Total Synthesis of (±)-Glyceollin II and a Dihydro Derivative

Article abstract of DOI:10.1021/acs.jnatprod.5b00607

Stressed soybeans produce a group of phytoalexins that belong to the 6a-hydroxypterocarpan family of flavonoids. Certain of the more prominent members, such as the glyceollins I, II, and III, have demonstrated potential antidiabetic properties and promisi

Full text of DOI:10.1021/acs.jnatprod.5b00607

Article
pubs.acs.org/jnp
Total Synthesis of ( )-Glyceollin II and a Dihydro Derivative
Neha Malik,† Zhaoqi Zhang,‡ and Paul Erhardt*,‡
†Center for Molecular Innovation and Drug Discovery, Northwestern University, Evanston, Illinois 60208, United States
‡Center for Drug Design and Development, Department of Medicinal and Biological Chemistry, College of Pharmacy and
Pharmaceutical Sciences, University of Toledo, Toledo, Ohio 43606, United States
S
* Supporting Information
ABSTRACT: Stressed soybeans produce a group of phytoalexins that
belong to the 6a-hydroxypterocarpan family of flavonoids. Certain of
the more prominent members, such as the glyceollins I, II, and III,
have demonstrated potential antidiabetic properties and promising
cytotoxicity in both human breast and prostate cancer cell cultures
with preliminary studies in animals further demonstrating antitumor
effects in estrogen-dependent, human breast cancer cell implants.
Although syntheses of glyceollin I have been reported previously, this work constitutes the first total directed synthesis of
( )-glyceollin II. It involves 12 steps with an overall yield of 7% using practical methods that should be readily scalable to
produce quantities needed for advanced biological characterization. Highlights include a novel intramolecular benzoin
condensation, a chelation-controlled lithium aluminum hydride-mediated reduction, and an intramolecular cyclization via the
formation of a transient epoxide intermediate to cap the construction of the 6a-hydroxypterocarpan system. Additionally, a
dihydro analogue has been obtained, and several isolated intermediates have been made available for evaluation of their biological
properties and possible contributions toward elaborating key structure−activity relationship data among this family of promising
phytoalexins elicited from stressed soybeans.
cultures.4 Preliminary studies in animal models further
tressed soybeans produce a group of phytoalexins that
1
demonstrate antitumor effects on estrogen-dependent human
Sbelong to the 6a-hydroxypterocarpan family of flavonoids.
breast cancer cell implants.5
To date, 10 members have been identified of which three are
the most abundant.2 Called glyceollins (GLYs; Figure 1), the
We are developing synthetic routes to several of the GLY
family members to provide larger quantities of material to
enable more advanced pharmacological studies and confirm the
preliminary biological findings. At this point, both a
biomimetic4 and novel6,7 route to GLY I, as well as two
different methods to prepare the key phytochemical inter-
mediate GLO, have been devised.8,9 Although GLY II is
obtained in low yield as a side-product during the synthesis of
GLY I, no direct synthesis of GLY II has been developed.
Herein, the first direct syntheses of racemic GLY II and its
dihydro analogue by a new route that should be generally
applicable to all of the GLYs using practical methodology are
reported.
most prominent member of this group, GLY I, has been shown
to have potential antidiabetic properties3 and promising
cytotoxicity in both human breast and prostate cancer cell
RESULTS AND DISCUSSION
■
The prior two syntheses of GLY I are depicted in Scheme 1.
After eight steps, both routes take advantage of the same
isoflav-3-ene intermediate for introduction of the 6a-hydroxy
group by treatment with osmium tetroxide (OsO4) to form the
diol before closure of the benzofuran C-ring. The latter occurs
via cis-fusion of the quinone-methide with the neighboring
phenolic hydroxy group that is simultaneously exposed during
hydrogenolysis. Construction of ring A1, by aldol reaction with
Figure 1. Structures of the three most abundant 6a-hydroxypter-
ocarpans elicited from stressed soybeans, glyceollins I, II, and III,
shown as GLY I, GLY II, and GLY III, respectively. Ring designations
are indicated on GLY I and pertinent scaffold numbering is depicted
on GLY II. Also shown is the key phytochemical intermediate of the
GLYs, glycinol (GLO). The phytochemical pathways leading to all of
soy’s normal and elicited 6a-hydroxypterocarpans are depicted in the
Supporting Information.
Received: July 9, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Scheme 1. Previous Syntheses of GLY I Accompanied by Isolation of GLY II as a Side Product3,6,7
Scheme 2. Successful Strategy Devised to Eliminate the Use of OsO4 that Fortuitously Allowed for Closure of Ring C without
Relying upon a Quinone-Methide Intermediate when Deploying Glycinol (GLO) as a Model Compound
an unsaturated aldehyde deployed as its acetal, occurs in
approximately 50% isolated yield for TBDMS-protected GLY I.
Approximately 10% of TBDMS-GLY II can also be obtained as
a side-product after chromatographic separation. In principle,
two steps common to both routes could be altered
advantageously while devising a new synthetic strategy. The
first was to eliminate the need for deploying osmium tetroxide,
and the second involved direct construction of the specific
types of A1 rings present in the various GLYs at the outset
rather than as the penultimate step of the overall synthesis. To
accomplish the latter, an alternate way to generate ring C was
needed because a quinone-methide cannot be formed when the
7-oxygen is alkylated as part of a preformed A1-ring system.
The strategy to eliminate osmium tetroxide is depicted in
Scheme 2, where the retro-synthesis arrows represent
retrosynthetic manipulations, and the regular arrows are the
typical experimental manipulations, in this case afforded by the
unanticipated result during formation of the diol during model
studies. The retrosynthetic strategy involved reduction of an
appropriate 3-hydroxychromanone with the latter potentially
being accessible by an intramolecular benzoin condensation.
This chemistry was explored within the framework of racemic
GLO as a convenient model.9 Not only were the conditions for
these novel steps refined, but the reduction with lithium
aluminum hydride (LAH) occurs via chelation control to
produce a racemic mixture of only the anti-1,2-diol. Having
some literature precedent,10 this fortuitous outcome permitted
the formation of an epoxide from the diols that could then be
opened by the neighboring phenolic hydroxy group to form
ring C. Thus, the need to rely upon a quinone-methide-driven
ring closure was eliminated. In the case of the model GLO, this
closure actually became significantly higher yielding than that
obtained by the quinone-methide approach.9
The approach to address a specific construction of the A1-A
ring system of GLY II at the start of the synthesis is depicted in
retrosynthetic Scheme 3. On the basis of a literature
precedent,11,12 this involves manipulating 7-hydroxycoumarin
to form the target ring system and then takes advantage of the
previous model chemistry to produce a keto-aldehyde that is
poised for an intramolecular benzoin condensation.
With the understanding that final deprotection in the
presence of the 6a-hydroxy group would need to be
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envisioned in retrosynthetic Scheme 3, the A1-A ring system
was successfully constructed using an excess of Grignard
reagent followed by acid-catalyzed cyclization. Initially, catalytic
amounts of 15% H2SO4 were used to prompt cyclization but
yields were low and accompanied by degradation. Thus, mildly
acidic silica gel was employed in refluxing toluene, which
afforded 3a in 88% yield across the two steps. A Vilsmeier−
Haack reaction13 of 3a, however, proved problematic in that it
caused removal of the silyl protecting group followed by
formylation of the resulting hydroxy group to generate side-
product 4 as observed by NMR data of the crude reaction
mixture. This type of event has literature precedent, where the
Vilsmeier reagent has been used as an electrophile for one-pot
deprotection and formylation of O-silylated ethers to form O-
formates.14 As an alternative, 1 was protected as its benzyl
ether, which successfully underwent both the A1-A ring
construction and subsequent Vilsmeier−Haack formylation to
afford 5 as the exclusive product in greater than 80% yield
across all four steps.
Scheme 3. Retrosynthetic Strategy Proposed for
Constructing the A1-A Ring System at the Outset of the
Overall Synthetic Path
Deprotection of 5 was necessary for the next coupling
reaction with 7 to form keto-aldehyde 8. Variously protected
forms of partner 7, including the dibenzyl derivative, have been
used in several of the prior 6a-hydroxypterocarpan-related
syntheses.6−9 Because conventional hydrogenolysis over
palladium will also reduce the double bond in ring A1, excess
trifluoroacetic acid (TFA) in toluene was used to cleave the
benzyl group in 5 where it is thought to be effective in this
specific context because it chelates with the ortho-keto-phenol
functionality.15 Although the model chemistry9 suggested that
the formyl group in 6 might need to be masked as an acetal
prior to coupling, this was found to be unnecessary, and keto-
aldehyde 8 was obtained directly in 60% yield after
recrystallization. This is attributed to the lower reactivity of
the formyl group toward an intramolecular aldol side-reaction
due to the presence of the electron-donating group present on
the A ring. Nevertheless, caution was taken while performing
this step by monitoring the reaction periodically with TLC and
quenching it upon the first appearance of any suspected aldol
product. In line with the model chemistry,9 the key intra-
molecular benzoin condensation to form 9 as a racemic mixture
proceeded efficiently by again deploying the Rovis triazolium
catalyst 8a,16 and then this was successfully followed by
reduction of the C-4 carbonyl group with LAH to afford anti-
diol 10 with yields of 86 and 77%, respectively. The final steps
leading to both racemic GLY II and its dihydro analogue are
conveyed in Scheme 5.
accomplished under mild conditions,6 a TBDMS group was
deployed first. As shown in Scheme 4, commercially available 7-
hydroxycoumarin 1 was protected using t-butyldimethylsilyl
chloride (TBDMSCl) and base to give 2a in high yield. As
Scheme 4. Synthesis of the Reduced Benzoin Condensation
Product Needed for Cyclization to Form Ring C in GLY II
Deprotection of 10 was required next to construct the C-
ring. As anticipated, removal of the benzyl groups by catalytic
hydrogenolysis was accompanied by reduction of the double
bond in ring A1 to produce 11, which could be utilized as an
intermediate to obtain an interesting dihydro derivative of
racemic GLY II. The latter underwent cyclization smoothly
upon generation of the epoxide and provided the target
analogue in 40% overall yield from 10. The C-11a proton shift
1
at 5.24 ppm in the H NMR spectrum was diagnostic for the
anticipated cis-fusion of ring C into the pterocarpan
skeleton.6,17 Alternatively, boron trichloride (BCl3) in penta-
methylbenzene (PMB) represents a gentle method that might
be used to selectively remove the benzyl groups in 10 without
losing the ring A1 double bond.18 However, even at low
temperature (−78 °C), complete decomposition of the starting
material and generation of a complex mixture within 15 min
after the addition of BCl3 were observed. This may be
C
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Scheme 5. Completion of Syntheses Leading to Racemic GLY II and its Dihydro Analogue
attributed to the propensity of the trans-diol functionality to
readily undergo dehydration by an E1 mechanism that is
initially driven by relieving the steric strain associated with the
tertiary hydroxy group while simultaneously extending the
conjugation across the two aromatic systems. The resulting
transient keto−enol species subsequently participates in other
side reactions. Given the latter and this outcome from one of
the more gentle methods, other Lewis acid-mediated conditions
for selective debenzylation were not pursued. Instead, the
benzyl functionality in precursor 9 was removed, and the
phenolic hydroxy groups were subsequently protected with
TBDMS groups because the latter can be cleaved selectively in
the presence of double bonds using mild conditions.
Deprotection of 9 proceeded uneventfully, as did subsequent
reprotection with TBDMS to provide 12 in 72% yield after
both steps.
group within this family of pterocarpans. These conditions were
employed in the present case and provided racemic GLY II in
78% yield.
Structural confirmations of ( )-GLY II and its dihydro
derivative were initially assessed by 1H and 13C NMR
comparison to samples of GLO and GLY I and to the GLY
II material previously obtained as a side product during
synthesis of GLY I. The C-11a proton shift at 5.23 ppm and its
splitting pattern are particularly diagnostic of the cis-fused 6a-
hydroxypterocarpan system.3,6,7,17 Reverse phase LC-MS
chromatograms for standard samples of GLY I and GLY II
produced a single peak in each case. The retention time for
synthetic ( )-GLY II at 9.6 min matched that for the natural
enantiomer isolated from stressed soybeans (chromatograms
available in the Supporting Information). As has been found
previously for other members of this family, elemental analyses
indicate the presence of water entrained within the matrices for
both solid products with 0.6 mol equivalents for the dihydro
derivative and 1.1 mol equivalents for ( )-GLY II.
This work constitutes the first total directed synthesis of
racemic GLY II. The synthesis involves 12 steps with an overall
yield of 7%. It is highly practical and should be readily scalable
to amounts required for preliminary biological character-
izations, including in vivo studies in small animals. Highlights
include a novel intramolecular benzoin condensation that
allows the overall synthesis to be free of the need for an
osmium-mediated dihydroxylation step, chelation-controlled
LAH-mediated reduction that affords only the trans-diols and
bypasses the need for a quinone-methide cyclization step to
form the C-ring, which can also be used to conveniently effect
selective removal of a neighboring TBDMS protecting group,
and an intramolecular cyclization via the formation of a
transient epoxide intermediate to cap the construction of the
6a-hydroxypterocarpan system. In addition to providing an
analytical standard for quantitative determination of GLY II in
plant samples by LC-MS, a dihydro analogue has been obtained
along with several isolated intermediates that can be evaluated
for their biological properties and potential contributions to
Reduction of 12 with 1.1 equiv of LAH produced both the
anticipated diol 13 and its partially deprotected version 14. It is
not unusual for LAH to cleave TBDMS protecting groups
under these conditions.19 Again fortuitously, the para-
substitution in diol 13 always remained intact, suggesting that
the ortho-TBDMS moiety is much more susceptible to such a
reaction. This is similar to an earlier observation where LAH
tends to chelate with the tertiary hydroxy group9 and, in the
present context, allows for preferential removal of the nearby
ortho-situated moiety compared to the distant para-moiety due
to neighboring group participation.19 Taking advantage of this
inherent preference for reactivity, conditions were optimized to
provide 14 as the only product and from which closure of ring
C to the 6a-hydroxypterocarpan 15 occurred smoothly in 54%
when scaled to the 50 mg level. Final deprotection of the
remaining TBDMS group was carried out with extreme care
due to the possibility for an analogous E1 loss of water from the
6a−11a positions. During a previous multigram total synthesis
of GLY I,7 a mild protocol was devised for the deprotection of
20
TBDMS ethers using 3HF-NEt3 in pyridine. Pyridine served
as a buffer to maintain the reaction pH between ∼6−7, which
was demonstrated to be optimal for preserving the 6a-hydroxy
D
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toluene (30 mL) and adding silica gel to the solution (2.5 g). The
reaction mixture was heated under reflux for 12 h. The reaction was
then cooled to RT and filtered. The silica gel was extensively washed
with EtOAc (50 mL). The filtrate was evaporated to give the crude
product which was purified using a short silica gel column (hexanes) to
yield the desired product 3a as a clear oil (2.3 g, 88%): TLC Rf 0.67
structure−activity relationships (SAR) within this unique family
of promising phytoalexins derived from stressed soybeans.
EXPERIMENTAL SECTION
■
General Experimental Procedures. All chemical reactions were
conducted in oven-dried glassware under a gentle flow of argon in
anhydrous solvents unless stated otherwise. Reagents obtained from
commercial suppliers were used without purification except penta-
methylbenzene (PMB), which was recrystallized from MeOH.
Acetone was dried over 4 Å molecular sieves, and THF was distilled
under N2 over sodium-benzophenone. Hydrogenation reactions were
performed using a Parr hydrogenator and by shaking the hydro-
genation flask at pressures indicated. TLC was performed on 250 μm
fluorescent SiO2 TLC plates and visualized by UV light or iodine
vapor. Normal-phase column chromatography was performed using
silica gel (200−425 mesh 60 Å pore size) and ACS grade solvents.
Melting points are uncorrected. Optical rotation was measured on a
1
[EtOAc/hex (1:4)]; H NMR (600 MHz, CDCl3) δ 0.19 (s, 6 H),
0.97 (s, 9 H), 1.41 (s, 6 H), 5.47 (d, J = 9.8 Hz, 1 H), 6.27 (d, J = 9.8
Hz, 1 H), 6.31−6.32 (m, 1 H), 6.33−6.35 (m, 1 H), 6.82 (d, J = 8.1
Hz, 1 H); 13C NMR (150 MHz, CDCl3) δ −4.5, 18.2, 25.6, 27.9, 76.2,
108.4, 112.6, 115.3, 122.0, 126.7, 128.1, 153.9, 156.6; anal. (%) calcd
for C17H26O2Si, C 70.29, H 9.02, found C 70.38, H 8.89.
7-(Benzyloxy)-2,2-dimethyl-2H-1-benzopyran (3b). Methyl mag-
nesium chloride (4.4 mL, 13.2 mmol, 3 M solution in THF) was
added dropwise to a solution of 7-benzyloxycoumarin 2b (1.12 g, 4.4
mmol) in THF (30 mL) at 0 °C. The resulting yellow solution was
stirred at RT overnight. Saturated NH4Cl (20 mL) was added, and the
layers were separated. The aqueous layer was extracted twice with
EtOAc (20 mL). The organic layers were combined and washed with
H2O (20 mL), brine (20 mL), dried over anhydrous Na2SO4, and
evaporated to give a yellow oil that was used immediately in the next
step. The oil was dissolved in toluene (30 mL), and silica gel was
added to the solution (2 g). The reaction mixture was heated under
gentle reflux for 12 h. Upon cooling to RT, the reaction was filtered,
and the silica gel was washed extensively with EtOAc (50 mL). The
filtrate was evaporated to give a crude product that was purified using a
short silica gel column (hexanes) to yield the target product as a clear
oil that solidified to a white amorphous solid upon cooling at 4 °C
(1.23 g, 95%): TLC Rf 0.71 [EtOAc/hex (2:1)]; 1H NMR (600 MHz,
CDCl3) δ 1.42 (s, 6 H), 5.01 (s, 2 H), 5.47 (d, J = 9.8 Hz, 1 H), 6.27
(d, J = 10.0 Hz, 1 H), 6.45−6.46 (m, 1 H), 6.46−6.48 (m, 1 H), 6.88
(d, J = 8.1 Hz, 1 H), 7.30−7.33 (m, 1 H), 7.36−7.39 (m, 2 H), 7.40−
7.43 (m, 2 H); 13C NMR (150 MHz, CDCl3) δ 28.0, 69.9, 76.4, 102.9,
107.4, 114.8, 121.9, 121.8, 126.9, 127.5, 127.9, 127.9, 128.5, 136.9,
154.1, 159.8; anal. (%) C 81.17, H 6.81, calcd for C18H18O2, C 81.07,
H 7.13.
7-(Benzyloxy)-2,2-dimethyl-2H-1-benzopyran-6-carboxaldehyde
(5). POCl3 (0.45 mL, 4.79 mmol) was added dropwise to a flask
containing DMF (0.32 mL, 4.13 mmol) at 0 °C. The resulting solution
was stirred at 0 °C for 15 min followed by the addition of benzopyran
3b (423 mg, 1.6 mmol) in DMF (1.5 mL). The ice bath was removed,
and the reaction mixture was stirred at 60 °C for 3 h. Upon cooling,
EtOAc (10 mL) and H2O (15 mL) were added to the reaction
mixture. The layers were separated, and the aqueous layer was
extracted with EtOAc (10 mL). The combined organic layer was
washed with brine (15 mL), dried over anhydrous Na2SO4, and
evaporated to a give a residue that was purified by a short silica gel
column [hexanes then EtOAc/hex (1:4)] to give the target product as
a yellow oil that solidified to a yellow solid upon cooling overnight at 4
°C (433 mg, 93%): mp 56−58 °C; TLC Rf 0.46 [EtOAc/hex (1:5)];
1H NMR (600 MHz, CDCl3) δ 1.45 (s, 6 H), 5.13 (s, 2 H), 5.56 (d, J
Rudolf Research Automatic Polarimeter (Autopol IV). H and 13C
1
NMR spectra were recorded on a Bruker Avance600 spectropho-
tometer, chemical shifts are reported to second and first decimal
places, respectively. Peak locations were referenced using either TMS
or residual nondeuterated solvent as an internal standard. Proton
coupling constants are expressed in Hz. In some cases, overlapping
signals occurred in the 13C NMR spectra. The following abbreviations
1
were used to denote spin multiplicity for H NMR: s = singlet, d =
doublet, dd = doublet of doublets, t = triplet, m = multiplet. Elemental
analyses [anal. (%)] were determined by Atlantic Microlabs Inc.,
Norcross, GA. All results were within 0.4% of the theoretical values.
7-(tert-Butyl-dimethylsilyloxy)-chromen-2-one (2a). TBDMSCl
(181 mg, 1.2 mmol) was added to a stirred mixture of 7-
hydroxycoumarin 1 (162 mg, 1 mmol) and NEt3 (0.28 mL, 2
mmol) in DCM (2 mL). The reaction mixture was stirred at RT for 4
h. Saturated NH4Cl (5 mL) was added to the mixture, and the layers
were separated. The aqueous layer was extracted with DCM (10 mL).
The combined organic layer was washed with H2O (5 mL) and brine
(5 mL), dried over anhydrous Na2SO4, and evaporated under reduced
pressure to give an oil that was purified by a short silica gel column
[hexanes, then EtOAc/hex (1:5)] to yield the target product as a white
solid (249 mg, 90%): mp 53−55 °C; TLC Rf 0.84 [EtOAc/hex (1:2)];
1H NMR (600 MHz, CDCl3) δ 0.25 (s, 6 H), 0.99 (s, 9 H), 6.26 (d, J
= 9.5 Hz, 1 H), 6.76−6.78 (m, 1 H), 6.78−6.79 (m, 1 H), 7.34 (d, J =
8.3 Hz, 1 H), 7.64 (d, J = 9.5 Hz, 1 H); 13C NMR (150 MHz, CDCl3)
δ −4.4, 18.3, 25.6, 107.8, 113.2, 113.4, 117.5, 128.7, 143.4, 155.6,
159.4, 161.2; anal. (%) C 65.18, H 7.29, calcd for C15H20O3Si, C 65.25,
H 7.17.
7-Benzyloxychromen-2-one (2b). Benzyl bromide (1.3 mL, 11
mmol) was added to a stirred mixture of 7-hydroxycoumarin 1 (1.62 g,
10 mmol) and anhydrous K2CO3 (2.07 g, 15 mmol) in acetone (20
mL). The reaction mixture was heated under reflux for 4 h. Upon
cooling, 1 N HCl (∼15 mL) was added to neutralize the mixture (pH
7.0−7.5). The solid obtained was collected by vacuum filtration,
washed with H2O (20 mL), and dried to yield the target product as an
off-white solid (2.4 g, 95%): mp 154−156 °C (lit. mp:21 153−155
°C); TLC Rf 0.63 [EtOAc/hex (2:1)]; 1H NMR (600 MHz, CDCl3) δ
5.13 (s, 2 H), 6.26 (d, J = 9.3 Hz, 1 H), 6.89 (d, J = 2.4 Hz, 1 H), 6.92
(dd, J = 8.6, 2.4 Hz, 1 H), 7.34−7.45 (m, 6 H), 7.64 (d, J = 9.5 Hz, 1
H); 13C NMR (150 MHz, CDCl3) δ 70.5, 101.9, 112.7, 113.2, 113.3,
127.5, 128.4, 128.8, 128.7, 135.7, 143.4, 155.8, 161.2, 161.8; anal. (%)
C 76.18, H 4.79, calcd for C16H12O3, C 76.06, H 5.02.
2,2-Dimethyl-7-(tert-Butyl-dimethylsilyloxy)-2H-1-benzopyran
(3a). Methyl magnesium chloride (9 mL, 27 mmol, 3 M solution in
THF) was added dropwise to a solution of 2a (2.5 g, 9 mmol) in THF
(65 mL) at 0 °C. The resulting yellow solution was stirred at RT for
12 h. Saturated NH4Cl (30 mL) was then added and the layers
separated. The aqueous layer was extracted twice with EtOAc (20
mL). The organic layers were combined and washed with H2O (50
mL), brine (50 mL) and dried over anhydrous Na2SO4, and
evaporated to give the dialkylated acyclic intermediate as a yellow
oil that was used in the next step immediately by dissolving it in
= 9.8 Hz, 1 H), 6.31 (d, J = 10.0 Hz, 1 H), 6.45 (s, 1 H), 7.34−7.45
(m, 5 H), 7.53 (s, 1 H), 10.35 (s, 1 H); 13C NMR (150 MHz, CDCl3)
δ 28.5, 28.6, 70.5, 78.1, 100.7, 114.6, 118.9, 121.1, 126.5, 127.3, 128.3,
128.7, 128.9, 135.9, 160.3, 162.9, 188.1; anal. (%) C 77.18, H 6.22,
calcd for C19H18O3·0.06 EtOAc, C 76.80, H 6.31.
2,2-Dimethyl-7-Hydroxy-2H-1-benzopyran-6-carboxaldehyde
(6). To a solution of aldehyde 5 (3.5 g, 11.85 mmol) in toluene (60
mL) was added TFA (30 mL) in small portions at RT. The reaction
mixture was allowed to stir at RT for 12 h. Upon completion (1H
NMR), saturated NaHCO3 (75 mL) was added carefully. EtOAc (50
mL) was then added, and the layers were separated. The organic layer
was washed with saturated NaHCO3 (30 mL × 2) and H2O (30 mL),
dried over anhydrous Na2SO4, and evaporated under reduced pressure
to give an oily residue. The residue was taken up in EtOAc (20 mL)
and washed with 10% NaOH (20 mL × 4). The combined NaOH
extract was neutralized to pH 6.5−7.0 using 1 N HCl (∼100 mL).
EtOAc (50 mL) was then added, and the layers were separated. The
aqueous layer was washed with EtOAc (25 mL), and the combined
organic layers were washed with H2O (25 mL × 4) and brine (25 mL),
E
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dried over anhydrous Na2SO4, and evaporated under high vacuum to
yield the target product as a light brown solid (1.61 g, 67%): mp 94−
96 °C; TLC Rf 0.63 [EtOAc/hex (1:3)]; 1H NMR (600 MHz, CDCl3)
δ 1.45 (s, 6 H), 5.59 (d, J = 9.9 Hz, 1 H), 6.29 (d, J = 9.9 Hz, 1 H),
6.33 (s, 1 H), 7.11 (s, 1 H), 9.66 (s, 1 H), 11.43 (s, 1 H); 13C NMR
(150 MHz, CDCl3) δ 28.6, 104.2, 114.4, 115.2, 120.6, 128.9, 131.4,
161.2, 164.3, 194.1; anal. (%) C 70.57, H 5.92, calcd for C12H12O3, C
70.58, H 5.90.
103.6, 106.3, 115.3, 118.1, 122.4, 123.1, 128.3, 128.4, 128.5, 128.7,
129.0, 129.1, 129.2, 137.5, 138.0, 154.1, 155.4, 158.8, 159.9; anal. (%)
C 76.10, H 6.01, calcd for C34C32O6, C 75.91, H 6.10.
Racemic Dihydroglyceollin II [( )-Dihydro-GLY II]. To a
solution of diol 10 (120 mg, 0.22 mmol) in EtOH (5 mL) was added
10% Pd/C (30 mg). The resulting mixture was shaken at RT for 12 h
under H2 atmosphere (35 psi). The mixture was filtered through
Celite, and the latter was washed with EtOAc (10 mL). The filtrate
was evaporated, and the residue obtained was passed through a short
silica gel column [EtOAc/hex (1:1)] to afford debenzylated
intermediate 11, which was used in the next step without further
purification. To a solution of the debenzylated diol 11 (80 mg, 0.22
mmol) in THF (3 mL) were added Ms2O (77 mg, 0.44 mmol) and
anhydrous pyridine (50 μL, 0.66 mmol). The resulting suspension was
stirred at RT for 12 h after which the solvents were evaporated under
reduced pressure. The residue obtained was purified by silica gel
column chromatography [EtOAc/hex (1:3)]. The eluting solvents
were evaporated under reduced pressure and then lyophilized to
obtain the product, ( )-dihydro-GLY II as an off-white solid (30 mg,
7-[2-(2,4-Bisbenzyloxy)phenyl-2-oxoethoxy]-2,2-dimethyl-2H-
chromene-6-carboxaldehyde (8). To a solution of aldehyde 6 (467
mg, 2.26 mmol) in acetone (5 mL) was added anhydrous K2CO3 (317
mg, 2.29 mmol). The solution was heated. Upon reflux, α-iodoketone
7 (prepared separately according to refs 6−9; 0.89 g, 1.93 mmol) was
added, and the mixture was refluxed for 4−5 h (monitoring the
reaction progress by TLC every hour). The reaction mixture was
cooled to 0 °C, and H2O (5 mL) was added. The crude solid was
collected by vacuum filtration and recrystallized from EtOAc/hex [1:4,
5 mL] to yield compound 8 as an off-white solid (619 mg, 60%): mp
1
170−172 °C; TLC Rf 0.36 [EtOAc/hex (1:3)]; H NMR (600 MHz,
1
CDCl3) δ 1.44 (s, 6 H), 5.13 (s, 4 H), 5.14 (s, 2 H), 5.53 (d, J = 9.9
Hz, 1 H), 5.91 (s, 1 H), 6.28 (d, J = 9.9 Hz, 1 H), 6.67 (d, J = 2.2 Hz, 1
H), 6.70 (dd, J = 8.8, 2.2 Hz, 1 H), 7.36−7.39 (m, 1 H), 7.40−7.46
(m, 9 H), 7.50 (s, 1 H), 8.03 (d, J = 8.8 Hz, 1 H), 10.37 (s, 1 H); 13C
NMR (150 MHz, CDCl3) δ 28.7, 70.4, 71.2, 74.3, 77.9, 100.0, 100.3,
107.1, 114.5, 118.3, 118.9, 121.2, 126.4, 127.6, 128.1, 128.4, 128.6,
128.8, 128.9, 129.1, 133.4, 135.2, 135.9, 160, 160.5, 162.6, 164.5, 188.4,
191.8; anal. (%) C 74.63, H 5.78, calcd for C34H30O6·0.7 H2O, C
74.23, H 5.54.
40%): mp 105−108 °C; TLC Rf 0.39 [EtOAc/hex (1:1)]; H NMR
(600 MHz, acetone-d6) δ 1.27 (s, 3 H), 1.29 (s, 3 H), 1.80 (t, J = 6.8
Hz, 2 H), 2.76 (t, J = 6.6 Hz, 2 H), 4.02 (d, J = 11.4 Hz, 1 H), 4.08 (d,
J = 11.4 Hz, 1 H), 4.92 (s, 1 H), 5.26 (s, 1 H), 6.17 (s, 1 H), 6.24 (d, J
= 1.8 Hz, 1 H), 6.42 (dd, J = 8.1, 2.2 Hz, 1 H), 7.16 (s, 1 H), 7.20 (d, J
= 8.4 Hz, 1 H), 8.45 (s, 1 H); 13C NMR (150 MHz, acetone-d6) δ
22.0, 26.6, 26.8, 33.0, 70.3, 74.8, 76.5, 85.8, 98.3, 104.5, 108.5, 113.4,
115.7, 121.2, 124.9, 132.5, 154.9, 155.7, 160.4, 161.7; anal. (%) C
68.40, H 6.08, calcd for C20H20O5·0.6 H2O, C 68.11, H 5.96.
( )-7-[2,4-Bis(benzyloxy)phenyl]-7-hydroxy-2,2-dimethyl-7,8-di-
hydro-2H,6H-pyrano[3,2-g]chromen-6-one (9). To a solution of
keto-aldehyde 8 (534 mg, 1 mmol) in THF (16 mL) was added Rovis
triazolium salt 8a (47 mg, 0.1 mmol) followed by the addition of NEt3
(30 μL, 0.18 mmol). The mixture was stirred at RT for 8 h. After the
( )-7-[2,4-Bis(t-butyldimethylsilyloxy)phenyl]-7-hydroxy-2,2-di-
methyl-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-6-one (12). A
solution of 9 (535 mg, 1 mmol) and pentamethylbenzene (371 mg,
2.5 mmol) in DCM (20 mL) was stirred for 15 min at −78 °C. To this
mixture was added BCl3 (2 mL, 2 mmol, 1 M solution in DCM)
dropwise over 10 min via syringe at −78 °C. The reaction mixture was
left to stir at this temperature for 30 min upon which the TLC
indicated complete consumption of the starting material. The reaction
was quenched with saturated NaHCO3:MeOH (1:1, 20 mL) at −78
°C, and the resulting solution was stirred at 0 °C for 15 min. The
MeOH and DCM were evaporated, and the resulting suspension was
dissolved in EtOAc (10 mL). The organic layer was washed with H2O
(5 mL) and brine (5 mL), dried over anhydrous Na2SO4, and
evaporated under reduced pressure to give a residue that was subjected
to silica gel column chromatography [EtOAc/hex (1:2)] to yield the
debenzylated 3-hydroxychromanone intermediate as an amorphous
solid (265 mg), which was then used in the next step. NEt3 (0.14 mL,
1 mmol) and TBDMSCl (148 mg, 1 mmol) were added to a solution
of deprotected 3-hydroxychromanone intermediate (173 mg, 0.5
mmol) in DCM (4 mL) at 0 °C. The reaction mixture was warmed to
RT and stirred for 8 h. Additional TBDMSCl (37 mg, 0.25 mmol) and
NEt3 (40 μL, 0.29 mmol) were added followed by stirring of the
mixture at RT for another 8 h. Upon disappearance of starting material
(TLC), H2O (5 mL) and DCM (5 mL) were added, and the layers
were separated. The organic layer was washed with 0.1 N HCl (5 mL),
H2O (5 mL), and brine (5 mL), dried over anhydrous Na2SO4, and
evaporated to give a residue that was purified by silica gel column
chromatography [EtOAc/hex (1:4)] to afford compound 12 as a clear
oil (419 mg, 72% combined for both steps): TLC Rf 0.67 [EtOAc/Hex
1
reaction was complete (monitored by TLC and H NMR), H2O (5
mL) and EtOAc (10 mL) were added, and the layers were separated.
The organic layer was washed with brine (10 mL), dried over
anhydrous Na2SO4, concentrated under reduced pressure, and
subjected to silica gel column chromatography [EtOAc/hex (1:3)]
to afford compound 9 as a pale yellow solid (460 mg, 86%): mp 69−
1
72 °C; [α]22 0 (c 0.5, CHCl3); TLC Rf 0.58 [EtOAc/hex (1:1)]; H
D
NMR (600 MHz, CDCl3) δ 1.43 (s, 3 H), 1.45 (s, 3 H), 3.49 (s, 1 H),
4.24 (d, J = 11.7 Hz, 1 H), 4.87 (d, J = 11.7 Hz, 1 H), 4.94−5.00 (m,
2H), 5.02 (s, 2 H), 5.59 (d, J = 9.9 Hz, 1 H), 6.26 (d, J = 9.9 Hz, 1 H),
6.27 (s, 1 H), 6.58 (dd, J = 8.4, 2.2 Hz, 1 H), 6.59−6.61 (m, 1 H), 7.25
(d, J = 1.5 Hz, 2 H), 7.27−7.30 (m, 2 H), 7.31−7.35 (m, 1 H), 7.36−
7.42 (m, 6 H), 7.45 (d, J = 8.4 Hz, 1 H); 13C NMR (150 MHz,
CDCl3) δ 28.5, 28.6, 70.2, 70.7, 73.9, 74.3, 77.9, 101.3, 104.1, 105.8,
113.7, 116.3, 119.8, 121.2, 125.7, 127.5, 127.4, 127.9, 128.1, 128.4,
128.5, 128.6, 129.4, 135.9, 136.6, 156.9, 160.1, 160.3, 162.6, 190.9;
anal. (%) C 76.26, H 5.67, calcd for C34H36O6·0.05 H2O, C 75.88, H
5.80.
( )-7-[2,4-Bis(benzyloxy)phenyl]-2,2-dimethyl-7,8-dihydro-2H-
pyrano[3,2-g]chromene-6,7-diol (10). To a solution of hydroxyke-
tone 9 (267 mg, 0.5 mmol) in THF (5 mL) was added dropwise LAH
(0.55 mL, 0.55 mmol, 1 M solution in THF) at 0 °C. The reaction was
brought to RT and stirred for 4 h. The reaction was quenched with 0.1
N HCl (5 mL); EtOAc (10 mL) was added, and the layers were
separated. The organic layer was washed with 0.1 N HCl (5 mL), 10%
NaHCO3 (5 mL), and brine (5 mL), dried over anhydrous Na2SO4,
and evaporated under reduced pressure. The resulting residue was
subjected to a short silica gel column [EtOAc/hex (1:3)] to afford
compound 10 as a white solid (206 mg, 77%): mp 79−81 °C; TLC Rf
0.36 [EtOAc/Hex (1:3)]; 1H NMR (600 MHz, acetone-d6) δ 1.36 (s,
6 H), 4.22 (d, J = 5.1 Hz, 1 H), 4.28 (dd, J = 10.6, 1.5 Hz, 1 H), 4.33
(s, 1 H), 4.69−4.74 (m, 1 H), 4.83−4.86 (m, 1 H), 5.11 (s, 2 H), 5.27
(s, 2 H), 5.56 (d, J = 9.5 Hz, 1 H), 6.14 (s, 1 H), 6.33 (d, J = 9.5 Hz, 1
H), 6.60 (dd, J = 8.4, 2.6 Hz, 1 H), 6.81 (d, J = 2.6 Hz, 1 H), 6.90 (s, 1
H), 7.24 (d, J = 8.4 Hz, 1 H), 7.32−7.37 (m, 2 H), 7.38−7.44 (m, 4
H), 7.47 (d, J = 7.3 Hz, 2 H), 7.57 (d, J = 7.3 Hz, 2 H); 13C NMR
(150 MHz, acetone-d6) δ 27.9, 68.3, 69.9, 70.2, 71.1, 72.4, 76.4, 101.7,
1
(1:3)]; H NMR (600 MHz, CDCl3) δ 0.17 (s, 6 H), 0.22 (s, 3 H),
0.27 (s, 3 H), 0.95 (s, 18 H), 1.43 (s, 3 H), 1.45 (s, 3 H), 3.75 (s, 1 H),
4.25 (d, J = 11.4 Hz, 1 H), 4.96 (d, J = 11.7 Hz, 1 H), 5.60 (d, J = 9.9
Hz, 1 H), 6.30 (d, J = 0.7 Hz, 1 H), 6.32 (d, J = 9.9 Hz, 1 H), 6.35 (d, J
= 2.2 Hz, 1 H), 6.36−6.38 (m, 1 H), 7.15 (d, J = 8.4 Hz, 1 H), 7.58 (s,
1 H); 13C NMR (150 MHz, CDCl3) δ −4.4, −4.1, −3.8, 18.2, 18.5,
25.6, 25.7, 25.8, 25.9, 28.5, 28.6, 73.5, 74.2, 77.9, 104.2, 110.9, 112.5,
113.6, 116.4, 121.1, 125.7, 128.5, 129.6, 154.6, 156.6, 160.3, 162.8,
190.9; anal. (%) C 64.45, H 8.03, calcd for C32H46O6Si2·0.75 H2O, C
64.10, H 8.22.
( )-7-(2′-Hydroxy-4′-t-butyldimethylsilylphenyl)-2,2-dimethyl-
7,8-dihydro-2H-pyrano[3,2-g]chromene-6,7-diol (14). To a solution
of TBDMS-protected hydroxyketone 12 (100 mg, 0.17 mmol) in THF
F
DOI: 10.1021/acs.jnatprod.5b00607
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(1.5 mL) was added dropwise LAH (0.3 mL, 0.3 mmol, 1 M solution
in THF) at 0 °C. The reaction was brought to RT and stirred for 6 h.
The reaction was quenched with 0.1 N HCl (5 mL); EtOAc (10 mL)
was added, and the layers were separated. The organic layer was
washed with 0.1 N HCl (5 mL), 10% NaHCO3 (5 mL), and brine (5
mL), dried over anhydrous Na2SO4, and evaporated under reduced
pressure. The resulting residue was subjected to a short silica gel
column [EtOAc/hex (1:2)] to afford compound 14 as a white solid
(60 mg, 75%): mp 175−177 °C; TLC Rf 0.53 [EtOAc/hex (1:2)]; 1H
NMR (600 MHz, acetone-d6) δ 0.22 (s, 6 H), 0.99 (s, 9 H), 1.38 (s, 3
H), 1.39 (s, 3 H), 4.19 (dd, J = 11.2, 1.7 Hz, 1 H), 4.66−4.70 (m, 1
H), 4.74 (d, J = 11.4 Hz, 1 H), 5.59 (d, J = 9.5 Hz, 1 H), 6.20 (s, 1 H),
6.32−6.35 (m, 2 H), 6.35 (d, J = 7.7 Hz, 1 H), 6.96 (s, 1 H), 7.03 (d, J
= 8.4 Hz, 1 H); 13C NMR (150 MHz, acetone-d6) δ −4.2, 18.8, 26.1,
28.3, 28.4, 67.9, 69.6, 74.4, 76.9, 104.2, 109.5, 111.8, 116.1, 117.81,
120.2, 122.7, 129.1, 129.2, 129.8, 154.8, 155.3, 157.2, 158.8; anal. (%)
calcd for C26H34O6Si·0.8 H2O, C 64.38, H 7.40, found C 64.12, H
7.01.
Racemic 9-(t-Butyldimethylsilyloxy)glyceollin II [( )-TBDMS-GLY
II] (15). To a solution of diol 14 (60 mg, 0.13 mmol) in THF (2.5 mL)
was added Ms2O (45 mg, 0.26 mmol) and anhydrous pyridine (32 μL,
0.4 mmol). The resulting suspension was stirred at RT for 12 h after
which the solvents were evaporated. The residue obtained was purified
by silica gel column chromatography [EtOAc/hex (1:3)] to yield the
target product as an amorphous white solid (32 mg, 54%): TLC Rf
0.68 [EtOAc/hex (1:3)]; 1H NMR (600 MHz, acetone-d6) δ 0.20 (s, 6
H), 0.97 (s, 9 H), 1.37 (s, 3 H), 1.39 (s, 3 H), 4.03 (d, J = 11.4 Hz, 1
H), 4.15 (dd, J = 11.4, 0.7 Hz, 1H), 5.07 (s, 1 H), 5.27 (s, 1 H), 5.66
(d, J = 9.9 Hz, 1 H), 6.22 (s, 1 H), 6.28 (d, J = 2.2 Hz, 1 H), 6.41 (d, J
= 9.9 Hz, 1 H), 6.47 (dd, J = 8.1, 2.2 Hz, 1H), 7.15 (s, 1 H), 7.27 (d, J
= 8.1 Hz, 1 H); 13C NMR (150 MHz, acetone-d6) δ −4.3, 18.8, 26.1,
28.5, 28.40, 70.7, 76.8, 77.3, 85.8, 103.4, 104.9, 113.6, 114.3, 117.3,
122.4, 123.6, 125.2, 129.9, 130.1, 155.4, 156.9, 158.9, 161.8; anal. (%)
C 68.99, H 7.13, calcd for C26H32O5Si, C 68.72, H 6.94.
linear from 5−3,000 ng/mL, and the three QC samples were 30, 300,
and 1000 ng/mL.
LC-MS Conditions. An Alliance LC (model Waters 2795)
equipped with a quaternary pump, a degasser, an autosampler, and a
column oven from Waters Corporation (Milford, MA, USA) were
used. The detector was a mass spectrometry MICROMASS Quatro
Micro API from Waters Corporation. MassLynx V4.1 software from
Waters Corporation (Milford, MA, USA) was used for data acquisition
and processing. Quattro Micro V4.1 software from Waters
Corporation was used to set up MS, and Inlet Method V4.1 software
from Waters Corporation was used to set up LC. The LC method
used a flow rate of 350 μL/min with an injection volume of 10 μL. The
total run time was around 15 min. The samples were run through an
Ascentis Express C18 analytical column (reversed-phase, 2.1× 75 mm,
2.7 μm) purchased from Sigma-Aldrich (St. Louis, MO, USA) with a
guard column XBridge C18 (2.1× 10 mm, 3.5 μm). The mobile phase
was 55% 0.1% formic acid buffer and 45% MeOH. Column and sample
temperatures were set at 35
5 and 5
5 °C, respectively. MS
detection used an electrospray ionization (ESI) source in positive
mode; nitrogen was used as the desolvation gas and was set at a flow
rate of 750 and 50 L/h, respectively. The source and desolvation gas
temperature were 100 and 400 °C, respectively. The ESI source tip
(capillary) voltage was 3.5 kV, extractor was 3.0 V, ion energy was 0.5
V, cone voltage was 45 V, and collision energy was 2 V.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00607.
Soybean’s phytochemical pathways leading to normal
1
natural products and stressed phytoalexins, H and 13C
NMR spectra for all synthesized compounds, and LC-MS
chromatogram for final material compared to natural
materials (PDF)
Racemic Glyceollin II [( )-GLY II]. To a solution of 15 (30 mg,
0.06 mmol) in DCM (1 mL) were added Et3N·3 HF (28 μL, 0.16
mmol) and excess anhydrous pyridine (40 μL, 0.6 mmol). The
reaction mixture was stirred at RT for 6 h. Upon disappearance of
starting material (TLC), the solvents were evaporated, and the
resulting residue was directly applied to a silica gel column [EtOAc/
hex (1:1)]. The eluting fractions were collected; the solvents were
removed under reduced pressure, and the resulting residue was
lyophilized to obtain the target product ( )-GLY II as a white solid
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 1-419-530-2167. Fax: 1-419-530-7946. E-mail: paul.
erhardt@utoledo.edu.
1
(16 mg, 78%): mp 61−65 °C; TLC Rf 0.52 [EtOAc/hex (1:1)]; H
Notes
NMR (600 MHz, acetone-d6) δ 1.36 (s, 3 H), 1.39 (s, 3 H), 4.04 (d, J
= 11.3 Hz, 1 H), 4.13 (d, J = 11.7 Hz, 1 H), 4.96 (s, 1 H), 5.25 (s, 1
H), 5.65 (d, J = 9.5 Hz, 1 H), 6.21 (s, 1 H), 6.25 (d, J = 2.2 Hz, 1 H),
6.40−6.44 (m, 2 H), 7.14 (s, 1 H), 7.21 (d, J = 8.1 Hz, 1 H), 8.47 (s, 1
H); 13C NMR (150 MHz, acetone-d6) δ 28.3, 70.5, 76.6, 77.2, 85.7,
98.7, 104.8, 108.9, 114.5, 117.1, 121.3, 122.2, 125.1, 129.8, 155.2,
156.8, 160.7, 161.9; anal. (%) C 67.07, H 5.68, calcd for C20H18O5·1.1
H2O, C 66.86, H 5.57; HPLC was essentially a single peak (>97%
AUC) with a retention time identical to the authentic sample of the
natural form from the soybeans (retention time = 9.6 min).
Glyceollins Extraction and Sample Handling. Dried soybean
plant powder (100 mg) was placed in a 25 mL round-bottom flask
fitted with a water-cooled condenser. HPLC grade MeOH (10 mL)
was added, and the slurry was gently refluxed at 64 °C for 2 h. The
slurry was filtered, and the residue was extracted again with fresh
MeOH (10 mL). This process was repeated a third time. The extracts
were combined, filtered with a Titan2 PTFE syringe filter consisting of
a glass wool prefilter and a 0.45 μm final filter, and then a 500 μL
aliquot was vacuum centrifuged (the remaining sample can be stored
at 4 °C for several months without deterioration). The resulting
residue was dissolved in 150 μL mobile phase and placed in a 0.3 mL
HPLC microvial for LC-MS analysis. Quantification of GLY I can be
accomplished using authentic material to prepare a stock solution in
MeCN at 8 × 10−4 M from which dilutions can afford a standard curve
and various QC samples. The calibration curve prepared for GLY I was
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the USDA ARS through the
Southern Regional Research Center (SRRC) located in New
Orleans, LA, and the Ohio Soybean Council (OSC) located in
Worthington, OH.
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