Synthesis and SAR of b-annulated 1,4-dihydropyridines define cardiomyogenic compounds as novel inhibitors of TGFβ signaling

Article abstract of DOI:10.1021/jm301144g

A medium-throughput murine embryonic stem cell (mESC)-based high-content screening of 17000 small molecules for cardiogenesis led to the identification of a b-annulated 1,4-dihydropyridine (1,4-DHP) that inhibited transforming growth factor β (TGFβ)/Smad signaling by clearing the type II TGFβ receptor from the cell surface. Because this is an unprecedented mechanism of action, we explored the series' structure-activity relationship (SAR) based on TGFβ inhibition, and evaluated SAR aspects for cell-surface clearance of TGFβ receptor II (TGFBR2) and for biological activity in mESCs. We determined a pharmacophore and generated 1,4-DHPs with IC ₅₀s for TGFβ inhibition in the nanomolar range (e.g., compound 28, 170 nM). Stereochemical consequences of a chiral center at the 4-position was evaluated, revealing 10- to 15-fold more potent TGFβ inhibition for the (+)- than the (-) enantiomer. This stereopreference was not observed for the low level inhibition against Activin A signaling and was reversed for effects on calcium handling in HL-1 cells.

Full text of DOI:10.1021/jm301144g

Article
pubs.acs.org/jmc
Synthesis and SAR of b‑Annulated 1,4-Dihydropyridines Define
Cardiomyogenic Compounds as Novel Inhibitors of TGFβ Signaling
Dennis Schade,*,†,‡,§,⊥,# Marion Lanier,†,§,∥,# Erik Willems,*,‡,§ Karl Okolotowicz,† Paul Bushway,‡
Christine Wahlquist,‡ Cynthia Gilley,† 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: A medium-throughput murine embryonic stem cell
(mESC)-based high-content screening of 17000 small molecules for
cardiogenesis led to the identification of a b-annulated 1,4-
dihydropyridine (1,4-DHP) that inhibited transforming growth
factor β (TGFβ)/Smad signaling by clearing the type II TGFβ
receptor from the cell surface. Because this is an unprecedented
mechanism of action, we explored the series’ structure−activity
relationship (SAR) based on TGFβ inhibition, and evaluated SAR
aspects for cell-surface clearance of TGFβ receptor II (TGFBR2)
and for biological activity in mESCs. We determined a
pharmacophore and generated 1,4-DHPs with IC50s for TGFβ
inhibition in the nanomolar range (e.g., compound 28, 170 nM).
Stereochemical consequences of a chiral center at the 4-position was
evaluated, revealing 10- to 15-fold more potent TGFβ inhibition for
the (+)- than the (−) enantiomer. This stereopreference was not observed for the low level inhibition against Activin A signaling
and was reversed for effects on calcium handling in HL-1 cells.
INTRODUCTION
■
Developing small molecules as therapeutics for regeneration or
repair of damaged tissue in the heart is a long-standing goal of
regenerative medicine.1 However, only a small number of
chemical mediators of cardiogenesis have been reported, and
most are either not very potent or promiscuous in their action.2
We recently reported on a distinct class of condensed 1,4-
dihydropyridines (1,4-DHPs) that derived from a medium-
throughput phenotypic screen in mouse embryonic stem cells
Figure 1. Chemical structures of 1,4-DHPs: 1 = cardiogenic “hit” from
a mESC high-content screen, 2 = calcium channel blocker nifedipine, 3
(mESCs).3 The annulated 1,4-DHP “hit” compound 1 was
among the most effective candidates for driving mESC-derived
mesoderm to cardiac fate.
= b-annulated 1,4-DHPs with weak calcium modulating activity
reported in the literature.4−6
The 1,4-DHP heterocycle is a frequently found structural
feature of various bioactive molecules. Although 1,4-DHPs have
received the most attention as calcium channel modulators,
with nifedipine (2) as the first marketed (1975) calcium
antagonist (Figure 1), they are also known to possess
vasodilator, antihypertensive, bronchodilator, antiatheroscler-
otic, hepatoprotective, antitumor, antimutagenic, geroprotec-
tive, and antidiabetic properties.7,8 A few closely related
structural analogues of 1 have been reported to have calcium
channel modulating activity (e.g., 3, Figure 1).4−6 Along with 1,
1,4-DHPs such as 3 show moderate calcium channel blockade,
i.e., typically >100-fold lower potency and 2−5-fold less efficacy
(max effects) than nifedipine-type calcium channel modulators.
We previously reported that nifedipine (2) as well as a few 3-
type 1,4-DHPs with calcium inhibitory potency did not
promote cardiac differentiation from ESCs, thus excluding
calcium antagonism as the mechanism of action.3 The key
mode of action of 1,4-DHP “hit” compound 1 in ESC
differentiation is the inhibition of TGFβ signaling. TGFβ is a
cytokine that controls many cellular functions that underlie
normal and pathological processes, including cell proliferation,
differentiation, angiogenesis, and wound healing.9 The TGFβ
Received: August 3, 2012
© XXXX American Chemical Society
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superfamily of secreted proteins includes three isoforms of
TGFβ (i.e., TGFβ-1, TGFβ-2, TGFβ-3) as well as activins,
bone morphogenetic proteins (BMPs), myostatin, nodal, and
other growth and differentiation factors.10 All superfamily
members bind a complex of type I and type II transmembrane
receptors on the cell surface. For TGFβ, signaling occurs
following initial binding to the TGFβ receptor type II
(TGFBR2) that recruits TGFβ receptor type I (TGFBR1,
activin-like kinase 5, Alk-5) into an active signaling complex
that is internalized into the cell concomitant with phosphor-
ylation of the effectors, Smad2 and Smad3. These in turn
complex with Smad4 and in the nucleus recognize and activate
transcription from the promoters of TGFβ-responsive genes.10
The TGFβ pathway has been considered an attractive
therapeutic target for a variety of diseases and has been
clinically validated for distinct cancer types and fibrosis.11−13
However, TGFβ can either promote or suppress tumor growth
and metastasis depending on cell-type, cellular context
(microenvironment), and developmental stage of the tumor
and is important for tissue integrity.12 Thus, the generation of
an inhibitor with a selective molecular mechanism of action
would aid in understanding the potential for therapeutic
applications. The 1,4-DHPs reported herein show a distinct
mechanism of action because they do not inhibit TGFBR1/2
kinases directly but rather direct TGFβ type II receptors to the
proteasome, thereby promoting their selective degradation,
hence the name “inducers of TGFβ type II receptor
degradation” (ITDs).3 Given the novel mechanism of action,
we report the synthesis and structure−activity relationship
(SAR) studies of 50 selected 1,4-DHPs based on the initial “hit”
compound 1. The primary SAR studies were conducted based
on dose-dependent inhibition of TGFβ signaling. SAR
information was then evaluated in a mechanistic assay that
quantified the degree of TGFBR2 down-regulation in HEK-
293T cells and in complex mESC-based phenotypic assays that
included assays for cardiomyogenesis and mesoderm formation.
Herein, we describe key pharmacophoric elements of this
subclass of 1,4-DHPs for TGFβ inhibition and provide insight
into stereochemical requirements for this activity.
an aqueous/organic extraction, compounds were purified by a
combination of flash chromatography and PTLC to afford 20−
50% yield of the desired compounds 7−30. When the required
aldehyde 5 was not commercially available, intermediates 31
were prepared and further modified via Suzuki coupling under
microwave conditions to afford compounds 32−43 (Scheme
1B) in overall yields between 35 and 85%.
For synthesis of the free carboxylic acid 44, methyl ester 10
was treated with 1 M BCl3 in CH2Cl2 to afford 75% of the
desired product. N-Methylated 1,4-DHP 45 was prepared by
treating 30 with methyl iodide in DMSO.15 A third class of
compound synthesized included fully oxidized pyridine
analogues of 1,4-DHPs. For example, 46 was synthesized in
good yield by treating 30 with periodic acid and sodium nitrite
in the presence of wet silica gel.16
Because 1,4-DHPs contain a center of chirality, it was
important to prepare the (+)- and (−)-enantiomers and test
them separately in functional biological assays. Optically pure
(+)- and (−)-enantiomers of selected 1,4-DHPs were prepared
based on a modified procedure previously described by Shan et
al. (2002) that involved the separation of diastereomeric esters
(Scheme 2).17 Chiral β-ketoester 51 was prepared in two steps.
First, L-threonine was esterified and subsequently treated with
3-nitrobenzoyl chloride to yield benzamide 48. Treatment of
48 with diketene 49 or 2,2,6-trimethyl-4H-1,3-dioxin-4-one
(50) afforded the β-ketoester 51 (Scheme 2). Compound 51
was then treated under Hantzsch reaction conditions described
in Scheme 1 to afford 52−54. The diastereomeric mixtures 52−
54 were obtained in good yield (i.e., 50−70%), but a significant
amount of material was lost during chromatographic
purification. An isolated yield for a single diastereomer was
approximately 10%. Subsequent removal of the chiral ester with
DBU (yields: 39−88%) and re-esterification (yields: 76−95%)
afforded the desired enantiomers of 1, 23, and 57 in moderate
to good yields. For esterification, an alkylation procedure was
superior to other methods such as treatment of the acid in
alcohol with different catalysts. The 3-(5-indole)-substituted
1,4-DHP enantiomers (i.e., (+)- and (−)-43) were prepared by
synthesizing and separating diastereomers of the 3-bromo
intermediate 57 and conducting the required Suzuki coupling
with 1H-indol-5-ylboronic acid (58) in the last step (Scheme
2).
CHEMICAL SYNTHESIS
■
The 1,4-DHPs were prepared in a library fashion following a
procedure described by Ko et al. (2005).14 In a typical
preparation, 1 equiv of a dimedone derivative 4, 1 equiv of the
desired aldehyde 5, 1 equiv of a β-ketoester 6, 1 equiv of
ammonium acetate, and 0.3 equiv of iodine were stirred
overnight at room temperature in ethanol (Scheme 1A). After
RESULTS AND DISCUSSION
■
From over 200 1,4-DHPs that were synthesized and screened
against TGFβ in a transient Smad4-binding element (SBE4)-
reporter system in HEK-293T cells, about 50 1,4-DHPs were
selected for a detailed SAR investigation by examining
inhibition of TGFβ-2 signaling. The 50 1,4-DHPs were
selected based on their functional activities as TGFβ inhibitors
and/or structural features to gain insight into a pharmacophore.
The primary assay was TGFβ inhibition and, for key
compounds, we evaluated whether the SAR was similar in
biologically more complex cardiac differentiation and meso-
derm inhibition assays.
Scheme 1. Synthesis of Racemic 1,4-DHPs
To exclude the possibility that TGFβ inhibitory effects of 1,4-
DHPs could be mediated through cell toxicity, representative
1,4-DHPs were tested in cell viability assays (i.e., resazurin and
ATP quantification assays) and in an acute toxicity assay that
indicated cell membrane integrity by quantifying “leakage” of
glucose-6-phosphate dehydrogenase (G6PDH) (data not
shown). From these experiments, we concluded that in HEK-
293T and mESC cells, 1,4-DHPs had no apparent adverse
B
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Scheme 2. Synthesis of 1,4-DHP Enantiomers Using a Modification of a Literature Protocol (Shan et al., 2002)17
effects on cell viability as long as the final concentration did not
exceed 10 μM. Cellular concentrations of 1,4-DHPs should
ideally be between 2.5 and 7.5 μM for reliable comparison of
maximum effects.
modification of the center of chirality (i.e., 4-position of the 1,4-
DHP) was examined to determine the stereoselectivity and
SAR features of the key pharmacophore.
Our initial SAR studies focused on the bicyclic core. We
found that N-alkylation (45, inactive) largely abrogated TGFβ
inhibition potency, indicating an H-donor function at position
1 might be important (Figure 3). Aromatization (i.e., full
Inhibition of TGFβ and Activin A Signaling. An SBE4
reporter assay in HEK-293T cells was used to evaluate 1,4-
DHPs and controls for inhibition of TGFβ-2 and Activin A
signaling in dose−response (i.e., 0.001−5.0 μM). Activin A and
TGFβ bind homologous, yet different type I and type II
receptors and signal through common intracellular cascades. All
previously reported TGFβ inhibitors bind and inhibit the
structurally similar kinase domains and hence indiscriminately
block signaling from TGFBR1 (Alk-5) and ActR1 (Alk-4/
7).3,10
To gather more detailed SAR information of the synthetic b-
annulated 1,4-DHPs, “hit” scaffold 1 was theoretically divided
into constituent parts as shown in Figure 2.
Figure 3. Illustration of variations in the 1,4-DHP core region.
oxidation) to a pyridine derivative (46, inactive) caused
complete loss of functional activity, underscoring the
assumption that an H-donor at the 1-position and a tetrahedral
configuration at the 4-position (instead of a planar ring system)
were apparently required. Molecular dissection of the annulated
ring system led to ring-opened analogues (59, Figure 3) that
were devoid of TGFβ inhibitory potency. The evaluation of
TGFβ inhibition was extended to include nifedipine (2, Figure
1) that completely lacked TGFβ inhibitory and cardiogenic
activities,3 indicating that (a) a certain degree of rigidity and (b)
hydrophobic interactions of a moiety at the annulation site
were apparently required. Therefore, the 1,4,5,6,7,8-hexahy-
droquinoline (i.e., b-annulated 1,4-DHP) scaffold was retained
in design and construction of the pharmacophore, and
modifications of the R1-R5 residues (Figure 2) were systemati-
cally done.
Figure 2. Systematic substructure depiction of b-annulated 1,4-DHPs
for regions modified in the SAR studies of TGFβ inhibition.
From the initially evaluated 1,4-DHPs, we determined that 4-
phenyl substitution of 1,4-DHPs (highlighted in red, Figure 2)
was important for TGFβ inhibitory activity, and we thus
primarily modified the R1-substituents (orange) of the 4-phenyl
moiety, the 3- (green) and 2- (magenta) residues, as well as the
condensed ring system highlighted in blue. Finally, the effect of
Table 1 summarizes TGFβ inhibition data of 4′-R1-
substituted 1,4-DHPs and clearly showed that bulky aliphatic
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Table 1. Effect of the R1 Region (4′-Substituents) on TGFβ
Table 2. Effect of the R1 Region (4-Biphenyl Substituents)
on TGFβ Inhibition
a
a
Inhibition
compd
Y
Z
IC50 (μM)
% inhibition (2.5 μM)
10
13
14
34
35
36
37
38
H
H
H
F
1.49 0.43
1.84 0.35
1.35 0.16
0.86 0.15
0.73 0.41
1.52 0.18
0.89 0.01
2.06 0.09
44
59
78
88
91
73
87
58
5
1
1
2
6
1
2
1
4′′-CO2Me
4′′-Me
4′′-CF3
3′′-Cl
2′′-Me
2′′-F
H
H
H
H
H
H
a
Data represent means SD of n = 2−4 independent experiments.
Table 3. Effect of the R1 Region (3′-Phenyl Substituents) on
a
TGFβ Inhibition
a
Data represent means SD of n = 2−4 independent experiments.
For this compound a single experiment was done.
b
substituents (9, 32 IC50s = 0.56 and 0.59 μM, respectively)
were superior to aromatic ones. Five- or six-membered
heterocycles (11, 12 IC50s = 8.80 and 3.57 μM, respectively)
or the bicyclic indole substituent (33 IC50 = 4.05 μM) were less
potent. The conclusion was that a large 4′-substituent was
required for potent TGFβ inhibitory activity, and this was
supported by the lack of inhibition by 7 or 8 (IC50 >10 μM, <
10% inhibition at 2.5 μM). Presumably, considerable hydro-
phobic interactions were present in this part of the molecule for
potent TGFβ inhibitory activity.
A few aromatic substituents of the biphenyl derivative 10
(IC50 = 1.49 μM, Table 1) were synthesized and tested to
examine the effects of steric and electronic interactions (Table
2). A number of substituents at positions 2′′, 3′′, or 4′′
maintained or increased the potency for TGFβ inhibition. For
example, greater potency was observed with a small substituent
at the para-position (i.e., 4′′-CH3, 34, IC50 = 0.86 μM, or 4′′-
CF3, 35, IC50 = 0.73 μM). It is notable that, although the
examples are limited, the possibility for torsion/rotation within
the biphenyl group (i.e., around the 4′−1′′ bond) due to the 2′-
substituent (e.g., 2′′-CH3, 37, IC50 = 0.89 μM) did not
apparently affect TGFβ inhibition potency compared to the
unsubstituted biphenyl derivative 10.
To examine the effect of aryl substituents in greater detail, a
small set of 3′-aryl substituted 1,4-DHPs was investigated
(Table 3). Among the compounds examined, only the 3′-(5-
indolyl) derivative 43 (IC50 = 4.45 μM) showed modest TGFβ
inhibition. All other 3′-aryl derivatives examined (39−42) were
poorly potent inhibitors (IC50 > 10 μM).
a
Data represent means SD of n = 2−4 independent experiments.
Another important region for SAR-based optimization of
structure 1 was the 3-carboxylic ester moiety. Accordingly, a
series of esters were synthesized and tested and revealed that
TGFβ inhibition increased with lipophilicity in the order CH3 <
CH2CH3 < CH2CF3 < CH2CH(CH3)2 (i.e., 10, 1, 15, and 16
IC50s = 1.49, 0.85, 0.76, and 0.53 μM, respectively), suggesting
that a hydrophobic interaction with the cellular target was
critical in that region of the molecule (Table 4). Large R2
groups such as a 2-(4-methoxy)benzyl ester could apparently
still be accommodated by the molecular target and afforded
potent inhibitors (i.e., 18, IC50 = 0.83 μM). The carboxamide
D
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a
Table 4. Effect of the R2 Region (Carboxylic Esters and
Amides) on TGFβ Inhibition
Table 6. Effect of the R4,5 Region on TGFβ Inhibition
a
compd
R4
R5
IC50 (μM)
% inhibition (2.5 μM)
10
21
22
23
24
25
26
27
Me
H
H
H
H
H
H
H
Me
Me
Et
nPr
iPr
iBu
Ph
1.49 0.43
2.73 0.75
0.79 0.15
0.53 0.20
0.94 0.44
0.59 0.21
1.92 0.20
3.97 0.94
44
48
92
96
83
94
61
40
5
6
1
2
8
2
1
6
4-(N(Me)2)Ph
a
Data represent means SD of n = 2−4 independent experiments.
further increase in size of the R5 group by introducing phenyl-
substituents resulted in analogues that were less potent (i.e., 26
and 27, IC50s = 1.92 and 3.97 μM, respectively).
Mono- and dimethyl substitution (i.e., 10 and 21, IC50s =
1.49 and 2.73 μM) did not alter the potency of TGFβ
inhibition. This was even more apparent when the maximum
inhibitory effects (44% and 48% inhibition for 10 and 21,
respectively) were examined.
a
Data represent means SD of n = 2−4 independent experiments.
analogue 19 (IC50 = 4.06 μM) was also modestly potent but
about 4-fold less potent than simple alkyl esters. Consistent
with these findings, we observed that the free carboxylic acid
was not potent (i.e., 44, IC50 > 10 μM).
Next, the effect of the size of the 1,4-DHP-core side chain at
position 2 was examined. As shown in Table 5, increasing the
To elaborate the most potent TGFβ inhibitors, optimization
of TGFβ inhibition potency in the 1,4-DHP series required
combining optimal substituents from various first generation
compounds. A “mix-and-match” approach to optimizing
compounds was taken to examine the concept of improving
potency by additive substituent effects. Thus, a 1,4,5,6,7,8-
hexahydroquinoline was designed that comprised a bulky
aliphatic substituent in the 4′-(4-phenyl) position and the
single 7-(n-propyl) residue in combination with an ethyl or iso-
butylcarboxylic ester. Indeed, TGFβ inhibition potency was
further increased with this substitution pattern because both
ethyl (i.e., 28) and butyl (i.e., 29) carboxylic ester derivatives
had IC50 values of 170 nM. Optimized second-generation
structures 28 and 29 were almost as potent as the reported
TGFBR1 kinase inhibitor SB-431542 that had an IC50 of 66 nM
in the TGFβ assay (Figure 4) or GW788388 (IC50 = 93 nM in
a cell-based TGFβ assay)18 or LY364947 (IC50 = 40 nM in a
cell-based TGFβ assay).3,19
a
Table 5. Effect of the R3 Region on TGFβ Inhibition
compd
R3
IC50 (μM)
% inhibition (2.5 μM)
1
Me
nPr
0.85 0.30
0.83 0.09
83
88
8
1
20
a
An important determinant of the potency of 1,4-DHPs on
TGFβ signaling inhibition was stereopreference of the
molecules based on a center of chirality at the 4-position.
Along these lines, it is well-recognized that chirality was crucial
for the pharmacological properties of calcium channel
modulating 1,4-DHPs. For example, the (−)-(S)-enantiomer
of BAY K8644 is approximately 10-fold more potent as a
calcium channel agonist compared to the (+)-(R)-antipode that
acts as an antagonist.20
To understand the structural basis for the stereopreference of
the 1,4-DHP enantiomers on TGFβ inhibition, several
enantiomeric pairs of 1,4-DHPs were synthesized and tested.
To examine a possible range of stereoselective function, three
DHPs were selected that covered a broad range of TGFβ
inhibition potency and cardiogenic activity. Accordingly, 1,4-
Data represent means SD of n = 2−4 independent experiments.
bulk of the R3-position did not apparently affect the potency of
TGFβ inhibition. For example, the 3-propyl substituted 1,4-
DHP (i.e., 20, IC50 = 0.83 μM) was equipotent as the 3-methyl-
substituted analogue (i.e.,1, IC50 = 0.85 μM).
As described above, the annulated region of the 1,4-DHP
appeared to be important for TFGβ inhibition. Accordingly, the
R4,5 region of 1 was modified. As shown in Table 6, mono- and
disubstitution as well as size and branching of alkyl residues at
the 7-position influenced the potency of TGFβ inhibition.
Potency increased for monosubstituted (R5) compounds in the
order of increasing size CH3 < CH2CH3 < CH2CH2CH3 (i.e.,
21 < 22 < 23, IC50s = 2.73, 0.79, and 0.53 μM, respectively). A
E
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Figure 4. Effect of stereochemistry of 1,4-DHPs 1, 23, and 43 on TGFβ and Activin A inhibition. (A) Dose−response profiles of (+)- and
(−)-enantiomers for TGFβ and Activin A inhibition. (B) Table of TGFβ and Activin A inhibition parameters, data represent means SD of n = 2−5
independent experiments. SB = SB-431542, a literature-reported TGFβ inhibitor (Alk-4/5/7 inhibitor).21
DHPs 1, 23, and 43 were chosen as probe molecules and both
enantiomers of each were prepared in >96% ee as described
above. Compounds 1 (TGFβ IC50 = 0.85 μM) and 23 (TGFβ
IC50 = 0.53 μM) were among the most potent compounds
prepared, whereas 43 (TGFβ IC50 = 4.45 μM) represented a
moderately potent 1,4-DHP derivative.
the substitution pattern of the 4-phenyl group was found to be
critical. Our previous findings for instance showed that a 4-(4′′-
trifluoromethyl)biphenyl substituted DHP (compound 35)
exhibited significantly greater pathway selectivity than a 4-(4′′-
methyl)biphenyl substituted analogue (compound 34).3 Here,
we observed that 3′-substitution decreased TGFβ inhibitory
potency as well as selectivity over Activin A and diminished the
selective biological activity of the (+)- and (−)-enantiomers.
Biological Activity in Secondary Functional Assays
Correlates with TGFβ Inhibition but Not with Calcium
Antagonism. We next asked if the SAR profiles of the 1,4-
DHPs for TGFβ inhibition correlated with their stem cell
activity. To address this question, we focused primarily on the
enantiomers of 1, 23, and 43, as well as selected 1,4-DHPs that
covered a wide range of TGFβ inhibition potency. In the
mESC-based phenotypic assays, we made use of the biphasic
role that we discovered for this class of DHPs, i.e., (1)
inhibition of mesoderm formation and, consequently, inhibition
of cardiogenesis when 1,4-DHPs were administered to cells
early during differentiation (days 1−3) and (2) promotion of
cardiogenesis when given during later phases of differentiation
(days 3−5).3
Effects of 1,4-DHP Enantiomers on Cardiogenesis in
mESCs. 1,4-DHPs were administered to mESCs on days 3−5
of differentiation and α-myosin heavy chain-green fluorescent
protein (Myh6-GFP) levels, as an index of cardiogenesis, were
quantified by imaging on day 10. Figure 5A shows a
representative dose−response curve for cardiogenesis for (+)-
and (−)-enantiomers of 1. On the basis of the increase in
fluorescence intensity at day 10, the amount of Myh6-GFP was
significantly increased for (+)-1 compared to (−)-1.
Stimulation of cardiomyocyte differentiation by (+)-1 reached
a maximum at 5 μM and then decreased with increasing
concentration of (+)-1. This type of dose−response curve has
also been observed for other cardiogenic compounds (i.e.,
inhibitors of the canonical Wnt pathway) in human ESCs.22,23
We speculate this may arise due to a number of contributions
including very strong TGFβ inhibition that interferes with cell
differentiation or proliferation. It is unlikely that the sigmoidal
effect on cell growth was due to cell toxicity because overall cell
Figure 4 summarizes the effect of the compounds on TGFβ
inhibition relative to Activin A inhibition. The functionally
active (+)-enantiomers were 10- to 15-fold more potent than
their (−)-antipodes, with IC50s of 0.46 μM for (+)-1 and 0.40
μM for (+)-23 compared to 6.90 and 4.06 μM for their
(−)-enantiomers (Figure 4B). Stereopreference for and
potency of Activin A inhibition, however, was much less than
inhibition of TGFβ suggesting a different, presumably non-
selective mode of action. Interestingly, in contrast to 1 and 23,
neither enantiomer of 43 showed any selectivity for TGFβ or
Activin A inhibition. These findings indicated that the
substitution pattern of the 4-phenyl group was not only critical
with regards to potency of TGFβ inhibition (Tables 1−3) but
also for pathway selectivity (i.e., selective inhibition of TGFβ
over Activin A). In the case of 43, the 3′-(indol-5-yl)
substituent decreased TGFβ inhibitory potency as well as
selectivity over Activin A and diminished the selective biological
activity of the (+)- and (−)-enantiomers. For 1 and 23,
(+)-stereochemistry at the 4-position was apparently critical for
greater potency in TGFβ inhibition. For compounds 21−27
(Table 6), a second center of chirality at the 7-position was
present. However, on the basis of data for 7-mono- and 7,7-
disubstituted 1,4-DHPs, it appeared unlikely that this position
would be as crucial for stereoselective binding as that of the 4-
position. Thus, we did not investigate a role of the
stereochemistry of the 7-position on TGFβ or Activin A
inhibition.
In summary, the key pharmacophoric structural elements for
TGFβ inhibition included a 1,4,5,6,7,8-hexahydroquinoline
core, a 4-phenyl substituent with bulky aliphatic residues at
the 4′-position, and a lipophilic carboxylic ester. Evaluation of
the center of chirality at the 4-position revealed that TGFβ
inhibition was associated with the (+)-enantiomer. Moreover,
with regards to pathway selectivity for TGFβ versus Activin A,
F
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Figure 5. Characterization of 1,4-DHP enantiomers in secondary functional assays. (A) Cardiogenesis in Myh6-GFP mESC cells: lower panel
illustrates representative whole-well images of mESCs on day 10 of differentiation after treatment with (+)- or (−)-enantiomers of 1 compared to
media and DMSO controls, upper panel shows the corresponding plot after image analysis (mean SD, normalized to DMSO = 1-fold). (B)
Cardiogenesis in Myh6-GFP mESC cells: (+)- or (−)-enantiomers of 23 and 43 compared to (rac)-1 at 2.5 μM (mean SD, normalized to DMSO
= 1-fold). (C) TGFBR2 degradation (down-regulation) in HEK-293T cells: % HA-tagged TGFBR2 positive cells after treatment with (+)- or
(−)-enantiomers of 1, 23, or 43 at 3 μM doses (mean SD, normalized to DMSO = 100%). (D) Mesoderm inhibition with J1 Brachyury/T-GFP
mESC cells: % T-GFP positive cells after treatment with (+)- or (−)-enantiomers of 1, 23, or 43 at 1 and 3 μM (mean SD, normalized to DMSO
= 100%). (E) 3D correlation plot: x-axis, TGFβ/Smad inhibition values (log IC50s); y-axis, mesoderm inhibition with J1 Brachyury/T-GFP mESC
cells at 3 μM 1,4-DHP doses (% Brachyury/T-GFP positive cells, normalized to DMSO = 100%); z-axis, TGFBR2 degradation values at 3 μM 1,4-
DHP doses (% HA-TGFBR2 positive cells, normalized to DMSO = 100%). (F) Calcium transient peak values in HL-1 cells: comparison of Ca2+
transient peak values (Fluo4 levels, average pixel intensity) in the presence of (+)- and (−)-enantiomers of 1 and 23 (mean SEM).
viability was normal, as assessed by viability assays in mESCs
and HEK-293T cells.3 Accordingly, determination of mean-
ingful SAR information (i.e., IC50 or EC50 values) deduced from
10-day phenotypic read-outs were complex to interpret but
normalized data at lower concentrations afforded reliable and
reproducible data. It was apparent that the optimum
concentration for 1,4-DHPs to promote cardiogenesis in
mESCs was in the range from 2.5 to 7.5 μM for (+)-1.
Similar to the enantiomers of 1, selective stimulation of
cardiomyogenesis was observed for the (+)-enantiomer of 23
compared to the (−)-enantiomer of 23 (Figure 5B). Moreover,
data in Figure 5B suggested a difference in efficacy of
cardiogenesis for the 1,4-DHPs 1, 23, and 43 that correlated
with their potency for TGFβ inhibition.
Effects of 1,4-DHP Enantiomers on TGFBR2 Degrada-
tion. To examine if the SAR profile for TGFβ inhibition
correlated with that for receptor clearance, we studied the
clearance of TGFBR2 from the membrane using an
extracellular hemagglutinin (HA)-tagged TGFBR2 in HEK-
293T cells. Compared to DMSO-treated control cells, through
live immunostaining of the extracellular HA-tag, flow cytometry
analysis allowed quantification of cell surface TGFBR2 in 1,4-
DHP-treated HEK-293T cells. In the presence of 2.5 μM of 1
or 23, the (+)-enantiomers selectively induced a greater
clearance of TGFBR2 from the cell surface compared to their
(−)-antipodes (Figure 5C). In good agreement with the data of
Figure 4, the enantiomers of 43 showed no detectable
stereopreference for TGFBR2 clearance (Figure 5C). In
summary, the TGFBR2 clearance data (Figure 5C) was in
excellent agreement with TGFβ inhibition data (Figure 4) for
1, 23, and 43 with regard to potency and stereopreference of
(+)-versus (−)-enantiomers.
Effects of 1,4-DHP Enantiomers on Mesoderm
Formation in mESCs. To further validate the ability of 1,4-
DHPs to functionally inhibit TGFβ, the effect of 1, 23, or 43
and their enantiomers on mesoderm formation was examined
using a J1 mESC line with a stably integrated Brachyury/T
promoter-eGFP reporter construct (Brachyury/T is a marker
for mesoderm).24 Treatment of mESCs on day 1−2 showed a
dose-dependent inhibition of mesoderm formation based on a
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proportional to the inhibition of cellular calcium transients.
Inhibition of calcium transients was about 3−5-fold greater for
the (−)-enantiomers compared to the (+)-enantiomers of 1
and 23. Because the (−)-enantiomers examined were not
cardiomyogenic, this finding supports the suggestion that
TGFβ inhibition and not calcium inhibition is largely
responsible for the mechanism of action for 1,4-DHPs in
mESC cardiogenesis. The opposite stereopreference results for
calcium inhibition vice versa reinforce the view that calcium
inhibition is not a mechanism of action for 1,4-DHP
cardiomyogenesis.
Table 7. Effect of SAR-Optimized Substitution Pattern on
TGFβ Inhibition
a
compd
R
IC50 (μM)
% inhibition (2.5 μM)
28
29
Et
0.17 0.05
0.17 0.02
98
94
1
1
iBu
CONCLUSIONS
■
a
Data represent means SD of n = 2−4 independent experiments.
1,4-DHP 1 was a potent “hit” in a medium-throughput mESC-
based screen of small molecules for the ability to drive
cardiomyocyte formation from mESCs. An SAR model was
constructed for a b-annulated subclass of 1,4-DHPs based
primarily on the ability of 1,4-DHPs to inhibit TGFβ/Smad
signaling through induction of TGFβ receptor type II
degradation (hence the name ITDs). Key structural features
of the 1,4-DHP pharmacophore included a 1,4,5,6,7,8-
hexahydroquinoline core, a 4-phenyl substituent with bulky
aliphatic residues at the 4′-position, and a lipophilic carboxylic
ester. The substitution pattern of the 4-phenyl group was found
critical not only for potent TGFβ inhibition but also for
selectivity in favor of TGFβ signaling versus the closely related
Activin A pathway. In contrast to alkyl- and aryl-substitution at
the 4′-position, 3′-substitution decreased TGFβ inhibitory
potency as well as selectivity over Activin A and diminished the
selective biological activity of the (+)- and (−)-enantiomers.
Evaluation of the center of chirality at the 4-position for certain
selected 1,4-DHPs revealed that potent cardiogenesis activity
was most commonly associated with the (+)-enantiomer.
Interestingly, this stereopreference of 1,4-DHP enantiomers
was reversed for inhibition of calcium transients in HL-1 cells,
with the (−)-enantiomers showing a greater inhibition
compared to the (+)-antipodes. In addition, a correlation of
data from secondary functional assays in mESCs (i.e.,
mesoderm inhibition assay) as well as for TGFβ receptor II
degradation with IC50 values (TGFβ/Smad inhibition)
confirmed and validated the SAR model.
There have been a number of small molecule TGFβ
inhibitors reported in the literature to target TGFBR1 kinase
(Alk-5) selectively. However, these all show similar potency
against signaling by Activin A or other growth factors that act
through the closely related Alk4 receptor (and Alk7 in most
cases), thus, the 1,4-DHPs are the first selective TGFβ
inhibitors to our knowledge. Also, the selective targeting of
TGFBR2 for clearance from the cell surface is a novel
mechanism of action. Although TGFBR2 trafficking to the
proteasome occurs during receptor cycling, it does so with
TGFBR1, and we could also not find any reports of small
molecule or biological reagents that dissociate proteasomal
trafficking of TGFBR2 and TGFBR1. Thus, in summary, the
1,4-DHPs described herein provide conceptually novel
chemical probes that may be useful to explore the role of
TGFBR2-mediated signaling in basic biology and for the
molecular basis of disease. The 1,4-DHP molecules not only
represent useful reagents for cardiac regenerative medicine but
also provide excellent probes for studying TGFβ signaling in
various biological systems.
decrease in Brachyury/T-GFP-positive cells on day 4 (Figure
5D). Inhibition of mesoderm formation was selective regarding
stereochemistry for 1 and 23 and followed the selectivity of
TGFβ inhibition (i.e., (+)-enantiomer > (−)-enantiomer). In
contrast to the lack of selective TGFβ inhibition in the SBE4/
Smad reporter assay (Figure 4), compound 43 showed a slight
stereopreference of the (+)-enantiomer for mesoderm inhib-
ition (Figure 5D). We conclude that, at least for 43, mesoderm
inhibition at day 1 occurs by a distinct and presumably more
complex mechanism than that for TGFβ inhibition in both the
reporter gene assay (Figure 4) and the induction of
proteasomal TGFBR2 degradation (Figure 5C).
Correlation of TGFβ/Smad4 Signaling, TGFBR2 Deg-
radation, and Mesoderm Inhibition. A 3D-correlation plot
depicting TGFβ inhibition (log IC50s, x-axis), inhibition of
Brachyury/T-positive cells (%, y-axis), and TGFBR2 clearance
from the cell surface by DHP compounds (%HA-TGFBR2
positive cells, z-axis) was generated for 13 selected 1,4-DHPs
that covered a broad range of biological activity (i.e., from
inactive to highly potent) as well as pairs of (+)- and
(−)-enantiomers (i.e., enantiomers of 1, 23, and 43) (Figure
5E). Here, the stereopreference of enantiomers of 1 and 23 was
apparent as the less active (−)-enantiomers (gray dots) can be
found in the upper right corner, whereas the highly potent
(+)-enantiomers (red dots) are clustered in the lower left
corner. In contrast, the 43 enantiomers are present in the
center of the graph, underscoring their lack of stereopreference
and modest potency in all assays examined. The robust overall
correlation from these biologically and technically distinct
functional assays validated the SAR profile of 1,4-DHPs as
TGFβ inhibitors, considering that the common thread of these
studies was inhibition of TGFβ signaling.
Effects of 1,4-DHPs on Calcium Transients. We also
evaluated the (+)- and (−)-enantiomers of 1 and 23 for
modulation of calcium transients in HL-1 cells (immortalized
murine atrial cardiomyocytes) because it has been reported that
1,4-DHP enantiomers can have opposing effects on calcium
channels (i.e., agonistic or antagonistic activity20). Figure 5F
shows the calcium transient peak values in the presence of 10
μM 1 or 23 enantiomers or nifedipine (NFP, 2). The results
showed that the (+)-enantiomers (i.e., (+)-1, 44% inhibition,
(+)-23, 58% inhibition) inhibited calcium transients less
effectively than nifedipine (i.e., 66% inhibition), whereas the
(−)-enantiomers were stronger inhibitors (i.e., (−)-1, 87%
inhibition, (−)-23, 85% inhibition). The stereopreference
observed for TGFβ inhibition (in three different assays, Figure
5E) and cardiomyogenesis (Figure 5A) is thus inversely
H
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pound was obtained according to the general procedure but replacing
dimedone by 5-(n-propyl)cyclohexane-1,3-dione and methyl acetoa-
cetate by isobutyl acetoacetate and using 4-tert-butylbenzaldehyde as
the aldehyde. Because of the presence of two centers of chirality (4-
EXPERIMENTAL SECTION
■
General. All reagents and solvents were of the purest grade
available from commercial sources and used as received. Synthetic
materials were purified using a flash column chromatography system
(CombiFlash Rf 200, Teledyne ISCO, Lincoln) and preparative thin
layer chromatography (PTLC) with UV indicator. NMR spectra were
recorded at 300 MHz (1H) on a Varian Mercury 300 (at HBRI, San
Diego), Bruker ARX 300 (at Christian-Albrechts University Kiel), or at
500 MHz (1H) and 125 MHz (13C) on a Bruker AMX-500 II
(NuMega Resonance Lab, San Diego). Chemical shifts were reported
as ppm (δ) relative to the solvent (CDCl3 at 7.26 ppm, CD3OD at
3.31 ppm, DMSO-d6 at 2.50 ppm) or TMS as internal standard.
Optical rotations were recorded on a Jasco P-1010 polarimeter (not
thermostatted). Low resolution mass spectra were obtained using a
Hitachi M-8000 mass spectrometer with an ESI source. Purity of final
compounds was determined with a Hitachi 8000 LC-MS using reverse
phase chromatography (C18 column, 50 mm × 4.6 mm, 5 μm,
Thomson Instrument Co., Oceanside, CA). Compounds were eluted
using a 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% 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 synthetic final products
was ≥95%.
Multiwell plates were from Greiner Bio-One (Monroe, USA).
Recombinant human TGFβ-2 from Merck-Millipore (Billerica, USA)
and Activin A from R&D Systems (Minneapolis, MN) were
purchased. Gelatin, fibronectin, SB-431542, Claycomb medium,
streptomycin, L-glutamine, norepinephrine, and Hoechst 33342 were
purchased from Sigma-Aldrich (St. Louis, MO). FBS, HEPES buffer,
and probenecid were from Life Technologies.
Chemistry. General Procedure for the Preparation of Racemic
1,4-DHPs (1, 7−31). Compounds were prepared in a library fashion
following a procedure described by Ko et al. (2005).14 In a typical
reaction, 28 mg (0.2 mmol) of dimedone 4, 0.2 mmol of the desired
aldehyde 5, 0.2 mmol of β-ketoester 6, 15 mg (0.2 mmol) of NH4OAc,
and 15 mg (0.06 mmol) of iodine were stirred overnight at room
temperature in 0.5 mL of EtOH. Solvent was evaporated, and the
residue was dissolved in 1 mL of EtOAc. The organic layer was washed
with 0.5 mL of a saturated solution of NaS2O3, dried over Na2SO4, and
concentrated under reduced pressure. The crude material was purified
by PTLC with hexanes/EtOAc to afford 20−50% yield of the final
compound.
Methyl 4-(Biphenyl-4-yl)-2-methyl-5-oxo-7-(n-propyl)-1,4,5,6,7,8-
hexahydroquinoline-3-carboxylate (23). The title compound was
obtained according to the general procedure, but replacing dimedone
by 5-(n-propyl)cyclohexane-1,3-dione and using 4-biphenylcarbox-
aldehyde as the aldehyde. Because of the presence of two centers of
chirality (4- and 7-positions), a mixture of diastereomers was obtained.
1H NMR (500 MHz, CDCl3): δ 0.85−0.89 (m, 3H), 1.18−1.34 (m,
4H), 1.98−2.48 (m, 5H), 2.40 + 2.41 (2 × s, 3H), 3.63 (s, 3H), 5.11 +
5.15 (2 × s, 1H), 6.04 + 6.11 (2 × s, 1H), 7.20 (t, J = 8.4 Hz, 1H),
7.33−7.44 (m, 6H), 7.52 (br d, J = 7.9 Hz, 2H). LRMS calcd for
C27H29NO3 415.22 [M]+, found 416.35 [M + H]+. HPLC purity
95.8%, tR = 7.03 min.
Ethyl 4-(4-tert-Butylphenyl)-2-methyl-5-oxo-7-(n-propyl)-
1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (28). The title com-
pound was obtained according to the general procedure, but replacing
dimedone by 5-(n-propyl)cyclohexane-1,3-dione and methyl acetoa-
cetate by ethyl acetoacetate and using 4-tert-butylbenzaldehyde as the
aldehyde. Because of the presence of two centers of chirality (4- and 7-
1
and 7-positions), a mixture of diastereomers was obtained. H NMR
(500 MHz, CDCl3): δ 0.76−0.80 (m, 6H), 0.85−0.91 (m, 3H), 1.25
(s, 9H), 1.28−1.36 (m, 4H), 1.81−1.87 (m, 1H), 1.96−2.07 (m, 2H),
2.16−2.21 (m, 1H), 2.28−2.44 (m, 1H), 2.37 (s, 3H), 3.71−3.76 (m,
1H), 3.81−3.85 (m, 1H), 5.02 + 5.06 (2 × s, 1H), 5.70 + 5.75 (2 × s,
1H), 7.19 (br s, NH), 7.20 (br s, 4H). LRMS calcd for C28H39NO3
437.3 [M]+, found 438.75 [M + H]+. HPLC purity 95.1%, tR = 9.11
min.
General Procedure for Suzuki Coupling of 1,4-DHPs (32−43). In a
typical reaction, to a 2−5 mL microwave vial, 50 mg of bromo
intermediate 31, 25 mg of desired boronic acid, 17 mg of palladium
tetrakis, and 0.15 mL of a 2 M Na2CO3 in water in dioxane/water (2/
1.5 mL) was heated in a microwave for 10 min at 150 °C. Solvents
were evaporated, the crude mixture was dissolved in EtOAc and
washed with water, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by PTLC with hexanes/
EtOAc to afford 35−85% yield of the final compound.
Methyl 4-(4-Cyclohexylphenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-
hexahydroquinoline-3-carboxylate (32). The title compound was
obtained according to the general procedure using cyclohexylboronic
acid as the boronic acid. 1H NMR (500 MHz CDCl3): δ 0.95 (s, 3H),
1.07 (s, 3H), 1.18−1.21 (m, 2H), 1.34 (t, J = 10.3 Hz, 4H), 1.71−1.85
(m, 4H), 2.16−2.39 (m, 4H), 2.35 (s, 3H), 3.62 (s, 3H), 3.75 (t, J =
6.4 Hz, 1H), 5.03 (s, 1H), 5.95 (s, 1H), 7.01 (d, J = 6.9 Hz, 2H), 7.17
(d, J = 6.9 Hz, 2H). LRMS calcd for C26H33NO3 407.25 [M]+, found
408.07 [M + H]+. HPLC purity 100%, tR = 8.06 min.
Methyl 4-(3-(1H-Indol-5-yl)phenyl)-2,7,7-trimethyl-5-oxo-
1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (43). The title com-
pound was obtained according to the general procedure using 1H-
indol-5-ylboronic acid as the boronic acid. 1H NMR (500 MHz,
CDCl3): δ 0.97, 1.08 (2 × s, 3H), 2.15−2.32 (m, 4H), 2.38 (s, 3H),
3.63 (s, 3H), 5.16 (s, 1H), 6.05 (br s, 1H), 6.58 (br t, 1H), 7.20 - 7.29
(m, 6H), 7.56 (mc, 1H), 7.78 (mc, 1H), 8.30 (br s, 1H). LRMS calcd
for C28H28N2O3 440.21 [M]+, found 463.27 [M + Na]+. HPLC purity
97.2%, tR = 6.40 min.
1,4-DHP Ester Hydrolysis: 4-(Biphenyl-4-yl)-2,7,7-trimethyl-5-oxo-
1,4,5,6,7,8-hexahydroquinoline-3-carboxylic Acid (44). First, 10 mL
of BCl3 (1 M DCM solution) was added to a cooled solution of 1 g
(2.49 mmol) of 10 in 20 mL dry DCM. Then the mixture was stirred
at room temperature overnight and diluted in ice water/EtOAc. The
organic layer was concentrated and the product purified by column
chromatography with DCM/MeOH (95/5). Yield: 700 mg (75%) of a
1
white solid. H NMR (500 MHz, DMSO-d6): δ 0.86 (s, 3H), 1.02 (s,
3H), 2.02−2.40 (m, 4H), 3.33 (s, 3H), 4.88 (s, 1H), 7.20−7.59 (m,
9H), 8.89 (s, NH), 11.70 (br s, 1H, COOH). LRMS calcd for
C25H25NO3 387.18 [M]+, found 343.42 [M-COOH]+. HPLC purity
99.1%, tR = 4.61 min.
1,4-DHP N-Methylation: Methyl 4-(Biphenyl-4-yl)-2-ethyl-1,7,7-
trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (45).
This synthesis was done according to a modified protocol of Cozzi et
al. (1993).15 First, 50 mg (0.12 mmol) of 30 was dissolved in 0.2 mL
of DMSO. Then 8 μL (1.1 equiv) of methyl iodide and 7 mg (1 equiv)
of KOH were added, and the mixture was stirred at room temperature
for 72 h in a sealed reaction vessel. The reaction mixture was diluted
with 3 mL of EtOAc, washed once with water (0.5 mL), dried with
Na2SO4, and purified by PTLC (hexane/EtOAc 1:1, Rf = 0.7) to give a
1
pale-yellow solid. H NMR (300 MHz, CDCl3): δ 1.03 (s, 3H), 1.08
1
(s, 3H), 1.25 (t, J = 9.0 Hz, 3H), 2.28 (d, J = 1.5 Hz, 2H), 2.54 (d, J =
1.5 Hz, 2H), 3.26 (s, 3H), 3.67 (s, 3H), 5.28 (s, 1H), 7.24−7.44 (m,
7H), 7.54 (d, J = 9.0 Hz, 2H). LRMS calcd for C28H31NO3 429.23
[M]+, found 430.75 [M + H]+. HPLC purity 99.7%, tR = 8.40 min.
1,4-DHP Oxidation: Methyl 4-(Biphenyl-4-yl)-2-ethyl-7,7-dimeth-
yl-5-oxo-5,6,7,8-tetrahydroquinoline-3-carboxylate (46). The title
compound was prepared using a literature protocol for the DHP
oxidation.16 A mixture of 41 mg (0.1 mmol) of 30, 45 mg (2 equiv)
HIO4, 14 mg (2 mmol) of Na2NO2, and 50 mg of wet silica gel was
positions), a mixture of diastereomers was obtained. H NMR (500
MHz, CDCl3): δ 0.85−0.91 (m, 3H), 1.19 (t, J = 7.3 Hz, 2H), 1.28 (s,
9H), 1.28−1.36 (m, 4H), 1.98−2.09 (m, 2H), 2.17−2.23 (m, 2H),
2.27−2.44 (m, 1H), 2.36 (s, 3H), 4.07 (q, J = 7.3 Hz, 2H), 5.02 + 5.06
(2 × s, 1H), 5.80 + 5.85 (2 × s, 1H), 7.18 (br s, NH), 7.19 (br s, 4H).
LRMS calcd for C26H35NO3 409.26 [M]+, found 410.48 [M + H]+.
HPLC purity 95.1%, tR = 9.11 min.
Isobutyl 4-(4-tert-Butylphenyl)-2-methyl-5-oxo-7-(n-propyl)-
1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (29). The title com-
I
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stirred in DCM at room temperature overnight. After filtration of the
silica gel, the residue was purified by PTLC (hexanes/EtOAc, 7/3). 1H
NMR (CDCl3): δ 1.14 (s, 6H), 1.34 (t, J = 9.0 Hz, 3H), 2.50 (s, 2H),
2.84 (q, J = 9.0 Hz, 2H), 3.13 (s, 2H), 3.49 (s, 3H), 7.20 (d, J = 8.4
Hz, 1H), 7.50−7.35 (m, 4H), 7.66−7.60 (m, 4H). LRMS calcd for
C27H27NO3 413.20 [M]+, found 414.35 [M + H]+. HPLC purity
99.9%, tR = 8.70 min.
Synthesis of 1,4-DHP Single Enantiomers of 1, 23, and 43. A
synthetic procedure by Shan et al. (2002) was used in a modified
form.17 Below, we provide experimental details for the preparation of
(+)-1 and (−)-1, whereas data for 23 and 43 enantiomers can be
found in the Supporting Information.
Diastereomer 52b. Yield: 650 mg (14%). TLC: Rf = 0.28 (tol/
EtOAc, 1:1); α2D3 = +24.4 (c = 0.4 in CHCl3); % de = ≥99% (1H NMR
according to 4-CH, 2-CH3 and COOCH3). 1H NMR (500 MHz,
CDCl3): δ 0.90 (s, 3H), 1.08 (s, 3H), 1.15 (d, J = 6.4 Hz, 3H), 2.16−
2.34 (m, 4H), 2.45 (s, 3H), 3.81 (s, 3H), 4.91 (dd, J = 8.7 and 3.5 Hz,
1H), 5.00 (s, 1H), 5.39−5.44 (dq, J = 3.5 and 6.4 Hz, 1H), 5.90 (s,
NH), 6.99 (d, J = 8.7 Hz, 1H), 7.14−7.42 (m, 9H), 7.50 (t, J = 8.0 Hz,
1H), 8.05 (dt, J = 1.1 and 7.7 Hz, 1H), 8.25 (ddd, J = 1.0, 2.3, and 8.2
Hz, 1H), 8.65 (br t, J = 1.9 Hz, 1H). 13C NMR (125 MHz, CDCl3): δ
17.3, 19.9, 27.4, 29.5, 33.0, 36.5, 41.5, 50.9, 53.2, 57.2, 70.5, 104.7,
112.8, 123.0, 125.6, 126.5, 127.0, 127.2, 128.3, 128.9, 130.2, 132.9,
135.6, 139.0, 141.0, 145.7, 146.1, 147.5, 148.5, 165.7, 166.6, 170.4,
195.7. LRMS calcd for C37H37N3O8 651.26 [M]+, found 652.19 [M +
H]+ and 652.35 [M + H]+ and 371.28 [M − C12H13N2O6 (4-methoxy-
3-(3-nitrobenzamido)-4-oxobutan-2-oyl) + H]+. HPLC purity 97.5%,
tR = 8.04 min.
General Procedure for the Synthesis of Carboxylic Acids 44, 55,
56. First, 0.406 mmol of each diastereomer was dissolved and stirred
in 11 mL of MeOH. Then 186 μL of DBU (1.24 mmol) was added
dropwise, and then the solution turned yellow. Stirring was continued
for another 3 h at room temperature until all starting material was
consumed as determined by TLC. After evaporation of the solvent, the
residue was taken up in 40 mL of water, acidified to pH 2−3 with 1 N
HCl (fine precipitate formed), and the title compound was extracted
with EtOAc (3×). The organic phase was dried (Na2SO4) and
concentrated to dryness.
(2S,3R)-Methyl 2-(3-nitrobenzamido)-3-(3-oxobutanoyloxy)-
butanoate (51). Method a: 10 g (35 mmol) of the L-threonine-
derived starting material 4817 was dissolved in 20 mL of dry THF and
3.8 mL of diketene (49, 50 mmol) was added while stirring. The
mixture was stirred at room temperature, and 214 mg of 4-DMAP
(1.75 mmol, 5 mol %) was added carefully (exothermic reaction).
Stirring was continued overnight, after which the organic solvent was
evaporated to dryness, taken up with DCM, and washed with 1%
aqueous HCl and brine. The organic phase was dried (Na2SO4),
concentrated in a vacuum, and purified by flash chromatography
(SiO2, hexane/EtOAC) to give 10 g of a colorless oil (78%, crystallized
upon standing for few days). Method b: 3 g (10.7 mmol) of the L-
threonine starting material 48 was dissolved in 7 mL of xylenes and
1.52 g of 2,2,6-trimethyl-4H-1,3-dioxin-4-one (50, 10.7 mmol) was
added. The mixture was heated to 150 °C for 30 min while stirring
vigorously. After evaporation, the crude mixture was purified by flash
chromatography (SiO2, hexane/EtOAc) to yield 2.2 g of a colorless oil
4-(Biphenyl-4-yl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroqui-
noline-3-carboxylic Acid Enantiomers (44a,b). The title enantiomers
were obtained according to the general procedure, starting from 52
with 87−88% yields. The crude mixture was purified by flash
chromatography (SiO2, DCM/MeOH: 0−5%) to afford 137 mg for
44a (87%) and 139 mg for 44b (88%) as off-white solids. TLC: Rf =
1
(86%). TLC: Rf = 0.36 (hexane/EtOAc, 1:1). H NMR (500 MHz,
CDCl3): δ 1.39 (d, J = 6.4 Hz, 3H), 2.29 (s, 2H), 3.53 (d, J = 8.4 Hz,
2H), 3.80 (s, 3H), 4.00 (dd, J = 8.9 and 2.5 Hz, 1H), 5.57 (qd, J = 6.9
and 2.5 Hz, 1H), 7.48 (d, J = 8.9 Hz, NH), 7.68 (t, J = 7.7 Hz, 1H),
8.01 (br s, OH), 8.30 (br d, J = 7.9 Hz, 1H), 8.39 (br d, J = 8.4 Hz,
1H), 8.80 (t, J = 2.0 Hz, 1H). LRMS calcd for C16H18N2O8 366.11
[M]+, found 265.95 [M − C4H5O3(acetoacetate) + H]+. HPLC purity
95.3%, tR = 6.47 min.
General Protocol for Synthesis and Resolution of Diastereomers
52−54. A mixture of 2.56 g (7 mmol) of (2S,3R)-methyl 2-(3-
nitrobenzamido)-3-(3-oxobutanoyloxy)butanoate (51), 1.28 g of 4-
biphenylcarboxaldehyde (5) (7 mmol), 981 mg of dimedone (4) (7
mmol), 540 mg of dry NH4OAc (7 mmol), and 533 mg of iodine (2.1
mmol) were stirred overnight in 4 mL of EtOH at room temperature.
Then 150 mL of EtOAc was added to the brown slurry and washed
with aqueous saturated Na2S2O3 (2×) and brine (1×). The organic
layer was dried (Na2SO4) and concentrated in a vacuum to afford 5.8 g
of a yellow solid foam. The crude mixture was purified by flash
chromatography (SiO2) using a short column with a hexane/EtOAc
gradient (20−60%) followed by a second gravity flow chromatography
on a larger column (250 g SiO2) with toluene/EtOAc (2:1) as the
eluent to separate the diastereomers.
1
0.14 (DCM/MeOH, 95:5). LC/MS and H NMR were identical for
both enantiomers and the same as the racemic compound (see above
for 44).
General Procedure for the Synthesis of Carboxylic Ester
Enantiomers 1, 23, 57. 137 mg (0.353 mmol) of the respective
carboxylic acid enantiomer (44), 56.8 mg of K2CO3, and 45 mg of
ethyl bromide (0.41 mmol) was stirred in 10 mL of dry DMF
overnight at room temperature. For workup, 50 mL of water was
added and the mixture extracted with EtOAc (3×). The organic layer
was washed with water (1 × 20 mL) and brine (1 × 20 mL), dried
(Na2SO4), and concentrated to dryness. The crude mixture (>95%
pure by TLC) was further purified by flash chromatography (SiO2,
hexanes/EtOAc: 0−60%).
Ethyl 4-(Biphenyl-4-yl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahy-
droquinoline-3-carboxylate Enantiomers (1a,b). Enantiomer 1a.
Yield: 140 mg (95%) of a pale-yellow solid. TLC: Rf = 0.33 (hexanes/
1
EtOAc, 1:1); α2D3 = −70.3 (c = 0.4 in CHCl3). H NMR (500 MHz,
CDCl3): δ 0.96 (s, 3H), 1.09 (s, 3H), 1.23 (t, J = 7.2 Hz, 3H), 2.15−
2.39 (m, 4H), 2.38 (s, 3H), 4.08 (q, J = 7.2 Hz, 2H), 5.10 (s, 1H), 6.49
(br s, NH), 7.25−7.54 (m, 9H). 13C NMR (125 MHz, CDCl3): δ 14.3
(OCH2CH3), 19.5 (2-CH3), 27.3 (7-CH3), 29.4 (7-CH3), 32.3 (7-C),
36.5 (4-CH), 41.3 (8-CH2), 50.7 (6-CH2), 59.9 (OCH2), 106.2 (3-C),
112.3 (4a-C), 126.8 (ArCH), 127.0 (ArCH), 128.4 (ArCH), 128.6
(ArCH), 138.8 (ArC), 141.3 (ArC), 143.2 (2-C), 146.1 (1′-C), 147.7
(8a-C), 167.4 (COOMe), 195.4 (CO). LRMS calcd for C27H29NO3
415.22 [M]+, found 416.68 [M + H]+. HPLC purity 99.0%, tR = 8.19
min.
(2R,3S)-4-Methoxy-3-(3-nitrobenzamido)-4-oxobutan-2-yl 4-(Bi-
phenyl-4-yl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-
3-carboxylate Diastereomers (52a,b). Diastereomer 52a. Yield: 550
mg (12%). TLC: Rf = 0.32 (tol/EtOAc, 1:1); α2D3 = +67.8 (c = 0.4,
CHCl3); % de = ≥99% (1H NMR according to 4-CH, 2-CH3 and
1
COOCH3). H NMR (500 MHz, CDCl3): δ 0.89 (s, 3H), 1.07 (s,
3H), 1.38 (d, J = 6.4 Hz, 3H), 2.18−2.34 (m, 4H), 2.49 (s, 3H), 3.59
(s, 3H), 4.74 (dd, J = 4.1 and 8.6 Hz, 1H), 5.10 (s, 1H), 5.54−5.59
(dq, 4.1 and 6.5 Hz, 1H), 5.94 (br s, NH), 6.51 (br d, J = 8.6 Hz, 1H),
7.14−7.36 (m, 10H), 7.79 (dt, J = 1.1 and 7.7 Hz, 1H), 8.14 (ddd, J =
1.0, 2.3, and 8.4 Hz, 1H), 8.40 (br t, J = 1.9 Hz, 1H). 13C NMR (125
MHz, CDCl3): δ 18.1, 20.1, 27.4, 29.6, 33.0, 35.9, 41.3, 51.0, 52.9,
57.5, 69.2, 104.2, 112.9, 122.8, 125.5, 126.3, 126.9, 127.1, 127.3, 128.0,
128.8, 129.8, 133.0, 135.4, 128.9, 140.7, 145.7, 146.4, 147.7, 148.3,
165.7, 166.8, 170.1, 195.9. LRMS calcd for C37H37N3O8 651.26 [M]+,
found 652.08 [M + H]+ and 371.28 [M − C12H13N2O6 (4-methoxy-3-
(3-nitrobenzamido)-4-oxobutan-2-oyl) + H]+. HPLC purity 99.8%, tR
= 8.23 min.
Enantiomer 1b. Yield: 137 mg (93%) as a pale-yellow solid. TLC:
Rf = 0.33 (hexanes/EtOAc, 1:1); α2D3 = +68.05 (c = 0.4 in CHCl3).
NMR data was identical to 1a. LRMS calcd for C27H30NO3 415.22
[M]+, found 416.62 [M + H]+. HPLC purity 100%, tR = 8.28 min.
In both cases, the optical purity of (+)-1a and (−)-1b was
determined to be ≥96% ee by 1H NMR spectroscopy. In this
experiment, 1.5 mg (+)Eu(hfc)3 dissolved in 50 μL (CDCl3) was
added to 1.5 mg of the respective enantiomer in 550 μL (CDCl3).
NMR resonances of the 2-CH3 and 7-CH3 groups as well as for the 4-
CH group shifted differently upon addition of the chiral reagent for
each enantiomer. The shifted resonances of the 7-methyl groups,
J
dx.doi.org/10.1021/jm301144g | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
however, were best suited to estimate the % ee (see also Supporting
Information).
containing 20 mM HEPES buffer and 2.5 mM probenecid. After
washing twice with Tyrode’s solution (140 mM NaCl, 6 mM KCl, 1
mM MgCl2, 5 mM HEPES, 2 mM CaCl2, 10 mM glucose, pH 7.4),
cells were incubated with indicated compounds diluted in Tyrode’s
solution for 10 min at room temperature before beginning Ca2+
recordings. Ca2+ transient recordings were done using a kinetic image
cytometer IC 100 (Vala Sciences, San Diego).30 Video streams of 10 s
of the Fluo-4 green channel were collected for each well at 33 frames
per second. Autofocus by the IC 100 was done prior to video
acquisition using the nuclear channel. All image capture was conducted
with a 20 × 0.50 numerical aperture (NA) objective. Cytometric
calcium kinetic parameters were determined by image analysis using
CyteSeer software.
Biology. Smad4 Binding Element (SBE-4)-Based Transient
Luciferase Reporter Gene Assay (TGFβ/Activin A Assay). An SBE-
4-firefly luciferase plasmid25 was cotransfected with a TK-driven
Renilla luciferase plasmid (for data normalization) into HEK-293T
(culture media: DMEM, 2% FBS). Cells were transfected in bulk with
SBE-4 and Rluc plasmids. After incubation for 12−14 h, cells were
counted, plated on 96-well plates at 25000 cells/well (media: DMEM,
1% FBS), and incubated for 2 h before addition of test compound or
DMSO or TGFβ-2 or Activin A (10 ng/mL) addition. Cells were
treated with different amounts of 1,4-DHP test compounds (0.001−5
μM), and the data was compared to cells treated with DMSO as a
control, nontreated cells, or SB-431542-treated cells. Each condition
was done in triplicate. Plates were incubated for 20−22 h, media was
aspirated and Promega cell lysis buffer was added. Firefly and Renilla
luciferase activities were determined using the Dual Luciferase Assay
Kit (Promega, Madison, USA) according to the manufacturer
instructions. The assay conditions were optimized to give a dynamic
range of typically >100-fold pathway induction and Z′ values between
0.75 and 0.95. Data was derived from a minimum of two independent
experiments unless otherwise stated. GraphPad Prism 5 software was
used for data evaluation: log(inhibitor)/normalized response for
TGFβ assays, log(inhibitor)/response for Activin A assays.
TGFBR2 Degradation Assay. The assay was done as described
previously.3 Briefly, in a 6-well format, a plasmid expressing
extracellularly hemagglutinin (HA)-tagged TGFBR226 was transfected
in HEK-293T cells. Cells were allowed to recover from transfection for
>6 h. 1,4-DHPs were added at a final concentration of 3 μM, and cells
were incubated for another 20 h before analysis by flow cytometry.
Cell populations were dissociated to single cell suspensions and
analyzed on a LSR Fortessa for HA-positive cells, using an anti-HA
antibody (Covance, San Diego) and an APC-labeled secondary
antibody (Life Technologies). Data analysis was performed using
FlowJo (Tree Star, Ashland).
ASSOCIATED CONTENT
* Supporting Information
■
S
Analytical data for 1, 7−22, 24−30, and 33−42; synthetic
procedures and analytical data for the (+)- and (−)-enan-
tiomers of 23, 43, and synthetic intermediates 48−57; 1D and
2D NMR spectra of 1; 1H NMR spectra of 52a,b
1
diastereomers; representative H NMR spectra of (+)Eu(hfc)3
shift reagent experiments for (rac)-1 and (+)- and (−)-1;
representative calcium transient curves (HL-1 cells) for (+)-
and (−)-enantiomers of 1 and 23. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*For D.S.: phone, +49-231-755-7067; E-mail, dennis.schade@
tu-dortmund.de, dschade@hbri.org. For E.W.: phone, +1-858-
646-3100; E-mail, ewillems@sanfordburnham.org.
Present Addresses
⊥TU Dortmund University, Faculty of Chemistry & Chemical
Biology, Otto-Hahn-Strasse 6, D-44227 Dortmund, Germany.
∥Takeda California, Inc., 10410 Science Center Drive, San
Diego, California 92121, United States.
Mouse Embryonic Stem Cell Culture and Differentiation Assays.
The assays were done as described previously.3,27 Here, brief
descriptions of the assays are provided.
mESC Cardiogenesis Assay. For the HCS assay with the mESC
lines CGR8 carrying a Myh6-GFP reporter,27,28 cells were seeded in
LIF-free DMEM (containing 10% FBS) with high glucose in black
384-well μClear plates to allow differentiation. On day 3 of
differentiation, different amounts of 1,4-DHPs (and controls) were
added and media was changed every other day, thereby attaining a
compound exposure window from days 3−5. On days 10 or 11 of
differentiation, cells were analyzed by image analysis on an INCell
1000 system (GE Healthcare, Little Chalfont) and quantified with
Cyteseer software (Vala Sciences, San Diego).
mESC Mesoderm Inhibition Assay. For the mesoderm inhibition
assay J1 Brachyury/T-GFP reporter cells were used as suspension
cultures and were conducted in serum-containing media. Embryoid
bodies (EBs) were allowed to form in differentiation media and were
incubated in the presence of 1,4-DHPs (and controls) at day 1 of
differentiation, just before the initiation of mesoderm. Analysis was
done by flow cytometry on day 4. Cell populations were dissociated to
single cell suspensions and analyzed on a FACSCanto for GFP-
positive cells. Data analysis was performed using FlowJo.
Calcium Transient Assay. HL-1 cells were used in these studies and
represent an immortalized murine atrial cell line that can be serially
passaged while retaining a differentiated cardiac phenotype and
displaying spontaneous contractions.29 For experiments, cells were
seeded on 96-well glass bottom plates coated with 0.02% gelatin and 5
μg/mL fibronectin at a density of 25000 cells/well and were
maintained in Claycomb Medium supplemented with 10% FBS, 100
units/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 0.1
mM norepinephrine. For Ca2+ analysis, cells were loaded with 200 ng/
mL Hoechst 33342 and Fluo-4 for 30 min at 37 °C followed by 30
min at room temperature. Fluo-4 NW calcium assay kit (Life-
Technologies/Molecular Probes) was prepared according to the
manufacturers’ instructions in 1× Hank’s Balanced Salt Solution
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Author Contributions
#These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge the NIH (R01HL059502 and R33HL088266
to M.M.) and the California Institute for Regenerative
Medicine (CIRM, RC1−000132 to M.M.), the Leducq
Transatlantic Alliance (SHAPEHEART to M.M.), and the T
Foundation (to J.R.C.) for their generous financial support. We
gratefully acknowledge postdoctoral fellowships from the
CIRM (TG-0004) and American Heart Association (to E.W.)
and the German Research Foundation (DFG, SCHA1663/1−1
and SCHA1663/2−1 to D.S.). We thank Alyssa Morgosh at
HBRI for her technical assistance with preparation of
compounds, Drs. Zebin Xia and Marcia I. Dawson (SBMRI)
for helpful discussions, and Dr. Kim Janda (The Scripps
Research Institute) for providing access to a polarimeter.
ABBREVIATIONS USED
1,4-DHP, 1,4-dihydropyridine; TGFβ, transforming growth
factor β; TGFBR2, TGFβ receptor type II; ITD, inducer of
■
K
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