Design of Development Candidate eFT226, a First in Class Inhibitor of Eukaryotic Initiation Factor 4A RNA Helicase

Article abstract of DOI:10.1021/acs.jmedchem.0c00182

Dysregulation of protein translation is a key driver for the pathogenesis of many cancers. Eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase, is a critical component of the eIF4F complex, which regulates cap-dependent protein synthesis. The flavagline class of natural products (i.e., rocaglamide A) has been shown to inhibit protein synthesis by stabilizing a translation-incompetent complex for select messenger RNAs (mRNAs) with eIF4A. Despite showing promising anticancer phenotypes, the development of flavagline derivatives as therapeutic agents has been hampered because of poor drug-like properties as well as synthetic complexity. A focused effort was undertaken utilizing a ligand-based design strategy to identify a chemotype with optimized physicochemical properties. Also, detailed mechanistic studies were undertaken to further elucidate mRNA sequence selectivity, key regulated target genes, and the associated antitumor phenotype. This work led to the design of eFT226 (Zotatifin), a compound with excellent physicochemical properties and significant antitumor activity that supports clinical development.

Full text of DOI:10.1021/acs.jmedchem.0c00182

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Article
Design of Development Candidate eFT226, a First in Class Inhibitor
of Eukaryotic Initiation Factor 4A RNA Helicase
Justin T. Ernst,* Peggy A. Thompson, Christian Nilewski, Paul A. Sprengeler, Samuel Sperry,
Garrick Packard, Theodore Michels, Alan Xiang, Chinh Tran, Christopher J. Wegerski, Boreth Eam,
Nathan P. Young, Sarah Fish, Joan Chen, Haleigh Howard, Jocelyn Staunton, Jolene Molter, Jeff Clarine,
Andres Nevarez, Gary G. Chiang, Jim R. Appleman, Kevin R. Webster, and Siegfried H. Reich
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sı Supporting Information
ABSTRACT: Dysregulation of protein translation is a key driver for the pathogenesis of many cancers. Eukaryotic initiation factor
A (eIF4A), an ATP-dependent DEAD-box RNA helicase, is a critical component of the eIF4F complex, which regulates cap-
4
dependent protein synthesis. The flavagline class of natural products (i.e., rocaglamide A) has been shown to inhibit protein synthesis
by stabilizing a translation−incompetent complex for select messenger RNAs (mRNAs) with eIF4A. Despite showing promising
anticancer phenotypes, the development of flavagline derivatives as therapeutic agents has been hampered because of poor drug-like
properties as well as synthetic complexity. A focused effort was undertaken utilizing a ligand-based design strategy to identify a
chemotype with optimized physicochemical properties. Also, detailed mechanistic studies were undertaken to further elucidate
mRNA sequence selectivity, key regulated target genes, and the associated antitumor phenotype. This work led to the design of
eFT226 (Zotatifin), a compound with excellent physicochemical properties and significant antitumor activity that supports clinical
development.
INTRODUCTION
UTRs that are dependent on enhanced eIF4A RNA helicase
1
■
−6,12
activity for translation.
Several natural product classes
The dysregulation of messenger RNA (mRNA) translation is a
common feature in malignancies, demonstrated by an
upregulation of oncoproteins, growth factors, and signal
transduction proteins associated with proliferation, survival,
have been reported to inhibit eIF4A-mediated translation and
exhibit antiproliferative and antitumor phenotypes both in vitro
13−20
and in vivo.
The flavaglines, exemplified by silvestrol (1)
1
−4
and rocaglamide A (2), have been shown to bind and stabilize a
translation incompetent RNA/eIF4A complex. Recent studies
demonstrate that the formation of a ternary complex between
eIF4A, mRNA, and rocaglamide A is specific for polypurine
and metastasis.
The expression of oncogenic drivers is held
under tight translational control and is regulated by the
eukaryotic translation initiation factor 4F (eIF4F) complex.
5
,6
The eIF4F complex consists of the 5′-mRNA cap binding
protein eIF4E, a large scaffolding protein eIF4G and the DEAD
box RNA helicase eukaryotic initiation factor 4A (eIF4A).
eIF4A functions in an ATP-dependent manner to unwind an
mRNA secondary structure to enable ribosome scanning and
motifs in the 5′-UTR of select mRNAs resulting in a small
21,22
molecule sequence-selective translational repressor.
Hip-
puristanol (3), an oxygenated steroid, has been shown to bind
an allosteric site in the C-terminal domain of eIF4A and
stabilizes a conformation that is incompatible with RNA
6
translation initiation. The eIF4F subunits are frequently
overexpressed in various malignancies; therefore, targeting the
components of the eIF4F complex to renormalize dysregulated
translation is an emerging strategy in anticancer drug
Received: February 1, 2020
2
,7−11
discovery.
Our efforts focused on the identification of
inhibitors of eIF4A-mediated translation.
Activation of eIF4F has a direct role in tumorigenesis due to
increased synthesis of oncogenes with highly structured 5′-
©
XXXX American Chemical Society
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A
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2
3
binding. Pateamine A (4) has been reported to irreversibly
bind eIF4A, resulting in the stabilization of an RNA/eIF4A
2
4
complex discordant with translation. Numerous drug
discovery efforts have been invested in the flavaglines, however,
to date, none of these programs have led to the advancement of
a development candidate into human clinical studies. Major
challenges include overcoming poor drug-like properties such
as metabolic instability and poor solubility, as well as
developing a robust synthetic methodology to produce the
drug substance on a scale necessary to support clinical
development. Thus, one of our main tactics in exploiting the
flavagline class of molecules was to focus on improving
physicochemical properties. The optimization of properties
such as log P and aqueous solubility were important to support
controlled intravenous delivery of an optimal therapeutic dose.
Herein we report on our design efforts that led to the
identification of eFT226, the first eIF4A inhibitor to enter
human clinical studies.
Figure 1. Modeled binding mode of rocaglamide A (pink) to a
complex formed by poly-U RNA (light blue) and eIF4A3 (dark blue).
Residues of eIF4A shown to confer resistance to rocaglamide
analogues are highlighted in (yellow), PDB ID 2HYI.
be pi-stacking between the mRNA bases, the benzofuran and
one of the phenyl rings of the natural product; (2) the phenyl
ring distal to the mRNA binds in a pocket of quite limited size
and thus might accommodate limited substitution; (3) the
potential importance of the hydrogen bond between Gln 195
and the small molecule; and (4) that silvestrol’s dioxane ring
likely binds in an open site on the opposite side of the RNA
from the rocaglamide core and thus may not be an efficient
binding element. Although this model was useful, it was
considered low resolution, prompting the development of
additional computational methodologies for assessing com-
pound designs.
We were intrigued at the outset of the program by the unique
three-dimensional structure of the flavagline core, a result of five
contiguous stereocenters in the molecule on a five-membered-
ring. Unlike six-membered-rings, the conformations of five-
membered rings are much less predictable because they
undergo pseudorotation. Understanding the preferred con-
formation of this system and how this is related to the observed
phenotypes and potency SAR for this class of molecules was
deemed critical for the optimization process that could offer a
ligand-based modeling methodology for evaluating potential
compounds. Ab initio, density functional theory (DFT)
calculations performed on rocaglamide A predicted the low
energy conformation to have an intramolecular hydrogen bond
between the secondary hydroxyl and the methoxy group at the
8-position. Importantly, the preferred low energy torsional
angle between the phenyl D and E rings was found to be
approximately 40° (Figure 2). We hypothesized that this
preferred torsion might be a prerequisite for potent binding to
the protein−RNA complex and might be utilized to prioritize
potential targets. DFT calculations that drove the aryl−aryl
torsional angle between ±60° at 5° increments of a flavagline
molecule produced an energy profile, an example of which is
shown in Figure 2 for rocaglamide A. It was discovered that
cores that embodied a 40-degree aryl−aryl torsion as a
preferred low-energy conformation tended to display highest
potency while cores without a low energy 40° torsion generally
did not produce potent molecules. This model was used
prospectively to filter out molecular designs lacking the desired
energy profile.
OPTIMIZATION OF THE FLAVAGLINE-BASED
NATURAL PRODUCTS TO YIELD EFT226
■
From the known flavagline-based derivatives, we focused our
initial attention on rocaglamide A (2) which has published
literature supporting its mechanism for inhibiting eIF4A-
13,25,26
mediated translation.
During the design and develop-
ment of eFT226, an X-ray crystal structure of a rocaglamide
derivative/mRNA/eIF4A1 complex had not been published.
Utilizing reported eIF4A1 mutational data (mutations confer-
25
ring resistance to flavagline derivatives), a small molecule
crystal structure of a flavagline from the Cambridge Structural
Database (CSD), and an X-ray structure of related isoform
27
eIF4A3 bound to polyuracil RNA (2HYI), a molecular model
was devised to analyze the potential binding complex formed
between RNA, rocaglamide A, and eIF4A1 (Figure 1). In this
model, the phenyl A and D rings of rocaglamide A are
positioned to promote pi-stacking with the RNA bases at the
RNA/eIF4A1 interface (see Table 1 for compound ring
assignment). The phenyl D and E rings are proposed to bind
into two adjacent hydrophobic pockets on eIF4A1 which
contain amino acid residues that have been shown through
mutational studies to be critical for small molecule binding. A
predicted hydrogen bond between an N−H side chain of Gln
Recently, an X-ray crystal structure (2 Å resolution) of the
ternary complex formed between eIF4A1, rocaglamide A, and a
2
2
1
95 and rocaglamide A’s carbonyl oxygen of the dimethyl
polypurine RNA sequence was disclosed (Figure 3).
amide group provides additional protein ligand stabilization.
This model allowed us to make key inferences to aid in design,
namely: (1) a key contributor to the binding interaction could
Interestingly, the binding mode of rocaglamide A in the crystal
structure is quite analogous to our proposed model and
confirms the 40° aryl−aryl torsion hypothesis. The X-ray
B
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Table 1. SAR Data for Flavagline Derivatives 2−23
compound
X
R8
R6
R4′
cell IC50 nM
clog P
LE
LLE
2
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
(-)
C
N
C
N
N
N
C
C
C
C
C
N
N
N
N
N
N
N
N
N
OMe
OMe
OMe
OMe
OMe
Cl
CN
Cl
Cl
OMe
CN
CN
Cl
CN
Cl
Cl
Cl
Cl
Cl
Cl
Cl
OMe
OMe
OMe
CN
CN
CN
OMe
CN
CN
OMe
CN
OMe
OMe
Me
20 ± 9
990
3.72
3.05
3.73
2.56
2.92
1.73
4.49
4.01
3.24
3.40
2.91
3.40
2.22
3.98
4.19
3.62
3.67
4.36
4.51
1.84
0.29
0.25
0.28
0.27
0.29
0.25
0.31
0.33
0.29
0.30
0.31
0.27
0.25
0.29
0.26
4.0
3.3
3.3
4.2
4.1
4.7
3.1
4.0
4.2
4.3
5.0
3.1
4.0
2.9
2.6
(rac)
(rac)
(-)
(-)
(-)
H
198 ± 51
159 ± 84
87 ± 26
385 ± 147
52 ± 15
19 ± 3
68 ± 18
38 ± 1
26 ± 2
283 ± 142
601 ± 266
143 ± 24
160 ± 46
>1000
241 ± 58
320 ± 68
603 ± 167
>1000
0 (rac)
1 (rac)
2 (rac)
3 (rac)
4 (rac)
5 (-)
6 (-)
7 (-)
8 (-)
9 (-)
0 (-)
1 (-)
2 (-)
3 (-)
H
H
H
H
H
Cl
F
CF H
0.26
0.25
0.23
2.9
0.21
1.7
2
CF3
OCF3
SO Me
2
Figure 2. Conformational analysis of rocaglamide A.
structure discloses an additional hydrogen bonding interaction
between the rocaglamide tertiary hydroxyl and N7 of an
adjacent RNA guanine base. This interaction is hypothesized to
impart sequence selectivity for the stabilization of purine
sequences to eIF4A1.
Initial efforts focused on optimization of the phenyl A ring of
rocaglamide A to promote pi-stacking with the RNA base and
impart sequence selectivity. We reasoned that the pi-stacking
interaction with RNA could potentially accommodate a
heterocycle in place of the phenyl A ring, a strategy that
would lower the lipophilicity of the core. Replacement of the
phenyl A ring in the rocaglamide system with a heterocycle is
unprecedented in the literature. We focused on utilizing
pyridine as a replacement as this would be a minimal structural
change and one that could greatly reduce the log P(−0.67) of
the scaffold. Incorporation of the pyridine nitrogen at the 8-
Figure 3. X-ray structure of a ternary complex formed between
rocaglamide A, eIF4A1, and a polypurine RNA sequence showing the
hydrogen bond from the tertiary hydroxyl group to the purine N7,
22
PDB ID 5ZC9.
C
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position of the core was explored first because the nitrogen at
this position could potentially mimic the methoxy group’s
hydrogen bond acceptor properties and eliminating one of the
methoxy groups would further reduce the lipophilicity of the
system. As stated earlier, conformational analysis of rocagla-
mide A indicated that the methoxy oxygen at the 8-position
forms a 7-membered intramolecular hydrogen bond with the
secondary hydroxyl at the 1-position, reinforcing the bioactive
conformation of the natural product core. Replacement with a
pyridine would enable a six-membered intramolecular hydro-
gen bonding arrangement. Pyridine 5 (Table 1) was
synthesized and was found to be 25-fold less potent than
rocaglamide A in the MDA-MB-231 breast cancer cell
proliferation assay. It should also be noted that for racemates,
the potencies are assumed to be 50% as potent as the active
enantiomer (the opposite enantiomers are completely inactive;
data not shown). To fully evaluate this structural change, the
des-8-methoxy derivative of rocaglamide A was also prepared
Figure 5. X-ray structure for compound 8 highlighting the intra-
molecular hydrogen bond between the secondary hydroxyl and the
pyridine nitrogen and the 40° Ar−Ar torsion.
(
6) and was found to be 5-fold more potent than 5.
Interestingly, lipophilic ligand efficiency (LLE) analysis showed
that the loss in potency for the pyridine system is entirely driven
by a reduction in clog P (5 and 6 are iso-LLE). To further
reduce the lipophilicity and potentially improve potency [as
well as ligand efficiency (LE) and LLE], the 4′-methoxy group
was replaced with a nitrile, a modification previously reported in
the literature to improve the potency of the rocaglamide
1
4
system. Nitrile analogue 7 was found to have threefold
improved potency versus 5, an increase similar to that reported
for this modification in related chemotypes. Modeling of
compound 7 in the binding pocket suggested that the nitrile
group fits into a narrow groove formed by the RNA and eIF4A
and is positioned to potentially interact with the side chain of
Asn 167 (Figure 4). We were encouraged at this point because
Figure 4. Modeling of compound 7 in the binding pocket formed from
eIF4A in complex with poly-U RNA, PDB ID 2HYI.
Figure 6. LLE plot for ring A phenyl series. Iso-LLE changes between
compounds are shown with yellow arrows while changes resulting in
LLE improvement are shown in green.
compound 7 has similar LE and LLE values as rocaglamide A,
however with a significant overall reduction in clog P (−1.2
logs). A review of the literature suggested that replacement of
the 6-position methoxy of the rocaglamide core with either a
chloro or nitrile group could also improve potency. Chloro
analogue 8 showed slightly improved potency versus 7 while
maintaining LLE, whereas the nitrile analogue 9 exhibited
modestly reduced potency, however, with an improved LLE. An
X-ray structure was obtained for compound 8 and confirmed
the intramolecular hydrogen bond between the secondary
hydroxyl and the pyridine nitrogen (Figure 5). The structure
also showed a torsional angle of 40° between the D and E
phenyl rings, consistent with earlier conformational analyses
conducted with rocaglamide A.
The decreased potency with nitrile 9 was the first indication
of divergence of potency SAR between the pyridine and phenyl
A-ring systems. Comparison of phenyl analogues 6, 10−14 with
an LLE analysis (Figure 6) indicated that the increase in
potency with 6-chloro substitution is driven by a lipophilicity
increase, whereas both 4′-nitrile and 6-nitrile substitutions lead
to potency increases via enthalpic contributions. Interestingly,
the analogous plot with pyridines 5, 7−9, and 15−16 (Figure
7) indicated that the potency increase with 4′-nitrile
substitution is enthalpically driven, but both the increase in
potency with 6-chloro substitution and the decrease in potency
with 6-nitrile substitution follow iso-LLE shifts, indicative of
lipophilic-driven potency changes. It should be noted that the
permeability properties of the 6-nitrile analogues were similar to
14
D
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Table 2. Modifications to the 2-Position of the Flavagline
Core
compound
R
cell IC50 nM
2
2
2
2
2
2
3
3
3
4 (rac)
5 (-)
6 (-)
7 (rac)
8 (rac)
9 (rac)
0 (-)
1 (-)
2 (rac)
H
670 ± 12
32 ± 4
90 ± 24
236 ± 63
304 ± 73
2037 ± 978
3018 ± 110
239 ± 123
1465 ± 1657
CONHMe
CONH2
2-oxazole
SO N(Me)2
SO NHMe
SO Me
CH N(Me)2
CH OH
2
2
2
2
2
utilizing the methoxy substitution at the 8-position, was found
to be 2−3-fold more potent than 8 (equal LE and improved
LLE vs 8) and within 2-fold of the potency of rocaglamide A.
This was a striking result given previous reports of the
importance of 6-position substitution (phenyl A ring chemo-
14
type) for potency. Also intriguing, the 5-pyridyl isomer (36)
is inactive, further demonstrating that subtle electronic
differences in the A-ring can have a dramatic impact on
potency. Modeling of 36 in the RNA/eIF4A binding site
indicated that the lone pair of electrons of the 5-pyridine would
be in close proximity to the polar RNA backbone, a situation
that would be highly disfavored for binding (Figure 8).
Figure 7. LLE plot for ring A pyridine series. Iso-LLE changes between
compounds are shown with yellow arrows while changes resulting in
LLE improvement are shown in green.
the 6-methoxy analogues; thus, variations in cell permeability
were ruled out as possible explanations for the observed
potency SAR (data not shown). One explanation for the SAR
differences between the pyridine and phenyl systems could be
subtle differential effects of ring substitution on the pi stacking
interactions with RNA in the ternary complex.
Utilizing chloro analogue 8 the 4′-position was further
interrogated (analogues 17−23 in Table 1), focusing on
functional groups previously unexplored in the literature at this
position of the molecule. Ultimately, all changes at this position
resulted in a loss of potency versus the nitrile substituent.
Modifications to the dimethyl amide functionality at the 2-
position of compound 8 were also explored (Table 2).
Complete removal of the amide side chain (24) resulted in a
significant reduction in potency. Interestingly, the secondary
Based on the improved potency and LLE relative to 8,
compound 35 was selected for further optimization. Analysis of
the DMPK properties of 35 (Table 4) revealed poor CACO
permeability and aqueous solubility; however, a good rat PK
profile was observed following intravenous dosing. The poor
aqueous solubility was a concern as this could potentially create
formulation challenges for clinical studies utilizing IV infusions.
Therefore, a strategy was sought that could greatly improve the
aqueous solubility of the scaffold as well as improve
permeability properties. Replacement of the dimethyl amide
group at the 2-position with a solubilizing basic amine side
chain was revisited in the context of analogue 35. Previously,
replacement with the dimethylamino-methylene side chain in
the 8-pyridyl series resulted in a 2−3-fold loss of potency (31 vs
7). The analogous analogue was prepared in the 6-pyridyl series
(37) and was found to be within 2-fold of the potency of
dimethyl amide 35 (Table 4). The basic amine side chain
greatly improved the aqueous solubility of the scaffold (>20
mg/mL for 37 vs 0.22 mg/mL for 35) while maintaining the
overall LE. The LLE of 37, however, was slightly reduced versus
35. Compound 37 showed no inhibition in a hERG functional
patch-clamp assay, a liability often associated with basic
molecules. However, incorporation of the basic amine did
further increase Pgp-mediated efflux and resulted in higher rat
IV clearance (Table 4). Given the solubility advantages of the
amine side chain, an extensive effort was conducted to optimize
this group to maintain the attractive solubility, potency, and
hERG off-target profiles and mitigate issues with efflux and IV
clearance. A focused library (compounds 38−52) was
(
25) and primary amide (26) derivatives had slightly improved
or equal potency, respectively. However, in both cases the
−
6
permeability was reduced (CACO AB = 0.5 × 10 cm/s)
versus tertiary amide 8 (CACO AB = 1.8 × 10− cm/s). Several
amide isosteres were also tested (27−30), as well as the amino
and alcohol analogues 31 and 32, respectively, and all were
found to be less potent than the parent system.
6
At this point, further exploration of the pyridine A ring was
conducted to improve potency while continuing to optimize the
physicochemical properties of the scaffold. Analogues 33−36
were synthesized to probe the optimal pyridine regioisomers of
this system (Table 3). The 7-position pyridyl isomers, having
either cyano substitution at the 6-position (33) or methoxy
substitution at the 8-position (34), demonstrated modest
potency. Interestingly, the 6-position pyridyl isomer (35),
generated in which the sterics, lipophilicity, and pK properties
a
E
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Table 3. Profiles of Pyridine Analogues 33−36
compound
cell IC50 nM
clog P
LE
LLE
3
3
3
3
3 (-)
4 (rac)
5 (-)
308 ± 84
314 ± 19
33 ± 9
1.93
2.96
2.56
2.96
0.26
0.27
0.29
4.6
3.8
4.9
6 (rac)
>1000
might have improved permeability due to increased shielding of
the pyridine nitrogen. It was also reasoned that a potency
increase was possible by combining substitutions at the 8 and 6-
positions of the A-ring (mimicking the original substitution
pattern of rocaglamide A). Thus, compound 53 was prepared
and found to be 3−4 fold more potent versus 37 (equal LE and
improved LLE; Table 4) and maintained excellent aqueous
solubility. Compound 53 also showed an improved CACO
profile, however, the efflux ratio was still deemed to be too high
(
efflux was viewed as a risk for potential MDR-based resistance
mechanisms). Analogue 53 was evaluated in rat IV PK and
found to have reduced clearance versus 37 (Table 4). To
mitigate the efflux liability of this system, the PSA was reduced
by replacing the 6-position nitrile with a methoxy group. This
modification ultimately led to the discovery of eFT226, an
analogue with similar potency as compound 53, however with
improved LE, LLE, CACO permeability, and reduced efflux
(Table 4). It is interesting to note that despite significant
optimization efforts at the 8 and 6-positions, ultimately eFT226
retained the A-ring bis-methoxy substitution pattern found in
rocaglamide A. As stated previously, two prioritized goals for
the program were to identify a development candidate with
reduced lipophilicity and increased aqueous solubility versus
the rocaglamide natural products. The experimentally measured
log P and log D 7.4 values for eFT226 are 2.6 and 1.5,
respectively, thus demonstrating a high level of lipophilic
optimization (eFT226 LLE = 6.5). The thermodynamic
aqueous solubility of eFT226, driven in large part by the
Figure 8. Binding model for compound 36. The 3.9 Å contact between
the pyridine nitrogen of 36 and one of the phosphate ether oxygens of
the RNA backbone is shown, PDB ID 2HYI.
of the amine side chain of 37 were modulated (Table 5).
Analogues with similar basicity as 37 (i.e., 40, 41, 49, 50, 52),
regardless of sterics and lipophilicity of the amine, were found
to have poor permeability properties. Reducing the pK of the
a
amine (i.e., analogues 42−46, 51) in general reduced efflux, but
also reduced antiproliferative potency. Analysis of hERG SAR
indicated a strong correlation to lipophilicity (preferred range
clog P < 2.5) but not to pK (comparing analogues 41 and 42).
a
Ultimately, the library was unsuccessful in identifying amine-
containing candidates in the 6-pyridyl series with the desired
potency, hERG, and permeability properties.
At this stage it was decided to analyze the 2-position amino
side chain in the context of the 7-pyridyl system (represented
by analogues 33 and 34), with the expectation that this system
basic dimethylamino side chain (pK = 8.6), is > 20 mg/mL (as
a
Table 4. Potency, Solubility, CACO Permeability and in Vivo Rat PK (1 mg/kg) Properties for 35, 37, 53, and eFT226
solubility
mg/mL
CACO AB
CACO
ER
hERG
IC50 μM
Cl
Vdss
L/kg
1 × 10− cm/s
6
mL/min/kg
T1/2
h
AUC h μg/mL
compound
cell IC50 nM
LE
LLE
3
3
5
5 (-)
7 (-)
3 (-)
33 ± 9
54 ± 4
15 ± 3
0.29
0.29
0.29
0.31
4.9
4.6
5.3
6.5
0.22
>20
>20
>20
0.65
0.21
1.0
5
22
11
4
18
39
26
48
1.1
5.2
10.3
13.0
2.0
0.95
0.44
0.73
0.39
>10
>10
>10
2.9
11.0
4.4
eFT226 (-)
10.6 ± 0.9
2.5
F
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Table 5. SAR Studies for 2-Position Amine Analogues
HCl salt), representing a significant improvement versus
rocaglamide A. Both of these properties greatly enhance the
drug-ability of eFT226 facilitating intravenous delivery.
CHARACTERIZATION OF CLINICAL CANDIDATE
EFT226
■
Sequence recognition motifs within the 5′-UTR of select target
genes have been shown to drive sensitivity to rocaglate
2
1,22,28
inhibition of eIF4A1.
eFT226 for polypurine (AGAGAG), G-quadruplex like
GGCGGC), or control 5′-UTR recognition motifs was tested
The sequence specificity of
(
using surface plasmon resonance. Direct binding studies
showed that eIF4A1 alone bound weakly (3−8 μM) to RNA
and only in the presence of ATP. Addition of eFT226 induced
the formation of an RNA sequence-dependent ternary complex
between eIF4A1 and polypurine (AGAGAG) RNA motifs in a
nucleotide (ATP, ADP) independent manner. eFT226
increased the binding affinity (K ) of eIF4A1 to an AGAGAG
D
polypurine RNA oligonucleotide by >100-fold in the presence
of ATP (K 21−69 nM) due to a change in binding kinetic
D
rates resulting in a slow off rate and prolonged residence time
for eIF4A1 bound to the polypurine RNA motif (Figure 9), a
Figure 9. eFT226 enhances formation of eIF4A1/RNA complex.
Sensograms of eIF4A1 binding to rAGA surface in the presence (solid)
or absence (dashed) of eFT226. The sensograms were fitted using a
global fitting function to determine the equilibrium dissociation
constant and binding kinetics. Running buffer contained 14.4 mM
Hepes pH 7.4, 108 mM NaCl, 1 mM MgCl , 0.36 mM TCEP, 1 mM
2
ATP, and 1% dimethyl sulfoxide (DMSO). Data were collected at 25
°
C.
Figure 10. eFT226 inhibition of reporter gene expression is dependent
upon the 5′ UTR sequence. Relative luciferase expressed by luciferase
reporter mRNAs containing 5′-UTRs encoding unique 6-mer
sequence motifs. The luciferase reporter gene constructs were
transiently transfected into the MDA-MB-231 cell line and treated
with increasing concentrations of eFT226 for 4 h in triplicate. Data
were fitted to a four-parameter dose response curve. AGAGAG
(green); GGCGGC (blue); CCGCCG (red) and CAACAA (black).
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mechanism of inhibition similar to that reported for
Rocaglamide A (Table 6). eFT226 did not induce a stable
Additional translational inhibitors were evaluated in the
luciferase reporter assay (Table 7). Rocaglamide A demon-
strated 5′-UTR sequence dependent inhibition of luciferase
reporter gene expression similar to eFT226, consistant with
interacting at the same binding site. Hippuristanol, an initiation
inhibitor which binds at the C-terminus of eIF4A and blocks
RNA binding, inhibited luciferase reporter gene expression with
an IC of 200−400 nM; however, inhibition was independent
of the sequence motif in the 5′-UTR (Table 7). Inhibition by
cycloheximide (CHX), an elongation inhibitor, also showed no
dependence on 5′-UTR sequence. These findings are consistent
with eFT226 inhibition converting eIF4A into a sequence-
selective translational repressor.
21
Table 6. Binding Characteristics of eFT226-Induced
Ternary Complex Formation with Different RNA Sequences
KD (μM) with KD (μM) without KD (μM) with
RNA sequence
eFT226
eFT226
Roc A
5
0
AGAGAG + ATP
0.021 (±0.001)
8.0 (±0.9)
0.156
(
±0.007)
GGCGGC + ATP 3.19 (±0.03)
8.0 (±0.3)
3.27 (±0.06)
3.78 (±0.05)
NA
NA
NA
CCGCCG + ATP
CAACAA + ATP
9.6 (±0.4)
2.43 (±0.01)
RNA sequence
half-life t1/2, s
The selectivity of eFT226 for eIF4A was shown by
introducing a mutation into the putative drug binding site of
the eIF4A1 gene in the eFT226 sensitive HAP1 cell line. Based
on literature and molecular modeling of the putative drug
binding site, a mutation was introduced in eIF4A1 converting
AGAGAG + ATP + eFT226
AGAGAG + AMP-PNP + eFT226
79
2311
ternary complex with RNA oligonucleotides containing
GGCGGC, CCGCCG, or CAACAA sequence motifs (Table
), nor was binding observed for eIF4A1 to DNA oligo
sequences (Supporting Information), demonstrating the
selectivity of the eFT226 induced ternary complex.
25,29
phenylalanine 163 to leucine (F163L).
Treatment of tumor
6
cell lines with eFT226 (see Figure 11) downregulates the
The binding kinetics for the formation of the ternary complex
with eIF4A1−eFT226−AGAGAG RNA was evaluated when
ATP was replaced with AMP-PNP, a nonhydrolysable analogue
of ATP. After formation of the ternary complex, the SPR
surfaces were washed with buffer containing only ATP or AMP-
PNP to evaluate the ternary complex dissociation. The
dissociation rate of eIF4A1 from the polypurine RNA was
∼
30-fold slower when using the nonhydrolysable ATP
analogue, suggesting that ATP hydrolysis results in a conforma-
tional change that destabilizes the eIF4A1−eFT226−poly-
purine RNA complex (Table 6). Collectively, these results
demonstrate that eFT226 is a potent and reversible inhibitor of
eIF4A through the formation of a stable ternary complex with
select polypurine RNA motifs.
To determine whether the sequences that facilitate the
formation of the ternary complex enable selective inhibition of
protein synthesis with eFT226 treatment, MDA-MB-231 cells
were transiently transfected with a luciferase reporter system
containing specific tandem sequence motif repeats in the 5′-
UTR (see Table 7). Treatment with eFT226 inhibited
Figure 11. Downregulation of eIF4A target genes by eFT226 is
rescued with eIF4A1 F163L knock-in mutation. Protein levels of
eIF4A sensitive genes after treatment with eFT226 for 24 h in HAP1
wt or eIF4A1 F163L mutant cells.
expression of several key oncogenic proteins, including c-MYC,
MCL-1, and Cyclin D1. To examine if this effect of eFT226 is
altered in eIF4A1 F163L cells, HAP1 wt and eIF4A1 F163L
cells were treated for 24 h with either DMSO or increasing
concentrations of eFT226 followed by Western blot analysis. A
dose-dependent decrease in the protein levels of eFT226 target
genes such as c-MYC, MCL-1, and Cyclin D1 was observed in
the parental HAP1 cells; however, the protein levels remained
unchanged in the eFT226 treated HAP1 eIF4A1 F163L mutant
clone (Figure 11). These results indicate that mutation of
eIF4A1 suppresses the ability of eFT226 to diminish steady-
state protein levels, supporting that eIF4A is the cellular target
of this compound.
Table 7. Sequence Specificity of eFT226 Translational
Regulation in a Cell-Based Assay
5
′-UTR
eFT226
Roc A
IC50 (nM)
hippuristanol
CHX
IC50 (nM)
sequence motif IC50 (nM)
IC (nM)
50
AGAGAG
GGCGGC
CCGCCG
CAACAA
1.5 ± 0.4
13.8 ± 2.0
92 ± 18
4.7
47
135
231
426
369
210
204
226 ± 55
101 ± 10
87 ± 6
218 ± 55
142 ± 12
To investigate if eFT226-dependent growth inhibition is
affected by the F163L eIF4A1 mutation, proliferation assays
were conducted with parental HAP1 and eIF4A1 F163L
mutant HAP1 cells. 72 h treatment with eFT226 yielded
striking differences in proliferation between the parental and
eIF4A1 F163L HAP1 cells, as shown in a representative
experiment in Figure 12. The eIF4A1 F163L clone was ∼60-
fold less sensitive to the antiproliferative effects of eFT226
(average CI50 = 371 vs 6.3 nM in parental HAP1 cells).
Importantly, treating with hippuristanol, a structurally distinct
eIF4A inhibitor, led to equivalent growth arrest in parental and
translation of each reporter construct in a dose-dependent
manner (Figure 10). However, eFT226 was 16-145-fold more
effective at inhibiting luciferase reporter gene expression for the
GGCGGC and AGAGAG constructs, respectively, versus a
CAACAA sequence element (Table 7). These data are
consistent with the order of binding affinity observed in vitro
and the antiproliferative potency of eFT226 in the MDA-MB-
2
31 tumor cell line. In addition, eFT226 was not effective at
inhibiting translation for the reporter construct containing a
complementary CCGCCG motif.
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Figure 12. eIF4A1 mutation (F163L) rescues eFT226 activity linking antitumor phenotype to eIF4A1 inhibition. (A) Potent inhibition of cell
growth was observed with 72 h eFT226 treatment of HAP1 wild-type cells. The HAP1 eIF4A1 F163L mutant cells were resistant to eFT226
treatment. (B) eIF4A1 F163L mutation demonstrated resistance only to eFT226. Mutation at this binding site did not rescue the antiproliferative
effect of hippuristanol.
Figure 13. eFT226 modulates key drivers of tumorigenesis in a dose-dependent manner. (A) Cell cycle progression was assessed by DNA staining
using DRAQ5 and monitoring fluorescence by flow cytometry. Tumor cells treated with increasing concentrations of eFT226 for 24 h resulted in G2
arrest. (B) Incubation of MDA-MB-231 tumor cells with increasing concentrations of eFT226 for 24 h were analyzed by annexin (V+) and PI flow
cytometry. A dose-dependent increase in cells undergoing apoptosis and cell death (V+ PI+) was observed with eFT226 treatment. (C) Treatment
of MDA-MB-231 cancer cells with eFT226 downregulates the protein levels of eFT226-sensitive target genes (Cyclin D1 and BCL2) as analyzed by
Western blot.
Figure 14. eFT226 efficacy in orthotopic MDA-MB-231 TNBC model. (A) Athymic nude mice (10 mice/group) implanted in the mammary fat pad
with MDA-MB-231 cells were treated once weekly (Q1W) with either vehicle or eFT226 by IV for 14 days to assess effect on tumor growth.
Treatment with eFT226 results in tumor regression during the treatment duration. (B) Effect of vehicle or drug treatment on body weight change
over the course of treatment in tumor bearing mice.
eIF4A1 F163L HAP1 cells, consistent with unique binding sites
for these different class of compounds targeting eIF4A1. These
data indicate that eFT226 mediates growth repression through
eIF4A1. Although both eIF4A1 and eIF4A2 have been reported
to cycle through the eIF4F complex in vitro, the ability of an
eIF4A1 selective mutation to rescue eFT226’s cellular
phenotype supports eIF4A1 as the anticancer target. The
mutational studies reported here also suggest that eIF4A1 is the
target of eFT226; however, because eIF4A2 is expressed at a
much lower level than eIF4A1 in this cell line, we cannot rule
out the role of eIF4A2.
eFT226 was also evaluated in a CEREP panel of 114 in vitro
radioligand binding and enzyme assays covering a diverse range
of off-target enzymes, receptors, ion channels, and transporters
at a concentration of 10 μM. Significant activity, defined as
exceeding 50% binding or inhibition, was detected for only one
2
+
target (54% inhibition of Ca channel; L, diltiazem site),
indicating that eFT226 is a very selective ligand for eIF4A.
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The antiproliferative activity and mechanism of action were
further evaluated in the MDA-MB-231 breast cancer cell line.
The concentration for 50% inhibition of MDA-MB-231 cell
proliferation (CI ) with eFT226 treatment was determined to
once weekly dosing, supportive of its advancement into human
clinical studies.
SYNTHETIC CHEMISTRY
5
0
■
be 10.6 ± 0.9 nM (Table 4). Cell cycle analysis showed a dose-
dependent increase in the G2/M population, indicating cell
cycle arrest in response to eFT226 treatment (Figure 13A). A
dose-dependent increase in the percentage of apoptotic and
dead cells measured by annexin V and PI (propidium iodide)
staining was also seen with eFT226 treatment (Figure 13B).
Exposure of MDA-MB-231 cells to eFT226 for 24 h
confirmed that eIF4A-dependent oncoproteins were rapidly
downregulated at the protein level relative to control. Potent
downregulation of Cyclin D1 and BCL2 was observed whereas
the protein level of housekeeping genes (i.e., GAPDH) were
unchanged (Figure 13C). Modulation of these target genes is
consistent with the observed block in cell cycle progression and
induction of apoptosis.
Several synthetic approaches toward flavagline natural products,
such as silvestrol (1), and rocaglamide A (2), as well as
analogues thereof, have been published in recent
14−19,30,31
years.
Out of these, the [3 + 2]-photocycloaddition
approach disclosed by Porco and co-workers is arguably the
most efficient one, as it provides rapid access to the flavaglines’
1
9,32−36
sterically congested cyclopenta[b]benzofuran core.
Three steps consisting of a [3 + 2]-photocycloaddition of
substituted flavonoles with methyl cinnamates, followed by
base-induced ketol rearrangement and directed reduction,
constitute the key sequence of this biomimetic strategy
(
Scheme 1).
The conciseness and efficiency of Porco’s approach
prompted us to investigate its utility in the synthesis of the
novel rocaglamide-inspired eIF4A inhibitors discussed in this
The antitumor efficacy of eFT226 was assessed in the MDA-
MB-231 orthotopic xenograft model treated once weekly for 2
weeks with drug or vehicle control administered IV. Treatment
with 1 mg/kg of eFT226 Q1W led to 122% tumor growth
inhibition or regression throughout the duration of the study
3
7
paper (Schemes 2−24). A salient feature of the work
described herein is the successful application of this strategy
to the synthesis of analogues featuring heterocyclic A-rings (cf.
5
, 7−9, 15−53, eFT226; see 1 and 2 in Scheme 1 for labeling
(
Figure 14A). eFT226 was well tolerated as seen by a lack of
of the rings and numbering).
body weight loss (Figure 14B). eFT226 demonstrated good
cross-species IV PK and low plasma protein binding (Table 8).
The aqueous solubility of eFT226 is excellent (>20 mg/mL),
allowing for simple formulations to be utilized for intravenous
delivery.
Analogues Containing Carbocyclic A-Rings. The
general synthesis of 8-unsubstituted, A-carbocyclic rocaglamide
analogues 6 and 10−14, featuring electron withdrawing as well
as electron donating substituents in the 6- and 4′-positions of
the rocaglamide scaffold, is shown in Scheme 2.
While the overall synthetic strategy shown in Scheme 2 was
applied to the synthesis of all A-ring carbocyclic targets, it is
noteworthy that the exact timing of the incorporation of the
desired 6- and 4′-substituents depended on the target and was
largely dictated by synthetic feasibility as well as availability of
the required starting materials. More specifically, and as shown
in Schemes 3 and 4, the synthesis of compounds rac-6 and rac-
Table 8. Nonclinical eFT226 Plasma Protein Binding and
Pharmacokinetic Parameters after a Single Intravenous
Bolus Dose
T1/2
CL
Vss
plasma protein binding
species
(h)
(mL/min/kg)
(L/kg) % bound
mouse
rat
dog
monkey
human
10.9
4.39
NR
14.9
NA
15.8
48.3
<12.4
11.7
NA
12.1
13
17.8
12.2
NA
74.8
73.4
81.2
63.5
66.8
1
0 relied on an early installation of the desired 6- and 4′-
substituents. Thus, the synthesis of rac-6 commenced with the
regioselective O-methylation of resacetophenone (92% yield),
followed by aldol condensation (90% yield) (Scheme 3a). The
so-obtained chalcone was then treated with hydrogen peroxide
under basic conditions, which induced an oxidative Algar−
a
a
a
a
T1/2 not reported (NR) due to insufficient terminal phase
characterization. In lieu of V estimate, V (dose/C ) was calculated
38−40
ss
d
0
Flynn−Oyamada (AFO) cyclization
modest but sufficient yield of 20%.
to give 69 in a
and reported in this table. CL estimated from AUC0−t.
At this point, flavonol 69 was subjected to a [3 + 2]-
cycloaddition with methyl cinnamate (55) under photo-
chemical conditions. In general, it proved favorable to simply
remove the excess methyl cinnamate chromatographically upon
completion of the reaction and move the otherwise unpurified
crude product through the sequence of base induced ketol-
rearrangement and directed ketone reduction to ultimately give
compound 70, which was obtained in 36% yield over three
steps. Saponification of the ester in 70 (84% yield) followed by
amide coupling (33% yield) completed the synthesis of rac-6.
The synthesis of rac-10 followed a closely related synthetic
strategy as detailed in Scheme 3b, starting from appropriately
substituted acetophenone 71 with the 6-Cl substituent in place.
It is noteworthy that in this case, the yields of both the AFO
cyclization to give 72 (63%) and the key photocycloaddition/
ketol-rearrangement/directed reduction sequence (72 → 73,
52% over three steps) were significantly improved compared to
the sequence leading to rac-6, an observation that might point
at the impact of the electronic nature of the chalcone and the
CONCLUSIONS
■
Targeting dysregulated translation is a growing strategy in
oncology-based drug discovery. The expression of many key
oncoproteins is translationally controlled by eIF4A and the
eIF4F complex. The flavagline natural product class,
represented by rocaglamide A, is known to inhibit translation
through the stabilization of a ternary complex between the
natural product, eIF4A, and select mRNA sequences. Herein we
describe the optimization of rocaglamide A, focusing on
improving the drug-like properties of this system, to yield the
development candidate eFT226 (Zotatifin). eFT226 is a
potent, highly selective inhibitor of eIF4A-mediated translation
with excellent physicochemical properties. Similar to rocagla-
mide A, specificity for polypurine RNA sequences was
demonstrated. eFT226 revealed good cross-species IV PK
and was shown to be highly efficacious and well tolerated in a
triple negative breast cancer orthotopic xenograft model with
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Scheme 1. Key Steps in Porco’s Synthetic Approach toward Silvestrol (1) and Rocaglamide a (2) Using a Biomimetic
Photocycloaddition Strategy
Scheme 2. General Synthetic Route toward Carbocyclic A-
Ring Analogues 6 and 10−14
otherwise followed the same strategy as discussed for
compounds rac-6 and rac-10 (Scheme 4). Compound rac-11
was obtained in an eight-step sequence, starting with the aldol
condensation of methyl ketone 71 and para-bromobenzalde-
hyde (quantitative), followed by AFO cyclization (47% yield)
(
Scheme 4a). The subsequent conversion of flavonol 74 to
intermediate 75 was achieved via the previously established
photocycloaddition/ketol-rearrangement/directed reduction
sequence, which proceeded in an overall yield of 31%. Ester
hydrolysis (93%), EDC/HOBt-mediated amide coupling
(
85%), and palladium-catalyzed 4′-cyanation (14%) afforded
analogue rac-11.
Analogues rac-12 and rac-13 were successfully synthesized
starting from monobenzylated resacetophenone 76 (Scheme
4
b). In the case of rac-12, compound 76 was successfully
transformed into flavonol 77 (18% overall), which was
converted into 78 in three steps utilizing the established
phtotochemical protocol (29% overall). As detailed in Scheme
4
b, compound 78 was then subjected to straightforward ester
hydrolysis, amide coupling, hydrogenolytic debenzylation, O-
methylation to install the 6-methoxy group, and finally
palladium-catalyzed cyanation to install the 4′-CN moiety
and provide rac-12. Compound rac-13 was synthesized in
analogous 10 steps, starting from 76, as described in detail in
Scheme 4b. In this case, aldol condensation with para-
methoxybenzaldehyde followed by AFO reaction gave 79
(
47% overall), photocycloaddition/ketol-rearrangement/direc-
ted reduction gave 80 (63% overall), and ester hydrolysis,
amide coupling, debenzylation, triflation, and palladium-
catalyzed cyanation afforded compound rac-13.
Finally, analogue rac-14 could be synthesized starting from a
key intermediate in the synthesis of rac-12, that is, 78 (Scheme
5
). Thus, the 4′-CN group could be successfully introduced via
Pd-catalyzed cyanation of 78 (81%). In a three-step sequence
involving hydrogenolytic debenzylation, triflation, and Pd-
catalyzed cyanation, the 6-benzyloxy group was then replaced
with the 6-CN group in 36% overall yield. Ester saponification
(69%) followed by amide coupling (41%) completed the
synthesis of rac-14.
flavonol system on the efficiency of the AFO reaction and
photocycloaddition, respectively.
The synthesis of analogues rac-11−rac-13 relied on a late
stage introduction of substituents at the 4′- and 6-positions but
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Scheme 3. (A) Synthesis of rac-6; (a) MeI, Acetone, rt
dehyde followed by AFO cyclization gave 91 in 23% yield over
two steps. The crucial photocycloaddition/ketol rearrange-
ment/directed reduction sequence then diastereoselectively
transformed 91 into ester 92 (24% yield over three steps),
which in turn was then converted into final target rac-5 via ester
saponification and amide coupling. Importantly, the successful
synthesis of rac-5 using the photochemical strategy validated
this approach for the synthesis of flavaglines featuring
heterocyclic A-rings.
(
92%); (b) p-MeO-benzaldehyde, NaOH, MeOH, 90%; (c)
NaOH, H O , Ethanol, 20%; (d) Methyl Cinnamate (55),
2
2
4
00 W UV, DCM, MeOH, MeCN; (e) NaOMe, MeOH, 100
°
C; (f) NaBH(OAc) , AcOH, MeCN, 36% over Three Steps;
g) LiOH, MeOH, Water, 84%; (h) HNMe −HCl, EDC,
3
(
2
HOBt, DIPEA, DCM, 33%; (B) Synthesis of rac-10. (a) p-
MeO-benzaldehyde, NaOH, MeOH, 89%; (b) NaOH, H O ,
Ethanol, Water, 63%; (c) Methyl Cinnamate (55), 400 W
2
2
UV, DCM, MeOH, MeCN; (d) NaOMe, MeOH, 80 °C; (e)
Compound (−)-7 was obtained in a similar fashion, which
suggested that differently substituted azaflavonols could be
successfully used as photocycloaddition partners. Thus, the
synthesis of (−)-7 commenced with the aldol condensation of
NaBH(OAc) , AcOH, MeCN, 52% over Three Steps; (f)
3
LiOH, MeOH, Water, 86%; (g) HNMe −HCl, EDC, HOBt,
2
DIPEA, DCM, 90%
9
0 with para-bromobenzaldehyde. AFO reaction then afforded
azaflavonol 93 and was followed by photochemical, stepwise
conversion of the latter intermediate into 94. Although this
sequence proceeded in only low yields (7% over five steps), it
enabled the preparation of enough material for the completion
of this target, which was accomplished in a three-step sequence
followed by chiral HPLC separation as outlined in Scheme 7b.
The synthesis of 8-aza 6-Cl analogues 8, 15, and 17−23
featuring electron-donating as well as electron-withdrawing
substituents in the 4′-position started from 2-acetyl-3-hydroxy-
5
-chloropyridine (98), which could be readily prepared in three
steps from 2-cyano-3-nitro-5-chloro pyridine (95) (Scheme
a).
With key pyridine 98 in hand, the previously established
8
strategy featuring a photocycloaddition-ketol rearrangement
sequence was envisioned for the construction of the sterically
congested cyclopenta[4,5]furo[3,2-b]pyridine cores of 8-aza 6-
Cl analogues 8, 15, and 17−23 (Schemes 8b,c).
With respect to compounds 15 and 17−23, an early-stage
introduction of the desired 4′-substituent via aldol condensa-
tion of 98 with an appropriately substituted benzaldehyde 61
proved synthetically most favorable (Scheme 8b). All but one
required benzaldehyde (cf. 61) were commercially available, the
exception being the somewhat unstable para-difluoromethyl
benzaldehyde, which was freshly prepared from 1-bromo-4-
(
difluoromethyl)benzene via bromo-lithium exchange followed
by quenching with DMF (see the Experimental Section for
details).
While all of these analogues (15 and 17−23) were
successfully obtained using the strategy outlined in Scheme 8,
it is noteworthy that the exact yields for each of the following
steps (in particular the AFO reaction: 14−78%, and the
photocycloaddition/ketol rearrangement/directed reduction
sequence: 7−26%) appeared to depend on the nature of the
4′ substituents, although no clear trend with respect to the
effect of electron-donating or electron-withdrawing character of
those substituents on the yields became evident (see
Experimental Section for details). The two final steps (ester
saponification, amide coupling; see steps f-g, Scheme 8b)
generally proceeded in a rather uneventful fashion and afforded
the desired targets as a racemic mixture of both enantiomers,
which could easily be separated by chiral HPLC (steps h,
Scheme 8b).
Compound (−)-8 was synthesized in a similar fashion, but in
this case the synthesis relied on a late stage introduction of the
4'-cyano substituent (Scheme 8c). Initially, aldol condensation
of 98 with para-bromobenzaldehyde followed by oxidative
AFO cyclization led to azaflavonol 102 in 19% overall yield.
Photocycloaddtion, ketol rearrangement, and diastereoselective
ketone reduction led to key intermediate 103 in good yields
Synthesis of 8-Aza Analogues Featuring Different
Substituents in the 6- and 4′-Positions. Focusing on the
expansion of the photochemical synthetic strategy to include
systems featuring heterocyclic A-rings, we envisioned the
general strategy outlined in Scheme 6 for the synthesis of 8-
aza analogues.
As evident from this general approach, the initial develop-
ment of a concise synthetic route toward functionalized 2-
acetyl-3-hydroxypyridine analogues (cf. 83, Scheme 6) was
essential. In this context, the synthesis of analogues rac-5 and
(−)-7 required 2-acetyl-3-hydroxy-5-methoxypyridine (90),
which could be obtained in four straightforward steps starting
from commercially available 2-cyano-3,5-difluoropyridine (86),
as detailed in Scheme 7a. This intermediate was successfully
utilized in the synthesis of analogues rac-5 and (−)-7 (Scheme
7
b). Thus, aldol condensation of 90 with para-methoxybenzal-
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Scheme 4. (A) Synthesis of rac-11; (a) p-Br-benzaldehyde, NaOH, MeOH, 90 °C, Quant.; (b) NaOH, H O , Ethanol, 47%; (c)
2
2
Methyl Cinnamate (55), 400 W UV, DCM, MeOH, MeCN; (d) NaOMe, MeOH, 90 °C; (e) NaBH(OAc) , AcOH, MeCN, 31%
3
over Three Steps; (f) LiOH, MeOH, Water, 93%; (g) HNMe −HCl, EDC-HCl, HOBt, DIPEA, DCM, 85%; (h) Zn(CN) ,
2
2
Pd(PPh ) , DMF, 120 °C, 14%; (B) Synthesis of rac-12 and rac-13; (a) p-MeO-benzaldehyde, NaOH, MeOH, Reflux, 40%; (b)
3
4
NaOH, H O , Ethanol, 46%; (c) Methyl Cinnamate (55), 400 W UV, DCM, MeOH, MeCN; (d) NaOMe, MeOH, 90 °C; (e)
2
2
NaBH(OAc) , AcOH, MeCN, 29% over Three Steps; (f) LiOH, THF, Water, 71%; (g) HNMe −HCl, EDC, HOBt, DIPEA,
3
2
DCM, 69%; (h) Pd(OH) , Ethyl Acetate, rt; (i) MeI, K CO , Acetone, rt, 77% over Two Steps; (j) Zn(CN) , Pd(PPh ) , DMF,
2
2
3
2
3 4
1
20 °C, 45%; (k) p-MeO-benzaldehyde, NaOH, MeOH, Reflux, 81%; (l) NaOH, H O , Ethanol, 58%; (m) Methyl Cinnamate
2 2
(55), 400 W UV, DCM, MeOH, MeCN; (n) NaOMe, MeOH, 80 °C; (o) NaBH(OAc) , AcOH, MeCN, 63% over Three Steps;
(p) LiOH, MeOH, Water; (q) HNMe −HCl, EDC, HOBt, DIPEA, DCM, 95% over Two Steps; (r) H , Pd(OH) , EtOAc; (s)
3
2
2
2
Tf O, DCM, 56%; (t) Zn(CN) , Pd(PPh ) , DMF, 110 °C, 25%
2
2
3 4
Scheme 5. Synthesis of rac-14; (a) Zn(CN) , Pd(PPh ) ,
Scheme 6. Envisioned Synthetic Route toward 8-Aza
Analogues rac-5 and Enantiopure 7−9 and 15−23
2
3 4
DMF, 100 °C, 81%; (b) H , Pd(OH) , EtOAc, 81%; (c)
2
2
Tf O, DIPEA, DCM, THF, 51%; (d) Zn(CN) , Pd(PPh ) ,
2
2
3 4
DMF, 120 °C, 86%; (e) LiOH, MeOH, Water, 69%; (f)
HNMe −HCl, EDC, HOBt, DIPEA, DCM, 41%
2
(
55%). Ester hydrolysis to give acid 104 and subsequent amide
coupling (86% overall) was then followed by high-yielding Pd-
catalyzed 4′-cyanation (83%) to provide access to analogue 8 in
racemic form. The racemate was then resolved using chiral
HPLC to give enantiopure analogue (−)-8.
Compounds (−)-9 and (−)-16 were synthesized as detailed
in Scheme 9. Key pyridine 109 was synthesized in four steps
from commercially available 2-cyano-3,5-dichloropyridine
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Scheme 7. (A) Synthesis of Key Intermediate 90; (a) NaOMe, MeOH, Reflux (85%); (b) MeMgCl, THF, −20 °C to rt, 65%; (c)
HBr, AcOH, 150 °C, 35%; (d) MeI, K CO , Acetone 0 °C to rt; (B) Synthesis of analogues rac-5 and (−)-7; (a) para-
2
3
Methoxybenzaldehyde, NaOH, MeOH, Reflux; (b) H O , aq NaOH, EtOH, 0 °C to rt, 23% over Two Steps; (c) Methyl
2
2
Cinnamate (55), MeCN, MeOH, DCM, rt, hν; (d) NaOMe, MeOH, 80 °C; (e) NaHB(OAc) , AcOH, MeCN, rt, 24% over Three
3
Steps; (f) LiOH, MeOH, H O, rt, 77%; (g) EDC, HOBt, DIPEA, Me NH−HCl, DCM, 0 °C to rt, 8%; (h) 4-
2
2
Bromobenzaldehyde, NaOH, MeOH, Reflux; (i) H O , aq NaOH, EtOH, 0 °C to rt; (j) Methyl Cinnamate, MeCN, MeOH,
2
2
DCM, rt, hν; (k) NaOMe, MeOH, 80 °C; (l) NaHB(OAc) , AcOH, MeCN, rt, 7% over Five Steps; (m) Zn, Zn(CN) , Pd dba ,
3
2
2
3
dppf, DMF, 140 °C; (n) LiOH, MeOH, H O, rt; (o) EDC, HOBt, DIPEA, Me NH−HCl, DCM, 0 °C to rt, 15% over Three
2
2
Steps; (p) Chiral HPLC Separation
(
105) (Scheme 9a) via S Ar replacement of both chlorides
2-(dimethylamino)methyl-substituted analogue (−)-31
could be conveniently prepared from acid 104 via EDC-
mediated amide coupling (76% yield) and amide reduction
with borane−DMS complex (61% yield) to give amine 117,
followed by Pd-catalyzed cyanation (46%) and chiral HPLC
separation (Scheme 11). 2-Oxazole-substituted analogue rac-27
was prepared from acid 104 in four steps, starting with the
coupling of 104 with amine primary 118 to give amide 119 in
64% yield (step e, Scheme 11). Hydrolysis of the acetal with
aqueous hydrochloric acid (step f) followed by cyclodehydra-
tion (step g; 21%, two steps) and Pd-catalyzed cyanation (step
h; 23%) gave rac-27.
The synthesis of rac-32, (−)-25, and (−)-26 is shown in
Scheme 12. Reduction of ester 103 with LAH, followed by
cyanation gave access to triol rac-32 (Scheme 12). Introduction
of the 4′-cyano group (step c; 78%) followed by ester
saponification (step d; 78%) led to acid 120. High-yielding
EDC/HOBt-mediated amide coupling using the HCl salts of
ammonia and methylamine, followed by chiral HPLC
separation, provided (−)-26 and (−)-25, respectively.
It was discovered that α,β-unsaturated β-aryl sulfonamides
were suitable alkene components for the photocycloaddition
with azaflavonols, as detailed in Scheme 13. Thus, photo-
cycloaddition of previously synthesized azaflavonol 102 with
α,β-unsaturated sulfonamides 121 and 123, followed by base-
induced ketol rearrangement and directed reduction, gave 122
in 2% overall yield and 124 in 9% overall yield, respectively.
Although the observed yields for this sequence are substantially
lower than the yield observed for the analogous sequence of
102 with methyl cinnamate (55) (cf. Scheme 8c, steps c−e, 56%
overall yield), the approach still allowed for the preparation of
enough material of 122 and 124 to complete the synthesis of
rac-28 and rac-29, respectively. Thus, 122 was successfully
transformed into rac-28 via Pd-catalyzed 4′-cyanation in low
N
with benzylalcohol (step a), followed by Grignard-addition to
the nitrile to install the methyl ketone moiety (step b),
hydrogenolytic removal of both benzyl groups (step c, 89%),
and regioselective monobenzylation of the 5-OH group over
the 3-OH group, taking advantage of the fact that the latter is
engaged in a hydrogen bond with the methyl ketone in the 2-
position (step d, 94%).
At this point, enantiopure (−)-9 and (−)-16 could be
obtained utilizing the same strategy that had successfully been
applied to other 8-aza analogues (Scheme 9b), beginning with
aldol condensation of 109 with a suitable benzaldehyde and
subsequent AFO reaction. Thus, 110 was prepared from 109 in
two steps (steps a,b; 28% overall) and further transformed into
1
11 (steps c−e, 38% overall). A series of functional group
manipulations as well as chiral HPLC separation then
completed the synthesis of (−)-9. Analogously, azaflavonol
1
12 was could be prepared from 109 (steps m,n) and
subsequently converted into 113 in three steps (steps o−q).
Again, the functional group manipulations including debenzy-
lation (step r; 7% over six steps), triflation of the resulting C6
phenol, and subsequent cyanation followed by stepwise
conversion of the C2 ester to the corresponding dimethyl
amide (steps s−v; 19% over four steps) afforded rac-16, which
was subjected to chiral HPLC separation to provide
enantiopure (−)-16.
Modification of the 2-Position in 8-Aza Analogues.
Acid 104, which had been previously prepared in the context of
the synthesis of analogue (−)-8 (Scheme 8c, step f), proved to
be a valuable intermediate in the synthesis of 2-unsubstituted
and various 2-substituted 8-aza analogues. Thus, 2-unsubsti-
tuted analogue rac-24 was synthesized from 104 via Barton-
decarboxylation (Scheme 10, steps a,b), followed by Pd-
catalyzed cyanation (step c).
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Scheme 8. (A) Synthesis of Key Intermediate 98; (a) BnOH, NaH, THF, rt, 84%; (b) MeMgBr, THF, −30 °C to rt, 64%; (c) H ,
2
Pd(OH) , EtOAc, 65%; (B) Synthesis of Analogues 15 and 17−23. (a) NaOH, 4-Substituted Benzaldehyde 61, MeOH, Δ; (b)
2
H O , NaOH, EtOH/DCM, rt; (c) Methyl Cinnamate, MeCN, MeOH, DCM, hν; (d) NaOMe, MeOH, Δ; (e) NaHB(OAc) ,
2
2
3
AcOH, MeCN, rt; (f) LiOH, THF/Water, rt; (g) EDC, HOBt, DIPEA, Me NH−HCl, DCM; (h) Chiral HPLC Separation; (c)
2
Synthesis of analogue 8. (a) para-Bromobenzaldehyde, NaOH, MeOH, Reflux, 61%; (b) H O , aq NaOH, EtOH, DCM, rt, 31%;
2
2
(
c) Methyl Cinnamate (55), MeCN, MeOH, DCM, rt, hν; (d) NaOMe, MeOH, 70 °C; (e) NaHB(OAc) , AcOH, MeCN, rt, 55%
3
over Three Steps; (f) LiOH, MeOH, H O, rt, Quant.; (h) EDC, HOBt, DIPEA, DCM, 0 °C, Then Me NH−HCl, rt, 86%; (i) Zn,
Zn(CN) , Pd dba , dppf, DMF, 140 °C, 83%; (j) Chiral HPLC Separation
2
2
2
2
3
yields (7%). 124 was subjected to high yielding acidic N-
debenzylation (92%, followed by Pd-catalyzed cyanation, which
againas in the case of rac-28proceeded in rather low yields
of 8% to give rac-29.
In the case of analogue (−)-30, a different approach for the
installation of the methyl sulfone moiety was identified
Aldol condensation and AFO reaction then led to azaflavonol
132. Using the established photochemistry/ketol rearrange-
ment/directed reduction protocol, 132 was transformed into
133 in three steps (steps f−h, 28% overall). Ester hydrolysis
followed by amide coupling proceeded as expected to give 134.
The subsequent 4′,6-dicyanation (step k) occurred in 36% yield
using conditions closely related to those utilized in the synthesis
of (−)-8 and was followed by chiral HPLC separation to
provide (−)-33.
Along the same lines, 7-aza analogue rac-34 was synthesized
as detailed in Scheme 16. In this case, the required pyridine 139
was obtained in three steps from 2-methoxy-4-aminopyridine
135 via electrophilic bromination (step a, 92%), Pd-catalyzed
coupling with enol ether stannane 137, which was followed by
acidic hydrolysis of the resulting enolether to the desired
methyl ketone (step b, 49%) and conversion of the amino
group into a hydroxy group via the transient diazonium salt
(step c, 37%). From this point on, a familiar sequence (steps d−
k) was pursued to complete the synthesis of rac-34 (see Scheme
16 for details). It is noteworthy that the three-step conversion
of 140 to 141 proceeded in unusually low yields (ca. 1%
overall). While suboptimal electronic match between aza-
flavonol 140 and methyl cinnamate (55) in the photo-
(
Scheme 14). Krapcho-decarboxylation with the 1-ketone in
place gave 126 (Scheme 14) in 33% yield. Copper(II)bromide-
41
mediated sulfonylation with sodium methanesulfinate fol-
lowed by directed reduction with sodium triacetoxyborohy-
dride provided methyl sulfone 127 in 4% overall yield. 4′-
Cyanation (72%) followed by chiral separation completed the
synthesis of (−)-30.
Synthesis 5-, 6-, and 7-Aza analogues. The synthesis of
other 5-, 6-, and 7-aza analogues was achieved utilizing the same
photochemical strategy that had successfully been used for the
synthesis of 8-aza analogues discussed above (Scheme 6),
highlighting the generality of this approach in the synthesis of
rocaglamide analogues featuring A-ring pyridines. The synthesis
of rac-35 is shown in Scheme 15. The required acetyl pyridine
131 was synthesized in three steps from commercially available
acid 128 via Weinreb amide formation, Grignard addition to
obtain the methyl ketone, and O-demethylation (steps a−c).
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Scheme 9. (A) Synthesis of Key Intermediate 109; (a) BnOH, NaH, THF, 0 °C to rt; (b) MeMgBr, THF, −30 °C to rt; (c) H ,
2
Pd(OH) , EtOAc, rt, 89%; (d) K CO , BnBr, Acetone, 94%; (B) Synthesis of Analogues (−)-9 and (−)-16; (a) 4-
2
2
3
Bromobenzaldehyde, NaOH, MeOH, 85 °C, 59%; (b) H O , NaOH, EtOH/DCM, rt, 53%; (c) Methyl Cinnamate (55), MeCN,
2
2
MeOH, DCM, rt, hν; (d) NaOMe, MeOH, 70 °C; (e) NaHB(OAc) , AcOH, MeCN, rt, 38% over Three Steps; (f) Zn, Zn(CN) ,
3
2
Pd dba , dppf, DMF, 125 °C (97%); (g) H , Pd(OH) , EtOAc, rt, 97%; (h) Tf O, DIPEA, DCM, −78 °C to 0 °C, 77%; (i) Zn,
2
3
2
2
2
Zn(CN) , Pd dba , dppf, DMF, 125 °C; (j) LiOH, THF/Water, 37% over Two Steps; (k) TEA, Me NH−HCl, T3P, DCM/
2
2
3
2
EtOAc, rt, 31%; (l) Chiral HPLC Separation; (m) NaOH, 4-Methoxybenzaldehyde, MeOH, Reflux; (n) H O , NaOH, EtOH/
2
2
DCM, rt; (o) Methyl Cinnamate (55), MeCN, MeOH, DCM, hν; (p) NaOMe, MeOH, 70 °C; (q) NaHB(OAc) , AcOH, MeCN,
3
rt; (r) H , Pd(OH) , EtOAc, 7% over Six Steps; (s) Tf O, DIPEA, DCM, −78 °C to rt; (t) Zn, Zn(CN) , Pd dba , dppf, DMF, 85
2
2
2
2
2
3
°
C; (u) LiOH, THF/Water, rt; (v) EDC, HOBt, DIPEA, Me NH−HCl, DCM, 0 °C to rt, 19% over Four Steps; (w) Chiral
2
HPLC Separation
Scheme 10. Synthesis of rac-24; (a) 114, DCC, DCM, rt; (b)
AIBN, Bu SnH, C H , 80 °C, 10% over Two Steps; (c) Zn,
more forcing conditions (80 °C) to give 144, which was then
reacted with methylmagnesium bromide to afford, upon
treatement with aqueous HCl, key pyridine 145. The
established two step procedure (aldol condensation, AFO
reaction) transformed 145 into azaflavonol 146. The latter was
successfully transformed into aza-rocaglate intermediate 147
using the well-established three step procedure featuring the
photocycloaddition with methyl cinnamate (55) as a key step,
which proceeded with an acceptable overall yield of 35% over
three steps. Functional group modifications followed by chiral
separation ultimately afforded analogue (−)-35.
3
6
6
Zn(CN) , Pd dba , dppf, DMF, 140 °C, 16%
2
2
3
The synthesis of analogue rac-36 required the preparation of
key pyridone 153 (Scheme 18). This was achieved in a five-step
sequence starting from 2,6-dichloronicotinic acid (150),
involving regioselective S Ar reaction with methanol in the
N
presence of potassium t-butoxide (step a), followed by Weinreb
amide formation (step b) and Grignard addition (step c) to
provide methyl ketone 152, S Ar with deprotonated PMB-
N
alcohol (step d), and acidic PMB deprotection (step e).
Transformation of 153 into 154 via aldol condensation and
AFO reaction proceeded as expected; however, the three-step
conversion of 154 to 155 was associated with very low yields of
only 3%, again suggesting an inefficient photocycloaddition,
potentially as a result of suboptimal electronic match of both
cycloaddition partners. The remaining steps (k−m) proceeded
as expected to provide 5-aza analogue rac-36.
Synthesis of 6-Aza Analogues Featuring Basic Amines
in the 2-Position. The majority of analogues featuring basic
amine substituents in the 2-position, that is, enantiopure 37,
40−44, 46−48, 50, and 51, as well as racemic 38 and 39, could
cycloaddition step might contribute to this outcome, this
observation is difficult to rationalize at this point, given the
moderate to good yields for this sequence observed in other
cases described herein. The remaining three steps proceeded as
expected to give rac-34.
The synthesis of 6-aza analogue (−)-35 is shown in Scheme
7. 3,5-Dichloro-4-cyanopyridine was subjected to a selective
1
SNAr reaction with sodium para-methoxybenzylate at 0 °C,
leading to intermediate 143 (76%). Displacement of the second
chloride under S Ar with sodium methoxide occurred under
N
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Scheme 11. Synthesis of rac-27, (−)-31, and rac-32; (a) NHMe −HCl, EDC, HOBt, DIPEA, DCM, 0 °C to rt, 76%; (b) BH -
2
3
DMS, THF, 0 °C to rt, Then MeOH, 0 °C to Reflux, 61%; (c) Zn, Zn(CN) , Pd dba , dppf, DMF, 130 °C, 46%; (d) Chiral HPLC
2
2
3
Separation; (e) EDC, HOBt, DIPEA, 0 °C, Then 118, 0 °C to 35 °C, 64%; (f) HCl(aq), THF, rt; (g) PPh , I , TEA, DCM, rt,
3
2
2
1% over Two Steps; (h) Zn, Zn(CN) , Pd dba , dppf, DMF, 150 °C, 23%
2 2 3
Scheme 12. Synthesis of rac-32, (−)-25, and (−)-26; (a)
LAH, THF, 0 °C to rt, 49%; (b) Zn, Zn(CN) , Pd dba , dppf,
compound was first trifluoroacetylated. Subsequent amide
reduction then gave 160, which was subjected to Pd-catalyzed
cyanation (24% yield over three steps) to give rac-45. Chiral
separation then provided analogue (+)-45.
Amine analogue (−)-49 was prepared from rac-48 (Scheme
21). In situ TBS protection of the secondary alcohol followed by
reductive amination with concomitant desilylation (step a,
55%) was followed by chiral HPLC separation to obtain
enantiopure (−)-49. It is noteworthy that the initial TBS
protection was crucial for the success of the sequence, as in the
absence of a protective group the secondary alcohol at C1 reacts
with the iminium generated in situ in the reductive amination
step to form a cyclic aminal.
2
2
3
DMF, Water, MW, 100 °C, 71%; (c) Zn, Zn(CN) , Pd dba ,
2
2
3
dppf, DMF, 125 °C, 78%; (d) LiOH, MeOH, Water, rt, 78%;
(
(
e) EDC, HOBt, DIPEA, DCM, 0 °C, Then NH Cl, rt, 78%;
4
f) Chiral HPLC Separation; (g) EDC, HOBt, DIPEA, DCM,
0
°C, Then MeNH −HCl, rt, 90%; (h) Chiral HPLC
2
Separation
The final route toward analogue rac-52 is shown in Scheme
2. Homologation of acid 148 was achieved in a four-step
2
sequence, involving borane reduction (step a), mesylation of
the resulting primary alcohol (step b), displacement of the
mesylate with cyanide (step c), and basic nitrile hydrolysis (step
d) to give 163 in 41% overall yield. Initial attempts to achieve
an amide coupling with 163 only led to formation of lactone
1
64 (92%), which could then be transformed into amide 165 in
a separate step in 47% yield. Amide reduction to give 166
71%) and subsequent Pd-catalyzed cyanation (19%) com-
(
pleted the synthesis of rac-52.
Synthesis of (−)-53 and eFT226. 6,8-disubstituted 7-aza
analogues (−)-53 and eFT226 were synthesized using closely
related routes (Schemes 23 and 24). The synthesis of the key
azaflavonol intermediates 174 and 175 is shown in Scheme 23
and commenced with the regioselective transformation of 2,4,6-
be prepared in a straightforward way starting from key
carboxylic acid 148 (Scheme 19), which had been prepared
in the context of the synthesis of (−)-35 (Scheme 17). Thus, in
each of these cases, amide coupling of 148 with an appropriate
amine gave access to amide 157. Borane reduction then
transformed the amide into the corresponding amine 158,
which was subjected to 4′-cyanation and, for 37, 40−44, 46−
trichloropyridine (167) into 168 via S
benzylate at 0 °C (72% yield). A second S
N
Ar with sodium
Ar reaction at
N
elevated temperatures was used to install the methoxy group
and afforded 169 in 56% yield. Regioselective, directed
lithiation followed by quenching with acetaldehyde led to
secondary alcohol 170 (65%), which was oxidized to ketone
(171, 75%). Hydrogenolytic debenzylation was carried out in
ethyl acetate to avoid reductive dechlorination and provided
hydroxypyridine 172 in 88% yield. Aldol condensation with
para-bromobenzaldehyde followed by AFO cyclization gave
174, a critical intermediate in the synthesis of (−)-53 (see
below), in 19% overall yield. It was found that modification of
the 6-position and installation of the methoxy group that is
4
8, 50, and 51, chiral separation to provide the desired final
compound (for details, see the Experimental Section).
The remaining 6-aza analogues of the focused amine library,
that is, (+)-45, (−)-49, and rac-52, were synthesized as shown
in Schemes 20−22. The synthesis of (+)-45 utilized 158b, an
intermediate in the synthesis of rac-38 (Scheme 20). This
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Scheme 13. Synthesis of rac-28 and rac-29. (a) 121, DCM, MeOH, MeCN, rt, hν; (b) NaOMe, MeOH, 80 °C; (c) NaHB(OAc)3,
AcOH, MeCN, rt, 2% over Three Steps; (d) Zn, Zn(CN) , Pd dba , dppf, DMF, 140 °C, 7%; (e) 123, DCM, MeOH, MeCN, rt,
2
2
3
hν; (f) NaOMe, MeOH, 80 °C; (g) NaHB(OAc) , AcOH, MeCN, rt, 9% over Three Steps; (h) TFA, Triflic acid, DCM, 92%; (i)
3
Zn, Zn(CN) , Pd dba , dppf, DMF, 125 °C, 8%
2
2
3
Scheme 14. Synthesis of (−)-30; (a) LiCl, DMSO, 150 °C,
3%; (b) Sodium Methanesulfinate, CuBr , DBU, DMSO, rt;
present in eFT226 was synthetically most feasible at this stage
of the synthesis, while its installation later on proved
challenging. Thus, treatment of 174 with sodium methoxide
in MeOH/DMF at 80 °C yielded azaflavonol 175, a key
intermediate in the synthesis of eFT226.
3
2
(
c) NaHB(OAc) , AcOH, MeCN, rt, 4% over Two Steps; (d)
Zn, Zn(CN) , Pd dba , dppf, DMSO, 150 °C, 72%; (e)
3
2
2
3
Chiral HPLC Separation
Both key azaflavonols 174 and 175 were utilized in the
synthesis of (−)-53 and (−)-eFT226, respectively, as outlined
in Scheme 24. Thus, the established three step sequence of
photocycloaddition/ketol rearrangement/directed reduction
transformed 174 into 176 in 27% yield. Ester hydrolysis,
amide coupling and amide reduction (75% overall) gave 177.
Pd-catalyzed 4′,6-dicyanation (63%) and chiral HPLC
separation completed the synthesis of (−)-53. eFT226 was
synthesized using the same strategy. In the three-step sequence
converting 175 to 178 best results were obtained when
chloroform/2,2,2-trifluoroethanol (TFE) was used as the
19
solvent system in the photocycloaddition step, in which
case the overall yield over three steps was 40%. Installation of
the dimethylaminomethyl sidechain via ester hydrolysis/amide
coupling/amide reduction (178 → 179) was achieved in 76%
yield over three steps. Pd-catalyzed 4′-cyanation led to racemic
Scheme 15. Synthesis of (−)-33; (a) EDC, HOBt, DIPEA, 129-HCl, DCM, 0 °C to rt, 63%; (b) MeMgBr, THF, 0 °C to rt, 88%;
(
c) AcOH, HCl (aq), 100 °C, 68%; (d) 4-Bromobenzaldehyde, NaOH, MeOH, DCM, 90 °C, 97%; (e) H O , aq NaOH, MeOH,
2 2
rt, 30%; (f) Methyl Cinnamate (55), MeCN, MeOH, DCM, rt, hν; (g) NaOMe, MeOH, 90 °C; (h) NaHB(OAc) , AcOH, MeCN,
3
rt, 28% over Three Steps; (i) LiOH, THF, H O, rt, 91%; (j) EDC, HOBt, DIPEA, Me NH−HCl, DCM, 0 to 40 °C, 71%; (k) Zn,
2
2
Zn(CN) , Pd dba , dppf, DMF, 150 °C, 36%; (l) Chiral HPLC Separation
2
2
3
R
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Scheme 16. Synthesis of rac-34; (a) NBS, DCM, 0 °C, 92%; (b) 137, cat. PdCl (PPh ) , Toluene, 100 °C, Then HCl (aq) 49%;
2
3 2
(
c) NaNO , H SO , Dioxane, Water, 0 to 50 °C, 37%; (d) 4-Bromobenzaldehyde, NaOH, MeOH, Reflux, 68%; (e) H O , aq
2 2 4 2 2
NaOH, EtOH, 0 °C to rt, 32%; (f) Methyl Cinnamate (55), MeCN, MeOH, DCM, rt, hν; (g) NaOMe, MeOH, 80 °C; (h)
NaHB(OAc) , AcOH, MeCN, rt, 1% over Three Steps; (i) LiOH, THF, H O, rt, 67%; (j) EDC, HOBt, DIPEA, Me NH−HCl,
3
2
2
DCM, 0 °C to rt, Quant.; (k) Zn, Zn(CN) , Pd dba , dppf, DMF, 140 °C, 19%
2
2
3
Scheme 17. Synthesis of (−)-35; (a) PMBOH, NaH, THF, 0 °C to rt, 76%; (b) NaOMe, MeOH, 80 °C, 46%; (c) MeMgBr, THF,
°C to rt; Then HCl(aq), rt, 52%; (d) para-bromobenzaldehyde, NaOH, MeOH, Reflux, 80%; (e) H O , aq NaOH, EtOH,
0
2
2
DCM, 0 °C to rt, 29%; (f) Methyl Cinnamate (55), MeCN, MeOH, DCM, rt, hn; (g) NaOMe, MeOH, 80 °C; (h) NaHB(OAc)3,
AcOH, MeCN, rt, 35% over Three Steps; (i) LiOH, MeOH, H O, rt, 79%: (j) NHMe −HCl, EDC, HOBt, DIPEA, DCM, 0 °C to
2
2
rt, 65%; (k) Zn, Zn(CN) , Pd dba , dppf, DMF, 140 °C; 50%; (l) Chiral HPLC Separation
2
2
3
Scheme 18. Synthesis of rac-36. (a) t-BuOK, MeOH, 80 °C; (b) EDC, HOBt, DIPEA, 129-HCl, DCM, 0 °C to rt, 75% over Two
Steps; (c) MeMgBr, Et O, THF, 0 °C to rt; (d) PMB-OH, NaH, THF, 0 °C to rt, 47% over Two Steps; (e) TFA, DCM, 0 °C to rt;
2
(
f) 4-Bromobenzaldehyde, NaOH, MeOH, 80 °C; (g) H O , aq NaOH, EtOH, DCM, rt, 40% over Three Steps; (h) Methyl
2 2
Cinnamate (55), MeCN, MeOH, DCM, rt, hν; (i) NaOMe, MeOH, 80 °C; (j) NaHB(OAc) , AcOH, MeCN, rt, 3% over Three
3
Steps; (k) LiOH, MeOH, H O, rt, 93%; (l) EDC, HOBt, DIPEA, Me NH−HCl, DCM, 0 °C to rt, 38%; (m) Zn, Zn(CN) ,
2
2
2
Pd dba , dppf, DMF, 140 °C, 22%
2
3
S
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Scheme 19. General Synthesis of 6-Aza-2-amine analogues Enantiopure 37, 40−44, 46−48, 50, 51, and Racemic 38, 39; (a)
R NH, EDC, HOBT, DCM, or R NH, HATU, DIPEA, DMF; (b) BH -DMS, THF, Δ, Then MeOH, Δ; (c) Zn, Zn(CN) ,
2
2
3
2
Pd dba , dppf, DMF, Δ; (d) Chiral HPLC Separation (for 37, 40−44, 46−48, 50, and 51)
2
3
Scheme 20. Synthesis of (+)-45. (a) TFAA, TEA, DCM, 0 °C to rt; (b) BH -DMS, THF, 60 °C, Then MeOH, Δ; (c) Zn,
3
Zn(CN) , Pd dba , dppf, DMF, MW, 140 °C, 24% over Three Steps; (d) Chiral HPLC Separation
2
2
3
Scheme 21. Synthesis of (−)-49. (a) TBSOTf, 2,6-Lutidine,
EXPERIMENTAL SECTION
■
o
DCM, 0 C-rt; Then (H CO) , NaOAc, NaBH(OAc) , rt,
2
n
3
Reagents. Hippuristanol and Rocaglamide A were synthesized and
QCed at Julilant Life Sciences (Uttar Pradesh, India). Cycloheximide
was purchased from Sigma-Aldrich (St. Louis, MO).
55%; (b) Chiral HPLC Separation
Liver Microsome Stability. Incubations were conducted at 1 μM
test article (TA), 0.5 mg/mL liver microsomal protein in 100 mM
phosphate buffer (PB; pH 7.4), 2 mM NADPH at 37 °C with a final
organic concentration of 0.1% DMSO, and 0.9% acetonitrile (MeCN).
Liver microsomes used in these experiments were obtained from
BioIVT (Hicksville, NY).
Each TA stock solution (10 mM DMSO) was diluted to 1 mM with
DMSO and then diluted an additional 10-fold with MeCN to 100 μM.
Stock microsomes (20 mg/mL) are thawed and diluted into PB to
0
.633 mg/mL. A solution of NADPH was made in PB at 8.33 mg/mL.
eFT226 in 73% yield, which was then subjected to chiral HPLC
separation to afford enantiopure (−)-eFT226. A crystal
structure analysis of (−)-eFT226 single crystals was success-
fully obtained and confirmed the structural assignment (Figure
The diluted microsomes were added to a 96-well plate (98.75 μL)
designated as master plate. The 100 μM TA (1.25 μL) was spiked into
the master plate and, following a 10 min preincubation period,
reactions were initiated by transferring equal aliquots from the master
plate (40 μL) to two clean plates. To one of these plates (T0 control
plate), 100 μL of MeCN containing an internal standard (IS) was
1
5).
Scheme 22. Synthesis of rac-52; (a) BH -DMS, THF, 0 °C to rt, Then MeOH, 70 °C, 93%; (b) MsCl, Pyridine, 0 °C to rt, 80%;
3
(
c) KCN, DMSO, 80 °C, 58%; (d) 10% NaOH (aq), THF, 125 °C, 94%; (e) Me NH−HCl, EDC-HCl, HOBT, DIPEA, DCM, 0
2
°C to rt, 92%; (f) Me NH, THF, T3P, EtOAc, 80 °C, 47%; (g) BH -DMS, THF, 0 °C to rt, Then MeOH, 0 °C to 70 °C, 71%; (h)
2
3
Zn, Zn(CN) , Pd dba , dppf, DMF, 140 °C, 19%
2
2
3
T
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Scheme 23. Synthesis of Azaflavonols 174 and 175; (a) BnOH, NaH, DMF, 0 °C, 72%; (b) NaOMe, MeOH, Toluene, 70 °C,
5
6%; (c) n-BuLi, THF, −75 °C, Then Acetaldehyde, 65%; (d) DMP, DCM, 75%; (e) H , Cat. Pd/C, EtOAc, 88%; (f) para-
2
Bromobenzaldehyde, NaOMe, MeOH, DMF, Quant.; (g) NaOH (aq), H O (aq), EtOH, DCM, Water, 19%; (h) NaOMe,
2
2
MeOH, DMF, 80 °C, 98%
Scheme 24. Completion of the Synthesis of (−)-53 and (−)-eFT226; (a) Methyl Cinnamate (55), MeCN, MeOH, DCM (R =
Cl) or Methyl Cinnamate (55), CHCl , TFE (R = OMe), 0 °C, hν; (b) NaOMe, MeOH, 60 °C; (c) NaHB(OAc) , AcOH, MeCN,
3
3
rt, 27% (R = Cl), 40% (R = OMe) (Yield over Three Steps); (d) LiOH, MeOH, Water, 50 °C (R = Cl), 90%, or LiOH(aq),
MeOH, THF, rt (R = OMe), 93%; (e) HATU, DIPEA, Me NH, THF, DMF, rt, 95% (R = Cl), 92% (R = OMe); (f) BH -DMS,
2
3
THF, 40 °C, Then MeOH, 65−70 °C, 88% (R = Cl), 89% (R = OMe); (g) Zn, Zn(CN) , Pd dba , dppf, DMF, Water, MW, 120
2
2
3
°C, 63% (R = Cl), 73% (R = OMe); (h) Chiral HPLC Separation
AB SCIEX TripleTOF 5600+ operated in electrospray ionization TOF
MS (+) mode. All experiments were run in duplicate, and the mean
percent remaining at 30 min was determined as follows.
%
remaining at 30 min
mean at 30 min(analyte peak area/IS peak area)
mean at 0 min(analyte peak area/IS peak area)
=
Caco-2 Permeability and Efflux Studies. CACOReady 96-well
kits were purchased from ADMECell (Alameda, CA). Two weeks after
seeding, ADMECell ships the kits in a proprietary semi-solid medium.
The media was changed after arrival, and all assays were performed on
day 21 post-seeding. A 10 mM stock solution of each TA was prepared
and stored at −20 °C until needed for experimentation. A working
buffer was prepared in Hanks’ balanced salt solution (HBSS) with 10
mM HEPES and 15 mM glucose at pH 7.4. An acceptor buffer was
prepared from the working buffer with an addition of 1% bovine serum
albumin. The dosing buffer was prepared from the working buffer with
the addition of TA resulting in a final concentration of 5 μM TA and
1% DMSO. Prior to the start of the assay cell junction, integrity was
assessed by transepithelial electrical resistance (TEER). For use in the
Figure 15. X-ray structure of (−)-eFT226.
added. To the other plate (T30 reaction plate), NADPH solution (10
μL) was added to initiate the reaction. To the T0 control plate,
NADPH solution was added (10 μL) and the plate was vortexed at
1
000 rpm for 5 min to precipitate protein. After the 30 min incubation
period, the T30 plate was taken from the incubator, and 100 μL MeCN
with IS was added. Subseqeuntly the plate was vortexed at 1000 rpm
for 5 min to quench reactions. Precipitated proteins were removed by
centrifugation, and supernatants were transferred to new 96-well
polypropylene plates. Plates were capped with pierceable septa and
subjected to bioanalysis by LC/MS/MS. The MS model used was an
2
assay, wells required a TEER measurement of at least 500 Ω·cm .
Apical and basolateral sides of the cell monolayers were washed once
with HBSS. Apical-to-basolateral (A-to-B) experiments were dosed
with 65 μL of dosing buffer (75 μL final volume) on the apical side of
the cell monolayer and 250 μL of acceptor buffer on the basolateral
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Table 9. 5′-UTR Sequence Motifs
CAACAA
AGAGAG
CCGCCG
GGCGGC
5′-AAGCTTGAA(CAACAA) CACCCCATGG-3′
13
5′-AAGCTTGAA(AGAGAGCAACAA) AGAGAGCACCCCATGG-3′
6
5′-AAGCTTGAA(CCGCCGCAACAA) CCGCCGCACCCCATGG-3’
6
5′-AAGCTTGAA(GGCGGCCAACAA) GGCGGCCACCCCATGG-3′
6
side of the cell monolayer. Basolateral-to-apical (B-to-A) experiments
were dosed with 250 μL of dosing buffer on the basolateral side of the
cell monolayer and 65 μL of acceptor buffer (75 μL final volume) on
the apical side of the cell monolayer. Cell monolayers were incubated
pK /log P. Ionization constants (pK ’s) and octanol water partition
a
a
coefficients (log P, log D 7.4) were determined at Pion, Inc (Billerica,
MA). A PULSE instrument (Pion Inc.) was used to measure pH of the
solutions. The instrument was equipped with three precision
dispensers (capable of adding a minimum volume of 0.042 μL and a
high-impedance (1015 Ω) pH circuit. The double junction
combination pH electrode was standardized using the Avdeef−Bucher
four−parameter equation. The standard aqueous method based on
at 37 °C with 5% CO in a humidified incubator for 2 h. After the
2
assay, cell junction integrity was again tested for damage with a Lucifer
yellow permeability assay. In order for cells to be of considered viable,
Lucifer yellow paracellular flux needed to be less than 0.3%. Post assay,
concentrations of TA from acceptor and donor compartments were
determined by LC/MS/MS using standard calibrators ranging in
concentration from 0.001 to 5 μM. The MS model used was a Sciex
API 4000 operated in electrospray ionization MRM-(+) mode.
Calculations of Papp and efflux were performed according to the
following formulas
potentiometric titration routinely determines pK values from 3 to 11.
a
The cosolvent method is narrower by about 1 pH unit, specifically due
to electrode calibration in the presence of organic solvents.
Furthermore, if precipitation of the compound occurred during pH-
metric titrations, that may affect the results increasing the error limits
on the pK determination. In these cases, the pK value cannot be
a
a
reliably determined. The aqueous pK values were extrapolated from
a
P
app,BA
dQ
dt
1
the pK values in cosolvents as a reciprocal function of the dielectric
a
P
=
×
; efflux =
app
A × C0
P
constant (ε) of each of the co-solvent mixture using Yasuda−
Shedlovsky extrapolation procedure. The extrapolation plots are used
to calculate the aqueous pK from the pK (p K ) measured in
app,AB
where dQ/dt: the amount of TA in the acceptor (nmol) over the
duration of the assay (seconds). A: area of transwell (cm ). C :
a
a
s a
2
cosolvent. However, if p K values are outside of the pH region
0
s a
concentration of TA in the donor well at the start of the assay (μM).
specified for the method, the aqueous pK cannot be determined. log
P/log D values are calculated based on the difference between
a
Papp,BA: apparent permeability of TA in the B-to-A direction. Papp,AB
:
apparent permeability of the TA in the A-to-B direction.
accurately determined aqueous pK values and pKa determined in the
a
Assignment of specific P-gp efflux was accomplished for some
compounds (e.g., eFT226) by conducting additional bi-direction
permeability assays which included a P-gp inhibitor (valspodar, 5 μM)
in the dosing buffer. These experiments consistently attenuated efflux.
In Vivo Pharmacokinetics. Jugular vein-catheterized Spague
dawley rats of both genders were administered a single IV bolus
dose of each TA via the saphenous vein, at a nominal level of 1 mg/kg
presence of 1-octanol (p K ). In cases when the p K or/and pK
o
a
o
a
a
values cannot be reliably determined, the log P/log D results are
reported as “Not Available” (N/A). The pK experiments for the
a
compound have been performed using the potentiometric and the UV-
metric methods. The log P/log D experiments were performed using
the potentiometric method.
Biacore Binding Studies. RNA 18-mer oligonucleotides were
custom synthesized from GE Healthcare Dharmacon Inc (Chicago,
IL) and biotinylated on the 3′ end and contained the following repeat
sequences:
(
free base) in a suitable formulation. Following dosing, blood was
collected from all animals via jugular vein into microtubes containing
K EDTA at 0.083, 0.25, 0.50, 1, 2, 4, 8, and 24 h post-dose. Blood
2
samples were initially stored on ice and subsequently centrifuged to
separate plasma. Plasma samples were kept on ice prior to storage in a
(AGAGAG): 5′-A.G.A.G.A.G.A.G.A.G.A.G.A.G.A.G.A.G-Bi-3′;
(GGCGGC): 5′G.G.C.G.G.C.G.G.C.G.G.C.G.G.C.G.G.C−Bi-3′;
(CCGCCG): 5′C.C.G.C.C.G.C.C.G.C.C.G.C.C.G.C.C.G-Bi-3′;
and
(CAACAA): 5′-C.A.A.C.A.A.C.A.A.C.C.A.C.C.A.C.C.A-Bi-3′
Full length recombinant eIF4A1 protein was prepared at Beryllium
Discovery (Bainbridge Island, WA) and was kept at −80 °C until use
in the following storage buffer: 20 mM Tris pH 7.5, 200 mM NaCl, 1%
glycerol, and 1 mM TCEP. Biacore studies were conducted at
Biosensor Tools (Salt Lake City, UT) on a ProteOn XPR36 protein
interaction array system BioRad (Hercules, CA) using NLC sensor
chips functionalized with streptavidin for capturing biotinylated
biomolecules. Running buffer contained 14.4 mM Hepes pH 7.4,
−80 °C freezer until time of processing for bioanalysis. Plasma
bioanalysis was performed on all time points using bioanalytical
methods that included using protein precipitation followed by liquid
chromatography−mass spectrometry (LC/MS) analysis. The MS
model used was an AB SCIEX TripleTOF 5600+ operated in
electrospray ionization TOF MS (+) mode. The analyte quantified was
the respective free base form of each TA. Bioanalytical time versus
concentration data were imported into Phoenix 64 WinNonlin
software and PK parameters calculated using noncompartemental
analysis.
Thermodynamic Solubility. Dry powders of each TA were
weighed into 1 dram vials. Deionized water was added to each vial to
reach the desired nominal free base concentration of 20 mg/mL.
Compounds isolated as the HCl salt (i.e., 37, 53, eFT226)
incorporated a salt correction factor of ∼1.08 mg TA per mg of free
base during the weighing process. Teflon-coated StirStix were added to
each dram vial and then capped. Samples were placed on a plate heater
108 mM NaCl, 1 mM MgCl , 0.36 mM TCEP, 1 mM ATP, and 1%
2
DMSO. Data were collected at 25 °C. The formation of the ternary
complex was tested with eIF4A1 and eFT226 in the presence or
absence of nucleotides (ATP and AMP-PNP). Once the complex was
formed on the RNA surface, dissociation of eIF4A1 was determined by
washing the surface for 110 s in the presence of compound and ATP,
followed by a wash without compound (ATP only). Response data for
the full concentration series were fit globally to a 1:1 interaction model
using the data processing and analysis software Scrubber, BioLogic
Software (Canberra, Australia).
(
Watlow, St. Louis, MO) with the temperature set to 37C and covered
with a Styrofoam cover. Samples were heated and stirred on a
magnetized paddles stirrer (V&P Scientific, San Diego, CA) for 18 h.
Post incubation, samples were filtered in an AcroPrep Advance 1 mL
0
.2 μm Supor filter plate (Pall Corporations, Ann Arbor, MI). The
Cell-Based Reporter Assay. Seven tandem repeats of various
sequence motifs (see Table 9) were cloned into the 5′-UTR of the
luciferase reporter vector pGL3 (Promega Corp., Madison, WI) at
Genewiz (San Diego). DNA templates were PCR amplified using
Phusion High-fidelity PCR Master Mix (New England Biolabs,
Ipswich, MA) from the reporter vector using the forward and reverse
primers described in Table 10 that incorporates the T7 promoter
filtrate (60 μL) was added to 20 μL of DMSO and analyzed by HPLC-
UV (Agilent 1100 series, Santa Clara, CA) with appropriate
standard(s) to determine soluble concentration in each experiment.
TAs solutions determined to be above the standard(s) concentration
were further diluted using a 3:1 deionized water/DMSO solution to
below the highest standard concentration.
V
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Table 10. Reporter Construct Forward and Reverse qPCR Primers.
forward (CAACAA)
forward (AGAGAG)
forward (CCGCCG)
forward (GGCGGC)
reverse (Firefly)
5′-GGGCACTAATACGACTCACTATAGGAAGCTTGAA(CAA) CACC-3′
5′-GGGCACTAATACGACTCACTATAGGAAGCTTGAA(AGAGAGCAACAA) AGAGAGCACC-3′
5′-GGGCACTAATACGACTCACTATAGGAAGCTTGAA(CCGCCGCAACAA) CCGCCGCACC-3′
5′-GGGCACTAATACGACTCACTATAGGAAGCTTGAA(GGCGGCCAACAA) GGCGGCCACC-3′
26
6
6
6
5′-TTACACGGCGATCTTTCCGCCCTT-3′
sequence for subsequent RNA transcription. Reactions were purified
using the Qiagen PCR clean up kit and eluted in 50 μL water. RNA
was transcribed from the DNA templates using the mMESSAGE
mMACHINE T7 Ultra kit (Ambion ThermoFisher, Waltham, MA).
RNA was purified with the RNA Mega Clear Kit and eluted with 30 μL
elution buffer (Ambion ThermoFisher, Waltham, MA). Using the
manufacturer’s instructions, RNA was transiently transfected in bulk
into exponentially growing MDA-MB-231 cancer cells using the
TransIT mRNA transfection kit (Mirus Bio, Madison, WI), and the
transfected cells were then seeded into 96-well plates. The transfected
cells were treated with various concentrations of translation inhibitors
Technology (Danvers, MA) supplemented with a final concentration
of 1 mM PMSF. Protein concentrations in cell lysates were quantitated
by BCA protein assay from Thermofisher Scientific (Waltham, MA)
and equal amounts of total protein were resolved by SDS-PAGE,
immunoblotted with the indicated antibodies and visualized by LI-
COR Odyssey imager.
Cell Proliferation Assay. Exponentially growing cells were seeded
at 2000−5000 cells per well in 96-well flat bottom white polystyrene
plates (Thermo Fisher) and cultured overnight. The following day,
compound was added in a 3-fold dilution series along with a DMSO
control. The final DMSO concentration was 0.1%. Cells were
(
(
eFT226, RocA, hippuristanol, and cycloheximide) or control
DMSO) for 4 h at 37 °C. Cells were rinsed with 100 μL of PBS
incubated for 72 h at 37 °C in a CO incubator. Baseline viability of
2
untreated cells was measured on the day of treatment and proliferation
was measured after 72 h of treatment using CellTiter-Glo reagent from
Promega (Madison, WI) according to manufactures instructions.
Dose−response curves were plotted using Prism 6 software, and CI
and lysed using 50 μL of 1x passive lysis buffer (Promega Corp.,
Madison, WI). After shaking for 20 min at room temperature, 100 μL
of luciferase assay reagent (Promega Corp., Madison, WI) was added,
and luminescence was quantitated using a Victor (PerkinElmer) plate
reader. The data were fitted using GraphPad Prism (GraphPad
Software, La Jolla, CA) and a four-parameter dose response equation.
Generation of HAP1 eIF4A1 F163L Cell Line. The pSpCas9 BB-
5
0
values were calculated using a 4 parameter, variable slope nonlinear
regression model. Calculation of % inhibition: % inhibition = 1 −
(((cells + inhibitor) − baseline)/((cells + DMSO) − baseline))) ×
100.
2
A-Puro (PX459) v2.0 vector containing the Cas9 nuclease and a
sgRNA targeting the sequence surrounding F163 in human eIF4A1
5′-CATATCAAACACACG GCCAG-3′) was purchased from Gene-
script (Piscataway, NJ). The ssODN donor template
5′GCCTGTATCGGGGGCACCAACGTGCGTGCTGAGGTG-
Analysis of Cell Cycle. Exponentially growing cells were seeded in
96-well plates and cultured overnight. Cells were then treated with
eFT226 or DMSO for 24 h. Cells were washed once with PBS,
dissociated from plates by adding Trypsin−EDTA (0.25%) from
Thermofisher Scientific (Waltham, MA), and incubated at 37 °C for 3
min. Trypsin is neutralized with culture medium containing 20% FBS,
and the cell suspension is transferred to a round bottom 96-well plate.
Cells are left to rest at room temperature for 15 min before washing
with PBS. Cells are stained with LIVE/DEAD Fixable Dead Cell Stain
kit from Thermo Fisher Scientific (Waltham, MA) and formalin fixed.
Cell cycle was assessed by DNA staining using DRAQ5 from Thermo
Fisher Scientific (Waltham, MA). Fluoresence of the cells was analyzed
by flow cytometry using Attune NxT flow cytometer from Thermo-
fisher Scientific (Waltham, MA).
Analysis of Apoptosis and Cell Death. For analysis of apoptosis
by Annexin V staining, exponentially growing cells were seeded at
10,000 cells per well in a 96-well tissue culture treated plates and
cultured overnight. Cells were then treated with eFT226 or DMSO for
24 h. Apoptosis and cell death are measured using BD Annexin V
FITC Apoptosis Detection Kit from BD Biosciences (San Jose, CA)
according to the manufacturer’s instructions. Briefly, cells were washed
once with PBS, dissociated from plates by adding Trypsin−EDTA
(0.25%) from Thermofisher Scientific (Waltham, MA), and incubated
at 37 °C for 3 min. Trypsin is neutralized with culture medium
containing 20% FBS, and the cell suspension is transferred to a round
bottom 96-well plate. Cells are left to rest at room temperature for 15
min before washing once with PBS and resuspended in 1× annexin V
binding buffer containing annexin V and PI. Cells were incubated at
room temperature for 15 min followed by dilution in 1× annexin V
binding buffer. Fluorescence of the cells was analyzed by flow
cytometry using Attune NxT flow cytometer Thermo Fisher Scientific
(Waltham, MA).
(
(
CAGAAACTGCAGAT GGAAGCTCCCCACATCATCGTGGG-
TACACCTGGCCGTGTGCTCGATATGCTTAACCGGAGA-
TACCTGTGTGAGTAATTCGGTTCTCCAATCCCCTGGGT-
CACTTTGCTCTTGTGCACG-3′; PAM mutation is italicized,
F163L mutation underlined and novel TaqαI restriction site is in
bold) was purchased from Integrated DNA Technologies (Coralville,
IA). The vector and ssODN were cotransfected into exponentially-
growing HAP1 cells (Horizon Discovery, Cambridge, UK) using X-
tremeGENE 9 (Roche; Basel, Switzerland), and 2 days later, a single
cell suspension was transferred into 96-well plates at approximately 1
cell/well. After 2 weeks, following gDNA extraction each clone was
assayed for the presence of the TaqαI restriction site by PCR
amplification and subsequent restriction enzyme digest of the region of
interest in human eIF4A1 (Herculase DNA polymerase from Agilent
Technologies; Santa Clara, CA and TaqαI from New England Biolabs;
Ipswich, MA). The undigested PCR product from potential positive
clones was ligated into the TOPO-Blunt vector (ThermoFisher,
Waltham, MA) and subjected to Sanger sequencing to verify correct
nucleotide changes.
Cell Culture, Antibodies, and Immunoblotting. MDA-MB-
31 (ATCC, Manassas, VA) and HAP1 cells were maintained in
2
DMEM supplemented with 10% FBS and 1× penicillin/streptomycin.
The GAPDH antibody was purchased from Santa Cruz Biotechnology
(
(
Dallas, TX). The c-MYC antibody was purchased from Abcam
Cambridge, UK). The Cyclin D1 antibody was purchased from Cell
Marque (Rocklin, CA). Antibodies against MCL-1, BCL2, and eIF4A1
were purchased from Cell Signaling Technology (Danvers, MA).
Secondary antibodies (Goat anti-rabbit IRDye-680, Goat anti-mouse
IRDye-800, Donkey anti-rabbit IRDye-800CW, and Donkey anti-
mouse IRDye-680RD) for Odyssey infrared imaging were purchased
from LI-COR (Lincoln, NE).
In Vivo Efficacy Studies. All animal studies were carried out in
accordance with the guidelines established by the Institutional Animal
Care and Use Committee at Explora BioLabs (San Diego, CA). For
MDA-MB-231 xenografts, athymic nude ((NCr) nu/nu fisol,
Simonsen) were implanted orthotopically in the mammary fat pad
For immunoblotting, exponentially growing cells were seeded into
-well plates at 0.5 million cells/mL and cultured overnight followed
6
6
with 5 × 10 cells in 0.1 mL of unsupplemented DMEM and an equal
by treatment with the indicated concentrations of eFT226 or DMSO
for 24 h. Cells were pelleted by centrifugation, washed once with ice-
cold PBS and lysed in 1× cell lysis buffer from Cell Signaling
volume (1:1) ratio of Matrigel (BD Biosciences) for tumor
development. When the mean tumor sizes reached approximately
170 mm (MDA-MB-231), the mice were randomized and size-
3
W
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matched into vehicle and treatment groups. The tumor size was
measured in length and width with a caliper twice a week. The tumor
volume was calculated by the formula L × W × W/2 according to NCI
standards. Body weights were collected prior to study start, and twice a
week during the study. eFT226 was formulated in 5% dextrose in
water (D5W), heated and/or vortexed to make a clear solution.
Computation. Rocaglamide A binding model. The protein
employed for the model was the PDB ID 2HYI structure of eIF4E3
from the pdb database because this was the only eIF4A structure at the
500, and Bruker AV-500 spectrometer. Coupling constants (J) are
reported in hertz (Hz). Chemical shifts (δ) of NMR spectra are
reported in parts per million (ppm) and are calibrated using residual
1
1
undeuterated solvent for H NMR [ H = 7.26 (CHCl ), 2.50 (D H-
3
5
1
3
13
DMSO) ppm] and C deuterated solvent for C NMR [39.52
(DMSO-d ) ppm] as an internal reference at 298 K. The following
42
6
abbreviations were used to designate the multiplicities: s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, b = broad.
High-resolution mass spectrometry (HRMS) was performed using a
Triple TOF 5600+ mass spectrometer (hybrid quadrupole time-of-
flight platform; AB Sciex) connected to a 1290 UHPLC system
(Agilent). The mass spectrometer was operated in electrospray
positive ionization mode (ESI+). Acquisition was a full scan from
m/z 100 to 1000 with a pulser frequency of 18.092 kHz and
accumulation time of 250 ms.
27
time that had an RNA strand bound. The initial conformation of
rocaglamide A was obtained using a rocaglamide analogue structure
from the Cambridge Structural Database (CSD), FROJM03. The
structure of rocaglamide A was constructed in 3D from this starting
model using the editing tools available in Benchware 3D and
minimized in the gas phase using the MMFF. The resulting 3D
model was placed by hand into the 2HYI protein structure with a
Connelly surface representation displayed to reveal potential binding
pockets of the protein in the region supported by the mutational
Optical rotations were measured using an Optical activity ltd
polarimeter (model: polAAr 3002).
Single-crystal X-ray diffraction studies were carried out on a Bruker
25
135
data. Only one pose was deemed reasonable for further study, and it
was minimized in the presence of the protein using the MMFF and
Benchware. Subsequent models of compounds 7 and 36 were
generated from the rocaglamide A model by editing the structure
with the 3D tools of Benchware and minimizing in the gas phase using
the MMFF; no re-optimization to the protein was performed.
Torsional modeling. All torsional studies were performed using
Spartan ’14 version 1.1.8 from Wavefunction, Inc. For each derivative
of interest, the structure was constructed in 3D using the editing tools
in Spartan and the equilibrium geometry calculated using DFT and
either EDF2 or B3LYP at the 6-31G* level of theory. The torsional
energy surface was then generated by driving the C1′−C3a−C3−C1″
from 60° to −30 or −60 in 5° increments using the energy profile
function. Plots were generated with the torsional angle value on the x-
axis and the relative energy on the y-axis using the smooth function to
draw a line.
D8 Platinum CCD diffractometer equipped with Cu Kα radiation (λ
= 1.5478).
Photocycloadditions were carried out using the following equip-
ment: Medium Pressure Mercury Vapor Lamp (Vendor: SAIC,
INDIA; model: MVL4; Power Supply model: MVL41, wattage: 400
W, diameter: 22 mm); Amit Scientific UV reactor (250 mL/500 mL/
1000 mL/5 L/10 L).
Synthesis of rac-(1R,2R,3S,3aR,8bS)-1,8b-Dihydroxy-6-methoxy-
3a-(4-methoxyphenyl)-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahy-
Chemistry. All reagents and solvents were used as purchased from
commercial sources unless otherwise noted. Microwave reactions were
performed with a Biotage Initiator focused beam microwave reactor.
Reactions were monitored, and analytical purities were assessed on the
following instruments: Waters Acquity UPLC with SQD mass
equipped with Acquity UPLC BEH C-18 (2.1 × 100 mm, 1.7 μm)
and Acquity UPLC HSS T3 (2.1 × 100 mm, 1.8 μm) columns
dro-1H-cyclopenta[b]benzofuran-2-carboxamide (rac-6). 1-(2-Hy-
droxy-4-methoxyphenyl)ethan-1-one (S1). To a solution of 1-(2,4-
dihydroxyphenyl)ethan-1-one (68) (50.0 g, 329 mmol) in acetone
(
500 mL), potassium carbonate (136.0 g, 984.0 mmol) was added at
room temperature. The reaction mixture was stirred for 15 min at
room temperature, and then methyl iodide (20 mL, 46 g 0.32 mol) was
added. The reaction mixture was stirred at room temperature for 16 h,
after which acetone was removed under reduced pressure, and the
solid obtained was washed with ice cold water 3 times and dried under
high vacuum, affording 1-(2-hydroxy-4-methoxyphenyl)ethan-1-one
(LCMS); Shimadzu LC-10AD liquid chromatograph system equipped
with a Shimadzu SCL-10A System controller and SPD-10A UV/Vis
detector, a PerkinElmer Series 200 Autosampler and a Phenomenex
Gemini NX-C18 column (3 μm, 30 × 4.6 mm) (LCMS). Analytical
chiral separations and determinations of enantiomeric excesses were
performed using a Waters Alliance 2695 Separations Module with
Waters 2998 Photo Diode Array Detector (HPLC) or a Waters
Acquity UPCC with a Waters Photo Diode Array detector (UPCC/
analytical SFC) equipped with Diacel Chiralpak IA (4.6 × 250 mm, 5
μm), Diacel Chiralpak IC (4.6 × 250 mm, 5 μm), or Diacel Chiralpak
IG (4.6 × 250 mm, 5 μm) columns. Final compounds for biological
evaluation were obtained in ≥95% purity as determined by LCMS.
Flash column chromatography was performed with a Teledyne
ISCO CombiFlash Rf200+ system or a Yamazen Smart Flash EPLC
W-Prep 2XY system using normal-phase silica columns (230−400
mesh). HPLC purification was performed using a Waters automated
purification system with Waters 2767 sample controller, 2545 binary
gradient pump, and 2998 Photo Diode Array Detector/2489 UV
Detector; or a Gilson automated purification system equipped with a
Gilson 215 liquid handler and 333 and 334 pumps using Phenomenex
Gemini NX-C18 columns (10 μm, 250 × 30 mm or 10 μm, 250 × 50
mm). SFC purification was performed on a Waters SFC 200q system
with waters 2489 UV/vis detector. Chiral SFC separations were
performed using Diacel Chiralpak IA (21 × 250 mm, 5 μm), Diacel
Chiralpak IC (30 × 250 mm, 5 μm), and Diacel Chiralpak IG (21 ×
(
1
=
68) as a white solid. Yield: 50.0 g (0.301 mol, 92%). MS (ESI) m/z:
−
1
65.07 [M − 1] ; H NMR (400 MHz, DMSO-d ): δ/ppm 7.80 (d, J
6
8.8 Hz, 1H), 6.49−6.44 (m, 2H), 3.79 (s, 3H), 2.55 (s, 3H).
E)-1-(2-Hydroxy-4-methoxyphenyl)-3-(4-methoxyphenyl)prop-
-en-1-one (S2). To a solution of 1-(2-hydroxy-4-methoxyphenyl)-
(
2
ethan-1-one (S1) (50.0 g, 301 mmol) in methanol (250 mL), sodium
hydroxide (36.0 g, 900 mmol) was added followed by 4-
methoxybenzaldehyde (55.0 g, 404 mmol). The reaction was then
heated to reflux for 1 h. Afterward, the reaction mix was cooled and the
solid was obtained by filtration, washed with water, and dried under
high vacuum to afford (E)-1-(2-hydroxy-4-methoxyphenyl)-3-(4-
methoxyphenyl)prop-2-en-1-one (S2) as a yellow solid. Yield: 77.0 g
+
1
(271 mmol, 90%). MS (ESI) m/z: 285.2 [M + 1] ; H NMR (400
MHz, DMSO-d ): δ/ppm 8.22 (d, J = 8.5 Hz, 1H), 7.91−7.76 (m,
6
4H), 7.01 (d, J = 7.9 Hz, 2H), 6.52−6.47 (m, 2H), 3.82 (s, 6H).
3-Hydroxy-7-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-One
(69). To a solution of (E)-1-(2-hydroxy-4-methoxyphenyl)-3-(4-
methoxyphenyl)prop-2-en-1-one (S2) (77.0 g, 271 mmol) in ethanol
(500 mL) at room temperature, sodium hydroxide (32.0 g, 800 mmol,
10% aq solution) was added, followed by hydrogen peroxide (19 mL,
810 mmol). The reaction mixture was stirred for 2 h at room
temperature (reaction is exothermic), after which the reaction mixture
was cooled and neutralized to pH ≈ 7 by the addition of 5 N
hydrochloric acid. The precipitate was collected by filtration and dried
2
50 mm, 5 μm) columns.
1H and 13C NMR spectra were recorded on a Bruker 400 MHz
Avance III HD Nanobay with a BBFO probe, Bruker 400 MHz Avance
II with a Dual 13C probe, Agilent MR400 spectrometer, Bruker DRX
X
https://dx.doi.org/10.1021/acs.jmedchem.0c00182
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
under vacuum to afford 3-hydroxy-7-methoxy-2-(4-methoxyphenyl)-
2-carboxylic acid (S5) as an off-white solid. Yield: 7.0 g (16 mmol,
84%). MS (ESI) m/z: 449.02 [M + 1] . H NMR (400 MHz, DMSO-
+
1
4
H-chromen-4-one (69) as a pale yellow solid. Yield: 16.0 g (53.6
+
1
mmol, 20%). MS (ESI) m/z: 299.10 [M + 1] ; H NMR (400 MHz,
d ): δ/ppm 12.05 (br s, 1H), 7.29 (d, J = 8.36 Hz, 1H), 7.06−7.00 (m,
6
DMSO-d ): δ/ppm 9.31 (br s, 1H), 8.18 (d, J = 7.9 Hz, 2H), 7.97 (d, J
5H), 6.88 (d, J = 7.3 Hz, 2H), 6.63−6.55 (m, 3H), 6.51−6.44 (m, 1H),
5.64−5.58 (m, 1H), 5.28−5.20 (m, 1H), 4.73 (d, J = 7.1 Hz, 1H), 4.01
(d, J = 7.1 Hz, 1H), 3.77−3.73 (m, 4H), 3.62 (s, 3H).
6
=
8.5, 1H), 7.25 (s, 1H), 7.10 (d, J = 8.1, 2H), 7.02 (d, J = 8.1, 1H),
3.90 (s, 3H), 3.84 (s, 3H).
rac-Methyl (3S,4S,5R)-5-Hydroxy-8-methoxy-2-(4-methoxyphen-
rac-(1R,2R,3S,3aR,8bS)-1,8b-Dihydroxy-6-methoxy-3a-(4-me-
thoxyphenyl)-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-
cyclopenta[b]benzofuran-2-carboxamide (rac-6). To a solution of
rac-(1R,2R,3S,3aR,8bS)-1,8b-dihydroxy-6-methoxy-3a-(4-methoxy-
phenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-
2-carboxylic acid (S5) (7.0 g, 14 mmol) in dichloromethane (140 mL),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (8.9 g,
yl)-10-oxo-3-phenyl-2,3,4,5-tetrahydro-2,5-methanobenzo[b]-
oxepine-4-carboxylate (S3). A solution of 3-hydroxy-7-methoxy-2-(4-
methoxyphenyl)-4H-chromen-4-one (16.0 g, 53.6 mmol) and methyl
cinnamate (86.9 g, 536 mmol) in dichloromethane (200 mL),
acetonitrile (100 mL) and methanol (100 mL) was placed in a UV
reactor flask. The reaction mixture was irradiated under 400 watts UV
light for 15 h. After completion, the solvent was removed under
reduced pressure, and the residue was purified over a plug of silica gel
by eluting the compound with 5% methanol in dichloromethane. The
desired fractions were concentrated under reduced pressure to afford
methyl rac-(3S,4S,5R)-5-hydroxy-8-methoxy-2-(4-methoxyphenyl)-
47 mmol), hydroxybenzotriazole (6.3 g, 47 mmol), and Hunig’s base
̈
(16.8 mL, 12.5 g, 96.4 mmol) were added at 0 °C. The reaction
mixture was stirred for 5 min. Dimethylamine·HCl (6.34 g, 77.8
mmol) was then added at the same temperature and the reaction was
stirred for 16 h at room temperature. After completion, the reaction
mass was diluted with dichloromethane and washed with cold water.
The organic layer was separated, dried over sodium sulfate, filtered,
and concentrated to give the crude product which was purified by silica
gel column chromatography eluting with 70−90% EtOAc in hexanes
to afford rac-(1R,2R,3S,3aR,8bS)-1,8b-dihydroxy-6-methoxy-3a-(4-
methoxyphenyl)-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-
cyclopenta[b]benzofuran-2-carboxamide (rac-6) as an off-white solid.
1
0-oxo-3-phenyl-2,3,4,5-tetrahydro-2,5-methanobenzo[b]oxepine-4-
carboxylate (S3) as a brown solid. Yield: 18.0 g, crude.
rac-Methyl (2R,3S,3aR,8bR)-8b-Hydroxy-6-methoxy-3a-(4-me-
thoxyphenyl)-1-oxo-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta-
[
5
b]benzofuran-2-carboxylate (S4). The crude rac-methyl 8a-hydroxy-
-methoxy-2a-(4-methoxyphenyl)-8-oxo-2-phenyl-2,2a,8,8a-tetrahy-
dro-1H-cyclobuta[b]chromene-1-carboxylate (S3) (18.0 g) was
suspended in methanol (250 mL), treated with 25% sodium
methoxide in methanol (90 mL. 85 g solution; 21 g, 0.39 mol
NaOMe), and heated to 100 °C for 2 h. The solvent was then removed
under reduced pressure. The crude product was then diluted with
ammonium chloride solution and extracted with ethyl acetate. The
organic phase was separated, dried over sodium sulphate, and
concentrated under reduced pressure to afford rac-methyl
+
Yield: 2.2 g (4.6 mmol, 33%). MS (ESI) m/z: 476.01 [M + 1] ; HRMS
+
1
(
ESI): calcd, 476.2068 [M + H] ; found, 476.2069; H NMR (400
MHz, DMSO-d ): δ/ppm 7.38 (d, J = 8.3 Hz, 1H), 7.09 (d, J = 2.0 Hz,
6
2
6
8
3
H), 7.03−6.95 (m, 3H), 6.78 (d, J = 6.7 Hz, 2H), 6.67−6.64 (m, 3H),
.51−6.48 (m, 1H), 5.44 (d, J = 4 Hz, 1H), 5.12 (s, 1H), 4.88 (t, J =
.3 Hz, 1H), 4.12 (d, J = 13.2 Hz, 1H), 3.94 (dd, J = 8.6, 13.1 Hz, 1H),
13
.78 (s, 3H), 3.64 (s, 3 H), 3.17 (s, 3H), 2.71 (s, 3H); C NMR (101
(
2R,3S,3aR,8bR)-8b-hydroxy-6-methoxy-3a-(4-methoxyphenyl)-1-
MHz, DMSO-d ): δ/ppm 168.61, 161.18, 159.02, 157.55, 139.04,
6
oxo-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-
carboxylate (S4) as a brown solid. Yield: 18.0 g, crude.
1
1
3
29.04, 128.73, 128.18, 127.70, 127.24, 125.69, 122.69, 112.14,
06.90, 100.71, 95.96, 90.94, 77.52, 55.34, 54.80, 54.34, 46.92, 36.38,
5.07.
rac-Methyl (1R,2R,3S,3aR,8bS)-1,8b-Dihydroxy-6-methoxy-3a-
(
[
4-methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta-
Synthesis of rac-(1R,2R,3S,3aR,8bS)-6-Chloro-1,8b-dihydroxy-3a-
b]benzofuran-2-carboxylate (70). A solution of crude rac-methyl
(
4-methoxyphenyl)-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-
(
2R,3S,3aR,8bR)-8b-hydroxy-6-methoxy-3a-(4-methoxyphenyl)-1-
oxo-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-
carboxylate (18.0 g) in acetonitrile (360 mL) was cooled to 0 °C, and
acetic acid (22.35 mL, 390.8 mmol) and sodium triacetoxyborohy-
dride (49.77 g, 234.8 mmol) were added at 0 °C. The mixture was
stirred for 12 h at room temperature. Afterward, the reaction mixture
was partitioned between saturated aqueous sodium bicarbonate
solution and ethyl acetate. The organic layer was separated and
dried over sodium sulphate, filtered, and concentrated to give the
crude product. The crude product was then purified by silica gel
column chromatography eluting with 2−3% in methanol in dichloro-
methane. The desired fractions were concentrated to afford rac-methyl
1H-cyclopenta[b]benzofuran-2-carboxamide (rac-10). (E)-1-(4-
Chloro-2-hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one
(S6). To a solution of 1-(4-chloro-2-hydroxyphenyl)ethan-1-one (71)
(10.0 g, 58.6 mmol) in methanol (60 mL), sodium hydroxide (7.0 g,
180 mmol) and 4-methoxybenzaldehyde (7.9 g, 58 mmol) were
successively added. The reaction mixture was heated to reflux for 30
min, then cooled. The resulting solid was collected by filtration,
washed with water, and dried under high vacuum to afford (E)-1-(4-
chloro-2-hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (S6)
as a yellow solid. Yield: 15.0 g (52.1 mmol, 89%). MS (ESI) m/z:
(
1R,2R,3S,3aR,8bS)-1,8b-dihydroxy-6-methoxy-3a-(4-methoxyphen-
yl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-car-
boxylate (70) as a white solid. Yield: 8.0 g (19 mmol, 36% over three
−
1
steps). MS (ESI) m/z: 461.01 [M − 1] ; H NMR (400 MHz,
DMSO-d ): δ/ppm 7.30 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 9.4 Hz, 1H),
6
7
6
1
3
.05−7.00 (m, 5H), 6.82 (d, J = 7.0 Hz, 2H), 6.64−6.62 (m, 3H),
.52−6.47 (m, 1H), 5.73 (t, J = 8.4 Hz, 1H), 5.28 (s, 1H), 4.75 (t, J =
2.5 Hz, 1H), 4.05−4.00 (m, 1H), 3.89−3.83 (m, 1H), 3.77 (s, 3H),
.74 (s, 1H), 3.67 (s, 3H), 3.46 (s, 3H).
−
1
287.06 [M − 1] ; H NMR (400 MHz, DMSO-d ): δ/ppm 8.10−7.93
6
(m, 2H), 7.81 (d, J = 8.2 Hz, 2H), 7.76−7.68 (m, 1H), 7.02 (d, J = 8.4
rac-(1R,2R,3S,3aR,8bS)-1,8b-dihydroxy-6-methoxy-3a-(4-me-
Hz, 2H), 6.87 (br s, 2H), 3.82 (s, 3H).
thoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxylic Acid (S5). To a solution of rac-methyl
7-Chloro-3-hydroxy-2-(4-methoxyphenyl)-4H-chromen-4-One
(72). To a solution of (E)-1-(4-chloro-2-hydroxyphenyl)-3-(4-
methoxyphenyl)prop-2-en-1-one (S6) (15.0 g, 52.1 mmol) in ethanol
(100 mL) and water (60 mL) at room temperature, sodium hydroxide
(8.3 g, 0.21 mol, 10% aq solution) was added, followed by hydrogen
peroxide (14.6 g, 129 mmol, 30% aq solution) at room temperature.
The reaction mixture was stirred for 1 h at room temperature
(exothermic) and then cooled and neutralized to pH ≈ 7 by addition
of 6 N hydrochloric acid. The resulting precipitate was collected by
filtration and dried under vacuum to afford 7-chloro-3-hydroxy-2-(4-
methoxyphenyl)-4H-chromen-4-one (72) as a pale yellow solid. Yield:
(
1R,2R,3S,3aR,8bS)-1,8b-dihydroxy-6-methoxy-3a-(4-methoxyphen-
yl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-car-
boxylate (70) (8.0 g, 19 mmol) in methanol/water (3:1, 200 mL),
lithium hydroxide (9.97 g, 416 mmol) was added. The reaction was
stirred for 16 h at room temperature. The reaction mixture was then
cooled to 0 °C and acidified to pH ≈ 3 with 1 N hydrochloric acid. The
precipitate was collected by filtration and dried under high vacuum to
afford (1R,2R,3S,3aR,8bS)-1,8b-dihydroxy-6-methoxy-3a-(4-methoxy-
phenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-
Y
https://dx.doi.org/10.1021/acs.jmedchem.0c00182
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
+
1
1
(
8
0.0 g (33.0 mmol, 63%). MS (ESI) m/z: 302.42 [M + 1] ; H NMR
2H), 7.05 (s, 1H), 7.0 (t, J = 7.3 Hz, 2H), 6.96−6.90 (m, 4H), 6.57 (d,
J = 8.7 Hz, 2H), 5.29 (s, 1H), 4.36 (d, J = 5.4 Hz, 1H), 4.10 (d, J = 13.5
Hz, 1H), 3.59 (s, 3H).
400 MHz, DMSO-d ): δ/ppm 9.61 (s, 1H), 8.21 (d, J = 8.7 Hz, 2H),
.09 (d, J = 8.5 Hz, 1H), 7.98 (s, 1H), 7.50 (d, J = 8.8 Hz, 1H), 7.13 (d,
6
J = 8.7 Hz, 2H), 3.85 (s, 3H).
rac-(1R,2R,3S,3aR,8bS)-6-Chloro-1,8b-dihydroxy-3a-(4-methoxy-
phenyl)-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-
cyclopenta[b]benzofuran-2-carboxamide (rac-10). To a solution of
(1R,2R,3S,3aR,8bS)-6-chloro-1,8b-dihydroxy-3a-(4-methoxyphenyl)-
3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-carbox-
ylic acid (S9) (2.5 g, 5.5 mmol) in dichloromethane (55 mL) at 0 °C,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.1 g,
rac-methyl (3S,4S,5R)-8-Chloro-5-hydroxy-2-(4-methoxyphenyl)-
1
4
0-oxo-3-phenyl-2,3,4,5-tetrahydro-2,5-methanobenzo[b]oxepine-
-carboxylate (S7). A solution of 7-chloro-3-hydroxy-2-(4-methox-
yphenyl)-4H-chromen-4-one (72) (10.0 g, 33.0 mmol) and methyl
cinnamate (53.6 g, 330 mmol) in dichloromethane (200 mL),
acetonitrile (100 mL), and methanol (100 mL) was irradiated under
400 W UV light for 24 h in a UV reactor flask. The solvent was then
16 mmol), hydroxybenzotriazole (2.2 g, 16 mmol), and Hunig’s base
̈
removed under reduced pressure, and the residue was purified by
eluting through a plug of silica gel with ethyl acetate. The desired
fractions were concentrated under reduced pressure to afford rac-
methyl 5-chloro-8a-hydroxy-2a-(4-methoxyphenyl)-8-oxo-2-phenyl-
(5.6 mL, 32 mmol) were added. The mixture was stirred for 5 min at 0
°C, after which dimethylamine hydrochloride (2.25 g, 27.6 mmol) was
added at same temperature. Then, the reaction mixture was stirred for
8 h at room temperature, after which it was diluted with
dichloromethane and washed with cold water. The organic layer was
separated, dried over anhydrous sodium sulfate, filtered, and
concentrated to give the crude product, which was purified by silica
gel column chromatography eluting with 70−90% ethyl acetate in
hexanes to afford rac-(1R,2R,3S,3aR,8bS)-6-chloro-1,8b-dihydroxy-3a-
2,2a,8,8a-tetrahydro-1H-cyclobuta[b]chromene-1-carboxylate (S7) as
a brown solid. Yield: 15.0 g, crude.
rac-Methyl (2R,3S,3aR,8bR)-6-Chloro-8b-hydroxy-3a-(4-methox-
yphenyl)-1-oxo-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxylate (S8). The crude rac-methyl (3S,4S,5R)-8-
chloro-5-hydroxy-2-(4-methoxyphenyl)-10-oxo-3-phenyl-2,3,4,5-tetra-
hydro-2,5-methanobenzo[b]oxepine-4-carboxylate (S7) (15.0 g) was
suspended in methanol (150 mL), treated with 25% sodium methoxide
in methanol (50 mL solution; 12 g NaOMe, 0.22 mol), and then
heated at 70 °C for 2 h. Afterward, the solvent was removed under
reduced pressure and the remaining crude product was treated with
saturated aqueous ammonium chloride solution and extracted with
ethyl acetate. The organic phase was dried over anhydrous sodium
sulfate, filtered, and concentrated under reduced pressure to afford
methyl (2R,3S,3aR,8bR)-6-chloro-8b-hydroxy-3a-(4-methoxyphenyl)-
(
4-methoxyphenyl)-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-
cyclopenta[b]benzofuran-2-carboxamide (rac-10) as a white solid.
+
Yield: 2.4 g (5.0 mmol, 90%). MS (ESI) m/z: 480.10 [M + 1] ; UPLC:
+
9
8.93%; HRMS (ESI): calcd, 480.1572 [M + H] ; found, 480.1590;
1
H NMR (400 MHz, DMSO-d ): δ/ppm 7.46 (d, J = 8.0 Hz, 1H), 7.22
6
(
7
d, J = 10.0 Hz, 1H), 7.16 (d, 1.4, 2H), 7.08−6.97 (m, 4H), 6.79 (d, J =
.0 Hz, 2H), 6.67 (d, J = 8.7 Hz, 2H), 5.60 (d, J = 6.1 Hz, 1H), 5.42 (s,
1
8
H), 4.89 (t, J = 6.5 Hz, 1H), 4.14 (d, J = 13.2 Hz, 1H), 3.98 (dd, J =
13
.2 Hz, 13.1 Hz, 1H), 3.64 (s, 3H), 3.18 (s, 3H), 2.71 (s, 3H);
C
NMR (101 MHz, DMSO-d ): δ/ppm 168.40, 158.68, 157.67, 138.71,
6
1
-oxo-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-
1
1
3
33.67, 129.85, 129.79, 128.69, 127.73, 127.65, 127.30, 125.79,
20.30, 112.20, 110.67, 101.05, 90.83, 77.42, 54.81, 54.49, 46.95,
6.40, 35.09.
carboxylate (S8) as a brown solid. Yield: 14.0 g, crude.
rac-Methyl (1R,2R,3S,3aR,8bS)-6-Chloro-1,8b-dihydroxy-3a-(4-
methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxylate (73). The crude rac-methyl
Synthesis of rac-(1R,2R,3S,3aR,8bS)-6-Chloro-3a-(4-cyanophen-
yl)-1,8b-dihydroxy-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-
(2R,3S,3aR,8bR)-6-chloro-8b-hydroxy-3a-(4-methoxyphenyl)-1-oxo-
3
-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-carbox-
ylate (S8) (14.0 g) was added to a solution of sodium
triacetoxyborohydride (38.0 g, 179 mmol) in acetonitrile (300 mL)
and acetic acid (18.0 mL). This mixture was stirred for 4 h at room
temperature. After completion, the reaction mixture was partitioned
between saturated aqueous sodium bicarbonate solution and ethyl
acetate. The organic layer was separated, dried over anhydrous sodium
sulfate, filtered, and concentrated under reduced pressure to obtain the
crude product, which was purified by silica gel (100−200 mesh)
column chromatography eluting with 50% ethyl acetate in hexanes.
The desired fractions were concentrated under reduced pressure to
afford rac-methyl (1R,2R,3S,3aR,8bS)-6-chloro-1,8b-dihydroxy-3a-(4-
methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxylate (73) as a white solid. Yield: 8.0 g (17.1
1H-cyclopenta[b]benzofuran-2-carboxamide (rac-11). (E)-3-(4-Bro-
mophenyl)-1-(4-chloro-2-hydroxyphenyl)prop-2-en-1-one (S10).
To a solution of 1-(4-chloro-2-hydroxyphenyl)ethan-1-one (71) (5.0
g, 29 mmol) in methanol (10 mL), sodium hydroxide (3.5 g, 88 mmol)
was added, followed by 4-bromobenzaldehyde (5.4 g, 30 mmol). The
reaction mixture was heated to reflux at 90 °C for 2 h. Then, the
mixture was cooled and the solid was collected by filtration, washed
with water, and dried under high vacuum to afford (E)-3-(4-
bromophenyl)-1-(4-chloro-2-hydroxyphenyl)prop-2-en-1-one (S10)
as a yellow solid. Yield: 10.0 g (29.6 mmol, quant.). MS (ESI) m/z:
−
1
mmol, 52% over three steps). MS (ESI) m/z: 465.31 [M − 1] ; H
NMR (400 MHz, DMSO-d ): δ/ppm 7.38 (d, J = 8.0 Hz, 1H), 7.14 (s,
6
1
H), 7.05−6.98 (m, 6H), 6.84 (d, J = 7.16 Hz, 2H), 6.63 (d, J = 8.4
Hz, 2H), 5.85 (d, J = 5.6 Hz, 1H), 5.57 (s, 1H), 4.74 (t, J = 6.3 Hz,
H), 4.07 (d, J = 13.9 Hz, 1H), 3.90 (dd, J = 13.7 Hz, 7.0 Hz, 1H), 3.62
1
+
1
3
1
37.12 [M + 1] ; H NMR (400 MHz, DMSO-d ): δ/ppm 8.56 (d, J =
6.1 Hz, 1H), 7.66−7.47 (m, 5H), 7.17 (d, J = 9.9 Hz, 1H), 6.22 (d, J
(
s, 3H), 3.52 (s, 3H).
rac-(1R,2R,3S,3aR,8bS)-6-Chloro-1,8b-dihydroxy-3a-(4-methoxy-
6
phenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxylic Acid (S9). To a solution of rac-methyl
= 12.7 Hz, 1H), 5.91 (d, J = 7.4 Hz, 1H).
2-(4-Bromophenyl)-7-chloro-3-hydroxy-4H-chromen-4-one (74).
To a solution of (E)-3-(4-bromophenyl)-1-(4-chloro-2-
hydroxyphenyl)prop-2-en-1-one (S10) (10.0 g, 29.6 mmol) in ethanol
(10 mL) at room-temperature, sodium hydroxide (3.5 g, 88 mmol,
10% aq solution) and subsequently hydrogen peroxide (30% aq
solution, 2.0 mL, 2.22 g, 0.66 g H O , 19.6 mmol) were added. The
(
3
1R,2R,3S,3aR,8bS)-6-chloro-1,8b-dihydroxy-3a-(4-methoxyphenyl)-
-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-carbox-
ylate (S8) (3.0 g, 6.4 mmol) in methanol/water (3:1, 120 mL), lithium
hydroxide (3.70 g, 154 mmol) was added. The reaction mixture was
stirred for 4 h at room temperature and then cooled to 0 °C and
acidified with 5 N hydrochloric acid to pH ≈ 3. The resulting
precipitate was collected by filtration and dried under high vacuum to
afford rac-(1R,2R,3S,3aR,8bS)-6-chloro-1,8b-dihydroxy-3a-(4-me-
thoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxylic acid (S9) as an off-white solid. Yield: 2.5 g
2
2
reaction mixture was stirred for 2 h at room temperature (reaction is
exothermic). Then, it was cooled and neutralized to pH ≈ 7 by
addition of 6 N hydrochloric acid. The resulting precipitate was
collected by filtration and dried under vacuum to afford 2-(4-
bromophenyl)-7-chloro-3-hydroxy-4H-chromen-4-one (74) as a pale
yellow solid. Yield: 5.0 g (14 mmol, 47%). MS (ESI) m/z: 351.09 [M +
−
1
(
5.5 mmol, 86%). MS (ESI) m/z: 450.99 [M − 1] ; H NMR (400
+
1
MHz, DMSO-d ): δ/ppm 7.28 (d, J = 7.9 Hz, 1H), 7.11 (d, J = 7.2 Hz,
1] ; H NMR (400 MHz, DMSO-d ): δ/ppm 8.18 (d, J = 8.6 Hz, 2H),
6
6
Z
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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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Article
8
.10 (d, J = 8.6 Hz, 1H), 7.99 (s, 1H), 7.78 (d, J = 8.6 Hz, 2H), 7.51 (d,
rac-(1R,2R,3S,3aR,8bS)-3a-(4-Bromophenyl)-6-chloro-1,8b-dihy-
droxy-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta-
J = 8.5 Hz, 1H).
[
b]benzofuran-2-carboxamide (S14). To a solution of rac-
rac-Methyl (3S,4S,5R)-2-(4-Bromophenyl)-8-Chloro-5-hydroxy-
1
4
0-oxo-3-phenyl-2,3,4,5-tetrahydro-2,5-methanobenzo[b]oxepine-
(1R,2R,3S,3aR,8bS)-3a-(4-bromophenyl)-6-chloro-1,8b-dihydroxy-3-
phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-carbox-
ylic acid (S13) (2.0 g, 4.0 mmol) in dichloromethane (50 mL), 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.15 g,
-carboxylate (S11). A solution of 2-(4-bromophenyl)-7-chloro-3-
hydroxy-4H-chromen-4-one (74) (5.0 g, 14 mmol) and methyl
cinnamate (55) (23.0 g, 142 mmol) in dichloromethane (100 mL),
acetonitrile (60 mL), and methanol (60 mL) was irradiated under 400
W UV light for 48 h in a UV reactor flask. Then, the solvent was
removed under reduced pressure, and the residue was purified over a
plug of silica gel eluting with 5% methanol in dichloromethane. The
desired fractions were concentrated under reduced pressure to afford
methyl (3S,4S,5R)-2-(4-bromophenyl)-8-chloro-5-hydroxy-10-oxo-3-
phenyl-2,3,4,5-tetrahydro-2,5-methanobenzo[b]oxepine-4-carboxylate
6.00 mmol), hydroxybenzotriazole (0.9 g, 7 mmol) and Hunig’s base
̈
(1.5 g, 12 mmol) were added at 0 °C. The reaction mixture was stirred
for 5 min at this temperature. Dimethylamine hydrochloride (3.25 g,
39.9 mmol) was then added at same temperature. The mixture was
stirred for 16 h at room temperature. It was next diluted with
dichloromethane and washed with cold water. The organic layer was
separated and dried over sodium sulfate, filtered and concentrated to
give the crude product, which was purified by silica gel column
chromatography eluting with 70−90% ethyl acetate in hexane to afford
(1R,2R,3S,3aR,8bS)-3a-(4-bromophenyl)-6-chloro-1,8b-dihydroxy-
N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxamide (S14) as yellow solid. Yield: 1.8 g (3.4
(S11) as a brown solid. Yield: 3.5 g, crude.
rac-Methyl (3S, 3aR, 8bR)-3a-(4-Bromophenyl)-6-Chloro-8b-
hydroxy-1-oxo-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxylate (S12). The crude rac-methyl (3S,4S,5R)-
2-(4-bromophenyl)-8-chloro-5-hydroxy-10-oxo-3-phenyl-2,3,4,5-tetra-
+
1
hydro-2,5-methanobenzo[b]oxepine-4-carboxylate (S11) (3.5 g) was
suspended in methanol (20 mL), treated with 25% sodium methoxide
in methanol (8.0 mL, 7.6 g solution; 1.9 g NaOMe, 35 mmol), and
heated at 90 °C for 3 h. Then, the solvent was removed under reduced
pressure. The crude product was treated with ammonium chloride
solution and extracted with ethyl acetate. The organic phase was
separated, dried over sodium sulfate, and concentrated under reduced
pressure to afford rac-methyl (3S,3aR,8bR)-3a-(4-bromophenyl)-6-
chloro-8b-hydroxy-1-oxo-3-phenyl-2,3,3a,8b-tetrahydro-1H-
cyclopenta[b]benzofuran-2-carboxylate (S11) as a brown solid. Yield:
mmol, 85%). MS (ESI) m/z: 528.15 [M + 1] ; H NMR (400 MHz,
DMSO-d ): δ/ppm 7.44 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 8.6 Hz, 2H),
6
7.18 (d, J = 1.6 Hz, 1H), 7.12 (d, J = 8.6 Hz, 2H), 7.05−6.99 (m, 4H),
6.84 (d, J = 7.1 Hz, 2H), 5.61 (d, J = 6.0 Hz, 1H), 5.57 (s, 1H), 4.87 (t,
J = 7.3 Hz, 1H), 4.23 (d, J = 13.1 Hz, 1H), 4.08−4.02 (m, 1H), 3.20 (s,
3H), 2.71 (s, 3H).
rac-(1R,2R,3S,3aR,8bS)-6-Chloro-3a-(4-cyanophenyl)-1,8b-dihy-
droxy-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta-
[
(
b]benzofuran-2-carboxamide (rac-11). To a solution of
1R,2R,3S,3aR,8bS)-3a-(4-bromophenyl)-6-chloro-1,8b-dihydroxy-
N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxamide (S14) (1.0 g, 1.9 mmol) in N,N-
dimethylformamide (10 mL), zinc cyanide (0.33 g, 2.8 mmol) was
added. The mixture was purged with argon for 5 min, after which
tetrakis(triphenylphosphine)palladium(0) (0.43 g, 0.37 mmol) was
added. The mixture was purged with argon for an additional 5 min and
then stirred for 2 h at 120 °C. Afterward, the reaction mixture was
cooled to room temperature and filtered through Celite. The filtrate
was washed with water and the organic layer was concentrated under
reduced pressure. The crude product was purified by preparative
HPLC to afford rac-(1R,2R,3S,3aR,8bS)-6-chloro-3a-(4-cyanophen-
yl)-1,8b-dihydroxy-N,N-dimethyl-3-phenyl 2,3,3a,8b-tetrahydro-1H-
cyclopenta[b]benzofuran-2-carboxamide (rac-11) as an off-white
solid. Yield: 0.13 g (0.27 mmol, 14%). MS (ESI) m/z: 475.17 [M +
3
.0 g, crude.
rac-Methyl (1R,2R,3S,3aR,8bS)-3a-(4-Bromophenyl)-6-Chloro-
,8b-dihydroxy-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
1
benzofuran-2-carboxylate (75). A solution of crude rac-methyl
(
3S,3aR,8bR)-3a-(4-bromophenyl)-6-chloro-8b-hydroxy-1-oxo-3-phe-
nyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-carboxylate
S12) (3.0 g) in acetonitrile (50 mL) was cooled to 0 °C, and acetic
(
acid (3.3 mL, 3.5 g, 58 mmol) and sodium triacetoxyborohydride (7.4
g, 35 mmol) were added. The resulting mixture was stirred for 6 h at
room temperature. Then, the mixture was partitioned between
saturated aqueous sodium bicarbonate solution and ethyl acetate.
The organic layer was separated and dried over sodium sulfate, filtered,
and concentrated to give the crude product which was purified by silica
gel column chromatography eluting with 2−3% in methanol in
dichloromethane. The desired fractions were concentrated to afford
rac-methyl (1R,2R,3S,3aR,8bS)-3a-(4-bromophenyl)-6-chloro-1,8b-
dihydroxy-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxylate (75) as a yellow solid. Yield: 2.2 g (4.3
+
+
1
4
] ; UPLC 97.95%; HRMS (ESI): calcd, 475.1419 [M + H] ; found,
1
75.1431; H NMR (400 MHz, DMSO-d ): δ/ppm 7.56 (d, J = 8.4
6
Hz, 2H), 7.43 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.20 (d, J =
1
.6 Hz, 1H), 7.07−6.96 (m, 4H), 6.84 (d, J = 7.2 Hz, 2H), 5.68 (s,
+
1
1H), 5.63 (d, J = 6.0 Hz, 1H), 4.88 (t, J = 6.8 Hz, 1H), 4.30 (d, J = 13.2
mmol, 31% over three steps). MS (ESI) m/z: 517.12 [M + 1] ; H
NMR (400 MHz, DMSO-d ): δ/ppm 7.35 (d, J = 8.0 Hz, 1H), 7.25
Hz, 1H), 4.14 (dd, J = 13.1, 7.8 Hz, 1H), 3.22 (s, 3H), 2.73 (s, 3H);
6
13
C NMR (101 MHz, DMSO-d ): δ/ppm 168.16, 158.48, 141.84,
6
(
d, J = 8.4 Hz, 2H), 7.16 (s, 1H), 7.07−7.00 (m, 6H), 6.90 (d, J = 7.2
1
1
4
38.10, 134.20, 133.88, 130.58, 129.33, 129.10, 128.56, 127.56,
27.52, 126.06, 120.74, 110.72, 109.17, 101.10, 91.67, 77.54, 55.09,
7.19, 36.46, 35.15.
Hz, 2H), 5.93 (d, J = 5.6 Hz, 1H), 5.84 (d, J = 5.8 Hz, 1H), 4.72 (t, J =
6
.1 Hz, 1H), 4.19 (d, J = 13.0, 1H), 4.09−4.00 (m, 2H), 3.54 (s, 3H).
rac-(1R,2R,3S,3aR,8bS)-3a-(4-Bromophenyl)-6-Chloro-1,8b-dihy-
Synthesis of rac-(1R,2R,3S,3aR,8bS)-3a-(4-Cyanophenyl)-1,8b-
droxy-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxylic Acid (S13). To a solution of rac-methyl
dihydroxy-6-methoxy-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahy-
(1R,2R,3S,3aR,8bS)-3a-(4-bromophenyl)-6-chloro-1,8b-dihydroxy-3-
phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-carboxy-
late (75) (2.2 g, 4.3 mmol) in methanol/water (3:1, 10.3 mL), lithium
hydroxide (2.04 g, 85.4 mmol) was added. The reaction mixture was
stirred for 16 h at room temperature, then cooled to 0 °C and acidified
with 1 N hydrochloric acid to pH ≈ 3. The resulting precipitate was
collected by filtration and dried under high vacuum to afford rac-
(1R,2R,3S,3aR,8bS)-3a-(4-bromophenyl)-6-chloro-1,8b-dihydroxy-3-
phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-carbox-
dro-1H-cyclopenta[b]benzofuran-2-carboxamide (rac-12). (E)-1-(4-
ylic acid (S13) as an off-white solid. Yield: 2.0 g (4.0 mmol, 93%). MS
(
Benzyloxy)-2-hydroxyphenyl)-3-(4-bromophenyl)prop-2-en-1-one
+
1
(
ESI) m/z: 503.02 [M + 1] ; H NMR (400 MHz, DMSO-d ): δ/ppm
(S15). To a solution of 1-(4-(benzyloxy)-2-hydroxyphenyl)ethan-1-
one (76) (50.0 g, 206 mmol) in methanol (300 mL), sodium
hydroxide (24.79 g, 619.8 mmol) was added followed by 4-
bromobenzaldehyde (38.29 g, 207.0 mmol). The reaction mixture
was refluxed for 1 h. Then, it was cooled, and the solid was obtained by
6
1
2.18 (s, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.26 (d, J = 10.2 Hz, 2H), 7.23
s, 1H), 7.15−6.99 (m, 6H), 6.91 (d, J = 7.2 Hz, 2H), 5.76 (t, J = 5.8
Hz, 1H), 5.68 (d, J = 11.6 Hz, 1H), 4.68 (t, J = 5.9 Hz, 1H), 4.18−4.02
m, 1H), 3.93−3.79 (m, 1H).
(
(
AA
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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
filtration, washed with water, and dried under high vacuum to afford
1H), 5.75 (t, J = 3.7 Hz, 1H), 5.46 (s, 1H), 5.21 (s, 2H), 4.71 (t, J = 6.4
Hz, 1H), 4.14 (d, J = 13.9 Hz, 1H), 4.05−3.96 (m, 1H), 3.52 (s, 3H).
rac-(1R,2R,3S,3aR,8bS)-6-(Benzyloxy)-3a-(4-bromophenyl)-1,8b-
dihydroxy-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxylic Acid (S18). To a solution of rac-methyl
(
E)-1-(4-(benzyloxy)-2-hydroxyphenyl)-3-(4-bromophenyl)prop-2-
1
en-1-one (S15) as a yellow solid. Yield: 34.0 g (83.1 mmol, 40%). H
NMR (400 MHz, DMSO-d ): δ/ppm 8.22 (d, J = 15.3 Hz, 1H), 8.10
6
(
7
d, J = 9.4 Hz, 1H), 7.79 (d, J = 8.2 Hz, 2H), 7.64 (d, J = 8.2 Hz, 3H),
.45−7.34 (m, 6H), 6.42 (s, 2H), 5.15 (s, 2H).
-(Benzyloxy)-2-(4-bromophenyl)-3-hydroxy-4H-chromen-4-one
77). To a solution of (E)-1-(4-(benzyloxy)-2-hydroxyphenyl)-3-(4-
(
1R,2R,3S,3aR,8bS)-6-(benzyloxy)-3a-(4-bromophenyl)-1,8b-dihy-
droxy-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-
carboxylate (78) (2.6 g, 4.4 mmol) in tetrahydrofuran and water (3:1,
7
(
8
0 mL), lithium hydroxide (2.5 g, 104 mmol) was added. The reaction
bromophenyl)prop-2-en-1-one (S15) (34.00 g, 83.07 mmol) in
ethanol (500 mL), sodium hydroxide (11.32 g, 283.0 mmol, 10% aq
solution) and hydrogen peroxide (6.6 mL, 0.28 mol) were
subsequently added at room temperature. The reaction mixture was
stirred for 2 h at room temperature (reaction is exothermic!).
Afterward, the reaction mixture was cooled and neutralized to pH ≈ 7
by addition of 5 N hydrochloric acid. The solid precipitate was
collected by filtration and dried under vacuum to afford 7-(benzyloxy)-
mixture was stirred for 16 h at room temperature, then cooled to 0 °C
and acidified with 1 M hydrochloric acid to pH ≈ 3. The precipitate
was collected by filtration and dried under vacuum to afford rac-
(
1R,2R,3S,3aR,8bS)-6-(benzyloxy)-3a-(4-bromophenyl)-1,8b-dihy-
droxy-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-
carboxylic acid (S18) as an off-white solid. Yield: 1.79 g (3.12 mmol,
−
7
1%); MS (ESI) m/z: 571.27 [M − 1] .
rac-(1R,2R,3S,3aR,8bS)-6-(Benzyloxy)-3a-(4-bromophenyl)-1,8b-
2
-(4-bromophenyl)-3-hydroxy-4H-chromen-4-one (77) as a pale
dihydroxy-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-
cyclopenta[b]benzofuran-2-carboxamide (S19). To a solution of
rac-(1R,2R,3S,3aR,8bS)-6-(benzyloxy)-3a-(4-bromophenyl)-1,8b-di-
hydroxy-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-
yellow solid. Yield: 16.0 g (37.8 mmol, 46%). MS (ESI) m/z: 422
+
1
[
M + 1] ; H NMR (400 MHz, DMSO-d ): δ/ppm 9.75 (s, 1H), 8.16
6
(
7
d, J = 8.2 Hz, 2H), 8.01 (d, J = 8.7 Hz, 1H), 7.77 (d, J = 8.2 Hz, 2H),
.51−7.36 (m, 6H), 7.11 (d, J = 8.3 Hz, 1H), 5.27 (s, 2H).
rac-(3R,4S,5S)-8-(Benzyloxy)-2-(4-bromophenyl)-5-hydroxy-10-
2
-carboxylic acid (S18) (1.79 g, 3.12 mmol) in dichloromethane (25
mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
1.797 g, 9.374 mmol), 1-hydroxybenzotriazole (1.29 g, 9.55 mmol),
and N,N-diisopropylethylamine (3.19 mL, 18.3 mmol) were added at 0
oxo-3-phenyl-2,3,4,5-tetrahydro-2,5-methanobenzo[b]oxepin-4-yl
Acetate (S16). A solution of 2-(4-bromophenyl)-7-chloro-3-hydroxy-
(
4H-pyrano[3,2-c]pyridin-4-one (77) (7.00 g, 16.5 mmol) and methyl
°C. The mixture was stirred for 5 min. Dimethylamine hydrochloride
(1.29 g, 15.8 mmol) was then added at the same temperature, and the
cinnamate (55) (26.8 g, 165 mmol) in dichloromethane (100 mL),
acetonitrile (50 mL), and methanol (50 mL) was irradiated in a UV
reactor flask for 24 h under 400 W UV light. The solvent was then
removed under reduced pressure and the remaining solid was purified
over a plug of silica gel by eluting the compound with ethyl acetate.
The desired fractions were concentrated under reduced pressure to
afford rac-(3R,4S,5S)-8-(benzyloxy)-2-(4-bromophenyl)-5-hydroxy-
reaction was stirred for 16 h at room temperature. Then, the reaction
mixture was diluted with dichloromethane and washed with cold
water. The organic layer was separated and dried over anhydrous
sodium sulfate, filtered, and concentrated to give the crude product,
which was recrystallized from ethanol to afford rac-
(
1R,2R,3S,3aR,8bS)-6-(benzyloxy)-3a-(4-bromophenyl)-1,8b-dihy-
1
0-oxo-3-phenyl-2,3,4,5-tetrahydro-2,5-methanobenzo[b]oxepin-4-yl
droxy-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxamide (11) as a white solid. Yield: 1.29 g (2.14
acetate (S16). Yield: 8.8 g, crude.
rac-Methyl (2R,3S,3aR,8bR)-6-(benzyloxy)-3a-(4-bromophenyl)-
+ 1
mmol, 69%.) MS (ESI) m/z: 600.02 [M + 1] ; H NMR (400 MHz,
8
[
b-hydroxy-1-oxo-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta-
DMSO-d ): δ/ppm 7.47 (d, J = 7.2 Hz, 2H), 7.42 (t, J = 7.1 Hz, 2H),
6
b]benzofuran-2-carboxylate (S17). The crude rac-(3R,4S,5S)-8-
7
.36−7.26 (m, 4H), 7.13 (d, J = 8.5 Hz, 2H), 7.03−6.96 (m, 3H), 6.82
d, J = 7.2 Hz, 2H), 6.75 (d, J = 2.0 Hz, 1H), 6.59 (dd, J = 2.0 Hz, 8.3
Hz, 1H), 5.47 (d, J = 5.9 Hz, 1H), 5.29 (s, 1H), 5.14 (s, 2H), 4.87 (t, J
(
benzyloxy)-2-(4-bromophenyl)-5-hydroxy-10-oxo-3-phenyl-2,3,4,5-
(
tetrahydro-2,5-methanobenzo[b]oxepin-4-yl acetate (S16) (8.8 g) was
suspended in methanol (50 mL), treated with sodium methoxide (25%
in methanol, 8.8 mL, 8.3 g solution; 2.1 g, 38 mmol, NaOMe), and
heated to 90 °C for 3 h. Next, the solvent was removed under reduced
pressure. The crude product was treated with ammonium chloride
solution and extracted with ethyl acetate. The organic phase was
separated, dried over anhydrous sodium sulphate, and concentrated
under reduced pressure to give rac-methyl (2R,3S,3aR,8bR)-6-
=
7.16 Hz, 1H). 4.21 (d, J = 13.1 Hz, 1H), 4.05−3.99 (m, 1H), 3.20 (s,
3H), 2.67 (s, 3H).
rac-(1R,2R,3S,3aR,8bS)-3a-(4-Bromophenyl)-1,6,8b-trihydroxy-
N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxamide (S20). A flask containing ethyl acetate
(
(
40 mL) was charged with rac-(1R,2R,3S,3aR,8bS)-6-(benzyloxy)-3a-
4-bromophenyl)-1,8b-dihydroxy-N,N-dimethyl-3-phenyl-2,3,3a,8b-
(
2
benzyloxy)-3a-(4-bromophenyl)-8b-hydroxy-1-oxo-3-phenyl-
,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-carboxylate
tetrahydro-1H-cyclopenta[b]benzofuran-2-carboxamide (S19) (1.08
g, 1.80 mmol). Palladium hydroxide (0.246 g, 1.75 mmol, 50% wet)
was added at room temperature under nitrogen. The reaction was
flushed with hydrogen gas twice and stirred at room temperature for 6
h under hydrogen pressure. The reaction mixture was then passed
through Celite, and the filtrate was concentrated under reduced
pressure to afford (1R,2R,3S,3aR,8bS)-3a-(4-bromophenyl)-1,6,8b-
trihydroxy-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-
(
S17) as brown oil. Yield: 8.9 g, crude.
rac-Methyl (1R,2R,3S,3aR,8bS)-6-(benzyloxy)-3a-(4-bromophen-
yl)-1,8b-dihydroxy-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta-
[
b]benzofuran-2-carboxylate (78). To a solution of sodium
triacetoxyborohydride (18.41 g, 86.86 mmol) and crude rac-methyl
2R,3S,3aR,8bR)-6-(benzyloxy)-3a-(4-bromophenyl)-8b-hydroxy-1-
(
oxo-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-
carboxylate (S17) (8.30 g) in acetonitrile (100 mL) at 0 °C, acetic acid
cyclopenta[b]benzofuran-2-carboxamide (12) as a brown oil. Yield:
+
1
.0 g (crude), MS (ESI) m/z: 509.98 [M + 1] .
(8.64 mL, 9.06 g, 150 mmol) was added. The resulting mixture was
rac-(1R,2R,3S,3aR,8bS)-3a-(4-Bromophenyl)-1,6,8b-trihydroxy-
stirred for 4 h at room temperature and then partitioned between
saturated aqueous sodium bicarbonate solution and ethyl acetate. The
organic layer was separated, dried over anhydrous sodium sulfate, and
concentrated under reduced pressure to obtain the crude product
which was purified by silica gel column chromatography by eluting
with 30% ethyl acetate in hexanes. The desired fractions were
concentrated to afford rac-methyl (5aR,6S,7R,8R,8aS)-5a-(4-bromo-
phenyl)-3-chloro-8,8a-dihydroxy-6-phenyl-5a,7,8,8a-tetrahydro-6H-
cyclopenta[4,5]furo[3,2-c]pyridine-7-carboxylate (78) as an off-white
solid. Yield: 2.6 g (4.4 mmol, 29% over three steps). MS (ESI) m/z:
N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxamide (S21). To a solution of rac-
(1R,2R,3S,3aR,8bS)-3a-(4-bromophenyl)-1,6,8b-trihydroxy-N,N-di-
methyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-
carboxamide (S20) (0.8 g, 2 mmol) in acetone (30 mL), methyl iodide
(0.300 g, 2.11 mmol), and potassium carbonate (0.552 g, 3.99 mmol)
were added. The mixture was stirred for 16 h at room temperature,
then diluted with dichloromethane, and washed with cold water. The
organic layer was separated and dried over anhydrous sodium sulphate,
filtered, and concentrated to give crude rac-(1R,2R,3S,3aR,8bS)-3a-(4-
bromophenyl)-1,6,8b-trihydroxy-N,N-dimethyl-3-phenyl-2,3,3a,8b-
tetrahydro-1H-cyclopenta[b]benzofuran-2-carboxamide (S21) as an
+
1
5
85 [M − 1] ; H NMR (400 MHz, DMSO-d ): δ/ppm 7.47 (d, J =
6
7
.3 Hz, 2H), 7.41 (t, J = 7.2 Hz, 2H), 7.35−7.23 (m, 4H), 7.07−6.99
1
(m, 5H), 6.88 (d, J = 7.2 Hz, 2H), 6.73 (s, 1H), 6.61 (d, J = 2.0 Hz,
off-white solid. Yield: 0.800 g (1.53 mmol, 77%); H NMR (400 MHz,
AB
https://dx.doi.org/10.1021/acs.jmedchem.0c00182
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
+
1
DMSO-d ): δ/ppm 7.35 (d, J = 8.3 Hz, 1H), 7.28 (d, J = 8.4 Hz, 2H),
18.0 g (48.1 mmol, 58%); MS (ESI) m/z: 375.27 [M + 1] ; H NMR
6
7
6
.12 (d, J = 8.4, 2H), 7.05−6.96 (m, 3H), 6.83 (d, J = 7.1 Hz, 2H),
.67 (d, J = 1.8 Hz, 1H), 6.53−6.50 (dd, J = 2.1 Hz, 8.3 Hz, 1H), 5.47
(400 MHz, DMSO-d ): δ/ppm 8.18 (d, J = 7.8 Hz, 2H), 8.00 (d, J =
5.9 Hz, 1H), 7.51−7.49 (m, 1H), 7.42−3.88 (m, 6H), 7.17−7.12 (m,
2H), 5.27 (s, 2H), 3.85 (s, 3H).
6
(d, J = 6.0 Hz, 1H), 5.29 (s, 1H), 4.86 (t, J = 6.9 Hz, 1H), 4.20 (d, J =
1
3
3.3, 1H), 4.04−3.83 (m, 1H), 3.78 (s, 3H), 3.24 (s, 3H), 2.72 (s,
rac-Methyl (3S,4S,5R)-8-(Benzyloxy)-5-hydroxy-2-(4-methoxy-
phenyl)-10-oxo-3-phenyl-2,3,4,5-tetrahydro-2,5-methanobenzo-
H).
[
b]oxepine-4-carboxylate (S23). A solution of 7-(benzyloxy)-3-
rac-(1R,2R,3S,3aR,8bS)-3a-(4-Cyanophenyl)-1,8b-dihydroxy-6-
methoxy-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-
cyclopenta[b]benzofuran-2-carboxamide (rac-12). To a solution of
rac-(1R,2R,3S,3aR,8bS)-3a-(4-bromophenyl)-1,8b-dihydroxy-6-me-
thoxy-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxamide (S21) (0.20 g, 0.38 mmol) in N,N-
dimethylformamide (2 mL), zinc cyanide (0.134 g, 1.14 mmol) was
added. The mixture was degassed with argon for 5 min. Tetrakis-
hydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one (79) (9.0 g, 24
mmol) and methyl cinnamate (55) (39.0 g, 240 mmol) in
dichloromethane (200 mL), acetonitrile (100 mL), and methanol
(100 mL) was irradiated with 400 W UV light in a UV reactor flask for
15 h. Then, the solvent was removed under reduced pressure, and the
residue was purified by eluting through a plug of silica gel with ethyl
acetate. The desired fractions were concentrated under reduced
pressure to afford rac-methyl (3S,4S,5R)-8-(benzyloxy)-5-hydroxy-2-
(4-methoxyphenyl)-10-oxo-3-phenyl-2,3,4,5-tetrahydro-2,5-
methanobenzo[b]oxepine-4-carboxylate (S23). Yield: 15.0 g, crude.
rac-Methyl (2R,3S,3aR,8bR)-6-(Benzyloxy)-8b-hydroxy-3a-(4-me-
thoxyphenyl)-1-oxo-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta-
(triphenylphosphine)palladium(0) (0.044 g, 0.038 mmol) was added
to the reaction mixture which was then degassed for another 5 min and
then heated at 120 °C for 1 h. Afterward, the reaction was cooled to
room temperature and filtered through Celite. The filtrate was
concentrated and treated with ice-cold water. The resulting crude
precipitate was collected by filtration and purified by column
chromatography in 100−200 silica mesh by eluting with 20−70%
ethyl acetate in hexanes. Then, the desired fractions were concentrated
under vacuum to afford (1R,2R,3S,3aR,8bS)-3a-(4-cyanophenyl)-1,8b-
dihydroxy-6-methoxy-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-
[
b]benzofuran-2-carboxylate (S24). The crude rac-methyl
3S,4S,5R)-8-(benzyloxy)-5-hydroxy-2-(4-methoxyphenyl)-10-oxo-3-
phenyl-2,3,4,5-tetrahydro-2,5-methanobenzo[b]oxepine-4-carboxylate
S23) (12.8 g) was suspended in methanol (100 mL), treated with
(
(
2
8
5% sodium methoxide in methanol (20 mL solution, 4.7 g NaOMe,
7 mmol), and heated at 70 °C for 1 h. The solvent was then removed
1H-cyclopenta[b]benzofuran-2-carboxamide (rac-12) as an off-white
under reduced pressure, and the crude product was treated with
ammonium chloride solution and extracted with ethyl acetate. The
organic phase was separated, dried over sodium sulfate, filtered, and
concentrated under reduced pressure to afford methyl
solid. Yield: 0.08 g (0.17 mmol, 45%). HRMS (ESI): calcd, 471.1914
+
1
[
M + H] ; found, 471.1929; H NMR (400 MHz, DMSO-d ): δ/ppm
6
7
.55 (d, J = 8.3 Hz, 2H), 7.36 (dd, J = 8.3 Hz, 14.7 Hz, 3H), 7.04−6.95
(m, 3H), 6.83 (d, J = 7.2 Hz, 2H), 6.69 (d, J = 2 Hz, 1H), 6.52 (dd, J =
(
2R,3S,3aR,8bR)-6-(benzyloxy)-8b-hydroxy-3a-(4-methoxyphenyl)-1-
2
6
3
.2 Hz, 8.4 Hz,1H), 5.49 (d, J = 5.9 Hz, 1H), 5.39 (s, 1H), 4.88 (t, J =
oxo-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-
carboxylate (S24). Yield: 10.0 g, crude.
.4 Hz, 1H), 4.27 (d, J = 13.2 Hz, 1H), 4.08 (dd, J = 8.0, 13.2 Hz, 1H),
1
3
.78 (s, 3H), 3.21 (s, 3H), 2.72 (s, 3H); C NMR (101 MHz, DMSO-
rac-Methyl (1R,2R,3S,3aR,8bS)-6-(Benzyloxy)-1,8b-dihydroxy-3a-
d6): δ/ppm 168.37, 161.33, 158.84, 142.40, 138.41, 130.53, 128.64,
(
[
4-methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta-
1
1
28.62, 127.52, 127.47, 125.97, 121.89, 118.91, 108.98, 107.31,
00.80, 95.98, 91.75, 77.62, 55.39, 54.93, 47.10, 36.44, 35.13.
Synthesis of rac-(1R,2R,3S,3aR,8bS)-6-Cyano-1,8b-dihydroxy-3a-
b]benzofuran-2-carboxylate (80). The crude rac-methyl
(2R,3S,3aR,8bR)-6-(benzyloxy)-8b-hydroxy-3a-(4-methoxyphenyl)-1-
oxo-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-
carboxylate (S24) (10.0 g) was added to a solution of sodium
triacetoxyborohydride (23.6 g, 111 mmol) in acetonitrile (200 mL)
and acetic acid (11 mL, 190 mmol). The resulting mixture was stirred
for 4 h at room temperature. Then, it was partitioned between
saturated aqueous sodium bicarbonate solution and ethyl acetate. The
organic layer was separated, dried over sodium sulfate, and
concentrated under reduced pressure to obtain the crude product
which was purified by silica gel column chromatography eluting with
(
4-methoxyphenyl)-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-
60% ethyl acetate in hexanes. The desired fractions were concentrated
1
H-cyclopenta[b]benzofuran-2-carboxamide (rac-13). (E)-1-(4-
Benzyloxy)-2-hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-
one (S22). To a solution of 1-(4-(benzyloxy)-2-hydroxyphenyl)ethan-
-one (76) (25.0 g, 103 mmol) in methanol (117 mL), sodium
hydroxide (12.4 g, 310 mmol) and p-methoxybenzaldehyde (14.0 g,
03 mmol) were added successively. The reaction mixture was refluxed
under reduced pressure to afford rac-methyl (1R,2R,3S,3aR,8bS)-6-
(benzyloxy)-1,8b-dihydroxy-3a-(4-methoxyphenyl)-3-phenyl-
2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-carboxylate (80)
as a pale yellow solid. Yield: 7.0 g (13.0 mmol, 63% over three steps).
(
1
+
1
MS (ESI) m/z: 539.33 [M + 1] ; H NMR (400 MHz, DMSO-d ): δ/
6
1
ppm 7.47 (d, J = 7.0 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.35 (d, J = 7.0
Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.05−7.01 (m, 4H), 6.82 (d, J = 7.2
Hz, 2H), 6.72 (s, 1H), 6.63 (d, J = 8.6 Hz, 2H), 6.58 (d, J = 8.2 Hz,
1H), 5.73 (d, J = 5.8, 1H), 5.29 (s, 1H), 5.13 (s, 2H), 4.75 (t, J = 6.6
Hz, 1H), 4.02 (s, 1H), 3.86 (dd, J = 7.2 Hz, 13.9 Hz, 1H), 3.62 (s, 3H),
3.51 (s, 3H).
for 30 min and then cooled. The precipitated solid was collected by
filtration, washed with water, and dried under high vacuum to afford
(
E)-1-(4-(benzyloxy)-2-hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-
en-1-one (S22) as a yellow solid. Yield: 30.0 g (83.2 mmol, 81%). MS
+
1
(
ESI) m/z: 361 [M + 1] ; H NMR (400 MHz, DMSO-d ): δ/ppm
6
8
.1 (br s, 1H), 7.97 (d, J = 14.4 Hz, 1H), 7.85 (d, J = 7.0 Hz, 2H), 7.75
d, J = 15.1 Hz, 1H), 7.47−7.34 (m, 6H), 7.02 (d, J = 7.5 Hz, 2H),
.50 (s, 2H), 5.18 (s, 2H), 3.82 (s, 3H).
-(Benzyloxy)-3-hydroxy-2-(4-methoxyphenyl)-4H-chromen-4-
one (79). To a solution of (E)-1-(4-(benzyloxy)-2-hydroxyphenyl)-3-
4-methoxyphenyl)prop-2-en-1-one (S22) (30.0 g, 83.2 mmol) in
ethyl acetate (100 mL) at room temperature, sodium hydroxide (6.6 g,
rac-(1R,2R,3S,3aR,8bS)-6-(Benzyloxy)-1,8b-dihydroxy-3a-(4-me-
thoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxylic Acid (S25). To a solution of methyl rac-
(1R,2R,3S,3aR,8bS)-6-(benzyloxy)-1,8b-dihydroxy-3a-(4-methoxy-
phenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-
2-carboxylate (80) (4.0 g, 7.4 mmol) in methanol/water (3:1, 120
mL), lithium hydroxide (4.2 g, 180 mmol) was added. The reaction
mixture was stirred for 3 h at room temperature, then cooled to 0 °C
and acidified with 5 N hydrochloric acid to pH ≈ 3. The resulting
precipitate was collected by filtration and dried under high vacuum to
afford rac-(1R,2R,3S,3aR,8bS)-6-(benzyloxy)-1,8b-dihydroxy-3a-(4-
methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxylic acid (S25) as an off-white solid. Yield: 6.0 g
(
6
7
(
1
3
70 mmol, 10% aq solution) and hydrogen peroxide (14 g, 0.41 mol,
0% aq solution) were added. The reaction mixture was stirred for 2 h
at room temperature (reaction is exothermic). The reaction mixture
was then cooled and neutralized to pH ≈ 7 by addition of 6 N
hydrochloric acid. The resulting precipitate was collected by filtration
and dried under vacuum to afford 7-(benzyloxy)-3-hydroxy-2-(4-
methoxyphenyl)-4H-chromen-4-one (79) as a pale yellow solid. Yield:
−
1
(crude). MS (ESI) m/z: 523.1 [M − 1] ; H NMR (400 MHz,
AC
https://dx.doi.org/10.1021/acs.jmedchem.0c00182
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
DMSO-d ): δ/ppm 7.47 (d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.7 Hz, 2H),
additional 5 min and then heated at 110 °C for 1 h. Next, the reaction
mixture was cooled to room temperature and filtered through Celite.
The filtrate was concentrated and treated with ice-cold water, and the
resulting precipitate was collected by filtration. The crude product was
purified by silica gel column chromatography eluting with 90% ethyl
acetate in hexanes to afford rac-(1R,2R,3S,3aR,8bS)-6-cyano-1,8b-
dihydroxy-3a-(4-methoxyphenyl)-N,N-dimethyl-3-phenyl-2,3,3a,8b-
tetrahydro-1H-cyclopenta[b]benzofuran-2-carboxamide (rac-13) as an
off-white solid. Yield: 0.10 g (0.21 mmol, 25%), MS (ESI) m/z: 471.17
6
7
6
2
3
.34 (d, J = 7.2 Hz, 1H), 7.21 (d, J = 8.2 Hz, 1H), 7.06−6.96 (m, 7H),
.65 (s, 1H), 6.58 (d, J = 8.8 Hz 2H), 6.53 (d, J = 9.5 Hz, 1H), 5.11 (s,
H), 5.03 (s, 1H), 4.45 (d, J = 4.92 Hz, 1H), 4.02 (d, J = 13.2 Hz, 1H),
.60 (s, 3H), 3.51−3.42 (m, 1H).
rac-(1R,2R,3S,3aR,8bS)-6-(Benzyloxy)-1,8b-dihydroxy-3a-(4-me-
thoxyphenyl)-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-
cyclopenta[b]benzofuran-2-carboxamide (S26). To a solution of
rac-(1R,2R,3S,3aR,8bS)-6-(benzyloxy)-1,8b-dihydroxy-3a-(4-methox-
yphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-
+
+
[
M + 1] ; UPLC: 96.39%; HRMS (ESI): calcd, 471.1914 [M + H] ;
2
-carboxylic acid (4.0 g, 7.6 mmol) in dichloromethane (40 mL), 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (4.38 g,
2.8 mmol), hydroxybenzotriazole (3.09 g, 20.2 mmol), and DIPEA
8.0 mL, 45.9 mmol) were added at 0 °C. This mixture was stirred for
min at 0 °C. Then, dimethylamine hydrochloride (3.09 g, 38.16
1
found, 471.1927; H NMR (400 MHz, DMSO-d ): δ/ppm 7.63 (d, J =
6
7
2
2
4
3
.7 Hz, 1H), 7.56 (s, 1H), 7.41 (d, J = 7.3 Hz, 1H), 7.08 (d, J = 8.6 Hz,
H), 7.04−6.98 (m, 3H), 6.80 (d, J = 7.1 Hz, 2H), 6.67 (d, J = 8.8 Hz,
H), 5.70 (d, J = 6.0 Hz, 1H), 5.66 (s, 1H), 4.91 (t, J = 7.0 Hz, 1H),
2
(
5
.15 (d, J = 13.1 Hz, 1H), 4.0 (dd, J = 7.88 Hz, 13.1 Hz, 1H), 3.64 (s,
mmol) was added, and the mixture was stirred for 8 h at room
temperature. Afterward, the mixture was diluted with dichloromethane
and washed with cold water. The organic layer was separated and dried
over sodium sulfate, filtered, and concentrated to give the crude
product which was purified by silica gel column chromatography
eluting with 70−90% ethyl acetate in hexane to afford rac-
13
H), 3.19 (s, 3H), 2.71 (s, 3H); C NMR (101 MHz, DMSO-d ): δ/
6
ppm 168.24, 157.79, 157.75, 138.51, 136.50, 129.71, 128.69, 127.74,
1
1
27.34, 127.31, 125.86, 124.67, 119.01, 113.79, 112.24, 111.75,
00.95, 91.06, 77.52, 54.82, 54.70, 47.05, 36.41, 35.10.
Synthesis of rac-(1R,2R,3S,3aR,8bS)-6-Cyano-3a-(4-cyanophen-
yl)-1,8b-dihydroxy-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-
(
1R,2R,3S,3aR,8bS)-6-(benzyloxy)-1,8b-dihydroxy-3a-(4-methoxy-
phenyl)-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta-
b]benzofuran-2-carboxamide (S26) as a white solid. Yield: 4 g (7.2
[
+
1
mmol, 95% over two steps). MS (ESI) m/z: 552.25 [M + 1] ; H
NMR (400 MHz, DMSO-d ): δ/ppm 7.48 (d, J = 7.3 Hz, 2H), 7.43−
6
.29 (m, 4H), 7.07 (d, J = 8.4 Hz, 2H), 7.03−6.97 (m, 3H), 6.78 (d, J
(
s, 3H), 3.17 (s, 3H), 2.71 (s, 3H).
rac-(1R,2R,3S,3aR,8bS)-1,6,8b-trihydroxy-3a-(4-methoxyphenyl)-
1H-cyclopenta[b]benzofuran-2-carboxamide (rac-14). rac-Methyl
(1R,2R,3S,3aR,8bS)-6-(Benzyloxy)-3a-(4-cyanophenyl)-1,8b-dihy-
droxy-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxylate (81). To a solution of rac-methyl
N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxamide (S27). To a solution of
(
1R,2R,3S,3aR,8bS)-6-(benzyloxy)-3a-(4-bromophenyl)-1,8b-dihy-
(
1R,2R,3S,3aR,8bS)-6-(benzyloxy)-1,8b-dihydroxy-3a-(4-methoxy-
phenyl)-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta-
b]benzofuran-2-carboxamide (S26) (2.0 g, 3.6 mmol) in ethyl acetate
100 mL), palladium hydroxide (0.66 g, 4.7 mmol) was added. The
droxy-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]benzofuran-2-
carboxylate (78) (1.50 g, 2.55 mmol) in N,N-dimethylformamide (5.0
mL), zinc cyanide (0.899 g, 7.65 mmol) was added. The reaction
mixture was degassed with argon for 5 min. Then, tetrakis-
[
(
reaction mixture was stirred under a hydrogen atmosphere for 2 h at
room temperature and then filtered through Celite and concentrated
under vacuum to afford rac-(1R,2R,3S,3aR,8bS)-1,6,8b-trihydroxy-3a-
(triphenylphosphine)palladium (0.442 g, 0.382 mmol) was added.
The mixture was degassed for an additional 5 min and then incubated
at 100 °C for 1 h in a microwave reactor. The mixture was then filtered
through Celite and washed with ethyl acetate. The filtrate was
concentrated, ice water was added, and the resulting precipitate was
collected by filtration and washed with ethanol and pentane to afford
methyl (1R,2R,3S,3aR,8bS)-6-(benzyloxy)-3a-(4-cyanophenyl)-1,8b-
dihydroxy-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxylate (81) as a white solid. Yield: 1.1 g (2.1 mmol,
(
4-methoxyphenyl)-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-
cyclopenta[b]benzofuran-2-carboxamide (S27) as an off-white solid.
Yield: 1.5 g, crude. MS (ESI) m/z: 460.4 [M − 1] .
−
rac-(1R,2R,3S,3aR,8bS)-2-(dimethylcarbamoyl)-1,8b-dihydroxy-
a-(4-methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-
3
cyclopenta[b]benzofuran-6-yl Trifluoromethanesulfonate (S28).
To a solution of crude rac-(1R,2R,3S,3aR,8bS)-1,6,8b-trihydroxy-3a-
−
1
(
4-methoxyphenyl)-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-
81%). MS (ESI) m/z: 332.3 [M − 1] ; H NMR (400 MHz, DMSO-
cyclopenta[b]benzofuran-2-carboxamide (S27) (1.4 g) in dichloro-
methane (100 mL) at 0 °C, DIPEA (1.28 mL, 7.35 mmol) was added.
Triflic anhydride (3.34 mmol) diluted in dichloromethane (10 mL)
was added dropwise to the reaction mixture while it was stirred at −40
d ): δ/ppm 7.51 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 7.2 Hz, 2H), 7.41 (t, J
6
= 7.1 Hz, 2H), 7.36−7.30 (m, 3H), 7.25 (d, J = 8.4 Hz, 1H), 7.07−
6.98 (m, 3H), 6.89 (d, J = 7.1 Hz, 2H), 6.76 (d, J = 1.9 Hz, 1H), 6.61
(d, J = 8.3 Hz, 1H), 5.73 (s, 1H), 5.57 (s, 1H), 5.13 (s, 2H). 4.71 (t, J =
6.5 Hz, 1H), 3.54 (s, 3H).
°C for 1 h. Then, the reaction mixture was neutralized with saturated
sodium bicarbonate solution and washed with cold water. The organic
layer was separated, dried over sodium sulfate, filtered, and
concentrated to afford (1R,2R,3S,3aR,8bS)-2-(dimethylcarbamoyl)-
rac-Methyl (1R,2R,3S,3aR,8bS)-3a-(4-Cyanophenyl)-1,6,8b-trihy-
droxy-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-2-carboxylate (S29). A flask containing ethyl acetate (10
mL) was charged with rac-methyl (1R,2R,3S,3aR,8bS)-6-(benzyloxy)-
1
1
,8b-dihydroxy-3a-(4-methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-
H-cyclopenta[b]benzofuran-6-yl trifluoromethanesulfonate (S28) as
3
1
a-(4-cyanophenyl)-1,8b-dihydroxy-3-phenyl-2,3,3a,8b-tetrahydro-
H-cyclopenta[b]benzofuran-2-carboxylate (81) (1.1 g, 2.1 mmol),
an off-white solid. Yield: 1.0 g (1.7 mmol, 56% over two steps). MS
−
and palladium hydroxide (50% wet, 0.579 g, 4.12 mmol) was added at
room temperature. The reaction mixture was purged with hydrogen
twice and stirred under hydrogen pressure for 16 h. Then, the reaction
mixture was filtered through Celite. The filtrate was concentrated
under reduced pressure to afford rac-methyl (1R,2R,3S,3aR,8bS)-3a-
(4-cyanophenyl)-1,6,8b-trihydroxy-3-phenyl-2,3,3a,8b-tetrahydro-1H-
cyclopenta[b]benzofuran-2-carboxylate (S29) as a white solid. Yield:
(
ESI) m/z: 592.0 [M − 1] .
rac-(1R,2R,3S,3aR,8bS)-6-Cyano-1,8b-dihydroxy-3a-(4-methoxy-
phenyl)-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-
cyclopenta[b]benzofuran-2-carboxamide (rac-13). To a solution of
rac-(1R,2R,3S,3aR,8bS)-2-(dimethylcarbamoyl)-1,8b-dihydroxy-3a-
(4-methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-cyclopenta[b]-
benzofuran-6-yl trifluoromethanesulfonate (S28) (0.50 g, 0.84 mmol)
in N,N-dimethylacetamide (20 mL), zinc cyanide (0.3 g, 3 mmol) was
added. The reaction mixture was degassed with argon for 15 min.
Tetrakis(triphenylphosphine)palladium(0) (0.09 g, 0.084 mmol) was
added to the reaction mixture. The mixture was degassed for an
−
1
0.76 g (1.7 mmol, 81%). MS (ESI) m/z: 442.00 [M − 1] ; H NMR
(400 MHz, DMSO-d ): δ/ppm 9.52 (s, 1H), 7.49 (d, J = 8.1 Hz, 2H),
6
7.25 (d, J = 8.3 Hz, 2H), 7.13 (d, J = 8.1 Hz, 1H), 7.06−6.99 (m, 3H),
6.87 (d, J = 7.2 Hz, 2H), 6.42 (d, J = 1.7 Hz, 1H), 6.37 (d, J = 8.32 Hz,
AD
https://dx.doi.org/10.1021/acs.jmedchem.0c00182
J. Med. Chem. XXXX, XXX, XXX−XXX