Synthesis of a Coumarin-Based
Analogue of Schweinfurthin
F
The natural schweinfurthins are stilbenes with significant antiproliferative activity and an uncertain mechanism of action. To obtain a fluorescent analogue with minimal deviation from the natural structure, a coumarin ring system was annulated to the D-ring, creating a new analogue of schweinfurthin F. This stilbene was prepared through a convergent synthesis, with a Horner–Wadsworth–Emmons condensation employed to form the central stilbene olefin. After preparation of a tricyclic phosphonate via a recent and more efficient modification of the classic Arbuzov reaction, condensation was attempted with an appropriately substituted bicyclic aldehyde but the coumarin system did not survive the reaction conditions. When olefin formation preceded generation of the coumarin, the stilbene formation proceeded smoothly and ultimately allowed access to the targeted coumarin-based schweinfurthin analogue. This analogue displayed the desired fluorescence properties along with significant biological activity in the National Cancer Institute’s 60-cell line bioassay, and the pattern of this biological activity mirrored that of the natural product schweinfurthin F. This approach gives facile access to new fluorescent analogues of the natural schweinfurthins and should be applicable to other natural stilbenes as well.
Introduction<!><!>Introduction<!><!>Introduction<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!>General Section<!>4-Bromo-3,5-dihydroxybenzoic
Acid (23)<!>Methoxymethyl 4-Bromo-3,5-bis(methoxymethoxy)benzoate (24)<!>[4-Bromo-3,5-bis(methoxymethoxy)phenyl]methanol (25)<!>2-Bromo-1,3-bis(methoxymethoxy)-5-(methoxymethyl)benzene
(21)<!>2,6-Bis(methoxymethoxy)-4-(methoxymethyl)benzaldehyde
(19)<!>2-Hydroxy-6-(methoxymethoxy)-4-(methoxymethyl)benzaldehyde
(26)<!>3-Acetyl-5-(methoxymethoxy)-7-(methoxymethyl)chromen-2-one
(28)<!>5-(Methoxymethoxy)-7-(methoxymethyl)-3-(2-methyl-1,3-dioxolan-2-yl)chromen-2-one
(29)<!>5-(Methoxymethoxy)-7-methyl-3-(2-methyl-1,3-dioxolan-2-yl)chromen-2-one
(30)<!>(7R,8aR,10aR)-4-Methoxy-7-(methoxymethoxy)-8,8,10a-trimethyl-6,7,8a,9-tetrahydro-5H-xanthene-2-carbaldehyde
(32)<!>[(7R,8aR,10aR)-4-Methoxy-7-(methoxymethoxy)-8,8,10a-trimethyl-6,7,8a,9-tetrahydro-5H-xanthen-2-yl]methanol (17)<!>[(2R,4aR,9aR)-7-(Diethoxyphosphonylmethyl)-5-methoxy-1,1,4a-trimethyl-3,4,9,9a-tetrahydro-2H-xanthen-2-yl]oxymethanol
(15)<!>Attempted Preparation of 7-[(E)-2-[(7R,8aR,10aR)-4-Methoxy-7-(methoxymethoxy)-8,8,10a-trimethyl-6,7,8a,9-tetrahydro-5H-xanthen-2-yl]vinyl]-3-(2-methyl-1,3-dioxolan-2-yl)chromen-2-one
(33)<!>[4-Bromo-3,5-bis(methoxymethoxy)phenyl]methoxy tert-Butyldimethylsilane (34)<!>4-(((tert-Butyldimethylsilyl)oxy)methyl)-2,6-bis(methoxymethoxy)benzaldehyde
(35)<!>tert-Butyl-[[4-(1,3-dioxolan-2-yl)-3,5-bis(methoxymethoxy)phenyl]methoxy]dimethylsilane
(36)<!>[4-(1,3-Dioxolan-2-yl)-3,5-bis(methoxymethoxy)phenyl]methanol
(37)<!>4-(1,3-Dioxolan-2-yl)-3,5-bis(methoxymethoxy)benzaldehyde
(38)<!>(2R,4aR,9aR)-7-[(E)-2-[4-(1,3-Dioxolan-2-yl)-3,5-bis(methoxymethoxy)phenyl]vinyl]-5-methoxy-2-(methoxymethoxy)-1,1,4a-trimethyl-3,4,9,9a-tetrahydro-2H-xanthene (39)<!>4-[(E)-2-[(7R,8aR,10aR)-7-Hydroxy-4-methoxy-8,8,10a-trimethyl-6,7,8a,9-tetrahydro-5H-xanthen-2-yl]vinyl]-2,6-dihydroxybenzaldehyde (40)<!>7-[(E)-2-[(7R,8aR,10aR)-7-Hydroxy-4-methoxy-8,8,10a-trimethyl-6,7,8a,9-tetrahydro-5H-xanthen-2-yl]vinyl]-3-acetyl-5-hydroxychromen-2-one (41)<!>Ethyl 7-Methyl-3-oxo-6-octenoate
(42)<!>5-(Methoxymethoxy)-7-(methoxymethyl)-3-(5-methylhex-4-enoyl)chromen-2-one
(43)<!>
<p>The schweinfurthins
(Figure 1) are a small
group of natural products isolated, at least
thus far, from plants of the genus Macaranga (Euphorbiaceae) directly1,2 or indirectly from propolis
produced by bees visiting Macaranga plants.3 The combination of an unusual
pattern of differential activity in the National Cancer Institute's
(NCI's) 60-cell line screen4,5 and isolation
efforts that have resulted in limited and sometimes poorly reproducible
quantities6 has encouraged us to pursue
efforts to synthesize these compounds and a variety of analogues.
To date, we have reported the total synthesis of several natural schweinfurthins
that include the hexahydroxanthene system [A (1),7 B (2),8 E (3),8 F (5),9 G (6),10 and vedelianin (4)],11 including one as both enantiomers to allow determination
of the absolute stereochemistry of the natural products (Figure 1). We also have prepared
approximately 90 analogues that have been evaluated in the NCI's
60-cell line screen for structure activity studies.7−9,12,13 Results of these studies
indicate that the A/B/C ring system and a stilbene in the trans orientation are essential to the selective antiproliferative
activity of these compounds, while modifications of the D-ring are
generally better tolerated. Of special significance, studies of structure–activity
relationships and chemical stability have revealed that the D-ring
resorcinol may limit the schweinfurthins' stability. Thus,
optimum placement of a coumarin system might preserve the biological
activity and simultaneously improve the chemical stability. Methylation
of one of the symmetric D-ring phenolic groups has been shown to significantly
increase chemical stability and testing in the NCI-60 assay revealed
that there is little or no loss in activity relative to the corresponding
non-methylated compounds. Furthermore, the indole analogues 7 and 8 also have shown significant activity,
suggesting a tolerance for substitution at a single phenolic position.14</p><!><p>Relevant natural schweinfurthins and two indole analogues.</p><!><p>The NCI created the COMPARE algorithm to associate
bioactivity
data from each candidate in the NCI-60 bioassay with those from other
compounds that have also been through the screening process and function
by a similar mechanism of action rather than by structural similarity.15 In the COMPARE analysis, the schweinfurthin
family did not pose biological resemblance to that of any chemotherapeutic
agent currently in use, but rather, the activity of the schweinfurthin
family most closely resembles that of the cephalostatins (e.g. 9), the ritterazines (e.g. 10), the stellettins
(e.g. 11), and OSW-1 (12, Figure 2).13 Deeper investigation into the mechanism of action of the schweinfurthins
by several groups has not yet provided a complete and clear mode of
action. Studies have suggested interactions between several targets
including oxysterol binding proteins,16−18 trans-Golgi-network
trafficking,19 and the production and export
of cholesterol20 and other products of
isoprenoid biosynthesis.21</p><!><p>Natural products that
display biological activity similar to that
of schweinfurthins, based on COMPARE analyses.</p><!><p>To increase understanding of the mechanism of action for the schweinfurthins,
it might be useful to prepare a fluorescent analogue, as long as that
analogue displays significant biological activity. To maximize the
possibility of activity, it appeared prudent to anneal a coumarin
ring to the D-ring, as suggested in structure 13 (Figure 3). Compound 13 would preserve the complete hexahydroxanthene system of
a mono-methylated schweinfurthin F (14), the trans-stilbene, and one free phenol in the D-ring. Thus,
preparation of the schweinfurthin analogues in the form of structure 13 became our goal.</p><!><p>Comparison of a coumarin-containing schweinfurthin
(13) with a mono-methylated schweinfurthin F (14).</p><!><p>Although
several disconnections have been explored for schweinfurthin
assembly,1,22,23 we have favored
use of late-stage Horner–Wadsworth–Emmons (HWE) condensation
to form the central stilbene olefin because this approach allows a
highly convergent synthesis. From this perspective, the coumarin-containing
schweinfurthin F analogue 13 could be seen arising from
an HWE olefination between phosphonate 15 and aldehyde 16 (Scheme 1). Phosphonate 15 may be formed from the known tricyclic
alcohol 17,10 which can be
prepared in high enantiomeric excess from commercial vanillin (18) through an enantioselective Shi epoxidation.24 If the HWE condensation were postponed to the
end of the synthetic sequence, the complementary aldehyde 16 would be required. Coumarin 16 could be prepared via
a Knoevenagel condensation between the aldehyde 19 and
a β-ketoester of the general structure 20, with
ethyl acetoacetate providing the methyl ketone and extended acetoacetates
giving larger analogues. Aldehyde 19 could be seen to
arise from bromide 21 by halogen metal exchange followed
by reaction with dimethylformamide (DMF). Finally, the benzyl methyl
ether 21 could be obtained from commercial 3,5-dihydroxybenzoic
acid (22).</p><p>Initial synthetic efforts were focused on the coumarin component
because previous syntheses of other schweinfurthins provided confidence
that an appropriate tricyclic component could be prepared. When compounds 13 and 16 include a methyl ketone, this group
can be viewed as a mimic of the prenyl group in schweinfurthin F and
a homoprenyl ketone could be imagined to mimic the geranyl substituent
of larger schweinfurthins. Our efforts began with the prenyl mimic
where R is a methyl group because this allowed use of commercially
available ethyl acetoacetate as the ketoester 20.</p><p>Although the brominated resorcinol 23 is commercially
available, it can be easily prepared in virtually quantitative yield
by treatment of 3,5-dihydroxybenzoic acid (22) with bromine
(Scheme 2). The benzoic
acid 23 has been converted to the benzylic alcohol 25 through a three-step sequence via the methyl ester,25,26 but it also was possible to accomplish this overall transformation
in just two steps by formation of the acyloxyester 24 while concurrently protecting the phenols as MOM ethers, followed
by reduction of this intermediate to the desired alcohol 25. Protection of the benzylic alcohol 25 as the methyl
ether 21 proceeded smoothly,27 and halogen metal exchange followed by treatment with DMF afforded
the desired aldehyde 19.</p><p>With the aldehyde 19 in hand, attention was turned
to formation of the desired coumarin ring system through a Knoevenagel
condensation. All efforts to form a coumarin directly from the bis-MOM-protected
compound 19 went unrewarded. Fortunately, treatment of
compound 19 with sodium bisulfate on silica resulted
in cleavage of a single MOM protecting group in reasonably good yield
(70%, Scheme 3).28,29 Grinding the resulting ortho-hydroxy benzaldehyde 26 with ethyl acetoacetate (27) and piperidine
resulted in condensation and cyclization to afford the coumarin 28. Because ketones can undergo reaction with DDQ via their
tautomeric enol forms, the ketone 28 was protected as
its acetal 29. Subsequent reaction with DDQ gave the
desired coumarin aldehyde 30 in modest yield. Preparation
of the tricyclic phosphonate 15 then was investigated
because aldehyde 30 appeared to be an appropriate substrate
for an HWE condensation.</p><p>The tricyclic phosphonate 15 was employed in our original
synthesis of schweinfurthin F,10 where
it was prepared from the corresponding alcohol 17 in
an overall yield of 62% via a classical approach involving formation
of the mesylate, displacement by sodium iodide, and an Arbuzov reaction
with triethyl phosphite. Instead, a shortened Arbuzov approach was
followed. After the aldehyde 31 was prepared via DDQ
oxidation of the corresponding benzyl methyl ether, the C-2 alcohol
was protected as the MOM ether (32) and the aldehyde
was reduced to the benzylic alcohol 17 (Scheme 4). Then the alcohol 17 simply was treated with zinc iodide and triethyl phosphite, modernized
Arbuzov conditions30 for benzylic alcohols,
which gave phosphonate 15 in a single step and 69% isolated
yield.</p><p>From all perspectives, the
HWE condensation of phosphonate 15 and aldehyde 30 was expected to be straightforward,
and parallel reactions have been employed in multiple schweinfurthin
syntheses. To our disappointment, this specific HWE condensation failed
despite repeated attempts (Scheme 5) perhaps because the coumarin subunit was not stable
under the reaction conditions. Whatever the root cause, the failure
of this reaction necessitated a redesign of the synthetic sequence.</p><p>To take full advantage of the intermediates in hand and the experience
gained with the successful reactions described above, introduction
of the coumarin ring system was postponed until after the formation
of the central stilbene. Therefore, the alcohol 25 was
protected as its TBS ether 34 to avoid potential side-reactions
during DDQ oxidation (Scheme 6). Compound 34 undergoes lithium halogen exchange
under standard conditions, and a subsequent reaction with DMF gave
the aldehyde 35. After protection of the carbonyl group
as its acetal 36, treatment with TBAF generated the primary
alcohol 37. Final MnO2 oxidation provided
the new HWE coupling partner, aldehyde 38.</p><p>The HWE condensation of phosphonate 15 and aldehyde 38 proceeded smoothly upon treatment with sodium hexamethyldisilazide,
affording the schweinfurthin analogue 39 in quantitative
yield based on recovered phosphonate 15 (Scheme 7). The three MOM acetals and
the ethylene glycol-protected aldehyde underwent hydrolysis under
acidic conditions to afford the aldehyde 40. After aldehyde 40 was combined with ethyl acetoacetate (27)
and piperidine and the reaction was allowed to stir in anhydrous MeOH,
the desired fluorescent coumarin schweinfurthin analogue 41 was obtained in high enantiomeric excess.10 The spectral data for coumarin 41 indicates that the
compound has an absorption maximum at ∼420 nm and an emission
maximum at ∼590 nm, showing the expected fluorescence.</p><p>In principal, synthesis of
other coumarin-based schweinfurthin
analogues could be based on C-alkylation of the methyl ketone in compound 41. However this would certainly require protection of the
free phenol and probably the C-2 hydroxyl group as well. A more attractive
approach might involve extension of ethyl acetoacetate (27) prior to the Knoevenagel condensation. To test this possibility,
the β-ketoester 42 was prepared via alkylation
of the ethyl acetoacetate dianion (Scheme 8).31 When compound 42 was ground in a mortar with a pestle in the presence of
piperidine to induce condensation and cyclization by mechanochemical
means, the extended coumarin 43 was obtained in an attractive
yield. Although synthesis of compound 43 demonstrates
the accessibility of more extended coumarins, pursuit of additional
schweinfurthin analogues was postponed pending the results of bioassays
on the new analogues in hand.</p><p>Both schweinfurthin analogues 40 and 41 were tested in the NCI-60 cell line
bioassay. Both compounds were
first tested in a single-dose assay and demonstrated sufficient activity
to warrant testing in the full five-dose assay. The aldehyde 40 shows modest activity against SF-295 with a GI50 of 3.0 Î¼M (Table 1 and Supporting Information). This
activity is 270 times less potent than that of natural schweinfurthin
A (1). Although aldehyde 40 is not as active
as most schweinfurthins and schweinfurthin analogues sent to the NCI,
it does show a pattern of activity similar to that of other schweinfurthins,
with a Pearson correlation coefficient of 0.66 to schweinfurthin A.32 The GI50 against each cell line in
the NCI-60 assay for compound 40 shows a pattern similar
to that of other schweinfurthins. Of the cell lines tested, analogue 40 also has the greatest activity against the CNS cancer cell
line SF-539 with a GI50 of 1.3 Î¼M, which aligns with
our expectation that the schweinfurthins have selective activity against
CNS malignancies.</p><!><p>Figure 4.</p><!><p>The five-dose assay of coumarin 41 also
shows selective
activity toward some cancer cell lines over others (c.f. Supporting Information) in a pattern of activity
strikingly similar to that of other schweinfurthins that carry a substituent
para to the stilbene linkage (i.e. more substituted variations on
the parent compound 44, Figure 4). Among the most
sensitive cell lines were the SF-295 and SF-539 human-derived glioblastoma
lines, with GI50 values of 0.51 and 0.98 Î¼M respectively,
but cells in the leukemia (RPMI-8226, 0.32 Î¼M) and renal (RXF
393, 0.43 Î¼M) panels also were sensitive as is often the case
with other schweinfurthins. Conversely, the ovarian cancer panel was
uniformly resistant, which is also typical of the schweinfurthins.
The three-fold increase in potency of the analogue 41 relative to its precursor, aldehyde 40, also is striking
and encouraging.</p><!><p>Parent compound 44.</p><!><p>In conclusion, two new schweinfurthin analogues, coumarin 41 and its immediate precursor aldehyde 40, have
been synthesized and tested for biological activity in the NCI-60
cell bioassay. Although the initial approach to the central stilbene
demonstrated only that this coumarin system did not survive the standard
HWE reaction conditions, it proved possible to incorporate the coumarin
ring system after formation of the central stilbene. Both traditional
Knoevenagel condensation and mechanical grinding of an ortho hydroxy
aldehyde with a β-ketoester allowed formation of the coumarin
system. Furthermore, the target compound 41 displays
both significant antiproliferative activity in the NCI 60-cell line
screen and fluorescent properties that may help illuminate the mechanism
of action for the schweinfurthins. Finally, the reactions and strategies
reported here might be applicable to preparation of fluorescent analogues
of other natural stilbenes, including compounds such as combretastatin,33 resveratrol34,35 and its myriad
derivatives,36 the chiricanines,37 the arachidins and arahypins,38 and the pawhuskins.39</p><!><p>Diethyl ether (Et2O) and
tetrahydrofuran (THF) were distilled from sodium and benzophenone,
and dichloromethane (CH2Cl2) was distilled from
calcium hydride prior to use. Solutions of n-BuLi
were purchased from commercial sources and titrated with diphenylacetic
acid to determine molar concentrations prior to use. All other reagents
and solvents were purchased from commercial sources and used without
further purification. The nuclear magnetic resonance (NMR) spectra
were obtained on 300, 400, or 600 MHz Bruker spectrometers with Si(CH3)4 (1H, Î´ 0.00), CDCl3 (1H, Î´ 7.26; 13C, Î´ 77.2), CD3CN (1H, Î´ 1.94; 13C, Î´ 118.3,
1.32), or (CD3)2CO (1H, Î´ 2.05; 13C, Î´ 206.3, 29.8) as internal standards. To assign
signals as C, CH, CH2, or CH3, DEPT-135 NMR
spectra were obtained. 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 column chromatography.
The UV–vis spectra were obtained on a Cary UV–vis NIR
spectrophotometer, and fluorescence data were collected on HORIBA
Scientific FluroMax-4. A quartz (200–2500 nm) 1400 μL
Hellma Analytics cuvette (semi-micro cell type 114F-QS) with a 10
mm × 4 mm path length fitted with a PTFE stopper was used for
UV–vis and fluorometry.</p><!><p>To an oven-dried and argon-purged
round-bottom flask containing 3,5-dihydroxybenzoic
acid (22, 10.0 g, 64.5 mmol) was added aqueous 20% HCl
(110 mL) followed by a dropwise addition of bromine (3.31 mL, 64.5
mmol). The reaction was heated in an oil bath under reflux for 3 h
and then was quenched by addition of ice. The solution was washed
with Et2O (3 × 50 mL), and the combined organic layers
were dried (Na2SO4) and then filtered through
a bed of Celite, and the filtrate was concentrated on a rotary evaporator
to afford aryl bromide 23 as an off-white solid (14.9
g, 99%). Both the 1H NMR and 13C NMR spectra
were in agreement with the reported data.40</p><!><p>To an oven-dried and argon-purged round-bottom
flask containing the carboxylic acid 23 (400 mg, 1.7
mmol) in CH2Cl2 (20 mL) at 0 °C was added
dropwise DIPEA (1.0 mL, 6.0 mmol) followed by a dropwise addition
of MOMCl (520 Î¼L, 6.9 mmol). The reaction was allowed to stir
at 0 °C for 2.5 h, and then the reaction was quenched by addition
of saturated NH4Cl (10 mL) and the organic compounds were
extracted into CH2Cl2 (3 × 20 mL). The
combined organic layers were washed with 3 N NaOH (15 mL) and dried
(Na2SO4), and the solids were removed by filtration.
The filtrate was concentrated on a rotary evaporator to afford compound 24 as a white solid (0.58 g, 93%): 1H NMR (400
MHz, CDCl3): Î´ 7.51 (s, 2H), 5.46 (s, 2H), 5.30 (s,
4H), 3.53 (s, 3H), 3.52 (s, 6H); 13C{1H} NMR
(101 MHz, CDCl3): Î´ 165.3, 154.9 (2C), 130.0, 110.2
(2C), 109.9, 95.1 (2C), 91.2, 57.9, 56.6 (2C). HRMS (ESI) m/z: [M + Na]+ calcd for C13H17O7BrNa, 387.0055; found, 387.0060.</p><!><p>To an oven-dried and argon-purged round-bottom flask containing
compound 24 (4.56 g, 12.5 mmol) in THF (60 mL) was slowly
added solid NaBH4 (4.02 g, 106 mmol), and the reaction
was allowed to stir at 65 °C for 15 min. After MeOH (60 mL) was
added dropwise, the reaction was heated under reflux in an oil bath
for an additional 2 h. After cooling to rt, the reaction was quenched
by dropwise addition of saturated NH4Cl (50 mL). The organic
compounds were extracted into EtOAc, the combined organic layers were
dried over Na2SO4, and the solids were removed
by filtration. The filtrate was concentrated on a rotary evaporator
to afford the benzylic alcohol 25 as a white solid (2.76
g, 72%): 1H NMR (400 MHz, CDCl3): Î´ 6.85
(s, 2H), 5.26 (s, 4H), 4.64 (s, 2H), 3.52 (s, 6H); 13C{1H} NMR (100 MHz, (CD3)2CO): Î´
154.8 (2C), 143.6, 107.3 (2C), 101.15, 95.4 (2C), 63.3, 55.6 (2C).25</p><!><p>To a solution of the alcohol 25 (8.66
g, 28.2 mmol) in THF (200 mL) was slowly added 60% NaH in oil (1.27
g, 53.0 mmol), and the reaction then was allowed to stir at 0 °C
for 5 min. To the solution was added iodomethane (2.19 mL, 35.3 mmol),
and the solution was allowed to stir for 2 h. After the reaction was
quenched by addition of H2O, it was extracted with EtOAc.
The combined organic layers were washed with 1 N NaOH, dried over
Na2SO4, and filtered, and the filtrate was concentrated
on a rotary evaporator to afford the methyl ether 21 as
colorless crystals (8.71 g, 96%): 1H NMR (400 MHz, (CD3)2CO): Î´ 6.88 (s, 2H), 5.30 (s, 4H), 4.40
(s, 2H), 3.49 (s, 6H), 3.36 (s, 3H); 13C{1H}
NMR (100 MHz, (CD3)2CO): Î´ 154.9 (2 C),
152.0 (C), 139.8 (C), 108.0 (2 CH), 94.9 (2 CH2), 73.5
(CH2), 57.4 (CH3), 55.6 (2 CH3).27</p><!><p>To a flame-dried and argon-purged round-bottom
flask containing n-BuLi (2.48 M, 5.5 mL, 13 mmol)
at −78 °C was added a −78 °C solution of the
aryl bromide 21 (3.55 g, 11.1 mmol) in Et2O (200 mL) using a cannula. The solution was allowed to stir for
15 min and then DMF (0.94 mL, 12 mmol) was added dropwise. Once the
reaction reached rt, it was quenched by addition of NH4Cl and extracted with Et2O. The combined organic layers
were dried over Na2SO4 and filtered, and the
filtrate was concentrated on a rotary evaporator to afford a yellow
oil. Final purification was achieved using an ISCO auto-column (0%–100%
EtOAc in hexanes) to afford aldehyde 19 as a yellow oil
(1.52 g, 50%): 1H NMR (400 MHz, CDCl3): Î´
10.51 (s, 1H), 6.82 (s, 2H), 5.27 (s, 4H), 4.42 (s, 2H), 3.50 (s,
6H), 3.41 (s, 3H); 13C{1H} NMR (100 MHz, CDCl3): Î´ 188.5 (CH), 159.4 (2 CH), 147.1 (C), 114.9 (C),
106.6 (2 C), 94.6 (2 CH2), 73.8 (CH2), 58.3
(CH3), 56.3 (2 CH3). HRMS (ESI) m/z: [M + Na]+ calcd for C13H18O6Na, 293.0996; found, 293.0995.</p><!><p>A mixture of compound 19 (1.38
g,
5.09 mmol) and NaHSO4 on SiO2 (3.06 g)28,29 was allowed to stir in CH2Cl2 (75 mL) at rt
for 10 min. The mixture was filtered through Celite, washed with CH2Cl2, and the filtrate was concentrated on a rotary
evaporator to afford the phenol 26 (806 mg, 70%): 1H NMR (300 MHz, CDCl3): Î´ 11.85 (s, 1H),
10.23 (s, 1H), 6.51 (s, 1H), 6.44 (s, 1H), 5.23 (s, 2H), 4.31 (s,
2H), 3.43 (s, 3H), 3.32 (s, 3H); 13C{1H} NMR
(75 MHz, CDCl3): Î´ 193.7 (CH), 163.3 (C), 160.0 (C),
150.3 (C), 110.3 (C), 108.4 (CH), 102.2 (CH), 94.5 (CH2), 73.7 (CH2), 58.3 (CH3), 56.4 (CH3). HRMS (ESI) m/z: [M + H]+ calcd for C11H15O5, 227.0920;
found, 227.0913.</p><!><p>The aldehyde 26 (515 mg, 2.28
mmol)
was combined with ethyl acetoacetate (27, 580 Î¼L,
4.55 mmol) and piperidine (nine drops) in a mortar, and the solution
was ground with a pestle for 30 min. The residue was transferred to
a round-bottom flask with EtOAc, and the solvent was removed using
a rotary evaporator. Final purification by crystallization from hot
EtOH and H2O gave the coumarin 28 (329 mg,
50%): 1H NMR (300 MHz, CDCl3): Î´ 8.87
(s, 1H), 6.96 (s, 1H), 6.93 (s, 1H), 5.33 (s, 2H), 4.50 (s, 2H), 3.51
(s, 3H), 3.44 (s, 3H), 2.70 (s, 3H); 13C{1H}
NMR (75 MHz, CDCl3): Î´ 195.3 (C), 159.6 (C), 157.2
(C), 156.5 (C), 149.1 (C), 142.2 (CH), 123.6 (C), 109.9 (C), 108.1
(CH), 108.0 (CH), 95.1 (CH2), 74.3 (CH2), 59.0
(CH3), 57.0 (CH3), 30.4 (CH3). HRMS
(ESI) m/z: [M + H]+ calcd
for C15H17O6, 293.1020; found, 293.1019.</p><!><p>To a round-bottom flask containing the
ketone 28 (250 mg, 850 Î¼mol) in benzene (20 mL)
were added ethylene glycol (0.34 mL, 6.0 mmol) and pyridinium p-toluenesulfonate (43 mg, 0.17 mmol). The solution was
heated in an oil bath under reflux with a Dean–Stark trap in
place for 18 h. After it had cooled to rt, the reaction was quenched
by addition of saturated NaHCO3 (5 mL) and extracted with
EtOAc, and the combined organic layers were dried (Na2SO4) and filtered. The filtrate was concentrated by rotary evaporation.
Final purification was achieved through flash column chromatography
(20% EtOAc in hexanes) to afford the acetal 29 as colorless
crystals (110 mg, 39%) and recovered ketone 28 (44 mg,
18%). For the acetal: 1H NMR (400 MHz, CDCl3): Î´ 8.27 (s, 1H), 6.96 (s, 1H), 6.93 (s, 1H), 5.33 (s, 2H),
4.40 (s, 2H), 4.10 (m, 2H), 3.92 (m, 2H), 3.55 (s, 3H), 3.44 (s, 3H),
1.83 (s, 3H); 13C{1H} NMR (100 MHz, CDCl3): Î´ 159.4 (C), 154.9 (C), 154.2 (C), 143.8 (C), 133.6
(CH), 126.7 (C), 109.0 (C), 108.1 (CH), 107.2 (CH), 107.1 (C), 94.9
(CH2), 74.0 (CH2), 65.0 (2 CH2),
58.5 (CH3), 56.6 (CH3), 24.4 (CH3). HRMS (ESI) m/z: [M + H]+ calcd for C17H21O7, 337.1282;
found, 337.1278.</p><!><p>To a solution of the methyl ether 29 (110 mg, 330 Î¼mol), CH2Cl2 (5
mL), and H2O (0.5 mL) was added DDQ (210 mg, 900 Î¼mol),
and the mixture was allowed to stir at rt for 24 h. The mixture was
quenched by addition of saturated NaHCO3, extracted with
CH2Cl2, and the combined organic layers were
washed with brine, dried (Na2SO4), and filtered.
The filtrate was concentrated on a rotary evaporator. Final purification
was achieved through flash column chromatography (20% EtOAc in hexanes)
to afford the aldehyde 30 as a yellow oil (45 mg, 43%): 1H NMR (400 MHz, CDCl3): Î´ 10.00 (s, 1H),
8.29 (s, 1H), 7.48 (s, 1H), 7.43 (s, 1H), 5.38 (s, 2H), 4.12 (m, 2H),
3.95 (m, 2H), 3.53 (s, 3H), 1.82 (s, 3H); 13C{1H} NMR (100 MHz, CDCl3): Î´ 190.7 (CH), 158.5 (C),
154.8 (C), 154.7 (C), 138.8 (C), 132.7 (CH), 129.8 (C), 118.1 (C),
111.9 (CH), 107.4 (CH), 107.0 (C), 95.1 (CH2), 65.1 (2
CH2), 56.8 (CH3), 24.5 (CH3). HRMS
(ESI) m/z: [M + H]+ calcd
for C16H16O7Na, 343.0788; found,
343.0796.</p><!><p>To a vial containing aldehyde 31(8) (177 mg, 580 μmol) in CH2Cl2 (5 mL) at 0 °C was added dropwise DIPEA (110
Î¼L, 0.64 mmol). The solution was allowed to stir for 10 min,
and then MOMCl (50 Î¼L, 0.64 mmol) was added dropwise. After
the solution was allowed to stir for 72 h, it was quenched by addition
of NH4Cl and was extracted with CH2Cl2. The combined organic layers were washed with 1 N NaOH, dried (Na2SO4), and filtered, and the filtrate was concentrated
in vacuo to afford aldehyde 32 (140 mg, 70%): 1H NMR (400 MHz, CDCl3): δ 9.80 (s, 1H), 7.26–7.23
(m, 2H), 4.79 (d, J = 6.9 Hz, 1H), 4.66 (d, J = 7.0 Hz, 1H), 3.90 (s, 3H), 3.41 (s, 3H), 3.31–3.24
(m, 1H), 2.80–2.78 (m, 2H), 2.18 (td, J =
12.9, 3.6 Hz, 1H), 2.02 (dq, J = 14.4, 4.0 Hz, 1H),
1.82 (ddd, J = 13.8, 13.8, 4.1 Hz, 1H), 1.72 (dd, J = 11.3, 7.5 Hz, 1H), 1.65–1.58 (m, 1H), 1.28 (s,
3H), 1.10 (s, 3H), 0.92 (s, 3H); 13C NMR (101 MHz, CDCl3): Î´ 191.2, 149.6, 148.9, 129.0, 127.4, 122.6, 107.4,
96.3, 83.8, 78.5, 56.1, 55.7, 46.7, 38.4, 37.5, 27.5, 25.4, 23.1,
20.1, 15.2. HRMS (ESI) m/z: [M +
H]+ calcd for C20H29O5, 349.2015; found, 349.2006.</p><!><p>To an
oven-dried and argon-purged round-bottom flask containing the aldehyde 32 (350 mg, 1.00 mmol) in THF (10 mL) and MeOH (2 mL) at 0
°C was added solid NaBH4 (234 mg, 1.60 mmol). The
solution was allowed to stir for 40 min, was quenched by addition
of H2O, and then was extracted with EtOAc. The combined
organic layers were washed with saturated NaHCO3 and brine
and dried (MgSO4). After filtration, the filtrate was concentrated
on a rotary evaporator to afford the benzylic alcohol 17 (352 mg, 100%) as a colorless oil. Both the 1H and 13C NMR spectra were in agreement with data reported in the
literature.10</p><!><p>To an oven-dried and argon-purged round-bottom
flask containing ZnI2 (1.1 g, 3.5 mmol) and triethyl phosphite
(400 Î¼L, 2.3 mmol) in THF (15 mL) was added benzylic alcohol 17 (410 mg, 1.2 mmol). The reaction was heated in an oil bath
under reflux for 17 h. The solution was concentrated in vacuo to 1
mL, and then the residue was dissolved in Et2O, which caused
formation of a solid that was removed by filtration. After the filtrate
was washed with 1 N NaOH (0.5 mL), the organic layer was dried (Na2SO4) and filtered, and the filtrate was concentrated
on a rotary evaporator. Excess triethyl phosphite was removed using
high vacuum to afford phosphonate 15 (370 mg, 69%) as
a colorless oil. Both the 1H and 31P NMR spectra
were in agreement with data reported for compound 15 prepared
by a traditional Arbuzov sequence.10</p><!><p>To a flame-dried round-bottom flask containing
the phosphonate 15 (78 mg, 0.17 mmol) in THF (1 mL) at
0 °C was added NaH (60% dispersion in oil, 10 mg, 0.25 mmol).
To the stirring solution was added the aldehyde 30 (8.6
mg, 26 Î¼mol) in THF (2 mL). The reaction was allowed to warm
to rt naturally and then was quenched by addition of H2O. The organic compounds were extracted into EtOAc, dried (Na2SO4), and filtered, and the filtrate was concentrated in vacuo. The desired stilbene 33 could not
be detected in the resulting material.</p><!><p>To an
oven-dried
and argon-purged round-bottom flask containing alcohol 25 (1.3 g, 3.4 mmol) in anhydrous CH2Cl2 (200
mL) was added imidazole (470 mg, 6.9 mmol) followed by TBSCl (560
mg, 3.8 mmol), and the reaction was allowed to stir at rt for 14 h.
After the reaction was quenched by addition of water, the aqueous
layer was extracted with CH2Cl2. The combined
organic layers were washed with brine, dried (Na2SO4), and filtered through a pad of Celite, and the filtrate
was concentrated under reduced pressure to afford the silyl ether 34 as a yellow oil (1.4 g, 97%): 1H NMR (400 MHz,
CDCl3): Î´ 6.84 (s, 2H), 5.24 (s, 4H), 4.68 (s, 2H),
3.52 (s, 6H), 0.94 (s, 9H), 0.10 (s, 6H). 13C{1H} NMR (75 MHz, CDCl3): Î´ 158.5 (2 C), 144.4 (C),
107.3 (2 CH), 103.5 (C), 94.7 (2 CH2), 64.9 (CH2), 56.1 (2 CH3), 26.1 (3 CH3), 18.6 (C), −5.1
(2 CH3).41</p><!><p>An oven-dried round-bottom flask containing
aryl bromide 34 (5.6 g, 13 mmol) in Et2O (150
mL) was cooled to −78 °C for 20 min. To the solution was
added n-BuLi (7.4 mL, 18 mmol, 2.4 M). Immediately
after the addition was complete, anhydrous DMF (1.4 mL, 18 mmol) was
added dropwise and the reaction was allowed to stir and warm to rt
overnight. After the reaction was quenched by addition of saturated
NH4Cl (50 mL), the organic compounds were extracted into
Et2O (3 × 50 mL). The combined organic layers were
dried (Na2SO4), the solids were removed by filtration,
and the filtrate was concentrated on a rotary evaporator. Final purification
was achieved by ISCO normal-phase auto-chromatography (0–5%
EtOAc in hexanes), which gave aldehyde 35 as a yellow
oil (2.61 g, 53%). Both the 1H and 13C NMR spectra
match the literature data for material prepared by a different route.421H NMR (300 MHz, CDCl3): Î´ 10.34 (s, 1H), 6.70 (s, 2H), 5.09 (s, 4H), 4.56 (s, 2H),
3.32 (s, 6H), 0.80 (s, 9H), 0.05 (s, 6H).</p><!><p>A round-bottom flask containing aldehyde 35 (680 mg, 1.8 mmol), ethylene glycol (660 Î¼L, 11 mmol),
and PPTS (110 mg, 370 Î¼mol) in benzene (50 mL) was fitted with
a Dean–Stark trap and heated in an oil bath under reflux. The
reaction progress was monitored periodically by thin-layer chromatography.
After 1 h, the reaction was allowed to cool to rt, quenched by addition
of saturated NaHCO3 (5 mL), and extracted with EtOAc (3
× 20 mL). The combined organic layers were dried (Na2SO4), the solid was removed by gravity filtration, and
the resulting filtrate was concentrated on a rotary evaporator to
afford acetal 36 (720 mg, 96%): 1H NMR (400
MHz, CDCl3) 6.74 (2H, s), 6.40 (1H, s), 5.09 (4H, s), 4.63
(2H, s), 4.11 (2H, m), 3.89 (2H, m), 3.39 (6H, s), 0.89 (9H, s), 0.03
(6H, s); 13C NMR (101 MHz, CDCl3) 157.4 (C),
145.0 (C), 114.1 (2 CH), 106.5 (CH), 98.9 (2 C), 94.7 (2 CH2), 65.6 (2 CH2), 64.4 (CH2), 56.1 (2 CH3), 26.2 (3 CH3), 18.4 (C), −5.4 (2 CH3). HRMS (ESI) m/z: [M +
H]+ calcd for C20H35O7Si, 417.2147; found, 415.2145.</p><!><p>To a flask containing the silyl ether 36 (2.5 g, 6.0 mmol) in THF (150 mL) was added TBAF (1.0 M
in THF, 6.0 mL, 6.0 mmol), and the solution was allowed to stir at
0 °C and naturally warm to rt over 1 h. The reaction was quenched
by addition of water, and the organic compounds were extracted into
EtOAc (3 × 100 mL), washed with brine, and dried (Na2SO4). The solids were removed by gravity filtration, and
the filtrate was concentrated on a rotary evaporator to afford alcohol 37 as a pale yellow solid (1.7 g, 94%): 1H NMR
(400 MHz, CDCl3) 6.81 (2H, s), 6.48 (1H, s), 5.20 (4H,
s), 4.57 (2H, s), 4.24–4.19 (2H, m), 4.04–4.01 (2H,
m), 3.50 (6H, s); 13C NMR (101 MHz, CDCl3):
Î´ 157.3 (2 C), 144.2 (C), 114.8 (C), 106.9 (2 CH), 98.5 (CH),
94.7 (2 CH2), 66.1 (2 CH2), 65.4 (CH2), 56.3 (2 CH3). HRMS (ESI) m/z: [M + H]+ calcd for C14H21O7, 301.1282; found, 301.1280.</p><!><p>To a flame-dried and argon-purged round-bottom
flask containing alcohol 37 (210 mg, 690 Î¼mol)
in anhydrous CH2Cl2 (20 mL) was slowly added
manganese dioxide (1.5 g, 14 mmol). The resulting mixture was allowed
to stir at rt for 26 h. The mixture then was filtered through a bed
of Celite, and the filtrate was concentrated to afford aldehyde 38 (180 mg, 86%): 1H NMR (400 MHz, CDCl3): Î´ 9.89 (s, 1H), 7.29 (s, 2H), 6.50 (s, 1H), 5.24 (s, 4H),
4.23–4.20 (m, 2H), 4.04–4.00 (m, 2H), 3.49 (s, 6H); 13C NMR (101 MHz, CDCl3): δ 191.7 (CH), 158.0
(2 C), 138.1 (C), 121.5 (C), 109.7 (2 CH), 98.1 (CH), 94.5 (2 CH2), 66.5 (2 CH2), 56.6 (2 CH3). HRMS
(ESI) m/z: [M + H]+ calcd
for C14H19O7, 299.1125; found, 299.1127.</p><!><p>To a flame-dried and
argon-purged round-bottom flask containing the phosphonate 15 (116 mg, 250 Î¼mol) and the aldehyde 38 (61 mg,
0.21 mmol) in THF (12 mL) at 0 °C was added dropwise NaHMDS (1.0
M in THF, 680 Î¼L, 680 Î¼mol), and the mixture was allowed
to stir at 0 °C for 32 h. The reaction was quenched by a dropwise
addition of saturated NH4Cl to reach a pH of 8, and the
organic compounds were extracted into EtOAc (3 × 20 mL). The
combined organic layers were washed with brine and dried (MgSO4), the solids were removed by filtration, and the filtrate
was concentrated on a rotary evaporator. Final purification was achieved
by column chromatography (15–100% EtOAc in hexanes) to afford
the recovered phosphonate 15 (98 mg, 83%) and the desired
stilbene 39 as a fluorescent yellow oil (25 mg, 16%): 1H NMR (400 MHz, CDCl3): δ 6.98 (d, J = 14.8 Hz, 1H), 6.92 (s, 2H), 6.90–6.84 (m, 3H),
6.47 (s, 1H), 5.22 (s, 4H), 4.77 (d, J = 7.1 Hz,
1H), 4.65 (d, J = 6.4 Hz, 1H), 4.24–4.19 (m,
2H), 4.02–3.99 (m, 2H), 3.89 (s, 3H), 3.51 (s, 6H), 3.41 (s,
3H), 3.28 (dd, J = 11.3, 3.5 Hz, 1H), 2.71 (d, J = 8.6 Hz, 1H), 2.15 (d, J = 12.9 Hz,
1H), 2.02–1.92 (m, 1H), 1.84–1.75 (m, 1H), 1.75–1.69
(m, 1H), 1.65–1.54 (m, 2H), 1.25 (s, 3H), 1.10 (s, 3H), 0.91
(s, 3H); 13C NMR (101 MHz, CDCl3): Î´ 157.5
(2C), 149.3, 142.9, 140.6, 130.1, 126.1, 122.7, 121.1, 114.8, 109.7,
107.0, 106.8 (2C), 98.8, 96.5, 94.9 (2C), 84.1, 66.0 (2C), 56.3, 56.0,
55.6, 47.0, 38.3, 37.6, 29.8, 27.5, 25.4, 23.1, 19.9, 15.2, 14.2.
HRMS (ESI) m/z: [M + H]+ calcd for C34H47O17, 615.3164;
found, 615.3171.</p><!><p>To the acetal 39 (35 mg, 57 Î¼mol) was
added 5 M HCl (5 mL) and anhydrous MeOH (15 mL); the solution was
allowed to stir at rt for 69 h, and then was quenched by addition
of saturated NaHCO3 to a pH of 7. The organic compounds
were extracted into EtOAc (2 × 25 mL), the combined organic layers
were dried (MgSO4), and the solids were removed by filtration.
The filtrate was concentrated to afford the aldehyde 40 as an orange solid (26 mg, 100%): 1H NMR (400 MHz, CDCl3): Î´ 10.27 (s, 1H), 7.05 (d, J = 15.9
Hz, 1H), 6.85 (m, 2H), 6.76 (d, J = 15.4 Hz, 1H),
6.47 (s, 2H), 3.87 (s, 3H), 3.44 (d, J = 6.5 Hz,
1H), 2.71 (d, J = 8.6 Hz, 2H), 2.18–2.07 (m,
2H), 1.92–1.80 (m, 1H), 1.73–1.49 (m, 2H), 1.24 (s,
3H), 1.09 (s, 3H), 0.87 (s, 3H); 13C NMR (101 MHz, CDCl3): Î´ 193.1 (CH), 149.1 (2 C), 148.1, 143.7, 133.2, 128.0,
125.1 (2 C), 123.0, 121.7, 109.5, 107.4, 105.2, 78.4, 77.5, 56.1,
46.8, 38.5, 37.6, 29.7, 28.2, 27.3, 23.1, 19.9, 14.3. HRMS (ESI) m/z: [M – H]– 
calcd for C26H29O6, 437.1964; found,
437.1965.</p><!><p>To a flame-dried round-bottom flask containing
the aldehyde 40 (19 mg, 41 Î¼mol) in anhydrous MeOH
(2 mL) were added ethyl acetoacetate (27, 5.2 Î¼L,
41 Î¼mol) and piperidine (2.0 Î¼L, 20 Î¼mol) and the
sides of the flask were washed with 1 mL of anhydrous MeOH. The solution
was allowed to stir in a foil-covered flask at rt for 75 h, and then
the reaction was quenched by addition of H2O (10 mL). The
organic compounds were extracted into CH2Cl2 (3 × 20 mL), the combined organic layers were dried (Na2SO4), and the solids were removed by filtration.
The filtrate was concentrated on a rotary evaporator, and the resulting
material was purified by column chromatography (50–100% EtOAc
in hexanes). Final purification was achieved by washing the solid
with pentane (3 × 2 mL) to afford coumarin 41 as
a fluorescent orange solid (22 mg, 100%): 1H NMR (400 MHz,
CD3CN): δ 8.66 (s, 1H), 7.27 (d, J = 15.9 Hz, 1H), 7.07–7.01 (m, 3H), 6.94 (s, 2H), 3.86 (s,
3H), 3.71–3.67 (m, 1H), 2.76 (d, J = 8.8 Hz,
2H), 2.61 (s, 3H), 1.80–1.71 (m, 3H), 1.71–1.65 (m,
2H), 1.22 (s, 3H), 1.09 (s, 3H), 0.87 (s, 3H); 13C NMR
(151 MHz, CD3CN): Î´ 195.3, 169.0, 166.5, 160.8, 159.1,
156.2, 149.4, 148.9, 141.7, 133.2, 124.3, 122.8, 121.9, 121.1, 108.1,
108.0, 107.7, 104.4, 77.1, 76.9, 55.2, 46.5, 38.2, 37.5, 29.2, 26.7,
25.9, 23.4, 19.3, 13.9. HRMS (ESI) m/z: [M + Na]+ calcd for C30H32O7Na, 527.2046; found, 527.2051.</p><!><p>To an oven-dried and argon-purged round-bottom
flask containing NaH
(2.08 g, 51.9 mmol) in THF (400 mL) at 0 °C was added ethyl acetoacetate
(27, 6.02 mL, 47.2 mmol), and the solution was stirred
for 10 min. To the reaction flask was added dropwise n-BuLi (21.6 mL, 51.9 mmol) followed by a dropwise addition of prenyl
bromide (6.00 mL, 51.9 mmol). The reaction was stirred at rt for 20
min and then quenched by addition of saturated NH4Cl and
extracted with Et2O. The combined organic layers were dried
(Na2SO4) and filtered, and the filtrate was
concentrated on a rotary evaporator. Final purification was achieved
by column chromatography (10% EtOAc in hexanes) to afford the β-ketoester 42 as a pale yellow oil (4.69 g, 50%). Both the 1H and 13C NMR spectra matched those in the literature.43</p><!><p>To a mortar were added aldehyde 26 (806 mg, 3.56 mmol), the β-ketoester 42 (1.41
g, 7.12 mmol), and piperidine (three drops). The solution was ground
with a pestle intermittently over 12 h. Purification was achieved
by column chromatography (20–40% EtOAc in hexanes). The desired
fractions were combined and heated (oil bath) under reflux in benzene
with a Dean–Stark trap overnight. After cooling to rt, the
resulting solution was concentrated in vacuo to afford compound 43 (1.12 g, 87%): 1H NMR (400 MHz, CDCl3): Î´ 8.83 (s, 1H), 6.93 (s, 1H), 6.90 (s, 1H), 5.30 (s, 2H),
5.13 (tt, J = 7.2, 1.3 Hz, 1H) 4.47 (s, 2H), 3.49
(s, 3H), 3.41 (s, 3H), 3.12 (t, J = 7.2 Hz, 2H),
2.34 (q, J = 7.2 Hz, 2H), 1.64 (s, 3H), 1.60 (s,
3H); 13C{1H} NMR (100 MHz, CDCl3):
Î´ 197.7 (C), 159.2 (C), 156.1 (C), 155.6 (C), 147.5 (CH), 142.8
(C), 132.5 (C), 123.0 (CH), 122.3 (C), 109.0 (C), 107.6 (CH), 107.0
(CH), 94.7 (CH2), 73.8 (CH2), 58.7 (CH3), 56.7 (CH3), 42.6 (CH2), 25.7 (CH3), 22.6 (CH2), 17.7 (CH3). HRMS (ESI) m/z: [M + H]+ calcd for C20H25O6, 361.1646; found, 361.1624.</p><!><p>1H and 13C NMR spectra for all
new compounds and full tables of the bioassay data (PDF)</p><p>jo1c02046_si_001.pdf</p><p>The authors declare no
competing financial interest.</p>