Chasing the Elusive Benzofuran Impurity of the THR Antagonist NH-3: Synthesis, Isotope Labeling, and Biological Activity

Article abstract of DOI:10.1021/acs.joc.5b02665

We have synthesized and established the structure of a long-suspected, but hitherto unknown, benzofuran side product (EBI) formed during the synthesis of NH-3. Understanding the mechanism of its formation has enabled isotope (D) labeling. We further devel

Full text of DOI:10.1021/acs.joc.5b02665

Article
pubs.acs.org/joc
Chasing the Elusive Benzofuran Impurity of the THR Antagonist NH-
3: Synthesis, Isotope Labeling, and Biological Activity
Latika Singh,† Brandon Pressly,† Brenda J. Mengeling,‡ James C. Fettinger,§ J. David Furlow,‡
Pamela J. Lein,∥ Heike Wulff,† and Vikrant Singh*,†
†Department of Pharmacology, University of California, Davis, California 95616, United States
‡Department of Neurobiology, Physiology, and Behavior, University of California Davis, California 95616, United States
§Department of Chemistry, University of California, Davis, California 95616, United States
∥Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616, United
States
S
* Supporting Information
ABSTRACT: We have synthesized and established the structure of a long-suspected, but hitherto unknown, benzofuran side
product (EBI) formed during the synthesis of NH-3. Understanding the mechanism of its formation has enabled isotope (D)
labeling. We further developed a highly efficient method for separating EBI from NH-3. Interestingly, EBI was found to be a very
potent thyroid hormone receptor (THR) agonist, while NH-3 is an antagonist. In this process, we have also achieved a
significantly improved synthesis of NH-3.
following a long synthetic route originally reported by Nguyen
et al.6 In our hands, the synthesis of NH-3 following this
procedure resulted in samples of NH-3 that contained high
amounts of an inseparable benzofuran impurity (EBI). Through
deuterium-labeling experiments, we have demonstrated that the
presence of EBI is the result of a base-mediated annulation of
an o-alkylnylphenol precursor. The mechanism of formation of
benzofurans from o-alkylnylphenols by Pd-,7a,b Cu-,7c Ru-,7d
and Pt-catalyzed7e reactions has previously been studied.
Jacubert et al.7f have reported an acid-mediated annulation of
o-alkylnylphenol methyl ethers and in one case used deuterium
labeling to establish the electrophilic nature of the trans-
formation. Our experiment reveals that NH-3 converts into EBI
via base-mediated intramolecular annulation without a
transition-metal catalyst. There have been speculations about
EBI by other research groups, but it could never previously be
identified.8 Therefore, we decided to identify, synthesize, and
study the biological activity of EBI and investigate its
mechanism of formation along with developing an improved
and higher-yielding synthesis of NH-3.9
INTRODUCTION
■
Thyroid hormones regulate growth, metabolism, and homeo-
stasis of vertebrates. They bind to thyroid hormone receptors
(THRs) found in almost all cells of the body and exert their
effect by altering gene expression.1 Various developmental and
metabolic disorders can result from under- or over-activity of
the thyroid. Thus, both THR agonists and antagonists have
immense medicinal importance.2,3 NH-3 (Figure 1) is currently
the most potent synthetic small-molecule THR antagonist.4,5
For a study investigating disrupting effects of environmental
toxicants on thyroid hormone regulated neurodevelopmental
processes, NH-3 was required as a reference compound. NH-3,
which is not commercially available, can only be obtained by
Figure 1. Structures of the THR antagonist NH-3 and the benzofuran
EBI.
Received: November 20, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.joc.5b02665
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a
Scheme 1. Synthesis of Key Intermediates 9 and 10
a
Reagents and conditions: (a) Br2, DCM, 0 °C, 6 h, 81%; (b) MOM-Cl, Hunigs’ base, DCM, 0 °C to rt, 18 h, 82%; (c) TiPS-CI, DMAP, Et3N,
THF, 0 °C to rt, 2 h, 90%; (d) Mg, THF, 3, 0 °C to rt, 8 h, 78%; (e) H2, Pd/C, EtOH/AcOH, rt, 6 h, 76%; (f) TBAF, THF, rt, 10 min, quantitative;
(g) BrCH2CO2Me, Cs2CO3, DMF, rt, 2 h, quantitative; (h) HCl, MeOH/THF (1:1), rt, 24 h, 72%; (i) ICI, Et3N, THF, −78 °C, 2 h, 71%; (j)
MOM-CI, Hunigs’ base, DCM, 0 °C to rt, 24 h, 80%.
a
Scheme 2. Attempted Synthesis of NH-3
a
Reagents and conditions: (a) PdCI2(PPh3)2, CuI, 4-nitrophenylacetylene, Et3N, rt, 40 h, 78%; (b) HCl, MeOH/THF (1:1), rt, 12 h, 79%; (c)
LiOH·H2O, MeOH/THF, rt, 16 h.
protected to give 5. At this stage, we made another significant
change to avoid the use of n-BuLi by fashioning a carbon−
carbon bond between aryl halide 3 and benzaldehyde derivative
5 via a Grignard reagent derived from 3. Using this method,
multigram quantities of benzyl alcohol 6 were obtained in very
good yield (78%). Reduction of compound 6 using hydrogen in
the presence of palladium and charcoal worked well, and after
following a series of functional group transformations and
protection/deprotection steps, the key intermediate 8 was
synthesized in good yield.7 In our hands, phenol 8 proved to be
a solid, and high-quality crystals were obtained from a
chloroform/hexane mixture. Single-crystal X-ray diffraction
RESULTS AND DISCUSSION
■
Our synthesis of the key intermediates 9 and 10 is depicted in
Scheme 1. This sequence is similar to the one reported by
Nguyen et al.6 which includes several steps involving the use of
n-BuLi, a pyrophoric substance, posing a safety hazard. The use
of BuLi not only necessitates extremely careful handling but
also adversely affects reaction scale-up in academic laboratory
settings. Therefore, we decided to minimize or eliminate the
use of n-BuLi from the sequence and made some changes in the
synthetic strategy. Thus, the commercially available phenol 1
was brominated (2) and subsequently MOM-protected to
furnish 3, and the commercially available aldehyde 4 was TiPS-
B
DOI: 10.1021/acs.joc.5b02665
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Scheme 3. Mechanism of Benzofuran Formation under Basic Conditions
a
Scheme 4. Unambiguous Synthesis of EBI
a
Reagents and conditions: (a) PdCI2(PPh3)2, 4-nitrophenylacetylene, CuI, Et3N, rt, 3 h, 78%; (b) LiOH·H2O, MeOH/THF, rt, 15 min, 97%.
(Supporting Information p S-29) not only confirmed the
structure of 8 but also that of its precursors 2, 3, and 6. The
reaction of ICl as an electrophilic iodine source with phenol 8
at a low temperature furnished 9, which in turn was
transformed into 10 by base-mediated MOM protection of
the phenol.
then set for a base-mediated transformation of 17 into EBI.
This proved to be a very fast reaction, giving the desired
material EBI in almost quantitative yield (Scheme 4).
1
The structure of EBI was easily determined based on its H
and 13C NMR spectra. High-quality crystals of EBI for single-
crystal X-ray diffraction were obtained by careful crystallization
from chloroform and hexane. X-ray diffraction of these crystals
clearly showed the presence of a benzofuran ring and confirmed
the proposed structure of EBI (Supporting Information p S-
32).
After the iodo-compound 10 was obtained, the stage was set
to attempt the Sonogashira coupling10 to combine the two
“parts” of NH-3. At this point, we thought about the possibility
of coupling 10 directly with 4-nitrophenylacetylene. If executed
successfully, this reaction would have eliminated additional
synthetic transformations from the reported synthetic se-
quence, rendering it much shorter and more efficient. The
Pd-catalyzed reaction worked well to furnish 78% of coupling
product 11, which was then converted into the methyl ester 12
in good yield. In the last step, the ester 12 was saponified in the
hope of directly obtaining NH-3. However, the 1H NMR
spectrum of the product showed a mixture of two compounds
with NH-3 as the minor component. At this stage, despite
various attempts, NH-3 could not be purified from this mixture
by means of column or thin-layer chromatography. It was a
disastrous culmination of a long synthetic endeavor. A
thorough analysis of the NMR data of the mixture revealed
that the major product of the reaction was EBI (Scheme 2).
The structural elucidation of EBI led us to the mechanism of
this transformation. In all likelihood, the phenoxide ion derived
from phenol 13 under basic conditions attacks the acetylenic
triple bond (14), resulting in a favorable 5-endo-dig-type
cyclization11 to generate a carbanion (15), which in turn
abstracts a proton from the solvent to form the benzofuran
derivative of type 16 (Scheme 3).
Having achieved the synthesis of EBI, we focused our
attention toward further testing the validity of our proposed
mechanism. We hypothesized that ester 12 as well as NH-3
should furnish EBI upon base treatment. Thus, a solution of
ester 12 in methanol and THF was treated with aq LiOH. This
reaction furnished EBI in excellent yield (85%), providing
further credence to the proposed mechanism (Scheme 5).
Scheme 5. Synthesis of EBI from 12
We further reasoned that the step of incorporating a proton
in the 3-position of the newly formed furan ring can easily be
manipulated to obtain deuterium- or tritium-labeled EBI by
merely changing the proton source to a deuterium or tritium
source, in this case, water. Thus, the treatment of a solution of
12 in MeOD-d4 and THF-d8 with LiOD in D2O (prepared by a
careful addition of Li metal in D2O) under exactly the same
conditions as described above furnished EBI-d in excellent yield
On the basis of the premise that under basic conditions a
favorable 5-endo-dig cyclization would generate the benzofuran,
we explored the possibility of synthesizing EBI directly from
iodo-compound 9. We postulated that under the basic
conditions required for Pd-catalyzed Sonogashira coupling,10
the transformation would follow the coupling step as the
phenol moiety was left unprotected. To our delight, the
reaction furnished 17 in very good yield (78%). The stage was
1
(Scheme 6). The absence of a singlet at δ 7.08 from the H
C
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of NH-3 from 11. Thus, saponification of ester 11 furnished the
carboxylic acid derivative 19. The phenolic hydroxyl group of
19 is not available for participation in the benzofuran
formation, which prevents any side reaction. At this stage, the
only transformation required to obtain NH-3 was the removal
of the MOM protecting group. After some experimentation, we
found that silica-supported NaHSO4 was the best reagent14 to
cleave the MOM group of 19 in a remarkably fast and easy
manner to render NH-3 in excellent yield (Scheme 8). Our
synthesis of NH-3 thus represents an improved, shorter,
efficient, and high-yielding sequence compared to the
previously reported methods.
We next evaluated the receptor binding and transactivator
activity of NH-3 and EBI using GH3.TRE-Luc cells, a female
rat pituitary tumor derived cell line, which has an integrated
thyroid hormone receptor response element (TRE) upstream
of a luciferase reporter gene.15 As expected, NH-3 was found to
be an effective and potent antagonist (IC50 = 55.2 nM). To our
surprise, EBI exerted the opposite effect and displayed potent
agonistic activity with an EC50 of 65.3 nM. We expect that this
finding could lead to a better understanding of the structure−
activity relationship of THR modulators and suggest that EBI
could potentially be useful as a thyromimetic, much sought-
after agent for various metabolic disorders.16
Scheme 6. Synthesis of Deuterium-Labeled EBI
NMR spectrum of EBI-d clearly showed incorporation of
deuterium on the furan moiety (Supporting Information p S-
17). This proved to be a remarkably easy, inexpensive, and
efficient way of generating isotope-labeled material, which
could potentially be used for radioisotope labeling, as well. On
the basis of this labeling experiment, we propose that tritium-
labeled EBI, a potentially useful material for pharmacokinetic/
pharmacodynamics studies, tissue labeling experiments, and
binding assays, may be obtained simply by employing tritiated
water to quench the reaction. Another attractive feature of this
reaction is that it could be monitored by NMR because all of
the solvents used are deuterated.
Having solved the problems related to the synthesis of EBI,
we turned our attention toward the synthesis of NH-3.
Obtaining NH-3 from 12 proved to be unreliable under basic
conditions. Therefore, we explored the possibility of trans-
forming the ester into an acid by employing a Lewis acid. Thus,
the reaction of 12 with BBr3 under an inert atmosphere was
Separation of NH-3 and EBI from Their Mixture. While
NH-3 and EBI have opposite biological activities, separating
them from their mixture has thus far proved to be a daunting
exercise because their mixture is not amenable to column or
preparative thin-layer chromatographic purification. Different
research groups have reported their failure to achieve this
objective.7−9 In view of the above-mentioned opposite
biological activity, it became imperative that a method to
purify both of these entities from their mixture be developed.
We confronted this challenge by undertaking an extensive study
of the solubility profile of the two compounds and found that,
in methanol, EBI is only sparingly soluble while NH-3 is highly
soluble. We exploited this difference in solubility to achieve
purification from the hitherto intractable mixture of NH-3 and
EBI. Thus, a 1:1 mix of EBI and NH-3 (2.6 mg each) was made
using pure compounds, and the 1H NMR spectrum was
recorded (Supporting Information p S-25). The solvent was
then removed, and the solid residue was extracted with 100 μL
of methanol. The soluble portion was separated and kept at 0
°C for 15 min. It was then centrifuged, and 90 μL of solution
was collected. The 1H NMR of this fraction (Supporting
Information p S-26) indicated recovery of pure NH-3 (2.0 mg,
77%). The insoluble portion was washed again with 120 μL of
explored.12 The purified product showed an interesting H
1
NMR spectrum (Supporting Information p S-18). The two sets
of methyl protons, attached directly to the phenyl ring bearing
ester, which appear as a singlet integrating for six protons in the
NMR of 12 (Supporting Information p S-8) changed into two
singlets integrating for three protons each in the NMR spectra
of 18. This was a clear indication of desymmetrization
occurring on the said phenyl moiety. Indeed, a detailed analysis
1
of H and 13C NMR spectra (Supporting Information pp S-18
and S-19) indicated the formation of the benzofuranone
derivative 18 presumably formed through an intramolecular
Friedel−Crafts acylation13 (Scheme 7). Though this reaction
Scheme 7. Synthesis of Furanone 18
1
cold methanol, dried, and dissolved in CDCl3. Its H NMR
(Supporting Information p S-27) revealed a recovery of
analytically pure EBI (2.5 mg, 96%). The remaining NH-3
was obtained by combining 10 μL of the first extraction and all
of the second wash, which was also quite pure and showed a
presence of less than 20% contamination of EBI (Supporting
Information p S-28). Thus, we have developed a remarkably
easy and highly efficient method of purification of NH-3 and
EBI that is suitable even for a biology laboratory without any
access to analytical chemistry facilities that requires a very small
quantity of NH-3 or EBI and wants to ensure their purity.
failed to yield the intended product NH-3, it furnished an
interesting entity 18 which is currently under investigation for
its bioactivity and is expected to provide important clues about
the SAR of thyromimetics.
There is literature precedence8 that confirms our observation
that the preparation of NH-3 from 12 under basic conditions is
not a reliable method because it also produces varying amounts
of benzofuran impurity. Therefore, the sequence suggested by
Gopinathan and Rehder8b was adopted to finish the synthesis
CONCLUSIONS
■
We have successfully synthesized and characterized EBI which
has been a long-elusive benzofuran side product of NH-3. The
discovery that EBI is a very potent THR agonist rather than an
D
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a
Scheme 8. Synthesis of NH-3
a
Reagents and conditions: (a) LiOH·H2O, MeOH/THF, rt, 2 h, 95%; (b) NaHSO4·SiO2, DCM, rt, 80 min, 96%.
Methyl 2-(4-(4-Hydroxy-3-iodo-5-isopropylbenzyl)-3,5-
dimethylphenoxy)acetate (9).8a To a solution of 8 (1.25 g, 3.65
mmol) in dry THF (70 mL) was added Et3N (7 mL). This solution
was then cooled to −78 °C, and ICl (1 M in DCM, 4.38 mL, 4.38
mmol) was added. After 2 h of stirring at −78 °C, the reaction was
quenched with 1 N HCl and washed with a saturated solution of
sodium thiosulfate. The organic phase was separated and dried over
anhyd Na2SO4. The solvent was evaporated under vacuum, and the
residue was purified by column chromatography on silica using a
gradient of ethyl acetate in hexane (5%−45%), which afforded 9 as a
yellow liquid. Yield: 1.3 g (71%). 1H NMR (800 MHz, CDCl3): δ 6.98
(s, 1H), 6.86 (s, 1H), 6.64 (s, 2H), 5.17 (s, 1H), 4.64 (s, 2H), 3.87 (s,
2H), 3.83 (s, 3H), 3.24 (septet, J = 7.0 Hz, 1H), 2.20 (s, 6H), 1.18 (d,
J = 7.0 Hz, 6H). 13C NMR (201 MHz, CDCl3): δ 169.7, 155.9, 149.9,
138.5, 135.1, 133.9, 133.7, 130.0, 126.8, 114.2, 87.1, 65.3, 52.3, 33.3,
28.5, 22.4, 20.5.
antagonist like NH-3 has important implications. An easy
access to stable and radioisotope-labeled material could find a
potential use in the detailed biological studies currently
underway. This work will help us to better understand the
SAR of thyromimetics. Our work also offers a shorter, more
efficient, and significantly higher-yielding synthetic route to
NH-3.
EXPERIMENTAL METHODS
■
General Remarks. All reactions were performed in flame-dried
glassware under nitrogen unless otherwise noted. Tetrahydrofuran and
diethyl ether were distilled from sodium/benzophenone ketyl under
nitrogen. Dichloromethane and triethylamine were distilled from
calcium hydride under nitrogen. Melting points were determined using
a heated block melting point apparatus and are uncorrected. For
HRMS analysis, samples were analyzed by flow-injection analysis into
an Orbitrap XL operated in profile mode. Source parameters were 5
kV spray voltage, capillary temperature of 275 °C, and a sheath gas
setting of 20. Spectral data were acquired at a resolution setting of
100 000 fwhm with the lockmass feature which typically results in a
mass accuracy <2 ppm. 1H NMR and 13C NMR spectra were recorded
on both an 800 MHz and a 500 MHz spectrometer with the the
above-mentioned solvents. Because of the need to probe reaction
mechanism and isotope-labeling experiments, the NMR spectra of
compound 12, EBI, and NH-3 were recorded and reported in more
than one solvent, either CDCl3 and methanol-d4 or a methanol-d4/
THF-d8 mixture. Chemical shifts are reported in parts per million
(ppm) on the δ scale and were referenced to the appropriate solvent
peaks (CDCl3 referenced to CHCl3 at δH 7.26 ppm and MeOD-d4
referenced to MeOH at 3.31 ppm). Signals are designated as follows: s
(singlet), d (doublet), t (triplet), septet (septet), m (multiplet). Thin-
layer chromatography (TLC) was performed on silica gel 60F254
coated aluminum-backed plates and visualized with UV light (254 nm)
and other common stains. Chromatographic separation was performed
using either silica gel (35−70 μm) or aluminum oxide (activated,
neutral, and Brockmann I).
Methyl 2-(4-(3-Isopropyl-4-(methoxymethoxy)-5-((4-
nitrophenyl)ethynyl)benzyl)-3,5-dimethylphenoxy)acetate
(11).6 To a solution of 10 (prepared from 9 and used without
purification) (595 mg, 1.1 mmol) in Et3N (25 mL) kept under
nitrogen were added [PdCl2(PPh3)2] (35 mg, 0.05 mmol) and CuI
(19 mg, 0.1 mmol). After 2 min, 1-ethynyl-4-nitrobenzene (250 mg,
1.7 mmol) was added, and the resulting mixture was stirred at rt for 40
h. The mixture was then filtered and washed with Et3N (10 mL). The
solvent was evaporated under vacuum, and the residue was purified by
column chromatography on silica using a gradient of ethyl acetate in
hexane (10%−40%), which afforded 11 as a yellow solid. Yield: 481
1
mg (78%). H NMR (800 MHz, CDCl3): δ 8.19 (d, J = 8.0 Hz, 2H),
7.62 (d, J = 8.0 Hz, 2H), 7.05 (s, 1H), 6.83 (s, 1H), 6.65 (s, 2H), 5.22
(s, 2H), 4.64 (s, 2H), 3.93 (s, 2H), 3.82 (s, 3H), 3.61 (s, 3H), 3.40
(septet, J = 7.0 Hz, 1H), 2.21 (s, 6H), 1.19 (d, J = 7.0 Hz, 6H). 13C
NMR (201 MHz, CDCl3): δ 169.8, 156.1, 154.4, 147.0, 142.4, 138.8,
136.2, 132.2, 130.4, 129.9, 129.7, 128.1, 123.8, 115.7, 114.3, 100.1,
92.6, 91.1, 65.4, 57.8, 52.4, 33.9, 26.6, 23.5, 20.7.
Methyl 2-(4-(4-Hydroxy-3-isopropyl-5-((4-nitrophenyl)-
ethynyl)benzyl)-3,5-dimethylphenoxy)acetate (12).6 To a sol-
ution of 11 (700 mg, 1.3 mmol) in a mixture of methanol/THF (1:1,
30 mL) was added 1 N HCl (9.0 mL), and the resulting mixture was
left stirring at rt for 24 h. Twenty-five milliliters of water was then
added to the mixture, and the solvents were evaporated under vacuum.
The aqueous phase was extracted using chloroform (3 × 20 mL). The
organic phase was separated and dried over anhyd Na2SO4. The
solvent was then evaporated under vacuum, and the residue was
purified by column chromatography on silica using a gradient of ethyl
acetate in hexane (5%−45%), which afforded 12 as a yellow solid.
Yield: 507 mg (79%). 1H NMR (800 MHz, CDCl3): δ 8.20 (d, J = 8.8
Hz, 2H), 7.63 (d, J = 8.8 Hz, 2H), 7.04 (s, 1H), 6.72 (s, 1H), 6.65 (s,
2H), 5.72 (s, 1H), 4.65 (s, 2H), 3.90 (s, 2H), 3.83 (s, 3H), 3.27
(septet, J = 7.0 Hz, 1H), 2.22 (s, 6H), 1.23 (d, J = 7.0 Hz, 6H). 13C
NMR (201 MHz, CDCl3): δ 169.8, 156.0, 152.5, 147.2, 138.7, 134.7,
132.2, 132.0, 130.1, 129.6, 128.8, 127.4, 123.8, 114.3, 108.03, 94.0,
89.6, 65.3, 52.4, 33.7, 27.6, 22.4, 20.6. 1H NMR (800 MHz, methanol-
d4/THF-d8, 3:1): δ 8.21 (d, J = 8.8 Hz, 2H), 7.74 (d, J = 8.8 Hz, 2H),
7.02 (s, 1H), 6.72 (s, 1H), 6.67 (s, 2H), 4.68 (s, 2H), 3.92 (s, 2H),
3.78 (s, 3H), 3.28−3.33 (septet, merged with quintet of methanol-d4, J
Methyl 2-(4-(4-Hydroxy-3-isopropylbenzyl)-3,5-
dimethylphenoxy)acetate (8).8a To a solution of 7 (2.3 g, 3.65
mmol) in a mixture of methanol/THF (1:1, 60 mL) was added conc
HCl (3.0 mL), and the resulting mixture was left stirring at rt for 24 h.
Twenty-five milliliters of water was added to the mixture, and the
solvents were evaporated under vacuum. The aqueous phase was
extracted using chloroform (3 × 20 mL). The organic phase was
separated and dried over anhyd Na2SO4, and the solvent was
evaporated under vacuum. The residue was purified by column
chromatography on silica using a gradient of ethyl acetate in hexane
(5%−45%), which afforded 8 as a solid. Yield: 1.3 g (72%), mp =
99.5−101.2 °C. 1H NMR (800 MHz, CDCl3): δ 6.91 (s, 1H), 6.62 (s,
2H), 6.64 (s, 2H), 6.58 (d, J = 8.0 Hz, 1H), 6.55 (d, J = 8.0 Hz, 1H),
4.66 (s, 1H), 4.63 (s, 2H), 3.89 (s, 2H), 3.82 (s, 3H), 3.15 (septet, J =
7.0 Hz, 1H), 2.20 (s, 6H), 1.21 (d, J = 7.0 Hz, 6H). 13C NMR (201
MHz, CDCl3): δ 169.9, 155.8, 150.9, 138.7, 134.3, 132.2, 131.0, 126.3,
125.5, 115.3, 114.2, 65.4, 52.4, 33.8, 27.2, 22.7, 20.6.
E
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= 6.9 Hz, 1H), 2.21 (s, 6H), 1.20 (d, J = 6.9 Hz, 6H). LRMS (ESI):
m/z calcd for C29H28NO6 (M − H)− 486.19, found 486.15.
5-(4-Hydroxy-3-isopropyl-5-((4-nitrophenyl)ethynyl)-
benzyl)-4,6-dimethylbenzofuran-3(2H)-one (18). To a stirred
solution of 12 (17 mg, 0.03 mmol) in dry DCM (10 mL) was added a
1.0 mL solution of BBr3 in DCM (1% v/v). The resulting mixture was
stirred at rt for 6 h. It was then filtered and washed with DCM (10
mL). The solvent was evaporated under vacuum, and the residue was
purified by column chromatography on silica using a gradient of ethyl
acetate in hexane (20%−40%), which afforded 18 as a yellow liquid.
Yield: 5.0 mg (35%). 1H NMR (800 MHz, CDCl3): δ 8.21 (d, J = 8.9
Hz, 2H), 7.64 (d, J = 8.8 Hz, 2H), 7.02 (s, 1H), 6.84 (s, 1H), 6.68 (s,
1H), 5.71 (s, 1H), 5.29 (s, 1H), 4.60 (s, 2H), 3.95 (s, 2H), 3.26
(septet, J = 6.8 Hz, 1H), 2.56 (s, 3H), 2.29 (s, 3H), 1.23 (d, J = 6.8 Hz,
6H). 13C NMR (201 MHz, CDCl3): δ 200.4, 173.2, 152.5, 148.9,
147.1, 138.0, 134.8, 132.0, 131.1, 130.9, 129.2, 128.4, 127.0, 123.6,
117.5, 112.1, 107.9, 93.9, 89.1, 74.7, 32.6, 27.4, 22.2, 21.8, 13.9. HRMS
(ESI): m/z calcd for C28H24NO5 (M − H)− 454.1655, found
454.1649.
Methyl 2-(4-((7-Isopropyl-2-(4-nitrophenyl)benzofuran-5-
yl)methyl)-3,5-dimethylphenoxy)acetate (17). To a solution of
9 (180 mg, 0.4 mmol) in Et3N (7 mL) kept under nitrogen were
added [PdCl2(PPh3)2] (10 mg, 0.014 mmol) and CuI (4.5 mg, 0.023
mmol). After 2 min, 1-ethynyl-4-nitrobenzene (74 mg, 0.5 mmol) was
added, and the resulting mixture was stirred at rt for 3 h. The mixture
was then filtered and washed with Et3N (5 mL). The solvent was
evaporated under vacuum, and the residue was purified by column
chromatography on silica using a gradient of ethyl acetate in hexane
(20%−50%), which afforded 17 as a yellow solid. Yield: 146 mg
(78%), mp = 183 °C (dec). 1H NMR (800 MHz, CDCl3): δ 8.29 (d, J
= 8.8 Hz, 2H), 7.95 (d, J = 8.8 Hz, 2H), 7.08 (s, 1H), 7.01 (d, J = 1.7
Hz, 1H), 6.87 (d, J = 1.7 Hz, 1H), 6.67 (s, 2H), 4.66 (s, 2H), 4.06 (s,
2H), 3.84 (s, 3H), 3.44 (septet, J = 7.0 Hz, 1H), 2.24 (s, 6H), 1.43 (d,
J = 7.0 Hz, 6H). 13C NMR (201 MHz, CDCl3): δ 169.8, 156.0, 152.9,
152.3, 147.1, 138.8, 136.7, 135.6, 132.3, 130.7, 125.1, 124.4, 123.8,
117.3, 114.3, 105.5, 65.4, 52.4, 34.4, 29.4, 22.7, 20.7, 20.6.
2-(4-(3-Isopropyl-4-(methoxymethoxy)-5-((4-nitrophenyl)-
ethynyl)benzyl)-3,5-dimethylphenoxy) Acetic Acid (19).8b To a
stirred solution of 11 (530 mg, 1 mmol) in methanol (20 mL) and
THF (5.0 mL) was added a solution of LiOH·H2O (200 mg, 5.2
mmol) in water (2.5 mL). The resulting mixture was stirred at rt for 2
h. The solvent was then removed under vacuum, and the residue was
diluted with DCM (30 mL), acidified with 1 N aq HCl, and extracted
with DCM (2 × 15 mL). The combined organic phase was washed
with brine (20 mL) and dried over Na2SO4 (anhyd). The solvent was
evaporated under vacuum, and the residue was purified by column
chromatography on a short silica column using a gradient of methanol
in DCM (5%−15%), which afforded 19 as a yellow solid. Yield: 483
2-(4-((7-Isopropyl-2-(4-nitrophenyl)benzofuran-5-yl)-
methyl)-3,5-dimethylphenoxy) Acetic Acid (EBI). Synthesis
from 17. To a stirred solution of 17 (68 mg, 0.14 mmol) in
methanol (5 mL) and THF (2.0 mL) was added a solution of LiOH·
H2O (50 mg, 1.2 mmol) in H2O (2 mL). The resulting mixture was
stirred at rt for 15 min. The solvent was then removed under vacuum,
and the residue was diluted with DCM (20 mL), acidified with 1 N aq
HCl, and extracted with DCM (2 × 10 mL). The combined organic
phase was washed with brine (20 mL) and dried over Na2SO4
(anhyd). The solvent was evaporated under vacuum, and the residue
was purified by column chromatography on a short silica plug using a
gradient of methanol in DCM (5%−40%), which afforded EBI as a
yellow solid. Yield: 61 mg (97%), mp = 195.5 °C (dec).
1
mg (95%). H NMR (500 MHz, CDCl3): δ 8.19 (d, J = 8.8 Hz, 2H),
7.62 (d, J = 9.0 Hz, 2H), 7.04 (s, 1H), 6.84 (s, 1H), 6.67 (s, 2H), 5.23
(s, 2H), 4.69 (s, 2H), 3.94 (s, 2H), 3.62 (s, 3H), 3.41 (septet, J = 6.9
Hz, 1H), 2.23 (s, 6H), 1.20 (d, J = 6.9 Hz, 6H).
Synthesis from 12. To a stirred solution of 12 (17 mg, 0.03
mmol) in methanol (5 mL) and THF (2.0 mL) were added LiOH·
H2O (8 mg, 0.2 mmol) and H2O (10 μL, 0.10 mmol). The reaction
mixture was stirred at rt for 30 h, acidified with 1 N aq HCl to pH 6,
and diluted with ethyl acetate (15 mL). The organic portion was
washed with brine (2 × 15 mL) and dried over Na2SO4 (anhyd). The
solvent was evaporated under vacuum, and the residue was purified by
column chromatography on silica using a gradient of methanol in
DCM (5%−40%), which afforded EBI as a yellow solid. Yield: 14 mg
(94%), mp = 195.5 °C (dec). 1H NMR (800 MHz, CDCl3): δ 8.29 (d,
J = 8.8 Hz, 2H), 7.95 (d, J = 8.8 Hz, 2H), 7.08 (s, 1H), 7.01 (s, 1H),
6.86 (s, 1H), 6.69 (s, 2H), 4.70 (s, 2H), 4.07 (s, 2H), 3.45 (septet, J =
7.0 Hz, 1H), 2.25 (s, 6H), 1.43 (d, J = 7.0 Hz, 6H). 13C NMR (201
MHz, CDCl3): δ 172.0, 155.5, 152.9, 152.4, 147.1, 139.0, 136.7, 135.5,
132.4, 131.2, 128.8, 125.1, 124.4, 123.8, 117.2, 114.3, 105.5, 65.0, 34.4,
29.4, 22.7, 20.7. 1H NMR (800 MHz, methanol-d4/THF-d8, 55:45): δ
8.34 (d, J = 8.9 Hz, 2H), 8.13 (d, J = 8.9 Hz, 2H), 7.38 (s, 1H), 7.09
(s, 1H), 6.97 (s, 1H), 6.71 (s, 2H), 4.63 (s, 2H), 4.11 (s, 2H), 3.46
(septet, J = 6.9 Hz, 1H), 2.25 (s, 6H), 1.45 (d, J = 6.9 Hz, 6H). 13C
NMR (201 MHz, methanol-d4/THF-d8, 55:45): δ 172.2, 157.8, 154.3,
153.5, 148.6, 139.5, 137.9, 137.3, 133.3, 131.3, 130.4, 126.3, 125.4,
124.7, 118.6, 115.3, 106.9, 65.9, 35.2, 30.6, 23.2, 20.8. HRMS (ESI):
m/z calcd for C28H26NO6 (M − H)− 472.1755, found 472.1743.
Deuterated EBI (EBI-d). To a solution of 12 (5 mg, 0.14 mmol) in
MeOD-d4 (300 μL) and THF-d8 (150 μL) was added a saturated
solution of LiOH·H2O in D2O (40 μL). The resulting mixture was
stirred at rt for 30 h. The solvent was then removed under vacuum,
and the remaining residue was diluted with DCM (2 mL) and acidified
with 1 N aq HCl. The organic phase was dried over Na2SO4 (anhyd).
The solvent was evaporated under vacuum, leaving EBI-d as a yellow
solid. Yield: 4.2 mg (85%). 1H NMR (800 MHz, CDCl3): δ 8.29 (d, J
= 8.8 Hz, 2H), 7.94 (d, J = 8.8 Hz, 2H), 7.00 (s, 1H), 6.85 (s, 1H),
6.69 (s, 2H), 4.69 (s, 2H), 4.07 (s, 2H), 3.45 (septet, J = 7.0 Hz, 1H),
2.25 (s, 6H), 1.43 (d, J = 7.0 Hz, 6H). 13C NMR (201 MHz, CDCl3):
δ 170.8, 155.4, 152.9, 152.4, 147.1, 139.1, 136.7, 135.5, 132.4, 131.2,
128.8, 125.1, 124.4, 123.7, 117.2, 114.3, 65.0, 34.5, 29.4, 22.7, 20.7.
HRMS (ESI): m/z calcd for C28H25DNO6 (M − H)− 473.1823, found
473.1830.
2-(4-(4-Hydroxy-3-isopropyl-5-((4-nitrophenyl)ethynyl)-
benzyl)-3,5-dimethylphenoxy) Acetic Acid (NH-3).6,8b To a
stirred solution of 19 (70 mg, 0.14 mmol) in dry DCM (10 mL) kept
under nitrogen was added silica-supported NaHSO4 (50 mg), and the
resulting mixture stirred at rt for 80 min. It was then filtered and
washed with DCM (10 mL). The solvent was evaporated under
vacuum, and the residue was purified by column chromatography on
silica using a gradient of methanol in dichloromethane (5%−20%),
which afforded NH-3 as a yellow solid. Yield: 59 mg (96%). 1H NMR
(800 MHz, CDCl3): δ 8.21 (d, J = 8.8 Hz, 2H), 7.64 (d, J = 8.8 Hz,
2H), 7.03 (s, 1H), 6.71 (s, 1H), 6.67 (s, 2H), 4.69 (s, 2H), 3.91 (s,
2H), 3.27 (septet, J = 7.0 Hz, 1H), 2.23 (s, 6H), 1.23 (d, J = 7.0 Hz,
6H). 13C NMR (201 MHz, CDCl3): δ 172.9, 155.5, 152.6, 147.3,
139.0, 134.8, 132.3, 131.9, 130.6, 129.5, 128.8, 127.4, 123.9, 114.3,
1
108.0, 94.1, 89.6, 64.9, 33.7, 27.7, 22.4, 20.7. H NMR (800 MHz,
methanol-d4): δ 8.21 (d, J = 8.8 Hz, 2H), 7.74 (d, J = 8.8 Hz, 2H), 7.01
(s, 1H), 6.70 (s, 1H), 6.68 (s, 2H), 4.38 (s, 2H), 3.90 (s, 2H), 3.30−
3.27 (septet, merged with quintet of methanol-d4, J = 6.9 Hz, 1H),
2.19 (s, 6H), 1.19 (d, J = 6.9 Hz, 6H). HRMS (ESI): m/z calcd for
C28H26NO6 (M − H)− 472.1755, found 472.1742.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.5b02665.
1H and 13C NMR spectra of new compounds and
important intermediates (PDF)
Crystal structure of 8 (CIF)
Crystal structure of EBI (CIF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: vssingh@ucdavis.edu.
F
DOI: 10.1021/acs.joc.5b02665
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by The United States Environmental
Protection Agency (Grant RD835550 to P.L.). The authors
also thank The National Science Foundation (Grant DBIO-
722538) for the NMR instrument at the University of
California, Davis, NMR facility.
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