds a new structural class to the
limited series of BH3 mimetics targeting BCL-XL and BCL-2
that have a mechanistically validated mode of action and
provides new structural insights into the development of either
selective inhibitors of BCL-XL or dual inhibitors of BCL-XL/
BCL-2. This has the potential to guide the development of
further selective inhibitors or protein degraders targeting this
family in the future.
High-resolution mass spectrometry (HRMS) analyses were carried
out at the Monash University Mass Spectrometry Facility using an
Agilent 6224 TOF LC/MS mass spectrometer coupled to an Agilent
1290 Infinity (Agilent, Palo Alto, CA). All data were acquired and
reference-mass-corrected via a dual-spray electrospray ionization
(ESI) source. Acquisition was performed using Agilent Mass Hunter
Data Acquisition software version B.06.01 Build 6.01.6157, and
analysis was performed using Mass Hunter Qualitative Analysis
version B.07.00 Build Service Pack 1.
Compound 55 was provided by AMT (Dr. Seb Marcuccio,
Melbourne).
Synthesis and Characterization of Compounds. Synthesis of
Compound (2). N-[(4-Methylphenyl)methyl]-4-phenylbenzamide
(32). To a stirred solution of biphenyl-4-carboxylic acid 31 (1.0 g,
5.0 mmol) in 12.5 mL of anhydrous DMF were added HBTU (3.8 g,
10 mmol) and DIEA (1.74 mL, 10 mmol) and the reaction was stirred
under N2 at room temperature for 15 min. 4-Methylbenzylamine
(1.30 mL, 10 mmol) was then added, and the reaction was allowed to
proceed at room temperature for 18 h. After this time, the reaction
mixture was poured over an ice/water mixture and the off-white solid
precipitate formed was collected by vacuum filtration, washed
copiously with cold water, and then dried in a 30 °C oven in
vacuo. The resulting solid was purified via flash chromatography and
eluted with ethyl acetate (EtOAc)/hexane (25:75) to afford
EXPERIMENTAL SECTION
■
1
compound 32 as fine white needles (1.46 g, 97%). H NMR (300
Chemistry. Materials and Methods. All nonaqueous reactions
were performed in oven-dried glassware under an inert atmosphere,
unless otherwise specified. Tetrahydrofuran, dichloromethane, diethyl
ether, and ethanol were obtained from a solvent drying apparatus
(MBraun). Anhydrous dimethylformamide (DMF), dioxane, and
acetonitrile were obtained from Aldrich and used without further
purification. All other solvents were of reagent grade. Petroleum ether
MHz, CDCl3) δ 7.86 (d, J = 8.67 Hz, 2H), 7.65 (d, J = 8.58 Hz, 2H),
7.61 (dd, J = 8.25, 1.32 Hz, 2H), 7.49−7.43 (m, 2H), 7.41−7.36 (m,
1H), 7.27 (d, J = 7.92 Hz, 2H), 7.18 (d, J = 7.92 Hz, 2H), 6.41 (br s,
1H), 4.63 (d, J = 5.52 Hz, 2H), 2.36 (s, 3H). 13C NMR (75 MHz,
CDCl3) δ 166.9, 144.4, 140.0, 137.4, 135.2, 133.1, 129.5, 128.9, 128.0,
127.5, 127.2, 127.2, 44.0, 21.1. MS (ES+), m/z 302.1, (M + H).
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HRMS (ES+ time-of-flight (TOF)) calcd for C21H19NO (M + H):
302.1539; found 302.1532.
white solid was obtained (85 mg, 17%). 1H NMR (300 MHz, CDCl3)
δ 9.17 (t, J = 5.3 Hz, 2H), 7.60−7.57 (m, 4H), 7.49−7.22 (m, 10H),
7.07 (d, J = 7.9 Hz, 2H), 6.95 (d, J = 8.1 Hz, 2H), 4.98 (s, 2H), 3.75
(s, 2H), 3.52 (q, J = 5.7 Hz, 2H), 2.64 (t, J = 6.8 Hz, 2H), 2.31 (s,
3H). 13C NMR (75 MHz, CDCl3) δ 175.0, 155.0, 143.5, 139.9, 138.1,
136.9, 134.9, 134.8, 129.2, 129.0, 128.9, 128.6, 128.1, 127.2, 127.1,
127.1, 126.6, 49.9, 39.8, 35.9, 30.9, 21.1. MS (ES+), m/z 495.1 (M +
H). HRMS (ES+ TOF) calcd for C31H30N2O2S (M + H): 495.2101;
found 495.2110.
Synthesis of Compound (5). L-Cysteine, N-[(1,1-Dimethylethoxy)-
carbonyl]-S-(phenylmethyl)-, Methyl Ester (18). To a stirred solution
of Boc-S-benzyl-L-cysteine (17, 623 mg, 2.00 mmol) in anhydrous
MeOH (3.00 mL) at 0 °C was added thionyl chloride (219 μL, 3.00
mmol); then, the reaction was allowed to warm to room temperature
and proceed for 1 h. After this time, di-tert-butyl dicarbonate (459 μL,
2.00 mmol) was added, and the reaction was allowed to proceed for a
further 6 h at room temperature. After this time, the reaction mixture
was concentrated under reduced pressure, and the residue was taken
in EtOAc (10 mL) and poured over water (20 mL). The two phases
were separated, and the aqueous phase was extracted with EtOAc (3
× 20 mL). The combined organic layers were washed with aqueous
saturated NaHCO3, dried over MgSO4, filtered, and concentrated
under reduced pressure to afford the title compound as a yellow oil
(607 mg, 93%). Spectral data obtained corresponded to those
reported in the literature.51
Biphenylcarbonyl(4-methylbenzyl)carbamic Chloride (33). To a
stirred solution of amide 32 (100 mg, 0.33 mmol) in 1 mL of
anhydrous Et2O was added triethylamine (50 μL, 0.36 mmol),
followed by TMSOTf (65 μL, 0.36 mmol), and the reaction was
stirred under N2 at room temperature for 30 min. After this time,
phosgene (660 μL, 2 mL/mmol amide starting material, 20% w/v in
toluene, CAUTION!) was added to the yellow-brown solution at 0
°C, which was then allowed to warm to room temperature and stirred
under N2 for 1.5 h. After this time, concentration in vacuo (using an
in-line solvent trap) over a room-temperature water bath afforded the
off-white solid carbamoyl chloride residue. This compound was used
in the next step without further purification.
O-Trimethylsilyl-S-benzyl-L-cysteine (39). To a stirred suspension
of S-benzyl-L-cysteine (34, 105 mg, 0.50 mmol) in 2 mL of anhydrous
acetonitrile was added 1 mL of propylene oxide and bistrimethylsi-
lylacetamide (183 μL, 0.75 mmol) and the reaction was stirred under
N2 at room temperature for 1.5 h. This compound was used in the
next step without further purification.
(2R)-3-(Benzylsulfanyl)-2-({[(4-methylphenyl)methyl][(4-
phenylphenyl)carbonyl]carbamoyl}amino)propanoic Acid (2).
Under a N2 atmosphere, the carbamoyl chloride 33 was redissolved
in 1 mL of anhydrous acetonitrile and then transferred to the stirred
suspension of the in situ-protected amino acid 39 at 0 °C. The
reaction was allowed to warm to room temperature and stirred at
room temperature for 1.5 h. After this time, the reaction was diluted
with EtOAc and poured onto aqueous 1 M HCl. The reaction mixture
was extracted three times with EtOAc. The combined organic layers
were washed with water and then brine, dried over MgSO4, filtered,
and concentrated under reduced pressure. Purification via flash
chromatography and eluting with methanol/dichloromethane (0:100
to 0.5:99.5 to 1:99 to 2:98, etc. to 4:96) afforded benzoylurea 2 as a
colorless glassy solid (149.1 mg, 83%). 1H NMR (300 MHz, CDCl3)
δ 9.68 (d, J = 6.9 Hz, 1H), 7.63−7.54 (m, 4H), 7.50−7.35 (m, 5H),
7.35−7.19 (m, 5H), 7.06 (d, J = 7.8 Hz, 2H), 6.95 (d, J = 8.0 Hz,
2H), 5.00 (s, 2H), 4.77 (dd, J = 12.1, 6.6 Hz, 1H), 3.78 (s, 2H), 3.02
(dd, J = 14.2, 5.3 Hz, 1H), 2.93 (dd, J = 14.0, 6.7 Hz, 1H), 2.29 (s,
3H). 13C NMR (75 MHz, CDCl3) δ 175.3, 174.2, 155.5, 143.9, 140.0,
137.7, 137.2, 134.6, 134.5, 129.5, 129.2, 129.1, 128.8, 128.3, 127.4,
127.4, 127.3, 127.3, 126.7, 53.6, 50.2, 36.8, 32.9, 21.2. MS (ES+), m/z
539.1, (M + H). HRMS (ES+ TOF) calcd for C32H30N2O4S (M + H):
539.1999; found 539.2000.
(R)-Methyl 2-Amino-3-(benzylthio)propanoate Hydrochloride
(19). To a sample of 18 (247 mg, 0.76 mmol) at room temperature
under N2 was added a solution of HCl (4 M) in 1,4-dioxane (1.5 mL).
The reaction was allowed to proceed for 1.5 h at room temperature;
then, the reaction mixture was carefully concentrated under reduced
pressure to yield the title compound as an off-white powdery solid
(192 mg, 97%). Spectral data obtained corresponded to those
reported in the literature.52
(R)-Methyl 2-(3-([1,1′-Biphenyl]-4-carbonyl)-3-(4-methylbenzyl)-
ureido)-3-(benzylthio)propanoate (5). Compound 33 was prepared
according to the method described for compound 2 using 32 (30 mg,
0.10 mmol). 33 was dissolved in 400 μL of anhydrous dichloro-
methane and transferred dropwise to a stirred solution of 19 (30 mg,
0.12 mmol), triethylamine (30 μL, 0.22 mmol), and propylene oxide
(192 μL, 2.4 mmol) in 460 μL of anhydrous dichloromethane at 0 °C.
The reaction was allowed to warm to room temperature and stirred
for 0.5 h. After this time, the reaction was diluted with dichloro-
methane and poured onto an aqueous 10% citric acid solution. The
reaction mixture was extracted three times with dichloromethane. The
combined organic layers were washed with water and then brine,
dried over MgSO4, filtered, and concentrated under reduced pressure.
The crude material was purified via flash chromatography and eluted
with EtOAc/cyclohexane (0:100−10:90), followed by further
purification via mass-directed preparative HPLC. A white solid was
Synthesis of Compound (3). 2(R)-(3-(Biphenylcarbonyl)-3-(4-
methylbenzyl)ureido)-3-(methylthio)propanoic Acid (3). The pro-
cedure used to prepare compound 2 was followed using compound
32 (301 mg, 1.0 mmol) and S-methyl-L-cysteine (37, 136 mg, 1.0
mmol). The crude material was purified by mass-directed preparative
1
HPLC. An orange oil was obtained (57 mg, 12%). H NMR (300
1
obtained (27 mg, 51%). H NMR (300 MHz, CDCl3) δ 9.64 (d, J =
MHz, CDCl3) δ 9.70 (d, J = 7.0 Hz, 1H), 8.01 (br s, 1H), 7.60−7.57
(m, 4H), 7.49−7.43 (m, 2H), 7.41−7.36 (m, 1H), 7.08 (d, J = 7.9 Hz,
2H), 6.96 (d, J = 9.1 Hz, 2H), 5.01 (s, 2H), 4.83 (q, J = 7.0 Hz, 1H),
3.14−3.01 (m, 2H), 2.31 (s, 3H), 2.18 (s, 3H). 13C NMR (75 MHz,
CDCl3) δ 175.2, 155.2, 143.8, 139.8, 137.0, 134.5, 134.3, 129.3, 129.0,
128.1, 127.3, 127.2, 127.1, 126.5, 53.4, 50.1, 35.9, 21.1, 16.2. MS
(ES+), m/z 463.1 (M + H). HRMS (ES+ TOF) calcd for
C26H26N2O4S (M + H): 463.1686; found 463.1691.
Synthesis of Compound (4). N-(2-(Benzylthio)ethylcarbamoyl)-
N-(4-methylbenzyl)biphenyl-4-carboxamide (4). Compound 33 was
prepared according to the method described for compound 2 using
32 (301 mg, 1.0 mmol). 33 was dissolved in 2.5 mL of anhydrous
acetonitrile and added to a solution of 2-(benzylthio)ethanaminium
chloride (204 mg, 1.0 mmol) and triethylamine (279 μL, 2 mmol) in
2.5 mL of anhydrous dichloromethane at 0 °C. The reaction was
warmed to room temperature and stirred for 1 h. After this time, the
reaction was diluted with EtOAc and poured onto aqueous 1 M HCl.
The reaction mixture was extracted three times with EtOAc. The
combined organic layers were washed with water and then brine,
dried over Na2SO4, and concentrated under reduced pressure. The
crude material was purified by mass-directed preparative HPLC. A
7.3 Hz, 1H), 7.64−7.53 (m, 4H), 7.53−7.20 (m, 11H), 7.07 (d, J =
7.9 Hz, 2H), 6.97 (d, J = 8.1 Hz, 2H), 5.00 (s, 2H), 4.80 (td, J = 6.6,
5.1 Hz, 1H), 3.77 (s, 3H), 3.76 (s, 2H), 2.94 (qd, J = 14.0, 5.8 Hz,
2H), 2.30 ppm (s, 3H). 13C NMR (75 MHz, CDCl3) δ 175.2, 171.1,
155.0, 143.8, 140.0, 137.8, 137.0, 134.8, 134.7, 129.4, 129.2, 129.1,
128.7, 128.2, 127.4, 127.3, 127.2, 126.7, 53.7, 52.8, 50.1, 36.7, 33.4,
21.2 ppm. One Ar−CH peak is not observed due to overlap. MS
(ES+), m/z 553.2 (M + H), HRMS (ES+ TOF) calcd for
C33H32N2O4S (M + H): 553.2156; found 553.2166.
Synthesis of Compound (6). N-Propylbiphenyl-4-carboxamide
(46). 4-Bromo-N-propylbenzamide (45, 484 mg, 2 mmol), Pd(PPh3)4
(120 mg, 0.1 mmol), phenylboronic acid (270 mg, 2.2 mmol), and 2
mL of an aqueous Na2CO3 solution were stirred for 20 h at 100 °C in
1 mL of ethanol. After this time, EtOAc was added along with an
ammonia solution (25% aqueous). The mixture was extracted three
times with EtOAc. The combined organic layers were washed twice
with brine, dried over MgSO4, filtered, and concentrated under
reduced pressure. Purification via flash chromatography and eluting
with petroleum ether/dichloromethane (20:80−100:0) and then
petroleum ether/EtOAc (100:0−80:20) afforded the title compound
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as a white crystalline solid (338 mg, 71%). H NMR (600 MHz,
CDCl3) δ 7.85 (d, J = 7.8 Hz, 2H), 7.63 (d, J = 7.8 Hz, 2H), 7.59 (d, J
= 7.5 Hz, 2H), 7.45 (t, J = 7.4 Hz, 2H), 7.38 (t, J = 7.0 Hz, 1H), 6.40
(br s, 1H), 3.44 (dd, J = 13.0, 6.4 Hz, 2H), 1.66 (sext, J = 7.2 Hz,
2H), 1.00 (t, J = 7.3 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 167.4,
144.2, 140.2, 133.7, 129.0, 128.0, 127.5, 127.3, 41.9, 23.1, 11.6. One
Ar−CH peak is not observed due to overlap. MS (ES+), m/z 240.5
(M + H). HRMS (ES+ TOF) calcd for C16H17NO (M + H):
240.1383; found 240.1384.
extracted with EtOAc (2 × 20 mL). The combined organic phases
were washed with brine, dried over MgSO4, filtered, and concentrated
under reduced pressure. Purification of the crude material via flash
chromatography and eluting with petroleum ether/EtOAc (100:0−
90:10) afforded the title compound as a colorless oil (492 mg, 95%).
1H NMR (300 MHz, CDCl3): δ 7.80−7.76 (m, 2H), 7.65 (br d, J =
6.5 Hz, 1H), 7.43−7.38 (m, 6H), 7.29−7.26 (m, 1H), 7.03 (d, J = 7.8
Hz, 1H), 3.91 (s, 3H), 3.63 (br s, 4H), 2.92 (t, J = 5.6 Hz, 2H), 2.72
(t, J = 5.7 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 167.1, 141.6,
139.7, 134.5, 133.4, 133.1, 130.9, 130.9, 130.5, 130.2, 130.1, 130.0,
128.5, 128.5, 127.7, 127.3, 126.8, 126.6, 59.4, 55.5, 52.0, 50.1, 29.0.
MS (ES+), m/z 392.1 (M + H). HRMS (ES+ TOF) calcd for
C24H22ClNO2 (M + H): 392.1412; found 392.1428.
2-((4′-Chlorobiphenyl-2-yl)methyl)-1,2,3,4-tetrahydroisoquino-
line-6-carboxylic Acid (53). LiOH·H2O (80 mg, 1.91 mmol) was
added to a solution of 52 (188 mg, 0.48 mmol) in a mixture of
methanol (0.60 mL), water (0.60 mL), and THF (1.8 mL). The
reaction was stirred at room temperature for 16 h. TLC (petroleum
ether/EtOAc 90:10) indicated full conversion. The reaction mixture
was concentrated under reduced pressure; then, water (10 mL) was
added and the reaction was acidified to pH ∼1.0 with ∼1.9 mL of 1 M
HCl. The resulting off-white precipitate was filtered, washed with a
small volume of water, and dried in a vacuum oven at 40 °C
overnight. This afforded the HCl salt of 53 as an off-white powdery
solid (146 mg, 74%). 1H NMR (600 MHz, CD3OD) δ 7.83 (d, J = 8.1
Hz, 1H), 7.81 (s, 1H), 7.78−7.75 (m, 1H), 7.60−7.54 (m, 2H), 7.45
(dd, J = 8.3, 0.9 Hz, 2H), 7.43−7.40 (m, 1H), 7.35 (dd, J = 8.2, 0.8
Hz, 2H), 7.12 (d, J = 8.1 Hz, 1H), 4.46 (s, 2H), 4.16 (s, 2H), 3.36 (t,
J = 5.6 Hz, 2H), 3.08 (t, J = 6.3 Hz, 2H). 13C NMR (75 MHz,
CD3OD) δ 167.6, 142.8, 138.2, 133.8, 132.4, 131.1, 130.9, 130.8,
130.5, 129.7, 129.7, 128.6, 128.6, 127.7, 127.5, 126.8, 55.6, 52.4, 49.2,
24.6. One Ar−CH peak is not observed due to overlap. MS (ES+), m/
z 378.2 (M + H). HRMS (ES+ TOF) calcd for C23H20ClNO2 (M +
H): 378.1255; found 378.1254.
(R)-3-(Benzylthio)-2-(3-(biphenylcarbonyl)-3-propylureido)-
propanoic Acid (6). The procedure used to prepare compound 2 was
followed using compound 46 (96 mg, 0.50 mmol) and S-benzyl-L-
cysteine (127 mg, 0.55 mmol). Purification via flash chromatography
and eluting with methanol/dichloromethane/acetic acid (0.5:99:0.5)
1
afforded benzoylurea 6 as a colorless glass (196 mg, 82%). H NMR
(600 MHz, CDCl3) δ 10.29 (br s, 1H), 9.66 (d, J = 7.0 Hz, 1H), 7.67
(d, J = 8.1 Hz, 2H), 7.61 (d, J = 7.3 Hz, 2H), 7.54 (d, J = 8.1 Hz, 2H),
7.46 (t, J = 7.7 Hz, 2H), 7.42−7.36 (m, 1H), 7.36−7.27 (m, 4H),
7.27−7.21 (m, 1H), 4.76 (td, J = 6.9, 5.0 Hz, 1H), 3.79 (s, 2H),
3.77−3.70 (m, 2H), 3.00 (dd, J = 14.1, 4.9 Hz, 1H), 2.91 (dd, J = 14.1
Hz, 6.8, 1H), 1.58 (sext., J = 7.5 Hz, 2H), 0.76 (t, J = 7.4 Hz, 3H). 13C
NMR (151 MHz, CDCl3) δ 175.5, 175.2, 155.2, 143.7, 139.9, 137.6,
134.8, 129.2, 129.1, 128.7, 128.2, 127.4, 127.4, 127.3, 127.2, 53.5,
49.1, 36.7, 32.9, 23.1, 11.1. MS (ES+), m/z 476.8 (M + H), 99% pure.
HRMS (ES+ TOF) calcd for C27H28N2O4S (M + H): 477.1843;
found 477.1849.
Synthesis of Compound (7). (R)-3-(Benzylthio)-2-(3-(4-chloro-
[1,1′:2′,1″:3″,1″′-quaterphenyl]-4″′-carbonyl)-3-(4-methylbenzyl)-
ureido)propanoic Acid (7). The procedure used to prepare
compound 2 was followed using compound 55 (125 mg, 0.26
mmol) and S-benzyl-L-cysteine (34, 65 mg, 0.31 mmol). The crude
material was purified by mass-directed preparative HPLC to afford the
1
title product as a white solid (53 mg, 28%). H NMR (300 MHz,
CDCl3) δ 9.70 (d, J = 7.0 Hz, 1H), 7.52−7.16 (m, 19H), 7.13−7.03
(m, 4H), 6.94 (d, J = 8.1 Hz, 2H), 4.98 (s, 2H), 4.75 (td, J = 6.7, 5.1
Hz, 1H), 3.78 (d, J = 1.3 Hz, 2H), 2.97 (qd, J = 14.1, 5.9 Hz, 2H),
2.30 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 175.3, 173.0, 155.6,
143.7, 141.8, 140.2, 139.6, 139.6, 137.7, 137.2, 134.6, 134.4, 132.9,
131.4, 130.7, 130.6, 129.6, 129.5, 129.2, 128.9, 128.8, 128.4, 128.2,
128.0, 127.4, 127.2, 126.6, 125.6, 77.4, 53.5, 50.2, 36.8, 32.8, 21.2.
Four Ar−CH peaks are not observed due to overlap. MS (ES+), m/z
726 (M + H). HRMS (ES+ TOF) calcd for C44H37ClN2O4S (M +
H): 725.2235; found 725.2258.
Synthesis of Compound (8). 4′-Chloro-[1,1′-biphenyl]-2-carbal-
dehyde (51). 2-Bromobenzaldehyde (50, 740 mg, 4.0 mmol), 4-
chloro-phenylboronic acid (751 mg, 4.8 mmol), tetra-n-butylammo-
nium bromide (13 mg, 1 mol %), and K2CO3 (1.66 g, 12.0 mmol) in
8 mL of a solution of acetone/water (1:1) were flushed with N2 and
stirred at room temperature for 30 min. Pd(OAc)2 (45 mg, 5 mol %)
was then added, and the reaction was allowed to stir for 30 min at 40
°C. After this time, the reaction mixture was concentrated under
reduced pressure and filtered through celite; then, water (20 mL) and
diethyl ether (20 mL) were added and the two layers were separated.
The aqueous phase was extracted with diethyl ether (2 × 20 mL), and
the combined organic phases were washed with water and brine, dried
over MgSO4, filtered, and concentrated under reduced pressure.
Purification of the crude material via flash chromatography and
eluting with a gradient of EtOAc/cyclohexane (0:100−5:95) afforded
the title product as a yellow oil (703 mg, 81%). Analytical data
obtained corresponded to reported literature values.53
2-((4′-Chlorobiphenyl-2-yl)methyl)-N-(4-methylbenzyl)-1,2,3,4-
tetrahydro-isoquinoline-6-carboxamide (54). The procedure used
to prepare compound 33 was followed using compound 53 (99 mg,
0.26 mmol), DIEA (183 μL, 1.05 mmol), and 4-methylbenzylamine
(67 μL, 0.52 mmol). The reaction mixture was poured onto ice-water
whereupon an orange solid precipitated. This compound was taken up
in EtOAc, and the two phases separated. The aqueous phase was
further extracted twice with EtOAc; then, the pooled organic phases
were washed with brine, dried over MgSO4, filtered, and concentrated
under reduced pressure. The resulting crude brown oil was purified
via flash chromatography and eluted with petroleum ether/EtOAc
(100:0−0:100) affording the title compound as a white solid (105 mg,
1
83%). H NMR (300 MHz, CDCl3) δ 7.53−7.45 (m, 1H), 7.43 (s,
1H), 7.39 (dd, J = 7.8, 1.5 Hz, 1H), 7.31−7.19 (m, 6H), 7.19−7.10
(m, 3H), 7.06 (d, J = 7.9 Hz, 2H), 6.89 (d, J = 8.0 Hz, 1H), 6.28 (t, J
= 5.3 Hz, 1H), 4.48 (d, J = 5.6 Hz, 2H), 3.46 (d, J = 4.4 Hz, 4H), 2.75
(t, J = 5.7 Hz, 2H), 2.56 (t, J = 5.8 Hz, 2H), 2.24 (s, 3H). 13C NMR
(75 MHz, CDCl3) δ 167.4, 141.6, 139.9, 138.9, 137.4, 135.6, 135.4,
135.1, 133.2, 132.4, 131.0, 130.2, 130.2, 129.6, 128.2, 128.1, 127.7,
127.6, 127.3, 126.9, 124.1, 59.8, 55.7, 50.3, 44.0, 29.4, 21.2. MS (ES+),
m/z 481.0 (M + H). HRMS (ES+ TOF) calcd for C31H29ClN2O (M
+ H): 481.2041; found 481.2021.
(R)-3-(Benzylthio)-2-(3-(2-((4′-chloro-[1,1′-biphenyl]-2-yl)-
methyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonyl)-3-(4-
methylbenzyl)ureido)propanoic Acid (8). The procedure used to
prepare compound 2 was followed using compound 54 (125 mg, 0.25
mmol) and S-benzyl-L-cysteine (34, 65 mg, 0.31 mmol), with the
following modifications: the reaction times following addition of
TMSOTf and phosgene were lengthened to 1.5 and 3 h, respectively,
and a small amount of the yellow oil that formed in the reaction
mixture was removed prior to phosgene addition. Purification via
mass-directed preparative HPLC afforded benzoylurea 8 as a white
Methyl 2-((4′-Chlorobiphenyl-2-yl)methyl)-1,2,3,4-tetrahydroiso-
quinoline-6-carboxylate (52). To a stirred solution of 1,2,3,4-
tetrahydroisoquinoline-6-carboxylate hydrochloride (300 mg, 1.3
mmol) and triethylamine (367 μL, 2.6 mmol) in anhydrous
dichloromethane (3 mL) at room temperature under a N2
atmosphere was added 51 (285 mg, 1.3 mmol) followed by sodium
triacetoxyborohydride (559 mg, 2.6 mmol). The reaction was allowed
to proceed for 18 h at room temperature, after which time aqueous
saturated NaHCO3 (10 mL) was added, followed by EtOAc (20 mL).
The layers were separated, and the aqueous phase was further
1
glassy solid (6 mg, 3%). H NMR (600 MHz, CD3OD) δ 7.85−6.91
(m, 20H), 4.89 (dd, J = 32.7, 15.7 Hz, 2H), 4.47 (dd, J = 6.7, 4.9 Hz,
1H), 4.36 (s, 2H), 4.03 (s, 2H), 3.69 (q, J = 13.0 Hz, 2H), 3.25−3.13
(m, 2H), 2.98−2.85 (m, 3H), 2.80 (dd, J = 13.9, 7.1 Hz, 1H), 2.26 (s,
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3H). 13C NMR (151 MHz, CD3OD) δ 174.5, 173.8, 157.0, 144.1,
139.9, 139.4, 138.2, 137.0, 135.8, 135.1, 132.9, 132.6, 132.3, 132.2,
132.1, 130.9, 130.1, 130.1, 130.0, 129.9, 129.5, 129.5, 128.5, 128.3,
128.1, 128.0, 126.6, 57.7, 55.1, 54.1, 50.6, 49.6, 37.2, 34.1, 26.5, 21.1.
One Ar−CH peak is not observed due to overlap. HRMS (ES+ TOF)
calcd for C42H40ClN3O4S (M + H): 718.2501; found 718.2495.
Synthesis of Compound (9). (S)-2-(3-([1,1′-Biphenyl]-4-carbon-
yl)-3-(4-methylbenzyl)ureido)-5-phenylpentanoic Acid (9). The
procedure used to prepare compound 2 was followed using
compound 32 (50 mg, 0.16 mmol) and L-2-amino-5-phenylpentanoic
acid (35, 46 mg, 0.24 mmol). Purification via flash chromatography
and eluting with a gradient of methanol/dichloromethane (0:100−
butyl dicarbonate (0.417 mL, 1.82 mmol) was added and the reaction
was stirred for a further 24 h at room temperature. Ethanol was
removed under reduced pressure, the residue was taken up in EtOAc
(20 mL), and then 10% aqueous citric acid solution (20 mL) was
added. The two phases were separated, the aqueous phase was further
extracted with EtOAc (2 × 10 mL), and the combined organic phases
were washed with brine, dried over MgSO4, filtered, and concentrated
under reduced pressure to yield the title compound as a colorless oil
(378 mg, 72%). Spectral data obtained corresponded to those
reported in the literature.54
(R)-2-((tert-Butoxycarbonyl)amino)-3-((cyclohexylmethyl)-
sulfonyl)propanoic Acid (22). To a stirred solution of compound 21
(314 mg, 0.99 mmol) in CH2Cl2 (10 mL) at 0 °C was added m-CPBA
(max 77%, 444 mg, approx. 1.98 mmol), and the reaction allowed to
warm to room temperature and proceed for 3 h. After this time, the
reaction was diluted with CH2Cl2 (20 mL); washed with 10%
aqueous sodium metabisulfite, 10% aqueous NaHCO3, and brine;
dried over MgSO4; filtered; and concentrated under reduced pressure.
Purification via flash chromatography and eluting with a gradient of
methanol/dichloromethane (0:100 to 5:95 to 1:90) afforded the title
1
4:96) afforded benzoylurea 9 as a colorless glass (60 mg, 72%). H
NMR (600 MHz, CDCl3) δ 9.46 (d, J = 6.9 Hz, 1H), 7.70−7.50 (m,
4H), 7.50−7.33 (m, 5H), 7.24 (dt, J = 16.9 Hz, 7.8, 5H), 7.05 (d, J =
7.9 Hz, 2H), 6.92 (d, J = 7.9 Hz, 2H), 4.99 (s, 2H), 4.61 (dd, J = 12.7,
7.3 Hz, 1H), 2.73−2.61 (m, 2H), 2.30 (s, 3H), 2.07−1.83 (m, 2H),
1.83−1.73 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 176.5, 175.4,
155.3, 143.8, 141.6, 139.9, 137.1, 134.7, 134.6, 129.4, 129.1, 128.6,
128.2, 127.3, 127.3, 126.6, 126.1, 53.8, 50.1, 35.5, 31.5, 27.4, 21.2.
Two Ar−CH peaks (range δ 129.8−126.0 ppm) are not observed due
to overlap. MS (ES+), m/z 521.4 (M + H). HRMS (ES+ TOF) calcd
for C33H32N2O4 (M + H): 521.2435; found 521.2440.
1
compound as a colorless oil (307 mg, 89%). H NMR (300 MHz,
CDCl3) δ 7.07 (br s, 1H), 5.77 (d, J = 6.3 Hz, 1H), 4.68 (dd, J = 12.1,
5.1 Hz, 1H), 3.66 (d, J = 3.8 Hz, 2H), 2.95 (d, J = 6.4 Hz, 2H), 2.07
(qdd, J = 9.9, 6.5, 3.6 Hz, 1H), 1.92 (d, J = 12.7 Hz, 2H), 1.78−1.58
(m, 3H), 1.45 (s, J = 19.9, 9H), 1.39−0.97 (m, 5H). 13C NMR (75
MHz, CDCl3) δ 172.6, 155.8, 81.4, 77.4, 61.7, 54.8, 49.9, 33.3, 32.4,
28.4, 25.9, 25.8. MS (ES−), m/z 348.2 (M − H). HRMS (ES+ TOF)
calcd for C15H27NO6S (M + Na): 372.1451; found 372.1452.
(R)-1-Carboxy-2-((cyclohexylmethyl)sulfonyl)ethanaminium
Chloride (23). To a sample of 22 (261 mg, 0.75 mmol) at room
temperature under N2 was added a solution of HCl (4 M) in 1,4-
dioxane (2.0 mL), and the stirred reaction was allowed to proceed for
14 h at room temperature. The fine white precipitate was filtered,
washed with a small volume of Et2O, and then dried in vacuo for 24 h
to afford the title compound as a fine off-white powder (165 mg,
77%). 1H NMR (300 MHz, CD3OD) δ 4.57 (d, J = 6.6 Hz, 1H), 3.89
(d, J = 14.7 Hz, 1H), 3.60 (dd, J = 13.5, 9.0 Hz, 1H), 3.20 (d, J = 6.1
Hz, 2H), 2.09 (br s, 1H), 1.96 (d, J = 12.2 Hz, 2H), 1.72 (t, J = 16.7
Hz, 3H), 1.47−1.08 (m, 5H). 13C NMR (75 MHz, CD3OD) δ 60.9,
53.8, 34.1, 34.0, 33.5, 26.9, 26.9. One peak is not observed due to
overlap. MS (ES−), m/z 248.3 (M − H). HRMS (ES+ TOF) calcd for
C10H19NO4S (M + H): 250.1108; found 250.1100.
(R)-2-(3-([1,1′-Biphenyl]-4-carbonyl)-3-(4-methylbenzyl)ureido)-
3-((cyclohexylmethyl)sulfonyl)propanoic Acid (12). The procedure
used to prepare compound 2 was followed using compound 32 (100
mg, 0.33 mmol). Compound 23 (67 mg, 0.24 mmol) was protected in
situ using the method described for compound 19, with the
modification that anhydrous dichloromethane was used as the
reaction solvent and triethylamine (33 μL, 0.24 mmol) was first
added to neutralize the amino acid. Compound 33 was dissolved in
anhydrous dichloromethane (1.0 mL) and transferred to the in situ-
protected amino acid 41 at 0 °C, and the reaction was allowed to
warm to room temperature and proceed for 2 h. Purification via flash
chromatography and eluting with a gradient of methanol/dichloro-
methane (0:100−5:95) afforded compound 12 as a colorless glass
(85.1 mg, 94%). 1H NMR (600 MHz, CDCl3) δ 9.89 (d, J = 6.8 Hz,
1H), 9.76 (br s, 1H), 7.57 (d, J = 7.9 Hz, 4H), 7.48−7.36 (m, 5H),
7.06 (d, J = 7.7 Hz, 2H), 6.95 (d, J = 7.7 Hz, 2H), 5.09−4.91 (m,
3H), 3.72 (qd, J = 14.8, 5.4 Hz, 2H), 2.94 (d, J = 6.4 Hz, 2H), 2.29 (s,
3H), 2.14−2.04 (m, 1H), 1.91 (d, J = 12.1 Hz, 2H), 1.69 (dd, J =
12.6, 2.5 Hz, 2H), 1.64 (d, J = 12.8 Hz, 1H), 1.30 (ddd, J = 25.5, 12.6,
2.6 Hz, 2H), 1.14 (ddt, J = 25.1, 12.6, 3.3 Hz, 1H), 1.06 (dd, J = 24.0,
12.1 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ 175.3, 172.5, 155.5,
144.0, 139.9, 137.1, 134.4, 134.1, 129.5, 129.1, 128.2, 127.5, 127.3,
127.2, 126.6, 61.3, 54.5, 50.2, 49.8, 33.2, 32.3, 25.9, 25.8, 21.2. MS
(ES+), m/z 577.9, (M + H). HRMS (ES+ TOF) calcd for
C32H36N2O6S (M + H): 577.2367; found 577.2375.
Synthesis of Compound (10). (S)-2-(3-([1,1′-Biphenyl]-4-carbon-
yl)-3-(4-methylbenzyl)ureido)-3-(benzyloxy)propanoic Acid (10).
The procedure used to prepare compound 2 was followed using
compound 32 (100 mg, 0.33 mmol) and O-benzyl-L-serine (38, 97
mg, 0.50 mmol), with the following modification: due to the presence
of a white insoluble residue in the carbamoyl chloride/acetonitrile
suspension, the solution of in situ-protected amino acid was instead
transferred to the carbamoyl chloride solution at 0 °C, and the
reaction was allowed to proceed for 3 days. Purification via flash
chromatography and eluting with a gradient of methanol/dichloro-
methane (0:100−5:95) afforded benzoylurea 10 as a colorless glass
1
(140 mg, 81%). H NMR (300 MHz, CDCl3) δ 9.61 (d, J = 7.3 Hz,
1H), 7.59 (dd, J = 7.0, 1.4 Hz, 4H), 7.52−7.22 (m, 10H), 7.06 (d, J =
7.8 Hz, 2H), 6.95 (d, J = 6.9 Hz, 2H), 5.00 (s, J = 17.4 Hz, 2H),
4.86−4.69 (m, 1H), 4.59 (s, 2H), 3.99 (dd, J = 9.5, 3.8 Hz, 1H), 3.79
(dd, J = 9.7, 3.9 Hz, 1H), 2.30 (s, 3H). 13C NMR (75 MHz, CDCl3) δ
174.9, 173.9, 155.4, 143.7, 139.9, 137.3, 136.9, 134.6, 134.5, 129.3,
128.9, 128.5, 128.0, 127.9, 127.8, 127.2, 127.2, 127.1, 126.5, 73.5,
68.9, 54.1, 50.0, 21.1. MS (ES+), m/z 523.1, (M + H). HRMS (ES+
TOF) calcd for C32H30N2O5 (M + H): 523.2227; found 523.2234.
Synthesis of Compound (11). (R)-2-(3-([1,1′-Biphenyl]-4-carbon-
yl)-3-(4-methylbenzyl)ureido)-3-(benzylsulfonyl)propanoic Acid
(11). To a stirred solution of compound 2 (151 mg, 0.28 mmol) in
anhydrous THF (1 mL) at room temperature was added m-CPBA
(77% max, 69 mg, approx. 0.31 mmol). The reaction was stirred at 0
°C for 20 min, forming a mixture of sulfoxide and sulfone products.
Concentration of the reaction mixture in vacuo followed by
purification via flash chromatography and eluting with a gradient of
methanol/dichloromethane (0:100−3:97) afforded the sulfone
1
product 11 as a colorless glass (58 mg, 36%). H NMR (300 MHz,
CDCl3) δ 9.87 (d, J = 6.8 Hz, 1H), 7.57 (dd, J = 7.6, 2.0 Hz, 4H),
7.51−7.32 (m, 10H), 7.03 (d, J = 8.2 Hz, 2H), 6.95 (d, J = 8.0 Hz,
2H), 5.09−4.86 (m, 3H), 4.26 (s, 2H), 3.79−3.49 (m, 2H), 2.25 (s,
3H).[a] 13C NMR (75 MHz, CDCl3) δ 175.2, 171.7, 155.4, 144.0,
139.7, 137.1, 134.2, 133.9, 130.9, 129.4, 129.2, 129.0, 128.9, 128.1,
127.5, 127.2, 127.1, 126.5, 61.2, 51.9, 50.2, 49.6, 21.0. One Ar−CH
peak is not observed due to overlap. MS (ES+) m/z 571.2, (M + H).
HRMS (ES+ TOF) calcd for C32H30N2O6S (M + H): 571.1897;
found 571.1882.
Synthesis of Compound (12). (R)-2-((tert-Butoxycarbonyl)-
amino)-3-((cyclohexylmethyl)thio)propanoic Acid (21). Compound
21 was prepared according to a literature procedure.54 Briefly, to a
stirred solution of L-cysteine (20, 200 mg, 1.65 mmol) in a 1:1
solution of NaOH (2 N) and EtOH (total 3.4 mL) was added
(bromomethyl)cyclohexane (0.253 mL, 1.82 mmol) followed by
tetrabutylammonium iodide (TBAI) (18 mg, 3 mol %). The reaction
was allowed to proceed for 3 days at room temperature; then, di-tert-
Synthesis of Compound (13). (3R,5R,7R)-Adamantan-1-ylmethyl
Trifluoromethanesulfonate (25). Compound 25 was synthesized
according to a literature procedure.55 Briefly, to a stirred solution of 1-
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adamantanemethanol (24, 233 mg, 1.40 mmol) and pyridine (0.886
mL, 9.29 mmol) in anhydrous dichloromethane (8.0 mL) at 0 °C was
added dropwise over a period of 10 min a solution of trifluoro-
methane sulfonic anhydride (0.471 mL, 2.80 mmol) in anhydrous
dichloromethane (8.0 mL). The reaction was allowed to proceed for 2
h at room temperature; diluted with dichloromethane (10 mL);
washed with 1 M aqueous HCl, 5% aqueous NaHCO3, and brine;
dried over MgSO4; filtered; and concentrated under reduced pressure
to yield the title compound as a pale-yellow oil (408 mg, 98%). The
crude material was used directly in the next step without further
purification. Spectral data obtained corresponded to those reported in
the literature.55
(R)-tert-Butyl 2-((tert-Butoxycarbonyl)amino)-3-mercaptopropa-
noate (27). Compound 27 was synthesized based on a literature
procedure, but substituting trimethylphosphine solution in THF for
tri-n-butylphosphine.56 Briefly, to a solution of L-cystine, N,N′-
bis[(1,1-dimethylethoxy)carbonyl]-1,1′-bis(1,1-dimethylethyl) ester
(26, 723 mg, 1.31 mmol) in THF (6.5 mL) was added
trimethylphosphine (1.1 equiv, 1.44 mL of a 1 M solution in
THF). The mixture was allowed to stir at room temperature for 2
min, and then water (158 μL) was added. After stirring overnight, the
reaction mixture was concentrated under reduced pressure.
Purification of the crude oil via flash chromatography and eluting
with a gradient of EtOAc/cyclohexane (0:100−5:95) afforded the
title compound as a colorless oil, which formed colorless needles on
standing (683 mg, 94%). Spectral data obtained corresponded to
those reported in the literature.56 The compound was used directly in
the next step to avoid oxidation over time.
(R)-tert-Butyl 3-(((3R,5R,7R)-Adamantan-1-ylmethyl)thio)-2-
((tert-butoxycarbonyl)amino)propanoate (28). To a solution of
27 (429 mg, 1.55 mmol) in anhydrous acetonitrile (31 mL) was
added 25 (553 mg, 1.85 mmol) followed by K2CO3 (641 mg, 4.64
mmol) and 18-crown-6 (1.22 g, 6.64 mmol). The mixture was stirred
at room temperature for 16 h, diluted with water (30 mL), and
extracted with EtOAc (3 × 20 mL), and the combined organic layers
were dried over MgSO4, filtered, and concentrated under reduced
pressure. The resulting brown-orange oil was purified via flash
chromatography, and eluting with a gradient of EtOAc/cyclohexane
(0:100−5:95) afforded the title compound as a colorless oil (418 mg,
64%). 1H NMR (600 MHz, CDCl3) δ 5.34 (d, J = 7.5 Hz, 1H), 4.42−
4.33 (m, 1H), 2.99−2.84 (m, 2H), 2.36 (q, J = 12.4 Hz, 2H), 1.96 (br
s, 3H), 1.69 (d, J = 12.1 Hz, 3H), 1.61 (d, J = 12.5 Hz, 3H), 1.53 (d, J
= 2.3 Hz, 6H), 1.48 (s, 9H), 1.45 (s, 9H). 13C NMR (75 MHz,
CDCl3) δ 170.3, 155.3, 82.5, 79.9, 77.4, 54.2, 48.6, 41.9, 37.0, 36.9,
34.3, 28.7, 28.5, 28.2. MS (ES+), m/z 426.4, (M + H). HRMS (ES+
TOF) calcd for C23H39NO4S (2M + Na): 873.5092; found 873.5108.
(R)-tert-Butyl 3-(((3R,5R,7R)-Adamantan-1-ylmethyl)sulfonyl)-2-
((tert-butoxycarbonyl)amino)propanoate (29). To a stirred solution
of 28 (204 mg, 0.48 mmol) in anhydrous dichloromethane (3 mL) at
0 °C was added m-CPBA (max 77%) (215 mg, approx. 0.96 mmol, as
a solution in 2 mL of dichloromethane) dropwise over 5 min. The
solution was allowed to stir at 0 °C for 1 h and then at room
temperature for 17 h. After this time, the reaction was diluted with
dichloromethane (50 mL), washed with 10% aqueous sodium
metabisulfite (2 × 40 mL) to ensure any remaining m-CPBA was
destroyed and then twice with 10% aqueous NaHCO3 and brine,
dried over MgSO4, filtered, and concentrated under reduced pressure
to yield the title compound as a colorless oil, containing minor
impurities (231 mg, quant.). 1H NMR (600 MHz, CDCl3) δ 5.59 (d,
J = 5.5 Hz, 1H), 4.48 (d, J = 5.5 Hz, 1H), 3.66−3.50 (m, 2H), 2.84 (s,
2H), 2.00 (br s, 3H), 1.92−1.76 (m, 6H), 1.69 (dd, J = 24.6, 12.1 Hz,
6H), 1.53−1.39 (m, 18H). 13C NMR (151 MHz, CDCl3) δ 168.3,
155.4, 83.6, 80.6, 67.5, 57.3, 50.8, 42.2, 36.6, 34.7, 28.5, 28.0, 27.1. MS
(ES+), m/z 457.9, (M + H). HRMS (ES+ TOF) calcd for
C23H39NO6S (M + Na): 480.2390; found 480.2390. This compound
was used directly in the next step without further purification.
(R)-3-(((3R,5R,7R)-Adamantan-1-ylmethyl)sulfonyl)-2-aminopro-
panoic Acid (30). A sample of 29 (215 mg, 0.47 mmol) was dissolved
in neat TFA (2.0 mL) in a small glass vial, and the reaction was
allowed to proceed at room temperature for 1 h, swirling every 15
min. The pale-yellow reaction mixture was then carefully concentrated
under a fine stream of N2 over a 40 °C heat block (caution!) and then
under reduced pressure. The off-white residue was taken up in
methanol (4 mL) and then purified via capture/release by passing
over a cation-exchange column (MEGA BE-SCX, 1 g, Varian), the
column was washed with methanol (2 × 4 mL), and the product was
eluted with 2 M ammonia in methanol (15 mL). The eluent was
concentrated under reduced pressure to yield compound 30 as the
1
free amine as a fine white powder (131 mg, 92%). H NMR (300
MHz, d6-DMSO) δ 3.77−3.57 (m, 2H), 3.53−2.91 (m, 5H), 1.94 (s,
3H), 1.79 (d, J = 2.4 Hz, 6H), 1.72−1.52 (m, 6H). 13C NMR (75
MHz, d6-DMSO) δ 63.6, 56.9, 48.3, 41.5, 41.3, 36.1, 33.7, 27.8. MS
(ES+), m/z 302.2, (M + H). HRMS (ES+ TOF) calcd for
C14H23NO4S (M + H): 302.1421; found 302.1427.
(R)-2-(3-([1,1′-Biphenyl]-4-carbonyl)-3-(4-methylbenzyl)ureido)-
3-(((3R,5R,7R)-adamantan-1-ylmethyl)sulfonyl)propanoic Acid
(13). The procedure used to prepare compound 2 was followed
using compound 32 (30 mg, 0.10 mmol). Compound 30 (45 mg,
0.15 mmol) was protected in situ using the method described for
compound 39 using anhydrous dichloromethane as the reaction
solvent. Compound 33 was dissolved in 1.0 mL of anhydrous
dichloromethane and transferred to the in situ-protected amino acid
at 0 °C, and the reaction was allowed to warm to room temperature
and proceed for 0.5 h. Purification via flash chromatography and
eluting with a gradient of methanol/dichloromethane (0:100−5:95)
1
afforded compound 13 as a colorless glass (51 mg, 81%). H NMR
(600 MHz, CDCl3) d 9.87 (d, J = 6.7 Hz, 1H), 7.57 (d, J = 8.1 Hz,
4H), 7.45 (t, J = 6.8 Hz, 4H), 7.38 (t, J = 7.3 Hz, 1H), 7.07 (d, J = 7.3
Hz, 2H), 6.95 (d, J = 7.0 Hz, 2H), 5.00 (s, 2H), 4.97 (dd, J = 12.1, 5.9
Hz, 1H), 3.69 (qd, J = 14.9, 5.1 Hz, 2H), 2.88 (s, 2H), 2.30 (s, 3H),
1.98 (s, 3H), 1.83 (s, 6H), 1.68 (q, J = 12.2 Hz, 6H). 13C NMR (151
MHz, CDCl3) δ 175.3, 172.2, 155.5, 144.0, 139.9, 137.2, 134.4, 134.1,
129.5, 129.1, 128.3, 127.6, 127.3, 127.2, 126.6, 66.8, 56.3, 50.2, 49.8,
42.1, 36.5, 34.7, 28.5, 21.2. MS (ES+), m/z 629.3, (M + H). HRMS
(ES+ TOF) calcd for C36H40N2O6S (M + H): 629.2680; found
629.2694.
Synthesis of Compound (14). (R)-3-(Benzylsulfonyl)-2-((tert-
butoxycarbonyl)amino)propanoic Acid (48). To a stirred solution
of Boc-S-benzyl-L-cysteine (17, 500 mg, 1.61 mmol) in anhydrous
dichloromethane (15 mL) at 0 °C was added m-CPBA (max 77%)
(720 mg, approx. 3.21 mmol, as a solution in 10 mL of
dichloromethane) dropwise over 10 min. The solution was allowed
to stir at 0 °C for 1 h and then at room temperature for 17 h. After
this time, the reaction was concentrated under reduced pressure
directly onto silica. Purification via flash chromatography and eluting
with a gradient of methanol/dichloromethane (0:100−10:90)
afforded the title compound as a white crystalline solid (418 mg,
1
76%). H NMR (600 MHz, CDCl3) δ 7.40 (s, 5H), 5.73 (d, J = 4.7
Hz, 1H), 4.72 (br s, 1H), 4.36−4.24 (m, 2H), 3.55 (ddd, J = 18.2,
15.5, 4.2 Hz, 2H), 1.46 (s, 9H). 13C NMR (151 MHz, CDCl3) δ
172.7, 155.8, 131.0, 129.4, 129.3, 127.3, 81.6, 61.8, 52.1, 49.8, 28.4.
MS (ES−), m/z 342.4, (M − H). HRMS (ES+ TOF) calcd for
C15H21NO6S (M + Na): 366.0982; found 366.0988.
(R)-2-Amino-3-(benzylsulfonyl)propanoic Acid Hydrochloride
(49). A sample of 48 (304 mg, 0.89 mmol) was dissolved in
anhydrous 1,4-dioxane (15 mL), and HCl (g) was bubbled through
the solution, produced by adding conc. H2SO4 dropwise to NaCl in a
separate flask. The reaction was allowed to proceed at room
temperature under N2 for 1.5 h, and then N2 (g) was bubbled
through the solution overnight to concentrate. A fine white solid
precipitated, which was filtered, washed with a small volume of 1,4-
dioxane, and dried in a vacuum oven to yield the title compound as a
fine white powder (144 mg, 51%). 1H NMR (600 MHz, d6-DMSO) δ
8.95 (br s, 1H), 7.52−7.33 (m, 5H), 4.71 (dd, J = 35.7, 13.7, 2H),
4.14 (d, J = 3.1, 1H), 3.79 (dd, J = 15.0, 3.8, 1H), 3.46 (dd, J = 15.0,
7.8, 1H). 13C NMR (151 MHz, d6-DMSO) δ 167.8, 131.2, 128.5,
128.0, 58.6, 51.5, 47.6. One Ar−CH peak is not observed due to
overlap. MS (ES+), m/z 244.4, (M + H). HRMS (ES+ TOF) calcd for
C10H13NO4S (M + H): 244.0638; found 244.0637.
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(R)-3-(Benzylsulfonyl)-2-(3-(2-((4′-chloro-[1,1′-biphenyl]-2-yl)-
methyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonyl)-3-(4-
methylbenzyl)ureido)propanoic Acid (14). The procedure used to
prepare compound 2 was followed using compounds 54 (50 mg, 0.10
mmol) and 49 (44 mg, 0.16 mmol), with the following modifications:
the reaction times following addition of TMSOTf and phosgene were
lengthened to 1 and 3 h, respectively, and anhydrous dichloromethane
was utilized as the reaction solvent for resuspension of the carbamoyl
chloride intermediate and in situ protection of the amino acid. The
crude material was purified via flash chromatography and eluting with
methanol/dichloromethane (0:100 to 5:95 to 15:85), followed by
further purification via mass-directed preparative HPLC. This
(br s, 1H), 7.85 (d, J = 7.3 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H), 7.41 (t, J
= 7.5 Hz, 1H), 7.38 (dd, J = 8.2, 1.5 Hz, 2H), 7.27 (d, J = 8.9 Hz,
2H), 7.23 (dd, J = 8.2, 1.4 Hz, 2H), 7.06−6.88 (m, 6H), 6.84 (d, J =
7.8 Hz, 1H), 4.73 (dd, J = 52.1, 15.4 Hz, 2H), 4.64 (dd, J = 10.3, 5.5
Hz, 1H), 4.30 (d, J = 11.5 Hz, 1H), 4.10 (d, J = 11.9, 1H), 3.85−3.64
(m, 3H), 3.60 (dd, J = 14.1, 5.4 Hz, 1H), 3.06−2.74 (m, 6H), 2.27 (s,
3H), 1.95 (br s, 3H), 1.79 (br s, 6H), 1.71−1.60 (m, 6H). 13C NMR
(151 MHz, CDCl3) δ 173.7, 172.9, 154.8, 142.2, 139.1, 136.8, 135.1,
134.9, 133.9, 132.8, 131.6, 131.0, 130.5, 129.3, 129.0, 128.9, 127.1,
126.9, 126.8, 124.6, 66.7, 57.1, 57.0, 53.1, 51.4, 49.7, 49.3, 42.1, 36.6,
34.5, 28.5, 26.6, 21.2. Three Ar−CH peaks are not observed due to
overlap. MS (ES+), m/z 808.3, (M + H). HRMS (ES+ TOF) calcd for
C46H50ClN3O6S (M + H): 808.3182; found 808.3198.
1
afforded compound 14 as a white solid (6 mg, 8%). H NMR (300
MHz, CD3OD) δ 7.74−7.64 (m, 1H), 7.63−7.27 (m, 12H), 7.23 (dd,
J = 8.0, 1.6, 1H), 7.15 (d, J = 1.1, 1H), 7.11−6.92 (m, 5H), 5.04−4.77
(m, 2H), 4.68 (dd, J = 7.5, 4.0, 1H), 4.29 (s, 2H), 4.15 (s, 2H), 3.87
(s, 2H), 3.59 (dd, J = 14.8, 4.0, 1H), 3.42 (dd, J = 14.7, 7.6, 1H), 3.00
(t, J = 6.2, 2H), 2.85 (t, J = 6.0, 2H), 2.20 (s, 3H). 13C NMR (75
MHz, CD3OD) δ 174.6, 173.6, 157.0, 143.8, 140.4, 138.1, 136.4,
136.0, 134.8, 134.4, 133.8, 132.5, 132.3, 132.0, 131.8, 131.4, 130.2,
129.8, 129.7, 129.6, 129.1, 128.5, 128.3, 127.9, 126.3, 60.7, 58.5, 54.8,
54.7, 52.2, 50.7, 50.5, 48.7, 48.4, 48.2, 27.4, 21.1. Two Ar−CH peaks
are not observed due to overlap. MS (ES+), m/z 750.1 (M + H).
HRMS (ES− TOF) calcd for C42H40ClN3O6S (M − H): 748.2254;
found 748.2227.
Synthesis of Compound (15). (R)-2-(3-(2-((4′-Chloro-[1,1′-
biphenyl]-2-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonyl)-
3-(4-methylbenzyl)ureido)-3-((cyclohexylmethyl)sulfonyl)-
propanoic Acid (15). The procedure used to prepare compound 2
was followed using compound 54 (47 mg, 0.10 mmol). Compound
23 (41 mg, 0.45 mmol) was protected in situ using the method
described for compound 12, with the modifications that anhydrous
dichloromethane as the reaction solvent and triethylamine (20 μL,
0.15 mmol) were first added to neutralize the amino acid. Compound
57 was dissolved in anhydrous dichloromethane (1.0 mL) and
transferred to the in situ-protected amino acid 41 at 0 °C, and the
reaction was allowed to warm to room temperature and proceed for
0.5 h. Purification via flash chromatography and eluting with a
gradient of methanol/dichloromethane (0:100−5:95) afforded
compound 15 as a colorless glass (44 mg, 60%). 1H NMR (300
MHz, CD3OD) δ 7.72−7.65 (m, 1H), 7.53−7.28 (m, 7H), 7.21 (dd, J
= 8.0, 1.7 Hz, 1H), 7.12 (d, J = 1.2 Hz, 1H), 7.09−7.01 (m, 4H), 6.99
(d, J = 8.1 Hz, 1H), 4.99−4.77 (m, 2H), 4.67 (dd, J = 7.6, 4.0 Hz,
1H), 4.10 (s, 2H), 3.85 (s, 2H), 3.62 (dd, J = 14.9, 4.0 Hz, 1H), 3.47
(dd, J = 14.8, 7.6 Hz, 1H), 3.08−2.92 (m, 4H), 2.85 (t, J = 5.5 Hz,
2H), 2.28 (s, J = 6.5 Hz, 3H), 2.08−1.93 (m, 1H), 1.88 (d, J = 12.5
Hz, 2H), 1.66 (dd, J = 18.4, 8.7 Hz, 3H), 1.40−0.99 (m, 5H). 13C
NMR (75 MHz, CD3OD) δ 174.9, 173.7, 156.9, 143.7, 140.5, 138.0,
136.3, 136.1, 135.0, 134.8, 134.0, 132.3, 132.0, 131.9, 131.8, 130.1,
130.0, 129.7, 129.5, 128.4, 128.2, 127.9, 126.1, 61.0, 58.7, 56.5, 55.1,
52.2, 50.8, 50.5, 34.2, 34.1, 33.4, 27.7, 27.0, 26.9, 21.1. One additional
CH peak is observed due to apparent peak splitting. MS (ES+), m/z
756.9, (M + H). HRMS (ES+ TOF) calcd for C42H46ClN3O6S (M +
H): 756.2869; found 756.2881.
Synthesis of Compound (16). (R)-3-(((3R,5R,7R)-Adamantan-1-
ylmethyl)sulfonyl)-2-(3-(2-((4′-chloro-[1,1′-biphenyl]-2-yl)methyl)-
1,2,3,4-tetrahydroisoquinoline-6-carbonyl)-3-(4-methylbenzyl)-
ureido)propanoic Acid (16). The procedure used to prepare
compound 2 was followed using compound 54 (150 mg, 0.31
mmol) but using a reduced volume of phosgene (200 μL, 0.64 mL/
mmol amide starting material, 20% w/v in toluene, CAUTION!).
Compound 30 (141 mg, 0.45 mmol) was protected in situ using the
method described for 39 using anhydrous dichloromethane as the
reaction solvent. Compound 57 was dissolved in anhydrous
dichloromethane (1.0 mL) and transferred to the in situ-protected
amino acid 44 at 0 °C; then, the reaction was allowed to warm to
room temperature and proceed for 0.5 h. Purification via flash
chromatography and eluting with a gradient of methanol/dichloro-
methane (0:100−5:95), followed by further purification via mass-
directed preparative HPLC, afforded compound 16 as an opaque
white glassy solid (32 mg, 13%). 1H NMR (600 MHz, CDCl3) δ 9.10
Recombinant Proteins and Synthetic Peptides. Protein
Expression and Purification. Human BCL-XL (ΔC25), human/
mouse chimeric MCL-1 (ΔN170, ΔC23), human BCL-2 (ΔC22),
human BCL-W (ΔC23, C29S/A128E), and mouse A1 (ΔC20,
P104K/C113S) were expressed and purified as described pre-
viously.32,34,46,57−62 BCL-XL, MCL-1, and A1 were expressed as
glutathione S-transferase (GST)-fusion proteins in the pGEX-6P-3
vector (Amersham Biosciences). BCL-2 and BCL-W were expressed
with an N-terminal 6-His tag in the pQE-9 vector (Qiagen). For use
in the AlphaSCREEN assay, GST-BCL-XL and GST-MCL-1 were
purified as intact fusion proteins, whereas for the SPR assay, the GST
tag was first removed via on-column cleavage on glutathione−
sepharose columns using PreScission protease prior to gel filtration.
Purified proteins were snap-frozen and stored at −80 °C prior to use,
with the exception of BCL-2, which was briefly stored at 4 °C for
immediate use in SPR experiments. Expression and purification of
human BCL-XL (ΔC24), human/mouse chimeric MCL-1 (ΔN170,
ΔC23), and loop-deleted human BCL-XL (Δ27-82, ΔC24) for
crystallization studies were performed as described previ-
ously.32,34,46,57,58,63,64
Synthetic Peptides. Biotinylated wild-type mouse BIMBH3 26mer
peptide (Biotin-DLRPEIRIAQELRRIGDEFNETYTRR) for use in
the AlphaSCREEN assay was purchased from Auspep. Wild-type
mBIMBH3 (DLRPEIRIAQELRRIGDEFNETYTRR) peptide
(“mBIM”) and an inert mBIMBH3 (DLRPEIREAQEERREGDEENE-
TYTRR) mutant peptide (“mBIM4E”) for use in SPR studies were
purchased from Mimotopes. All synthetic peptides were purified by
reverse-phase HPLC to >90% purity.
AlphaSCREEN Assay. Relative binding affinities of compounds
for GST-BCL-XL and GST-MCL-1 were determined using a solution
competition assay based on an AlphaSCREEN GST donor/acceptor
detection system (PerkinElmer Lifesciences) as described in detail
elsewhere.21,32,34
GST-BCL-XL and GST-MCL-1 proteins were expressed and
purified as described earlier, and biotinylated-GST (for the counter-
screen) was purchased (PerkinElmer). These were stored as stock
solutions at −80 °C. Biotinylated-BAK and biotinylated-BIM 26mer
peptides (Auspep) were stored as 500 μM stock solutions in 100%
DMSO at −20 °C. AlphaSCREEN glutathione S-transferase (GST)
detection kits were obtained from PerkinElmer Lifesciences. White,
384, low-volume assay plates and polypropylene, 384, compound
storage plates were obtained from Greiner Bio One and Matrical,
respectively. Optical-grade, adhesive plate seals for assay plates were
obtained from Applied Biosystems, while adhesive aluminum foil seals
used for compound plate storage were obtained from Beckman-
Coulter. DMSO was purchased from AnalaR. Assay buffer (25 mM
hydroxyethyl piperazineethanesulfonic acid (HEPES)/25 mM Tris−
HCl/50 mM NaCl, pH 7.5) stock was warmed to room temperature
from 4 °C, followed by the addition of 5 mM 1,4-dithiothreitol
(DTT), 0.1 mg/mL casein sodium salt, and 0.03% v/v Tween 20 (all
final concentrations).
Screening of the test compounds was performed using the
AlphaSCREEN glutathione S-transferase (GST) detection kit system
(PerkinElmer Lifesciences). Assays were performed in 384-well white
Optiplates (PerkinElmer Lifesciences), in an assay buffer consisting of
50 mM HEPES pH 7.4, 10 mM DTT, 100 mM NaCl, 0.05% Tween
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20, and 0.1 mg/mL casein, with a final DMSO concentration of 1.0%
(v/v). All bead solutions were prepared under green light.
tubes, and tubes were centrifuged (1500 rpm, 5 min, 4 °C). The
FACS buffer consisted of KDS BSS (150 mM NaCl, 3.7 mM KCl, 2.5
mM CaCl2, 1.2 mM MgSO4, 7.4 mM HEPES·NaOH, 1.2 mM
KH2PO4, and 0.8 mM K2HPO4) supplemented with 2% FCS. After
centrifugation, the supernatant was decanted, propidium iodide (PI)
(Sigma) was added (100 μL per tube, 0.1% (v/v) diluted in FACS
buffer), and cells were resuspended ready for FACS analysis.
Flow Cytometry Analysis. Cells were identified by forward and
side scatter and assessed for PI positivity (FL3 channel) using a
FACSCalibur cytometer. In sum, 1000 events were acquired (Cell
Quest), and data were analyzed with FlowJo software (Treestar). The
percentage of live cells (PI negative) in duplicate wells was
determined, normalized to 100% based on an internal DMSO-only
vehicle control for each compound titration series and cell type, and
Briefly, test compounds were titrated in an assay buffer (5 μL per
well, from 20 mM compound stock in 100% DMSO). To each well
was then added 10 μL of a prosurvial protein/acceptor bead solution
(containing GST-tagged BCL-XL ΔC25 protein and anti-GST
acceptor beads, coincubated previously for 30 min at room
temperature, and final assay concentrations of 0.6 nM and 15 μg/
mL, respectively). Plates were sealed and incubated at room
temperature for 30 min. Next was added 10 μL of a BH3 peptide/
donor bead solution containing biotinylated-BIMBH3-26mer peptide,
biotin-DLRPEIRIAQELRRIGDEFNETYTRR (final concentration:
5.0 nM), and streptavidin donor beads (final concentration: 15 μg/
mL), coincubated previously for 30 min at room temperature
respectively). Plates were sealed with foil and incubated in the dark
for a further 4 h at room temperature before reading on a PerkinElmer
Fusion Alpha plate reader (Ex 680, Em 520−620 nM).
plotted (mean of three independent experiments
SEM) as a
function of compound concentration using Prism software (version
6.0h for Macintosh OS X, GraphPad). EC50 values were determined
by curve-fitting using nonlinear regression.
The conditions for the assay with GST-MCL-1 were essentially the
same using the following final concentrations: GST-MCL-1 (0.8 nM)
and biotinylated-BAK peptide (4 nM). The counter-screen assay was
performed using the donor and acceptor beads in the presence of
biotinylated-GST (final concentration 2 nM).
Cytochrome c Release Assay (FACS). Cells were seeded in 12-
well cell culture plates (Corning) at a concentration of 1.5 × 105
cells/well in 1 mL of FMA medium containing only 1% FCS. Cells
were incubated for 3 h prior to compound addition. Test compounds
were prepared by diluting compound stocks (1 mM for ABT-737 (1)
and 10 mM for all other compounds, in neat DMSO) in FMA
medium containing 1% FCS (final DMSO concentration 0.25%). A
DMSO-only vehicle control was also included. Following aspiration of
the media, diluted test compounds were added to the cells (1 mL per
well) and test plates were incubated for a further 18 h. After this time,
the media from each well (containing dead cells) was aspirated and
transferred to fluorescence-assisted cell sorting (FACS) tubes (on
ice). Trypsin (200 μL per well, 0.5% trypsin) was added to each well,
and once cells had detached (∼5 min), the FMA medium was added
(200 μL per well) and cells were resuspended and transferred to each
corresponding FACS tube. FACS buffer KDS BSS (150 mM NaCl,
3.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 7.4 mM HEPES·
NaOH, 1.2 mM KH2PO4, and 0.8 mM K2HPO4) (1 mL) was added
to all tubes, and tubes were centrifuged (1500 rpm, 5 min, 4 °C).
After centrifugation, the supernatant was decanted and cells were
permeabilized by gently resuspending in a permeabilization buffer
(200 μL per sample) (consisting of 0.025% digitionin (Calbiochem)
in 20 mM HEPES pH 7.2, 100 mM KCl, 5 mM MgCl2, 1 mM EDTA,
1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic
acid (EGTA), and 250 mM sucrose, supplemented with a protease
inhibitor (cOmplete, Roche)) and incubating for 5 min on ice. Cells
were washed with ice-cold FACS buffer (1 mL) and then centrifuged
(1500 rpm, 5 min, 4 °C). The supernatant was decanted, and cells
were fixed by resuspending in 1% paraformaldehyde in phosphate-
buffered saline (Gibco PBS, ThermoFisher) (100 μL per sample) and
incubating at room temperature for 5 min. Cells were washed twice
with ice-cold FACS buffer (1 mL), each time pelleting cells via
centrifugation (1500 rpm, 5 min, 4 °C) and decanting the
supernatant. Cells were then incubated with the primary antibody,
mouse anticytochrome c (1:100, clone 6H2.B4, #556423, BD
Pharmingen), in Saponin buffer (100 μL per sample) (consisting of
a 1/3 volume of 1% saponin (Sigma) in phosphate-buffered saline
(Gibco PBS, ThermoFisher) and a 2/3 volume of the FACS buffer)
for 45 min on ice. After this time, Saponin buffer (3 mL) was added to
each sample, cells were pelleted by centrifugation (1500 rpm, 5 min, 4
°C), and the supernatant was decanted. Cells were then incubated
with the secondary antibody, PE (R-phycoerythrin) conjugated goat
antimouse IgG (#1031-09, SouthernBiotech) in Saponin buffer (100
μL per sample) for 45 min on ice. After this time, Saponin buffer (3
mL) was added to each sample, cells were pelleted by centrifugation
(1500 rpm, 5 min, 4 °C), and the supernatant was decanted. Cells
were resuspended in FACS buffer (150 μL per sample) ready for
FACS analysis.
SPR Competition Assay. Solution competition assays to
determine IC50 values for BCL-XL, BCL-2, BCL-W, MCL-1, and A1
were performed using a Biacore 3000 instrument as described
previously.21,32,46,65,66 Briefly, prosurvival proteins (10 nM) were
incubated with varying concentrations of compounds (12.5−0.0001
μM, 10-step half-log-fold serial dilution) for 2 h in running buffer [10
mM HEPES, 150 mM NaCl, 3.4 mM ethylenediaminetetraacetic acid
(EDTA), and 0.005% (v/v) Tween 20, pH 7.4] prior to injection
onto a CM5 sensor chip onto which either a wild-type mBIMBH3
(DLRPEIRIAQELRRIGDEFNETYTRR) peptide or an inert
mBIMBH3 (DLRPEIREAQEERREGDEENETYTRR) mutant peptide
(“BIM4E”) (mutations in bold) was immobilized (via N-hydrox-
ysuccinimide coupling) to a density of approximately 100 RU. Specific
binding of the prosurvival protein to the surface in the presence and
absence of compounds was quantified by subtracting the signal from
the BIM4E mutant channel from that obtained on the wild-type BIM
channel, following the “global analysis” baseline correction for each
using BIAevaluation software (version 4.1, Biacore). IC50 values were
calculated by nonlinear curve-fitting of the data with Prism software
(version 6.0h for Macintosh OS X, GraphPad).
Cell Culture and Cell Viability Assay: MEFs. Cultured wild-
type (WT), MCL-1−/−, and MCL-1−/−/BAX−/−/BAK−/− mouse
embryonic fibroblast (MEFs) cells were maintained at 37 °C in a
humidified 5% CO2 incubator, in FMA mediuma high-glucose
DME medium supplemented with 10% (v/v) fetal calf serum (FCS)
(Bovogen), 250 mM L-asparagine (Sigma), and 50 mM 2-
mercaptoethanol (Sigma).
Cells were seeded in 24-well cell culture plates (Corning) at a
concentration of 1 × 104 cells/well in 500 μL of FMA medium as
described above but containing only 1% FCS. Cells were incubated
for 24 h prior to compound addition. A titration series was prepared
for all compounds at twice the final assay concentration, by first
diluting compound stocks (in 100% DMSO) 250-fold in the FMA
medium containing 1% FCS, followed by serial dilution in FMA
medium containing 1% FCS and 0.4% DMSO. For ABT-737 (1), a
four-point 15-fold serial dilution was used (final assay concentrations
0.168−0.00005 μM), and for all other compounds, a four-point 2-fold
serial dilution was used (final assay concentrations 25−3.13 μM). A
DMSO-only vehicle control was also included. Diluted test
compounds were then added to the cells in the existing media (500
μL per well, in duplicate, final DMSO concentration 0.2%), and test
plates were incubated for a further 24 h. After this time, the total
media from each well (containing dead cells) was aspirated and
transferred to fluorescence-assisted cell sorting (FACS) tubes (on
ice). Trypsin (200 μL per well, 0.5% trypsin) was added to each well,
and once cells had detached (∼5 min), the FMA medium was added
(200 μL per well) and cells were resuspended and transferred to each
corresponding FACS tube. FACS buffer (5 mL) was added to all
Flow Cytometry Analysis. Cells were identified by forward and
side scatter and assessed for the level of intracellular cytochrome c
(high/low) based on PE fluorescence (FL2 channel) using a
FACSCalibur cytometer. In sum, 10 000 events were acquired (Cell
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Quest), and data were analyzed with FlowJo software (Treestar). For
each treatment, the percentage of cells for which cytochrome c had
been released from mitochondria (PE low) was determined and
plotted and analyzed using Prism software (version 6.0h for
Macintosh OS X, GraphPad). Using data from four independent
experiments, statistical significance was determined by performing a
multiple unpaired t test using the Holm−Sidak method, with α = 0.01.
Each row was analyzed individually, without assuming a consistent
SD.
ligand geometries were first minimized using Sybyl 7.0 software
(Tripos Associates); from these geometries, ligand parameters were
generated using the CCP4 program libcheck. Parameters were then
manually curated to ensure accurate molecular representation. The
overall structure was validated using MOLPROBITY.75 All graphical
depictions of crystal structures were created using Pymol (The
̌
́
́
̈
PyMOL Molecular Graphics System, Version 1.7.4.0, Schrodinger,
LLC) unless otherwise indicated.
Structure Determination of the BCL-XL:7 Complex. Crystals of the
BCL-XL:7 complex were optimized from an initial screening hit
obtained in a cocrystallization screen at the CSIRO C3 facility.
Subsequent in-house fine-screening using the hanging drop method as
for the BCL-XL:2 complex and optimization using streak-seeding
ultimately yielded diffraction-quality crystals. The reservoir solution
consisted of 0.9 M trisodium citrate and 0.1 M 2-(N-morpholino)-
ethanesulfonic acid (MES) pH 6.0, 5% (v/v) PEG400. Crystals were
equilibrated into a cryoprotectant consisting of a reservoir solution
supplemented with 25% (v/v) ethylene glycol and flash-frozen in N2
(liq). X-ray diffraction data were collected on the MX2 beamline at
the Australian Synchrotron.76 Diffraction data were processed using
XDS.77 The structure was solved and the model was built, refined, and
validated as described for the BCL-XL:2 complex, with the exception
that the search model used for molecular replacement was BCL-XL
from the previously solved BCL-XL:8 complex (with the ligand
removed). The asymmetric unit contained three copies of the BCL-XL
monomer (one domain-swapped dimer within the asymmetric unit
and a further domain-swapped dimer between the third copy of BCL-
XL and its symmetry mate), each with a single copy of compound 7
occupying the hydrophobic groove in essentially identical con-
formations.
Structure Determination of the BCL-XL:8 Complex. Crystals of the
BCL-XL:8 complex were grown by the hanging drop method with a
reservoir solution consisting of 1.3 M ammonium sulfate and 0.1 M
HEPES pH 6.5. Crystals were equilibrated into cryoprotectant
consisting of a reservoir solution supplemented with 25% (v/v)
ethylene glycol and flash-frozen in N2 (liq). X-ray diffraction data
were collected on the MX2 beamline at the Australian Synchrotron.
The crystal of the BCL-XL:8 complex diffracted to 1.9 Å resolution.
Diffraction data were processed using HKL2000.69 The structure was
solved with Phaser and the model was built, refined, and validated as
described for the BCL-XL:2 complex, with the exception that the
search model for molecular replacement was BCL-XL from the BCL-
XL:ABT-737 (1) complex (with the ligand removed) (PDB code:
2YXJ).37 The asymmetric unit contained two copies of the BCL-XL
monomer, arranged as domain-swapped dimers within the asymmetric
unit, each with a single copy of compound 8 occupying the
hydrophobic groove in essentially the same conformation.
Structure Determination of the BCL-XL:12 Complex. Crystals of
the BCL-XL:12 complex were obtained using the hanging drop
method as for the BCL-XL:2 complex, using a reservoir solution
consisting of 1.4 M ammonium sulfate and 0.1 M MES pH 6.0. X-ray
diffraction data were collected, the structure was solved, and the
model was built, refined, and validated as described for the BCL-XL:7
complex, but using the BCL-XL coordinates from the BCL-XL:2
complex (with the ligand removed) as the search model for molecular
replacement. The asymmetric unit contained 12 copies of the BCL-XL
monomer, arranged as six domain-swapped dimers. In each monomer,
a single copy of the ligand was observed to occupy the hydrophobic
groove in essentially identical conformations.
Structure Determination of the BCL-XL:13 Complex. Crystals of
the BCL-XL:13 complex were obtained using the hanging drop
method as for the BCL-XL:2 complex, using a reservoir solution
consisting of 1.7 M ammonium sulfate and 0.1 M MES pH 6.0. X-ray
diffraction data were collected, the structure was solved, and the
model was built, refined, and validated as described for the BCL-
XL:12 complex. The asymmetric unit contained 12 copies of the BCL-
XL monomer, arranged as six domain-swapped dimers. In 11 of the 12
copies, the ligand was observed to occupy the groove in a
conformation similar to that of compound 12 in the BCL-XL:12
complex; in the final copy, the ligand was observed to occupy an
Cell Culture and Cell Viability Assay: COLO205 Cells. Wild-
type (WT) COLO205 (ATCC CCL222) and BAK−/−/BAX−/−
COLO205 were maintained at 37 °C in a humidified 5% CO2
incubator, in Roswell Park Memorial Institute (RPMI)-1640 medium
supplemented with 1% penicillin−streptomycin (base medium) and
10% fetal calf serum (FCS, SAFC Biosciences #F9423).67 Cell line
identity was verified using the CellBank Australia short tandem repeat
(STR) profiling service and confirmed to be mycoplasma-free using
the MycoAlert mycoplasma detection kit (Lonza #LT-07).
Cells were seeded in 96-well white polystyrene flat-bottom plates
(Greiner #655083) at 3 × 103 cells/well in the base medium
containing either 10 or 1% fetal calf serum (FCS) and treated one day
later with serially diluted concentrations of BH3 mimetics (0.0003−
10 μM, six-point, 1:8 dilutions) or benzoylurea compounds 6 or 15
(0.1−25 μM, six-point, 1:3 dilutions). BH3 mimetics used were A-
1331852 (synthesized by D Nhu and G Lessene, WEHI),24,36 ABT-
199 (venetoclax, Chemgood #C-1008), S638458 (Active Biochem
#A-6044), or ABT-737 (1)15 (Chemgood #C-1010). Cell viability
was determined 48 h later using the CellTiter-Glo reagent (Promega
#G9241) according to the manufacturer’s instructions. Percentage cell
viability was calculated by normalizing to the viability of cells treated
with an equivalent concentration of DMSO (vehicle control).
GraphPad Prism software was used to calculate the half-maximal
effective concentration (EC50).
Protein Crystallization, Data Collection, and Structure
Determination. Complexes of compounds with BCL-XL for
crystallization were prepared as described previously.21,32,37,63 BCL-
XL, a C-terminally truncated, “loop-deleted” form of human BCL-XL
(Δ27-82 ΔC24),64 was combined with a 1.5 molar excess of the
relevant compound in 20 mM Tris−HCl, pH 8.0, 150 mM NaCl,
supplemented with 2% DMSO. In general, the complex was then
concentrated to approximately 10 mg/mL (10 kDa molecular weight
cutoff (MWCO) Amicon Ultra-0.5 Concentrator) for crystallization
experiments.
The “loop-deleted” BCL-XL construct (with the large, flexible, and
disordered α1−2 loop removed) crystallizes in the form of a domain-
swapped dimer, involving a reciprocal exchange of the α1-helix of one
monomer of BCL-XL into the neighboring monomer (on the opposite
face of the protein from the canonical hydrophobic groove to which
small molecules are targeted).63,64,68 The increased symmetry and
stability of these dimers appear to facilitate crystallization.
Structure Determination of the BCL-XL:2 Complex. Crystals of the
BCL-XL:2 complex were obtained by the vapor diffusion (hanging
drop) method at 295 K by mixing 1 μL of the complex at 10 mg/mL
with 1 μL of reservoir solution containing 1.4 M ammonium sulfate
and 0.1 M HEPES pH 6.5. Crystals were equilibrated into a
cryoprotectant consisting of a reservoir solution supplemented with
25% (v/v) ethylene glycol and flash-frozen in N2 (liq). X-ray data
were collected at 100 K using an in-house RAXIS-IV++ detector with
a micro-max007 rotating anode X-ray source. The crystal of the BCL-
XL:2 complex diffracted to 2.15 Å resolution. Data were processed
using HKL2000.69 Each structure was solved by molecular
replacement with PHASER70 using the BCL-XL coordinates (PDB
code: 1MAZ)40 as a search model. The model was further improved
using several iterative rounds of manual model building in COOT71
followed by refinement with PHENIX72 and BUSTER73 incorporat-
ing an initial round of simulated annealing in the PHENIX refinement
protocol to minimize model bias.
Ligand geometry restraint (cif) files for compounds were generated
from SMILES strings using the GRADE web server74 using automatic
geometry optimization, except for compounds 2 and 8, for which
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alternative conformation in which the adamantyl moiety did not bind
into the cryptic p5 pocket of BCL-XL but rather bridged across to
another BCL-XL monomer. This interaction mediated a crystal
contact between two BCL-XL monomers and appeared to be a result
of crystallization, unlikely to represent the structure in solution.
Structure Determination of the BCL-XL:15 Complex. Crystals of
the BCL-XL:15 complex were obtained using the hanging drop
method as for the BCL-XL:2 complex using a reservoir solution
consisting of 1.3 M ammonium sulfate and 0.1 M Tris pH 7.0. X-ray
diffraction data were collected, the structure was solved, and the
model was built, refined, and validated as described for the BCL-
XL:12 complex. The asymmetric unit contained four copies of the
BCL-XL monomer, arranged as two domain-swapped dimers. In each
monomer, a single copy of the ligand was observed to occupy the
hydrophobic groove in essentially identical conformations.
Department of Medical Biology, The University of Melbourne,
Parkville, VIC 3050, Australia
Brian J. Smith − The Walter and Eliza Hall Institute of
Medical Research, Parkville, VIC 3052, Australia;
Department of Medical Biology, The University of Melbourne,
Parkville, VIC 3050, Australia; orcid.org/0000-0003-
0498-1910
Richard W. Birkinshaw − The Walter and Eliza Hall Institute
of Medical Research, Parkville, VIC 3052, Australia;
Department of Medical Biology, The University of Melbourne,
Parkville, VIC 3050, Australia
Hong Yang − The Walter and Eliza Hall Institute of Medical
Research, Parkville, VIC 3052, Australia; Department of
Medical Biology, The University of Melbourne, Parkville, VIC
3050, Australia
ASSOCIATED CONTENT
■
Houda Abdo − The Walter and Eliza Hall Institute of
Medical Research, Parkville, VIC 3052, Australia;
Department of Medical Biology, The University of Melbourne,
Parkville, VIC 3050, Australia
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01771.
Overview of the benzoylurea scaffold (Figure S1),
detailed overview of the BCL-XL:2 complex (Figure
S2), overview of the SPR competition experiment
(Figure S3), detailed overview of adamantyl substituent
binding in the cryptic p5 pocket of BCL-XL (Figure S5),
additional cellular data for inhibitors (Figure S6);
cytochrome c release assay using MEFs (Figure S7);
describing data collection and refinement statistics for
protein crystallography (Tables S1 and S2); chemistry,
1H and 13C NMR spectra; and purity of key compounds
by HPLC-UV (PDF)
Christine. A. White − The Walter and Eliza Hall Institute of
Medical Research, Parkville, VIC 3052, Australia;
Department of Medical Biology, The University of Melbourne,
Parkville, VIC 3050, Australia
David Segal − The Walter and Eliza Hall Institute of Medical
Research, Parkville, VIC 3052, Australia
David C. S. Huang − The Walter and Eliza Hall Institute of
Medical Research, Parkville, VIC 3052, Australia;
Department of Medical Biology, The University of Melbourne,
Parkville, VIC 3050, Australia
Jonathan B. Baell − The Walter and Eliza Hall Institute of
Medical Research, Parkville, VIC 3052, Australia;
Department of Medical Biology, The University of Melbourne,
Parkville, VIC 3050, Australia; orcid.org/0000-0003-
2114-8242
Peter M. Colman − The Walter and Eliza Hall Institute of
Medical Research, Parkville, VIC 3052, Australia;
Department of Medical Biology, The University of Melbourne,
Parkville, VIC 3050, Australia
Molecular formula SMILES strings (CSV)
Accession Codes
PDB accession codes for BCL-XL in complex with 2, 7, 8, 12,
13, and 15 are 6UVD, 6UVE, 6UVC, 6UVF, 6UVG, and
6UVH, respectively. Authors will release the atomic
coordinates and experimental data upon article publication.
AUTHOR INFORMATION
Corresponding Authors
■
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01771
Peter E. Czabotar − The Walter and Eliza Hall Institute of
Medical Research, Parkville, VIC 3052, Australia;
Department of Medical Biology, The University of Melbourne,
Parkville, VIC 3050, Australia; Email: czabotar@
wehi.edu.au
Guillaume Lessene − The Walter and Eliza Hall Institute of
Medical Research, Parkville, VIC 3052, Australia;
Department of Medical Biology and Department of
Pharmacology and Therapeutics, The University of
Melbourne, Parkville, VIC 3050, Australia; orcid.org/
0000-0002-1193-8147; Email: glessene@wehi.edu.au
Author Contributions
G.L., P.E.C., and P.M.C. devised and supervised the project.
M.J.R., A.V., and G.L. designed and synthesized inhibitors.
M.J.R., T.O., R.W.B., H.Y., H.A. performed binding studies and
biophysical characterization. M.J.R. and P.E.C. performed the
crystallization and solved the crystal structures. M.J.R. and
C.A.W. performed cellular studies, under guidance by D.S. and
D.C.S.H. B.J.S. performed modeling. M.J.R. wrote the
manuscript with contributions from other authors. All authors
have given approval to the final version of the manuscript.
Authors
Funding
Michael J. Roy − The Walter and Eliza Hall Institute of
Medical Research, Parkville, VIC 3052, Australia;
Department of Medical Biology, The University of Melbourne,
Parkville, VIC 3050, Australia; orcid.org/0000-0003-
0198-9108
Amelia Vom − The Walter and Eliza Hall Institute of Medical
Research, Parkville, VIC 3052, Australia; Department of
Medical Biology, The University of Melbourne, Parkville, VIC
3050, Australia
This work was supported by fellowships and grants from the
Australian Government (Australian Postgraduate Award to
M.J.R.), the Australian Research Council (fellowship to
P.E.C.), the National Health and Medical Research Council
(NHMRC, fellowships to P.E.C., G.L., D.C.S.H., B.J.S., J.B.B.,
P.M.C.; Project Grant GNT1025138; Development Grant
305536; and Program Grants 257502, 461221, and 1016701),
the Leukemia and Lymphoma Society (Specialized Center of
Research Grants 7015 and 7413), the Cancer Council of
Victoria (fellowship to P.M.C., Grant-in-Aid 461239), and the
Toru Okamoto − The Walter and Eliza Hall Institute of
Medical Research, Parkville, VIC 3052, Australia;
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Notes
The authors declare the following competing financial
interest(s): M.J.R., A.V., B.J.S., R.W.B., H.Y., H.A., C.A.W.,
D.S., D.C.S.H., J.B.B., P.M.C., P.E.C., and G.L. are, or have
been, employees of the Walter and Eliza Hall Institute, which
has an agreement with Genentech and AbbVie and receives
milestone and royalty payments related to venetoclax.
Employees of Walter and Eliza Hall Institute may be eligible
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P.E.C., and G.L. receive such a financial benefit as a result of
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ACKNOWLEDGMENTS
■
We gratefully acknowledge our colleagues at WEHI and
elsewhere for their pioneering work into the BCL-2 family. We
would like to thank Grant Dewson, Nadia Kershaw, Adeline
Robin, Mark van Delft, Ryan Brady, Romina Lessene, Brad
Sleebs, Paul Stupple, Stef Chappaz, and Ben Kile for
discussions; Ahmad Wardak, Chris Riffkin, Dana Buczek,
Wilco Kersten, Pat Novello, and Kate Jarman for technical
assistance; Seb Marcuccio (AMT); Jason Dang and colleagues
at the Monash University Mass Spectrometry Facility; and
Janet Newman and colleagues at the CSIRO C3 crystallization
facility. This research was undertaken in part using the MX1
and MX2 crystallography beamlines at the Australian
Synchrotron, Victoria, Australia, and made use of the ACRF
detector.
ABBREVIATIONS
■
BCL-2, B-cell lymphoma 2; BCL-XL, B-cell lymphoma
extralarge; MCL-1, myeloid cell leukemia 1; BH3, Bcl-2
homology domain 3; BAX, Bcl-2-associated X protein; BAK,
Bcl-2 homologous antagonist killer; BIM, BCL-2-interacting
mediator of cell death; PPI, protein−protein interaction;
PROTAC, proteolysis-targeting chimera; SAR, structure−
activity relationship; α, amplified luminescent proximity
homogenous assay; AS, AlphaSCREEN; IC50, half-maximal
inhibitory concentration; SPR, surface plasmon resonance;
MEF, mouse embryonic fibroblast; FCS, fetal calf serum; PI,
propidium iodide; SEM, standard error of mean; EC50, half-
maximal effective concentration; TBAB, tetrabutylammonium
bromide; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethy-
luronium hexafluorophosphate; DIEA, N,N-diisopropylethyl-
amine; DMF, dimethylformamide; DMSO, dimethyl sulfoxide;
EtOAc, ethyl acetate; TMSOTf, trimethylsilyl trifluorometha-
nesulfonate; m-CPBA, meta-chloroperoxybenzoic acid; TFA,
trifluoroacetic acid; LCMS, liquid chromatography−mass
spectrometry; HRMS, high-resolution mass spectrometry;
HEPES, hydroxyethyl piperazineethanesulfonic acid; DTT,
1,4-dithiothreitol
(12) Merino, D.; Kelly, G. L.; Lessene, G.; Wei, A. H.; Roberts, A.
W.; Strasser, A. BH3-Mimetic Drugs: Blazing the Trail for New
Cancer Medicines. Cancer Cell 2018, 34, 879−891.
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the mitochondria: therapeutic targeting of the BCL-2 family-driven
pathway. Br. J. Pharmacol. 2014, 171, 1973−1987.
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