Stilbenes as κ-selective, non-nitrogenous opioid receptor antagonists

Article abstract of DOI:10.1021/np4009046

The natural stilbene pawhuskin A has been shown to function as an opioid receptor antagonist, with preferential binding to the κ receptor. This finding encouraged assembly of a set of analogues to probe the importance of key structural features. Assays on these compounds determined that one (compound 29) shows potent opioid receptor binding activity and significantly improved selectivity for the κ receptor. These studies begin to illuminate the structural features of these non-nitrogenous opioid receptor antagonists that are required for activity.

Full text of DOI:10.1021/np4009046

Article
pubs.acs.org/jnp
Stilbenes as κ‑Selective, Non-nitrogenous Opioid Receptor
Antagonists
Alyssa M. Hartung,† John A. Beutler,‡ Hernan A. Navarro,§ David F. Wiemer,† and Jeffrey D. Neighbors*,†
́
†Department of Chemistry, The University of Iowa, Chemistry Building, Iowa City, Iowa 52242, United States
‡Molecular Targets Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, United States
§Discovery Sciences, RTI International, Research Triangle Park, North Carolina 27709, United States
S
* Supporting Information
ABSTRACT: The natural stilbene pawhuskin A has been
shown to function as an opioid receptor antagonist, with
preferential binding to the κ receptor. This finding encouraged
assembly of a set of analogues to probe the importance of key
structural features. Assays on these compounds determined
that one (compound 29) shows potent opioid receptor
binding activity and significantly improved selectivity for the κ
receptor. These studies begin to illuminate the structural
features of these non-nitrogenous opioid receptor antagonists
that are required for activity.
n 2004 Belofsky and co-workers reported a small set of
categorize the opioid receptor binding of dioflorin have not yet
1
Iprenylated stilbenes that they named the pawhuskins. This been reported. Bioassays with a series of other natural
flavonoids including catechin (5) and hesperetin (6) have
been conducted and demonstrate that this scaffold may have
considerable potential for development of opioid receptor
ligands.22 Other structural subtypes with opioid-binding activity
are becoming more common,23−25 including stilbenoids more
reminiscent of the pawhuskins such as resveratrol (7)26,27 and,
more recently, chlorophorin (8).28
family of compounds, exemplified by pawhuskins A (1) and C
(2), was isolated from the common North American purple
prairie clover (Dalea purpurea) collected near Pawhuska,
Oklahoma. Extracts of this plant reportedly have been made
into teas and used by Native American peoples as a
prophylactic and for treatment of various ailments.2 Belofsky’s
findings support this ethnomedical use, since the pawhuskins
were shown to modulate opioid receptors by displacement of a
nonselective radioactive antagonist in rat brain striatal tissue.1
Pawhuskin A was the most potent member of the family,
making it one of a small group of non-nitrogenous compounds
with effects on the opiate receptor system. As part of an
ongoing interest in natural prenylated stilbenes,3,4 we under-
took studies to elucidate the character and receptor subtype
selectivity of opioid modulation by pawhuskins. This effort
already has led to the synthesis of both pawhuskins A5 and C,6
and here we report the results of our further studies on this
class of compounds.
Several non-nitrogenous opioid receptor modulators have
been isolated from natural sources. The most studied
compound is salvinorin A (3), a potent hallucinogen isolated
from Salvia divinorum.7 Salvinorin A has been shown to be a κ-
opioid (KOP) receptor agonist, and KOP receptor ligands have
become of interest with respect to studies of addiction and
other disorders.8 Two total syntheses of salvinorin A have been
reported,9,10 but modifications of the isolated natural product
have driven more extensive structure−activity studies.11−17 The
non-nitrogenous compound dioflorin (4), a prenylated
flavonoid, was isolated from the Brazilian vine Dioclea
grandiflora through activity-guided fractionation18−20 and
shown to have analgesic activity.21 More extensive efforts to
Salvinorin A (3) has been shown to be a functional agonist.
Dioflorin (4) and other isolates of Dioclea display morphine-
like analgesia that is inhibited by naloxone, a nonspecific opioid
receptor antagonist, so they are presumably agonists as well.20
While the flavan-3-ol catechin (5) had good activity as an
antagonist at the KOP receptor (Ke = 320 nM), the flavanone
hesperetin (6) had no activity at the μ, δ, or κ receptors.22 The
work of Sobolev and co-workers on peanut phytoalexins such as
stilbene 8 determined the selectivity of these compounds
against each opioid receptor, but these compounds have not yet
been fully characterized using functional assays.28 Here we
report the opioid receptor binding affinity and selectivity of
pawhuskin A using a functional assay based on [35S]GTP-γ-S
binding. We also report initial results of structure−activity
relationship studies, which begin to illuminate the significance
of the phenols and the prenyl group for activity.
RESULTS AND DISCUSSION
■
We began this exploration of pawhuskin opioid activity by
probing the selectivity of pawhuskin A for human κ (KOP), μ
(MOP), and δ (DOP) receptors. Even at a 10 μM
Received: October 25, 2013
Published: January 23, 2014
© 2014 American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Structures of some non-nitrogenous opioid receptor modulators.
concentration, pawhuskin A was found to have no intrinsic
agonist activity at these receptors. However, further testing
showed antagonist activity at all three of the opioid receptor
subtypes. Furthermore, pawhuskin A caused a rightward shift in
the agonist concentration response curve, and its antagonism
was surmountable, suggesting a competitive mode of
antagonism (Figure 2).29 Pawhuskin A is modestly selective
for the κ receptor, with a Ke of 203 nM (δ/κ = 14.5, μ/κ = 2.9).
Pawhuskin C (2) also displayed some antagonist activity at the
KOP receptor, but was much less potent than compound 1.
While the natural product salvinorin A and many of its
analogues are KOP receptor agonists, there are only limited
examples of non-nitrogenous KOP receptor antagonists
including some flavanoids.22 Pawhuskin A rivals the potency
of the flavonoids, although catechin (5) displayed higher
selectivity versus the other opioid receptors (μ/κ > 31).
However improved KOP receptor selectivity might be
uncovered by a synthetic exploration involving the pawhuskin’s
stilbene scaffold. Synthetic efforts along these lines are
encouraged by the recent interest in KOP receptor antagonists
as potential treatments for stimulant abuse. Such agents might
be of particular value as potential preventatives for relapse.
While there has been some interest in using KOP agonists for
treatment of substance abuse, compounds such as salvinorin A
have been accompanied by serious side effects including potent
hallucinogen activity. There is a significant relationship between
relapse to stimulant abuse and stress.30 Indeed encounters with
stressors, and even images that induce stress, have been shown
to induce craving in stimulant abusers.31,32 The potent KOP
receptor antagonist JDTic33 has been shown to block stress-
induced cocaine seeking behavior and also has demonstrated
antidepressant-like activity.34 This result was confirmed and
expanded to show that pretreatment with the κ-opioid
antagonist arodyn prevented stress-related induction of
cocaine-conditioned place preference,35 which further height-
ens interest in κ-selective antagonists.
Our approach to exploration of the structure−activity
relationships of pawhuskin A analogues took advantage of a
core strategy used in the synthesis of other natural stilbenes
(Figure 3).36−38 A disconnection of the central olefinic moiety
(9) through a Horner−Wadsworth−Emmons transform allows
Figure 2. Representative graph of the antagonist activity of pawhuskin
A in the KOP receptor affinity assay. Each data point represents the
mean and SEM of duplicate samples.
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Figure 3. Synthetic strategy for pawhuskin analogues.
Scheme 1. Synthesis of the Aldehydes 14 and 16
choice of phosphonate coupling partners such as 10 and
aldehydes such as 11, although the reversed pairing is also
viable.39,40 This permits maximum convergence and provides
for divergence through condensations of one aldehyde with
several phosphonates or one phosphonate with several
aldehydes.41 To begin exploration of the pharmacophore of
pawhuskin A and the essential binding motifs for κ-selective
antagonist activity, we undertook syntheses aimed at
preparation of a small set of analogues through this strategy.
Phenolic H-bonding is important to the KOP receptor
selectivity of the antagonist JDTic and other members of the
phenylpiperidine class of opioid receptor modulators.42
Furthermore, as in past studies of salvinorin A,43 the lack of
a readily ionizable group that would form salt bridges with an
opioid receptor suggested that attention should be directed at
the H-bonding groups of pawhuskin A. Thus, we chose to
prepare various methylated analogues to assess the importance
of H-bond donation from the various hydroxy groups without a
significant change in electron donation.
To allow efficient preparation of several analogues, as well as
synthesis of regiospecifically methylated materials, the per-
methylated pawhuskin A analogue was pursued through
preparation of both coupling partners aldehyde 14 (Scheme
1) and phosphonate 19 (Scheme 2) rather than methylation of
the natural product. To access compound 14, halogen metal
exchange was carried out on the known bromide 12.44,45
Treatment of the lithiated arene with geranyl bromide afforded
the THP ether 13. Hydrolysis of the acetal protecting group
and oxidation of the resulting benzylic alcohol with MnO2 gave
aldehyde 14 in satisfactory yield. The alcohol 15 is known from
our synthesis of pawhuskin A,5 and oxidation of that benzylic
alcohol gave the methoxymethyl (MOM)-protected aldehyde
16.
Scheme 2. Synthesis of Phosphonates 18 and 19
To prepare the complementary phosphonate 19, the known
aldehyde 1746 was reduced to the corresponding alcohol, and
the alcohol was treated with mesyl chloride and Et3N, then
LiBr, and finally allowed to react with triethyl phosphite to
obtain the phosphonate 18. After hydrolysis of the MOM
protecting group, standard reaction with MeI and base afforded
the dimethylated phosphonate 19.
Coupling of aldehyde 14 and phosphonate 19 via Horner−
Wadsworth−Emmons condensation afforded the fully methy-
lated pawhuskin A analogue 20 in good yield (Scheme 3). With
this compound in hand, we employed our small library of
readily available phosphonates of type 10 to synthesize
additional pawhuskin analogues. Thus, coupling of aldehyde
14 with known phosphonate 2147 afforded the stilbene 22,
which upon deprotection gave stilbene 23. Exhaustive
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Scheme 3. Synthesis of Pawhuskin A Analogues 20, 23, 24, 26, 29, and 31
methylation of compound 23 then gave the permethylated
analogue 24. Condensation of compound 16 with phosphonate
21,38 followed by hydrolysis of the protecting groups in the
resulting stilbene, gave the free phenolic pawhuskin A analogue
26. To reposition the prenyl group so that it is isomeric to
pawhuskin A (1), phosphonate 27 was prepared from the
corresponding benzylic alcohol under standard conditions.
That prenylated benzylic alcohol could be prepared from
bromovanillin through reactions parallel to those reported in a
schweinfurthin synthesis.39 Condensation of phosphonate 27
with aldehyde 14 gave stilbene 28, and hydrolysis of the MOM
groups gave the dimethylated analogue 29. Finally, condensa-
tion of aldehyde 14 with phosphonate 18 gave the selectively
trimethylated pawhuskin A derivative 30. Hydrolysis of the
MOM acetal gave the specific phenol 31. This compound was
methylated to provide permethylated pawhuskin A (20) via a
different route.
Scheme 4. Pawhuskin C Analogues
Because pawhuskin C (2) showed activity,1 we tested several
analogues of this chemotype. This set includes the natural
product schweinfurthin J (32, Scheme 4), which was isolated
from the African plant Macaranga schweinfurthii48,49 and can be
viewed as lacking one phenolic hydroxy group and bearing a
farnesyl side chain in relation to pawhuskin C. We then used
the known phosphonate 3350,51 to access the prenylated
pawhuskin C analogue 37 and the natural product trans-
arachidin-2 (39). Condensation of phosphonate 33 with the
known aldehydes 34 and 3552 gave the protected stilbenes 36
and 38, respectively. Hydrolysis of the four MOM acetals of
compound 36 gave stilbene 37. Use of the MOM protecting
group for all the hydroxy groups of compound 38 allows for
deprotection to stilbene 39 in a single step, which is more
efficient than the previous synthesis.53
Of the various analogues studied for binding to opioid
receptors, only the pawhuskin A analogue 29 and schweinfur-
thin J (32) demonstrated appreciable activity (Table 1).
Schweinfurthin J with a 3 μM Ke for the MOP receptor and
limited selectivity (δ/κ = 0.67, μ/κ = 0.33) is the only stilbene
we have studied that shows selectivity for the μ-opioid receptor.
Interestingly, schweinfurthin J is also closely related to
chlorophorin (8), which was shown by Sobolev and co-workers
to lower agonist binding to the κ and δ receptors to an equal
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efforts to improve these molecules via synthesis. The isomeric
pawhuskin A analogue 29 exhibited greater affinity and
selectivity for the KOP receptor than pawhuskin A itself,
indicating that the shikimate-derived ring is a key for κ-opioid
receptor binding. Compound 29 shows significantly greater
selectivity for the KOP than PF-04455242, which was advanced
to phase 1 clinical trials for alcohol dependency, albeit with
significantly lower potency (∼150 nM vs 3 nM).54,55
Thus far, none of the new compounds reported here have
shown any agonist activity. While the study of KOP agonists for
treatment of pain and addiction has been moving forward, their
potential may be limited by side effects such as hallucinations
and dysphoria. This makes the discovery of additional classes of
KOP antagonists appealing. Therefore, this stilbene scaffold
may present new opportunities for the discovery of compounds
with utility in the treatment of addiction and depression.
Table 1. Apparent Affinity of Compounds Tested
apparent affinity of competitive
antagonists (Ke) in μM
selectivity
compd
KOP
0.20
>10
DOP
MOP
0.57
>10
δ/κ
μ/κ
Paw A (1)
2.9
>10
>10
>10
>10
>10
>10
>10
6
14.5
2.9
Paw C (2)
20
23
24
26
29
31
32
37
39
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
3
0.15
>67
0.67
>67
0.33
>10
9
>10
>10
>10
>10
>10
>10
extent but to have no substantial effect on the binding of
agonists to the μ-opioid receptor.28
EXPERIMENTAL SECTION
■
Of greater interest is analogue 29, which showed better
binding affinity to the κ receptor than pawhuskin A and also
demonstrated dramatically improved selectivity (δ/κ at least 4-
fold larger and μ/κ at least 20-fold larger for compound 29 than
for pawhuskin A). Indeed we could not find antagonist activity
at the μ or δ receptors for compound 29 up to the highest
concentrations typically tested (10 μM). This compound
demonstrates that methylation of the malonate-derived hydroxy
groups on the pawhuskin A scaffold does not abrogate the KOP
receptor antagonist activity on this stilbene scaffold. Compar-
isons to compounds 20, 23, and 24 indicate that the presence
and position of the prenyl substituent are important factors in
binding to the KOP receptor. These results point to the
importance of the shikimate-derived substructure and should
allow further design with the aim of introducing more drug-like
characteristics. Work on this strategy is currently under way.
General Experimental Procedures. Both THF and Et2O were
freshly distilled from Na/benzophenone. Both CH2Cl2 and Et3N were
distilled from CaH2 prior to use. Solutions of n-BuLi were purchased
from a commercial source and titrated with diphenylacetic acid prior
to use. All other reagents and solvents were purchased from
commercial sources and used without further purification. All reactions
in nonaqueous solvents were conducted in flame-dried glassware
under a positive pressure of Ar and with magnetic stirring. NMR
spectra were obtained at 300−500 MHz for 1H and 75−125 MHz for
13C with CDCl3 or CD3OD as solvent and (CH3)4Si (1H, 0.00 ppm)
or CDCl3 (13C, 77.0 ppm or 49.0 ppm) as internal standards unless
otherwise noted. The 31P chemical shifts were reported in ppm relative
to 85% H3PO4 (external standard). High-resolution mass spectra were
obtained at the University of Iowa Mass Spectrometry Facility. Silica
gel (60 Å, 0.040−0.063 mm) was used for flash chromatography.
2-Geranyl-3,5-dimethoxytetrahydropyranylbenzyl Alcohol
(13). To a stirred solution of TMEDA (0.58 mL, 3.9 mmol) and n-
BuLi (2.47 M solution in hexanes, 1.5 mL, 3.6 mmol) in Et2O (20 mL)
at −10 °C was added the bromide 12 (979 mg, 3.0 mmol) dissolved in
Et2O (4 mL). After 45 min of stirring, CuI (742 mg, 3.9 mmol) was
added and then geranyl bromide (840 mg, 3.9 mmol) was added
slowly over 8 min to the reaction. After the mixture was stirred
overnight, the reaction was quenched by addition of saturated aqueous
NH4Cl. The resulting mixture was extracted with EtOAc, and the
combined organic extracts were washed with brine, dried (MgSO4),
filtered, and concentrated in vacuo. Final purification by flash column
chromatography (4% EtOAc in hexanes) afforded compound 13 (364
mg, 32%) as a yellow oil: 1H NMR (500 MHz, CDCl3) δ 6.61 (d, J =
2.4 Hz, 1H), 6.42 (d, J = 2.5 Hz, 1H), 5.07−5.04 (m, 2H), 4.74 (d, J =
12.2 Hz, 1H), 4.70 (t, J = 3.7 Hz, 1H), 4.48 (d, J = 12.1 Hz, 1H),
3.94−3.90 (m, 1H), 3.80 (s, 3H), 3.79 (s, 3H), 3.37−3.29 (m, 2H),
2.06−1.80 (m, 5H), 1.80−1.54 (m, 5H), 1.75 (s, 3H), 1.65 (s, 3H),
1.57 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 158.6, 158.4, 138.0,
134.5, 131.2, 124.4, 123.3, 121.3, 105.0, 98.0 (2C), 66.9, 62.2, 55.7,
55.3, 39.8, 30.7, 26.8, 25.7, 25.5, 24.1, 19.5, 17.7, 16.1; HRMS (ESI)
m/z calcd for C24H36O4Na (M + Na)+ 411.2511, found 411.2495.
2-Geranyl-3,5-dimethoxybenzaldehyde (14). To a solution of
the THP acetal 13 (364 mg, 0.9 mmol) in MeOH (8 mL) at room
temperature was added TsOH (356 mg, 1.9 mmol). The solution was
stirred for 2.5 h and quenched by addition of NaHCO3. The mixture
was extracted with EtOAc, and the combined organic extracts were
dried (MgSO4), filtered, and concentrated in vacuo to afford the
benzylic alcohol as a yellow oil. This material was used in further
reactions without additional purification: 1H NMR (300 MHz,
CDCl3) δ 6.59 (d, J = 2.4 Hz, 1H), 6.43 (d, J = 2.2 Hz, 1H), 5.09−
5.02 (m, 2H) 4.64 (d, J = 3.9 Hz, 2H), 3.81 (s, 3H), 3.80 (s, 3H), 3.35
(d, J = 6.8 Hz, 2H), 2.10−1.94 (m, 4H), 1.76 (s, 3H), 1.65 (s, 3H),
1.57 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 158.7, 158.3, 140.6,
135.0, 131.4, 124.1, 123.5, 120.3, 104.0, 97.9, 63.3, 55.6, 55.3, 39.6,
CONCLUSIONS
■
This study has shown that the natural stilbene pawhuskin A is a
competitive antagonist with selectivity for the KOP receptor.
We also have shown that improved selectivity for the KOP
versus the DOP and MOP receptors is possible within the
constraints of the stilbene structure, which encourages further
Figure 4. Representative graph of the antagonist activity of compound
29 in the KOP receptor affinity assay. Each data point represents the
mean and SEM of duplicate samples.
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26.6, 25.6, 23.7, 17.6, 16.1; HRMS (EI) m/z calcd for C19H28O3 (M)+
304.2038, found 304.2044.
6.96 (dd, J = 8.7 Hz, JHP = 3.2 Hz, 1H), 6.68 (d, J = 8.8 Hz, 1H), 4.96
(t, J = 6.5 Hz, 1H), 3.99−3.86 (m, 4H), 3.79 (s, 3H), 3.70 (s, 3H),
3.42 (d, J = 6.5 Hz, 2H), 3.04 (d, JHP = 21 Hz, 2H), 1.72 (s, 3H), 1.61
(s, 3H), 1.18 (td, J = 7.5 Hz, JHP = 3.7 Hz, 6H).
To a stirred solution of the benzylic alcohol (285 mg, 0.9 mmol,
assuming 100% conversion in the previous step) in CH2Cl2 (15 mL)
was added activated MnO2 (815 mg, 9.4 mmol). The mixture was
stirred overnight and subsequently was filtered and concentrated in
vacuo. Final purification by flash column chromatography (12% EtOAc
in hexanes) afforded aldehyde 14 (146 mg, 52% from 13) as a yellow
oil: 1H NMR (300 MHz, CDCl3) δ 10.3 (s, 1H), 6.98 (d, J = 2.2 Hz,
1H), 6.68 (d, J = 1.9 Hz, 1H), 5.13−5.07 (m, 1H), 5.05−5.00 (m,
1H), 3.82 (s, 6H), 3.70 (d, J = 6.5 Hz, 2H), 2.24−1.90 (m, 4H), 1.76
(s, 3H), 1.64 (s, 3H), 1.56 (s, 3H); 13C NMR (75 MHz, CDCl3) δ
191.8, 158.8, 158.6, 135.2, 134.9, 131.4, 127.3, 124.0, 123.4, 104.8,
101.9, 55.8, 55.5, 39.5, 26.5, 25.6, 22.5, 17.6, 16.2; HRMS (ESI) m/z
calcd for C19H26O3Na (M + Na)+ 325.1780, found 325.1783.
2-Geranyl-3,5-bis(methoxymethoxy)benzaldehyde (16). Ac-
tivated MnO2 (644 mg, 7.1 equiv) was added to a solution of alcohol
155 (267 mg, 0.7 mmol) in CH2Cl2 (15 mL) at room temperature, and
the mixture was stirred overnight. The mixture was filtered, and the
filtrate was concentrated in vacuo. Final purification by flash column
chromatography (50% EtOAc in hexanes) afforded aldehyde 16 (256
2-Geranyl-3,5,3′,4′-tetramethoxy-2′-prenyl-(E)-stilbene (20).
To a stirred suspension of NaH (60% dispersion in mineral oil, washed
with hexanes, 33 mg, 0.8 mmol) in THF (2.5 mL) were added
phosphonate 19 (56 mg, 0.2 mmol), aldehyde 14 (39 mg, 0.1 mmol),
and 15-crown-5 (3 drops). The mixture was stirred for 2 h and
quenched by addition of saturated aqueous NH4Cl. The resulting
mixture was extracted with EtOAc, and the combined organic extracts
were washed with brine, dried (MgSO4), and concentrated in vacuo.
Final purification by flash column chromatography (10% EtOAc in
1
hexanes) afforded stilbene 20 (20 mg, 31%) as a yellow oil: H NMR
(500 MHz, CDCl3) δ 7.32 (d, J = 8.7 Hz, 1H), 7.12 (s, 2H), 6.80 (d, J
= 8.8 Hz, 1H), 6.71 (d, J = 2.5 Hz, 1H), 6.21 (d, J = 2.5 Hz, 1H),
5.16−5.11 (m, 2H), 5.07−5.04 (m, 1H), 3.88 (s, 3H), 3.84 (s, 3H),
3.81 (s, 3H), 3.81 (s, 3H), 3.52−3.50 (m, 2H), 3.42 (d, J = 6.7 Hz,
2H), 2.07−2.03 (m, 2H), 1.98−1.92 (m, 2H), 1.81 (s, 3H), 1.78 (s,
3H), 1.67 (s, 3H), 1.62 (s, 3H), 1.55 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ 158.5, 158.4, 152.3, 146.9, 138.4, 134.4, 134.0, 131.4, 131.2,
130.4, 128.0, 127.0, 124.4, 123.5, 123.3, 121.6, 121.1, 110.2, 101.6,
97.9, 60.7, 55.7 (2C), 55.3, 39.7, 26.8, 25.7, 25.6, 25.5, 24.3, 18.1, 17.6,
16.3; HRMS (EI) m/z calcd for C33H44O4 (M)+ 504.3240, found
504.3237.
1
mg, 96%) as a yellow oil: H NMR (300 MHz, CDCl3) δ 10.26 (s,
1H), 7.21 (d, J = 2.7 Hz, 1H), 7.04 (d, J = 2.2 Hz, 1H), 5.21 (s, 2H),
5.19 (s, 2H), 5.14−5.10 (m, 1H), 5.05−5.00 (m, 1H), 3.73 (d, J = 6.5
Hz, 2H), 3.48 (s, 6H), 2.08−1.93 (m, 4H), 1.86 (s, 3H), 1.73 (s, 3H),
1.65 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 191.6, 156.3, 156.2,
135.3 (2C), 131.5, 128.1, 124.1, 123.2, 109.3, 108.7, 94.7, 94.5, 56.2,
56.1, 39.6, 26.6, 25.6, 22.9, 17.6, 16.3; HRMS (ESI) m/z calcd for
C21H30O5Na (M + Na)+ 385.1991, found 385.1983.
2-Geranyl-3,5-dimethoxy-3′,4′-bis(methoxymethoxy)-(E)-
stilbene (22). To a stirred suspension of NaH (60% dispersion in
mineral oil, washed with hexanes, 28 mg, 0.7 mmol) in THF (2.5 mL)
were added aldehyde 14 (35 mg, 0.1 mmol), phosphonate 2138 (49
mg, 0.1 mmol), and 15-crown-5 (3 drops). The mixture was stirred for
2 h and quenched with saturated aqueous NH4Cl. The resultant
mixture was extracted with EtOAc, and the combined organic extracts
were washed with brine, dried (MgSO4), and concentrated in vacuo.
Final purification of the residue by flash column chromatography (10%
Diethyl{[4-methoxy-3-(methoxymethoxy)-2-(prenyl)-
phenyl]methyl} Phosphonate (18). To a stirred solution of
aldehyde 17 (1.16 g, 4.5 mmol) in MeOH (10 mL) at 0 °C was
added NaBH4 (282 mg, 7.5 mmol) as a single aliquot. This solution
was stirred for 30 min, and then H2O (50 mL) was added and the
resulting solution was extracted with EtOAc. After concentration in
vacuo, the resulting alcohol (1.07 g, 90%) was dissolved in THF (10
mL) and treated with Et3N (0.80 mL, 5.7 mmol) followed by
methanesulfonyl chloride (0.31 mL, 4.01 mmol). After 15 min, LiBr
(391 mg, 0.45 mmol) was added in THF (15 mL). The resulting
solution was stirred for an additional 45 min and quenched by addition
of H2O. This mixture was extracted with EtOAc, and then the
combined organic extracts were washed with brine, dried (MgSO4),
filtered, and concentrated in vacuo. The resulting yellow oil was
dissolved in triethyl phosphite (5 mL) and heated at reflux for 5 days.
Removal of excess triethyl phosphite in vacuo gave a yellow oil.
Purification by flash column chromatography (100% EtOAc) afforded
1
EtOAc in hexanes) gave stilbene 22 (38 mg, 67%) as a yellow oil: H
NMR (300 MHz, CDCl3) δ 7.32 (d, J = 1.6 Hz, 1H), 7.21 (d, J = 16.1
Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 7.10 (dd, J = 8.5 Hz, 1.7 Hz, 1H),
6.89 (d, J = 16.2 Hz, 1H), 6.72 (d, J = 2.2 Hz, 1H), 6.42 (d, J = 2.1 Hz,
1H), 5.28 (s, 2H), 5.26 (s, 2H), 5.14−5.03 (m, 2H), 3.85 (s, 3H), 3.81
(s, 3H), 3.54 (s, 3H), 3.53 (s, 3H), 3.43 (d, J = 6.7 Hz, 2H), 2.08−1.95
(m, 4H), 1.81 (s, 3H), 1.61 (s, 3H), 1.54 (s, 3H); 13C NMR (75 MHz,
CDCl3) δ 158.5, 158.3, 147.3, 146.8 137.8, 134.3, 132.4, 131.2, 129.7,
125.8, 124.3, 123.5, 121.1, 120.8, 116.5, 114.6, 101.2, 98.0, 95.4, 95.3,
56.2 (2C), 55.6, 55.3, 39.7, 26.7, 25.6, 24.3, 17.6, 16.2; HRMS (ESI)
m/z calcd for C30H41O6 (M + H)+ 497.2903, found 497.2918.
2-Geranyl-3,5-dimethoxy-3′,4′-dihydroxy-(E)-stilbene (23).
To a solution of bis(methoxymethyl) ether 22 (18 mg, 0.04 mmol)
in MeOH (2 mL) was added TsOH (29 mg, 0.15 mmol). After the
solution was stirred overnight, the reaction was quenched by addition
of saturated aqueous NaHCO3. The resultant mixture was extracted
with EtOAc, and the combined organic extracts were dried (MgSO4),
filtered, and concentrated in vacuo. Final purification of a portion
(25%) of the residual oil by preparative TLC (25% EtOAc in hexanes)
afforded the stilbene 23 (5 mg, 100% by NMR) as a yellow oil; the
remaining material (75%) was moved forward without additional
purification. For compound 23: 1H NMR (300 MHz, CDCl3) δ 7.20−
6.82 (m, 5H), 6.71 (m, 1H), 6.42 (m, 1H), 5.19−4.98 (m, 2H), 3.86
(s, 3H), 3.81 (s, 3H), 3.42 (d, J = 5.9 Hz, 2H), 2.07−1.97 (m, 4H),
1.80 (s, 3H), 1.62 (s, 3H), 1.55 (s, 3H); 13C NMR (75 MHz, CDCl3)
δ 158.5, 158.4, 143.8, 143.5, 137.9, 134.5, 131.3, 129.9, 125.1, 124.3,
123.5, 121.1, 119.9, 115.5, 113.1, 110.9, 101.4, 97.9, 55.7, 55.4, 39.7,
26.7, 25.6, 24.3, 17.7, 16.3; HRMS (ESI) m/z calcd for C26H33O4 (M
+ H)+ 409.2379, found 409.2374.
1
phosphonate 18 (622 mg, 40%) as a clear oil: H NMR (300 MHz,
CDCl3) δ 6.97−6.93 (m, 1H), 6.67−6.63 (m, 1H), 4.95 (br s, 3H),
3.92−3.86 (m, 4H), 3.70 (s, 5H), 3.56 (s, 3H), 3.01 (d, JHP = 21 Hz,
2H), 1.70 (s, 3H), 1.58 (s, 3H), 1.16−1.10 (m, 6H); 13C NMR (75
MHz, CDCl3) δ 150.8 (d, JCP = 3.6 Hz), 143.7 (d, JCP = 3.7 Hz), 134.8
(d, JCP = 6.7 Hz), 131.3, 126.3 (d, JCP = 5.2 Hz), 122.7 (d, JCP = 9.3
Hz), 122.5, 109.4 (d, JCP = 3.9 Hz), 98.6, 61.6 (d, JCP = 7.1 Hz, 2C),
57.1, 55.2, 29.5 (d, JCP = 139 Hz), 25.3, 25.2, 17.6, 16.0 (d, JCP = 5.8
Hz, 2C); 31P NMR (122 MHz, CDCl3) 27.2; HRMS (EI) m/z calcd
for C19H31O6P (M)+ 386.1858, found 386.1857.
Diethyl{[3,4-dimethoxy-2-(prenyl)phenyl]methyl} Phospho-
nate (19). To a stirred solution of MOM ether 18 (103 mg, 0.3
mmol) in EtOH (2.5 mL) was added TsOH (152 mg, 0.8 mmol). The
solution was stirred overnight, quenched by addition of saturated
aqueous NH4Cl, and extracted with EtOAc. The combined organic
extracts were washed with brine, dried (MgSO4), and concentrated in
vacuo to afford the phenol as a yellow oil. To a stirred solution of the
phenol (88 mg, 0.3 mmol) in acetone (6 mL) were added K2CO3 (242
mg, 1.8 mmol) and MeI (0.1 mL, 1.6 mmol). After the mixture was
heated to reflux and stirred overnight, it was quenched by addition of
H2O, and the mixture was extracted with EtOAc. The organic extracts
were washed with 2 M NaOH, dried (MgSO4), and concentrated in
vacuo. Without additional purification, methyl ether 19 (56 mg, 59%, 2
2-Geranyl-3,5,3′,4′-tetramethoxy-(E)-stilbene (24). To a
stirred solution of stilbene 23 in acetone (3 mL) was added K2CO3
(35 mg, 0.25 mmol) followed by MeI (38 μL, 0.61 mmol). The
mixture was stirred for 2 days, and the reaction was quenched with
H2O. The resulting mixture was extracted with EtOAc, and the
combined organic extracts were washed with brine, dried (MgSO4),
and concentrated in vacuo. Final purification by flash column
1
steps) was obtained as a yellow oil: H NMR (300 MHz, CDCl3) δ
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Journal of Natural Products
Article
chromatography (gradient of hexanes to 40% EtOAc in hexanes)
provided stilbene 24 (4 mg, 27%) as a yellow oil: 1H NMR (300 MHz,
CDCl3) δ 7.27−7.18 (m, 1H), 7.06−7.04 (m, 2H), 6.94−6.85 (m,
2H), 6.75 (d, J = 2.7 Hz, 1H), 6.43 (d, J = 2.4 Hz, 1H), 5.14 (t, J = 7.5
Hz, 1H), 5.06 (t, J = 7.5 Hz, 1H), 3.94 (s, 3H), 3.91 (s, 3H), 3.86 (s,
3H), 3.83 (s, 3H), 3.46−3.44 (m, 2H), 2.05−1.95 (m, 4H), 1.82 (s,
3H), 1.62 (s, 3H), 1.55 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 158.9,
149.1, 138.0, 134.2, 131.3, 130.8, 130.1, 125.2, 124.2, 123.4, 121.1,
119.9, 111..3, 108.8, 107.1, 105.8, 101.4, 97.9, 56.0, 55.8, 55.7, 55.4,
39.7, 26.8, 25.6, 24.4, 17.6, 16.3; HRMS (EI) m/z calcd for C28H36O4
(M)+ 436.2614, found 436.2606.
solution was stirred for an additional 22.5 h, the reaction was
quenched by addition of NaHCO3. The resultant mixture was
extracted with EtOAc, and the combined organic extracts were dried
(MgSO4), filtered, and concentrated in vacuo. Final purification by
preparative TLC (35% EtOAc in hexanes) provided stilbene 29 (14
mg, 47%) as a yellow oil: 1H NMR (500 MHz, CDCl3) δ 7.37 (d, J =
15.6 Hz, 1H), 6.94 (s, 1H), 6.84−6.80 (m, 2H), 6.71 (d, J = 2.1 Hz,
1H), 6.40 (d, J = 2.0 Hz, 1H), 5.45 (br s, 1H), 5.34 (t, J = 6.1 Hz, 1H),
5.11 (t, J = 6.7 Hz, 1H), 5.06−5.04 (m, 1H), 3.84 (s, 3H), 3.80 (s,
3H), 3.42 (d, J = 6.5 Hz, 2H), 3.37 (d, J = 7.1 Hz, 2H), 2.07−2.03 (m,
2H), 1.98−1.95 (m, 2H), 1.81−1.79 (m, 9H), 1.61 (s, 3H), 1.54 (s,
3H); 13C NMR (125 MHz, CDCl3) δ 158.5, 158.4, 144.2, 141.9,
138.0, 135.3, 134.4, 131.3, 130.6, 130.1, 127.3, 125.2, 124.3, 123.6,
121.6, 121.1, 120.5, 110.9, 101.5, 97.9, 55.7, 55.4, 39.7, 29.9, 26.8, 25.8,
25.6, 24.4, 17.9, 17.6, 16.3; HRMS (ESI) m/z calcd for C31H41O4
(M)+ 477.3005, found 477.2994.
2-Geranyl-3,5,3′,4′-tetrakis(methoxymethoxy)-(E)-stilbene
(25). To a solution of potassium hexamethyldisilazane (KHMDS) (0.5
M solution in toluene, 2.3 mL, 1.16 mmol) in THF (1.5 mL) were
added phosphonate 21 (46 mg, 0.13 mmol) and aldehyde 16 (35 mg,
0.10 mmol). After the solution was stirred for 4 h, the reaction was
quenched by addition of NH4Cl. The resulting mixture was extracted
with EtOAc, washed with brine, dried (MgSO4), and concentrated in
vacuo. Final purification by flash column chromatography (7% EtOAc
2-Geranyl-3,5-dimethoxy-2′-prenyl-3′-(methoxymethoxy)-
4′-methoxy-(E)-stilbene (30). To a stirred solution of aldehyde 14
(27 mg, 0.1 mmol) and 18 (51 mg, 0.1 mmol) in THF (1.5 mL) at
room temperature were added NaH (60% dispersion in mineral oil, 22
mg, 0.6 mmol) and 15-crown-5 (2 drops). After the mixture was
stirred overnight, the reaction was quenched by addition of NH4Cl.
The resulting mixture was extracted with EtOAc, and then the
combined organic extracts were dried (MgSO4), filtered, and
concentrated in vacuo. Final purification by flash column chromatog-
raphy (10% EtOAc in hexanes) provided 30 (18 mg) in 38% yield as a
1
in hexanes) provided stilbene 25 (46 mg, 86%) as a yellow oil: H
NMR (500 MHz, CDCl3) δ 7.31−7.11 (m, 4H), 6.96 (s, 1H), 6.88 (d,
J = 15.7 Hz, 1H), 6.74 (s, 1H), 5.27−5.05 (m, 10H), 3.54−3.48 (m,
14H), 2.04−1.97 (m, 4H), 1.82 (s, 3H), 1.61 (s, 3H), 1.54 (s, 3H);
13C NMR (125 MHz, CDCl3) δ 156.1, 155.8, 147.4, 146.9, 138.2,
134.5, 132.4, 131.3, 130.1, 125.5, 124.2, 123.3, 122.8, 120.9, 116.7,
114.8, 106.2, 102.9, 95.5, 95.4, 94.6 (2C), 56.2 (2C), 56.0 (2C), 39.7,
26.7, 25.6, 24.7, 17.6, 16.2; HRMS (EI) m/z calcd for C32H44O8 (M)+
556.3036, found 556.3056.
1
yellow oil: H NMR (300 MHz, CDCl3) δ 7.33 (d, J = 8.7 Hz, 1H),
7.11 (m, 2H), 6.80 (d, J = 8.6 Hz, 1H), 6.70 (d, J = 2.3 Hz, 1H), 6.41
(d, J = 2.5 Hz, 1H), 5.16−5.03 (m, 3H), 5.09 (s, 2H), 3.86 (s, 3H),
3.83 (s, 3H), 3.81 (s, 3H), 3.60 (s, 3H), 3.57 (d, J = 6.6 Hz, 2H), 3.42
(d, J = 6.7 Hz, 2H), 2.05−1.96 (m, 4H), 1.80 (s, 3H), 1.78 (s, 3H),
1.68 (s, 3H), 1.62 (s, 3H), 1.55 (s, 3H); 13C NMR (75 MHz, CDCl3)
δ 158.8, 158.7, 152.1, 144.1, 138.7, 134.8, 134.5, 131.6 (2C), 130.9,
128.4, 127.3, 124.7, 123.8, 123.6, 122.2, 121.4, 110.4, 101.8, 99.4, 98.2,
58.0, 56.1, 56.0, 55.6, 40.1, 30.0, 27.1, 26.1, 26.0 (2C), 24.7, 18.5, 18.0,
16.6; HRMS (EI) m/z calcd for C34H46O5 (M)+ 534.3345, found
534.3330.
2-Geranyl-3,5-dimethoxy-2′-prenyl-3′-hydroxy-4′-methoxy-
(E)-stilbene (31). To a stirred solution of MOM ether 30 (27 mg, 0.1
mmol) in MeOH (2.5 mL) was added TsOH (40 mg, 0.2 mmol). The
solution was stirred overnight, and the reaction was quenched by
addition of saturated aqueous NaHCO3. The resulting mixture was
extracted with EtOAc, and the organic extracts were dried (MgSO4),
filtered, and concentrated in vacuo. Stilbene 31 (22 mg, 88%) was
obtained as a yellow oil: 1H NMR (300 MHz, CDCl3) δ 7.14 (m, 2H),
7.10 (m, 1H), 6.76 (m, 1H), 6.73 (d, J = 2.4 Hz, 1H), 6.42 (d, J = 2.2
Hz, 1H), 5.21−5.04 (m, 3H), 3.90 (s, 3H), 3.84 (s, 3H), 3.81 (s, 3H),
3.52 (d, J = 6.5 Hz, 2H), 3.43 (d, J = 6.4 Hz, 2H), 2.09−1.96 (m, 4H),
1.82 (s, 3H), 1.78 (s, 3H), 1.68 (s, 3H), 1.62 (s, 3H), 1.55 (s, 3H); 13C
NMR (75 MHz, CDCl3) δ 158.9, 158.1, 146.2, 143.6, 138.7, 134.7,
131.9, 131.5, 131.0, 128.5, 127.5, 126.1, 124.7, 123.9, 123.0, 121.5,
117.5, 108.8, 101.9, 98.3, 56.0, 55.7, 55.3, 39.8, 26.8, 25.7, 25.6, 25.1,
24.4, 18.1, 17.6, 16.3; HRMS (EI) m/z calcd for C32H42O4 (M + H)+
490.3083, found 490.3087.
2-Geranyl-3,5,3′,4′-tetrahydroxy-(E)-stilbene (26). To a sol-
ution of stilbene 25 (23 mg, 0.04 mmol) in MeOH (4 mL) was added
TsOH (63 mg, 0.33 mmol). The solution was stirred for 24 h, and the
reaction was quenched by addition of NaHCO3. The resulting mixture
was extracted with EtOAc, dried (MgSO4), and concentrated in vacuo.
Final purification by preparative TLC (30% EtOAc in hexanes)
afforded stilbene 26 (6 mg, 38%) as a yellow oil: 1H NMR (500 MHz,
CD3OD) δ 6.96 (d, J = 16 Hz, 1H), 6.85 (d, J = 2.3 Hz, 1H), 6.71 (d, J
= 2 Hz, 1H), 6.70 (d, J = 1.7 Hz, 1H), 6.65 (d, J = 15.8 Hz, 1H), 6.63
(d, J = 8.3 Hz, 1H), 6.44 (d, J = 2.1 Hz, 1H), 6.13 (d, J = 2.3 Hz, 1H),
5.01−5.00 (m, 1H), 4.95−4.92 (m, 1H), 3.27 (d, J = 6.6 Hz, 2H),
1.97−1.92 (m, 2H), 1.88−1.85 (m, 2H), 1.70 (s, 3H), 1.47 (s, 3H),
1.42 (s, 3H); 13C NMR (125 MHz, CD3OD) δ 157.0, 156.7, 146.5,
146.4, 139.8, 134.5, 132.1, 131.6, 130.7, 125.8, 125.4 (2C), 120.0,
119.1, 116.4, 114.0, 104.3, 102.6, 40.8, 27.8, 25.8, 25.1, 17.7, 16.5;
HRMS (EI) m/z calcd for C24H28O4 (M)+ 380.1988, found 380.2014.
2 - G e ra n y l - 3 , 5 - d i m e t h o x y - 3 ′ - p r e n y l - 4 ′ , 5 ′ - b i s -
(methoxymethoxy)-(E)-stilbene (28). To a stirred solution of
aldehyde 14 (21 mg, 0.07 mmol) and phosphonate 27 (51 mg, 0.12
mmol) in THF (1.4 mL) at 0 °C was added KHMDS (0.5 M in
toluene, 0.69 mL, 0.35 mmol). The solution was stirred for 22 h at rt,
and the reaction was quenched with NH4Cl. The resultant mixture was
extracted with EtOAc, and the combined organic extracts were washed
with brine, dried (MgSO4), filtered, and concentrated in vacuo. Final
purification by preparative TLC (30% EtOAc in hexanes) gave stilbene
28 (36 mg, 92%) as a yellow oil: 1H NMR (500 MHz, CDCl3) δ 7.20
(d, J = 16.0 Hz, 1H), 7.14 (d, J = 1.8 Hz, 1H), 6.98 (d, J = 2.1 Hz,
1H), 6.87 (d, J = 15.7 Hz, 1H), 6.72 (d, J = 2.4 Hz, 1H), 6.41 (d, J =
2.6 Hz, 1H), 5.34−5.31 (m, 1H), 5.22 (s, 2H), 5.12 (s, 2H), 5.06−5.03
(m, 1H), 3.85 (s, 3H), 3.81 (s, 3H), 3.61 (s, 3H), 3.52 (s, 3H), 3.44−
3.41 (m, 4H), 2.06−2.02 (m, 2H), 1.98−1.95 (m, 2H), 1.81 (s, 3H),
1.76 (s, 3H), 1.74 (s, 3H), 1.60 (s, 3H), 1.53 (s, 3H); 13C NMR (125
MHz, CDCl3) δ 158.5, 158.4, 150.0, 144.4, 137.9, 136.1, 134.2, 133.9,
132.7, 131.2, 130.1, 126.3, 124.3, 123.5, 122.6, 121.5, 121.2, 112.1,
101.4, 99.1, 98.1, 95.2, 57.5, 56.2, 55.7, 55.4, 39.7, 28.6, 26.7, 25.8,
25.6, 24.4, 17.9, 17.6, 16.2; HRMS (ESI) m/z calcd for C35H49O6 (M
+ H)+ 565.3529, found 565.3524.
2-Geranyl-3,5,3′,4′-tetramethoxy-2′-prenyl-(E)-stilbene (20).
To a solution of stilbene 31 (11 mg, 0.02 mmol) in THF (3 mL) were
added NaH (60% dispersion in mineral oil, 6 mg, 0.2 mmol) and MeI
(2 drops). The mixture was stirred for 5 h, and the reaction was
quenched by addition of H2O. The resultant mixture was extracted
with EtOAc, and the combined organic extracts were washed with 2 M
NaOH, dried (MgSO4), and concentrated in vacuo. Stilbene 20 (6 mg,
1
55%) was obtained as a yellow oil, with H NMR data that were
identical to the data given above.
4-Prenyl-3,5,3′,4′-tetrakis(methoxymethoxy)-(E)-stilbene
(36). To a solution of KHMDS (0.5 M solution in toluene, 2.12 mL,
1.06 mmol) in THF (1.5 mL) were added phosphonate 2351 (46 mg,
0.11 mmol) and aldehyde 3452 (20 mg, 0.09 mmol). After the solution
was stirred for 2 h, the reaction was quenched by addition of NH4Cl.
The resulting mixture was extracted with EtOAc, washed with brine,
dried (MgSO4), and concentrated in vacuo. Final purification by flash
2-Geranyl-3,5-dimethoxy-3′-prenyl-4′,5′-dihydroxy-(E)-stil-
bene (29). To a solution of bis-MOM acetal 28 (36 mg, 0.06 mmol)
in MeOH (6.4 mL) was added TsOH (49 mg, 0.26 mmol). After the
solution was stirred for 19.5 h at rt, additional TsOH (25 mg, 0.13
mmol) was added due to incomplete conversion to product. After the
317
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Journal of Natural Products
Article
column chromatography provided stilbene 36 (25 mg, 58%) as a
yellow oil: H NMR (500 MHz, CDCl3) δ 7.32 (d, J = 1.9 Hz, 1H),
ASSOCIATED CONTENT
■
1
S
* Supporting Information
7.14−7.09 (m, 2H), 6.98−6.89 (m, 4H), 5.29 (s, 2H), 5.25−5.21 (m,
7H), 3.56 (s, 3H), 3.53 (s, 3H), 3.50 (s, 6H), 3.39 (d, J = 7.2 Hz, 2H),
1.79 (s, 3H), 1.66 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 155.8
(2C), 147.4, 146.8, 136.4, 132.1, 131.0, 127.7 (2C), 122.7, 121.0,
119.7, 116.6, 114.3, 106.0 (2C), 95.4 (2C), 94.5 (2C), 56.2 (2C), 56.0
(2C), 25.7, 22.7, 17.7; HRMS (EI) m/z calcd for C27H36O8 (M)+
488.2410, found 488.2416.
Supplementary data including the 1H and 13C NMR spectra for
the key intermediates and final products in this article are
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
4-Prenyl-3,5,3′,4′-tetrahydroxy-(E)-stilbene (37). After TsOH
(40 mg, 0.21 mmol) was added to a solution of stilbene 36 (13 mg,
0.03 mmol) in MeOH (2.5 mL), the solution was stirred for 24 h. The
reaction was quenched by addition of NaHCO3, and the resulting
mixture was extracted with EtOAc, dried (MgSO4), and concentrated
in vacuo. Final purification by preparative TLC (30% EtOAc in
Corresponding Author
*Tel: +1-319-335-1467. Fax: +1-319-335-1270. E-mail: jeffrey-
neighbors@uiowa.edu.
Notes
The authors declare no competing financial interest.
hexanes) gave stilbene 37 (4 mg, 50%) as a yellow oil. The 1H and 13
C
NMR spectra matched published data.56,57
ACKNOWLEDGMENTS
■
4-Prenyl-3,5,4′-tris(methoxymethoxy)-(E)-stilbene (38). To a
solution of KHMDS (0.5 M solution in toluene, 4.62 mL, 2.31 mmol)
in THF (1.5 mL) were added phosphonate 33 (99 mg, 0.24 mmol)
and aldehyde 3552 (32 mg, 0.19 mmol). After the solution was stirred
for 3 h, the reaction was quenched by addition of NH4Cl. The
resulting mixture was extracted with EtOAc, washed with brine, dried
(MgSO4), and concentrated in vacuo. Final purification of the residue
by flash column chromatography (4% EtOAc in hexanes) provided
stilbene 38 (33 mg, 40%) as a yellow oil: 1H NMR (500 MHz, CDCl3)
δ 7.44 (t, J = 2.7 Hz, 1H), 7.42 (t, J = 1.9 Hz, 1H), 7.03−7.01 (m, 2H),
6.98−6.95 (m, 1H), 6.92−6.90 (m, 1H), 6.92 (s, 2H), 5.24−5.19 (m,
5H), 5.19 (s, 2H), 3.50 (s, 6H), 3.49 (s, 3H), 3.39 (d, J = 7.2 Hz, 2H),
1.79 (s, 3H), 1.66 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 156.8,
155.8 (2C), 136.6, 131.3, 131.0, 127.7, 127.6 (2C), 127.2, 122.7, 119.6,
116.4 (2C), 106.0 (2C), 94.5 (2C), 94.4, 56.0 (3C), 25.8, 22.8, 17.8;
HRMS (EI) m/z calcd for C25H32O6 (M)+ 428.2199, found 428.2191.
trans-Arachidin-2 (39). To a solution of compound 38 (17 mg,
0.04 mmol) in MeOH (5 mL) was added TsOH (46 mg, 0.24 mmol).
After the solution was stirred for 24 h, the reaction was quenched with
NaHCO3. The resulting mixture was extracted with EtOAc, dried
(MgSO4), and concentrated in vacuo. Final purification by flash
column chromatography (12% EtOAc in hexanes) provided stilbene
We thank Dr. R. J. Barney for his assistance with preparation of
some early intermediates. Financial support from the National
Institutes of Health (DA02-6573) is gratefully acknowledged.
This research also was supported in part by the Intramural
Research Program of NIH, National Cancer Institute, Center
for Cancer Research, and the Roy J. Carver Charitable Trust as
a Research Program of Excellence.
DEDICATION
■
We dedicate this contribution to our valued colleague Paul J.
Klausmeyer, who isolated and characterized schweinfurthin J
and who was killed in an automobile accident on January 22,
2013.
REFERENCES
■
(1) Belofsky, G.; French, A. N.; Wallace, D. R.; Dodson, S. L. J. Nat.
Prod. 2004, 67, 26−30.
(2) Gillmore, M. Uses of Plants by the Indians of the Missouri River
Region; University of Nebraska Press: Lincoln, NE, 1977.
(3) Topczewski, J. J.; Kodet, J. G.; Wiemer, D. F. J. Org. Chem. 2011,
76, 909−919.
1
39 (12 mg, 100%) as a yellow oil. Both the H and 13C data matched
those of the known compound.56,57
Biological Assays. All compounds were initially screened for
intrinsic and antagonist activity at 10 μM in the [35S]GTP-γ-S binding
assay at the human κ and the μ and δ opioid receptors overexpressed
in CHO cells. These cell lines were kindly provided by Dr. Liu-Chen
(Temple University, κ) and Dr. Larry Toll (SRI International, μ and
δ). Compounds were identified as antagonist characterized for
functional antagonism (Ke) and selectivity by measuring the ability
of the test compounds to inhibit stimulated [35S]GTP-γ-S binding
produced by one of the selective agonists DAMGO (μ), DPDPE (δ),
or U69,593 (κ). Agonist concentration−response curves were run in
the presence or absence of a single concentration of test compound.
Briefly, the test compounds were assayed in duplicate in 1.4 mL
polypropylene tubes in a 96-well format. CHO membrane
homogenates (20−40 μg protein) were incubated with a positive
control or the test compound, 0.1 nM [35S]GTP-γ-S, and 1 μM GDP
in 50 mM HEPES buffer (pH 7.4) at room temperature for 1 h, after
which bound radioligand was separated from free radioligand via rapid
vacuum filtration over GF-B filters with a Brandel Scientific
(Gaithersburg, MD, USA) 96-well harvester. Bound radioactivity is
determined using a TopCount 12-detector instrument (Packard
Instruments) using standard scintillation counting techniques. Bound
radioactivity is normalized to samples containing vehicle (basal
binding). A four-parameter logistic function was fit to these data to
calculate the EC50 and Emax values using Prism (v. 6; Graph Pad
Software, San Diego, CA, USA). The Ke values were calculated using
the formula Ke = [L]/DR − 1, where [L] is the concentration of test
compound, and DR is the ratio of agonist EC50 value in the presence
or absence of test compound.
(4) Topczewski, J. J.; Wiemer, D. F. Tetrahedron Lett. 2011, 52,
1628−1630.
(5) Neighbors, J. D.; Buller, M. J.; Boss, K. D.; Wiemer, D. F. J. Nat.
Prod. 2008, 71, 1949−1952.
(6) Neighbors, J. D.; Salnikova, M. S.; Wiemer, D. F. Tetrahedron
Lett. 2005, 46, 1321−1324.
(7) Siebert, D. J. J. Ethnopharmacol. 1994, 43, 53−56.
(8) Lovell, K. M.; Vasiljevik, T.; Araya, J. J.; Lozama, A.; Prevatt-
Smith, K. M.; Day, V. W.; Dersch, C. M.; Rothman, R. B.; Butelman, E.
R.; Kreek, M. J.; Prisinzano, T. E. Bioorg. Med. Chem. 2012, 20, 3100−
3110.
(9) Scheerer, J. R.; Lawrence, J. F.; Wang, G. C.; Evans, D. A. J. Am.
Chem. Soc. 2007, 129, 8968−8969.
(10) Hagiwara, H.; Suka, Y.; Nojima, T.; Hoshi, T.; Suzuki, T.
Tetrahedron 2009, 65, 4820−4825.
(11) Harding, W. W.; Schmidt, M.; Tidgewell, K.; Kannan, P.;
Holden, K. G.; Dersch, C. M.; Rothman, R. B.; Prisinzano, T. E. Bioorg.
Med. Chem. Lett. 2006, 16, 3170−3174.
(12) Harding, W. W.; Schmidt, M.; Tidgewell, K.; Kannan, P.;
Holden, K. G.; Gilmour, B.; Navarro, H.; Rothman, R. B.; Prisinzano,
T. E. J. Nat. Prod. 2006, 69, 107−112.
(13) Holden, K. G.; Tidgewell, K.; Marquam, A.; Rothman, R. B.;
Navarro, H.; Prisinzano, T. E. Bioorg. Med. Chem. Lett. 2007, 17,
6111−6115.
(14) Simpson, D. S.; Katavic, P. L.; Lozama, A.; Harding, W. W.;
Parrish, D.; Deschamps, J. R.; Dersch, C. M.; Partilla, J. S.; Rothman,
R. B.; Navarro, H.; Prisinzano, T. E. J. Med. Chem. 2007, 50, 3596−
3603.
318
dx.doi.org/10.1021/np4009046 | J. Nat. Prod. 2014, 77, 311−319

Journal of Natural Products
Article
(15) Tidgewell, K.; Harding, W. W.; Lozama, A.; Cobb, H.; Shah, K.;
Kannan, P.; Dersch, C. M.; Parrish, D.; Deschamps, J. R.; Rothman, R.
B.; Prisinzano, T. E. J. Nat. Prod. 2006, 69, 914−918.
(16) Lozama, A.; Cunningham, C. W.; Caspers, M. J.; Douglas, J. T.;
Dersch, C. M.; Rothman, R. B.; Prisinzano, T. E. J. Nat. Prod. 2011, 74,
718−726.
(47) Cushman, M.; Nagarathnam, D.; Gopal, D.; Chakraborti, A. K.;
Lin, C. M.; Hamel, E. J. Med. Chem. 1991, 34, 2579−2588.
(48) Klausmeyer, P.; Van, Q. N.; Jato, J.; McCloud, T. G.; Beutler, J.
A. J. Nat. Prod. 2010, 73, 479−481.
(49) Singh, M.; Argade, N. P. Synthesis 2012, 44, 2895−2902.
(50) Mente, N. R.; Neighbors, J. D.; Wiemer, D. F. J. Org. Chem.
2008, 73, 7963−7970.
(17) Polepally, P. R.; White, K.; Vardy, E.; Roth, B. L.; Ferreira, D.;
Zjawiony, J. K. Bioorg. Med. Chem. Lett. 2013, 23, 2860−2862.
(18) Almeida, E. R.; Almeida, R. N.; Navarro, D. S.; Bhattacharryya,
J.; Silva, B. A.; Birnbaum, J. S. J. Ethnopharmacol. 2003, 88, 1−4.
(19) Bhattacharyya, J.; Majetich, G.; Jenkins, T. M.; Almeida, R. N. J.
Nat. Prod. 1998, 61, 413−414.
(51) Mente, N. R.; Wiemer, A. J.; Neighbors, J. D.; Beutler, J. A.;
Hohl, R. J.; Wiemer, D. F. Biorg. Med. Chem. Lett. 2007, 17, 911−915.
(52) Heynekamp, J. J.; Weber, W. M.; Hunsaker, L. A.; Gonzales, A.
M.; Orlando, R. A.; Deck, L. M.; Jagt, D. L. V. J. Med. Chem. 2006, 49,
7182−7189.
(53) Park, B. H.; Lee, H. J.; Lee, Y. R. J. Nat. Prod. 2011, 74, 644−
649.
(20) Batista, J. S.; Almeida, R. N.; Bhattacharyya, J. J. Ethnopharmacol.
1995, 45, 207−210.
(54) Verhoest, P. R.; Basak, A. S.; Parikh, V.; Hayward, M.;
Kauffman, G. W.; Paradis, V.; McHardy, F.; McLean, S.; Grimwood,
S.; Schmidt, A. W.; Vanase-Frawley, M.; Freeman, J.; Van Deusen, J.;
Cox, L.; Wong, D.; Liras, S. J. Med. Chem. 2011, 54, 5868−5877.
(55) Carroll, F. I.; Carlezon, W. A. J. Med. Chem. 2013, 56, 2178−
2195.
(56) Chang, J.-C.; Lai, Y.-H.; Djoko, B.; Wu, P.-L.; Liu, C.-D.; Liu, Y.-
W.; Chiou, R. Y. Y. J. Agric. Food Chem. 2006, 54, 10281−10287.
(57) Huang, C.-P.; Au, L.-C.; Chiou, R. Y. Y.; Chung, P.-C.; Chen, S.-
Y.; Tang, W.-C.; Chang, C.-L.; Fang, W.-H.; Lin, S.-B. J. Agric. Food
Chem. 2010, 58, 12123−12129.
(21) Almeida, R. N.; Navarro, D. S.; Almeida, E. R.; Majetich, G.;
Bhattacharyya, J. Pharm. Biol. 2000, 38, 394−395.
(22) Katavic, P. L.; Lamb, K.; Navarro, H.; Prisinzano, T. E. J. Nat.
Prod. 2007, 70, 1278−1282.
(23) Gao, J. T.; Leon, F.; Radwan, M. M.; Dale, O. R.; Husni, A. S.;
Manly, S. P.; Lupien, S.; Wang, X. N.; Hill, R. A.; Dugan, F. M.; Cutler,
H. G.; Cutler, S. J. J. Nat. Prod. 2011, 74, 1636−1639.
(24) Gao, J. T.; Radwan, M. M.; Leon, F.; Dale, O. R.; Husni, A. S.;
Wu, Y. S.; Lupien, S.; Wang, X. N.; Manly, S. P.; Hill, R. A.; Dugan, F.
M.; Cutler, H. G.; Cutler, S. J. J. Nat. Prod. 2013, 76, 824−828.
(25) Lovell, K. M.; Simpson, D. S.; Cunningham, C. W.; Prisinzano,
T. E. Future Med. Chem. 2009, 1, 285−301.
(26) Jang, M. S.; Cai, E. N.; Udeani, G. O.; Slowing, K. V.; Thomas,
C. F.; Beecher, C. W. W.; Fong, H. H. S.; Farnsworth, N. R.;
Kinghorn, A. D.; Mehta, R. G.; Moon, R. C.; Pezzuto, J. M. Science
1997, 275, 218−220.
(27) Gupta, Y. K.; Sharma, M.; Briyal, S. Methods Find. Exp. Clin.
Pharmacol. 2004, 26, 667−672.
(28) Sobolev, V. S.; Khan, S. I.; Tabanca, N.; Wedge, D. E.; Manly, S.
P.; Cutler, S. J.; Coy, M. R.; Becnel, J. J.; Neff, S. A.; Gloer, J. B. J.
Agric. Food Chem. 2011, 59, 1673−1682.
(29) Arunlakshana, O.; Schild, H. O. Br. J. Pharm. Chemother. 1959,
14, 48−58.
(30) Kreek, M. J.; Koob, G. F. Drug Alcohol Depend. 1998, 51, 23−47.
(31) Sinha, R.; Fuse, T.; Aubin, L. R.; O’Malley, S. S.
Psychopharmacology (Berlin, Ger.) 2000, 152, 140−148.
(32) McMahon, R. C. J. Subst. Abuse Treat. 2001, 21, 77−87.
(33) Thomas, J. B.; Atkinson, R. N.; Vinson, N. A.; Catanzaro, J. L.;
Perretta, C. L.; Fix, S. E.; Mascarella, S. W.; Rothman, R. B.; Xu, H.;
Dersch, C. M.; Cantrell, B. E.; Zimmerman, D. M.; Carroll, F. I. J. Med.
Chem. 2003, 46, 3127−3137.
(34) Beardsley, P. M.; Howard, J. L.; Shelton, K. L.; Carroll, F. I.
Psychopharmacology (Berlin, Ger.) 2005, 183, 118−126.
(35) Carey, A. N.; Borozny, K.; Aldrich, J. V.; McLaughlin, J. P. Eur. J.
Pharmacol. 2007, 569, 84−89.
(36) Neighbors, J. D.; Beutler, J. A.; Wiemer, D. F. J. Org. Chem.
2005, 70, 925−931.
(37) Kuder, C. H.; Neighbors, J. D.; Hohl, R.; Wiemer, D. F. Biorg.
Med. Chem. 2009, 17, 4718−4723.
(38) Neighbors, J. D.; Salnikova, M. S.; Beutler, J. A.; Wiemer, D. F.
Bioorg. Med. Chem. 2006, 14, 1771−1784.
(39) Mente, N. R.; Neighbors, J. D.; Wiemer, D. F. J. Org. Chem.
2008, 73, 7963−7970.
(40) Kodet, J. G. Ph.D. Thesis, University of Iowa, 2010.
(41) Ulrich, N. C.; Kodet, J. G.; Mente, N. R.; Kuder, C. H.; Beutler,
J. A.; Hohl, R. J.; Wiemer, D. F. Bioorg. Med. Chem. 2010, 18, 1676−
1683.
(42) Thomas, J. B.; Fix, S. E.; Rothman, R. B.; Mascarella, S. W.;
Dersch, C. M.; Cantrell, B. E.; Zimmerman, D. M.; Carroll, F. I. J. Med.
Chem. 2004, 47, 1070−1073.
(43) Prisinzano, T. E. J. Med. Chem. 2013, 56, 3435−3443.
(44) Tan, Y. L.; White, A. J. P.; Widdowson, D. A.; Wilhelm, R.;
Williams, D. J. J. Chem. Soc., Perkin Trans. 1 2001, 3269−3280.
(45) Zhou, Q.; Snider, B. B. J. Org. Chem. 2010, 75, 8224−8233.
(46) Li, Y. L.; Zhao, Y. L. Chin. Chem. Lett. 1994, 5, 935−938.
319
dx.doi.org/10.1021/np4009046 | J. Nat. Prod. 2014, 77, 311−319