Discovery of potent myeloid cell leukemia 1 (Mcl-1) inhibitors using fragment-based methods and structure-based design

Article abstract of DOI:10.1021/jm301448p

Myeloid cell leukemia 1 (Mcl-1), a member of the Bcl-2 family of proteins, is overexpressed and amplified in various cancers and promotes the aberrant survival of tumor cells that otherwise would undergo apoptosis. Here we describe the discovery of potent and selective Mcl-1 inhibitors using fragment-based methods and structure-based design. NMR-based screening of a large fragment library identified two chemically distinct hit series that bind to different sites on Mcl-1. Members of the two fragment classes were merged together to produce lead compounds that bind to Mcl-1 with a dissociation constant of <100 nM with selectivity for Mcl-1 over Bcl-xL and Bcl-2. Structures of merged compounds when complexed to Mcl-1 were obtained by X-ray crystallography and provide detailed information about the molecular recognition of small-molecule ligands binding Mcl-1. The compounds represent starting points for the discovery of clinically useful Mcl-1 inhibitors for the treatment of a wide variety of cancers.

Full text of DOI:10.1021/jm301448p

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pubs.acs.org/jmc
Discovery of Potent Myeloid Cell Leukemia 1 (Mcl-1) Inhibitors Using
Fragment-Based Methods and Structure-Based Design
Anders Friberg, Dominico Vigil, Bin Zhao, R. Nathan Daniels, Jason P. Burke, Pedro M. Garcia-Barrantes,
DeMarco Camper, Brian A. Chauder, Taekyu Lee, Edward T. Olejniczak, and Stephen W. Fesik*
Department of Biochemistry, Vanderbilt University School of Medicine, 2215 Garland Avenue, 607 Light Hall, Nashville, Tennessee
37232-0146, United States
S
* Supporting Information
ABSTRACT: Myeloid cell leukemia 1 (Mcl-1), a member of the Bcl-2 family of proteins, is
overexpressed and amplified in various cancers and promotes the aberrant survival of tumor
cells that otherwise would undergo apoptosis. Here we describe the discovery of potent and
selective Mcl-1 inhibitors using fragment-based methods and structure-based design. NMR-
based screening of a large fragment library identified two chemically distinct hit series that bind
to different sites on Mcl-1. Members of the two fragment classes were merged together to
produce lead compounds that bind to Mcl-1 with a dissociation constant of <100 nM with
selectivity for Mcl-1 over Bcl-xL and Bcl-2. Structures of merged compounds when complexed
to Mcl-1 were obtained by X-ray crystallography and provide detailed information about the
molecular recognition of small-molecule ligands binding Mcl-1. The compounds represent
starting points for the discovery of clinically useful Mcl-1 inhibitors for the treatment of a wide
variety of cancers.
targeting Mcl-1, we applied a combination of fragment-based
methods25 and structure-based design.26 A similar approach was
successfully utilized in the discovery of ABT-26327,28 and in the
discovery of inhibitors against other challenging protein targets,
such as Hsp9029 and BACE.30 Fragment-based drug discovery
(FBDD) takes advantage of sensitive biophysical methods to
detect the binding of molecular fragments (MW < 300 Da) that
bind weakly to their target proteins. This approach leads to a
more efficient sampling of chemical space and can provide hits
with higher ligand efficiencies.31,32 Furthermore, the affinity of
the initial fragment hits can be markedly increased by growing,
linking, or scaffold merging to obtain potent lead molecules.
Here, we describe the discovery of potent small-molecule Mcl-
1 inhibitors (Ki < 100 nM) that inhibit BH3-containing peptides
from binding to Mcl-1. An NMR-based fragment screen yielded
two distinct classes of hits that were shown by NMR to bind to
two different regions of Mcl-1. On the basis of NMR-derived
structural information, the hits from the two classes were merged
together to obtain potent Mcl-1 inhibitors that exhibited >100-
fold enhanced binding affinity over each component separately.
Improved analogues were rapidly optimized by exploiting the
SAR observed for the initial fragment hits. Crystal structures of
inhibitor-bound Mcl-1 complexes validate the design strategy
and also provide detailed structural information for the discovery
of more potent inhibitors.
INTRODUCTION
■
Mcl-1 (myeloid cell leukemia 1) is a member of the Bcl-2 family
of proteins that when dysregulated prevents cancer cells from
undergoing programmed cell death, a hallmark of cancer.1 By
overexpression of the Mcl-1 protein or amplification of the Mcl-1
gene, a cancerous cell can avoid death, the normal fate for cells
exhibiting abnormal and deregulated growth.2,3 Indeed,
amplification of Mcl-1 is one of the most common genetic
aberrations observed in human cancer,4,5 including lung, breast,
prostate, pancreatic, ovarian, and cervical cancers, as well as
melanoma and leukemia.6−13 Moreover, Mcl-1 overexpression
has emerged as a resistance mechanism against a number of
anticancer therapies including the widely prescribed micro-
tubule-targeted agents paclitaxel and vincristine14 as well as
gemcitabine,15 a first-line treatment option for pancreatic cancer.
Mcl-1 overexpression also confers resistance to ABT-263, a Bcl-
2/Bcl-xL inhibitor currently in clinical trials.16−18 Not
surprisingly, specific down-regulation of Mcl-1 by RNA
interference inhibits cell growth and colony formation, induces
apoptosis in pancreatic cancer cells in vitro, and markedly
decreases tumorigenicity in mouse xenograft models.15 Silencing
of Mcl-1 also potently kills particular subgroups of non-small-cell
lung cancer (NSCLC) cell lines.19 Together, these data suggest
that direct inhibition of Mcl-1 could be an effective therapeutic
option for a wide variety of cancers. In an attempt to target Mcl-1
for the treatment of cancer, small-molecule inhibitors20−22 and
stapled helix peptides23,24 that bind to Mcl-1 have been reported.
However, no Mcl-1 inhibitors have entered clinical trials. Indeed,
targeting Mcl-1 is extremely difficult, since Mcl-1 exerts its
activity through protein−protein interactions involving a large
binding interface. To overcome the challenges associated with
Received: October 5, 2012
Published: December 17, 2012
© 2012 American Chemical Society
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naphthyl groups as contained in compounds 17 and 18,
respectively, were found to be preferred for binding to Mcl-1.
NOE-Guided Fragment Docking. To determine how class
I and II fragments bind to Mcl-1, we performed NMR-based
structural studies of Mcl-1/fragment hit complexes. Using
double-labeled (15N, 13C) Mcl-1 protein, we acquired NOE-
derived distance restraints and used these to dock representative
fragments into a previously determined Mcl-1/Bim BH3 peptide
complex (PDB code 3KJ235). Compounds were docked using a
simulated annealing protocol in XplorNIH, followed by energy
minimization of such models in MOE 2011.10 (Chemical
Computing Group Inc.). Fragments representing classes I and II
exhibited different patterns of protein/ligand NOEs, confirming
that they bind to different regions of Mcl-1. The model structures
obtained by this method were consistent with the observed
NOEs. A model for Mcl-1 complexed to a compound
representative of class I (compound 5) is shown in Figure 2A.
Compound 5 displayed NOEs from A227, M231, and F270 to
the six-membered ring of the ligand. In this model the acid at the
2-position points toward R263. An NOE-derived model of a
fragment hit representing class II (compound 17) is depicted in
Figure 2B. In this case, NOEs from M250, F270, and V249 to the
two methyl groups of the 4-chloro-3,5-dimethylphenyl indicate
that this aromatic group sits deep in the pocket. Additional NOEs
from V253 to the aliphatic tether show that the carboxylic acid
sits at the surface of the pocket. The NMR-derived models
demonstrate that the hydrophobic portions of the two classes of
molecules bind to adjacent parts of a large pocket and that the
carboxylic acids of both series point toward and likely interact
with R263. These results explain the observation that class I and
class II compounds are not able to bind simultaneously to Mcl-1.
Fragment Merging and Linker Optimization. NOE-
guided molecular modeling positioned the class I heterocycles
above the hydrophobic pocket utilized by the class II
compounds. This arrangement suggests that the attachment of
class II aromatic groups with a two- to four-atom linker to the 3-
position of the class I 6,5-fused heterocycles would lead to
merged compounds that maintain the favorable hydrophobic
contacts of both fragments to Mcl-1 as well as the interaction
between the common carboxylic acid and R263 of Mcl-1.
Support for this linking strategy was also obtained from
compounds identified and tested from the Vanderbilt Institute
of Chemical Biology chemical collection, compounds 19 and 20
(Table 1), that exhibited Mcl-1 binding affinities of 62 and 87
μM, respectively. In order to test this hypothesis, the fragment
hits for which we obtained NMR-derived model structures
(Figure 2) were linked together. Scaffold merging produced a
compound with greater than 2 orders of magnitude improved
binding affinity (Figure 3). To determine the optimum linker
length, a 1-naphthyl group was connected to the benzothio-
phene- and benzofuran-2-carboxylic acid cores at the 3-position
using two- to four-atom linkers. Table 2 summarizes the Mcl-1
binding affinities for merged compounds with varying linker
lengths. The two series of compounds containing different core
units showed a parallel SAR, and all merged compounds 21−26
exhibited markedly enhanced binding affinity compared to the
unsubstituted cores 1 and 6, confirming the validity of the linking
strategy. Although the two- and three-atom linked compounds
led to a significant increase in potency, the four-atom linked
compounds (e.g., 23 and 26) yielded the most potent Mcl-1
inhibitors in the series, displaying submicromolar dissociation
constants. Moreover, these compounds exhibited selectivity for
binding to Mcl-1 over Bcl-xL and Bcl-2 (Table 2).
RESULTS
■
Hit Identification. Recombinant, isotopically labeled Mcl-1
(residues 172−327) was used to screen our curated fragment
library (>13 800 compounds) by recording SOFAST H−15N
1
HMQC spectra of Mcl-1 (Figure 1) incubated with mixtures of
1
Figure 1. H−15N HMQC spectra of Mcl-1 with (red) and without
(black) fragment 3. The NMR sample contained 50 μM 15N-labeled
protein and 800 μM ligand.
12 fragments. Deconvolution of the mixtures by screening
individual compounds yielded 132 hits (0.95% hit rate) of 11
distinct chemical classes. Of the initial hits, 93 inhibited Mcl-1
with Ki < 500 μM, and more than a quarter of the hits
demonstrated a ligand efficiency (LE) of 0.25 or greater.
Analogues were tested at a single concentration (400 μM) for
their ability to displace a FITC-labeled Mcl-1-derived BH3
peptide from binding to Mcl-1 in a fluorescence polarization
anisotropy (FPA) assay.
On the basis of their affinity and distinct chemical character-
istics, two classes of compounds were selected for follow-up
experiments (Table 1). Ki values were derived from competition
FPA assays measuring the disruption of Mcl-1 and a FITC-Mcl-
1-BH3 peptide or of Bcl-xL and Bcl-2 and a FITC-BAK-BH3
peptide. Class I fragment hits contained 6,5-fused heterocyclic
carboxylic acids. Analogues of the hits were obtained to explore
the SAR. Parent benzothiophene-, benzofuran-, and indole-2-
carboxylic acids were not identified in our initial screen because
of their weak affinity (1, 6, and 12). The class I fragment hits
identified in the screen contained one or two Cl and/or Me
substitutions that significantly improved their binding affinities.
Similar trends in the SAR for all three core series suggest that
they may display similar binding modes. The fragment SAR also
suggests that the 3-, 4-, and 6-positions are preferred sites for
substitutions and that multiple substitutions can be tolerated or
even beneficial in some combinations. For the indole series, a 1-
Me substitution exerted similar effects as 3-Cl or 3-Me on
benzothiophene and benzofuran scaffolds, suggesting that this
core unit could be flipped along its long axis when bound.
Interestingly, Mcl-1 inhibitors containing indole acids have also
been recently reported in the patent literature.33,34
Another structurally distinct group of fragment hits (class II)
consists of a hydrophobic aromatic system tethered by a linker to
a polar functional group, most often a carboxylic acid (Table 1).
Of the hits in this series, 4-chloro-3,5-dimethylphenyl and 1-
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Table 1. SAR of Class I and II Fragment Hits for Binding to Bcl-2 Family Proteins
SAR of the Merged Compounds. To further improve the
binding affinity to Mcl-1, the binding contribution of the
hydrophobic group of the class II fragments was investigated
(Table 3). A library of indoles (27−48) tethered by the
optimized four-atom linker to various aromatic groups
containing different substitution patterns and sizes was prepared.
The parent indole 27 with an unsubstituted phenyl group (Ki =
35 μM) showed a marginal increase in affinity over the initial
fragment hits in the class (13−15). Substitutions at the 2′-
position (28, 29) on the phenyl group yielded only a minor
increase in potency. However, potency was remarkably enhanced
(20-fold) by monosubstitution of small groups at the 3′-position,
as demonstrated by compounds 30 and 31 containing 3′-Me and
3′-CF3 substitutions, respectively. An increase in affinity was also
observed for 4′-Cl substitutions as exemplified by compound 33.
When these beneficial groups were combined, the effect was
additive. For example, compound 35 which contains a 3′-Me-4′-
Cl-phenyl group displays a 90-fold improved potency over the
parent molecule 27. Indole 37 containing a 3′,5′-di-Me-4′-Cl-
phenyl also exhibited a similar potency as 35, whereas increasing
the size of the 3′-substitution resulted in a slight reduction in
affinity as evidenced by compound 36.
Bicyclic aromatic groups, such as 1′-naphthyl, 1′-(4′-Cl-
naphthyl), and 1′-(5,6,7,8-tetrahydronaphthyl) as exemplified in
compounds 42, 44, and 45, exhibited submicromolar binding
affinities comparable to that of compound 37. Interestingly,
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pattern, and hydrophobicity of the aromatic group are all
essential for binding to Mcl-1. Interestingly, most of the optimal
groups identified from the data set were identical to the initial
class II fragment hits, demonstrating that the preliminary
fragment SAR can guide the optimization of the linked
compounds.
The initial fragment SAR indicated that substitutions at the 4-
or 6-position are beneficial. The same principles were applied to
further optimize the potency of the linked compounds. As
expected from the SAR of the fragment hits, 6-Cl adducts (52−
55) displayed a potency increase. In contrast, 4-Cl analogues
(49−51) exhibited mixed results, suggesting that the anchoring
unit might cause a subtle alteration in the binding conformation
of the core unit. Merged compounds comprising benzothio-
phene and benzofuran scaffolds revealed similar SAR patterns as
the indole series.
An additional linking strategy was explored by attaching
compounds to the 1-position of the indole core (Table 4).
Compounds 69−71 containing the optimized linker and anchor
units retained comparable potency as the corresponding 3-
substituted indoles. This demonstrates that the core indole can
bind in a conformation flipped along its long axis as previously
predicted from the SAR of the fragment hits. Finally, the
importance of the 2-carboxylic acid was evaluated by converting
71 to an amide as in compound 72. This single modification
resulted in a dramatic reduction in potency of at least 3 orders of
magnitude. In line with previous results, merged compounds
showed selectivity over Bcl-2 and Bcl-xL, respectively. Com-
pound 53, for example, is one of the most potent inhibitors of
Mcl-1 (Ki = 55 nM, Supporting Information, Figure S1) and
displays a 16-fold selectivity over Bcl-2 and 270-fold over Bcl-xL.
These results clearly demonstrate the effectiveness of
fragment-based methods and a structure-guided fragment
merging strategy as a very powerful approach for rapidly
obtaining potent binders against target proteins. Additionally,
the ability to translate the lessons learned from the SAR of the
fragment hits to our merged compounds aided the optimization
of our lead compounds.
Figure 2. Model structures of Mcl-1 complexed to two different classes
of fragment hits obtained using NMR-derived distance restraints.
Residues (labeled) with NOEs to the fragments (blue) are rendered as
sticks (gray). R263 is also labeled. (A) Class I: benzothiophene 5. (B)
Class II: tethered aromatic 17.
X-ray Structures of Merged Compounds Complexed to
Mcl-1. The three-dimensional structures of compounds
representing different series of merged compounds in complex
with Mcl-1 were obtained by X-ray crystallography (Table 5). As
shown in Figure 4 for a representative member of both the
benzothiophene and the indole series, the merged compounds
occupy both pockets identified by NMR. The Mcl-1 inhibitors
bind in the BH3 binding groove and explain why our compounds
compete with BH3-peptides for binding to Mcl-1. The overall
secondary structures of Mcl-1 in the Mcl-1/inhibitor complexes
did not change upon binding the small molecules.23,36 However,
ligand binding does result in several important structural changes
near the binding pocket (Figure 5A,B). In compound-bound
structures, a pronounced bend was observed in helix 4 (α4)
which helps accommodate the anchor unit of the ligand in the
deep hydrophobic pocket and places the top of α4 closer to the
ligand. In fact, the top of α4 protrudes slightly into the BH3
binding cleft. This causes the loop connecting helix 4−5 to be
pulled toward the ligand and allows changes in the charge−
charge interactions of R263 (Figure 5C,D). The guanidinium
group of R263 can now interact with the carboxylate moiety of
the merged compounds. These changes are not easily anticipated
from the inspection of Mcl-1 complexed with a peptide. In
peptide-bound structures, α4 is straight which keeps the BH3
binding cleft and the connecting loop open to better
Figure 3. Schematic illustrating the merging of fragments 2 and 17 to
produce compound 60. The affinity of each molecule to Mcl-1 is shown
as well as the ligand efficiencies.
compound 43 containing a 2′-naphthyl moiety was 20-fold less
potent than its regioisomer 42, indicating that the attachment
position to this group is important for optimal binding to Mcl-1.
The largest reduction in potency was observed for compounds
47 and 48 with nitrogen-containing heterocycles (6′-quinolinyl
and 4′-indole) showing a 190- and 40-fold lower affinity
compared to 42. These observations indicate that binding in
the lower part of the pocket is dictated by hydrophobic
interactions and that polar moieties are not accommodated in
the binding pocket of Mcl-1. Additional substitutions of the core
indole nitrogen are also tolerated as shown in compounds 56−
59. This SAR trend strongly suggests that size, substitution
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Table 2. Optimization of Linker Length in the Merged Compounds
a
b
Ki values are measured in triplicate. Ki values are measured in duplicate.
accommodate the peptide. The side chain of R263 also adopts a
different conformation so that it can interact with D218 of the
peptide (Figure 5C). It is also evident that the peptide does not
utilize the full capacity of the hydrophobic pocket. In the peptide
structure, L213 is only occupying the very upper part of this deep
pocket (Figure 6A).
In all of the structures obtained thus far, the hydrophobic unit
of the class II fragments binds in the lower part of the pocket
(Figure 4). Furthermore, all core units derived from class I
fragments are positioned in the upper part of the pocket,
directing the carboxylic acid toward R263, and forming an
interaction with M231, A227, F228, and F270 via the six-
membered ring (Figure 4B).
explanation for the selectivity of our compounds for binding to
specific Bcl-2 family proteins.
SYNTHESIS
■
Compounds 21−26 and 60−68 were prepared as outlined in
Scheme 1. Selective lithiation at the 3-Me position of compounds
80a,b with LDA at −10 °C followed by nucleophilic substitution
with 1-(bromoalkyl)naphthylenes or 1-(2-bromoethoxy)-
naphthalene gave compounds 21−26 with varying linker length
to the terminal 1-naphthyl group. 3-Methylbenzothiophene-2-
carboxylic acid cores 82a−c containing 4-, 6-, or 7-Cl
substitutions were synthesized from the corresponding chloro-
(2-fluorophenyl)ethanones (81a−c) by reacting with methyl
thioglycolate under DBU followed by saponification of the
produced esters. Compounds 82a−c and 80a were then
subjected to the alkylation condition described above to yield
compounds 60−66. Unlike unsubstituted benzothiophene 80a,
it is necessary to employ alkyl iodides as substrates to increase
yields because of reduced nucleophilicity of cores 82a−c by the
substituted chloride. 5-Cl-Benzofuran 84 was prepared from 83
by reacting with methyl bromoacetate. By use of similar
conditions, compounds 67 and 68 were obtained.
The SAR of our merged compounds can now be rationalized
from a more detailed analysis of the X-ray structures. Positions 1
and 7 of the core unit are surface exposed, and as shown,
substitutions here have little or no effect on compound binding.
In contrast, there is minimal space for any substitution at the 5-
position, which is consistent with results of the observed SAR.
The results obtained when adding groups at the 4-position could
be explained by slight differences in how the hydrophobic group
and the linker are positioned. These subtle changes may affect
how much room there is at the 4-position or how the entire core
unit is positioned in the binding pocket. Insights regarding the
improved affinity due to substitutions at the 6-position can be
derived from the X-ray structures. The up to 5-fold increase in
binding may be explained by the addition of a hydrophobic
group, which fits between M231 and A227 in a previously
unoccupied pocket (Figure 4B,C). Presumably the chlorine
keeps the core unit in a more favorable position so that the
carboxylic acid can make a better interaction with R263 (Figure
5D). The latter interaction also explains the requirement of a
negatively charged acidic group in our fragment hits as well as the
loss of affinity upon amidation of the carboxylic acid (71, 72).
The SAR of the class II hits was also analyzed in terms of the X-
ray structures. From the structures, it is evident why hydrophobic
groups are preferred over more hydrophilic aromatic ring
systems containing a nitrogen atom (compounds 47 and 48).
The pocket is lined with numerous nonpolar side chains, namely,
M231, L235, L246, V249, M250, L267, F270, V273, L290, and
I294. An overlay of compounds 53 and 60 when bound to Mcl-1
shows a precise overlap of the anchoring 3′,5′-di-Me-4-Cl-phenyl
group (Figure 4A), suggesting that this unit anchors the rest of
the ligand in the binding pocket. This binding pocket is present
in both Bcl-xL and Bcl-2; however, it is neither as deep nor as
large as it is in Mcl-1. Accessing parts of this pocket not present in
other members of the Bcl-2 family provides a possible
Mcl-1 inhibitors containing indole core 27−59 were
synthesized by the route shown in Scheme 2. Indoles 85a−c
were prepared utilizing the modified Japp−Klingemann
reaction.37 Selective reduction using excess BH3 gave the
corresponding alcohols which were then treated with a variety
of aromatic alcohols under Mitsunobu conditions to afford the
corresponding ether 86. The ester was then saponified to give
indoles 27−55. N-Substituted indoles 56−59 were produced
through alkylations of selected esters 86 followed by hydrolysis.
Class II fragments, binding in the deep hydrophobic pocket,
can also be linked to the indole nitrogen as depicted in Scheme 3.
Ethyl indole-2-carboxylate 88 was substituted at the 1-position
with ethyl 3-bromopropionate using K2CO3 as a base under
reflux in good yield. The adduct 89 then underwent the same
series of reactions to give compounds 69−71. Compound 71 was
further functionalized by amidation to produce 72.
DISCUSSION
■
Mcl-1 is one of the most frequently amplified genes in human
cancers and a common resistance factor to treatment with
current chemotherapeutic agents and existing Bcl-2 family
inhibitors (e.g., ABT-263 and ABT-199).4,38 Here, we describe
the discovery of small molecules that potently inhibit Mcl-1 using
fragment-based methods and structure-based design. The X-ray
structures of our merged compounds complexed with Mcl-1
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Table 3. SAR of Merged Compounds Containing an Optimized Four-Atom Linker
Ki (μM)
Bcl-xL
a
b
b
compd
A
R
Ar
Mcl-1
Bcl-2
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
23
61
62
63
64
65
66
67
26
68
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NMe
NMe
NMe
NBn
S
H
Ph
35 7.1
15 0.7
8.8 1.7
1.9 0.6
H
2-Me-phenyl
2-CF3-phenyl
3-Me-phenyl
3-CF3-phenyl
4-Me-phenyl
4-Cl-phenyl
4-CF3-phenyl
H
H
H
1.7 0.08
16 3.1
H
H
9.8 2.9
H
9.9 3.2
H
3-Me-4-Cl-phenyl
3-Et-4-Cl-phenyl
3,5-di-Me-4-Cl-phenyl
3-(1,1′-biphenyl)
4-(1,1′-biphenyl)
3-phenoxyphenyl
4-phenoxyphenyl
1-naphthyl
0.38 0.14
1.1 0.1
>15
>15
9.1
5.3
H
H
0.30 0.15
7.7 2.1
H
H
7.6 0.7
H
5.2 0.3
H
6.4 0.6
H
0.33 0.10
7.5 0.6
>15
2.7
H
2-naphthyl
H
1-(4-Cl-naphthyl)
1-(5,6,7,8-tetrahydronaphthyl)
5-(2,3-dihydroindenyl)
6-quinolinyl
0.48 0.01
0.30 0.10
2.9 0.1
H
H
H
62 17
H
4-indolyl
14 0.3
4-Cl
4-Cl
4-Cl
6-Cl
6-Cl
6-Cl
6-Cl
H
3-Me-4-Cl-phenyl
3,5-di-Me-4-Cl-phenyl
1-naphthyl
0.81 0.16
0.16 0.04
0.70 0.24
0.19 0.02
0.055 0.018
0.075 0.025
0.066 0.028
0.18 0.01
0.26 0.04
0.14 0.01
0.29 0.17
0.32 0.01
0.37 0.07
1.3 0.52
1.6 0.41
0.25 0.04
0.12 0.03
0.75 0.24
0.72 0.15
3.0 0.10
1.0 0.44
2.5 1.1
3-Me-4-Cl-phenyl
3,5-di-Me-4-Cl-phenyl
1-naphthyl
>15
>15
6.8
1.4
0.87
0.96
1-(5,6,7,8-tetrahydronaphthyl)
3,5-di-Me-4-Cl-phenyl
1-naphthyl
>15
>15
7.4
H
>15
6-Cl
H
3,5-di-Me-4-Cl-phenyl
1-naphthyl
>15
>15
15
>15
5.3
H
3,5-di-Me-4-Cl-phenyl
1-naphthyl
S
H
9.7
S
4-Cl
4-Cl
6-Cl
6-Cl
7-Cl
7-Cl
H
3,5-di-Me-4-Cl-phenyl
1-naphthyl
S
S
3,5-di-Me-4-Cl-phenyl
1-naphthyl
4.1
6.8
0.80
0.96
S
S
3,5-di-Me-4-Cl-phenyl
1-naphthyl
S
O
3,5-di-Me-4-Cl-phenyl
1-naphthyl
O
H
>15
4.3
O
5-Cl
1-naphthyl
a
b
Ki values are measured in triplicate. Ki values are measured in duplicate.
identify several opportunities to significantly improve the affinity
of the current lead molecules.
occupied by the compounds (Figure 6). Mutation of V216,
D218, and V220 to alanine reduces peptide binding affinity 50-,
5-, and 45-fold, respectively, demonstrating the importance of
these additional binding pockets.23 Expanding our lead
compounds to fill more of the peptide interface could yield
more potent Mcl-1 inhibitors. In support of this hypothesis is a
comparison of one of our current lead molecules bound to Mcl-1
Although current lead compounds are able to compete with
BH3-containing peptides for binding to Mcl-1, crystal structures
reveal that the compounds may not be maximally exploiting the
available binding opportunities. For example, pockets occupied
by the peptide residues V216, D218, and V220 are currently not
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a
Table 4. SAR of 2-Carboxylate Analogues on 1-Substituted Indoles
a
The asterisk (*) indicates that all Ki values are measured in triplicate.
a
Table 5. X-ray Data Collection and Refinement Statistics
ligand
b
parameter
53
60
16-mer peptide
4HW4
PDB code
4HW2
6
4HW3
12
no. chains
2
protein construct
Mcl-1 Δ5
Mcl-1 Δ5
Mcl-1 WT
P21
Data collection
space group
cell dimensions
a, b, c (Å)
α, β, γ (deg)
resolution (Å)
Rsym
P21212
P21
121.99, 134.32, 62.04
90.00, 90.00, 90.00
50.00−2.80 (2.85−2.80)
5.7 (29.7)
139.44, 58.76, 140.67
90.00, 90.71, 90.00
50.00−2.40 (2.44−2.40)
5.3 (29.9)
50.07, 48.35, 68.18
90.00, 93.83, 90.00
50.00−1.53 (1.56−1.53)
4.7 (40.8)
Rmerge
4.5 (41.1)
4.5 (36.4)
4.2 (31.3)
I/σ(I)
5.7 (1.9)
12.5 (4.9)
25.0 (2.5)
completeness (%)
redundancy
97.7 (95.0)
98.2 (96.3)
100 (100)
4.1 (3.4)
3.5 (2.7)
4.1 (3.4)
Structure Refinement
no. reflections
25 097
88 346
49 292
R
work/Rfree
no. atoms
protein
21.69/24.52
21.51/26.28
13.74/18.40
c
7181
190
14 259
300
4823
ligand
water
520
387
252
d
Ramachandran
preferred regions (%)
allowed regions (%)
generously allowed (%)
disallowed region (%)
90.1
7.4
89.9
8.4
94.5
5.1
1.5
1.2
0.3
1.0
0.6
0.0
rms deviations
bond length (Å)
bond angle (deg)
0.026
1.993
0.010
1.411
0.008
1.005
a
b
Values in parentheses represent results for the highest resolution shell. 16-mer Mcl-1 BH3-peptide: Ac-ALETLRRVGDGVQRNH-NH2.
Including hydrogen atoms. Backbone conformation was analyzed with PROCHECK.52
c
d
with ABT-737 complexed to Bcl-xL (Figure 6B). Our lead
compounds are much smaller than ABT-737 and only use a
fraction of the binding pockets.
To identify small molecules that bind to these additional
pockets, fragment-based screens of Mcl-1 in the presence of one
of our nanomolar leads represent a possible strategy for
improving potency. By blocking the initial site, fragments could
be identified that bind to these additional pockets of the protein.
These fragments could be rapidly incorporated into new
molecules to markedly improve binding affinity.
An important lesson learned from these studies is that the SAR
observed with the fragment hits is mirrored in the merged
compounds and quickly led to the design and synthesis of potent
lead molecules. In addition, the selectivity profile of the fragment
hits for binding to Bcl-2 family members is mimicked by the
merged compounds. The affinity of initial Mcl-1 fragment hits
against the closely related family members Bcl-xL and Bcl-2
revealed a 5- to 50-fold selectivity for binding to Mcl-1 which is
retained in the lead compounds.
In summary, this study demonstrates the successful targeting
of Mcl-1 by fragment-based and structure-guided methods to
quickly produce an initial set of high-affinity and Mcl-1 selective
inhibitors. The compounds described here serve as starting
points for the discovery of clinically useful Mcl-1 inhibitors for
the treatment of cancer.
EXPERIMENTAL SECTION
■
Protein Expression and Purification. A codon-optimized gene
sequence encoding residues 172−327 of human Mcl-1 (Uniprot code
Q07820) was purchased (Genscript) and cloned into a Gateway entry
vector (pDONR-221, Invitrogen) using the protocols provided
(Supporting Information, Table S1). This construct was further
subcloned into an expression vector (pDEST-HisMBP) containing a
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Figure 5. Several structural differences between peptide-bound (red)
and compound-bound (gray) Mcl-1 complexes were identified. The
Mcl-1-derived BH3-peptide is depicted in green and compound 60 in
blue. (A) To accommodate the hydrophobic fragments into the deep
pocket, helix 4 (α4) is slightly kinked in the structures of Mcl-1/
compound complexes compared to the Mcl-1/peptide structures. This
leads to further rearrangements of the loop, pulling it toward the merged
compound. (B) Close-up of the small-molecule binding site in Mcl-1.
Compared to the peptide-bound state, our merged compounds induce
changes in the position and/or in rotamer states of nearby residues. (C,
D) The network of charge−charge interactions and polar contacts
involving R263 are dramatically different in the two types of complexes.
(C) While bound to the peptide, R263 of Mcl-1 interacts closely with
D218 of the 16-mer, a residue shown to be important by alanine-
scanning.23 Moreover, R263 interacts with the side chain of D256 and
the backbone (bb) oxygen of V258 in Mcl-1. (D) When complexed with
merged compounds (e.g., 60), this network is replaced by an interaction
to the carboxylic acid of the small-molecule ligand and additional polar
contacts to the backbone oxygen atoms of S255 and D256.
Figure 4. X-ray structures of merged compounds bound to Mcl-1.
Compounds incorporating different scaffolds (benzothiophene and
indole) occupy the same binding pocket and adopt very similar binding
poses (inset). (A) Compounds 53 and 60 interact with Mcl-1 in the
BH3-peptide binding cleft between helices 3, 4, and 5. Shown is the
surface depiction of Mcl-1 when complexed to (B) 60 and (C) 53 that
illustrates how the merged compounds fill the pockets as predicted by
NMR and interact with R263.
maltose binding protein (MBP) tag for increased solubility, a tobacco
etch virus (TEV) protease recognition site for tag removal, and an N-
terminal His tag to facilitate purification. The integrity of all plasmids
was checked by sequencing. Soluble Mcl-1 protein was expressed in
Escherichia coli BL21 CodonPlus (DE3) RIL (Stratagene) using
ampicillin and chloramphenicol for selection.
implemented. The running buffer also acted as the Mcl-1 storage and
crystallography buffer (20 mM HEPES, pH 6.8, 50 mM NaCl, 3 mM
DTT, 0.01% NaN3). Purifications were done at 4 °C, and concentration
steps were performed in stirred ultrafiltration cells (Amicon, Millipore).
To improve protein sample quality and to increase crystal diffraction,
protein mutants were created by site-directed mutagenesis (Quik-
Change, Agilent Technologies). Primers for a C-terminal deletion (Δ5)
were designed using their online tool and ordered from Eurofins MWG
Operon. Mutations were made on the entry vector above, analyzed by
in-house sequencing, and subsequently transferred into the pDEST-
HisMBP expression vector. Mutant proteins were purified in the same
way as wild-type (WT) proteins.
In brief, a colony from a fresh transformation plate was picked to
inoculate 100 mL of LB medium (37 °C). The overnight culture was
used to start a 10 L fermentation (BioFlo 415, New Brunswick
Scientific) grown at 37 °C. For NMR studies, uniformly 15N and
15N/13C isotopically labeled protein samples were produced in minimal
M9 medium, where 15NH4Cl and [U-13C]-D-glucose were used as sole
nitrogen and carbon sources (Cambridge Isotope Laboratories). When
the cell density corresponded to OD600 = 2, the temperature was
lowered to 20 °C. After 1 h, protein expression was induced with 0.5 mM
IPTG. Cells were harvested after 16 h by centrifugation. Pellets were
frozen and redissolved in lysis buffer (20 mM Tris, pH 7.5, 300 mM
NaCl, 20 mM imidazole, 5 mM BME), approximately 100 mL/10 g
pellet, before the cells were broken by homogenization (APV-2000,
APV). Prior to application to an affinity column (140 mL, ProBond,
Invitrogen), lysate was cleared by centrifugation (18 000 rpm) and
filtration (0.44 μm). Bound protein was washed on the column and then
eluted by a gradient (20 mM Tris, pH 7.5, 300 mM NaCl, 500 mM
imidazole, 5 mM BME). To enhance TEV protease cleavage, samples
were buffer exchanged (50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM
BME) on three serially connected columns (HiPrep 26/10 Desalting,
GE Healthcare). TEV protease was added to a molar ratio of 1:10
(TEV/Mcl-1) and incubated at room temperature until cleavage was
complete. After adding 20 mM imidazole to the samples, they were
passed over a subtractive second nickel-column (120 mL, Ni-NTA
Superflow, Qiagen) to remove the MBP-tag, noncleaved protein, and
TEV protease. Mcl-1 protein for NMR screening was buffer exchanged
into an optimized NMR buffer (25 mM sodium phosphate, pH 6.3, 25
mM NaCl, 1 mM DTT, 0.01% NaN3). To achieve highly pure samples
(e.g., for crystal screening), a supplementary step of size-exclusion
chromatography (HiLoad 26/60, Superdex 75, GE Healthcare) was
NMR Experiments: Fragment Screening and NOE-Guided
Fragment Docking. Nuclear magnetic resonance (NMR) screening
experiments were performed at 30 °C, using either a Bruker Avance III
500 MHz or 600 MHz NMR spectrometer equipped with a 5 mm single-
axis z-gradient cryoprobe and a Bruker Sample Jet sample changer. Two-
1
dimensional, gradient-enhanced H,15N heteronuclear multiple-quan-
tum coherence (SOFAST-HMQC) spectra (32 scans, ∼12 min) were
used to track shift changes upon ligand binding.39 Spectra were
processed and analyzed in Topspin, version 3.1 (Bruker BioSpin). A
fragment library comprising ∼13 800 compounds that generally satisfy
the “rule of three” (MW ≤ 300, cLogP ≤ 3.0, no more than three
hydrogen bond donors)40 and having no more than four rotatable bonds
was screened as mixtures of 12 fragments. Samples (500 μL) contained
50 μM 15N-labeled Mcl-1, 400 μM of each fragment, and 5% DMSO-d6.
Deconvolution of hit mixtures was performed as single fragments.
Recycling of the protein used for screening was accomplished by a
simple buffer exchange with a yield of 50−80%.
NOE-derived distance restraints were acquired to enable NMR-based
docking of fragments into a previously determined X-ray structure of a
Mcl-1/peptide complex. Spectra were recorded on a Bruker 800 MHz
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of a Mcl-1/BIM peptide complex (PDB code 3KJ2) using the NMR-
derived restraints and a simulated annealing protocol using the program
Xplor-NIH.35,42 A square-well potential (FNOE = 50 kcal mol−1) was
employed to constrain NOE-derived distances. Five low energy models
from this process were energy minimized using MOE 2011.10
(Chemical Computing Group Inc., Montreal, Canada). The lowest
energy structures obtained by this method were consistent with the
observed NOEs.
Protein Crystallization, Data Collection, and Structure
Refinement. Fresh batches of Mcl-1 proteins, WT and Δ5, were
concentrated to 600 μM (10.7 mg/mL) and 1 mM (17.4 mg/mL),
respectively, and screened for crystallization conditions with a 1.2×
excess of ligand. Crystals were obtained by mixing 1 μL of protein with 1
μL of reservoir solution (25−30% PEG 3350, 0.1 M Bis-Tris, pH 6.5, 0.2
M MgCl2) as a hanging drop at 4 or 18 °C. Crystals appeared within the
first week and were flash frozen in liquid nitrogen after cryoprotection
using 10−20% glycol. Crystals of Mcl-1 bound to a 16-mer BH3-peptide
derived from Mcl-1 itself (residues 209−224, Ac-
ALETLRRVGDGVQRNH-NH2, GenScript) were grown in a slightly
different crystallization condition (25% PEG 3350, 0.1 M Bis-TRIS pH
5.5, 0.2 M NaCl). Crystals grew overnight at 18 °C.
Data were collected on the Life Sciences Collaborative Access Team
(LS-CAT) 21-ID-D beamline at the Advanced Photon Source (APS),
Argonne National Laboratory. Indexing, integration, and scaling were
performed with HKL2000.43 By use of a previously determined peptide-
bound structure (PDB code 3MK8), phasing was done by molecular
replacement with Phaser44 as implemented in CCP4.45 Refinement of
the structural models were performed with Phenix46 and Refmac47 and
included rounds of manual model building in COOT.48 Figures were
prepared in PyMOL49 and MOE.
FPA Competition Assays. Fluorescein isothiocyanate (FITC)
labeled Mcl-1-BH3 peptide (FITC-AHx-KALETLRRVGDGVQ-
RNHETAF-NH2)23 and FITC-Bak-BH3 peptide (FITC-AHx-
GQVGRQLAIIGDDINR-NH2) were purchased from GenScript and
used without further purification. FPA measurements were carried out
in 384-well, black, flat-bottom plates (Greiner Bio-One) using the
EnVision plate reader (PerkinElmer). All assays were conducted in assay
buffer containing 20 mM Tris, pH 7.5, 50 mM NaCl, 3 mM DTT, and
2% DMSO. To measure inhibition of the Mcl-1/FITC-Mcl-1-BH3
interaction, Mcl-1 and FITC-Mcl-1-BH3 peptide were each added at
250 nM. To measure inhibition of the FITC-Bak-BH3 interaction with
Bcl-2 family members, 10 nM FITC-Bak-BH3 peptide was incubated
Figure 6. (A) Comparison of Mcl-1 (gray surface) when complexed to
compound 60 (blue) and a 16-mer BH3-peptide derived from Mcl-1
(green). (B) Overlay of the X-ray structures of Mcl-1/60 complex and
Bcl-xL/ABT-737 (PDB code 2YXJ). The Mcl-1 protein is shown in gray,
60 in blue, and ABT-737 in green.
spectrometer equipped with a cryoprobe and pulsed field gradients. 300
μM 15N/13C-labeled samples of Mcl-1 were prepared in a D2O-based
NMR buffer and mixed with selected fragments at 1 mM. Side chain 1H
and 13C NMR signals were assigned from 13C-edited NOE and HCCH-
TOCSY experiments.41 NOE distance restraints were obtained from
three-dimensional 13C-edited NOESY spectra, as well as three-
dimensional 15N- and 13C-filter/edited NOESY spectra acquired with
a mixing time of 80 ms. Compounds were docked into a X-ray structure
Scheme 1. Synthesis of Benzothiophene- and Benzofuran-Containing Compounds
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Scheme 2. Synthesis of Indole-Based Compounds
Scheme 3. Synthesis of Compounds 69−72
with either 14 nM Mcl-1, 4 nM Bcl-xL, or 40 nM Bcl-2. For IC50
determination, compounds were diluted in DMSO in a 16-point, 2-fold
serial dilution scheme, added to assay plates, and incubated for 1 h at
room temperature. The change in anisotropy was measured and used to
calculate an IC50 (inhibitor concentration at which 50% of bound
peptide is displaced) by fitting the inhibition data using XLFit software
(Guildford, UK) to a single-site binding model. This was converted into
a binding dissociation constant (Ki) according to the formula50
washing, and chromatography were HPLC grade. All reagents were
purchased from chemical suppliers and used without purification.
3-(2-(Naphthalen-1-yl)ethyl)benzo[b]thiophene-2-carbox-
ylic Acid (21). General Procedure for Alkylation of 3-
Methylbenzothiophene or 3-Methylbenzofuran-2-carboxylic
Acid. To a stirred solution of 3-methylbenzo[b]thiophene-2-carboxylic
acid (101 mg, 0.53 mmol) in anhydrous THF (5.0 mL) was added LDA
(0.58 mL, 1.17 mmol, 2 M in THF) dropwise under Ar at −10 °C. The
reaction mixture was stirred for 30 min. Then a solution of 1-
(bromomethyl)naphthalene (116 mg, 0.525 mmol) in THF (2 mL) was
added dropwise. The reaction mixture was stirred for an additional 1 h at
−10 °C and then warmed to room temperature and stirred overnight.
The reaction was quenched by addition of saturated NH4Cl aqueous
solution and extracted with CH2Cl2 (2 × 20 mL). The organic extracts
were combined, concentrated and the residue was purified by reverse
phase preparative HPLC to give the product as a solid (103 mg, 0.310
mmol). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.37 (d, J = 8.2 Hz,
1H), 8.02 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 8.0 Hz, 2H), 7.79 (dd, J = 7.2,
1.8 Hz, 1H), 7.53 (m, 3H), 7.44 (m, 3H), 3.61 (m, 2H), 3.33 (m, 2H);
>98% at 215 nm, MS (ESI) m/z = 333.3 (M + H)+.
pep
pep
Ki = [I]50 /([L]50 /Kd + [P]0 /Kd + 1)
where [I]50 is the concentration of the free inhibitor at 50% inhibition,
[L]50 is the concentration of the free labeled ligand at 50% inhibition,
pep
[P]0 is the concentration of the free protein at 0% inhibition, and Kd
represents the dissociation constant of the FITC-labeled peptide probe.
Chemistry. General. All NMR spectra were recorded at room
1
temperature on a 400 MHz AMX Bruker spectrometer. H chemical
shifts are reported in δ values in ppm downfield with the deuterated
solvent as the internal standard. Data are reported as follows: chemical
shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br =
broad, m = multiplet), integration, coupling constant (Hz). Low
resolution mass spectra were obtained on an Agilent 1200 series 6140
mass spectrometer with electrospray ionization. All samples were of
≥95% purity as analyzed by LC−UV/vis−MS. Analytical HPLC was
performed on an Agilent 1200 series with UV detection at 214 and 254
nm along with ELSD detection. LC−MS parameters were as follows:
Phenomenex-C18 Kinetex column, 50 mm × 2.1 mm, 2 min gradient,
5% (0.1% TFA/MeCN)/95% (0.1% TFA/H2O) to 100% (0.1% TFA/
MeCN). Preparative purification was performed on a Gilson HPLC
(Phenomenex-C18, 100 mm × 30 mm, 10 min gradient, 5% → 95%
MeCN/H2O with 0.1% TFA) or by automated flash column
chromatography (Isco, Inc. 100sg Combiflash). Solvents for extraction,
3-(3-(Naphthalen-1-yl)propyl)benzo[b]thiophene-2-carbox-
ylic Acid (22). The general procedure for alkylation was followed using
3-methylbenzo[b]thiophene-2-carboxylic acid (86 mg, 0.45 mmol) and
1-(2-bromoethyl)naphthalene (105 mg, 0.45 mmol) to yield the title
1
compound (78 mg, 0.23 mmol). H NMR (400 MHz, DMSO-d6): δ
(ppm) 7.96 (m, 2H), 7.88 (m, 2H), 7.75 (d, J = 7.5 Hz, 1H), 7.48 (m,
3H), 7.40 (m, 3H), 3.41 (t, J = 7.7 Hz, 2H), 3.15 (t, J = 7.8 Hz, 2H), 1.99
(p, J = 7.7 Hz, 2H); >98% at 215 nm, MS (ESI) m/z = 369.1 (M + Na)+.
3-(3-(Naphthalen-1-yloxy)propyl)benzo[b]thiophene-2-car-
boxylic Acid (23). The general procedure for alkylation was followed
using 3-methylbenzo[b]thiophene-2-carboxylic acid (100 mg, 0.52
mmol) and 1-(2-bromoethoxy)naphthalene (131 mg, 0.52 mmol) to
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1
yield the title compound (101 mg, 0.28 mmol). H NMR (400 MHz,
DMSO-d6): δ (ppm) 8.18 (d, J = 7.8 Hz, 1H), 8.01 (t, J = 7.9 Hz, 2H),
7.85 (m, 1H), 7.48 (m, 4H), 7.37 (m, 2H), 6.89 (d, J = 7.4 Hz, 1H), 4.21
(t, J = 5.9, 2H), 3.54 (t, J = 7.6, 2H), 2.20 (m, 2H); >98% at 215 nm, MS
(ESI) m/z = 363.1 (M + H)+.
2H), 3.54 (t, J = 7.5, 2H), 2.21 (p, J = 7.1 Hz, 2H); MS (ESI) m/z =
397.0 (M + H)+.
3-(3-(Naphthalen-1-yl)propyl)benzofuran-2-carboxylic Acid
(24). The general procedure for alkylation was followed using 3-
methylbenzofuran-2-carboxylic acid and 1-(bromomethyl)naphthalene
to yield the title compound. 1H NMR (400 MHz, CDCl3): δ (ppm) 8.26
(d, 1H, J = 8.4 Hz), 7.91 (d, 1H, J = 8.4 Hz), 7.75 (d, 1H, J = 8.2 Hz),
7.32−7.62 (m, 8H), 3.48−3.59 (m, 4H); >98% at 215 nm, MS (ESI) m/
z = 317.0 (M + H)+.
3-(2-(nNaphthalen-1-yl)ethyl)benzofuran-2-carboxylic Acid
(25). The general procedure for alkylation was followed using 3-
methylbenzofuran-2-carboxylic acid and 1-(2-bromoethyl)naphthalene
to yield the title compound. 1H NMR (400 MHz, CDCl3): δ (ppm) 8.26
(d, 1H, J = 8.6 Hz), 7.32−7.97 (m, 10H), 3.42−3.59 (m, 2H), 3.17−3.36
(m, 2H), 2.18−2.30 (m, 2H); >98% at 215 nm, MS (ESI) m/z = 331.0
(M + H)+.
3-(3-(Naphthalen-1-yloxy)propyl)benzofuran-2-carboxylic
Acid (26). The general procedure for alkylation was followed using 3-
methylbenzofuran-2-carboxylic acid and 1-(2-bromoethoxy)-
naphthalene to yield the title compound. 1H NMR (400 MHz,
CDCl3): δ (ppm) 8.26 (d, 1H, J = 8.6 Hz), 7.32−7.97 (m, 10H), 4.01
(m, 2H), 3.32 (m, 2H), 2.28 (m, 2H); >98% at 215 nm, MS (ESI) m/z =
347.2 (M + H)+.
3-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)benzofuran-2-
carboxylic Acid (67). The general procedure for alkylation was
followed using 3-methylbenzofuran-2-carboxylic acid and 2-chloro-5-
(2-bromoethoxy)-1,3-dimethylbenzene to yield the title compound. 1H
NMR (400 MHz, CDCl3): δ (ppm) 7.71 (d, J = 8.4 Hz, 1H), 7.59 (d, J =
8.6 Hz, 1H), 7.49−7.54 (m, 1H), 7.28−7.34 (m, 1H), 6.62 (s, 2H), 3.99
(t, J = 6.0 Hz, 2H), 3.32 (t, J = 7.4 Hz, 2H), 2.34 (s, 6H), 2.23 (m, 2H);
>98% at 215 nm, MS (ESI) m/z = 359.1 (M + H)+.
5-Chloro-3-(3-(naphthalen-1-yloxy)propyl)benzofuran-2-
carboxylic Acid (68). The general procedure for alkylation was
followed using 5-chloro-3-methylbenzofuran-2-carboxylic acid51 and 1-
(2-bromoethoxy)naphthalene to yield the title compound: >98% at 215
nm, MS (ESI) m/z = 381.1 (M + H)+.
3-(3-Phenoxypropyl)-1H-indole-2-carboxylic Acid (27). Gen-
eral Procedure for Phenol Analogue Coupling and Saponifica-
tion. To a solution of ethyl 3-(3-hydroxypropyl)-1H-indole-2-
carboxylate (70 mg, 0.28 mmol), PPh3 (110 mg, 0.51 mmol), and
phenol (49 mg, 0.52 mmol) in THF (3.5 mL) was added Dt-BuAD (99
mg, 0.51 mmol) at 20 °C. The reaction mixture was stirred for 15 h at 20
°C and then concentrated in vacuo. The residue was purified by flash
chromatography (Combi-flash Rf hexane/EtOAc gradient 0−10%) to
give ethyl 3-(3-phenoxypropyl)-1H-indole-2-carboxylic acid, the title
compound (92 mg, 0.25 mmol), as a colorless oil: >98% at 215 nm, MS
(ESI) m/z = 324.1 (M + H)+.
To a solution of the ethyl ester (28 mg, 0.085 mmol) in EtOH (1.0
mL) was added 50% NaOH−H2O solution (50 μL) at 20 °C. The
reaction mixture was stirred for 15 h at 20 °C. The reaction mixture was
acidified with 1 N HCl solution, extracted with EtOAc, dried over
MgSO4, filtered, and concentrated in vacuo. The crude product was
purified by reverse phase preparative HPLC (H2O/CH3CN gradient to
95% CH3CN/0.5% TFA) to yield the title compound (23 mg, 0.078
mmol) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 11.43
(s, 1H), 7.64. (d, J = 8.0 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.20−7.31 (m,
3H), 7.00 (t, J = 7.2 Hz, 1H), 6.89−6.99 (m, 3H), 3.95 (t, J = 6.4 Hz,
2H), 3.21 (t, J = 6.4 Hz, 2H), 2.01−2.10 (m, 2H); >98% at 215 nm, MS
(ESI) m/z = 296.1 [M + H]+.
3-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)benzo[b]-
thiophene-2-carboxylic Acid (60). The general procedure for
alkylation was followed using 3-methylbenzo[b]thiophene-2-carboxylic
acid (97 mg, 0.50 mmol) and 5-(2-bromoethoxy)-2-chloro-1,3-
dimethylbenzene (133 mg, 0.50 mmol) to yield the title compound
(89 mg, 0.24 mmol). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.00 (d,
J = 8.1 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H), 7.42 (t,
J = 7.6 Hz, 1H), 6.74 (s, 2H), 3.98 (t, J = 6.2 Hz, 2H), 3.38 (t, J = 7.5,
2H), 2.26 (s, 6H), 2.01 (p, J = 7.2, 2H); >98% at 215 nm, MS (ESI) m/z
= 375.1 (M + H)+.
4-Chloro-3-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-
benzo[b]thiophene-2-carboxylic Acid (61). The general procedure
for alkylation was followed using 4-chloro-3-methylbenzo[b]thiophene-
2-carboxylic acid (57 mg, 0.25 mmol) and 2-chloro-5-(2-iodoethoxy)-
1,3-dimethylbenzene (78 mg, 0.25 mmol) to yield the title compound
(25.6 mg, 0.063 mmol). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.01
(dd, J = 7.7, 1.1 Hz, 1H), 7.49 (m, 2H), 6.73 (s, 2H), 4.05 (t, J = 6.2 Hz,
2H), 3.70 (m, 2H), 2.26 (s, 6H), 2.05 (m, 2H); >98% at 215 nm, MS
(ESI) m/z = 409.1 (M + H)+.
4-Chloro-3-(3-(naphthalen-1-yloxy)propyl)benzo[b]-
thiophene-2-carboxylic Acid (62). The general procedure for
alkylation was followed using 4-chloro-3-methylbenzo[b]thiophene-2-
carboxylic acid (57 mg, 0.25 mmol) and 1-(2-iodoethoxy)naphthalene
(75 mg, 0.25 mmol) to yield the title compound (9.9 mg, 0.025 mmol).
1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.15 (d, J = 8.0 Hz, 1H), 8.02
(m, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.46 (m, 6H), 6.96 (d, J = 7.2 Hz, 1H),
4.28 (t, J = 5.9, 2H), 3.87 (m, 2H), 2.23 (m, 2H); >98% at 215 nm, MS
(ESI) m/z = 419.1 (M + Na)+.
6-Chloro-3-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-
benzo[b]thiophene-2-carboxylic Acid (63). The general procedure
for alkylation was followed using 6-chloro-3-methylbenzo[b]thiophene-
2-carboxylic acid (57 mg, 0.25 mmol) and 2-chloro-5-(2-iodoethoxy)-
1,3-dimethylbenzene (78 mg, 0.25 mmol) to yield the title compound
(36 mg, 0.087 mmol). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.18
(d, J = 1.9 Hz, 1H) 7.97 (d, J = 8.7 Hz, 1H), 7.45 (dd, J = 8.7, 1.9 Hz,
1H), 6.70 (s, 2H), 3.96 (t, J = 6.2 Hz, 2H), 3.36 (m, 2H), 2.26 (s, 6H),
2.00 (p, J = 7.2 Hz, 2H); >98% at 215 nm, MS (ESI) m/z = 431.1 (M +
Na)+.
6-Chloro-3-(3-(naphthalen-1-yloxy)propyl)benzo[b]-
thiophene-2-carboxylic Acid (64). The general procedure for
alkylation was followed using 6-chloro-3-methylbenzo[b]thiophene-2-
carboxylic acid (57 mg, 0.25 mmol) and 1-(2-iodoethoxy)naphthalene
(75 mg, 0.25 mmol) to yield the title compound (9.9 mg, 0.025 mmol).
1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.17 (d, J = 1.9 Hz, 1H), 8.12
(d, J = 8.0 Hz, 1H), 8.02 (d, J = 8.8 Hz, 1H), 7.84 (d, J = 7.7 Hz, 1H),
7.48 (m, 3H), 7.38 (m, 2H), 6.89 (d, J = 7.4 Hz, 1H), 4.20 (t, J = 5.9,
2H), 3.52 (t, J = 7.6, 2H), 2.18 (m, 2H); >98% at 215 nm, MS (ESI) m/z
= 419.1 (M + Na)+.
7-Chloro-3-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-
benzo[b]thiophene-2-carboxylic Acid (65). The general procedure
for alkylation was followed using 7-chloro-3-methylbenzo[b]thiophene-
2-carboxylic acid (57 mg, 0.25 mmol) and 2-chloro-5-(2-iodoethoxy)-
1,3-dimethylbenzene (78 mg, 0.25 mmol) to yield the title compound
(53 mg, 0.13 mmol). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 7.97 (d,
J = 8.1 Hz, 1H) 7.64 (d, J = 7.6 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H), 6.70 (s,
2H), 3.97 (t, J = 6.1 Hz, 2H), 3.38 (m, 2H), 2.25 (s, 6H), 2.02 (p, J = 7.1
Hz, 2H); MS (ESI) m/z = 409.1 (M + H)+.
Compounds 28−55 were prepared following the general procedure
outlined above in library format. Purity of all final compounds was
determined by HPLC analysis and is >95%. All compounds were
isolated as solids.
3-(3-(o-Tolyloxy)propyl)-1H-indole-2-carboxylic Acid (28).
Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-2-carboxylate and
o-cresol yielded 28. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 11.39 (s,
1H), 7.64 (d, J = 8.0 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.22 (t, J = 7.2 Hz,
1H), 7.13 (t, J = 8.0 Hz, 1H), 7.02 (t, J = 7.2 Hz, 1H), 6.67−6.76 (m,
3H), 3.94 (t, J = 6.4 Hz, 2H), 3.20 (t, J = 6.4 Hz, 2H), 2.25 (s, 3H), 2.01−
2.11 (m, 2H); >98% at 215 nm, MS (ESI) m/z = 310.1 [M + H]+.
7-Chloro-3-(3-(naphthalen-1-yloxy)propyl)benzo[b]-
thiophene-2-carboxylic Acid (66). The general procedure for
alkylation was followed using 7-chloro-3-methylbenzo[b]thiophene-2-
carboxylic acid (57 mg, 0.25 mmol) and 1-(2-iodoethoxy)naphthalene
(75 mg, 0.25 mmol) to yield the title compound (25 mg, 0.062 mmol).
1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.05 (d, J = 8.2 Hz, 1H), 8.02
(d, J = 8.1 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H),
7.50 (m, 1H), 7.41 (m, 4H), 6.88 (d, J = 7.5 Hz, 1H), 4.20 (t, J = 5.8,
25
dx.doi.org/10.1021/jm301448p | J. Med. Chem. 2013, 56, 15−30

Journal of Medicinal Chemistry
Featured Article
3-(3-(2-(Trifluoromethyl)phenoxy)propyl)-1H-indole-2-car-
boxylic Acid (29). Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-
2-carboxylate and 2-(trifluoromethyl)phenol yielded 29. 1H NMR (400
MHz, DMSO-d6): δ (ppm) 11.43 (s, 1H), 7.64. (d, J = 8.0 Hz, 1H), 7.50
(t, J = 7.2 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.16−7.35 (m, 4H), 6.98 (t, J
= 7.2 Hz, 1H), 4.04 (t, J = 6.4 Hz, 2H), 3.22 (t, J = 6.4 Hz, 2H), 2.01−
2.12 (m, 2H); >98% at 215 nm, MS (ESI) m/z = 364.1 [M + H]+.
3-(3-(m-Tolyloxy)propyl)-1H-indole-2-carboxylic Acid (30).
Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-2-carboxylate and
m-cresol yielded 30. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 11.39 (s,
1H), 7.64. (d, J = 8.0 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.22 (t, J = 7.2
Hz, 1H), 7.13 (t, J = 8.0 Hz, 1H), 7.02 (t, J = 7.2 Hz, 1H), 6.67−6.76 (m,
3H), 3.94 (t, J = 6.4 Hz, 2H), 3.20 (t, J = 6.4 Hz, 2H), 2.25 (s, 3H), 2.01−
2.11 (m, 2H); >98% at 215 nm, MS (ESI) m/z = 310.1 [M + H]+.
3-(3-(3-(Trifluoromethyl)phenoxy)propyl)-1H-indole-2-car-
boxylic Acid (31). Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-
2-carboxylate and 3-(trifluoromethyl)phenol yielded 31. 1H NMR (400
MHz, DMSO-d6): δ (ppm) 11.43 (s, 1H), 7.64. (d, J = 8.0 Hz, 1H), 7.50
(t, J = 7.2 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.16−7.35 (m, 4H), 6.98 (t, J
= 7.2 Hz, 1H), 4.04 (t, J = 6.4 Hz, 2H), 3.22 (t, J = 6.4 Hz, 2H), 2.01−
2.12 (m, 2H); >98% at 215 nm, MS (ESI) m/z = 364.1 [M + H]+.
3-(3-(p-Tolyloxy)propyl)-1H-indole-2-carboxylic Acid (32).
Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-2-carboxylate and
p-cresol yielded 32. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 11.41 (s,
1H), 7.63 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.22 (t, J = 7.2 Hz,
1H), 7.06 (d, J = 8.0 Hz, 2H), 7.02 (t, J = 7.2 Hz, 1H), 6.79 (d, J = 8.4 Hz,
2H), 3.91 (t, J = 6.4 Hz, 2H), 3.19 (t, J = 6.4 Hz, 2H), 2.22 (s, 3H), 1.98−
2.09 (m, 2H); >98% at 215 nm, MS (ESI) m/z = 310.1 [M + H]+.
3-(3-(4-Chlorophenoxy)propyl)-1H-indole-2-carboxylic Acid
(33). Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-2-carboxylate
5H), 7.25−7.46 (m, 3H), 6.98−7.03 (m, 1H), 6.91 (d, J = 8.0 Hz, 1H),
4.05 (t, J = 6.4 Hz, 2H), 3.23 (t, J = 6.4 Hz, 2H), 2.01−2.10 (m, 2H);
>98% at 215 nm, MS (ESI) m/z = 372.1 [M + H]+.
3-(3-([1,1′-Biphenyl]-4-yloxy)propyl)-1H-indole-2-carboxylic
Acid (39). Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-2-
1
carboxylate and [1,1′-biphenyl]-4-ol yielded 39. H NMR (400 MHz,
DMSO-d6): δ (ppm) 11.42 (s, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.63−7.52
(m, 4H), 7.35−7.45 (m, 4H), 7.32 (t, J = 7.2 Hz, 1H), 7.22 (t, J = 7.2 Hz,
1H), 6.95−7.05 (m, 2H), 4.01 (t, J = 6.4 Hz, 2H), 3.23 (t, J = 6.4 Hz,
2H), 2.03−2.13 (m, 2H); >98% at 215 nm, MS (ESI) m/z = 372.2 [M +
H]+.
3-(3-(3-Phenoxyphenoxy)propyl)-1H-indole-2-carboxylic
Acid (40). Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-2-
1
carboxylate and 3-phenoxyphenol yielded 40. H NMR (400 MHz,
DMSO-d6): δ (ppm) 11.42 (s, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.33−7.42
(m, 2H), 7.20−7.31 (m, 4H), 7.16 (t, J = 7.2 Hz, 1H), 6.95−7.05 (m,
3H), 6.69 (d, J = 7.6 Hz, 1H), 6.52 (s, 1H), 3.93 (t, J = 6.4 Hz, 2H), 3.19
(t, J = 6.4 Hz, 2H), 1.95−2.10 (m, 2H); >98% at 215 nm, MS (ESI) m/z
= 388.2 [M + H]+.
3-(3-(4-Phenoxyphenoxy)propyl)-1H-indole-2-carboxylic
Acid (41). Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-2-
1
carboxylate and 4-phenoxyphenol yielded 41. H NMR (400 MHz,
DMSO-d6): δ (ppm) 11.42 (s, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.32−7.45
(m, 3H), 7.23 (t, J = 8.0 Hz, 1H), 7.09 (t, J = 7.6 Hz,1H), 6.90−7.03 (m,
7H), 3.95 (t, J = 6.4 Hz, 2H), 3.21 (t, J = 6.8 Hz, 2H), 2.02−2.10 (m,
2H); >98% at 215 nm, MS (ESI) m/z = 388.2 [M + H]+.
3-(3-(Naphthalen-1-yloxy)propyl)-1H-indole-2-carboxylic
Acid (42). Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-2-
1
carboxylate and 1-naphthol yielded 42. H NMR (400 MHz, DMSO-
d6): δ (ppm) 11.60 (s, 1H), 8.24 (d, J = 6.8 Hz, 1H), 7.87 (d, J = 7.2 Hz,
1H), 7.66 (d, J = 8.0 Hz, 1H), 7.30−7.55. (m, 5H), 7.25. (t, J = 8.0 Hz,
1H), 6.95 (t, J = 7.6 Hz, 1H), 6.87 (d, J = 7.6 Hz, 1H), 4.16 (t, J = 6.0 Hz,
2H), 3.30−3.40 (m, 2H), 2.15−2.25 (m, 2H); >98% at 215 nm, MS
(ESI) m/z = 346.1 [M + H]+.
1
and 4-chlorophenol yielded 33. H NMR (400 MHz, DMSO-d6): δ
(ppm) 11.42 (s, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H),
7.30 (d, J = 8.8 Hz, 2H), 7.22 (t, J = 7.2 Hz, 1H), 6.99 (t, J = 7.2 Hz, 1H),
6.93 (d, J = 8.8 Hz, 2H), 3.94 (t, J = 6.4 Hz, 2H), 3.19 (t, J = 6.4 Hz, 2H),
2.00−2.10 (m, 2H); >98% at 215 nm, MS (ESI) m/z = 330.1 [M + H]+.
3-(3-(4-(Trifluoromethyl)phenoxy)propyl)-1H-indole-2-car-
boxylic Acid (34). Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-
2-carboxylate and 4-(trifluoromethyl)phenol yielded 34. 1H NMR (400
MHz, DMSO-d6): δ (ppm) 11.43 (s, 1H), 7.60−7.69 (m, 3H), 7.39 (d, J
= 8.4 Hz, 1H), 7.22 (t, J = 7.2 Hz, 1H), 7.08 (d, J = 8.4 Hz, 2H), 6.99 (t, J
= 7.2 Hz, 1H), 4.04 (t, J = 6.4 Hz, 2H), 3.22 (t, J = 6.4 Hz, 2H), 2.03−
2.13 (m, 2H); >98% at 215 nm, MS (ESI) m/z = 364.1 [M + H]+.
3-(3-(4-Chloro-3-methylphenoxy)propyl)-1H-indole-2-car-
boxylic Acid (35). Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-
2-carboxylate and 4-chloro-3-methylphenol yielded 35. 1H NMR (400
MHz, DMSO-d6): δ (ppm) 11.42 (s, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.39
(d, J = 8.4 Hz, 1H), 7.18−7.30 (m, 2H), 7.00 (t, J = 7.6 Hz, 1H), 6.85 (s,
1H), 6.75 (d, J = 8.4 Hz, 1H), 3.94 (t, J = 6.4 Hz, 2H), 3.19 (t, J = 6.8 Hz,
2H), 2.24 (s, 3H), 1.95−2.10 (m, 2H); >98% at 215 nm, MS (ESI) m/z
= 344.1 [M + H]+.
3-(3-(4-Chloro-3-ethylphenoxy)propyl)-1H-indole-2-carbox-
ylic Acid (36). Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-2-
carboxylate and 4-chloro-3-ethylphenol yielded 36. 1H NMR (400
MHz, DMSO-d6): δ (ppm) 11.42 (s, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.39
(d, J = 8.4 Hz, 1H), 7.26 (d, J = 8.8 Hz, 1H), 7.22 (t, J = 8.0 Hz, 1H), 7.02
(t, J = 8.0 Hz,1H), 6.87 (s, 1H), 6.76 (d, J = 8.8 Hz, 1H), 3.95 (t, J = 6.4
Hz, 2H), 3.20 (t, J = 6.8 Hz, 2H), 2.61 (q, J = 7.6 Hz, 2H), 1.95−2.10 (m,
2H), 1.15 (t, J = 7.6 Hz, 3H); >98% at 215 nm, MS (ESI) m/z = 358.1
[M + H]+.
3-(3-(Naphthalen-2-yloxy)propyl)-1H-indole-2-carboxylic
Acid (43). Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-2-
1
carboxylate and 2-naphthol yielded 43. H NMR (400 MHz, DMSO-
d6): δ (ppm) 11.42 (s, 1H), 7.82 (d, J = 8.8 Hz, 1H), 7.73 (d, J = 8.0 Hz,
1H), 7.67 (d, J = 8.0 Hz, 1H), 7.35−7.52 (m, 3H), 7.34 (d, J = 7.2 Hz,
1H), 7.17−7.27 (2H, m), 6.98 (t, J = 7.2 Hz, 1H), 4.09 (t, J = 6.4 Hz,
2H), 3.26 (t, J = 6.4 Hz, 2H), 2.10−2.18 (m, 2H); >98% at 215 nm, MS
(ESI) m/z = 346.1 [M + H]+.
3-(3-((4-Chloronaphthalen-1-yl)oxy)propyl)-1H-indole-2-car-
boxylic Acid (44). Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-
1
2-carboxylate and 4-chloronaphthalen-1-ol yielded 44. H NMR (400
MHz, DMSO-d6): δ (ppm) 11.47 (S, 1H), 8.30 (d, J = 8.0 Hz, 1H), 8.11
(d, J = 8.0 Hz, 1H), 7.73 (t, J = 7.2 Hz, 1H), 7.62−7.69 (m, 2H), 7.56 (d,
J = 8.4 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.21 (t, J = 7.2 Hz, 1H), 6.95 (t,
J = 7.2 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 4.17 (t, J = 6.4 Hz, 2H), 3.28−
2,35 (m, 2H), 2.17−2.27 (m, 2H); >98% at 215 nm, MS (ESI) m/z =
380.1 [M + H]+.
3-(3-((5,6,7,8-Tetrahydronaphthalen-1-yl)oxy)propyl)-1H-in-
dole-2-carboxylic Acid (45). Coupling of ethyl 3-(3-hydroxypropyl)-
1H-indole-2-carboxylate and 5,6,7,8-tetrahydronaphthalen-1-ol yielded
45. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 11.42 (s, 1H), 7.63 (d, J =
8.0 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.20−7.28 (m, 1H), 6.92−7.07 (m,
2H), 6.60−6.70 (m, 2H), 3.94 (t, J = 6.4 Hz, 2H), 3.23 (t, J = 6.4 Hz,
2H), 2.69 (t, J = 0.60 Hz, 2H), 2.63 (t, J = 0.60 Hz, 2H), 2.01−2.10 (m,
2H), 1.63−1.80 (m, 4H); >98% at 215 nm, MS (ESI) m/z = 350.2 [M +
H]+.
3-(3-((2,3-Dihydro-1H-inden-5-yl)oxy)propyl)-1H-indole-2-
carboxylic Acid (46). Coupling of ethyl 3-(3-hydroxypropyl)-1H-
indole-2-carboxylate and 2,3-dihydro-1H-inden-5-ol yielded 46. 1H
NMR (400 MHz, DMSO-d6): δ (ppm) 11.42 (s, 1H), 7.64 (d, J = 8.0
Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 7.08 (d, J = 8.4
Hz, 1H), 7.03 (t, J = 7.6 Hz,1H), 6.75 (s, 1H), 6.65−6.70 (d, J = 8.4 Hz,
1H), 3.91 (t, J = 6.4 Hz, 2H), 3.33 (t, J = 6.4 Hz, 2H), 2.70−2.83 (m,
4H), 1.90−2.10 (m, 4H); >98% at 215 nm, MS (ESI) m/z = 336.1 [M +
H]+.
3-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-1H-indole-2-
carboxylic Acid (37). Coupling of ethyl 3-(3-hydroxypropyl)-1H-
1
indole-2-carboxylate and 4-chloro-3,5-dimethylphenol yielded 37. H
NMR (400 MHz, DMSO-d6): δ (ppm) 11.42 (s, 1H), 7.63 (d, J = 8.0
Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.22 (t, J = 7.6 Hz,1H), 7.00 (t, J = 7.6
Hz,1H), 6.74 (s, 2H), 3.93 (t, J = 6.4 Hz, 2H), 3.19 (t, J = 6.8 Hz, 2H),
2.27 (s, 6H), 1.95−2.10 (m, 2H); >98% at 215 nm, MS (ESI) m/z =
358.1 [M + H]+.
3-(3-([1,1′-Biphenyl]-3-yloxy)propyl)-1H-indole-2-carboxylic
Acid (38). Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-2-
1
carboxylate and [1,1′-biphenyl]-3-ol yielded 38. H NMR (400 MHz,
3-(3-(Quinolin-6-yloxy)propyl)-1H-indole-2-carboxylic Acid
(47). Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-2-carboxylate
DMSO-d6): δ (ppm) 11.43 (s, 1H), 7.55−7.75 (m, 3H), 7.31−7.52 (m,
26
dx.doi.org/10.1021/jm301448p | J. Med. Chem. 2013, 56, 15−30

Journal of Medicinal Chemistry
Featured Article
and quinolin-6-ol yielded 47. 1H NMR (400 MHz, DMSO-d6): δ (ppm)
11.47 (s, 1H), 8.92 (d, J = 4.0 Hz, 1H), 8.53 (d, J = 8.0 Hz, 1H), 8.04 (d, J
= 9.2 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.61 (d,
J = 9.2 Hz, 1H), 7.46 (s, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 9.2 Hz,
1H), 7.21 (t, J = 8.0 Hz, 1H), 6.97 (t, J = 8.0 Hz, 1H), 4.14 (t, J = 6.4 Hz,
2H), 3.26 (t, J = 7.2 Hz, 2H), 2.14−1.93 (m, 2H); >98% at 215 nm, MS
(ESI) m/z = 347.1 [M + H]+.
mmol) in DMF (0.8 mL) was added NaH (3.0 mg, 0.075 mmol, 60%
in mineral oil) at 20 °C under Ar. The reaction mixture was stirred for 20
min at 20 °C. Then MeI (34 mg, 0.24 mmol) was added. The reaction
mixture was stirred for an additional 1 h and then quenched by addition
of water. The reaction mixture was extracted with CH2Cl2. Combined
organic layer was concentrated in vacuo, and the residue was purified by
reverse phase preparative HPLC to give the product as a solid (12 mg,
0.03 mmol): >98% at 215 nm, MS (ESI) m/z = 400.2 [M + H]+.
The title compound was obtained by following the general procedure
for saponification outlined above as a white solid. 1H NMR (400 MHz,
DMSO-d6): δ (ppm) 7.67 (d, J = 8.0 Hz, 1H), 7.52. (d, J = 8.0 Hz, 1H),
7.32. (t, J = 7.2 Hz, 1H), 7.07 (t, J = 7.2 Hz, 1H), 6.75 (s, 2H), 3.91−3.99
(m, 5H), 3.18 (t, J = 7.2 Hz, 2H), 2.23 (s, 6H), 1.96−2.05 (m, 2H);
>98% at 215 nm, MS (ESI) m/z = 372.1 [M + H]+.
3-(3-((1H-Indol-4-yl)oxy)propyl)-1H-indole-2-carboxylic Acid
(48). Coupling of ethyl 3-(3-hydroxypropyl)-1H-indole-2-carboxylate
and quinolin-6-ol yielded 48. 1H NMR (400 MHz, DMSO-d6): δ (ppm)
11.45 (s, 1H), 11.04 (S, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.40 (d, J = 7.8 Hz,
1H), 7.15−7.30 (m, 2H), 6.91−7.07 (m, 3H), 6.48 (s, 1H), 6.39 (d, J =
7.2 Hz, 1H), 4.08 (t, J = 6.0 Hz, 2H), 3.26 (t, J = 6.0 Hz, 2H), 2.09−2.20
(m, 2H); >98% at 215 nm, MS (ESI) m/z = 335.1 [M + H]+.
4-Chloro-3-(3-(4-chloro-3-methylphenoxy)propyl)-1H-in-
dole-2-carboxylic Acid (49). Coupling of ethyl 4-chloro-3-(3-
hydroxypropyl)-1H-indole-2-carboxylate37 and 4-chloro-3-methylphe-
Compounds 57−59 were prepared following the general procedure
outlined above in library format. Purity of all final compounds was
determined by HPLC analysis and is >95%. All compounds were
isolated as solids.
1
1
nol yielded 49. H NMR (400 MHz, DMSO-d6): δ (ppm) H NMR
(400 MHz, DMSO-d6): δ 11.68 (s, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.26
(d, J = 8.4 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 7.07 (d, J = 7.6 Hz, 1H),
6.88−6.94 (m, 1H), 6.69−6.78 (m, 1H), 4.03 (t, J = 6.4 Hz, 2H), 3.45 (t,
J = 7.2 Hz, 2H), 2.27 (s, 3H), 2.01−2.08 (m, 2H); >98% at 215 nm, MS
(ESI) m/z = 378.1 [M + H]+.
1-Methyl-3-(3-(naphthalen-1-yloxy)propyl)-1H-indole-2-car-
boxylic Acid (57). Methylation of ethyl 3-(3-(naphthalen-1-yloxy)-
1
propyl)-1H-indole-2-carboxylate by MeI yielded 57. H NMR (400
MHz, DMSO-d6): δ 8.22 (d, J = 6.4 Hz, 1H), 7.87 (d, J = 6.4 Hz, 1H),
7.70 (d, J = 8.0 Hz, 1H), 7.36−7.58. (m, 5H), 7.30. (t, J = 7.6 Hz, 1H),
7.06 (t, J = 7.6 Hz, 1H), 6.87 (d, J = 7.6 Hz, 1H), 4.16 (t, J = 6.0 Hz, 2H),
3.97 (s, 3H), 3.30−3.40 (m, 2H), 2.15−2.25 (m, 2H); >98% at 215 nm,
MS (ESI) m/z = 360.1 [M + H]+.
6-Chloro-3-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-1-
methyl-1H-indole-2-carboxylic Acid (58). Methylation of ethyl 6-
chloro-3-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-1H-indole-2-car-
boxylate by MeI yielded 58. 1H NMR (400 MHz, DMSO-d6): δ 7.79−
7.57 (m, 2H), 7.07 (dd, J = 8.6, 1.7 Hz, 1H), 6.73 (s, 2H), 3.97−3.89 (m,
5H), 3.20−3.10 (m, 2H), 2.27 (s, 6H), 2.06−1.93 (m, 2H); >98% at 215
nm, MS (ESI) 1.83 min, m/z = 428 [M + Na]+.
1-Benzyl-3-(3-(naphthalen-1-yloxy)propyl)-1H-indole-2-car-
boxylic Acid (59). Benzylation of ethyl 3-(3-(naphthalen-1-yloxy)-
propyl)-1H-indole-2-carboxylate by benzyl bromide yielded 59. 1H
NMR (400 MHz, DMSO-d6): δ (ppm) 8.18−8.24 (m, 1H), 7.87 (d, J =
7.2 Hz, 1H), 7.72−7.80 (m, 1H), 7.42−7.56 (m, 3H), 7.35−7.40. (m,
1H), 7.15−7.30. (m, 5H), 6.95−7.10 (m, 3H), 6.83−6.90 (m, 1H), 5.84
(s, 2H), 4.17 (t, J = 6.0 Hz, 2H), 3.30−3.40 (m, 2H), 2.15−2.25 (m,
2H); >98% at 215 nm, MS (ESI) m/z = 436.2 [M + H]+.
1-(3-(4-Chloro-3-methylphenoxy)propyl)-1H-indole-2-car-
boxylic Acid (69). The title compound was obtained by following the
general procedure for phenol analogue coupling and saponification
outlined above using ethyl 1-(3-hydroxypropyl)-1H-indole-2-carbox-
ylate and 4-chloro-3-methylphenol as a white solid: >98% at 215 nm,
MS (ESI) m/z = 344.1 [M + H]+.
4-Chloro-3-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-1H-
indole-2-carboxylic Acid (50). Coupling of ethyl 4-chloro-3-(3-
hydroxypropyl)-1H-indole-2-carboxylate37 and 4-chloro-3,5-dimethyl-
phenol yielded 50. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 11.70 (s,
1H), 7.67. (d, J = 8.4 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.07 (d, J = 7.6
Hz, 1H), 6.69 (s, 2H), 4.20 (t, J = 6.4 Hz, 2H), 3.44 (t, J = 7.2 Hz, 2H),
2.28 (s, 6H), 1.97−2.08 (m, 2H); >98% at 215 nm, MS (ESI) m/z =
392.1 [M + H]+.
4-Chloro-3-(3-(naphthalen-1-yloxy)propyl)-1H-indole-2-car-
boxylic Acid (51). Coupling of ethyl 4-chloro-3-(3-hydroxypropyl)-
1H-indole-2-carboxylate37 and 1-naphthol yielded 51. H NMR (400
1
MHz, DMSO-d6): δ (ppm) 11.68 (s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 7.86
(d, J = 7.6 Hz, 1H), 7.35−7.57 (m, 5H), 7.21 (d, J = 8.0 Hz, 1H), 7.09 (d,
J = 7.2 Hz, 1H), 6.94 (d, J = 7.2 Hz, 1H), 6.69−6.78 (m, J = 7.2 Hz, 1H),
4.25 (t, J = 6.0 Hz, 2H), 3.60 (t, J = 7.6 Hz, 2H), 2.16−2.25 (m, 2H);
>98% at 215 nm, MS (ESI) m/z = 380.1 [M + H]+.
6-Chloro-3-(3-(4-chloro-3-methylphenoxy)propyl)-1H-in-
dole-2-carboxylic Acid (52). Coupling of ethyl 6-chloro-3-(3-
hydroxypropyl)-1H-indole-2-carboxylate37 and 4-chloro-3-methylphe-
nol yielded 52. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 11.64 (s, 1H),
7.66. (d, J = 8.8 Hz, 1H), 7.40 (s, 1H), 7.26 (d, J = 8.8 Hz, 1H), 7.02 (d, J
= 6.8 Hz, 1H), 7.07 (d, J = 7.6 Hz, 1H), 6.89 (s, 1H), 6.80 (d, J = 6.0 Hz,
1H), 3.93 (t, J = 6.4 Hz, 2H), 3.17 (t, J = 7.2 Hz, 2H), 2.27 (s, 3H), 1.95−
2.08 (m, 2H); >98% at 215 nm, MS (ESI) m/z = 378.1 [M + H]+.
6-Chloro-3-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-1H-
indole-2-carboxylic Acid (53). Coupling of ethyl 6-chloro-3-(3-
hydroxypropyl)-1H-indole-2-carboxylate37 and 4-chloro-3,5-dimethyl-
phenol yielded 53. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 11.60 (s,
1H), 7.66 (d, J = 8.4 Hz, 1H), 7.40 (s, 1H), 7.02 (d, J = 7.2 Hz, 1H), 6.73
(s, 2H), 3.92 (t, J = 6.4 Hz, 2H), 3.17 (t, J = 7.6 Hz, 2H), 2.27 (s, 6H),
1.95−2.05 (m, 2H); >98% at 215 nm, MS (ESI) m/z = 392.1 [M + H]+.
6-Chloro-3-(3-(naphthalen-1-yloxy)propyl)-1H-indole-2-car-
boxylic Acid (54). Coupling of ethyl 6-chloro-3-(3-hydroxypropyl)-
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-1H-indole-2-
carboxylic Acid (70). The title compound was obtained by following
the general procedure for phenol analogue coupling and saponification
outlined above using ethyl 1-(3-hydroxypropyl)-1H-indole-2-carbox-
ylate and 4-chloro-3,5-dimethylphenol as a white solid: >98% at 215 nm,
MS (ESI) m/z = 357.1 [M + H]+.
1-(3-(Naphthalen-1-yloxy)propyl)-1H-indole-2-carboxylic
Acid (71). The title compound was obtained by following the general
procedure for phenol analogue coupling and saponification outlined
above using ethyl 1-(3-hydroxypropyl)-1H-indole-2-carboxylate and 1-
1H-indole-2-carboxylate37 and 1-naphthol yielded 54. H NMR (400
1
MHz, DMSO-d6): δ (ppm) 11.62 (s, 1H), 8.19 (d, J = 7.6 Hz, 1H), 7.86
(d, J = 7.6 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.33−7.57 (m, 5H), 6.97 (d,
J = 8.0 Hz, 1H), 6.87 (d, J = 8.0 Hz, 1H), 4.15 (t, J = 6.4 Hz, 2H), 3.27−
3−37 (m, 2H), 2.16−2.25 (m, 2H); >98% at 215 nm, MS (ESI) m/z =
380.1 [M + H]+.
6-Chloro-3-(3-((5,6,7,8-tetrahydronaphthalen-1-yl)oxy)-
propyl)-1H-indole-2-carboxylic Acid (55). Coupling of ethyl 6-
chloro-3-(3-hydroxypropyl)-1H-indole-2-carboxylate37 and 5,6,7,8-tet-
rahydronaphthalen-1-ol yielded 55: >98% at 215 nm, MS (ESI) m/z =
384.1 [M + H]+.
1
naphthol as a white solid. H NMR (400 MHz, DMSO-d6): δ (ppm)
8.26 (d, J = 7,2 Hz, 1H), 7.87 (d, J = 7.2 Hz, 1H), 7.69 (d, J = 8.0 Hz,
1H), 7.37−7.57. (m, 4H), 7.35 (t, J = 8.0 Hz, 1H), 7.15−7.25 (m, 2H),
7.07 (t, J = 7.2 Hz, 1H), 6.85 (d, J = 7.2 Hz, 1H), 4.89 (t, J = 6.8 Hz, 2H),
4.23 (t, J = 7.2 Hz, 2H), 2.15−2.25 (m, 2H); >98% at 215 nm, MS (ESI)
m/z = 346.1 [M + H]+.
1-(3-(Naphthalen-1-yloxy)propyl)-1H-indole-2-carboxamide
(72). To a solution of 1-(3-(naphthalen-1-yloxy)propyl)-1H-indole-2-
carboxylic acid (15 mg, 0.043 mmol) in CH2Cl2 (1.0 mL) was added
oxalyl chloride (37 μL, 0.43 mmol) followed by a drops of N,N-
dimethylformamide at room temperature under Ar. The reaction
mixture was stirred for 3 h at room temperature and then concentrated
under vacuum. The residue was redissolved in CH2Cl2 (0.5 mL), and
3-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-1-methyl-1H-
indole-2-carboxylic Acid (56). General Procedure for Alkylation
of Indole NH. To a solution of ethyl 3-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-1H-indole-2-carboxylate (15 mg, 0.04
27
dx.doi.org/10.1021/jm301448p | J. Med. Chem. 2013, 56, 15−30

Journal of Medicinal Chemistry
Featured Article
concentrated NH4OH aqueous solution (0.67 mL) was added. The
reaction mixture was stirred for 2 h and then extracted with ethyl acetate.
The organic layer was concentrated in vacuo, and the residue was
purified by reverse phase preparative HPLC to give the product as a
white solid (11 mg, 0.032 mmol). 1H NMR (400 MHz, DMSO-d6): δ
(ppm) 8.24−8.30 (m, 1H), 7.99 (br, 2H), 7.82−7.90 (m, 1H), 7.63 (d, J
= 8.0, 1H), 7.47−7.60 (m, 3H), 7.42−7.46 (m, 1H), 7.35−7.40. (m,
1H), 7.19 (s, 1H), 7.14 (t, J = 7,2 Hz, 1H), 7.06 (t, J = 7,2 Hz, 1H), 6.84
(t, J = 7.2 Hz, 1H), 4.86 (t, J = 7.2 Hz, 2H), 4.12 (t, J = 6.0 Hz, 2H),
2.25−2.35 (m, 2H); >98% at 215 nm, MS (ESI) m/z = 345.1 [M + H]+.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Mcl-l protein sequence used for X-ray crystallography, 1H−15N
HMQC spectra of Mcl-1 with and without compound 53, FPA
dose−response curve of compound 53 displacing a FITC-labeled
BAK peptide from Mcl-1, and synthesis of intermediates. This
material is available free of charge via the Internet at http://pubs.
acs.org.
Accession Codes
Atom coordinates and structure factors for Mcl-1/ligand
complexes have been deposited at the Protein Data Bank
(4HW2, 4HW3, and 4HW4).
AUTHOR INFORMATION
Corresponding Author
*Phone: +1 (615) 322 6303. Fax: +1 (615) 875 3236. E-mail:
stephen.fesik@vanderbilt.edu.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
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ABBREVIATIONS USED
■
FBDD, fragment-based drug discovery; Mcl-1, myeloid cell
leukemia 1; Bcl-2, B-cell lymphoma 2; Bcl-xL, B-cell lymphoma
extra large; BH3, Bcl-2 homology domain 3; CSP, chemical shift
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cence polarization anisotropy
28
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