Wnt inhibition correlates with human embryonic stem cell cardiomyogenesis: A structure-activity relationship study based on inhibitors for the Wnt response

Article abstract of DOI:10.1021/jm2010223

Human embryonic stem cell-based high-content screening of 550 known signal transduction modulators showed that one "lead" (1, a recently described inhibitor of the proteolytic degradation of Axin) stimulated cardiomyogenesis. Because Axin controls canonical Wnt signaling, we conducted an investigation to determine whether the cardiogenic activity of 1 is Wnt-dependent, and we developed a structure-activity relationship to optimize the cardiogenic properties of 1. We prepared analogues with a range of potencies (low nanomolar to inactive) for Wnt/β-catenin inhibition and for cardiogenic induction. Both functional activities correlated positively (r ² = 0.72). The optimal compounds induced cardiogenesis 1.5-fold greater than 1 at 30-fold lower concentrations. In contrast, no correlation was observed for cardiogenesis and modulation of transforming growth factor β (TGFβ)/Smad signaling that prominently influences cardiogenesis. Taken together, these data show that Wnt signaling inhibition is essential for cardiogenic activity and that the pathway can be targeted for the design of druglike cardiogenic molecules.

Full text of DOI:10.1021/jm2010223

Article
pubs.acs.org/jmc
Wnt Inhibition Correlates with Human Embryonic Stem Cell
Cardiomyogenesis: A Structure−Activity Relationship Study Based
on Inhibitors for the Wnt Response
Marion Lanier,*,†,§,∥ Dennis Schade,†,‡,§,∥ Erik Willems,‡,§ Masanao Tsuda,‡ Sean Spiering,‡
Jaroslaw Kalisiak,† Mark Mercola,‡,§ and John R. Cashman†,§
†Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, California 92121-2804, United States
‡Sanford−Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, United States
§ChemRegen Inc., 11171 Corte Cangrejo, San Diego, California 92130, United States
S
* Supporting Information
ABSTRACT: Human embryonic stem cell-based high-con-
tent screening of 550 known signal transduction modulators
showed that one “lead” (1, a recently described inhibitor of the
proteolytic degradation of Axin) stimulated cardiomyogenesis.
Because Axin controls canonical Wnt signaling, we conducted
an investigation to determine whether the cardiogenic activity
of 1 is Wnt-dependent, and we developed a structure−activity
relationship to optimize the cardiogenic properties of 1. We
prepared analogues with a range of potencies (low nanomolar to inactive) for Wnt/β-catenin inhibition and for cardiogenic
induction. Both functional activities correlated positively (r2 = 0.72). The optimal compounds induced cardiogenesis 1.5-fold
greater than 1 at 30-fold lower concentrations. In contrast, no correlation was observed for cardiogenesis and modulation of
transforming growth factor β (TGFβ)/Smad signaling that prominently influences cardiogenesis. Taken together, these data
show that Wnt signaling inhibition is essential for cardiogenic activity and that the pathway can be targeted for the design of
druglike cardiogenic molecules.
capable of mobilizing endogenous cardiac progenitor cells to
regenerate damaged muscle in the adult heart.4
INTRODUCTION
■
The proposal to produce human heart muscle cells from stem
cells has attracted much attention because of the potential for
promising applications in medicine and drug discovery, ranging
from cell transplantation to in vitro pharmacological testing.
Use of human stem cell-derived cardiomyocytes as tools for
drug discovery and development (i.e., high-throughput assays,
specific disease models, target identification and validation, or
toxicity assessment) has many advantages over current assays
that rely on a noncardiomyocyte setup.1−3 Cardiomyocytes, the
key cells for cardiac safety evaluation at the preclinical stage, are
hard to come by. Scientists are usually limited to animal-derived
cells or tissues or human engineered cells (cell lines
heterologously expressing human cardiac ion channels, cardiac
cell cultures, isolated tissue preparations, and perfused animal
hearts) that have limited predictivity in humans. For example,
overexpression of the hERG channel in fibroblasts is commonly
used in drug development as a model to evaluate cardiotoxicity
of novel drugs. One major advantage of cardiomyocytes derived
from human embryonic stem cells is that they are of human
origin and can be maintained in culture for extended time
periods without losing their characteristics. Theoretically, they
represent an unlimited source for human cardiomyocytes for in
vitro testing. Moreover, the information gained from
developing small molecules for stem cell-derived cardiomyocyte
differentiation in vitro may lead to the development of drugs
Cell differentiation is a complex and still poorly understood
process. As for other tissue types, the development of human
myocardial cells requires close temporal control of inducing
factors to stimulate the stepwise progression from pluripotent
cells to uncommitted progenitor to committed precursor and
finally to myocardial cells including cardiomyocytes.4,5 Current
approaches to stimulate stem cell differentiation have included
naturally occurring factors, the introduction of lentiviral vectors
carrying transcription factors, the addition of growth factors,
and the use of small-molecule signaling pathway modulators.1
The approach described herein focuses on the latter, because
use of small molecules as differentiation reagents overcomes the
inherent high cost of biological factors or reagents for in vitro
applications, and they can be developed into drug candidates
for in vivo applications for therapeutic development.
We recently described a human embryonic stem cell (hESC)-
based high-content screen (HCS) of about 550 known pathway
modulators (i.e., InhibitorSelect and StemSelect, both from
EMD Chemicals Inc.) and used these compounds to identify
key signaling pathway(s) that control differentiation of
uncommitted cardiac progenitors to form cardiomyocytes.6
Received: July 30, 2011
Published: December 22, 2011
© 2011 American Chemical Society
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Only one small molecule [1 (IWR-1),7,8 Figure 1] was
identified as a “lead” from this library screen. The abbreviation
to improve the pharmacological properties of 1. Compound 1
was optimized for its ability to inhibit the Wnt pathway and
stimulate cardiogenesis. Newly designed compounds 10, 29,
and 34 showed decreased IC50 values for inhibition of the Wnt
pathway as well as increased cardiomyogenesis potency.
Compared to 1, compounds 10, 29, and 34 showed improved
functional activity and improved physiochemical properties,
thus rendering them more attractive for in vitro and possibly in
vivo studies.
Figure 1. Screening “lead” 1. Regions A, B, and C indicate the
different areas of the molecule that were systematically modified for
lead optimization.
CHEMICAL SYNTHESIS
■
The synthesis of 1 and certain analogues was recently described
in papers reporting new Wnt inhibitors for cancer.7,8 From a
structural perspective and as previously described by Lu et al.,8
compound 1 can be divided into three regions (A, B, and C in
Figure 1). The structure−activity relationships (SAR) of all
three regions of 1 were explored.
Regions A and C were modified via synthetic protocols
described by Chen et al.,7 (Schemes 1 and 2). Briefly, the
desired amino acid 6 was heated with anhydride 7 in N,N-
dimethylformamide (DMF) and afforded intermediates 8a−e
in good yields. The carboxylic acid moiety of 8a−e was then
activated in the presence of thionyl chloride and treated with
the appropriate amine (R3NH2) to give compounds 9−39 and
53−56 (Scheme 1). Compounds 41, 42c−f, 43g−k, and 44l−p
were prepared by treating the acid chloride of p-nitrobenzoic
acid with the appropriate amine, R3NH2. After hydrogenation
of the nitro group, intermediate 40 was subjected to a reductive
amination step or acylation or treatment with an anhydride to
give the desired products 41−44 (Scheme 2).
IWR stands for Inhibitor of the Wnt Response. Wnt is a hybrid
name of Int, a gene active in mouse mammary tumors, and
Wingless, a gene essential for wing development in Drosophila.
The Wnt pathway has been studied in a wide range of
organisms and is implicated in mammalian development and
cancer. Compound 1 was recently reported as an inhibitor of
the oncogenic canonical Wnt response at the Axin level.7,8
Besides being involved in cancer,9 Wnt and Wnt inhibition
regulate crucial processes during embryonic development.10
Wnt signaling plays a multiphasic role in heart development,
and Wnt inhibition is critical to form committed progenitor
cells.11,12 This has led to the use of the natural Wnt inhibitor
Dickkopf-1 (DKK-1) to enhance cardiogenesis in hESC
culture13 and the discovery of small-molecule tool compounds.2
Herein, we examined the effect of the previously reported
Wnt inhibitors 1−5 (Figure 2A) on human ESC-mediated
cardiomyocyte differentiation in parallel with a stepwise process
Figure 2. Inhibition of the Wnt pathway with reported inhibitors 1−5. (A) Structures of compounds 1−5. (B) Dose−response curves and IC50
values for Wnt inhibition for 1−5, representing different classes of Wnt inhibitors. Data is the mean
standard deviation of at least three
independent experiments. (C) Maximum cardiogenesis in the presence of Wnt inhibitors in hESCs (data from Willems et al.);6 human recombinant
a
DKK-1 = 200 ng/mL (7.75 nM). Cardiogenesis corresponds to MYH6-mCherry expression levels as fold induction over DMSO vehicle control
(=1-fold).
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a
Scheme 1. Synthesis of Analogues 9−39 and 53−56
Table 1. SAR for Central Region B of the Molecule for Wnt
Inhibition
a
(a) DMF, 120 °C, 16 h. (b) Thionyl chloride, DMF, 80 °C, 16 h. (c)
R3NH2, Py, DCE, room temp to 50 °C, 16 h.
The SAR of region B was explored by various synthetic
approaches. Compound 45 (Table 1) was prepared following
the same sequence of steps as in Scheme 1 but with 6 replaced
by 3-aminobenzoic acid. Compound 46 (Table 1) was prepared
following Scheme 2 with 4-nitrobenzoic acid replaced by 5-
nitro-2-furoic acid. Compound 48 was prepared in three steps
from 4-nitrophthalic anhydride (Scheme 3). Condensation of
5-nitroisobenzofuran-1,3-dione with 8-aminoquinoline followed
by reduction of the nitro group afforded 47. Condensation of
47 with cis−endo-dihydrocarbic anhydride under conditions
described in Scheme 1 afforded 48. For the reduced version 50
(Table 1), intermediate 49 was obtained in two steps by
reductive amination of 8-aminoquinoline with 4-nitrobenzalde-
hyde followed by reduction of the nitro group with sodium
dithionite. Compound 49 was treated with cis−endo-dihydro-
carbic anhydride under conditions described in Scheme 1 to
afford compound 50 (Scheme 4). Finally, the reverse amide 52
(Table 1) was synthesized via intermediate 51. Treatment of 4-
nitroaniline with carbic anhydride followed by hydrogenation
gave 51, and following addition of freshly prepared quinoline-8-
carbonyl chloride, treatment with aniline provided the desired
reverse amide (52, Scheme 5).
a
NM, not measured.
RESULTS AND DISCUSSION
■
Because 1, the lead compound from screening, was reported to
inhibit canonical Wnt signaling at the Axin level,7 we compared
1 to four mechanistically different Wnt inhibitors [2 (XAV939),
a
Scheme 2. Synthesis of Compounds 41−44
a
(a) Thionyl chloride, reflux, 2 h. (b) R3NH2, pyridine, reflux, 3 h. (c) H2, Pd/C, EtOH, room temp, 16 h. (d) Anhydride, TEA, DMF, 120 °C, 16 h.
(e) R4COCl, TEA, DCE, 70 °C, 16 h. (f) R4CHO, NaBH(OAc)3, AcOH, MgSO4, DCE, 50 °C, 72 h.
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a
Scheme 3. Synthesis of 48
a
(a) DMF, 120 °C, overnight. (b) H2, Pd/C, EtOH, room temp, overnight. (c) cis-Endodihydrocarbic anhydride, DMF, 120 °C, 10 h.
a
Scheme 4. Synthesis of 50
a
(a)NaBH3CN, AcOH, EtOH, room temp, 16 h. (b) Na2S2O4, EtOH/NaHCO3aqueous, room temp, 16 h. (c) cis-Endodihydrocarbic anhydride,
DMF, 120 °C, 16 h.
a
Scheme 5. Synthesis of 52
a
(a) cis-Endocarbic anhydride, DMF, 120 °C, 16 h. (b) H2, Pd/C, EtOH, room temp. (c) Quinoline-8-carbonyl chloride, TEA, 3 days, room temp.
3 (IWP-3), 4 (iCRT-5), and 5 (pyrvinium); Figure 2A] in a
Wnt inhibition assay (Figure 2B) and a hESC cardiogenesis
assay (Figure 2C) to examine the relation between Wnt
inhibition and cardiogenesis and to narrow down and identify
the part of the pathway blocked in the signaling cascade that
influenced cardiogenic activity. To ensure that the SAR analysis
was not confounded by cell toxicity, the five Wnt inhibitors (1−
5) and certain analogues of 1 were tested in parallel for
cytotoxicity (see Supporting Information, Figure S2). Com-
pound 1 blocks the Wnt pathway by stabilizing the Axin protein
complex via a direct interaction with Axin.7 Compound 2
stabilizes Axin by inhibiting the poly-ADP-ribosylating enzymes
tankyrase 1 and 2.14 Compound 3 inhibits the production of
Wnt by preventing the palmitoylation of Wnt proteins by
Porcupine.7 Compound 4 has been reported to inhibit the
interaction between β-catenin and TCF4 within the cell
nucleus.15 Compound 5 activates CK1α activation in the Wnt
pathway and promotes degradation of β-catenin and inhibited
Axin degradation.16 In our hands, 5 was a very modest Wnt
inhibitor (IC50 = 592 nM) and also quite cytotoxic, so it was
not tested in the cardiac assay. Compounds 3 and 4 were
similarly modest Wnt inhibitors (i.e., IC50 = 538 and 728 nM,
respectively) but were not toxic to HEK293T cells at
concentrations up to 20 μM (see Figure S2 in Supporting
Information). Compound 2, described to induce cardiac
differentiation in mouse ESCs,17 had an IC50 of 51 nM in the
Wnt assay and did not induce cell toxicity. Compound 1 had a
similar profile (i.e., IC50 = 26 nM and was not toxic to
HEK293T cells). As we showed previously,6 1, 2, and 3
induced cardiogenesis by ca. 1000-fold at their maximum
efficacious concentrations (i.e., 4, 2, and 2.5 μM respectively;
Figure 2C) over a dimethyl sulfoxide (DMSO) control but
differed in their potencies. Compound 2 was about 1.5-fold less
potent than 1 and 3. Compound 4 was weakly cardiogenic but
only at a higher concentration (50 μM). Compound 3 was not
selected as a lead candidate because its mode-of-action in
inhibiting Wnt production is reported to be at an upstream
position compared to 1. Taken together, these data suggested
that 1 was the most appealing structure as a lead candidate. It
was potent both as a Wnt inhibitor and as a cardiogenic agent
in the hESC studies and was not cytotoxic to the cells
examined.
Wnt Inhibition. Initially, a structurally diverse library of ca.
90 analogues of 1 was synthesized and tested for Wnt inhibition
at a standard concentration (i.e., 1 μM). Compounds that
showed inhibition above 50% at 1 μM were re-evaluated for
dose-dependent inhibition. Next, more focused libraries were
prepared to improve potency and physicochemical features of
promising analogues.
By use of synthetic strategies depicted in Schemes 1−5,
variations on the central region (region B) were prepared and
evaluated for Wnt inhibition (Table 1). Lu et al.8 previously
showed that the phenyl ring of 1 could be replaced by a trans-
cyclohexyl moiety and still maintain Wnt inhibition potency. As
shown in Table 1, an increase in Wnt inhibition potency was
observed with the completely saturated trans-cyclohexyl
analogue 10 (IC50 = 4 nM). Because a cyclohexyl ring can
adopt both cis and trans configurations, it was of interest to
examine the stereoselectivity of Wnt inhibition. The cis
analogue 11 was prepared and found to be potent (i.e., IC50
= 57 nM). It is possible that the potency of the cis-cyclohexyl
moiety can be explained by 11 adopting a boat conformation.
Next, modifications of the amide function revealed important
information about Wnt inhibition SAR. Reducing the amide
function (50, 45% inhibition at 1 μM), changing the amide
position to the meta position (45, 0% inhibition at 1 μM), or
replacing the central aryl substituent by a furan group (46, 0%
inhibition at 1 μM) decreased the potency of Wnt inhibition
and showed that both the amide bond and the 1,4-substitution
pattern for the central B region were essential for maximal
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inhibitory potency. A rigidified version (48) and a reverse-
amide version (52) were also prepared. Reversing the amide as
in 52 was tolerated but showed some loss of Wnt inhibitory
potency (IC50 = 119 nM), while the rigid analogue 48 was
inactive (15% inhibition at 1 μM). Compounds 9 (central
phenyl ring) and 10 (central trans-cyclohexyl ring) possessed
similar IC50 values (24 and 4 nM, respectively), which
suggested that the central ring itself likely seems to be a
simple placeholder and does not contribute to a discernible
biological interaction.
electronic properties and the position of the substituent
affected potency. A carbonyl-containing functionality at the
ortho position afforded the most potent analogues examined
and apparently mimicked the role of the quinoline nitrogen to
afford potent Wnt inhibition. A methyl ester at the 2-position
(27) had an IC50 of 67 nM, and the 2-methylketone (28) had
an IC50 of 6 nM. The tetralone analogue 29 was similarly
potent (IC50 = 7 nM). When increased steric bulk was
introduced by 9H-fluoren-9-one (30, IC50 = 194 nM), potency
decreased, suggesting steric hindrance in this region may have a
negative effect on Wnt inhibition (Table 3).
A number of unsubstituted heterocycles [2-pyrimidine, 2-
pyrazine, 3-(1,2,4-triazole), 2-thiazole, 2-pyridine, 3-pyridine,
and 4-pyridine] were synthesized as replacements for the
quinoline substituent. While the unsubstituted 2-, 3- and 4-
pyridines (compounds 31−33, Table 3) showed IC50 values in
the 100 nM range (IC50 = 109, 120, and 80 nM, respectively),
none of the other heterocycles described above were
functionally active (inhibition below 50% at 1 μM; data not
shown). Substituted pyridines were then prepared by
combining the optimal substituents identified from the data
of Table 3 with 3- and 4-pyridine derivatives. Compounds
combining the 3-pyridine with an acetyl group at the 2- or 6-
position were prepared and tested. Remarkably, the 2-acetyl-3-
pyridine analogue 34 gave an IC50 value of 7 nM, while the 6-
A systematic study of the effect of position of the nitrogen
atom in the quinoline heterocycle on Wnt inhibition was done
with compounds possessing a trans-cyclohexyl ring in the
central position. The results summarized in Tables 2 and 3
Table 2. SAR for Region C: Systematic Study of the Effect of
Nitrogen Position of the Quinoline Heterocycle on Potency
for Wnt Inhibition
acetyl-3-pyridine isomer 35 was significantly less potent (IC50
=
590 nM).
As reported (but not exemplified) by Lu et al.,8 region A
(Figure 1) was very sensitive to chemical modifications. In our
hands, any attempts to modify this region, either by replacing
the bicycle by bulky R groups (i.e., compounds 41 and 42c−f,
Scheme 2) or replacing the entire carbic moiety by various
groups (compounds 43g−k and 44l−p, Scheme 2), led to a
complete loss of potency (inhibition < 32% at 1 μM). Only
minor structural alterations such as saturation of the double
bond (9, IC50 = 24 nM) were well-tolerated; two-carbon-bridge
analogues (36, IC50 = 412 nM, and 37, IC50 = 314 nM; Table
4) and oxa analogue (39, IC50 = 605 nM) were >10-fold less
potent. Adding a methyl group to the carbic moiety (38, IC50
=1176 nM) also resulted in significant loss of potency,
highlighting the sensitivity of this region to substitution.
Chemical Stability. Certain selected compounds, as well as
1 for comparison purposes, were evaluated for chemical
stability in buffer before being tested for their cardiogenic
properties. hESC cardiogenesis studies were done in a cell-
based assay at pH 7.4, and biological activity was presumably
required for at least 24 h after compound addition, 4 days after
initiating hESC differentiation.6 Therefore, we determined the
aqueous stability of key compounds at pH 7.4 for at least 24 h.
Representatives of the various subseries, (compounds 1, 10, 28,
32, and 52), were incubated at 10 μM in phosphate buffer (pH
7.4) for up to 30 h. No degradation was observed as determine
by HPLC for any of the compounds except 52, whose half-life
was 3.8 h (Supporting Information, Table S1).
a
Compound 12 has a phenyl central ring instead of the trans-
cylcohexyl.
show that the 8-aminoquinoline isomer was the most potent
quinoline (10, IC50 = 4 nM). Potency decreased when the
distance between the quinoline nitrogen and the amide
function increased, suggesting either an intramolecular
interaction between the amide hydrogen and the quinoline
nitrogen or, alternatively, a specific interaction with a biological
target. Lu et al.8 showed that replacement of the quinoline with
a phenyl ring substituted at various positions by halogens or a
trifluoromethyl group usually led to a significant loss of
potency. The optimal aryl substitution pattern was for a 4-
bromophenyl and 2-methoxyphenyl (IC50 = 100 nM for both
compared to 200 nM for 1).8 Incorporation of electron-
donating groups (2-hydroxyl compound 19, 33% inhibition at 1
μM; 2- and 4-methoxy compounds 20 and 21, 50% inhibition
at 1 μM and IC50 = 215 nM; 4-dimethylamino compound 22,
25% inhibition at 1 μM) or electron-withdrawing groups (4-
cyano compound 23, IC50 = 317 nM; 4-acetyl compound 24,
41% inhibition at 1 μM) did not lead to a clear picture of the
effect of electronic substituents on Wnt inhibition (Table 3).
On the other end, the presence of a methoxy group at the ortho
position (compound 21, IC50 = 215 nM) or a p-methoxy
substituent (22, 50% inhibition at 1 μM) suggested that the
electronic nature of the substituent at the aryl ring was not
critical for Wnt inhibition. It is likely that shape rather than
Induction of Cardiogenesis. From the 180 synthetic
analogues tested in the Wnt inhibition assay, 26 were selected
to examine cardiogenic induction in the presence of hESCs.
This subset of analogues comprised a full range of inhibitory
potency in the Wnt assay and contained the most structurally
diverse scaffolds for evaluation of hESC cardiogenic activity. As
shown in Figure 3, analogues from the following series were
selected as representative members to investigate (a) variation
of the N-position of the quinoline C region, including phenyl
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Table 3. SAR of Region A: Effects of Phenyl and Pyridinyl Substituents on Potency for Wnt Inhibition
a
NM, not measured.
Table 4. SAR for Region A of the Molecule for Wnt Inhibition
versus pyridinyl substituents and o-carbonyl groups; (b)
variation of the central B region linker, including aromatic
versus aliphatic and cis- versus trans-configured linkers; and (c)
variation in the norbornene A region moiety. Figure 3A shows a
list of values for the potency of Wnt inhibition in HEK293T
cells and induction of cardiogenesis in hESCs. Three classes of
compounds stimulated hESC cardiogenesis. The first group of
compounds (32, 21, 35, 33, 31, 39, 37, 23, 52, and 12) was
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Figure 3. Cardiogenic activity and Wnt inhibition of selected analogues. (A) Data set for analogues tested in cardiac and Wnt assays; IC50 values
(nanomolar) were measured in the Wnt inhibition assay. Those highlighted in red are outlier compounds. (B) Maximum cardiogenic activity for
each compound tested relative to the maximum induction for 1 (set at 100%). (C) Representative dose−response curves of IWR analogues in hESCs
for cardiogenesis. (D) Correlation of cardiogenesis (based on the Z-score at 625 nM) and Wnt inhibition (IC50 values) in a double-logarithmic plot;
data points (in red) indicate outlier derivatives that were excluded from the correlation (this r2 is highlighted in red). The correlation coefficient r2
a
shown in black includes all data. Percent cardiogenesis represents normalized data for most effective (cardiac) concentration to the maximum
induction of 1 (100% at 2.5 μM). b Z-score at a dose of 625 nM. cMaximum cardiac activity = normalized to 1, no cardiac activity = Z-score of ≤0.
functionally inactive as inducers of cardiogenesis and possessed
uniformly large IC50 values in the Wnt inhibition assay (>200
nM). A subset of these compounds (31, 32, and 33), however,
had IC50 values in the 100 nM range as Wnt inhibitors. Further
studies need to be done to explain their biological properties.
Compound 52 contained a “reverse amide” and was a relatively
potent Wnt inhibitor (IC50 = 119 nM) but was inactive as a
cardiogenic inducer. The chemical half-life of 52 has been
estimated to be less than 5 h in the presence of assay buffer (see
Supporting Information, Table S1) and this could explain its
low cardiogenesis functional activity. While lack of aqueous
stability would not be expected to compromise functional
activity in the short time frame of the Wnt assay, the actual
concentration of 52 in the hESC assay may have declined
compared to the time necessary to induce cardiogenesis and
thus could explain its lack of potency as a cardiogenic stimulant.
The second class (14, 30, 1, 13, 56, 9, 11, 55, 54, 27, 18, and
53) contained compounds that elicited a dose-dependent
induction of cardiogenesis functional activity, without a
decrease in cardiogenesis at higher doses. These compounds
were also potent Wnt inhibitors on the basis of their IC50 values
in the Wnt assay (20−200 nM). Compound 30 exhibited
modest Wnt inhibition (IC50 = 194 nM) and solid cardiac
induction (104% cardiogenesis). We suspect that the high
lipophilicity of the 9-fluorene substituent causes low aqueous
solubility and extensive protein binding. This could possibly
explain the observed differences in these two distinct assays.
The third class of compounds (29, 34, 10, and 28) were potent
Wnt inhibitors and potent stimulators of cardiogenesis, even at
low concentrations (i.e., < 0.15 μM). They were distinguishable
from the second group of compounds by their dose−response
curves (Figure 3C). They showed very strong cardiogenesis at
the lowest concentrations, followed by a constant decrease of
percent cardiogenesis at increasing compound concentration in
the cell-based assay. This effect could be due to anticardiogenic,
antiproliferative, or toxic cellular effects caused by strong Wnt
inhibition. Compared to other analogues, compound 52 was
substantially less stable at pH 7.4 than other analogues (see
Supporting Information, Table S1) and thus its potency in the
hESC assay may be possibly underestimated. Compound 18
had a negative Z-score at 625 nM and thus appeared as an
outlier in our correlation, but it was cardiogenic at higher
concentrations and reached 24% maximum cardiogenesis at 10
μM (Figure 3A).
On the basis of the correlation analysis (Figure 3D), hESC
cardiomyocyte induction apparently strongly paralleled Wnt
inhibition, and that is apparent in SAR studies. Even minor
variations in the norbornene part of the molecule (region C)
led to a loss of functional activity for both Wnt inhibition and
hESC cardiogenesis. For example, the two-carbon-bridge
analogue 37 and oxa bridge analogue 39 had IC50 values of
314 and 605 nM, respectively, in the Wnt inhibition assay and
stimulated cardiogenesis only to the extent of less than 5%. The
more potent compounds 9 (one-carbon-bridge analogue) and
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10 (saturated version) had IC50 values of 24 and 4 nM,
respectively, in the Wnt assay and induced cardiogenesis of 75%
and 145%, respectively, compared to 1. The effect of
stereochemistry of the central portion of the molecule (region
B) on the stereoselectivity of the biological end points was
examined. The cis- and trans-configurations of the saturated
cyclohexyl linker (region B) had similar effects on cardiogenesis
and Wnt inhibition. For example, 28 (trans) and 56 (cis) had
cardiogenesis of 78% and 72%, respectively (Figure 3B), and
were relatively potent at Wnt inhibition (IC50 values of 6 and
23 nM respectively, Table 3). A number of alternate positions
of the nitrogen in the quinoline heterocycle were tolerated for
functional biological activity, but complete omission of the
nitrogen atom led to a dramatic decrease in induction of
cardiogenic potency (12, 41% Wnt inhibition at 1 μM and 8%
cardiogenesis). Finally, different phenyl- and pyridinyl-sub-
stituted analogues gave comparable functional activity for
induction of cardiogenesis and this showed that the quinoline
could be replaced while retaining cardiogenesis induction. In
particular, ortho substitution of phenyl and pyridinyl residues
with carbonyl-containing groups afforded highly potent
derivatives, and those analogues (28, 29, and 34) were
among the most potent and effective compounds tested.
Finally, we tested potent Wnt inhibitors for selective
inhibition of transforming growth factor β (TGFβ) signaling
to investigate possible off-target effects. TGFβ interacts with
Wnt to modulate stem cell fate,18 and inhibition of TGFβ
signaling can also be cardiogenic in mouse19 and human
ESCs.20 Therefore, 10 representative structurally distinct
compounds (1, 10, 28, 32, 34, 35, 29, 53, 34, and 35) were
incubated at concentrations varying from 0.01 to 5 μM (same
dose range used in the Wnt assay) in a Smad response
element−reporter assay in HEK293T cells stimulated with
human recombinant TGFβ2. No significant inhibition effect
was observed compared to DMSO vehicle controls even at the
highest concentrations examined (inhibition of Smad response
<20% at 0.01, 0.1, 1, and 5 μM; see Figure S3 in Supporting
Information), indicating that TGFβ inhibition is not involved in
the cardiac phenotype of the IWR-1 analogues examined.
Furthermore, because the functional biological activity of 1 has
been attributed to Axin stabilization, these data also indicate
that Axin stabilization does not inhibit TGFβ/Smad signaling.
human cardiac progenitor cells. Therefore, an important next
step will be to explore their utility for ex vivo expansion of adult
heart-derived progenitors and to nominate and evaluate optimal
compounds for their potential as in vivo regenerative
compounds in animal models of myocardial infarction.
EXPERIMENTAL SECTION
■
General. Reagents and solvents were used as received from
commercial sources. Compound 2 was purchased from Cayman
Chemicals, 4 from Princeton BioMolecular Research, and 5 from
Sigma−Aldrich. Synthetic products were isolated on a flash column
chromatography system (Teledyne ISCO, CombiFlash Rf) with UV
detection at 254 nm or PTLC (preparative thin-layer chromatog-
raphy) with UV indicator. NMR (nuclear magnetic resonance) spectra
were recorded at 300 MHz (1H) on a Varian Mercury 300 or at 125
MHz (13C) on a Bruker AMX-500 II (NuMega Resonance Lab, San
Diego, CA). Chemical shifts were reported as parts per million, ppm
(δ), relative to the solvent (CDCl3 at 7.26 ppm, CD3OD at 3.31 ppm,
DMSO-d6 at 2.50 and 3.52 ppm): s stands for singlet, d for doublet, t
for triplet, q for quadruplet, m for multiplet, and br for broad. Low-
resolution mass spectra were obtained on a Hitachi M-8000 mass
spectrometer with an electrospray ionization (ESI) source.
Purity of final products was determined with a Hitachi 8000 LC-MS
(Hitachi) using reverse-phase chromatography (C18 column, 50 × 4.6
mm, 5 μm, Thomson Instrument Co., Oceanside, CA). Compounds
were eluted with gradient elution of 95/5 to 5/95 A/B over 5 min at a
flow rate of 1.5 mL/min, where solvent A was water with 0.05%
trifluoroacetic acid (TFA), and solvent B was acetonitrile with 0.05%
TFA. For purity analysis, peak area percent for the TIC (total ion
count) at 254 nm and retention time (tR in minutes) were provided.
Purity of final products was ≥95%.
Biological Assays. Wnt Inhibition. Compounds were tested for
their ability to inhibit the β-catenin-dependent canonical Wnt pathway.
A Wnt assay was adapted from Chen et al.7 in our laboratories, with
HEK293T cells in a 96-well format. Briefly, the commercially available
Super(8×)TOPflash vector driven by a (7×)TCF-firefly luciferase
response element was transiently transfected into HEK293T cells
together with a TK-driven Renilla luciferase plasmid as an internal
control to normalize the luminescence signal and a Wnt3A-expressing
vector as the source of pathway activation. Because of the transient
nature of the assay and, consequently, variations between independent
experiments, 1 was included as a positive control in parallel with
untreated and DMSO-treated vehicle controls in experiments
conducted. Maximum inhibition of Wnt response in the assay format
was around 90%, with the most potent inhibitors at the highest doses
examined. Nonlinear regression analysis was performed with the
log(inhibitor)/normalized response equation of the Prism 5 software.
hESC Cardiogenesis. Human embryonic stem cell H9 lines carrying
MYH6-mCherry reporters were used in the hESC assay, as previously
described in detail6 and summarized in the Supporting Information. In
brief, embryoid bodies (EB) were grown until day 4, dissociated gently
to single cells, and transferred to 384-well plates. Concomitantly,
different dilutions of small molecules were added. At day 10, medium
was exchanged for a serum-free medium (SFM) containing the thyroid
hormone analogue triiodothyronine T3 to increase the red signal
driven by the MYH6 promoter for improved image analysis.21 Cells
were changed to phosphate-buffered saline (PBS) at day 14 for
imaging, and red fluorescence images were collected on a high-
throughput microscope. For quantifying the level of cardiac induction,
the total area and intensity of the MYH6-mCherry reporter was
measured in each well. Cardiac activity was reported by stating either
percent cardiogenesis normalized to 1 (set as 100% at its maximum
potency) or Z scores (relative to DMSO control) (Figure 3A and B).
Chemistry. Compounds 1 and 3 were synthesized following the
CONCLUSION
■
Enhancing the production of human cardiomyocytes offers a
wealth of opportunities for drug research and toxicological
assessment of new chemical entities. The Wnt pathway has
been associated with cardiogenesis, and using 1 as a “lead”, we
obtained small-molecule Wnt inhibitors with IC50 values
ranging from low nanomolar (4 nM) to the micromolar
range. We showed a direct (i.e., titratable) correlation between
Wnt inhibitory potency as determined by IC50 values in
mammalian cells and cardiogenic functional activity in a human
ESC-based assay. We were also able to improve the percentage
of cardiogenesis to 176% with compound 29 compared to
100% for the initial “lead” 1 at much lower concentrations (i.e.,
150 nM for 29 compared to 5000 nM for 1, equivalent to 30-
fold greater potency), rendering 29 more amenable to in vitro
and in vivo applications. Because adult hearts contain
cardiogenic progenitor cells that resemble the hESC-derived
progenitor cells used in assays described herein in terms of gene
expression profile and developmental potential,4 it is possible
that Wnt inhibition could promote differentiation of adult
1
procedure described in ref 7 and their H NMR compared to the
literature spectra.7
General Procedure for Intermediates 8. Triethylamine (0.92 mL,
6.6 mmol) and the desired anhydride 7 (6 mmol) were added to a
solution of 4-amino acid 6 (6 mmol) in 5 mL of N,N-
704
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Journal of Medicinal Chemistry
Article
dimethylformamide (DMF). The solution was heated for 16 h at 120
°C. After it returned to room temperature, the solvent was evaporated.
The residue was then dissolved in ethyl acetate (100 mL) and washed
with 1 N HCl (20 mL). The organic layer was washed with brine (25
mL) and dried with anhydrous magnesium sulfate. The solution was
filtered to yield the desired intermediate 8 that was then used directly
or purified by liquid chromatography.
(dd, J = 8.1 and 1.5 Hz, 1H), 8.72−8.82 (m, 2H), 9.89 + 10.07 (br s,
1H).
4-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(naphthalen-1-yl)benzamide (12). Off-white solid, 56% yield;
hexanes/EtOAc 1/1, Rf 0.6; LC-MS [1000 (+)-5.5−254−95:5] tR 3.81
min, 410.88 [M + H] 95.3% at 254 nm; 1H NMR (300 MHz, CDCl3)
1.41−1.51 (m, 2H), 1.58−1.79 (m, 4H), 2.89 (br s, 2H), 3.28 (br s,
2H), 7.46−7.61 (m, 5H), 7.77 (d, J = 8.7 Hz, 1H), 7.86−7.95 (m,
2H), 8−8.12 (m, 3H), 8.24 (br s, 1H).
4-endo-Dihydronorbornylbenzoic Acid (8a). Beige solid, 84%
yield; (DCM)/MeOH 9/1 Rf 0.5; 1H NMR (300 MHz, CD3OD) 1.41
(d, J = 8.4 Hz, 2H), 1.61−1.69 (m, 4H), 2.89 (br s, 2H), 3.26 (br s,
2H), 7.41 (d, J = 8.7 Hz, 2H), 8.04 (s, OH), 8.19 (d, J = 8.7 Hz, 2H).
4-(endo-Dihydronorbornyl)-trans-cyclohexanecarboxylic Acid
(8b). White solid, 35% yield; DCM/MeOH 9/1 Rf 0.8; LC-MS
[1000 (+)-5.5−254−95:5] tR 3.49 min, 246.02 [M − COOH], 291.75
4-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(isoquinolin-1-yl)-trans-cyclohexylcarboxamide (13). White solid,
51% yield; DCM/MeOH 9/1 Rf 0.7; LC-MS [1000 (+)-5.5−254−
1
95:5] tR 4.84 min, 418.02 [M + H] 99.6% at 254 nm; H NMR (300
MHz, CDCl3 + CD3OD) 1.22 (d, J = 8.4 Hz, 2H), 1.51−1.61 (m,
4H), 1.65−1.77 (m, 4H), 2.16 (d, J = 11.7 Hz, 2H), 2.21−2.44 (m
3H), 2.70 (br s, 2H), 2.98 (br s, 2H), 4.03 (tt, J = 12.1 and 3 Hz, 1H),
7.43 (br d, J = 5.4 Hz, 2H), 7.53−7.59 (m, 1H), 7.67 (dt, J = 6.9 and
0.9 Hz, 1H), 7.76 (d, J = 8.1 Hz, 1H), 8.06 (br s, 1H).
1
[M + H]; H NMR (300 MHz, CDCl3) 1.25 (d, J = 8.4 Hz, 2H),
1.46−1.69 (m, 8H), 2.13 (d, J = 11.7 Hz, 2H), 2.21−2.44 (m, 3H),
2.74 (br s, 2H), 2.99 (br s, 2H), 3.98 (tt, J = 12.1 and 3 Hz, 1H), 8.01
(s, OH).
4-(endo-Dihydronorbornyl)-cis-cyclohexanecarboxylic Acid (8c).
White solid, 73% yield; hexanes/EtOAc 1/1, Rf 0.4; LC-MS [1000
(+)-5.5−254−95:5] tR 3.49 min, 246.02 [M − COOH], 291.75 [M +
H]; 1H NMR (300 MHz, CDCl3) 1.23 (d, J = 8.4 Hz, 2H), 1.46−1.62
(m, 8H), 2.28−2.45 (m, 5H), 2.71 (br s, 2H), 2.97 (br s, 2H), 3.96 (tt,
J = 12.1 and 3 Hz, 1H), 8.01 (s, 1H).
4-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(isoquinolin-4-yl)-trans-cyclohexylcarboxamide (14). Yellow solid,
16% yield; DCM/MeOH 9/1 Rf 0.4; LC-MS [1000 (+)-5.5−254−
1
95:5] tR 4.10 min, 418.02 [M + H] 99.9% at 254 nm; H NMR (300
MHz, CDCl3+CD3OD) 1.25 (d, J = 8.4 Hz, 2H), 1.51−1.61 (m, 4H),
1.65−1.77 (m, 4H), 2.15 (br d, J = 12.3 Hz, 2H), 2.25−2.43 (m, 3H),
2.74 (br s, 2H), 2.99 (br s, 2H), 3.99 (tt, J = 12.3 and 3.9 Hz, 1H),
7.58−7.73 (m, 3H), 7.82 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 8.1 Hz, 1H),
8.04 (br s, 1H), 8.74 (s, NH).
4-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(quinolin-4-yl)-trans-cyclohexylcarboxamide (15). Off-white solid,
5% yield; LC-MS [1000 (+)-5.5−254−95:5] tR 2.75 min, 417.88 [M +
H] 99.4% at 254 nm.
4-cis-endo-(3a,4,7,7a-Tetrahydro-1H-4,7-ethanoisoindole-
1,3(2H)-dion-2-yl)-benzoic Acid (8d). Off-white solid, 99% yield;
1
DCM/MeOH 9/1 Rf 0.2; H NMR (300 MHz, DMSO) 1.61−1.38
(m, 4H), 2.16 (s, 2H), 2.92 (s, 2H), 7.41 (d, J = 8.4 Hz, 2H), 8.05 (d, J
= 8.7 Hz, 2H), 13.15 (br s, 1H).
4-cis−endo-[Hexahydro-1H-4,7-ethanoisoindole-1,3(2H)-dion-2-
yl]benzoic Acid (8e). White solid, 12% yield; DCM/MeOH 9/1 Rf
4-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(quinolin-5-yl)-trans-cyclohexylcarboxamide (16). White solid,
40% yield; DCM/MeOH 9/1, Rf 0.4; LC-MS [1000 (+)-5.5−254−
1
0.3; H NMR (300 MHz, CDCl3) 1.68−1.61 (m, 4H), 2.16 (s, 2H),
2.92 (s, 2H), 3.25−3.23 (m, 4H), 8.06−8.03 (m, 2H), 7.29−7.26 (m,
2H).
1
95:5] tR 4.20 min, 418.02 [M + H] 99.5% at 254 nm; H NMR (300
General Procedure for Compounds 9−39 and 53−56. Synthesis
of compounds 9−39 and 53−56 is illustrated in Scheme 1.
Compounds were prepared in a library fashion. One gram of the
appropriate intermediate 8 was heated for 16 h in 5 mL of thionyl
chloride at 70 °C. TLC (DCM/MeOH 9/1) of a reaction aliquot
diluted in methanol showed complete conversion to the methyl ester.
Excess thionyl chloride was removed to afford the acid chloride. The
acid chloride was dissolved in 20 mL of dichloroethane (DCE). A
portion (0.3 mL) of the DCE solution was added to 30 mg of the
appropriate amine and 0.1 mL of pyridine. Solutions were heated at 50
°C for 16 h, and the crude material was purified by liquid
chromatography.
MHz, CDCl3) 1.25 (d, J = 8.4 Hz, 2H), 1.53−1.64 (m, 4H), 1.71−1.85
(m, 4H), 2.17 (br d, J = 12.3 Hz, 2H), 2.21−2.49 (m, 3H), 2.72 (br s,
2H), 2.99 (br s, 2H), 4.08 (tt, J = 12.3 and 3.9 Hz, 1H), 7.42 (dd, J =
8.4 and 3.9 Hz, 1H), 7.66−7.83 (m, 2H), 7.98 (d, J = 7.5 Hz, 1H),
8.17 (d, J = 8.4 Hz, 1H), 8.92 (d, J = 4.5 Hz, 1H), 8.82 (br s, 1H).
4-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(isoquinolin-5-yl)-trans-cyclohexylcarboxamide (17). Yellow
glassy solid, 72% yield; DCM/MeOH 9/1, Rf 0.7; LC-MS [1000
(+)-5.5−254−95:5] tR 3.27 min, 418.02 [M + H] 92.9% at 254 nm;
1H NMR (300 MHz, CDCl3) 1.28 (d, J = 8.4 Hz, 2H), 1.53−1.64 (m,
4H), 1.71−1.85 (m, 4H), 2.22 (br d, J = 12.3 Hz, 2H), 2.24−2.58 (m,
3H), 2.71 (br s, 2H), 2.99 (br s, 2H), 4.08 (tt, J = 12.3 and 3.9 Hz,
1H), 7.55−7.64 (m, 2H), 7.79 (d, J = 8.4 Hz, 1H), 7.95 (s, 1H), 8.13
(d, J = 7.8 Hz, 1H), 8.53 (d, J = 6.3 Hz, 1H), 9.24 (s, 1H).
4-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(quinolin-8-yl)benzamide (9). White solid, 51% yield; hexanes/
EtOAc 3/7 Rf 0.6; LC-MS [1000 (+)-5.5−254−95:5] tR 2.79 min,
411.95 [M + H] 96.3% at 254 nm; 1H NMR (300 MHz, CDCl3) 1.49
(d, J = 8.4 Hz, 2H), 1.58−1.79 (m, 4H), 2.90 (br s, 2H), 3.29 (br s,
2H), 7.47−7.63 (m, 5H), 8.16−8.23 (m, 3H), 8.84 (dd, J = 4.2 and 1.5
Hz, 1H), 8.92 (dd, J = 6.9 and 1.8 Hz, 1H), 10.79 (br s, NH).
4-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(quinolin-8-yl)-trans-cyclohexylcarboxamide (10). Off-white solid,
55% yield; hexanes/EtOAc 1/1, Rf 0.4; LC-MS [1000 (+)-5.5−254−
4-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(isoquinolin-8-yl)-trans-cyclohexylcarboxamide (18). Yellow solid,
16% yield; DCM/MeOH 9/1, Rf 0.8; LC-MS [1000 (+)-5.5−254−
1
95:5] tR 4.04 min, 418.02 [M + H] 90.1% at 254 nm; H NMR (300
MHz, CDCl3) 1.27 (d, J = 6.9 Hz, 2H), 1.53−1.69 (m, 4H), 1.71−1.88
(m, 4H), 2.20 (br d, J = 12.4 Hz, 2H), 2.24−2.58 (m, 4H), 2.73 (br s,
2H), 3.00 (br s, 2H), 4.01−4.16 (m, 1H), 7.57−7.64 (m, 2H), 8.00−
8.06 (m, 1H), 8.20 (br s, 1H), 8.53 (d, J = 5.8 Hz, 1H), 9.43 (s, NH).
4-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(p-tolyl)benzamide (19). Off-white solid, 14% yield; hexanes/
EtOAc 1/1, Rf 0.6; LC-MS [1000 (+)-5.5−254−95:5] tR 4.55 min,
374.88 [M + H] 98.5% at 254 nm; 1H NMR (CDCl3) 1.46 (d, J = 8.4
Hz, 2H), 1.53−1.74 (m, 4H), 2.34 (s, 3H), 2.89 (br s, 2H), 3.27 (br s,
2H), 7.18 (d, J = 8.1 Hz, 2H), 7.41 (d, J = 8.7 Hz, 2H), 7.51 (d, J = 8.1
Hz, 2H), 7.79 (br s, 1H), 7.95 (d, J = 8.4 Hz, 2H).
1
95:5] tR 5.43 min, 418.02 [M + H] 96.3% at 254 nm; H NMR (300
MHz, CDCl3) 1.28 (d, J = 8.4 Hz, 2H), 1.53−1.64 (m, 4H), 1.71−1.85
(m, 4H), 2.21 (br d, J = 12.3 Hz, 2H), 2.42 (br q, J = 12.6 and 3.6 Hz,
2H), 2.56 (tt, J = 12 and 3.3 Hz, 1H), 2.76 (br s, 2H), 3.01 (br s, 2H),
4.09 (tt, J = 12.3 and 3.9 Hz, 1H), 7.43−7.55 (m, 3H), 8.15 (dd, J =
8.1 and 1.5 Hz, 1H), 8.76 (dd, J = 6.9 and 2.1 Hz, 1H), 8.81 (dd, J =
3.9 and 1.5 Hz, 1H), 9.92 (br s, 1H).
4-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(quinolin-8-yl)-cis-cyclohexylcarboxamide (11). Transparent oil,
50% yield; hexanes/EtOAc 1/1, Rf 0.4; LC-MS [1000 (+)-5.5−254−
4-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(4-methoxyphenyl) benzamide (20). Gray solid, 20% yield;
hexanes/EtOAc 1/1, Rf 0.4; LC-MS [1000 (+)-5.5−254−95:5] tR
3.73 min, 390.82 [M + H] 93.4% at 254 nm; 1H NMR (CDCl3) 1.19−
1.26 (m, 2H), 1.45−1.74 (m, 4H), 2.89 (br s, 2H), 3.27 (br s, 2H),
3.82 (s, 3H), 6.91 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.54
(d, J = 8.4 Hz, 2H), 7.78 (s, NH), 7.95 (d, J = 8.4 Hz, 2H).
1
95:5] tR 3.35 min, 418.02 [M + H] 97.4% at 254 nm; H NMR (300
MHz, CDCl3) 1.26 (d, J = 8.4 Hz, 2H), 1.46−1.59 (m, 4H), 1.71−1.85
(m, 4H), 2.18 (br d, J = 12.3 Hz, 2H), 2.34−2.75 (m, 5H), 2.94 (br s,
1H), 3.00 (br s, 1H), 3.98−4.09 (m, 1H), 7.43−7.55 (m, 3H), 8.13
705
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Journal of Medicinal Chemistry
Article
2-cis−endo-(1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(2-methoxyphenyl)benzamide (21). White solid, 91% yield;
hexanes/EtOAc 3/7, Rf 0.6; LC-MS [1000 (+)-5.5−254−95:5] tR
4-[cis−endo-(1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-
yl)]-N-(9H-fluoren-9-on-1-yl)-trans-cyclohexylcarboxamide (30).
Yellow solid, 46% yield; DCM/MeOH 95/5, Rf 0.7; LC-MS [1000
(+)-5.5−254−95:5] tR 4.38 min, 469 [M + H] 99.4% at 254 nm; H
1
1
4.12 min, 390.35 [M + H] 100% at 254 nm; H NMR (300 MHz,
NMR (300 MHz, CDCl3) 1.26 (d, J = 8.4 Hz, 2H), 1.47−1.74 (m,
8H), 2.18 (br d, J = 11.7 Hz, 2H), 2.26−2.48 (m, 3H), 2.75 (br s, 2H),
3.00 (br s, 2H), 4.04 (t, J = 12.6 Hz, 1H), 7.15 (t, J = 7.2 Hz, 1H),
7.25−7.31 (m, 1H), 7.38−7.47 (m, 3H), 7.58 (d, J = 7.5 Hz, 1H), 8.34
(d, J = 8.1 Hz, 1H), 10.17 (br s, NH).
CDCl3) 1.47 (d, J = 8.4 Hz, 2H), 1.58−1.79 (m, 4H), 2.89 (br s, 2H),
3.28 (br s, 2H), 3.92 (s, 3H), 6.92 (dd, J = 7.8 and 1.2 Hz, 1H), 6.99−
7.13 (m, 2H), 7.41−7.45 (m, 2H), 7.97−8.02 (m, 2H), 8.51 (dd, J =
7.8 and 1.2 Hz, 1H), 8.52 (br s, 1H).
2-cis−endo-(1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(4-(dimethylamino)phenyl)benzamide (22). Dark green solid, 36%
yield; DCM/MeOH 9/1, Rf 0.2; LC-MS [1000 (+)-5.5−254−95:5] tR
4-[cis−endo-(1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-
yl)]-N-(pyridin-2-yl)-trans-cyclohexylcarboxamide (31). Off-white
solid, 6% yield; DCM/MeOH 9/1, Rf 0.7; LC-MS [1000 (+)-5.5−
1
2.17 min, 403.95 [M + H] 99.6% at 254 nm; H NMR (CDCl3) 1.46
1
254−95:5] tR 1.27 min, 367.88 [M + H] 97.5% at 254 nm; H NMR
(d, J = 8.4 Hz, 2H), 1.59−1.76 (m, 4H), 2.88 (br s, 2H), 2.94 (s, 6H),
3.27 (br s, 2H), 6.74 (d, J = 9.3 Hz, 2H), 7.38 (d, J = 8.1 Hz, 2H), 7.48
(d, J = 9.3 Hz, 2H), 7.74 (s, NH), 7.94 (d, J = 8.1 Hz, 2H).
(300 MHz, CDCl3) 1.26 (d, J = 8.4 Hz, 2H), 1.51−1.74 (m, 9H), 2.09
(br d, J = 11.7 Hz, 2H), 2.26−2.39 (m, 2H), 2.75 (br s, 2H), 3.01 (br
s, 2H), 4.01−4.17 (m, 1H), 7.01−7.05 (m, 1H), 7.69 (dt, J = 9 and 2.1
Hz, 1H), 7.99 (br s, NH), 8.21 (d, J = 8.7 Hz, 1H), 8.24−8.27 (m,
1H).
2-cis−endo-(1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(4-cyanophenyl)benzamide (23). White solid, 48% yield; DCM/
MeOH 95/5, Rf 0.1; LC-MS [1000 (+)-5.5−254−95:5] tR 3.48 min,
1
4-[cis−endo-(1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-
yl)]-N-(pyridin-3-yl)-trans-cyclohexylcarboxamide (32). White solid,
32% yield; DCM/MeOH 9/1, Rf 0.8; LC-MS [1000 (+)-5.5−254−
347.68 [M + H] 90%; H NMR (CDCl3 + CD3OD) 1.33 (d, J = 8.4
Hz, 2H), 1.55−1.69 (m, 5H), 2.77 (br s, 2H), 3.20 (br s, 1H), 7.26 (d,
J = 8.2 Hz, 2H), 7.53 (d, J = 9.1 Hz, 2H), 7.77 (d, J = 9.1 Hz, 2H),
7.90 (d, J = 8.8 Hz, 2H).
2-cis−endo-(1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(4-acetylphenyl)benzamide (24). Off-white solid, 35% yield;
DCM/MeOH 95/5, Rf 0.4; LC-MS [1000 (+)-5.5−254−95:5] tR
2.98 min, 402.84 [M + H] 97.9% at 254 nm.
1
95:5] tR 2.46 min, 367.88 [M + H] 97.9% at 254 nm; H NMR (300
MHz, CDCl3 + 1 drop of CD3OD) 1.28 (d, J = 8.4 Hz, 2H), 1.53−
1.64 (m, 4H), 1.71−1.85 (m, 4H), 1.86 (br d, J = 12.3 Hz, 2H), 2.12−
2.38 (m, 3H), 2.76 (br s, 2H), 3.01 (br s, 2H), 4.08 (tt, J = 12.3 and
3.9 Hz, 1H), 7.21 (dd, J = 8.4 and 3.6 Hz, 1H), 8.14 (br d, J = 3.6 Hz,
1H), 8.20 (br d, J = 8.4 Hz, 1H), 8.42 (d, J = 2.1 Hz, 1H).
4-(cis−endo-1,3-dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(2-hydroxyphenyl)-trans-cyclohexylcarboxamide (25). Orange
solid, 43% yield; hexanes/EtOAc 1/1, Rf 0.4; LC-MS [1000
4-[cis−endo-(1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-
yl)]-N-(pyridin-4-yl)-trans-cyclohexylcarboxamide (33). Yellow solid,
8% yield; DCM/MeOH 9/1, Rf 0.7; LC-MS [1000 (+)-5.5−254−
1
(+)-5.5−254−95:5] tR 3.01 min, 418 [M + H] 99% at 254 nm; H
1
95:5] tR 1.68 min, 367.89 [M + H] 97.3% at 254 nm; H NMR (300
NMR (300 MHz, CD3OD) 1.23 (d, J = 8.4 Hz, 2H), 1.53−1.71 (m,
4H), 1.75−2.01 (m, 4H), 2.06 (br d, J = 12.3 Hz, 2H), 2.28−2.35 (m,
3H), 2.72 (br s, 2H), 2.98 (br s, 2H), 3.98−4.14 (m, 1H), 6.76−6.87
(m, 2H), 6.96 (dd, J = 7.98 and 1.5 Hz, 1H), 7.58 (dd, J = 7.98 and 1.5
Hz, 1H).
MHz, CDCl3) 1.25 (d, J = 8.4 Hz, 2H), 1.46−1.75 (m, 8H), 2.07 (br
d, J = 12.3 Hz, 2H), 2.23−2.37 (m, 3H), 2.75 (br s, 2H), 3.02 (br s,
2H), 4.08 (tt, J = 12.3 and 3.9 Hz, 1H), 7.31 (br s, NH), 7.48 (d, J =
6.3 Hz, 2H), 8.50 (br d, J = 6.3 Hz, 2H).
4-[cis−endo-(1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-
yl)]-N-(2-acetylpyridin-3-yl)-trans-cyclohexylcarboxamide (34). Off-
white solid, 17% yield; hexanes/EtOAc 7/3, Rf 0.2; LC-MS [1000
(+)-5.5−254−95:5] tR 4.35 min, 409.28 [M + H] 96.4% at 254 nm;
1H NMR (300 MHz, CDCl3) 1.26 (d, J = 8.8 Hz, 2H), 1.51−1.78 (m,
8H), 2.18 (br d, J = 11.8 Hz, 2H), 2.31−2.52 (m, 5H), 2.76 (br s, 1H),
2.80 (s, 3H), 3.03 (br s, 1H), 3.97−4.15 (m, 1H), 7.43−7.48 (m, 1H),
8.35 (dd, J = 4.4 and 0.9 Hz, 1H), 9.09 (dd, J = 8.8 and 0.9 Hz, 1H),
11.62 (br s, NH).
4-[cis−endo-(1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-
yl)]-N-(6-acetylpyridin-3-yl)-trans-cyclohexylcarboxamide (35). Off-
white solid, 18% yield; hexanes/EtOAc 7/3, Rf 0.2; LC-MS [1000
(+)-5.5−254−95:5] tR 3.46 min, 409.48 [M + H] 98.6% at 254 nm;
1H NMR (300 MHz, CDCl3) 1.28 (d, J = 8.4 Hz, 2H), 1.49−1.78 (m,
4-(cis_-endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(2-ethoxyphenyl)-trans-cyclohexylcarboxamide (26). White solid,
73% yield; hexanes/EtOAc 1/1, Rf 0.6; LC-MS [1000 (+)-5.5−254−
95:5] tR 3.46 min, 418 [M + H] 99% at 254 nm; 1H NMR (300 MHz,
CDCl3) 1.23 (d, J = 8.4 Hz, 2H), 1.53−1.71 (m, 10H), 2.06 (br d, J =
12.3 Hz, 2H), 2.28−2.35 (m, 4H), 2.72 (br s, 2H), 2.98 (br s, 2H),
3.98−4.14 (m, 3H), 6.83 (dd, J = 7.5 and 1.5 Hz, 1H), 6.87−7.11 (m,
2H), 7.85 (br s, NH), 8.34 (dd, J = 7.5 and 1.5 Hz, 1H).
Methyl 2-[4-(cis−endo-1,3-dioxooctahydro-2H-4,7-methanoi-
soindol-2-yl)]-N-trans-cyclohexanecarboxamido)benzoate (27).
White solid, 58% yield; DCM/MeOH 95/5 Rf 0.8; LC-MS [1000
1
(+)-5.5−254−95:5] tR 3.58 min, 418 [M + H] 92.4% at 254 nm; H
NMR (300 MHz, CDCl3) 1.24 (d, J = 8.4 Hz, 2H), 1.51−1.72 (m,
8H), 2.14 (br d, J = 11.7 Hz, 2H), 2.26−2.41 (m, 3H), 2.73 (br s, 2H),
2.99 (br s, 2H), 3.91 (s, 3H), 4.03 (t, J = 12.6 Hz, 1H), 7.05 (t, J = 7.8
Hz, 1H), 7.51 (t, J = 8.7 Hz, 1H), 8 (dd, J = 7.8 and 1.2 Hz, 1H), 8.71
(d, J = 8.7 Hz, 1H), 11.15 (br s, 1H).
8H), 2.17 (d, J = 11.7 Hz, 2H), 2.29−2.49 (m, 5H), 2.71 (s, 3H), 2.77
(br s, 1H), 2.99 (br s, 1H), 3.98−4.11 (m, 1H), 7.64 (d, J = 5.23 Hz,
1H), 8.47 (d, J = 5.53 Hz, 1H), 10.05 (s, 1H), 11.20 (s, NH).
4-cis_-endo-[3a,4,7,7a-Tetrahydro-1H-4,7-ethanoisoindole-
1,3(2H)-dion-2-yl]-N-quinolin-8-ylbenzamide (36). Yellow solid, 10%
yield; hexanes/EtOAc 1/1, Rf 0.3; LC-MS [1000 (+)-5.5−254−95:5]
4-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(2-acetylphenyl)-trans-cyclohexylcarboxamide (28). Brown solid,
38% yield; hexanes/EtOAc 1/1, Rf: 0.4; LC-MS [1000 (+)-5.5−254−
1
tR 3.87 min, 423.68 [M + H] 96% at 254 nm; H NMR (300 MHz,
1
95:5] tR 4.40 min, 408.28 [M + H] 94.7% at 254 nm; H NMR (300
CDCl3) 1.70−1.45 (m, 4H), 3.06 (s, 2H), 3.29 (br s, 2H), 6.33−6.31
(m, 2H), 7.60−7.41 (m, 5H), 8.21−8.14 (m, 3H), 8.85−8.83 (m, 1H),
8.93−8.90 (m, 1H), 10.74 (s, 1H).
MHz, CDCl3) 1.26 (d, J = 8.4 Hz, 2H), 1.53−1.85 (m, 8H), 2.16 (br
d, J = 12.3 Hz, 2H), 2.28−2.49 (m, 3H), 2.67 (s, 3H), 2.75 (br s, 2H),
3.01 (br s, 2H), 4.08 (tt, J = 12.3 and 3.9 Hz, 1H), 7.11 (t, J = 7.5 Hz,
1H), 7.55 (t, J = 8.7 and 1.5 Hz, 1H), 7.90 (dd, J = 7.8 and 1.2 Hz,
1H), 8.76 (d, J = 8.4 Hz, 1H), 11.84 (s, NH).
4-cis−endo-[Hexahydro-1H-4,7-ethanoisoindole-1,3(2H)-dion-2-
yl]-N-quinolin-8-ylbenzamide (37). White solid, 51% yield; hexanes/
EtOAc 3/7, Rf 0.6; LC-MS [1000 (+)-5.5−254−95:5] tR 2.79 min,
1
4-[cis−endo-(1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-
yl)]-N-(8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)-trans-cyclohexyl-
carboxamide (29). Brown oil, 51% yield; hexanes/EtOAc 1/1, Rf 0.6;
LC-MS [1000 (+)-5.5−254−95:5] tR 3.73 min, 434.48 [M + H] 97.5%
411.95 [M + H] 96.3% at 254 nm; H NMR (300 MHz, DMSO)
1.48−1.53 (m, 4H), 1.66−1.68 (m, 4H), 2.08−2.10 (m, 2H), 3.08−
3.10 (m, 2H), 7.55 (d, J = 8.8 Hz, 2H), 7.66−7.70 (m, 2H), 7.77 (dd, J
= 8.4 and 1.5 Hz, 1H), 8.17 (d, J = 8.8 Hz, 2H), 8.48 dd, J = 8.4 and
1.5 Hz, 1H), 8.73 (dd, J = 8.4 and 1.5 Hz, 1H), 8.98−8.99 (m, 1H),
10.70 (br s, NH).
4-(cis−endo-8-Methyl-1,3-dioxooctahydro-2H-4,7-methanoi-
soindol-2-yl)-N-quinolin-8-ylbenzamide (38). Orange solid, 64%
yield; hexanes/EtOAc 1/1, Rf 0.4; 1H NMR (300 MHz, DMSO)
1
at 254 nm; H NMR (300 MHz, CDCl3) 1.21 (d, J = 8.4 Hz, 2H),
1.53−1.67 (m, 8H), 2.07−2.15 (m, 4H), 2.19−2.54 (m, 3H), 2.61−
2.78 (m, 4H), 2.88−3.-02 (m, 4H), 3.98−4.14 (m, 1H), 6.91 (dd, J =
7.5 and 1.5 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 8.60 (dd, J = 7.5 and 1.5
Hz, 1H), 12.23 (br s, 1H).
706
dx.doi.org/10.1021/jm2010223 | J. Med. Chem. 2012, 55, 697−708

Journal of Medicinal Chemistry
Article
1.47 (d, J = 8.4 Hz, 1H), 1.58−1.79 (m, 1H), 1.83 (s, 3H), 3.28 (br s,
2H), 3.92 (s, 2H), 5.87 (s, 1H), 7.49−7.55 (m, 2H), 7.52−7.77 (m,
3H), 8.13 (d, J = 8.8 Hz, 2H), 8.45 (dd, J = 8.53 and 0.9 Hz, 1H), 8.69
(dd, J = 8.53 and 0.9 Hz, 1H), 8.95 (dd, J = 4.4 and 0.9 Hz, 1H).
4-[Hexahydro-1H-4,7-epoxyisoindole-1,3(2H)-dion-2-yl]-N-quino-
lin-8-ylbenzamide (39). 7-Oxabicyclo[2.2.1]heptane-2,3-dicarboxylic
anhydride was prepared according to literature procedures.22 Beige
solid, 68% yield; DCM/MeOH 9/1, Rf 0.8; LC-MS [1000 (+)-5.5−
254−95:5] tR 3.23 min, 414 [M + H] >99% at 254 nm; 1H NMR (300
MHz, CDCl3) 1.68−1.75 (m, 2H), 1.94−1.98 (m, 2H), 3.11 (s, 2H),
5.05 (m, 2H), 7.47−7.63 (m, 5H), 8.16−8.19 (m, 2H), 8.21 (br d, J =
1.67 Hz, 1H), 8.85 (dd, J = 1.67, 4.21 Hz, 1H), 8.91 (dd, J = 1.89, 7.08
Hz, 1H), 10.75 (s, 1H).
step a) was synthesized according to the procedure described for
intermediate 8 but starting from 1.64 g of carbic anhydride (10 mmol),
1.67 mL of triethylamine, and 1.52 g of 4-nitroaniline. Yellow solid,
52% yield; DCM/MeOH 9/1, Rf 0.6; LC-MS [1000 (+)-5.5−254−
1
95:5] tR 3.35 min, 96.8% at 254 nm; H NMR (300 MHz, CDCl3)
1.62−1.68 (m, 1H), 1.80−1.86 (m, 1H), 3.49 (dd, J = 1.63, 3.09 Hz,
2H), 3.55 (m, 2H), 6.27 (br t, J = 1.80 Hz, 2H), 7.40−7.44 (m, 2H),
8.26−8.31 (m, 2H).
N-(4-Aminophenyl)-(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-
1,3-dioxo-4,7-methano-2H-isoindole. As shown in Scheme 5, step b,
300 mg of 51 was dissolved in 30 mL of EtOH, and 40 mg of Pd/C
(10%) was added. The mixture was stirred at room temperature for 16
h under a hydrogen atmosphere. The solution was filtered through a
pad of Celite and concentrated to dryness. The crude product was
purified by flash chromatography (hexanes/EtOAc 1/1, Rf 0.2) to
afford 180 mg (67% yield). LC-MS [1000 (+)-10.0−254−95:5] tR
4-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(4-bromo-2-methoxyphenyl)-trans-cyclohexylcarboxamide (53).
Off-white solid, 11% yield; DMC/MeOH 9/1, Rf 0.8; LC-MS [1000
(+)-5.5−254−95:5] tR 3.98 min, 474.48−476.48 [M + H] 90.3% at
254 nm; 1H NMR (300 MHz, CDCl3) 1.11 (d, J = 8.4 Hz, 2H), 1.45−
1.77 (m, 6H), 2.04 (br d, J = 12 Hz, 4H), 2.08−2.41 (m, 4H), 2.74 (br
s, 2H), 2.99 (br s, 2H), 3.87 (s, 3H), 4.04 (t, J = 12.6 Hz, 1H), 7 (s,
1H), 7.07 (d, J = 7 Hz, 1H), 7.87 (br s, NH), 8.24 (d, J = 7 Hz, 1H).
4-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(5-hydroxylpyridin-3-yl)-trans-cyclohexylcarboxamide (54). Beige
solid, 21% yield; DCM/MeOH 9/1, Rf 0.7; LC-MS [1000 (+)-5.5−
1
3.14 min, 257 [M + H]; 98.8% at 254 nm. H NMR (300 MHz,
CDCl3): 1.43−1.72 (m, 6H), 2.85 (m, 2H), 3.21 (m, 2H), 3.81 (br s,
2H, NH2), 6.71−6.75 (m, 2H), 6.98−7.01 (m, 2H).
N-{4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-
methano-2H-isoindol-2-yl]phenyl}quinoline-8-carboxamide (52).
As shown in Scheme 5, step c, quinoline-8-carbonyl chloride was
freshly prepared by treating 135 mg (0.78 mmol) of quinoline-8-
carboxylic acid with an excess of thionyl chloride (622 μL) for 3 h at
60−70 °C. After the mixture returned to room temperature, excess
thionyl chloride was removed by evaporation. The mixture was added
dropwise to a solution of 100 mg of the aniline precursor from step b
(0.39 mmol) and 5 equiv of triethylamine (1.95 mmol) in 7 mL of dry
acetonitrile. The mixture was stirred at room temperature for 3 days,
concentrated in a vacuum, and purified by flash chromatography
(toluene/EtOAc/acetone 6/3/1, Rf 0.5). The product was obtained as
an off-white solid in 87% yield (140 mg). LC-MS [1000 (+)-5.5−
254−95:5] tR 3.45 min, 412 [M + H] >99% at 254 nm; 1H NMR (300
MHz, CDCl3) 1.48−1.75 (m, 6H), 2.88 (m, 2H), 3.25 (m, 2H), 7.27−
7.32 (m, 2H), 7.56 (dd, J = 4.3 and 8.3 Hz, 1H), 7.74 (dd, J = 7.45 and
7.50 Hz, 1H), 7.98−8.03 (m, 2H), 8.02 (br d, J = 1.6 Hz, 1H), 8.34
(dd, J = 1.8 and 8.4 Hz, 1H), 8.96 (dd, J = 1.6 and 7.4 Hz, 1H), 9.01
(dd, J = 1.8 and 4.3 Hz, 1H).
1
254−95:5] tR 1.47 min, 383.88 [M + H] 97.2% at 254 nm; H NMR
(300 MHz, CDCl3 + CD3OD) 1.06 (d, J = 8.4 Hz, 2H), 1.39−1.59
(m, 6H), 1.82 (br d, J = 11.7 Hz, 2H), 2.06−2.38 (m, 3H), 2.58 (br s,
2H), 2.88 (br s, 2H), 4.04 (t, J = 12.6 Hz, 1H), 7.59 (s, 1H), 7.65 (s,
1H), 7.87 (s, 1H).
4-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(2-chloropyridin-3-yl)-trans-cyclohexylcarboxamide (55). Pale
yellow solid, 35% yield; DCM/MeOH 9/1, Rf 0.6; LC-MS [1000
(+)-5.5−254−95:5] tR 2.91 min, 401.68 [M + H] 98.7% at 254 nm;
1H NMR (300 MHz, CDCl3) 1.26 (d, J = 8.4 Hz, 2H), 1.51−1.74 (m,
6H), 2.18 (br d, J = 11.7 Hz, 2H), 2.26−2.48 (m, 3H), 2.73 (br s, 2H),
3.00 (br s, 2H), 3.92−4.02 (m, 1H), 7.25 (dd, J = 8.4 and 4.9 Hz, 1H),
7.69 (br s, NH), 8.10 (dd, J = 4.9 and 1.8 Hz, 1H), 8.73 (dd, J = 8.4
and 1.8 Hz, 1H).
4-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-(2-acetylphenyl)-cis-cyclohexylcarboxamide (56). Beige wax, 40%
yield; hexanes/EtOAc 1/1, Rf 0.5; LC-MS [1000 (+)-5.5−254−95:5]
ASSOCIATED CONTENT
■
1
tR 4.06 min, 408.42 [M + H] 96.2% at 254 nm; H NMR (300 MHz,
S
* Supporting Information
CDCl3) 1.24 (d, J = 8.4 Hz, 2H), 1.48−1.79 (m, 7H), 2.29−2.46 (m,
5H), 2.64 (s, 3H), 2.72 (br s, 2H), 2.98 (br s, 2H), 3.97−4.08 (m,
1H), 7.07−7.13 (m, 1H), 7.54 (tt, J = 8.7 and 1.5 Hz, 1H), 7.89 (td, J
= 8.1 and 1.5 Hz, 1H), 8.76 + 8.87 (dd, J = 8.7 and 0.9 Hz, 1H), 11.80
+ 11.96 (br s, NH).
Additional text, three figures, and one table showing analytical
and spectral characterization data for all compounds obtained
from Schemes 2−4, detailed hESC assay procedures, and data
on cell viability/toxicity, TGFβ inhibition, and chemical
stability for select compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
3-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-quinolin-8-ylbenzamide (45). Compound 45 was synthesized
according to procedures described for compounds 9−39 and 53−56
but 4-aminobenzoic acid was replaced by 3-aminobenzoic acid. White
solid, 96% yield; hexanes/EtOAc 1/1, Rf 0.4; LC-MS [1000 (+)-5.5−
AUTHOR INFORMATION
■
1
254−95:5] tR 3.91 min, 411.82 [M + H] 98.9% at 254 nm; H NMR
Corresponding Author
(500 MHz, DMSO-d6) 1.32−1.37 (m, 2H), 1.57−1.64 (m, 3H), 1.70−
1.72 (m, 1H), 2.70−2.72 (m, 2H), 3.31−3.33 (m, 2H, overlaps with
residual H2O signal), 7.53−7.55 (m, 1H), 7.66−7.70 (m, 2H), 7.75−
7.79 (m, 2H), 7.88−7.89 (m, 1H), 8.10−8.12 (m, 1H), 8.47 (dd, J =
8.4 and 1.5 Hz, 1H), 8.69 (dd, J = 8.4 and 1.5 Hz, 1H), 8.96−8.97 (m,
1H), 10.68 (br s, NH, 1H).
*E-mail mlanier@hbri.org; telephone 001 (858) 458-9305; fax
001 (858) 458-9311.
Author Contributions
∥These authors contributed equally.
5-(cis−endo-1,3-Dioxooctahydro-2H-4,7-methanoisoindol-2-yl)-
N-quinolin-8-ylfuran-2-carboxamide (46). Compound 46 was
synthesized according to procedures described for compounds 41−
44 but 4-nitrobenzoic acid was replaced by 5-nitrofuroic acid. Orange
oil, 20% yield; DCM/MeOH 9/1, Rf 0.7; LC-MS [1000 (+)-5.5−254−
95:5] tR 4.12 min, 401.82 [M + H] 90% at 254 nm; 1H NMR (CDCl3)
1.2−1.82 (m, 5H), 2.92 (br s, 2H), 3.33 (br s, 2H), 7.36 (d, J = 3.6 Hz,
1H), 7.43−7.48 (m, 3H), 7.54−7.58 (m, 1H), 8.17 (dd, J = 8.25 and
1.65 Hz, 1H), 8.76 (dd, J = 6.9 and 2.1 Hz, 1H), 8.87 (dd, J = 3.9 and
1.5 Hz, 1H), 10.74 (br s, NH).
ACKNOWLEDGMENTS
■
We thank Alyssa Morgosh and Claire Johns at HBRI for their
technical assistance with compound preparation during their
internship at HBRI, and Drs. Zebin Xia and Marcia I. Dawson
(SBMRI) for kindly providing 3 for biological testing. We thank
the NIH (R01HL059502 and R33HL088266 to M.M.) and the
California Institute for Regenerative Medicine (CIRM, RC1-
000132 to M.M.) and the T Foundation (to J.R.C.) for their
generous financial support. We gratefully acknowledge
postdoctoral fellowships from the German Research Founda-
N-(4-Nitrophenyl)-(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-
dioxo-4,7-methano-2H-isoindole (51). Compound 51 (Scheme 5,
707
dx.doi.org/10.1021/jm2010223 | J. Med. Chem. 2012, 55, 697−708

Journal of Medicinal Chemistry
Article
inhibitors of the Wnt/wingless signaling pathway. Proc. Natl. Acad. Sci.
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Heart Association (to E.W.).
ABBREVIATIONS:
■
hERG, human ether-a-go-go-related gene; hESC, human
embryonic stem cells; HCS, high content screen; HEK293T,
human embryonic kidney 293T; IWR, inhibitor of Wnt
response
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