Chiral Dihydrobenzofuran Acids Show Potent Retinoid X Receptor-Nuclear Receptor Related 1 Protein Dimer Activation

Article abstract of DOI:10.1021/acs.jmedchem.5b01702

The nuclear receptor Nurr1 can be activated by RXR via heterodimerization (RXR-Nurr1) and is a promising target for treating neurodegenerative diseases. We herein report the enantioselective synthesis and SAR of sterically constricted benzofurans at RXR. The established SAR, using whole cell functional assays, lead to the full agonist 9a at RXR (pEC₅₀ of 8.2) and RXR-Nurr1. The X-ray structure shows enantiomeric discrimination where 9a optimally addresses the ligand binding pocket of RXR.

Full text of DOI:10.1021/acs.jmedchem.5b01702

Brief Article
pubs.acs.org/jmc
Chiral Dihydrobenzofuran Acids Show Potent Retinoid X Receptor−
Nuclear Receptor Related 1 Protein Dimer Activation
Henrik Sunden,†,⊥ Anja Schafer,†,§ Marcel Scheepstra,§ Seppe Leysen,§ Marcus Malo,† Jian-Nong Ma,‡
́
̈
Ethan S. Burstein,‡ Christian Ottmann,§ Luc Brunsveld,§ and Roger Olsson*,†,‡,∥
†Department of Chemistry and Molecular Biology, Medicinal Chemistry, University of Gothenburg, SE-41296 Gothenburg, Sweden
‡ACADIA Pharmaceuticals Inc., San Diego, California 92130, United States
§Department of Biomedical Engineering and Institute of Complex Molecular Systems, Laboratory of Chemical Biology, Technische
Universiteit Eindhoven, Den Dolech 2, 5612 AZ Eindhoven, The Netherlands
∥Chemical Biology & Therapeutics, Department of Experimental Medical Science, Lund University, Solvegatan 19, BMC DIO,
̈
SE-221 84 Lund, Sweden
S
* Supporting Information
ABSTRACT: The nuclear receptor Nurr1 can be activated by RXR via
heterodimerization (RXR−Nurr1) and is a promising target for treating
neurodegenerative diseases. We herein report the enantioselective
synthesis and SAR of sterically constricted benzofurans at RXR. The
established SAR, using whole cell functional assays, lead to the full agonist
9a at RXR (pEC50 of 8.2) and RXR−Nurr1. The X-ray structure shows
enantiomeric discrimination where 9a optimally addresses the ligand
binding pocket of RXR.
are therefore very important when it comes to developing
disease modifying drugs for neurodegenerative disorders.10 In
this context, the orphan receptor Nurr1 is highly interesting
because of its role in the proper development, function, and
survival of dopaminergic neurons.11−13 Thus, drugs that
activate Nurr1 have disease modifying potential in PD.
INTRODUCTION
■
Modulation of nuclear receptors, including retinoid X receptors
(RXRα, β, and γ), has neuroprotective effects in animal models
of neurodegenerative disorders such as Parkinson’s disease
(PD), Alzheimer’s disease (AD), and multiple sclerosis
(MS).1−5 RXRs are master regulator receptors that hetero-
dimerize with other RXR subtypes and several other nuclear
receptors, e.g., retinoid acid receptors (RARα, β, and γ),6 the
liver X receptors (LXRα and β),7 the peroxisome proliferator-
activated receptors (PPARα, β, and γ),8 and nuclear receptor
related 1 protein (Nurr1) and Nurr77,8 causing specific
physiological responses by modulating gene expression.
Recently, Cramer et al.3 showed that bexarotene, an RXR
agonist approved for cutaneous T-cell lymphoma,9 can improve
overall cognitive function in an AD mouse model. In AD,
misfolded amyloid β (Aβ) peptide accumulates in the brain.
Clearance of Aβ is usually facilitated by apolipoprotein E
(apoE). Expression of apoE is induced by activation of RXR.
When bexarotene was administered to AD mice, Aβ levels
decreased. The activity in the mouse Alzheimer’s models is
believed to be mediated through activation of PPARγ−RXR
and LXR−RXR heterodimers which induces the expression of
apoE and facilitates Aβ clearance, however, contributions by
other RXR heterodimers were not evaluated.3
Nurr1 deficiencies have been linked to familial PD14−17 and
Nurr1 knockout mice show a severe reduction in dopaminergic
neurons and perinatal lethality.18,19 Moreover, activation of
Nurr1 has been shown to inhibit expression of pro-
inflammatory neurotoxic mediators in microglia and astrocytes
and to promote neuronal survival by protecting them from
toxic insults.20 In contrast to the majority of nuclear receptors,
the ligand binding domain of Nurr1 adopts a “closed
conformation”21 and therefore may be difficult to target
directly with small molecule drugs. An indirect way to activate
Nurr1 would be to target its potential permissive hetero-
dimerization partner RXR. Using bioluminescence resonance
energy transfer (BRET2) assays, directly monitoring inter-
actions of Nurr1 with other nuclear receptors, we recently
demonstrated that bexarotene activates RXR−Nurr1 hetero-
dimers, rescues dopamine neurons, and restores behavioral
function in a rat model of PD.1 Synthetic ligands (Figure 1),
PD is caused by the degeneration of dopamine neurons in
the ventral midbrain. Factors that influence neuronal survival
Received: November 1, 2015
Published: January 28, 2016
© 2016 American Chemical Society
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DOI: 10.1021/acs.jmedchem.5b01702
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Journal of Medicinal Chemistry
Brief Article
RESULTS AND DISCUSSION
■
Synthesis. Having outlined the basis for the new scaffold
design, a set of compounds based on an asymmetric synthesis
of quadricyclic dihydrobenzofuran acids was commenced.26
The key intermediate allylic alcohol 4a−e (Scheme 1) was
synthesized starting with an amine-catalyzed iodo-Baylis−
Hillman reaction of α,β-unsaturated ketones, yielding the α-
iodo-carbonyls 2a−c in 56−82%.27,28 Iodo-carbonyls 2a−c
were easily functionalized through a Pd/C-catalyzed Suzuki
reaction to efficiently produce aryl ketones 3a−e in 65−93%
yield.29 To introduce the stereogenic center in an enantiose-
lective fashion, 3a and 3b were subjected to an asymmetric
oxaborolidine-catalyzed reduction to give cyclic allylic alcohols
in high yields and high enantiomeric excess. For example; R-
CBS catalyzed the reduction of ketone 3a and 3b to yield the
(S)-alcohols 4a and 4b in 94% ee and 99% ee, respectively. The
enantiomers ent-4a and ent-4b are available by reductions using
the S-CBS catalyst.30 The racemic allylic alcohols 4c−e were
obtained in excellent yields after reduction of the aryl ketones
3c−e under Luche conditions. Having established a highly
enantioselective and robust method, the cyclic allylic alcohols
were further reacted using a Mitsunobu reaction protocol with
iodophenol 5a to yield the ethers 6a−e (Scheme 2).31 The
reactions proceeded in moderate to good yields, however, due
to a competing reaction path, the formation of the stereogenic
center in 6b did not lead to complete inversion; in reactions
toward 6b, and ent-6b we could detect racemization. In spite of
the racemization, the arylethers could be obtained in their
enantiopure form after a recrystallization in methanol.
Subsequently, a palladium catalyzed intramolecular Mizoroki−
Heck coupling using Pd(OAc)2, PPh3, and Ag2CO3 converted
the ethers 6a−e to the quadricyclic benzofurans 7a−d (and
benzofuran 10, vide infra) in generally high yield and very high
dr in preference for the cis-fused ring system. The cis-fused ring-
system was determined by NOESY NMR experiments.32,33 In a
few cases, the Heck product was isolated together with trace
amounts of a double bond isomer. Interestingly, when
conducting the Mizoroki−Heck reaction with ring ether 6e,
the seven-membered carbocycle 10 is obtained as a 1:1 mixture
of diastereomers. The 1:1 trans/cis ratio of 10 is believed to be
the result of the higher flexibility of the cycloheptyl ring as
compared to cyclohexyl. Furthermore, the intramolecular Heck
reaction proceeds with full conversion of the starting material
to the product and consequently only filtration as a work up is
necessary. In the final step, the double bond was removed by
Pd/C under an atmosphere of hydrogen. In the case of the
benzyl protected compound 7d, this step yielded the free
phenolic compound 14. In all other cases, treatment with
aqueous sodium hydroxide produced the carboxylic acids 9a−c
in moderate to good yield (24−51%) and diastereomers 12 and
13 in 50% combined yield.
Figure 1. Nuclear receptor modulators.
such as HX600,22 HX603,22 and XCT0135908,23 the latter
found by Perlman and co-workers in a high throughput
screening, showed selectivity for RXR−Nurr1 over a broad
range of other RXR dimerization partners but with rather low
potency. Nevertheless, the latter yielded promising results in a
rat neuron PD model;24 the dopaminergic neurons were
selectively protected from degeneration, and the effect was only
seen in Nurr1-expressing neurons. Hence, RXR−Nurr1
agonists may have promise as future PD treatments but
improvements regarding potency and chemotype are urgently
required.
The dihydrobenzofuran scaffold is a recurring motif in
biologically active natural products, e.g., morphine and
galanthamine. We recently took advantage of the asymmetry
of the dihydrobenzofuran scaffold to design a nonplanar,
potent, and highly selective estrogen receptor β (ERβ) agonist
(Figure 1).25 Initial overlays with bexarotene indicated that the
chiral dihydrobenzofuran scaffold could also be used to
synthesize new RXR and RXR−Nurr1 ligands. The two cis-
fused enantiomers of the dihydrobenzofuran scaffold showed
good overlays with two separate low energy conformers of
bexarotene (see graphical abstract). Hence the dihydrobenzo-
furan scaffold presented an opportunity to design a ligand that
mimics only the active bexarotene conformer, avoiding
potential drawbacks originating from the in-active conformer,
e.g., nontarget effects. Of note, RO01 (Figure 1) was designed
on the dihydrobenzofuran enantiomer not matching the active
bexarotene conformer seen in the crystal structure (Figure 2).
Thus, each enantiomer of the dihydrobenzofuran scaffold
shows biological activities at different receptors of the nuclear
hormone receptor superfamily. In this study, we present a
highly potent and selective benzofuran scaffold as a promising
candidate for exploring the biological effects of RXR−Nurr1
modulation.
The heptacyclic diastereomers 12 (cis-fused) and 13 (trans-
fused) were separated on reversed-phase preparative HPLC,
and the stereochemistry was determined by NOESY NMR
experiments.
Pharmacological Evaluation. The dihydrobenzofuran
acids were evaluated in vitro using a luciferase reporter gene
assay and a BRET2 assay,1 using bexarotene as a reference
(Tables 1 and 2). The racemic forms of 9a−c and 12 showed
partial to full agonism with high potencies and efficacies ranging
from 47 to 119% at RXR. In addition, the compounds showed
selectivity; no activation was seen at estrogen receptors or RAR
1233
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Journal of Medicinal Chemistry
Brief Article
Figure 2. (A) Crystal structure of RXR (green) in complex with 9a (shown in orange sticks and surface representation). The cofactor peptide is
shown in cyan. (B) Interactions of 9a in the ligand binding pocket of RXR. The gray mesh shows the electron density for 9a, contoured at 0.61 e/Å3
(1.5σ). RXR residues making hydrophobic and hydrogen bonding interactions with 9a are shown as white and green sticks, respectively. For clarity,
residues 263−279 belonging to helix 3 are not shown. Of these, I268, A271, A272, and Q275 also make hydrophobic contacts with 9a. A 2D plot of
the interactions can be found in the SI. (C) Structural superposition of RXR in complex with bexarotene (purple) and 9a (orange) in the LBD of
RXR. The RXR molecules are shown in different shades of green. The arrow marks the structural difference between the ligands. (D) Overlay of 9a
(orange) and its enantiomer ent-9a (red) in the LBD of RXR. The arrow indicates how the enantiomer would sterically clash with RXR residue
A272.
(data not shown). The racemic 9a showed partial agonism
displaying a pEC50 value of 7.6 and efficacy of 65% at RXR
(Table 1, entry 2). When the alkyl ring is expanded from five to
six carbons (rac-9b), the efficacy increases to 96% (Table 1,
entry 3). In comparison, the ortho-fluorocarboxylic acid (rac-
9c) (Table 1, entry 4) has a similar activity as the
nonsubstituted rac-9b, indicating that modulating the carbox-
ylic acid by adding an ortho-fluoro had no beneficial effects on
activity. In addition, rac-9b and rac-9c are equipotent with a
pEC50 value of 7.3. The cis-fused seven-membered benzofuran
12 (Table 1, entry 5) displayed a slightly lower potency and a
severely reduced efficacy as compared to the other ring
congeners, indicating that the bulk of the ring may be of less
importance for potency but more important for efficacy
(compare to rac-9a). A reason for this observation might be
the increased flexibility of the seven-membered ring. Con-
versely, the trans-fused diastereomer 13 was inactive (Table 1,
entry 6), indicating that the molecular topology that is
determined by the stereochemistry at the two stereogenic
centers is critical for the activity of the benzofuran scaffold.
Thus, the enantiomers of the two most potent compounds
were investigated separately (Table 1, entries 8−11). The
pentacyclic enantiomer 9a turned out to be a full agonist and
the most potent compound in the series, with a pEC50 value of
8.2 and efficacy of 119%. By contrast, ent-9a displays an over
100-fold drop in potency, with a pEC50 value of 6.0. This trend
is accentuated in case of the six-membered ring where the most
potent enantiomer, 9b, has a pEC50 value of 7.7 (Table 1, entry
10) and the ent-enantiomer (ent-9b) is inactive (Table 1, entry
11). The fact that one of the enantiomers was inactive is not
surprising considering the built-in rigidity of the ligand, causing
the enantiomers to have a drastically different molecular
topology (vide infra crystallography). Replacing the carboxylic
acid with a hydroxyl group led to a completely inactive
compound (14), underlining the importance of the carboxylic
acid for RXR activity.
A BRET2 assay was used to monitor the interactions of the
enantiomers of the most active compounds in the dihydro-
benzofuran series with RXR−RXR and RXR−Nurr1 hetero-
dimers (Table 2). The pentacyclic dihydrobenzofuran 9a
displayed a 3-fold biased interaction with RXR−Nurr1, with a
pEC50 of 7.9 and 111% efficacy, as compared to RXR−RXR
pEC50 of 7.6 and 85% efficacy. ent-9a was 100-fold less potent
in recruiting the RXR−Nurr1 heterodimer and not active at
RXR−RXR, in accordance with the trends seen in the reporter
gene assay. The six-membered dihydrobenzofuran 9b displayed
a similar potency for both RXR−Nurr1 and RXR−RXR dimers
but is higher in efficacy for RXR−Nurr1. Again, the antipode
ent-9b was almost inactive.
1234
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Journal of Medicinal Chemistry
Brief Article
a
a
Scheme 1. Synthesis of 4a−e
Scheme 2. Synthesis of 9a−c, 12, 13, and 14
v
a
(a) I2, DABCO, 2a 56%, 2b 82%. (b) 2c: I2, pyridine, 59%. (c) Pd/C,
Na2CO3, ArB(OH)2, 3a 68%, 3b 93%, 3c 85%, 3d 91%, 3e 65%. (d)
(R)-CBS, BMS, 4a 52%, 94% ee, 4b 77%, 99% ee. (e) CeCl3·7H2O,
NaBH4, 4c 95%, 4d 77%, 4e 74%.
Structural Evaluation. To elucidate the structural basis for
the highly potent RXR interaction of the dihydrobenzofuran
compounds, we set up crystal screens with the most potent
compound 9a, RXRα and a TIF2 NR2 cofactor peptide (686-
KHKILHRLLQDSS-698; for details, see Supporting Informa-
tion (SI)).34 After five days, crystals were observed and directly
flash-cooled in liquid nitrogen for X-ray crystallographic
structure determination. 9a fills the classical ligand binding
pocket, with helix 12 adopting the typical agonist conformation
which facilitates binding of the coactivator peptide (Figure 2A).
The clear electron density for enantiomer 9a in the crystal
structure reveals how the molecule is accommodated in the
ligand binding pocket. Extensive hydrophobic interactions are
formed with residues located in RXRα helices 3, 5, 10, and 11,
among them Val 342, Val 349, Ile 310, Ile 268, and Leu 436
(Figure 2B and SI, Figure S1). The carboxylic acid functional
group of 9a creates a charge-assisted hydrogen bond with Arg
316 and a hydrogen bond with the backbone amide nitrogen of
Ala 327. These polar interactions are important for binding as
evidenced by the complete inactivity of hydroxyl group
congener rac-14. When 9a is superimposed with bexarotene
for comparison, the two molecules overlay nicely (Figure 2C).
A small but significant difference is that the five-membered
carbocycle occupies slightly more space than the ethylene
group, filling a cavity formed by amino acids of helix 5. The
cavity is most likely more spacious, as the SAR shows that both
six- and seven-membered rings also can be accommodated here.
Moreover, the X-ray structure provides a structural basis for the
observed enantioselectivity of the RXR agonist. Accordingly,
benzofuran ent-9a encounters a steric clash with amino acid Ala
272 from helix 2 and fails to establish the hydrogen bond with
the backbone nitrogen in Ala 327 and the salt bridge with Arg
316 (Figure 2D), explaining why ent-9a and ent-9b show little
or no activity in the biological testing.
a
(a) DIAD, PPh3, toluene, 0 °C to rt; 6a 100%, 98% ee; 6b 77%, >
99% ee; 6c 44%; 6d 49%, 6e 43%. (b) Pd(OAc)2, PPh3, Ag2CO3, 7a−
d crude yields 94−100%. (c) Pd/C, H2, 14 85%. (d) NaOH (9 M aq)
9a 51%, 9b 24%, 9c 32%, 12 and 13 50% yield (both diastereomers).
Table 1. Biological Evaluation: Luciferase Reporter Gene
Assay for RXR Binding
a
RXR
entry
compd
pEC50
efficacy (%)
1
bexarotene
rac-9a
rac-9b
rac-9c
rac-12
rac-13
rac-14
9a
7.5 0.1
7.6 0.1
7.3 0.1
7.3 0.1
6.9 0.1
NA
101
65
5
2
7
3
96
6
4
88 22
47
5
7
6
7
NA
8
8.2 0.1
6.0 0.1
7.7 0.1
NA
119 16
59
94 20
9
ent-9a
9b
4
10
11
ent-9b
a
NA = not active. pEC50 is the negative logarithm of EC50 in molar.
Agonist efficacies were compared to that of bexarotene (100%). Values
represent the mean SD of three or more independent experiments
(n ≥ 3).
CONCLUSION
■
In conclusion, we have presented an efficient seven-step
asymmetric synthesis for a new class of conformationally rigid
RXR agonists. A focused library of highly potent compounds
has been synthesized and pharmacologically evaluated using
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Journal of Medicinal Chemistry
Brief Article
62.45, 41.11, 35.89, 35.21, 35.12, 34.63, 34.06, 32.27, 32.13, 32.02,
32.00, 29.70, 24.83; m/z (ESI) 391 ([M + H]+, 100%), 279 (9), 186
(5); [α]2D0 = +16.1 (c 0.3, methanol). HPLC: purity, 97.1%; retention
time, 15.85 min. HRMS (ESI) calcd for C26H30O3 [M + H]+,
391.2267; found, 391.2243 (ent-9a); [α]2D0 = −20.8 (c 0.8, methanol).
HPLC: purity:, 97.4%; retention time, 15.85 min
Table 2. Biological Evaluation Using BRET2 Assays for
RXR−Nurr1 and RXR−RXR Dimers
a
RXR−Nurr1
pEC50 efficacy (%)
100
RXR−RXR
pEC50 efficacy (%)
entry
compd
1
2
3
4
5
bexarotene
9a
8.0 0.1
7.9 0.1
6.3 0.1
7.9 0.3
<5.5
3
7.5 0.2
7.6 0.2
100
85
7
4-[(11S,16R)-4,4,7,7-Tetramethyl-17-oxatetracyclo
[8.7.0.03,8.011,16]heptadeca-1(10),2,8-trien-11-yl]benzoic Acid
111 12
49 13
3
1
(9b). H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 8.6 Hz, 2H), 7.50
ent-9a
9b
NA
80
NA
(d, J = 8.6 Hz, 2H), 6.80 (s, 1H), 6.75 (s, 1H), 4.81 (t, J = 3.9 Hz,
1H), 2.38−2.26 (m, 1H), 2.07−1.98 (m, 1H), 1.92−1.83 (m, 1H),
1.75−1.49 (m, 9H), 1.27 (s, 6H), 1.19 (s, 3H), 1.13 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 169.70, 156.58, 151.46, 145.12, 137.58, 134.78,
130.06, 127.91, 126.99, 121.10, 107.30, 89.15, 52.12, 35.23, 35.11,
34.66, 34.11, 33.97, 32.23, 32.05, 32.01, 31.99, 26.49, 21.72, 19.78;
[α]2D0 = +16.1 (c 0.9, CHCl3); retention time HPLC (min), 16.53.
Purity: 95.0%. HRMS (ESI) calcd for C27H33O3 [M + H]+, 405.2423;
found, 405.2402 (ent-9b); [α]2D0 = −13.6 (c 0.5, CHCl3); retention
time HPLC (min), 16.53. Purity: 95.0%, 3 mg, 11% yield.
96
92
8
1
7.8 0.7
4
ent-9b
a
Pharmacological profiling in BRET2 assays. BRET2 assays performed
as described by Tan et al.35 using RXR and Nurr1 receptors tagged
with GFP and Renilla. luciferase as described in the methods. NA =
not active. pEC50 is the negative logarithm of EC50 in molar. Agonist
efficacies were compared to that of bexarotene (100%). Values
represent the mean SD of three or more independent experiments
(n ≥ 3)
2-Fluoro-4-{4,4,7,7-tetramethyl-17-oxatetracyclo
[8.7.0.03,8.011,16]]heptadeca-1(10),2,8-trien-11-yl}benzoic Acid
1
whole cell functional assays resulting in the finding of full
agonist 9a with pEC50 of 8.2 at RXR and RXR−Nurr1. The
established SAR showed that the five- and six-membered
benzofurans are more potent than the seven-membered
congeners and that the enantiomers 9a and 9b are more active
as compared to ent-9a and ent-9b. The crystal structural data,
which show that 9a nicely addresses the key hydrophobic and
polar interaction potential in the RXRα pocket, supports this
trend. The sterically constricted dihydrobenzofuran scaffold
imparts exactly the right molecular topology for activating
RXRα, as also addressed by bexarotene, but additionally
providing a stable framework for specific molecular optimiza-
tions. Given the specific molecular topology of the benzofuran
scaffold, this series of compounds provides a new platform for
further RXR−Nurr1 exploration.
(rac-9c). H NMR (400 MHz, CDCl3) δ 7.97 (t, J = 8.1 Hz, 1H),
7.26 (dd, J = 8.3, 1.8 Hz, 1H), 7.19 (dd, J = 12.9, 1.7 Hz, 1H), 6.80 (s,
1H), 6.76 (s, 1H), 4.77 (t, J = 4.1 Hz, 1H), 2.25 (dd, J = 14.1, 5.6 Hz,
1H), 1.99 (dd, J = 11.5, 6.0 Hz, 1H), 1.95−1.82 (m, 1H), 1.79−1.49
(m, 9H), 1.27 (s, 6H), 1.20 (s, 3H), 1.15 (s, 3H). 13C NMR (101
MHz, CDCl3) δ 167.90 (d, J = 6.5 Hz), 162.43 (d, J = 260.8 Hz),
156.60 (s), 154.75 (d, J = 7.9 Hz), 145.52 (s), 137.82 (s), 133.84 (s),
132.47 (s), 123.48 (d, J = 3.2 Hz), 121.03 (s), 116.46 (d, J = 23.5 Hz),
115.56−114.54 (m), 107.52 (s), 88.89 (s), 52.24 (s), 35.23 (s), 35.12
(s), 34.71 (s), 34.14 (s), 33.71 (s), 32.26 (s), 32.08 (s), 32.01 (s),
32.00 (s), 26.59 (s), 21.53 (s), 19.67 (s). 19F NMR (376 MHz,
CDCl3) δ −75.80 (s, ethyl trifluoroacetate), −107.75 (dd, J = 12.8, 7.9
Hz). Retention time HPLC (min): 16.18. Purity: 92.4%. HRMS (ESI)
calcd for C27H31FO3 [M + H]+, 423.2329; found, 423.2307
4-{13,13,16,16-Tetramethyl-9-oxatetracyclo[8.8.0.02,8.012,17]-
octadeca-1(18),10,12(17)-trien-2-yl}benzoic Acid (12 and 13).
12: 1H NMR (400 MHz, CDCl3) δ 8.01 (d, J = 8.7 Hz, 2H), 7.40 (d, J
= 8.7 Hz, 2H), 6.88 (s, 1H), 6.77 (s, 1H), 4.89 (dd, J = 7.3, 1.8 Hz,
1H), 2.41 (dd, J = 14.5, 10.3 Hz, 1H), 2.21 (m, 1H), 2.11 (dd, J =
14.42, 8.30 Hz, 1H), 1.91 (m, 1H), 1.64 (m, 8H), 1.42 (m, 1H), 1.29
(s, 3H), 1.27 (s, 3H), 1.25 (m, 1H), 1.22 (s, 3H), 1.20 (s, 3H). 13C
NMR (101 MHz; CDCl3) δ 170.46, 157.08, 155.61, 145.63, 137.08,
130.36, 126.82, 126.47, 122.69, 106.22, 93.69, 57.88, 38.19, 35.24,
35.17, 34.67, 34.05, 32.31, 32.16, 32.08, 32.01, 31.81, 31.04, 29.71,
25.27, 23.43; m/z (ESI) 419 ([M + H]+, 100%), 282 (40). Retention
time HPLC (min): 17.99. Purity: 93.5%. HRMS (ESI) calcd for
EXPERIMENTAL SECTION
■
General Information. Chemicals and solvents were purchased
from Sigma-Aldrich. 1H and 13C NMR spectra were recorded at 25 °C
in CDCl3 on a Varian 400 MHz spectrometer equipped with a Varian
OneNMRProbe with a proton observe frequency of 399.95 MHz.
Chemical shifts are reported in ppm with the solvent residual peak as
internal standard. 19F NMR was measured with ethyl trifluoroacetate
as internal standard (−75.8 ppm). Optical rotation was measured on a
PerkinElmer polarimeter 341 LC. Gas chromatography/mass spec-
trometry analyses were performed on a Varian Saturn 2000 GCMS
with a Supelco SLB-5 ms fused silica capillary column using helium as
carrier gas at an injector temperature of 300 °C. Temperature
program: 70−330 °C (12 °C/min) with 4 min hold time. The MS
detector consisted of an ion trap with 70 eV ionization. Purity was
measured at λ = 254 nm on a Waters 2690/996 photodiode array
detector using HPLC-grade solvents; water 0.1% TFA/acetonitrile
0.1% TFA with an Atlantis T3 5 μm, 4.6 mm × 250 mm column. The
purity of the target compounds was determined to be at least 95%.
The only exception was rac-9c and 12, which were determined to be
92.4% and 93.5% pure, respectively. Preparative HPLC was executed
on a Waters 600/2487 dual λ absorbance detector using HPLC-grade
solvents; water 0.1% TFA/acetonitrile 0.1% TFA with an Atlantis prep
T3 5 μm, 19 mm × 250 mm column. HRMS was measured on a
Thermo LTQ-OrbitrapXL nano-ES in positive ion mode.
1
C28H34O3 [M + H]+, 419.2580; found, 419.2600. 13: H NMR (400
MHz, CDCl3) δ 7.97 (d, J = 8.6 Hz, 2H), 7.56 (d, J = 8.6 Hz, 2H),
6.80 (s, 1H), 6.78 (s, 1H), 4.88 (dd, J = 11.5, 6.6 Hz, 1H), 3.02 (m,
1H), 2.24 (m, 2H), 1.89 (m, 3H), 1.59 (m, 6H), 1.47 (m, 1H), 1.25 (s,
3H), 1.21 (s, 3H), 1.18 (s, 3H), 1.04 (s, 3H), 0.90 (m, 1H). 13C NMR
(101 MHz; CDCl3) δ 171.41, 155.88, 149.44, 145.02, 137.78, 136.84,
130.23, 126.85, 126.40, 120.54, 107.76, 92.46, 54.40, 35.15, 35.01,
34.61, 34.07, 32.38, 32.03, 31.89, 31.77, 29.70, 27.09, 26.31, 25.02,
23.15; m/z (ESI) 419 ([M + H]+, 100%), 254 (48). Retention time
HPLC (min): 16.75. Purity: 98.9%. HRMS (ESI) calcd for C28H34O3
[M + H]+, 419.2580; found, 419.2597.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01702.
4-[(11S,15R)-4,4,7,7-Tetramethyl-16-oxatetracyclo
[8.6.0.03,8.011,15]hexadeca-1(10),2,8-trien-11-yl]benzoic Acid
1
(9a). H NMR (400 MHz, CDCl3) δ 8.05 (d, J = 8.4 Hz, 2H), 7.42
(d, J = 8.5 Hz, 2H), 6.87 (s, 1H), 6.71 (s, 1H), 5.14 (d, J = 5.8 Hz,
1H), 2.33 (dd, J = 9.47, 4.25 Hz, 2H), 2.15 (dd, J = 13.58, 6.22 Hz,
1H), 1.91 (m, 2H), 1.65 (m, 5H), 1.26 (s, 3H), 1.25 (s, 3H), 1.21 (s,
3H), 1.13 (s, 3H). 13C NMR (101 MHz; CDCl3) δ 171.82, 158.21,
153.02, 145.47, 137.44, 130.47, 130.36, 126.75, 122.23, 106.02, 96.19,
Detailed experimental procedures, analytical data (NMR,
MS, HRMS, optical rotation, HPLC methods), crystal-
lography (PDF)
Molecular formula strings (CSV)
1236
DOI: 10.1021/acs.jmedchem.5b01702
J. Med. Chem. 2016, 59, 1232−1238

Journal of Medicinal Chemistry
Brief Article
(8) Bardot, O.; Aldridge, T. C.; Latruffe, N.; Green, S. PPAR-RXR
Heterodimer activates a peroxisome proliferator response element
upstream of the bifunctional enzyme gene. Biochem. Biophys. Res.
Commun. 1993, 192, 37−45.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +46768874217. E-mail: roger.olsson@med.lu.se.
(9) Duvic, M.; Martin, A. G.; Kim, Y.; Olsen, E.; Wood, G. S.;
Crowley, C. A.; Yocum, R. C.; Aeling, J.; Bagot, M.; Bernard, P.; Bran,
E. L.; Breneman, D.; Burg, G.; Diez, L. I.; Duvic, M.; el-Azhary, R.;
Fivenson, D.; Foss, F.; Heald, P.; Joly, P.; Kim, Y.; Martin, A. G.;
Mehlmauer, M.; Miller, W.; Muglia, J.; Myskowski, P.; Olsen, E.;
Pluzanska, A.; Prince, H. M.; Rozkiewicz, J.; Tharp, M. D.; Thiers, B.;
Washenik, K.; Wood, G.; Zone, J. Phase 2 and 3 clinical trial of oral
bexarotene (targretin capsules) for the treatment of refractory or
persistent early-stage cutaneous t-cell lymphoma. Arch. Dermatol.
2001, 137, 581−593.
Present Address
⊥Chalmers University of Technology, Chemistry and Chemical
Engineering, Kemivagen 10, 412 96, Gothenburg.
̈
Author Contributions
The manuscript was written through contributions of all
authors./All authors have given approval to the final version of
the manuscript. H.S. and A.S. contributed equally.
Notes
The authors declare no competing financial interest.
(10) Lang, A. E. Clinical trials of disease-modifying therapies for
neurodegenerative diseases: the challenges and the future. Nat. Med.
2010, 16, 1223−1226.
ACKNOWLEDGMENTS
■
(11) Saucedo-Cardenas, O.; Conneely, O. Comparative distribution
of NURR1 and NUR77 nuclear receptors in the mouse central
nervous system. J. Mol. Neurosci. 1996, 7, 51−63.
The authors thank the Swedish Research Counsil (VR),
Michael J. Fox Foundation for Parkinson’s Research, The
Netherlands Organization for Scientific Research via Gravity
program 024.001.035 and ECHO grant 711011017 and Marie
Curie Action PIAPP-GA-2011-286418 14-3-3Stabs for financial
support.
́
(12) Wallen, Å.; Zetterstrom, R. H.; Solomin, L.; Arvidsson, M.;
̈
Olson, L.; Perlmann, T. Fate of mesencephalic AHD2-expressing
dopamine progenitor cells in Nurr1 mutant mice. Exp. Cell Res. 1999,
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development of dopaminergic neurons and Parkinson’s disease. Prog.
Neurobiol. (Oxford, U. K.) 2005, 77, 128−138.
ABBREVIATIONS USED
■
RXR, retinoid X receptor; PD, Parkinson’s disease; AD,
Alzheimer’s diseases; MS, multiple sclerosis; RAR, retinoid
acid receptors; LXR, liver X receptors; PPAR, peroxisome
proliferator-activated receptors; Nurr1, nuclear receptor related
1 protein; PEG, polyethylene glycol; Tris, Tris(hydroxymeth-
yl)-aminomethan; NiCo21, competent Escherischia coli strain
optimized for nickel columns; NR, nuclear receptor; LBD,
ligand binding domain; MME, monomethyl ether; TIF,
transcriptional intermediary factor; NucHRs, nuclear hormone
receptors; BRET, bioluminescence energy transfer; SAR,
structure−activity relationships; ER, estrogen receptor
(14) Le, W.-d.; Xu, P.; Jankovic, J.; Jiang, H.; Appel, S. H.; Smith, R.
G.; Vassilatis, D. K. Mutations in NR4A2 associated with familial
Parkinson disease. Nat. Genet. 2002, 33, 85−89.
(15) Le, W.; Pan, T.; Huang, M.; Xu, P.; Xie, W.; Zhu, W.; Zhang, X.;
Deng, H.; Jankovic, J. Decreased NURR1 gene expression in patients
with Parkinson’s disease. J. Neurol. Sci. 2008, 273, 29−33.
(16) Sleiman, P. M. A.; Healy, D. G.; Muqit, M. M. K.; Yang, Y. X.;
Van Der Brug, M.; Holton, J. L.; Revesz, T.; Quinn, N. P.; Bhatia, K.;
Diss, J. K. J.; Lees, A. J.; Cookson, M. R.; Latchman, D. S.; Wood, N.
W. Characterisation of a novel NR4A2 mutation in Parkinson’s disease
brain. Neurosci. Lett. 2009, 457, 75−79.
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Kordower, J. H. Nurr1 in Parkinson’s disease and related disorders. J.
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