Optimization of Peptidomimetics as Selective Inhibitors for the β-Catenin/T-Cell Factor Protein-Protein Interaction

Article abstract of DOI:10.1021/acs.jmedchem.9b00147

The β-catenin/T-cell factor (Tcf) protein-protein interaction (PPI) plays a critical role in the β-catenin signaling pathway which is hyperactivated in many cancers and fibroses. Based on compound 1, which was designed to target the Tcf4 G¹³ANDE¹⁷ binding site of β-catenin, extensive structure-activity relationship studies have been conducted. As a result, compounds 53 and 57 were found to disrupt the β-catenin/Tcf PPI with the K_i values of 0.64 and 0.44 μM, respectively, and exhibit good selectivity for β-catenin/Tcf over β-catenin/E-cadherin and β-catenin/adenomatous polyposis coli (APC) PPIs. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assays revealed that 56, the ethyl ester of 53, was more potent than 53 in inhibiting viability of most of the Wnt/β-catenin hyperactive cancer cells. Further cell-based studies indicated that 56 disrupted the β-catenin/Tcf PPI without affecting the β-catenin/E-cadherin and β-catenin/APC PPIs, suppressed transactivation of Wnt/β-catenin signaling in dose-dependent manners, and inhibited migration and invasiveness of Wnt/β-catenin-dependent cancer cells.

Full text of DOI:10.1021/acs.jmedchem.9b00147

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Article
Optimization of Peptidomimetics as Selective Inhibitors
for the #-Catenin/T-Cell Factor Protein-Protein Interaction
Zhen Wang, Min Zhang, Jin Wang, and Haitao Ji
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00147 • Publication Date (Web): 11 Mar 2019
Downloaded from http://pubs.acs.org on March 12, 2019
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Optimization of Peptidomimetics as Selective
Inhibitors for the β-Catenin/T-Cell Factor
Protein−Protein Interaction
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Zhen Wang, †,# Min Zhang, †,# Jin Wang, Haitao Ji *
†
†,‡,
†Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa,
Florida 33612–9497, United States
‡Departments of Oncologic Sciences and Chemistry, University of South Florida, Tampa, Florida
3
3620–9497, United States
ABSTRACT: The β-catenin/T-cell factor (Tcf) protein−protein interaction (PPI) plays a critical
role in the β-catenin signaling pathway which is hyperactivated in many cancers and fibroses.
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17
Based on compound 1, which was designed to target the Tcf4 G ANDE binding site of β-catenin,
extensive structure-activity relationship (SAR) studies have been conducted. As a result,
compounds 53 and 57 were found to disrupt the β-catenin/Tcf PPI with the K of 0.64 and 0.44
i
μM, respectively, and exhibit good selectivity for β-catenin/Tcf over β-catenin/E-cadherin and β-
catenin/adenomatous polyposis coli (APC) PPIs. The MTS tetrazolium cell viability assays
revealed that 56, the ethyl ester of 53, was more potent than 53 in inhibiting viability of most of
the Wnt/β-catenin hyperactive cancer cells. Further cell-based studies indicated that 56 disrupted
the β-catenin/Tcf PPI without affecting the β-catenin/E-cadherin and β-catenin/APC PPIs,
suppressed transactivation of Wnt/β-catenin signaling in dose-dependent manners, and inhibited
migration and invasiveness of Wnt/β-catenin dependent cancer cells.
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INTRODUCTION
The Wnt/β-catenin signaling pathway plays a critical role in regulation of cell proliferation,
differentiation, and survival.1-3 The aberrant activation of Wnt/β-catenin signaling has been
implicated in initiation and progression of many cancers and fibroses10-11. For instance, loss of
adenomatous polyposis coli (APC) function can lead to the inappropriate stabilization of β-catenin
and promote the formation of the constitutive complex between β-catenin and the T-cell factor
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(Tcf)/lymphoid enhancer-binding factor (Lef) family of transcriptional factors, which transcribes
specific Wnt target genes that produce crypt progenitor-like cells in the surface intestinal
epithelium, eventually causing sporadic colorectal cancer.4-5 The autocrine activation of Wnt
ligands can stabilize β-catenin into the dephosphorylated state and result in an increased level of
nuclear β-catenin to interact with Tcf/Lef to induce overexpression of Wnt target genes and cause
initiation and progression of triple negative breast cancers (TNBCs).8-9 Hyperactivation of β-
catenin signaling was detected in cancer stem cells, which control tumor growth, seed metastases,
and result in cancer recurrence after remission.12-14 In addition, activation of β-catenin signaling
+
was demonstrated to exclude CD8 T cells from the tumor microenvironment and promote
intratumoral regulator T cell (Treg) survival and infiltration, thus impairing antitumor immunity.15-
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Therefore, the suppression of this signaling pathway holds great promise for designing new
targeted cancer therapy. Further biological studies indicated that the formation of β-catenin/Tcf
complex in the cell nucleus is the penultimate step of the Wnt/β-catenin signaling pathway and the
activation of Wnt/β-catenin target genes is dependent on the formation of this complex.19-21
Therefore, the β-catenin/Tcf PPI has emerged as an appealing therapeutic target to suppress
hyperactive β-catenin signaling.
Extensive efforts have been made to identify several small-molecule inhibitors for this PPI.22-
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However, the binding mode of these compounds remains unknown, making it difficult for
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further optimization. The large peptides or peptide-based macrocycles have also been designed
as β-catenin/Tcf inhibitors.31-34 Hydrocarbon-stapled peptide, aStAx-35, designed based on the
Axin sequence and the phage display result, was reported to bind with the Axin-binding site of β-
catenin and inhibit the β-catenin/Tcf PPI.31 Further design offered a more cell-permeable
derivative, NLS-StAx-h, by substituting all six arginine residues with homoarginine and
introducing the nuclear localization sequence (NLS) to the N-terminus.33 Recently, Logan,
Kirshenbaum, and coworkers disclosed the design of peptoid-peptide macrocycles as β-catenin/Tcf
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inhibitors, which showed promising efficacy in prostate cancer models. In the previous studies,
our group reported small-molecule inhibitors for the β-catenin/Tcf PPI using different strategies.35-
37
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By targeting the Tcf4 G ANDE binding site, selective small-molecule inhibitors for β-
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catenin/Tcf PPI have been synthesized. The best compound 1 (UU-T02) disrupted β-catenin/Tcf
PPI with a K of 1.36 µM in the fluorescence polarization (FP) competitive inhibition assay and
i
displayed 175- and 64-fold selectivity over β-catenin/E-cadherin and β-catenin/APC, respectively.
However, this compound was almost inactive in cell-based assays unless converted into the ester
form, including MTS tetrazolium cell viability assays and TOPFlash/FOPFlash luciferase reporter
assays. Herein, we report our further medicinal chemistry efforts on optimization, synthesis, and
biological characterization of new derivatives.
RESULTS
Structure-activity relationship (SAR) studies. The indole scaffold represents as one of the
38
important privileged structures for the discovery of new drug candidates. However, its electron-
rich nature renders the indole ring susceptible to metabolism, often by oxidation at the C-3 position
3
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of indole, which has the highest electron density. Most of the reported indole-related drugs and
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probes contain substituents at indole 3-postion to block this metabolically labile site.38-40
Therefore, to minimize the potential issue with indole in 1, three different strategies were
employed at the initial stage of our inhibitor optimization. As a direct approach, compounds 2 and
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with indole C-3 position substitution were designed and synthesized. Both compounds showed
a great loss of potency (Table 1). Alternatively, various electron-deficient heterocycles were
introduced to replace the indole ring (4–8). While most compounds (4–7) in this series turned out
to be inactive, compound 8 showed a K of 22 µM for the β-catenin/Tcf PPI. In addition, we
i
designed 9 with the indole ring directly attaching to the amide group. This design is expected to
reduce the electron density of the indole ring, thus increasing compound metabolic stability.
Literature search revealed that this 1H-indole-2-carboxamido motif had been widely adopted in
various medicinal chemistry programs.41-44 The FP competitive inhibition assay 45 showed 9
inhibited the β-catenin/Tcf PPI with a promising K of 11 µM. Based on these results, we
i
envisioned compound 9 would represent a new starting point for further modification, and
extensive SAR studies on this new scaffold were conducted.
Preliminary SAR studies on 9 suggested both a Cl substituent at indole C-5 position is optimal
among all mono-substitution analogues (Table 1, 9–17). Switching the Cl substituent from C-5
position to C-4 (10) or C-6 (11) led to decreased activity. Meanwhile, the derivatives in which the
Cl atom at the C-5 position was replaced by the other groups, including both electron withdrawing
(
F (12), Br (13), CF (16), NO (17)) and donating (Me (14), OMe (15)) groups, were also made.
3 2
The results revealed that the electron withdrawing groups were more tolerated than the electron
donating groups, while all of them were less potent than 9. Two compounds with di-Cl
substitutions (18 and 19) were synthesized, and both showed improved potency, especially 19,
which inhibited the β-catenin/Tcf PPI with a K of 4.3 µM, ~3-fold potency improvement over 9.
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Table 1. FP Competitive Inhibition Assay Results of Inhibitors 1–19.a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
4
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6
0
1
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9
0
1
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9
0
1
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9
0
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9
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No.
R
1
IC50 ± SD (µM)
K
i
± SD (µM) No.
R
1
IC50 ± SD (µM)
i
K ± SD (µM)
1
15 ± 5.9
3.8 ± 1.5
> 100
11
12
60 ± 2.2
15 ± 0.56
89 ± 8.6
2
3
4
5
6
>400
>400
>400
>400
>400
349 ± 34
62 ± 6.6
>400
> 100
>100
>100
>100
13
14
15
16
16 ± 1.7
>100
>400
>100
47 ± 4.1
12 ± 1.0
7
8
9
>400
>100
17
18
19
67 ± 6.0
38 ± 4.1
17 ± 1.4
17 ± 1.5
9.7 ± 1.0
4.3 ± 0.34
88 ± 12
45 ± 3.6
60 ± 5.1
22 ± 3.0
11 ± 0.92
15 ± 1.3
1
0
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Each set of data was expressed as mean ± standard deviation (n = 3). The K value for the parent
i
1
3
17
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peptide G ANDE was determined to be 290 ± 1.3 µM.
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Next, to increase the stability of the C-terminal ester group that tends to be hydrolyzed under
relatively weak basic condition (as shown in Supplementary Note 1), a collection of derivatives
with amides, instead of the methyl ester, was designed and synthesized. As shown in Table 2,
Introduction of isobutyl (20) and phenyl (21) amides in replacement of the methyl ester resulted
in the compounds with increased activities. For instance, compound 21 inhibited the β-catenin/Tcf
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PPI with a K of 3.3 µM, which is 3.4-fold more potent than 9. Moreover, these more stable amides
i
overcame the instability problem of the methyl ester in 1 (Supplementary Note 1). Further
substitution of the phenyl group in 21 with benzyl and phenylethyl groups as in 22 and 23,
respectively, showed decreased activities. Next, compounds 24–29 were designed and synthesized
to explore the optimal substitution type on the phenyl group. As a result, compounds 24–26 with
F substitution showed the similar activities, while 27 and 28 were 3- to 4-fold more potent than
29, indicating the para- and meta-substitutions on the phenyl ring are preferable to the ortho
derivatives. Therefore, more substituents were explored for these two positions. Compounds 33–
3
8 were synthesized, and the results were summarized in Table 2. It was shown that most of these
compounds displayed slight improvement of the inhibitory potency, when compared with 21.
Specifically, compounds 30 and 34, featuring para-CF and para-OCF on the phenyl ring,
3
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respectively, were slightly more potent than the other compounds (the FP competitive inhibition
assay curves of 30 and 34 are shown in Supplementary Figure S3). Compounds with double
substitutions (36–38) were also explored at these two positions, but they did not show potency
improvement.
Table 2. FP Competitive Inhibition Assay Results of Inhibitors 20–38. a
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3
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0
1
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9
0
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9
0
1
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0
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0
No.
R
2
IC50 ± SD (µM)
K
i
± SD (µM) No.
R
2
IC50 ± SD (µM)
i
K ± SD (µM)
2
2
2
0
1
2
33 ± 3.8
8.5 ± 0.98
3.3 ± 0.64
7.6 ± 0.94
30
31
32
7.3 ± 0.78
1.9 ± 0.20
2.3 ± 0.17
2.6 ± 0.37
13 ± 2.5
30 ± 3.7
8.9 ± 0.68
10 ± 1.4
2
2
3
4
35 ± 3.6
8.9 ± 0.91
33
9.3 ± 1.2
2.4 ± 0.31
17 ± 3.4
13 ± 1.5
4.5 ± 0.88
3.3 ± 0.37
34
35
7.5 ± 0.51
8.5 ± 0.60
1.9 ± 0.13
2.2 ± 0.15
25
2
2
6
7
14 ± 2.9
3.6 ± 0.74
36
13 ± 1.1
3.3 ± 0.26
11 ± 2.0
12 ± 2.0
38 ± 8.4
2.7 ± 0.50
3.1 ± 0.51
9.6 ± 2.1
37
38
10 ± 0.81
9.8 ± 3.3
2.6 ± 0.20
2.5 ± 0.83
28
2
9
a
Each set of data was expressed as mean ± standard deviation (n = 3). The K value for the parent
i
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3
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peptide G ANDE was determined to be 290 ± 1.3 µM.
Further SAR studies on the naphthyl group were undertaken (Table 3). It was shown that most
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of the synthesized compounds were less potent than 21. Only compounds 40 and 46 exhibited
comparable activity, indicating that the large hydrophobic moieties are critical for maintaining the
activity at this site.
0
1
2
3
4
5
6
7
8
9
0
1
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4
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9
0
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Table 3. FP Competitive Inhibition Assay Results of Inhibitors 39–46.a
No.
R
3
IC50 ± SD (µM)
K
i
± SD (µM) No.
R
3
IC50 ± SD (µM)
K
i
± SD (µM)
3
4
9
0
273 ± 37
70 ± 9.5
43
44
45
37 ± 4.5
9.3 ± 1.2
14 ± 2.3
44 ± 3.9
3.7 ± 0.58
11± 1.0
79 ± 6.8
99 ± 7.5
20± 1.7
25± 1.9
41
42
55 ± 3.5
14± 0.89
46
11 ± 1.8
2.9 ± 0.45
a
Each set of data is expressed as mean ± standard deviation (n = 3). The K value for the parent
i
1
3
17
36
peptide G ANDE was determined to be 290 ± 1.3 µM.
Our extensive SAR studies revealed that the primary configuration (S, S, S) was optimal for
this series, since individually reversing the configuration of each amino acid resulted in decreased
activity (Table 4). Base on the inhibitory activity data, we can also conclude that the sensitivity of
these three chiral amino acids to the configurational change was decreased following the order of
glutamic acid (47), naphthyl substituted amino acid (49), and aspartic acid (48), with glutamic acid
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being the most sensitive residue, when compared with those in 33.
Table 4. FP Competitive Inhibition Assay Results of Inhibitors 47-49. a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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3
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9
0
1
2
3
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9
0
No.
R,S
IC50 ± SD (µM)
i
K ± SD (µM)
4
7
R, S, S
S, R, S
S, S, R
39 ± 2.9
26 ± 1.6
31 ± 1.7
10 ± 0.73
6.6 ± 0.42
8.0 ± 0.42
48
49
a
Each set of data is expressed as mean ± standard deviation (n = 3). The K value for the parent
i
1
3
17
36
peptide G ANDE was determined to be 290 ± 1.3 µM.
Our next modification was focused on the two carboxylic acid groups, with the aim of
46
improving both potency and cell permeability. One common bioisostere of carboxylic acid,
amide, was first used to replace the carboxylic acids in 34 individually. The resulting compounds
50 and 51 showed decreased activities. To our delight, introduction of tetrazole, another common
bioisostere of carboxylic acid, resulted in the derivatives with the improved potency. For instance,
compound 53, for the first time, displayed the submicromolar K (0.64 µM) for this challenging
i
PPI target. In an attempt to eliminate both carboxylic acids in 34, the amide was further introduced
into the tetrazole derivatives 52 and 53. However, the resulting compounds 54 and 55 displayed
3
- to 6-fold potency loss. The ester derivative (56) of compound 53 was also synthesized to
improve compound’s cell permeability. This compound inhibited the β-catenin/Tcf PPI with a Ki
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of 5.2 µM. In addition, compound 57 was synthesized based on the previous SAR results. This
compound inhibited the β-catenin/Tcf PPI with a K of 0.44 µM, which was the most potent
i
inhibitor of this series. The competitive FP inhibition assay curves of 52–57 are shown in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Supplementary Figure S3.
Table 5. FP Competitive Inhibition Assay Results of Inhibitors 50-57. a
No.
R
4
R
5
IC50 ± SD (µM)
26 ± 2.3
i
K ± SD (µM)
5
5
0
1
CH
2
CONH
2
COOH
6.7 ± 0.60
10 ± 0.88
CH
2
COOH
CONH
2
42 ± 3.4
52
COOH
5.9 ± 0.27
2.5 ± 0.48
19 ± 2.3
1.5 ± 0.066
0.64 ± 0.12
4.9 ± 0.58
3.9 ± 0.16
5.2 ± 0.070
5
5
5
5
3
4
5
6
CH
2
COOH
CONH
2
CH
2
CONH
2
16 ± 0.62
21 ± 0.28
CH
2
COOEt
57
1.7 ± 0.39
0.44 ± 0.098
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O
O
OEt
O
O
(
Et)-15
H
>400
>100
N
N
N
H
H
O
O
HN
O
OMe
OEt
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Each set of data was expressed as mean ± standard deviation (n = 3). The K value for the parent
i
1
3
17
36
peptide G ANDE was determined to be 290 ± 1.3 µM.
Inhibitor selectivity between β-catenin/Tcf, β-catenin/E-cadherin, and β-catenin/APC PPIs.
β-Catenin not only interacts with Tcf/Lef, BCL9/B9L, CBP/p300, etc. to culminate Wnt/β-catenin
signaling, but also forms the complexes with E-cadherin and APC, respectively, to play specific
roles in cellulo. The PPI between β-catenin and E-cadherin is essential for cell–cell adhesion, while
the β-catenin/APC PPI is critical for β-catenin phosphorylation and degradation. The
crystallographic analyses of β-catenin in complexes with Tcf, E-cadherin, and APC indicated that
β-catenin uses the same armadillo repeats to bind Tcf/Lef47-50, cadherin , and APC52-54.
Biochemical analyses confirmed that the binding mode of Tcf, cadherin, and APC with β-catenin
was identical and mutually exclusive.55-58 The selectivities of 53 and 57 between β-catenin/Tcf4,
β-catenin/E-cadherin, and β-catenin/APC-R3 interactions were quantified using the FP selectivity
51
2
9
assay. As shown in Table 6, the selectivities of 53 for β-catenin/Tcf4 over β-catenin/E-cadherin
and β-catenin/APC-R3 interactions are 50- and 137-fold, respectively. The selectivities of 57 for
β-catenin/Tcf4 over β-catenin/E-cadherin and β-catenin/APC-R3 interactions are 30- and 395-fold.
Table 6. The Selectivities of 53 and 57 for β-Catenin/Tcf over β-Catenin/E-Cadherin and β-
Catenin/APC Interactions. a
K
i
± SD (μM)
Selectivity
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Compounds
β-catenin/Tcf
0.64 ± 0.12
0.44 ± 0.098
β-catenin/E-cadherin β-catenin/APC
TCF/E-cadherin
TCF/APC
137
5
5
3
7
32 ± 2.8
88 ± 7.3
173 ± 15
50
31
13 ± 0.60
395
a
Each set of data was expressed as mean ± standard deviation (n = 3).
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0
Cell-based studies. MTS tetrazolium cell viability assays were performed to evaluate the effect
of β-catenin/Tcf inhibitors on growth of different cancer cell lines with hyperactive Wnt signaling,
including colorectal cancer cells (SW480 and HCT116) and TNBC cells (MDA-MB-231, MDA-
MB-468, and BT-20), and one cancer cell line with normal Wnt signaling, A549. The half maximal
inhibitory concentrations (IC ) of three representative compounds (53, 55, and 56) were
5
0
determined. An inactive analogue (Et)-15 (K >100 μM, Table 5) was also assessed in parallel. As
i
shown in Table 7 and Supplementary Figure S4, compounds 53, 55, and 56 except (Et)-15
inhibited viability of Wnt/β-catenin hyperactive cancer cells. Compound 56 is the most potent and
selective inhibitor for most of the Wnt/β-catenin hyperactive cancer cells over Wnt/β-catenin
normal cancer cells.
Table 7. The MTS Assay to Monitor the Inhibitory Activities of (Et)-15, 53, 55, and 56 on
Viability of Cancer Cells. a
MTs IC50 ± SD (μM)
Wnt/β-catenin
Wnt/β-catenin hyperactive
normal
No.
SW480
>200
HCT116
>200
MDA-MB-231 MDA-MB-468
BT-20
>200
A549
> 200
(Et)-15
>200
>200
5
3
40.5 ± 1.78 45.8 ± 1.37
26.4 ± 0.74
20.6 ± 0.72
24.7 ± 1.00
71.7 ± 2.66
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24.6 ± 1.01 57.8 ± 3.75
17.7 ± 0.54 60.6 ± 4.93
29.3 ± 2.15
16.6 ± 1.16
26.9 ± 1.54
10.6 ± 0.30
29.3 ± 2.20
20.1 ± 0.52
47.5 ± 2.63
67.9 ± 2.09
a
Each set of data was expressed as mean ± standard deviation (n = 3).
1
1
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The suppressing effect of the inhibitors on transactivation of β-catenin signaling was evaluated
by TOPFlash/FOPFlash luciferase reporter assays. As shown in Figure 1, the inactive compound
Et)-15 did not inhibit the TOPFlash (in which the luciferase reporter has three wild-type Tcf4
(
binding sites) luciferase activity at the concentration of up to 200 μM. Compounds 55 and 56
suppressed the TOPFlash luciferase activities in dose-dependent manners, while did not inhibit the
FOPFlash (with three mutant Tcf4 binding sites) luciferase reporter activities. However, it was
noted that 56 did not show the sigmoidal curve at high concentrations. The cause of this result
warrants further studies.
Figure 1. Wnt-responsive TOPFlash and FOPFlash luciferase reporter assay results of (Et)-15
(
negative control) and inhibitors 55 and 56 in β-catenin activated HEK293 cells. *P< 0.05, ** P
< 0.01, as determined by the unpaired, two-tailed Student t test.
Co-immunoprecipitation (co-IP) experiments were conducted to assess the effect of the
inhibitors for disruption of the interaction between β-catenin and Tcf using SW480 cell lysates.
The results were shown in Figure 2 and Supplementary Figure S5. Compound 53 can disrupt the
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interaction between full-length β-catenin and full-length Tcf in a dose-dependent manner after 4-
h incubation with SW480 cell lysates. The effect of 56 on disruption of the β-catenin/Tcf PPI and
on the selectivity between three PPIs in the cellular context were also evaluated by co-IP
experiments using HCT116 cells. As shown in Figure 2 and Supplementary Figure S5, compound
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0
5
6 dose-dependently inhibited the β-catenin/Tcf PPI, but had no effect on the β-catenin/E-cadherin
and β-catenin/APC PPIs at the concentrations that were sufficient to disrupt the β-catenin/Tcf PPI.
Figure 2. (A) Co-IP experiments to evaluate the disruption of the β-catenin/TCF PPI by 53 in
SW480 cell lysate. (B–D) Co-IP experiments to evaluate the disruption of the β-catenin/Tcf PPI
by 56 and the inhibitory selectivity of 56 between β-catenin/Tcf, β-catenin/E-cadherin, and β-
catenin/APC PPIs using HCT116 cells. IP, immunoprecipitation; IB, immunoblotting; input, 10%
of cell lysate. Each experiment was performed in duplicate.
β-Catenin signaling induces and maintains migration, invasion and metastasis of cancer cells,
including TNBC cells.59-70 Scratch wound healing and Matrigel invasion assays using TNBC
MDA-MB-231 cells were conducted. As shown in Figure 3, compound 56 can effectively inhibit
TNBC cell migration (Figure 3A) and invasion (Figure 3B) at 10 µM. The effects are comparable
7
1
with that of β-catenin/CBP inhibitor ICG-001 at 5 µM.
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Figure 3. (A) Wound-healing assays showed that 56 (10 µM) inhibited migration of human TNBC
MDA-MB-231 cells induced by serum (10% in media). Control, 0.2% DMSO in 10% FBS. The
β-catenin/CBP inhibitor ICG-001 (5 µM) was assessed in parallel. In all experiments, mitomycin
(10 µg/mL) was added to inhibit cell proliferation and allow examination of the effects on cell
migration. (B) Matrigel invasion assays showed that 56 (10 M) inhibited invasion of human
TNBC MDA-MB-231 cells. Control, 0.2% DMSO in 10% FBS. ICG-001 (5 M) was assessed in
parallel. **P < 0.01, as determined by the unpaired, two-tailed Student t test. Each set of data is
expressed as mean ± standard deviation (n = 3).
Chemistry. The synthetic routes for final products 20–21, 30–38, 53, and 55–57 are shown in
Schemes 1–3. The synthetic routes for the other final products and intermediates are shown in
Supplementary Schemes S1–S14. The synthetic routes for 20–21 and 30–38 are shown in Scheme
1
, in which CH Cl was employed as the solvent for the amide coupling reactions. The amide bond
2 2
coupling reaction between N-Cbz-L-glutamic acid 5-tert-butyl ester and various amines generated
intermediate 62, which underwent the hydrogenation reaction to remove the Cbz protecting group
and then coupled with N-Cbz-L-aspartic acid to yield 63. Hydrogenation of 63 and coupling with
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1
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1
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N-Cbz-L-2-naphthylalanine produced 64. Removal of the Cbz protecting group in 64 and then
coupling with 5-chloroindole-2-carboxylic acid gave 65, in which the tert-butyl ester protecting
group was removed by TFA in CH Cl solution to offer the final products.
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1. Synthesis of 20–21 and 30–38.
t
t
t
O
O Bu
O
O Bu
1. H , Pd/C, MeOH, rt
O
O Bu
t
2
1. H2, Pd/C, MeOH, rt
2.
O
O Bu
R2 NH2
HOAt, EDC•HCl
O
2.
O
H
N
NHCbz
H
N
O
Et3N, CH2Cl2
HO
H
N
H
NHCbz
HO
N
0
°C to rt
R2
NHCbz
R2
N
H
O
NHCbz
R2
N
H
O
NHCbz
O
O
79–87%
O
O
O
O
t
O Bu
62
63
t
HO
O Bu
NHCbz
64
t
EDC•HCl, HOAt
O Bu
Et3N, CH2Cl2, 0 °C to rt
O
82–88% (two steps)
EDC•HCl, HOAt
Et3N, CH2Cl2, 0 °C to rt
74–85% (two steps)
1. H2, Pd/C, MeOH, rt
2.
Cl
O
OH
O
OtBu
O
TFA, CH2Cl2, 0 °C
~99%
O
O
O
O
H
N
H
H
N
H
HO
N
N
N
H
R2
N
H
O
N
R2
N
H
O
N
H
H
EDC•HCl, HOAt, Et3N
CH2Cl2, 0 °C to rt
O
O
HN
O
O
HN
Cl
Cl
70–89% (two steps)
OtBu
OH
65
20–21, 30–38
F3C
O
R2 =
,
,
,
,
,
,
F3C
O
20
21
30
31
32
33
O
O
F3C
O
O
F3C
F3C
,
,
,
O
,
34
35
36 O
37
O
38
The synthetic routes for 53, 56, and 57 are shown in Scheme 2, in which DMF was used as the
solvent for all the amide coupling reactions. The amide bond coupling reactions between N-Cbz-
L-glutamic acid 5-tert-butyl ester or N-Cbz-L-glutamic acid 5-ethyl ester and various amines
produced intermediate 62g or 90, which underwent the hydrogenation reaction to remove the Cbz
protecting group and then the coupling reaction with (S)-2-((((9H-fluoren-9-
yl)methoxy)carbonyl)amino)-3-(2H-tetrazol-5-yl)propanoic acid to yield 91a,b. Removal of the
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Fmoc protecting group under the basic condition and then coupling with 68a,h gave 92, in which
the Boc (and tert-butyl) protecting group(s) was removed by TFA in CH Cl solution to offer the
2
2
final products.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2. Synthesis of 53, 56, and 57.
O
OR4
O
OR4
°
O
OEt
1. Et NH/DCM, 0 C
1
. Pd/C, H2, MeOH, rt
2
H2N
OCF3
O
2.
2
.
O
N
HOAt, EDC•HCl
DMF, 0 °C to rt
76-82%
H
N
NHFmoc
H
N
HO
N
NHFmoc
HO
NHCbz
N
H
N
68a or 68h
NHCbz
O
O
O
O
HN
F3CO
3
F CO
N
HN
HO
N
N
6
9
2g, R4 = t-Bu
0, R4 = Et
EDC•HCl, HOAt
Et3N, DMF, 0 °C to rt
60-69% (two steps)
N
H
O
BocN
91a, R4 = t-Bu
91b, R4 = Et
EDC•HCl, HOAt, Et3N
DMF, 0 °C to rt
R6
5
3-58%
O
OR4
O
OR5
(two steps)
O
N
O
O
N
O
H
N
H
N
N
53, R5 = H, R6 = 5-Cl
56, R5 = Et, R6 = 5-Cl
57, R5 = H, R6 = 4,6-diCl
H
N
H
TFA, CH2Cl2, 0 °C
7-83%
N
N
H
N
N
N
H
H
H
O
6
O
HN
O
HN
O
BocN
N
F3CO
F3CO
R6
HN
R6
N
92
N
The synthetic route for 55 is shown in Scheme 3. The amide bond coupling reaction between
N-Cbz-L-glutamine and 4-(trifluoromethoxy) aniline and then the deprotection of the Cbz
protecting group produced 95, which was coupled with (S)-2-((((9H-fluoren-9-
yl)methoxy)carbonyl)amino)-3-(2H-tetrazol-5-yl)propanoic acid to afford 96. Removal of the
Fmoc group in 96 and coupling with intermediate 86a gave 97. The Boc deprotection yielded final
product 55.
Scheme 3. Synthesis of 55.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
1
. Et2NH/DCM, rt, 62%
1
.
NHFmoc
O
NH2
H2N
OCF3
HO
NH2
N
2.
O
NH2
O
HOAt, EDC•HCl
DMF, 0 C-rt
5%
O
N
HN
H
N
86a

N
N
NHFmoc
N
H
N
O
7
H
N
HO
O
HO
NHCbz
NH2
EDC•HCl, HOAt F3CO
Et3N, DMF, 0 °C-rt
71%
N
2. Pd/C, H2, MeOH, rt
F3CO
HN
O
O
H
O
BocN
N
Cl
95
96
EDC•HCl, HOAt, Et3N
DMF, 0 °C to rt
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
65%
O
NH2
O
NH2
O
N
O
O
N
O
H
H
N
TFA, CH2Cl2, 0 °C
4%
H
N
H
N
N
N
H
N
N
N
H
N
N
H
9
H
O
HN
O
BocN
O
O
HN
Cl
Cl
F3CO
F3CO
HN
N
N
97
55
DISCUSSION AND CONCLUSIONS
The β-catenin signaling pathway is frequently overactivated in many cancers and fibroses. The
β-catenin/Tcf PPI represents an appealing therapeutic target to suppress this signaling pathway,
since the transcriptional overactivation of β-catenin signaling is dependent on the formation of the
key downstream effector, the β-catenin/Tcf complex, in the cell nucleus. However, selective
targeting of the β-catenin/Tcf PPI remains a great challenge. On one side, β-catenin and Tcf have
a large contacting surface area and the tight binding affinity (K = 7−10 nM), which makes it
d
challenging to achieve potent inhibition. One the other side, β-catenin uses the same armadillo
repeats to bind Tcf, cadherin, and APC, and the crystallographic studies show their binding mode
is identical, indicating the selectivity would be a major issue when designing β-catenin/Tcf
inhibitors. In this work, the SAR studies on compound 1 has yielded inhibitors (compounds 53 and
57) with improved activities for the β-catenin/Tcf PPI in this series. They also showed good
selectivity for β-catenin/Tcf over β-catenin/E-cadherin and β-catenin/APC PPIs in the FP
selectivity assay.
The structure–activity relationship (SAR) is to study the correlation between the chemical
structures of a series of derivatives and their biological activities. The SAR study has been widely
1
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used to facilitate step-by-step inhibitor optimization. Herein, extensive SAR studies on the
structure of 1 were conducted. It not only yielded more potent inhibitors for the β-catenin/Tcf PPI,
the SAR results obtained can also guide the future inhibitor optimization. This is quite important
given that no co-crystal structure of a small-molecule inhibitor with β-catenin has been reported.
For instance, the study revealed the naphthyl group of 1 can only be substituted by the large
hydrophobic groups. This gives us two directions to further investigate this site. One is to introduce
larger moieties to examine whether the compound can further improve potency and selectivity.
The other is to adopt nitrogen-containing benzoheterocyclic rings, which are large and
hydrophobic enough, and at the same time might capture potential polar interactions with the
protein and balance the physiochemical property of the inhibitors. This study revealed that the
indole moiety was crucial for inhibitor potency. The feasible method to further optimize this
substructure is to extensively modify the indole ring and the adjacent methylene group that
connects indole with the amide group. In addition, the SAR results confirmed the importance of
the two carboxylic acid groups, but they can be replaced by tetrazole, one of its bioisosteres, to
improve the potency. This prompts us to try the other bioisosteres of carboxylic acid in the future
optimization.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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7
8
9
0
1
2
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4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
The prodrug strategy was designed to address undesirable physicochemical properties of the
7
2
drug and has been widely applied in contemporary drug design and development. In this work,
we were interested whether this strategy could be applied to compound 53 to improve its cellular
uptake, since 53 has a carboxylic acid moiety, which could impair its cell permeability. Based on
this hypothesis, compound 56 was synthesized. The preliminary cell-based studies indicated that
5
6 was indeed more potent and selective than 53 in inhibiting growth of most cancer cells with
hyperactive Wnt/β-catenin signaling. Interestingly, the HPLC-MS based cell bioavailability
1
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2
2
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2
2
2
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assay revealed that compound 56 did not transform into 53 after 24-h incubation with SW480
cells in 5 mL of DMEM media with 10% FBS, suggesting 56 might not act as a prodrug of 53
(Supplementary Note 2 and Supplementary Figures S6–S8). Further analyses revealed compound
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
6 did achieve a higher cell-bound concentration than 53. For instance, the cell-bound
concentration of 56 at 37 °C was determined to be 1.4 nmol/million SW480 cells, which is 8-fold
higher than 0.17 nmol/million SW480 cells determined for 53, for the 3-h incubation in 5 mL of
DMEM media with 10% FBS, when the input concentration was set to 25 μM. The increased cell-
bound concentration might explain why 56 exhibited the better cellular activity than 53, although
it showed the higher K value in FP assays. Further cell-based co-IP experiments indicated that
i
compound 56 disrupted the β-catenin/Tcf PPI while leaving the β-catenin/E-cadherin and β-
catenin/APC PPIs unaffected, demonstrating the selectivity of new inhibitors on the cell-based
level. This compound also dose-dependently inhibited TOPFlash luciferase reporter activity
without affecting FOPFlash luciferase reporter activity. In contrast, the negative control (Et)-15
did not suppress Wnt specific TOPFlash luciferase reporter activity and did not inhibit growth of
the cancer cells with hyperactive Wnt/β-catenin signaling.
In summary, the β-catenin/Tcf PPI has emerged as an appealing therapeutic target to suppress
the overactivated β-catenin signaling for targeted cancer therapy. Extensive SAR studies were
1
3
17
conducted based on compound 1, which was designed to target the Tcf4 G ANDE binding site
of β-catenin. Compounds 53 and 57 were found to disrupt the β-catenin/Tcf PPI with the K of 0.64
i
and 0.44 μM, respectively, and showed good selectivity for β-catenin/Tcf over β-catenin/E-
cadherin and β-catenin/APC PPIs. Cell-based studies indicated that compound 56, the ethyl ester
derivative of 53, disrupted the β-catenin/Tcf interaction without affecting the β-catenin/E-cadherin
and β-catenin/APC interactions, dose-dependently suppressed transactivation of Wnt/β-catenin
2
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signaling, and inhibited viability, migration and invasiveness of Wnt/β-catenin dependent cancer
cells. The extensive SAR results offered the directions for future inhibitor optimization.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL SECTION
Chemical Synthesis. General Methods, Reagents, and Materials. All reagents were purchased
1
from commercial sources and used without further purification unless stated otherwise. H NMR
1
3
and C NMR spectra were recorded on Bruker AVANCEIIIHD 500 (500 MHz) spectrometers
1
3
(
125.7 MHz for C NMR spectra) in d -DMSO, d -acetone, d -methanol, and CDCl . Chemical
6 6 4 3
shifts were reported as values in parts per million (ppm), and the reference resonance peaks were
set at 7.26 ppm (CHCl ), 3.31 ppm (CD HOD), 2.50 ppm [(CD H) SO], 2.05 ppm [(CD H) CO]
3
2
2
2
2
2
1
for H NMR spectra and 77.23 ppm (CDCl ), 49.00 ppm (CD OD), 39.52 ppm (d -DMSO), and
3
3
6
1
3
2
9.84 ppm (d -acetone) for C NMR spectra. Low-resolution mass spectra were determined on an
6
Agilent 6120 single quadrupole mass spectrometer with 1220 infinity LC system (HPLC-MS) and
an ESI source. High-resolution mass spectra were determined on Agilent G6230BA TOF LCMS
Mass Spectrometer with a TOF mass detector. Thin-layer chromatography was carried out on E.
Merck pre-coated silica gel 60 F254 plates with a UV–visible lamp. Column chromatography was
@
performed with SilicaFlash F60 (230–400 mesh). The purity of final compounds 2–57 was
determined by HPLC analyses with two different conditions (see the Supporting Information).
The instrument was an Agilent 1260 Infinity II HPLC system with a quaternary pump, a vial
sampler, and a DAD detector. A Kromasil 300–5–C18 column (4.6 × 250 mm) was used. The
DAD detector was set to 220, 254, and 280 nm. The purity of all tested compounds was >95%.
°
General peptide coupling procedure. a. CH Cl as the solvent: At 0 C, to a suspension of
2
2
carboxylic acid (1 equiv), amine (1 equiv), EDC•HCl (2 equiv), and HOAt (1.5 equiv) in
2
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2
2
2
2
3
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6
dichloromethane (CH Cl ) was added triethylamine (3 equiv) dropwise. The reaction mixture was
2
2
warmed to room temperature and stirred overnight. After completion of the reaction, more CH Cl
2
2
was added. The CH Cl phase was washed by 1 M HCl, saturated NaHCO , and brine, and was
2
2
3
0
1
2
3
4
5
6
7
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9
0
1
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3
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9
0
1
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8
9
0
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4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
dried over Na SO and concentrated under reduced pressure. Column chromatography was used
2
4
°
to purify the target compound. b. DMF as the solvent: At 0 C, to a suspension of carboxylic acid
(
1 equiv), amine (1 equiv) in dimethylformamide (DMF) was added EDC•HCl (2 equiv) and HOAt
2 equiv). The reaction mixture was warmed to room temperature and stirred overnight. The
(
mixture was poured into water and the solid was collected. The pure compounds were obtained by
recrystallization using the hexane and ethyl acetate mixture or CH Cl .
2
2
General procedure for deprotection of the Cbz-protected amines. To the solution of the Cbz-
protected amine in methanol was added 10% Pd/C under Ar. The mixture was stirred overnight at
room temperature under H . The resulting product was collected by removal of the Pd/C catalyst
2
and used directly in next step without further purification.
°
General procedure for deprotection of tert-butyl ester or Boc-protected indoles. At 0 C, to a
solution of tert-butyl ester or Boc-protected indole in CH Cl (5 mL) was added 5 mL
2
2
°
trifluoroacetic acid (TFA) dropwise. The reaction was kept at 0 C for 4 h. Upon completion, the
solvent was removed under reduced pressure. TFA was completely removed by adding CH Cl
2
2
three times to afford the desired product.
°
General procedure for deprotection of Fmoc-protected amines. At 0 C, to a stirred solution of
the Fmoc protected amine (1 mmol) in dichloromethane (10 mL), diethylamine (10 mL) was added
°
dropwise. The reaction was kept at 0 C for 6 h until TLC showed no starting material left. Upon
completion, the mixture was evaporated under reduced pressure. The diethyl amine residue was
removed by adding dichloromethane at least three times. The residue was purified by flash column,
2
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except Asn and Gln-containing compounds, which were recrystallized in dichloromethane.
(S)-4-((S)-3-carboxy-2-((S)-2-(5-chloro-1H-indole-2-carboxamido)-3-(naphthalen-2-
1
yl)propanamido)propanamido)-5-(isopentylamino)-5-oxopentanoic acid (20). H NMR (500
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MHz, DMSO-d ) δ 12.27 (s, 2H), 11.63 (d, J = 2.2 Hz, 1H), 8.80 (d, J = 8.5 Hz, 1H), 8.66 (d, J =
6
7.6 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 1.6 Hz, 1H), 7.83 – 7.73 (m, 4H), 7.70 (d, J =
2.1 Hz, 1H), 7.56 (dd, J = 8.5, 1.7 Hz, 1H), 7.42 (pd, J = 6.8, 1.6 Hz, 2H), 7.36 (d, J = 8.7 Hz, 1H),
7.24 – 7.19 (m, 1H), 7.14 (dd, J = 8.7, 2.1 Hz, 1H), 4.99 – 4.78 (m, 1H), 4.62 (q, J = 7.2 Hz, 1H),
4.21 (td, J = 8.3, 5.0 Hz, 1H), 3.27 (d, J = 3.6 Hz, 1H), 3.20 – 2.96 (m, 3H), 2.78 (dd, J = 16.6, 6.1
Hz, 1H), 2.60 (dd, J = 16.7, 7.4 Hz, 1H), 2.24 (dd, J = 9.6, 7.0 Hz, 2H), 2.05 – 1.89 (m, 1H), 1.84
– 1.69 (m, 1H), 1.55 (dt, J = 13.4, 6.7 Hz, 1H), 1.29 (q, J = 7.2 Hz, 2H), 0.85 (d, J = 6.6 Hz, 6H).
13
C NMR (126 MHz, DMSO-d ) δ 174.4, 172.4, 172.0, 170.9, 170.7, 161.0, 136.5, 135.2, 133.4,
6
1
33.1, 132.2, 128.5, 128.3, 127.91, 127.90, 127.84, 127.80, 126.4, 125.8, 124.6, 123.9, 121.1,
114.3, 103.3, 54.8, 52.5, 50.2, 38.4, 37.9, 37.3, 36.4, 30.5, 27.9, 25.5, 22.83, 22.80. HRMS (ESI)
–
Calcd for C H ClN O (M – H) 704.2487, found 704.2492. HPLC purity 95.6%, t = 14.01 min
3
6
40
5
8
R
(
condition A2); 96.6%, t = 16.83 min (condition B2).
R
(S)-4-((S)-3-carboxy-2-((S)-2-(5-chloro-1H-indole-2-carboxamido)-3-(naphthalen-2-
1
yl)propanamido)propanamido)-5-oxo-5-(phenylamino)pentanoic acid (21). H NMR (500
MHz, DMSO-d ) δ 12.30 (s, 2H), 11.63 (d, J = 2.3 Hz, 1H), 9.86 (s, 1H), 8.81 (d, J = 8.5 Hz, 1H),
6
8
.67 (d, J = 7.6 Hz, 1H), 8.13 (d, J = 7.8 Hz, 1H), 7.85 (d, J = 1.6 Hz, 1H), 7.83 – 7.75 (m, 3H),
7.70 (d, J = 2.1 Hz, 1H), 7.62 (d, J = 1.4 Hz, 1H), 7.60 (d, J = 1.2 Hz, 1H), 7.56 (dd, J = 8.5, 1.7
Hz, 1H), 7.48 – 7.39 (m, 2H), 7.36 (d, J = 8.7 Hz, 1H), 7.33 – 7.27 (m, 2H), 7.21 (d, J = 2.0 Hz,
1
H), 7.15 (dd, J = 8.7, 2.1 Hz, 1H), 7.10 – 7.03 (m, 1H), 5.12 – 4.82 (m, 1H), 4.67 (td, J = 7.5, 6.0
Hz, 1H), 4.43 (td, J = 8.2, 5.0 Hz, 1H), 3.31 (d, J = 8.8 Hz, 1H), 3.16 (dd, J = 13.9, 11.0 Hz, 1H),
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1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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6
2
2
.81 (dd, J = 16.7, 6.0 Hz, 1H), 2.63 (dd, J = 16.6, 7.6 Hz, 1H), 2.32 (td, J = 10.2, 6.2 Hz, 2H),
1
3
.05 (ddt, J = 15.0, 9.9, 5.7 Hz, 1H), 1.89 (ddt, J = 13.6, 8.7, 4.7 Hz, 1H). C NMR (126 MHz,
DMSO-d ) δ 174.3, 172.4, 172.1, 171.1, 170.3, 161.1, 139.1, 136.5, 135.2, 133.4, 133.1, 132.2,
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
29.2, 128.5, 128.3, 127.9, 127.89, 127.85, 127.7, 126.4, 125.8, 124.6, 123.99, 123.92, 121.1,
19.9, 114.3, 103.3, 54.8, 53.3, 50.2, 38.0, 36.4, 30.6, 27.8. HRMS (ESI) Calcd for C H ClN O
8
3
7
34
5
–
(M – H) 710.2018, found 710.2024. HPLC purity 97.9%, t = 13.98 min (condition A2); 98.8%,
R
tR = 16.59 min (condition B2).
S)-4-((S)-3-carboxy-2-((S)-2-(5-chloro-1H-indole-2-carboxamido)-3-(naphthalen-2-
(
yl)propanamido)propanamido)-5-oxo-5-((4-(trifluoromethyl)phenyl)amino)pentanoic acid
1
(30). H NMR (500 MHz, DMSO-d ) δ 12.31 (s, 2H), 11.64 (s, 1H), 10.24 (s, 1H), 8.82 (d, J = 8.5
6
Hz, 1H), 8.67 (d, J = 7.6 Hz, 1H), 8.23 (d, J = 7.5 Hz, 1H), 7.98 – 7.74 (m, 6H), 7.74 – 7.59 (m,
3
H), 7.56 (d, J = 8.4 Hz, 1H), 7.48 – 7.30 (m, 3H), 7.21 (s, 1H), 7.14 (d, J = 8.8 Hz, 1H), 4.90 (d,
J = 8.1 Hz, 1H), 4.66 (q, J = 7.1 Hz, 1H), 4.42 (q, J = 7.1 Hz, 1H), 3.44 – 3.22 (m, 1H), 3.16 (t, J
12.5 Hz, 1H), 2.80 (dd, J = 16.8, 5.9 Hz, 1H), 2.62 (dd, J = 16.6, 7.6 Hz, 1H), 2.33 (td, J = 10.2,
=
1
3
6
1
1
3
.3 Hz, 2H), 2.11 – 2.03 (m, 1H), 1.95 – 1.80 (m, 1H). C NMR (126 MHz, DMSO-d ) δ 174.3,
6
72.4, 172.0, 171.2, 171.0, 161.1, 142.7, 136.5, 135.2, 133.4, 133.1, 132.2, 128.4, 128.3, 127.9,
27.7, 126.45, 126.40, 125.8, 124.6, 123.9, 121.1, 119.8, 114.3, 103.3, 54.8, 53.6, 50.2, 38.0, 36.5,
–
0.6, 27.5. HRMS (ESI) Calcd for C H ClF N O (M – H) 778.1892, found 778.1894. HPLC
3
8
33
3
5
8
purity 95.1%, t = 14.58 min (condition A2); 97.5%, t = 17.13 min (condition B2).
R
R
(S)-4-((S)-3-carboxy-2-((S)-2-(5-chloro-1H-indole-2-carboxamido)-3-(naphthalen-2-
yl)propanamido)propanamido)-5-oxo-5-((3-(trifluoromethyl)phenyl)amino)pentanoic acid
1
(31). H NMR (500 MHz, DMSO-d ) δ 12.32 (s, 2H), 11.63 (s, 1H), 10.23 (s, 1H), 8.81 (d, J = 8.6
6
Hz, 1H), 8.66 (d, J = 7.6 Hz, 1H), 8.19 (d, J = 7.6 Hz, 1H), 8.12 (s, 1H), 7.93 – 7.73 (m, 5H), 7.70
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(
s, 1H), 7.55 (t, J = 7.7 Hz, 2H), 7.47 – 7.33 (m, 4H), 7.21 (s, 1H), 7.15 (d, J = 8.8 Hz, 1H), 4.97
–
4.86 (m, 1H), 4.75 – 4.59 (m, 1H), 4.42 (t, J = 7.0 Hz, 1H), 3.40-3.31 (m, 1H), 3.16 (t, J = 12.5
Hz, 1H), 2.80 (dd, J = 16.8, 5.8 Hz, 1H), 2.63 (dd, J = 16.7, 7.8 Hz, 1H), 2.34 (dt, J = 16.2, 7.7
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
Hz, 2H), 2.15 – 2.03 (m, 1H), 1.95 – 1.76 (m, 1H). C NMR (126 MHz, DMSO-d ) δ 174.3, 172.3,
6
1
72.1, 171.2, 170.9, 161.1, 139.9, 136.5, 135.2, 133.4, 133.1, 132.2, 130.4, 128.5, 128.3, 127.9,
27.8, 127.7, 126.4, 125.8, 124.6, 123.9, 123.5, 121.1, 120.3, 115.9, 114.3, 103.3, 54.8, 53.5, 50.2,
1
–
3
8.0, 36.5, 30.5, 27.5. HRMS (ESI) Calcd for C H ClF N O (M – H) 778.1892, found
3
8
33
3
5
8
7
78.1898. HPLC purity 96.5%, t = 14.56 min (condition A2); 96.5%, t = 17.11 min (condition
R R
B2).
(S)-4-((S)-3-carboxy-2-((S)-2-(5-chloro-1H-indole-2-carboxamido)-3-(naphthalen-2-
1
yl)propanamido)propanamido)-5-((4-methoxyphenyl)amino)-5-oxopentanoic acid (32). H
NMR (500 MHz, DMSO-d ) δ 12.31 (s, 2H), 11.63 (d, J = 2.2 Hz, 1H), 9.72 (s, 1H), 8.81 (d, J =
6
8.5 Hz, 1H), 8.69 (d, J = 7.5 Hz, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.93 – 7.73 (m, 4H), 7.70 (d, J =
2
8
4
3
2
.1 Hz, 1H), 7.56 (dd, J = 8.5, 1.7 Hz, 1H), 7.54 – 7.48 (m, 2H), 7.47 – 7.39 (m, 2H), 7.36 (d, J =
.7 Hz, 1H), 7.21 (d, J = 2.2 Hz, 1H), 7.15 (dd, J = 8.7, 2.1 Hz, 1H), 6.91 – 6.81 (m, 2H), 4.93 –
.82 (m, 1H), 4.66 (q, J = 7.1 Hz, 1H), 4.40 (td, J = 8.3, 5.1 Hz, 1H), 3.71 (s, 3H), 3.30 (s, 1H),
.16 (dd, J = 13.9, 11.0 Hz, 1H), 2.81 (dd, J = 16.6, 6.0 Hz, 1H), 2.63 (dd, J = 16.6, 7.6 Hz, 1H),
1
3
.31 (dq, J = 16.6, 10.5 Hz, 2H), 2.11 – 1.99 (m, 1H), 1.87 (dq, J = 10.2, 5.3, 4.2 Hz, 1H). C
NMR (126 MHz, DMSO-d ) δ 174.3, 172.4, 172.1, 171.0, 169.7, 161.1, 155.9, 136.5, 135.2, 133.4,
6
133.1, 132.23, 132.20, 128.5, 128.3, 127.9, 127.89, 127.85, 127.7, 126.4, 125.8, 124.6, 123.9,
1
21.5, 121.1, 114.3, 103.3, 55.6, 54.8, 53.2, 50.2, 38.0, 36.4, 30.5, 27.9. HRMS (ESI) Calcd for
–
C H ClN O (M – H) 740.2123, found 740.2128. HPLC purity 96.1%, t = 13.87 min
3
8
36
5
9
R
(condition A2); 95.8%, t = 16.47 min (condition B2).
R
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(
S)-4-((S)-3-carboxy-2-((S)-2-(5-chloro-1H-indole-2-carboxamido)-3-(naphthalen-2-
1
yl)propanamido)propanamido)-5-((3-methoxyphenyl)amino)-5-oxopentanoic acid (33). H
NMR (500 MHz, DMSO-d ) δ 12.31 (s, 2H), 11.63 (d, J = 2.2 Hz, 1H), 9.86 (s, 1H), 8.81 (d, J =
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
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8
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
2
7
4
.5 Hz, 1H), 8.68 (d, J = 7.5 Hz, 1H), 8.11 (d, J = 7.8 Hz, 1H), 7.94 – 7.73 (m, 4H), 7.70 (d, J =
.1 Hz, 1H), 7.56 (dd, J = 8.4, 1.7 Hz, 1H), 7.42 (pd, J = 6.9, 1.6 Hz, 2H), 7.37 (d, J = 8.7 Hz, 1H),
.31 (t, J = 2.2 Hz, 1H), 7.25 – 7.18 (m, 2H), 7.17 – 7.10 (m, 2H), 6.65 (dd, J = 8.2, 2.5 Hz, 1H),
.91 (ddd, J = 11.8, 8.6, 3.7 Hz, 1H), 4.67 (q, J = 7.1 Hz, 1H), 4.41 (td, J = 8.3, 5.1 Hz, 1H), 3.71
(s, 3H), 3.32 (s, 1H), 3.17 (dd, J = 13.9, 11.0 Hz, 1H), 2.81 (dd, J = 16.7, 5.9 Hz, 1H), 2.63 (dd, J
=
16.7, 7.7 Hz, 1H), 2.31 (dq, J = 16.6, 10.4 Hz, 2H), 2.14 – 1.91 (m, 1H), 1.88 (dq, J = 10.3, 5.3,
1
3
4.3 Hz, 1H). C NMR (126 MHz, DMSO-d ) δ 174.3, 172.3, 172.1, 171.1, 170.3, 161.1, 159.9,
6
1
1
40.3, 136.5, 135.2, 133.4, 133.1, 132.2, 130.0, 128.5, 128.3, 127.9, 127.8, 127.7, 126.4, 125.8,
24.6, 123.9, 121.1, 114.3, 112.2, 109.5, 105.7, 103.3, 55.4, 54.9, 53.4, 50.2, 38.0, 36.4, 30.5, 27.8.
–
HRMS (ESI) Calcd for C H ClN O (M – H) 740.2123, found 740.2127. HPLC purity 95.3%,
3
8
36
5
9
t = 14.00 min (condition A2); 97.0%, t = 16.60 min (condition B2).
R
R
(
S)-4-((S)-3-carboxy-2-((S)-2-(5-chloro-1H-indole-2-carboxamido)-3-(naphthalen-2-
yl)propanamido)propanamido)-5-oxo-5-((4-(trifluoromethoxy)phenyl)amino)pentanoic
1
acid (34). H NMR (500 MHz, DMSO-d ) δ 12.30 (s, 2H), 11.64 (s, 1H), 10.07 (s, 1H), 8.82 (d, J
6
=
8.5 Hz, 1H), 8.67 (d, J = 7.5 Hz, 1H), 8.19 (d, J = 7.7 Hz, 1H), 7.97 – 7.65 (m, 7H), 7.56 (d, J =
.4 Hz, 1H), 7.50 – 7.26 (m, 5H), 7.23 – 7.01 (m, 2H), 4.91 (ddd, J = 11.9, 8.4, 3.7 Hz, 1H), 4.66
(q, J = 7.1 Hz, 1H), 4.40 (td, J = 8.1, 5.0 Hz, 1H), 3.31 (s, 1H), 3.16 (dd, J = 13.9, 10.9 Hz, 1H),
8
2
2
.80 (dd, J = 16.7, 6.0 Hz, 1H), 2.62 (dd, J = 16.6, 7.5 Hz, 1H), 2.32 (pd, J = 10.1, 3.2 Hz, 2H),
1
3
.05 (ddt, J = 15.0, 11.1, 5.8 Hz, 1H), 1.89 (dt, J = 14.5, 4.3 Hz, 1H). C NMR (126 MHz, DMSO-
d6) δ 174.3, 172.4, 172.1, 171.2, 170.5, 161.1, 138.3, 136.5, 135.2, 133.4, 133.1, 132.2, 128.5,
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Journal of Medicinal Chemistry
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28.3, 127.9, 127.88, 127.85, 127.7, 126.4, 125.8, 124.6, 123.9, 122.1, 121.3, 121.1, 114.3, 103.3,
–
4.8, 53.4, 50.2, 38.0, 36.5, 30.5, 27.6. HRMS (ESI) Calcd for C H ClF N O (M – H)
3
8
33
3
5
9
794.1841, found 794.1846. HPLC purity 100%, t = 10.24 min (condition A1); 100%, t = 12.59
R
R
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
min (condition B1).
S)-4-((S)-3-carboxy-2-((S)-2-(5-chloro-1H-indole-2-carboxamido)-3-(naphthalen-2-
(
yl)propanamido)propanamido)-5-oxo-5-((3-(trifluoromethoxy)phenyl)amino)pentanoic
1
acid (35). H NMR (500 MHz, DMSO-d ) δ 12.30 (s, 2H), 11.62 (d, J = 2.2 Hz, 1H), 10.16 (s,
6
1
5
H), 8.80 (d, J = 8.5 Hz, 1H), 8.66 (d, J = 7.5 Hz, 1H), 8.16 (d, J = 7.6 Hz, 1H), 7.97 – 7.76 (m,
H), 7.70 (d, J = 2.1 Hz, 1H), 7.55 (ddd, J = 10.6, 8.3, 1.8 Hz, 2H), 7.48 – 7.34 (m, 4H), 7.21 (d,
J = 2.2 Hz, 1H), 7.15 (dd, J = 8.7, 2.1 Hz, 1H), 7.05 (dd, J = 8.3, 2.3 Hz, 1H), 4.91 (ddd, J = 11.8,
8
.6, 3.7 Hz, 1H), 4.67 (td, J = 7.6, 5.7 Hz, 1H), 4.41 (td, J = 8.3, 5.2 Hz, 1H), 3.46 – 3.27 (m, 1H),
.16 (dd, J = 13.9, 10.9 Hz, 1H), 2.81 (dd, J = 16.7, 5.8 Hz, 1H), 2.63 (dd, J = 16.7, 7.8 Hz, 1H),
3
1
3
2.39 – 2.24 (m, 2H), 2.05 (td, J = 8.8, 4.5 Hz, 1H), 1.90 (ddt, J = 13.5, 8.7, 4.7 Hz, 1H). C NMR
126 MHz, DMSO-d ) δ 174.2, 172.3, 172.1, 171.2, 170.8, 161.1, 148.9, 140.7, 136.5, 135.2,
(
6
1
1
33.4, 133.1, 132.2, 130.9, 128.5, 128.3, 127.9, 127.87, 127.84, 127.7, 126.4, 125.8, 124.6, 123.9,
21.5, 121.1, 119.5, 118.5, 116.0, 114.3, 112.0, 103.3, 54.8, 53.5, 50.2, 38.0, 36.4, 30.5, 27.5.
–
HRMS (ESI) Calcd for C H ClF N O (M – H) 794.1841, found 794.1854. HPLC purity 98.2%,
3
8
33
3
5
9
t = 14.67 min (condition A2); 100%, t = 17.23 min (condition B2).
R
R
(S)-4-((S)-3-carboxy-2-((S)-2-(5-chloro-1H-indole-2-carboxamido)-3-(naphthalen-2-
yl)propanamido)propanamido)-5-((3,5-dimethoxyphenyl)amino)-5-oxopentanoic acid (36).
1
H NMR (500 MHz, DMSO-d ) δ 12.30 (s, 2H), 11.62 (d, J = 2.2 Hz, 1H), 9.84 (s, 1H), 8.81 (d, J
6
=
8.5 Hz, 1H), 8.68 (d, J = 7.5 Hz, 1H), 8.08 (d, J = 7.8 Hz, 1H), 7.94 – 7.74 (m, 4H), 7.70 (d, J =
2.0 Hz, 1H), 7.56 (dd, J = 8.5, 1.7 Hz, 1H), 7.49 – 7.32 (m, 3H), 7.21 (dd, J = 2.1, 0.9 Hz, 1H),
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.15 (dd, J = 8.7, 2.1 Hz, 1H), 6.87 (d, J = 2.3 Hz, 2H), 6.23 (t, J = 2.3 Hz, 1H), 5.01 – 4.83 (m,
H), 4.66 (td, J = 7.6, 5.8 Hz, 1H), 4.40 (td, J = 8.3, 5.1 Hz, 1H), 3.70 (s, 6H), 3.30 (d, J = 3.7 Hz,
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1H), 3.17 (dd, J = 13.9, 11.0 Hz, 1H), 2.80 (dd, J = 16.7, 5.8 Hz, 1H), 2.63 (dd, J = 16.7, 7.8 Hz,
0
1
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0
1
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3
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0
1
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7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
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7
8
9
0
13
1
H), 2.38 – 2.23 (m, 2H), 2.11 – 1.96 (m, 1H), 1.87 (ddt, J = 13.6, 8.7, 4.8 Hz, 1H). C NMR (126
MHz, DMSO-d ) δ 174.3, 172.3, 172.1, 171.0, 170.3, 161.1, 160.9, 140.7, 136.5, 135.2, 133.4,
6
1
1
33.0, 132.2, 128.5, 128.3, 127.9, 127.88, 127.85, 127.7, 126.4, 125.8, 124.6, 123.9, 121.1, 114.3,
03.4, 98.2, 96.0, 55.5, 54.9, 53.4, 50.2, 38.0, 36.4, 30.5, 27.8. HRMS (ESI) Calcd for
–
C H ClN O (M – H) 770.2229, found 770.2237. HPLC purity 98.1%, t = 14.08 min
3
9
38
5
10
R
(condition A2); 97.6%, t = 16.69 min (condition B2).
R
(S)-4-((S)-3-carboxy-2-((S)-2-(5-chloro-1H-indole-2-carboxamido)-3-(naphthalen-2-
yl)propanamido)propanamido)-5-((3-methoxy-5-(trifluoromethyl)phenyl)amino)-5-
1
oxopentanoic acid (37). H NMR (500 MHz, DMSO-d ) δ 12.30 (s, 2H), 11.62 (s, 1H), 10.19 (s,
6
1H), 8.80 (d, J = 8.5 Hz, 1H), 8.66 (d, J = 7.6 Hz, 1H), 8.16 (d, J = 7.6 Hz, 1H), 7.92 – 7.75 (m,
4
H), 7.74 – 7.68 (m, 1H), 7.66 (s, 1H), 7.55 (s, 1H), 7.51 – 7.35 (m, 4H), 7.31 – 7.19 (m, 1H), 7.15
(dd, J = 8.7, 2.1 Hz, 1H), 6.95 (s, 1H), 4.99 – 4.82 (m, 1H), 4.67 (q, J = 7.1 Hz, 1H), 4.40 (q, J =
7
1
1
1
.3 Hz, 1H), 3.80 (s, 3H), 3.42 – 3.28 (m, 1H), 3.25 – 3.05 (m, 1H), 2.81 (dd, J = 16.7, 5.7 Hz,
H), 2.63 (dd, J = 16.6, 7.9 Hz, 1H), 2.33 (td, J = 9.9, 6.3 Hz, 2H), 2.06 (td, J = 11.2, 8.9, 5.3 Hz,
1
3
H), 2.00 – 1.83 (m, 1H). C NMR (126 MHz, DMSO-d ) δ 174.2, 172.3, 172.1, 171.2, 170.9,
6
61.1, 160.4, 141.1, 136.5, 135.2, 133.4, 133.0, 132.2, 130.9 (d, J = 31.8 Hz), 128.5, 128.3, 127.9,
127.87, 127.86, 127.7, 126.4, 125.8, 125.4, 124.6, 123.9, 123.3, 121.1, 114.3, 108.9, 108.4, 105.9,
03.4, 56.1, 54.9, 53.6, 50.2, 37.9, 36.4, 30.5, 27.5. HRMS (ESI) Calcd for C H ClF N O (M –
1
3
9
35
3
5
9
–
H) 808.1997, found 808.2004. HPLC purity 99.6%, t = 14.68 min (condition A2); 98.3%, t =
R
R
17.28 min (condition B2).
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Journal of Medicinal Chemistry
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(
S)-5-(benzo[d][1,3]dioxol-5-ylamino)-4-((S)-3-carboxy-2-((S)-2-(5-chloro-1H-indole-2-
1
carboxamido)-3-(naphthalen-2-yl)propanamido)propanamido)-5-oxopentanoic acid (38). H
NMR (500 MHz, DMSO-d ) δ 12.30 (s, 2H), 11.62 (s, 1H), 9.76 (s, 1H), 8.80 (d, J = 8.4 Hz, 1H),
6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
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3
4
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4
4
4
4
4
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
.67 (d, J = 7.5 Hz, 1H), 8.09 (d, J = 7.7 Hz, 1H), 7.93 – 7.74 (m, 4H), 7.70 (s, 1H), 7.56 (d, J =
.4 Hz, 1H), 7.42 (p, J = 6.9 Hz, 2H), 7.37 (d, J = 8.7 Hz, 1H), 7.29 (s, 1H), 7.21 (s, 1H), 7.15 (d,
8
J = 8.8 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 6.84 (d, J = 8.3 Hz, 1H), 5.98 (s, 2H), 5.00 – 4.79 (m,
H), 4.66 (q, J = 7.1 Hz, 1H), 4.38 (q, J = 7.4 Hz, 1H), 3.31 (s, 1H), 3.16 (t, J = 12.5 Hz, 1H), 2.81
dd, J = 16.8, 6.0 Hz, 1H), 2.63 (dd, J = 16.7, 7.5 Hz, 1H), 2.39 – 2.21 (m, 2H), 2.10 – 1.95 (m,
1
(
1
3
1
H), 1.87 (dt, J = 15.3, 7.8 Hz, 1H). C NMR (126 MHz, DMSO-d ) δ 174.3, 172.4, 172.1, 171.0,
6
169.9, 161.1, 147.5, 143.6, 136.5, 135.2, 133.4, 133.3, 133.1, 132.2, 128.5, 128.3, 127.9, 127.88,
1
5
27.85, 127.7, 126.4, 125.8, 124.6, 123.9, 121.1, 114.3, 112.8, 108.5, 103.3, 102.1, 101.4, 54.8,
–
3.2, 50.2, 38.0, 36.4, 30.5, 27.8. HRMS (ESI) Calcd for C H ClN O (M – H) 754.1916, found
3
8
34
5
10
754.1924. HPLC purity 98.6%, t = 13.83 min (condition A2); 96.1%, t = 16.39 min (condition
R
R
B2).
S)-4-((S)-2-((S)-2-(5-Chloro-1H-indole-2-carboxamido)-3-(naphthalen-2-yl)propanamido)-
(
3
-(2H-tetrazol-5-yl)propanamido)-5-oxo-5-((4-(trifluoromethoxy)phenyl)amino)pentanoic
1
acid (53). Yield, 82%. H NMR (500 MHz, DMSO-d ) δ 12.14 (s, 1H), 11.69 – 11.53 (m, 1H),
6
1
0.25 (s, 1H), 8.79 (d, J = 8.3 Hz, 1H), 8.69 (d, J = 7.5 Hz, 1H), 8.31 (d, J = 7.4 Hz, 1H), 7.90 –
.64 (m, 8H), 7.53 (d, J = 8.4 Hz, 1H), 7.49 – 7.26 (m, 5H), 7.25 – 7.09 (m, 2H), 4.94 – 4.73 (m,
7
2H), 4.40 (td, J = 8.0, 5.1 Hz, 1H), 3.56 – 3.41 (m, 2H), 3.14 (dd, J = 13.9, 10.8 Hz, 2H), 2.39 –
13
2
.22 (m, 2H), 2.04 (ddt, J = 15.1, 10.6, 5.7 Hz, 1H), 1.89 (dtd, J = 14.5, 9.0, 5.8 Hz, 1H). C NMR
(126 MHz, DMSO-d ) δ 174.2, 172.1, 170.5, 170.4, 161.2, 144.3, 138.2, 136.3, 135.3, 133.4,
6
133.0, 132.2, 128.4, 128.2, 127.9, 127.8, 126.4, 125.8, 124.7, 124.0, 122.0, 121.5, 121.1, 114.3,
2
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ACS Paragon Plus Environment