Multigram synthesis of glyceollin i

Article abstract of DOI:10.1021/op200112g

Scaled-up procedures and preparation of glyceollin I in multigram quantities are described. The synthesis features construction of a cis-fused ring system in high enantiomeric excess after Sharpless asymmetric dihydroxylation of a key intermediate that is initially produced by an intramolecular Wittig reaction to afford the requisite alkene while simultaneously forming the first ring. The overall yield is 12% after 11 steps.

Full text of DOI:10.1021/op200112g

ARTICLE
pubs.acs.org/OPRD
Multigram Synthesis of Glyceollin I
Amarjit Luniwal,† Rahul Khupse,‡ Michael Reese,§ Jidong Liu,^ Mohammad El-Dakdouki,|| Neha Malik,†
Lei Fang,z and Paul Erhardt*,†
†Center for Drug Design and Development, University of Toledo,
College of Pharmacy and Pharmaceutical Sciences Department of Medicinal and Biological Chemistry, 2801 West Bancroft Street,
Toledo, Ohio 43606, United States
‡University of Findlay, College of Phamarcy, Findlay, Ohio 45840, United States
§Affymetrix, Inc., Anatrace Division, Maumee, Ohio 43537, United States
^ALDLAB Chemicals, LLC., Woburn, Massachusetts 01801, United States
Michigan State University, Department of Chemistry, East Lansing, Michigan 48824, United States
zNanjing Aosaikang Medicinal Group, Ltd., Jiangning, Nanjing, Jiangsu, China
S Supporting Information
b
ABSTRACT: Scaled-up procedures and preparation of glyceollin I in multigram quantities are described. The synthesis features
construction of a cis-fused ring system in high enantiomeric excess after Sharpless asymmetric dihydroxylation of a key intermediate
that is initially produced by an intramolecular Wittig reaction to afford the requisite alkene while simultaneously forming the first
ring. The overall yield is 12% after 11 steps.
’ INTRODUCTION
Owing in part to the lengthy, multistep routes needed to prepare
these contiguous ring systems while maintaining rigorous stereo-
control, only four members of this family have been synthesized
to date, namely pisatin,8 variabilin,9 and most recently in our
laboratories GLY I1,10 along with the GLY family’s key phyto-
chemical precursor glycinol11 (respectively structures 5, 6, 1, and
7 in Figure 1). Despite having already accomplished the total
synthesis of 1, it was additionally clear that scale-up from
milligram to gram quantities of product would require a highly
concerted team effort within our academic-based drug design
and development center’s laboratories in order to achieve
this goal according to an intentionally ambitious time frame
of ∼6 months.
Our collaborating laboratories have been examining the
possibility for stress-induced enhancement of anticancer natural
products within legumes that are staples of the U.S. agricultural
industry.1 Of particular note is the common soybean, Glycine
max, which produces a family of phytoalexins2 known as the
glyceollins (GLYs; Figure 1). Because phytoalexins typically are
present in very low amounts across select periods of time, supply
issues represent an additional challenge toward their potential
development as therapeutics. For example, the GLYs can be
elicited in only trace amounts and isolated as a complex mixture
when soybean plants are subjected to specific types of stress, such
as when soy cotyledons are infected with Aspergillus.3 Unlike
their abundant isoflavonoid relatives genistein and daidzein
which are normally present in soy, the GLYs exhibit marked
antiestrogenic activity in some tissues4 as well as unique
anticancer properties1,5 that hold promise for their use as
selective estrogen receptor modulators (SERMs).6 Among
the GLYs, GLY I (1) is the most prevalent and appears to be
the most interesting in terms of anticancer properties. To
further characterize its promising activity, we have devised an
advanced biological testing plan that requires several grams of
1. Although attempts to optimize the isolation of 1 from
natural sources remain ongoing, it was clear that a practical
synthesis would be highly desirable in order to more quickly
address the immediate supply needs.
’ BACKGROUND
From the onset of our synthetic studies in this area, we have
recognized that introduction of the distinctive 6a-hydroxy group
represents one of the most intricate chemical steps leading to
these natural products. Previous investigators typically have
utilized an isoflav-3-ene (8 in Scheme 1) from which dihydrox-
ylation and subsequent closure to the dihydrobenzofuran, 9,
produces the natural and more stable cis-fused ring system.12
Alternatively, as also shown in retrosynthetic Scheme 1, we
imagined that assembly of the GLYs’ central skeleton might be
accomplished by either of two routes. Both proceed from the
same isoflavone, 10, and subsequently take advantage of what we
perceived to be very accessible alkene intermediates (8 or 11) for
The pterocarpans, as generally represented by 4 in Figure 1,
are the second largest group of natural isoflavonoids.7 Their 6a-
hydroxy-substituted family, however, has only a few members.
Thus, the 6a-hydroxy group represents a very distinctive molec-
ular arrangement in these systems for which the accompanying
asymmetry must be properly accounted for during synthesis.
Special Issue: Asymmetric Synthesis on Large Scale 2011
Received: April 21, 2011
Published: June 23, 2011
r
2011 American Chemical Society
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Figure 1. Selected structures. The glyceollins, such as GLY I 1, GLY II 2, and GLY III 3, are phytoalexins produced by the soybean in response to specific
stress conditions. Structure 4 depicts the typical pterocarpan ring system and specifically denotes the “6a” position. Structures 5 and 6 are respectively
(+) pisatin and (+) variabilin; these compounds are 6a-hydroxypterocarpan natural products which have been previously synthesized by other
investigators. All of the GLYs are derived from the phytochemical intermediate glycinol 7. Structure 8 is an appropriately substituted isoflav-3-ene system
that serves as a key intermediate for both of our former total syntheses and for the present scale-up synthesis of GLY I. Structure 12 is lespedezol A1 which
we previously prepared and used as a model system to explore alternative synthetic entries toward GLY I (see text for details).
Scheme 1. Retrosynthetic approaches to the 6a-hydroxypterocarpan (9) required for GLY I
potential insertion of the 6a-hydroxy group. The diol route is
similar to that in the literature, while the other necessitates
adding a water molecule across the double bond of a pterocar-
pene (11) analogous to what has been reported for indene.13 To
examine the alternative strategy, we previously prepared lespe-
dazol A1 (12 in Figure 1)14 and used it as a model pterocarpene
that we then subjected to several different types of conditions,
none of which proved successful toward obtaining the 6a-
hydroxy-pterocarpan system 9 (Scheme 1). We thus adopted
the diol strategy and, in that regard, have previously explored
several ways to obtain the key isoflav-3-ene intermediate 8.
Isoflav-3-enes such as 8 are generally obtained from their
corresponding isoflavones 10. While several routes can be taken
to the latter, they are historically obtained, and seemingly still so in
the most generally consistent manner, from their correspondingly
substituted chalcones (13 in Scheme 2) through an acetal adduct
by using a thallium-mediated oxidative rearrangement.15 In addi-
tion to successfully utilizing this route, we attempted the
rearrangement via a newer method that employs Koser’s reagent16
but found that its net yields of 10ꢀ15% were considerably lower
than that for the thallium-mediated approach at ∼70%. Similarly,
attempts to assemble 10 via a Suzuki coupling17 also proved less
efficient, likely due to steric hindrance when R is a benzyl group on
the boron-containing partner (Scheme 2).Other seemingly attrac-
tive condensations such as that involving a deoxybenzoin,18
require more elaborate preassembly of the appropriately substi-
tuted coupling partners needed for eventual elaboration into our
specific target compound, GLY I (1). Alternatively, as also shown
in retrosynthetic Scheme 2, we imagined that assembly of 8 might
instead be accomplished without having to traverse the isoflavone,
namely via an intramolecular Wittig reaction somewhat analogous
to the latter’s deployment in several other types of ring-forming
systems.19 Examination of this alternative strategy was investigated
directly with very accessible starting materials that would be
needed later for synthesis of 1. All of these early-stage reactions,
including the novel Wittig-driven ring-closure, proved quite
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Scheme 2. Retrosynthetic approaches to the key isoflav-3-ene intermediate 8
Scheme 3. Previous10 total synthesis of GLY I utilizing a nonbiomimetic Wittig reaction (steps (a) and (b)) to obtain the key
intermediate isoflav-3-ene 8 (after adjustment of protecting groups)a
a Reagents and conditions: (a) PPh3 HBr, CH3CN, rt; (b) t-BuONa, MeOH, reflux, 78% (over two steps); (c) OsO4, (DHQD)2PHAL, CH2Cl2,
3
ꢀ78 ꢀC; (d) PdꢀC (10%), H2, Me2CO, 80%; (e) polymeric base, EtOH, molecular sieves 4 Å, 64%; f) 1,1-diethoxy-3-methyl-2-butene, picoline,
p-xylene, 130 ꢀC, 61%; (g) silica gel column chromatography, (CH2Cl2/MeOH); (h) Et3N 3HF, CH3CN, ꢀ20 to 4 ꢀC, 77%.
3
successful. Thus, from this backdrop two strategies became
available for potential scale-up of 1: a ‘biomimetic route’ in which
the initial intermediates involved chalcone and isoflavone-related
materials; and a ‘nonbiomimetic route’ in which the early assembly
utilized a Wittig reaction. Our early Wittig and biomimetic routes
to GLY I are depicted in Schemes 3 and 4, respectively.1,10
Interestingly, while both of these routes begin with the
same two starting materials such that this aspect of overall cost
considerations is not a factor, their initial building blocks become
reversed within the contiguous ring system during assembly of
the key isoflav-3-ene (note locations of rings “A” and “B” within 8
between Schemes 3 and 4). Likewise, both routes require a
similar number of overall synthetic steps and column chroma-
tography purifications, again equally balancing these types of
practical comparisons. Alternatively and much to its favor, the
Wittig approach was accomplished in ∼3% overall yield while
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Scheme 4. Previous1 biomimetic route to the key isoflav-3-ene intermediate 8a
a Reagents and conditions: (a) BnBr, K2CO3, CH3CN, reflux, 10 h, 88%; (b) MOMCl, K2CO3, Me2CO, rt, 24 h, 70%; (c) BnBr, K2CO3, CH3CN, reflux,
4 h, 80%; (d) piperidine, MeOH, 60 ꢀC, 6 h, 80%; (e) acetic anhydride, Et3N, rt, 92%; (f) Tl(NO3)3, 3H2O, MeOH:TMOF (1:1), rt, 8 h; (g) 10% HCl,
THF, reflux, 6 h, 68% (over two steps); (h) TBDMSCl, Et3N, CH2Cl2, 90%; (i) 1) LiBH4, THF, 0 ꢀC, 6 h; 2) 10% HCl, reflux, 2 h, 45% (over two steps).
Table 1. MOM deprotection experiments using the racemic
form of GLY I’s penultimate intermediate
that for the biomimetic approach was only about 1%. In addition,
the Wittig route eliminates the use of thallium which we felt
would be a very advantageous step closer to a ‘greener’ overall
procedure immediately from the outset of our scale-up activities.
reagent
pH temp. (ꢀC) yield (%)a time (h) ref
PPh3 HBr
4ꢀ6
4ꢀ6
4ꢀ6
4ꢀ6
rt
0
0ꢀ5
5ꢀ10
5ꢀ10
10ꢀ15
0
2ꢀ4
4ꢀ6
3ꢀ5
4ꢀ6
1ꢀ2
3ꢀ4
1ꢀ3
4ꢀ5
20
21
22
23
3
PPh3 HBr
3
’ STRATEGIC SCALE-UP STUDIES
NaHSO4 SiO2
rt
0
3
Before embarking on a step-by-step enhancement of the
Wittig route, five strategic questions were entertained experi-
mentally on small scale: (1) Could the initial MOM protecting
group be retained through the entire sequence even though we
previously had trouble removing it at the end due to concomitant
decomposition under acidic conditions (note that while the
TBDMS protecting group behaves very nicely during its removal
at the end of the overall synthesis, it is not compatible with our
earlier Wittig step such that its singular use across the entire route
is precluded)? (2) What is the chemical nature and physical
scope of the instability observed for certain of the intermediates
and, especially, for the final product GLY I? (3) Although pre-
liminary results had not been encouraging, could we perform the
asymmetric dihydroxylation step with less than an equal stoichi-
ometry of chiral ligand? (4) Could we coax spontaneous ring-
closure to the dihydrobenzofuran system as part of the debenzy-
lation step, particularly since preliminary data suggested that this
might be feasible? (5) By taking another look at the late-stage
formation of the final chromene ring needed for GLY I, could we
improve the regiochemistry so as to provide an even larger ratio
of the desired GLY I product over the GLY II side-product? Each
of these questions is briefly addressed below.
NaHSO4 SiO2
3
0.01 M HCl (MeOH) 2ꢀ3
rt
0
0.01 M HCl (MeOH) 2ꢀ3
0ꢀ5
0ꢀ5
0ꢀ5
MgBr2/EtSH
MgBr2/EtSH
ꢀ
ꢀ
rt
0
a Based on TLC and 1H NMR peak ratios. Major side product is
dehydro GLY I initially, followed by further degradation with time.
sensitive to acidic conditions. These studies used the less
precious racemic version of the penultimate intermediate that we
were able to take through the entire synthetic route to that point
uneventfully with a MOM protecting group. Several experi-
ments20ꢀ23 were then conducted on small scale so as to examine
a range of acidic pH levels, cold temperatures and abbreviated
reaction times, as well as in some cases deploying alternative
reagents such as magnesium bromide which is known to be
highly selective toward deprotection of MOM groups.23 Table 1
provides a further listing of the various deprotection studies.
None of these experiments were able to circumvent decomposi-
tion while simultaneously delivering enhanced product ratios
to the extent that it would become advantageous to eliminate
the earlier two-step switch of MOM to a TBDMS group imme-
diately after the Wittig-driven ring closure that forms the key
isoflav-3-ene 8a.
(1) An attempt was made to find a selectivity window that
would allow for removal of MOM while minimizing decomposi-
tion of GLY I, still suspecting that the latter may be particularly
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Scheme 5. Degradation of GLY I (1) and its dehydrated producta
a Note that the initial transient formation of the tertiary carbocation (additionally stabilized by its benzylic nature) via an E1 process is required because
the cis-ring fusion in GLY I precludes a simple E2 type of collapse in a concerted manner.
Accepting that it remains most desirable overall to start with a
MOM protecting group and then replace it with a TBDMS
group, we next investigated the possibility for accomplishing this
exchange in a ‘one-pot’ fashion. This would eliminate the need to
isolate the first isoflav-3-ene intermediate for which susceptibility
to spontaneous decomposition during workup was at least
suspected to contribute to lower yields (see further discussion
below). After taking advantage of our previously devised proce-
amount of warming such that its manipulations in basic or acidic
solvents should be kept to a minimum. Proposed mechanisms for
all of these decomposition pathways are provided within Scheme 5.
Verification of the postulated reactive intermediates has not been
undertaken experimentally.
Importantly, an appreciation of the propensity for these dehydro-
(or ‘ene-containing’) materials to undergo spontaneous decom-
position must also be extended to include analogous structures
such as the key intermediate 8 being envisioned for scale-up.
Even though 8 lacks the additional ring strain present in GLY I
and, itself, had not previously demonstrated any particularly
problematic stability issues during our prior total syntheses
studies, subsequent isoflavene-related molecules synthesized
more recently in our laboratories have demonstrated significant
sensitivity toward acidic conditions when subjected to, albeit,
somewhat harsher conditions.25
(3) Previous attempts to simultaneously reduce the levels of
both osmium tetroxide (OT) and chiral ligand utilized during the
Sharpless dihydroxylation step were unsuccessful, with ee falling-
off proportionately when anything less than full stoichiometric
amounts were deployed. However, these investigations were of a
preliminary nature and utilized a convenient chiral shift reagent
NMR protocol as an expeditious but crude (∼( 5% precision)
assay method.26 The diastereomeric proton shifts that become
diagnostic for the diol enantiomers in this practical, front-line
assay are tabulated within the Supporting Information. To
follow-up, we felt it would be worthwhile to develop an HPLC
assay method at this step for future quality control (QC) use, as
well as for repeating some of our prior efforts toward potentially
reducing the load of chiral ligand, if not also reducing the OT
load. Table 2 provides the distinct chromatographic retention
dure that uses PPh3 HBr in dichloromethane to gently remove
3
MOM within the course of a few hours,10 we added triethylamine
followed by TBDMSCl to the same flask and allowed the mix-
ture to stir overnight. This facile manipulation proved successful
while retaining a similar net yield across the former two-step
procedure. Its utility was then further demonstrated by success-
fully deploying it during our reported synthesis of glycinol, 7.11
(2) Prompted by the experimentally confirmed sensitivity
toward acidic conditions noted above, coupled with theoretical
concerns from the start that GLY I (1) might readily loose a water
molecule to form an inherently more stable material because the
resulting double bond then conjugates two aryl groups (Scheme 5),
we undertook HPLC stability studies of 1 to better define the
scope of its liability. GLY I appears to be adequately stable (for
synthetic-related purification manipulations) in protic media
above pH 4 and below pH 10 providing that temperatures are
not raised significantly past 40 ꢀC for prolonged periods, e.g.
during solvent removal on a rotavap.24 Quite unexpectedly,
however, even though the conjugated dehydro-GLY I may be
more thermodynamically stable, its chemical sensitivity appears
to be enhanced and its resulting solution stability becomes
diminished. The latter material is apparently capable of collap-
sing across essentially the entire pH range with just a modest
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Table 2. Chiral HPLC data for the diol intermediate and
for GLY I (1)a
Table 3. Different reaction conditions using chiral ligand
DHQD-CLB
b
b
cmpd
RT1
RT2
OsO4
DHQD-CLBa
(equiv)
addition
duration
reac. temp.
(equiv)
(ꢀC)
% ee
racemic (()-diol
chiral (+)-diolc
chiral (ꢀ)-diold
racemic (()-1e
natural (ꢀ)-1f
unnatural (+)-1e
10.38
10.35
ꢀ
11.74
11.75
ꢀ
15.31
ꢀ
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.1
2.5
4
all at once
10ꢀ15 min
10ꢀ15 min
10ꢀ15 min
10ꢀ15 min
5 h
ꢀ20
ꢀ20
ꢀ20
ꢀ80
ꢀ80
ꢀ80
ꢀ80
ꢀ80
80
15.27
13.51
ꢀ
83
82
4
89
13.35
5
92
a Methods for all compounds utilized a Chiracel OD 25 cm ꢁ 0.46 cm
column with UV detection at 230 nm on a Waters HPLC system. Mobile
phase for the diols was hexane/isopropanol (25:75) at 1.0 mL/min.
Mobile phase for 1 was hexane/isopropanol (10:90) at 1.5 mL/min.
5
94.5
98.2
97.8
6
10 h
6
5 h
c
a Dihydroquinidine p-chlorobenzoate.
b RT = retention time in minutes. Having stereochemistry eventually
needed for natural (ꢀ)-GLY I. d Having stereochemistry eventually
needed for unnatural (+)-GLY I. e Prepared as previously described.1,10
f Prepared as previously described1,10 and identical to authentic sample
isolated from elicited soybeans.
such as hydroquinidine 4-chlorobenzoate (exemplified in Table 3).
None of these studies identified a better ligand than (DHQD)2-
PHAL which was shown to consistently deliver enantiomeric excess
at 97% or higher and, wherein, we ultimately set our QC ee
specification (by HPLC assay) for this critical step in the planned
overall synthesis.
times for each of the diol enantiomers when examined by an
HPLC method that utilizes a chiral column and, ultimately,
provided ∼( 1% precision.
(4) Debenzylation of the diol to the tetrol affords the possi-
bility for spontaneous cyclization to form the requisite dihydro-
benzofuran via the dehydrated quinone-methide intermediate
shown within brackets in Scheme 3. Indeed, a ‘one-pot’ proce-
dure has been described28 and previously used during the
synthesis of pisatin.12b However, prior experience with this
approach during our total syntheses of GLY I produced only
small amounts of the cyclized product, and we eventually deemed
it more advantageous to first isolate and purify the tetrol. During
subsequent cyclization, we used a dilute solution in anhydrous
ethanol to promote the desired intramolecular reaction, a poly-
mer-bound base,29 and molecular sieves to trap the water side
product so as to additionally drive the cyclization forward. In the
end, a respectable 64% yield across the two steps was obtained.10
Nevertheless, upon additionally exploring this situation, we
found that by simply changing the solvent used during the
hydrogenolysis reaction from acetone to anhydrous ethanol,
and then increasing the hydrogen pressure from 15 to 35 psi
so as to maintain a similar net reaction time, at least 85% yields of
the cyclized material were obtained with the remaining mass
balance also being isolable and still available for separate cycliza-
tion. Again, the improved utility of this modified procedure was
further demonstrated by deploying it successfully during our
reported synthesis of glycinol, 7, wherein we also noted that the
pronounced solvent effect may be due to enhanced solvation of
the polar transition state during nucleophilic attack by the resor-
cinolic ortho hydroxy-oxygen atom on the quinone-methide,
as well as from the anhydrous conditions being better able to
accommodate the side-product water.11 In some subsequent
runs during further scale-up (later discussion), the desired
cyclization was found to be essentially quantitative.
Even though stoichiometric amounts of OT are typically
utilized during these types of dihydroxylations, and especially
so for the case of isoflavenes which bear neighboring oxygen
substituents that are thought to additionally chelate with the OT
intermediate complex,12b we previously were able to reduce the
amount of OT required during this reaction to just catalytic
amounts by adding N-methylmorpholine oxide as a secondary
oxidant plus methanesulfonamide27 as an agent to facilitate cleavage
of the osmateꢀester complexes so as to expedite recycling of
osmium.10 These conditions proved to be extremely useful when
we prepared racemic materials but became disappointing when
we tried to prepare the individual enantiomers by adding chiral
ligands at various equivalents in that no asymmetric bias was
observed. Thus, we adopted the more traditional approach of
using stoichiometric amounts of OT and the chiral ligand wherein
we were then able to achieve greater than 95% ee. Our initial choice
for the ligand upon using the Sharpless mnemonic model27 was
(DHQD)2PHAL, and it did produce the appropriate stereochem-
istry ultimately needed for natural GLY 1. Repetition of these earlier
experiments with less OT and various ligand loads performed on
small scale and utilizing the more refined HPLC assay method,
produced the same negative results relative to loss in ee. For
example, the data recorded in Table 3 indicates that while other
catalysts can be utilized, higher equivalents and lower tempera-
tures are required to obtain greater than 95% ee.
We next evaluated the possibility of reducing the quantity of
chiral ligand while still maintaining the full stoichiometry of OT,
but again this did not provide for adequately biased asymmetry
during the reaction. Presumably a subtle extension of our earlier
explanation10 for these negative results also pertains to these new
studies, namely that the chiral ligand needs to remain fully engaged
with the osmate ester-substrate complex in order to display its
enantioselectivity during a given molecular-level reaction event.
If this molecular arrangement is disrupted by either reducing OT
(in an attempt to allow for recycling) or a lack of chiral ligand,
then the asymmetric bias quickly becomes lost. Finally, an experi-
mental survey of other potential asymmetric ligands was con-
ducted in stoichiometric and even excess amounts, paying parti-
cular attention to materials that are considerably less expensive
(5) Finally, we took another look at both the stage and method
where we assemble the chromene ring. For our previous total
syntheses and related medicinal chemistry structureꢀactivity
relationship (SAR) studies, we felt that it was advantageous to
reserve construction of the isoprenyl-related chromene ring system
until the end because late-stage divergent chemistry could then
allow for GLY I, GLY II, and potentially GLY III analogues to be
generated from the same overall route. This incentive is not
present for our strategic scale-up considerations directed toward
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Scheme 6. Mechanism of the isoprenylation reaction utilized
to form the final chromene ring system within GLY I
have been firmly established for the critical asymmetric step, and
both a convenient front-line NMR assay and a practical follow-up
HPLC assay have been developed to assess ee. Performing a
retro-yield analysis suggested that in order to obtain ∼5 g of GLY
I, we would need to prepare ∼250 g of the central isoflav-3-ene
intermediate starting from ∼2 kg of the initial commercially
available building blocks. Toward such amounts, we felt that
scale-up for the early steps could proceed in increments of
100ꢀ250 mg initially and quickly move across gram into 100 g
quantities with limitations in glassware ultimately preventing
scale-up much past the 250ꢀ500 g level (2ꢀ5 L flasks) within
our academic-based center’s laboratories. We felt that the middle
steps could then be comfortably explored at the 10-mg range with
scale-up proceeding to the 100-mg and finally 1ꢀ50 g level.
However, because of the size limitations of our specialized
glassware and equipment, as well as for safety reasons within
our multipurpose academic laboratories, we envisioned caps on
the OT and hydrogenation reactions being set at the ∼10 g scale.
The last several steps would likely not be faced with such logistic
problems as the accumulated falloff in material will become more
limiting than either glassware size or safety considerations. Our
team approach placed two chemists at the front-end of the
composite of operations who were charged with optimizing reac-
tions and initiating enough scale-up to deduce satisfactory material
specifications for achieving high performance in each subsequent
step. One chemist was dedicated to developing analytical meth-
ods and performing much of the sample assays with assistance for
the latter from any of the others as needed. The remaining four
lab-based chemists performed scale-up reactions and repetitive
large-scale purification operations. ‘All-team-member’ meetings
were held weekly. In addition to continually monitoring and
potentially upgrading the technological considerations while
appropriately balancing the distributions of effort, these meetings
also ensured that coverage of critical overnight reactions and
column purifications were tended to in an equitably shared
manner across all of the team members. A summary of the actual
scale-up follows in the next section. A singular focus is provided
for the critical establishment of asymmetry associated with the
6a-hydroxy functionality which is so distinctive for all of the
glyceollin family members inclusive of our specific target
compound GLY I.
just the production of GLY I. Instead, for scale-up purposes it
would initially appear to be advantageous to move this promis-
cuous ring-forming reaction to the earliest stage possible in the
overall process with the thought that it might even then play the
role of a protecting group for the appropriate phenolic hydroxyl
functionality immediately from the start of the synthesis. Indeed,
we have deployed this type of strategy successfully in our prior
total synthesis of xanthohumol by an overall method that is quite
amenable to process chemistry, although in that case xanthohu-
mol contains a noncyclized isoprene unit linked directly to an
aryl-ring within the molecular framework of a chalcone.30 Adding
to the challenge within the context of GLY I’s structure, however,
is the chromene’s requirement for itself to ultimately display a
double bond. That type of double bond would be susceptible to
both the OT reaction at the dihydroxylation stage and to
potential reduction during hydrogenolysis of a remaining ortho-
gonally generated benzyl protecting group at the stage of the
dihydrobenzofuran ring formation, the latter now having been
nicely worked-out according to the experiments discussed above.
While temporarily masking the double bond in a preassembled
chromene system additionally can be imagined, we felt that these
extra steps and their less precedented behavior during scale-up
chemistry, detracted from moving the chromene ring’s construc-
tion to an earlier stage of the planned synthesis. Alternatively, we
did reinvestigate the two common methods used to form this
ring, namely a modified Aldol condensation31 and a Harfe-
nistꢀThom rearrangement of the propargyl ether.32 As before,10
introduction of a dimethylpropargyl group remained tedious
such that the Aldol route was still preferred. As shown in
Scheme 6, this reaction proceeds by condensing the tautomeric
keto-form of the phenol with the unsaturated aldehyde masked as
its acetal. Because this can occur with the keto-form by involving
either of the R-carbons, the reaction produces both the GLY I
and GLY II penultimate intermediates. However, GLY I is
produced in ∼5-fold higher amounts. Various attempts to
modify this to even higher ratios by adjustment of the reaction
conditions were not fruitful.
’ MULTIGRAM PREPARATION OF GLY I
Scale-Up to the Key Isoflav-3-ene Intermediate (8). Initial
attempts to regioselectively protect 14 had resulted in significant
amounts of dibenzylated material (30%) which we previously
were able to largely circumvent (<5%)10 by specifically deploying
sodium bicarbonate in acetonitrile.33 Likewise, subsequent re-
duction to alcohol 15 using sodium borohydride followed by
conventional workups also had proven to be problematic, likely
owing to ready formation of the quinone-methide under basic
and even acidic conditions. A review of the literature indicated
that despite being more elaborate, alternative methods are usually
employed for making salicylalcohol from salicylaldehyde.34
Nevertheless, during our prior small scale syntheses10 we even-
tually were able to conduct a standard sodium borohydride
reduction in methanol by adopting a workup wherein after eva-
poration of solvent, 0.1 N sulfuric acid was carefully added so as
to achieve an acidic pH of not less than 6.0. Addition of water
then conveniently precipitated high purity product in nearly 80%
yield. This convenient two-step process was found to be quite
Benefiting from these initial investigations, the intended scale-
up chemistry route was upgraded to that shown in Scheme 7. It is
two steps shorter than the original Wittig route, certain of its
reactions have already been improved in yield, and the need for
column chromatography purifications in at least some instances
have been completely removed. In addition, QC specifications
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Scheme 7. Final scale-up chemistry route used to prepare GLY 1 (1)a
a Specific reaction conditions: (a) i) BnBr, KHCO3, CH3CN, reflux, 15 h; ii) NaBH4, MeOH, 0 ꢀC then rt, 6 h, 61%, over two steps; (b) i) DIPEA,
CH3COCl, CH3OCH2OCH3, cat. Zn(OAc)2, EtOAc, 0 ꢀC then rt, 18 h; ii) BnBr, K2CO3, Me2CO, reflux, 18 h, 74% over two steps; (c) I2, Selectfluor,
CH2Cl2/MeOH, rt, 20 h, 79%; (d) K2CO3, Me2CO, reflux, 20 h, 72%; (e) i) PPh3 HBr, CH3CN, rt, 1 h; ii) t-BuOK, MeOH, reflux, 24 h, 76% over two
3
steps; (f) PPh3 HBr, CH2Cl2, rt, 2 h, then Et3N, TBDMS-Cl, rt, 12 h, 70%; (g) OsO4, (DHQD)2PHAL, CH2Cl2, ꢀ20 ꢀC, 20 h, 95%; (h) Cat. Pd/C, H2,
3
EtOH, rt, 2 h, 100%; (i) 1,1-diethoxy-3-methylbut-2-ene, 3-picoline, p-xylene, 110 ꢀC, 18 h, 60%; (j) Et3N 3HF, pyridine, CH2Cl2, rt, 5 h, 90%.
3
Scheme 8. Proposed mechanism for in situ generation of
MOM-Cl
MOM-Cl as being undesirable. Thus, this reagent was generated
in situ from ‘methylal’ and Zn2+.35 A proposed mechanism for the
latter is shown in Scheme 8. This safer procedure did not alter
regioselectivity, was scalable to the 200-g level, and in the end,
increased the yield for this step by nearly 20%. The ortho
hydroxyl was protected next by treatment with benzylbromide
using sodium carbonate in acetone to deprotonate the phenol.
This veritably useful reagent/solvent pair36 behaved appropri-
ately for ready incorporation of the second protecting group up
to similar 200 g levels of reaction.
In order to reduce costs related to using the expensive catalyst
Selectfluor, alternative reaction conditions for selective halogena-
tion of the diprotected ketone were quickly reinvestigated10 at
the gram level. Bromination by conventional methods such as
bromine in acetic acid, cupric bromide in dioxane, phenyltri-
methylammonium bromide, and polymer-supported tribromide,37
all resulted in a complex mixture of products associated with
additions to the electron rich aromatic ring and dibromination at
the R-position, as well as loss of the MOM protecting group
(Scheme 9). Among such approaches, bromine in acetic acid was
the only one that delivered a single, R-brominated product but
the latter had also lost MOM. Extensive investigations were then
directed toward altering the reaction conditions for selective
iodination, including an experimental survey to completely
replace the expensive catalyst.38 None of these studies identified
a superior system. Alternatively, upon scale-up from the 1- and
2-g levels to the 5- and 10-g levels, it was found that use of
scalable up to the 100 g level. A recrystallization of the protected
aldehyde intermediate from methanol to remove traces of
unreacted benzylbromide was found to be conducive to this
material’s better performance in the next step. During larger-
scale runs, sodium borohydride was added portion-wise and
0.5 N sulfuric acid was used for the workup to carefully adjust the
pH to 6ꢀ7. Over 900 g of intermediate 15 was conveniently
prepared in this manner with average yields across the two steps
consistently running at about 61%.
Orthogonal protection of the acetophenone 16 began with
regioselective introduction of a MOM group. We previously had
demonstrated on small scale while using MOM-Cl that this can
be accomplished with high selectivity for the para position
because the ortho hydroxy group forms a tight hydrogen bond
with the adjacent carbonyl so as to render it less reactive.10 At
larger scale, however, we regarded the direct manipulation of
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Scheme 9. Problematic bromination of diprotected ketone
Scheme 10. Proposed mechanism for Selectfluor-assisted selective r-iodinationa
a It is also suggested that the Selectfluor molecule may tend to form a ‘stacking’ arrangement with the benzene ring depending upon the nature of the
solvent and solutes’ concentrations.
methylene chloride and methanol (1:5) in 0.2 M concentration
was important for obtaining high yields of regioselectively
iodinated product 17. Addition of freshly prepared saturated
aqueous solutions of sodium thiosulfate pentahydrate to neu-
tralize traces of unused iodine was also found to be important
during workup. Scale-up proceeded in 2- to 5-fold increments up
to 124-g runs where apparatus size (larger than 2 L round-
bottom flask) with adequate stirring of the nonhomogeneous
reaction mixture became a limiting factor. Another problem
encountered during scale-up of this reaction was the observation
that there were periodic losses in yield which appeared to be
attributable to variable quality of the Selectfluor even from new
bottles having an identical batch number from the same com-
mercial source, perhaps reflecting sensitivity of the reagent to
historically undefined variations in storage and shipping condi-
tions. Because there were no readily discernible analytical
differences in these reagent batches, to circumvent this issue
we devised a standardized small-scale reaction (5 g, 17.5 mmol)
protocol as a functional QC measure to be undertaken in our
laboratories for each new bottle prior to its use in larger-scale
reactions. The progress of this reaction was followed by both
TLC and 1H NMR, with the appearance of a peak at ca. δ 4.4 for
the protons on the carbon bearing the iodine atom accompanied
by the loss of the methylketone peak at ca. δ 2.5 being particularly
diagnostic. A proposed mechanism for this interesting reaction is
shown in Scheme 10. Beyond knowing that the transformation
proceeds via a reactive iodinium species,39 further mechanistic
details have not been reported. Regioselective iodination at the
R-carbon in the presence of a benzene ring is very delicate,
especially when the latter’s electron density is increased by
substitution with electron-donating benzyloxy- and methoxy-
methyleneoxy groups. Perhaps in addition to just activating
elemental iodine toward electrophilic attack (Scheme 10), the
positive charges on this catalyst also serve to counter the overall
Table 4. Stability data determined by 1H NMR for 17 in
different solvents at different temperatures (t is time after
reaction set up)
approx. % degradation
solvent
CH2Cl2
t = 0 h
t = 6 h
t = 17 h
t = 24 h
a
reflux
70 ꢀC
rt
0
0
0
0
0
0
60
0
0
70
2.0
60
95
ꢀ
a
CH3CN
Me2CO
ꢀ
6
reflux
70 ꢀC
0
95
b
a
DMF
ꢀ
ꢀ
a Reaction was not performed up to this duration. b No observation was
made until t = 17 h time point.
electron density on the benzene ring by residing in a stacking
arrangement with the latter. While we have not pursued mechan-
istic studies per se, the specific solvent ratio and concentration
range found to be optimal for this reaction are thus less surprising
because such an intermolecular arrangement would be expected
to be highly sensitive to even subtle modifications of these
solvation shell variables.40
The ether linkage between 15 and 17 was formed under basic
conditions after refluxing the reaction mixture for 20ꢀ24 h in
acetone. Solutions of 17 were observed to undergo decomposi-
tion at elevated temperatures. Stability studies were undertaken
to ascertain the least harsh conditions needed to form the ether.
Although the identities of the degradation products were not
discernible from these studies, the integrity of 17 could be readily
assessed by proton NMR. The results are summarized in Table 4.
After examination of several different solvents, acetone remained
preferable across variations in temperature and time relative to
17’s stability. Scale-up readily proceeded from the ∼1ꢀ115 g
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(∼140 mmol) level. Reaction progress was again followed by
(DHQD)2PHAL with all of our orders suddenly being placed
on indefinite back-order status. Given our overall time frame
requirement, this situation made an in-house synthesis of the
chiral ligand imperative. The latter was accomplished by follow-
ing a two-step literature procedure41 further adapted to allow for
preparation of large quantities. QC measures were adopted to
establish the integrity of the synthesized ligand, namely by
comparison of melting point and NMR spectra with data from
the literature.41 Because of the observed potential for variation in
catalytic performance, however, a functional QC measure was
additionally established. This QC assay involved conducting a 50
mg standard reaction using separately QC-approved starting
material 8 and separately QC-approved OT, noting that OT is
itself capable of showing differences in performance between lots
(further discussed below). Satisfactory assessment of the product
from the standard reaction had to meet a specification of >97% ee
using our chiral HPLC assay as described above. Interestingly, in
the end, not only were the supply issues overcome by preparing
this catalyst ourselves, but a cost comparison also indicated that
after our synthesis of ∼200 g of chiral catalyst, we were able to
effect a savings of nearly $4000.00.
Another concern arising during scale-up at this step pertained
to safety issues for handling the OT because we would need to
use levels as high as 5 g during a given larger-scale reaction run.
OT was purchased as crystalline material in 1-g sealed vials. To
manipulate multiples of this highly toxic material into repeated
reaction runs, we devised a procedure wherein several vials could
be individually broken by a plunger extending into a small-
mouthed, thick-walled bottle already containing a specified
volume of toluene so as to make 100 mg/mL solutions of
reagent. An upper range of 50 to 100 mL volumes was typically
prepared in this manner. A sodium bisulfite ‘bath’ was addition-
ally deployed around the bottle to protect the environment
should any breakage of the device itself occur, although this never
became an issue across numerous deployments of this conve-
nient process. Scale-up proceeded to the 10 g (18 mmol) level
with average yields being ∼95%, somewhat higher than our lower
scale efforts at ∼86% and substantially higher than our prior
milligram-range total synthesis efforts at ∼70%.1,10 In addition,
for the highest scale runs 19 exhibited >98% ee even though the
temperature during the asymmetric dihydroxylation was able to
be raised to the more convenient ꢀ20 ꢀC compared to the
previous conditions that utilized ꢀ78 ꢀC.1,10 Purification again
exploited a high-yielding crystallization rather than column
chromatography. Final product specifications included: melting
point; TLC; proton NMR; elemental analysis data for carbon and
hydrogen; and importantly, >97% ee by HPLC analysis.
1
both TLC and H NMR, with complete disappearance of the
diagnostic peak at about δ 4.4 for the protons on the carbon
bearing the iodine atom in 17, serving to signal stoppage of the
reaction. Purification of 15 and 17 as well as the ether inter-
mediate 18 can be accomplished by crystallization in high yields.
As discussed in the Background section, cyclization of 18 to 8a
was accomplished by an intramolecular Wittig olefination reac-
tion. At the 1 g scale, substantial amounts of 18 were found to
degrade during initial preparation of the Wittig salt upon
treatment with triphenylphosphine hydrobromide. This oc-
curred even when formation of this precursor was attempted in
freshly distilled acetonitrile at room temperature.10 Analysis of
data collected for the major side-product confirmed that the
MOM protecting group was gradually being cleaved, an event
under these types of conditions that also has at least some
precedent within the literature.21 Careful monitoring of this
initial part of the reaction by TLC revealed that formation of
the Wittig salt could be completed in just 1 h of stirring at room
temperature. Even more importantly, as scale-up proceeded we
found that portionwise addition of the triphenylphosphine
hydrobomide was preferable to adding the entire reagent in
one lot. Thus, this reagent was added in five separate fractions of
0.2 equiv at ∼15ꢀ20 min apart. The overall timing of this
reaction can be easily monitored visually because 18 has limited
solubility in acetonitrile, whereas its Wittig salt is very soluble
such that, by addition of the last 0.2 equiv, the reaction mixture
becomes a homogeneously clear solution. Ultimately, we were
able to achieve complete conversion of 18 to its Wittig salt with
negligible formation of any side products including that involving
loss of MOM. This material was then used without further
purification in the intramolecular condensation by treatment
with sodium tert-butoxide in refluxing methanol, after which 8a
precipitates from the reaction medium. Scale-up proceeded to
the 30 g (58 mmol) level averaging ∼76% yields, somewhat
higher than the ∼70% observed during our early scale-up
chemistry efforts and comparable to those at 78% observed
previously during our initial total syntheses of GLY I that had
been accomplished at the mg scale.1,10
As already described in the prior section pertaining to our
initial strategic chemistry studies, protecting group modification
of 8a to 8 was optimized so as to become a high-yielding, one-pot
procedure. Scale-up proceeded to the 20 g (41 mmol) level with
average yields being maintained around 70%. Triphenylpho-
sphine was removed by filtering a slurry of the reaction mixture
in CH2Cl2/TFA/activated silica. Product purification was then
accomplished by silica pad filtration followed by crystallization
such that, to this point in the overall synthesis, the need for
column chromatography purification procedures had been com-
pletely removed.
Scale-Up of the Asymmetric Dihydroxylation Reaction to
Produce the Chiral Diol Intermediate (19). Optimization of
this critical step has already been described above in the discus-
sions about our strategic chemistry studies. The following
comments convey additional hurdles encountered during further
scale-up. Somewhat similar to our previous observations for the
iodination reagent, we also noted variation in the quality of
performance by the chiral catalyst needed for this step, although
in this case pertaining more toward when it was purchased from
different suppliers rather than from lot-to-lot and within lot
variations. Even more problematic, however, was the fact that we
seemed to have quickly depleted the commercial world supply of
Formation of the Last Two Rings and Final Deprotection
to Provide GLY I (1). Optimizations for debenzylation of 19 with
concomitant cyclization to the cis-fused dihydrobenzofuran system
in 20, and for formation of the final chromeme ring system in 21,
already have been discussed above in the strategic chemistry
development studies section. The following comments convey
additional observations encountered during further scale-up. The
latter was performed up to the range of ∼5 g (8.5 mmol) and 3 g
(6.6 mmol) in ∼93% and 60% yields for 20 and 21, respectively.
Because the chromene regioisomers for both GLY I and GLY II
are produced in an ∼5:1 ratio during the course of this last ring-
forming reaction, their separation by gravity column chromatog-
raphy at this late stage represents the first use of this purification
technique needed during the course of the entire synthesis.
Specifications for both 20 and 21 include melting point, TLC,
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Table 5. Optical rotation data for key chiral intermediates
and for GLY I
chromatography purifications, and the optimized synthesis requir-
ing 11 steps with only two column chromatography purifications
that occur at the end of the overall route. For the key asymmetric
dihydroxylation step, it is necessary to have a stoichiometric
amount of both OT and chiral ligand present throughout the
course of the reaction. Favorable to deploying this type of
asymmetric reaction within these types of molecular constructs,
scale-up from milligram to low-gram and then to mid-gram range
was accompanied by increasing yields while retaining excellent
ee. Preparation of the chiral ligand can be highly advantageous
from both a supply and cost perspective. Safe ways to handle both
of the reagents needed for this reaction in a very practical manner
on multigram scale have been devised and conveyed. In the end,
nearly 5 g of material was prepared after several runs conducted
by a team of six synthetic medicinal chemists and one bioanaly-
tical chemist working over a period of ∼6 months. It is felt that by
following this [now] well-trodden path,44 the overall procedure
should be quite amenable to even further scale-up within quicker
timeframes while demanding less person-power. Of course,
alternative paths always remain inviting as well.
cmpd
[R]25
solvent
conc. [c, (g/100 mL)]
D
diol 19
20
+6.7
ꢀ209.5
ꢀ202.6
ꢀ201.0
MeOH
MeOH
EtOAc
EtOAc
1.6
0.3
synthetic 1
naturala1
0.15
0.10
a Authentic sample obtained by isolation from stressed soybean plants.
Table 6. Yield and scale comparison for synthesis of GLY I
before optimization
after optimization
reaction
step
av % yielda
largest scalea
av % yield
largest scale
a
b
c
d
e
f
61b
56
70
72
78
69
70
54
61
77
1 mmol (0.14 g)
1 mmol (0.15 g)
1 mmol (0.29 g)
1 mmol (0.41 g)
1 mmol (0.51 g)
1 mmol (0.48 g)
1 mmol (0.54 g)
0.1 mmol (39 mg)
0.1 mmol (40 mg)
0.1 mmol (43 mg)
61b
74
0.72 mol (100 g)
1.3 mol (200 g)
0.43 mol (124 g)
0.28 mol (116 g)
58 mmol (30 g)
41 mmol (20 g)
18 mmol (10 g)
8.5 mmol (5 g)
6.6 mmol (3 g)
2.2 mmol (1 g)
79
72
76
’ EXPERIMENTAL SECTION
70
g
h
i
95
General. Chemical reactions were conducted under nitrogen
in anhydrous solvents unless stated otherwise. Anhydrous sol-
vents were purchased from commercial sources and were used
without additional purification except for: (i) acetone (Me2CO)
which was further dried over 3 Å molecular sieves; and
(ii) tetrahydrofuran (THF) which was further distilled under
nitrogen over sodium benzophenone. All other reagents obtained
from commercial suppliers were used without further purifica-
tion. Thin layer chromatography (TLC) was done on 250 μm
fluorescent TLC plates (Baker-flex, Silica Gel IB-F from VWR
International, LLC) and visualized by using UV light or iodine
vapor. Normal-phase flash and gravity column chromatography
purifications were performed using silica gel (200ꢀ425 mesh
60 Å pore size) and ACS grade solvents. A RediSep flash column
(12 g) was used for flash separation on a Combiflash companion
unit from Teledyne Isco, Inc. Optical rotations were determined
on a Rudolph Research model AUTOPOL III automatic polari-
meter. Melting points (mps) were determined on an Electro-
thermal digital melting point apparatus and are uncorrected.
NMR spectra were recorded on either a Varian Inova-600 spec-
trometer at 600 MHz, or a Unity-400 spectrometer at 400 MHz.
Peak locations were referenced using either tetramethylsilane
(TMS) or residual nondeuterated solvent as an internal standard.
Proton coupling constants are expressed in hertz. 13C NMR
chemical shifts are reported to the first decimal place unless peaks
are very close, wherein for such instances values are reported to a
second decimal place. Spin multiplicity for 1H NMR are reported
using the following abbreviations: s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet, quin = quintet, br = broadened,
dd = doublet doublet, dt = doublet triplet, dq = doublet quartet
and other combinations derived from those listed. Elemental
analyses were performed by Atlantic Microlab, Inc., Norcross,
GA, U.S.A., and were regarded as acceptable when experimental
values are within (0.4% of the calculated values.
100
60
j
90
overall
2.7c
∼25 mg after 13 steps
11.5c
∼5 g after 11 steps
a Data from previous synthesis.10 b Across two steps. c Across longest
linear flow of steps.
proton NMR and chiral HPLC data with preservation of at least
97% ee. In the final step, deprotection with basic reagents like
silica-supported TBAF or TAS-F42 had largely resulted in
degradation of the initially formed product.10 Subsequent stabi-
lity studies (see prior discussion above) have shown that forma-
tion of a dehydro-species can occur which, in turn, is even more
susceptible toward additional decomposition (Scheme 5). Mildly
acidic reagents were further explored as part of the scale-up.
These studies indicated that while NEt3 3 HF43 should be
3
retained as the reagent of choice, it is important to buffer the
reaction mixture with excess pyridine. The latter significantly
reduces formation of the dehydro-GLY I side-product and raises
the yield for this step to ∼90% of the target compound 1 when it
is run at the 1-g (2.2 mmol) level. Purification of the final material
was accomplished by either flash column chromatography or by
deployment of a Combiflash companion unit. After combination
and evaporation of eluted samples, residues can be conveniently
manipulated via methanol which, in turn, can be either lyophi-
lized or evaporated and dried so as to provide a free-flowing solid
product. Complete specifications for 1 are provided in the
Experimental Section. A summary of the optical rotation data
for the asymmetric intermediates and final compound 1 are
provided in Table 5. Chiral HPLC chromatograms for 1 are
provided within the Supporting Information.
’ SUMMARY
4-Benzyloxy-2-hydroxybenzaldehyde (15a). To a solution
of the dihydroxybenzaldehyde 14 (100 g, 0.67 mol) in CH3CN
(1 L), BnBr (112.4 g, 0.67 mol) and NaHCO3 (90.8 g, 0.67 mol)
were added. The reaction mixture was refluxed for 48 h and was
monitored by TLC. Upon completion, the white precipitate was
Table 6 provides a side-by-side comparison of the yields and
reaction amounts for each of the steps before and after optimiza-
tion and scale-up. The overall yields are ∼3% versus nearly 12%
with the former route requiring 13 steps plus several column
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filtered and the filtrate was evaporated under vacuum. The
residue was dissolved in MeOH (500 mL) and was precipitated
at 0 ꢀC. The precipitates were further recrystallized from MeOH
to obtain 15a (127.5 g, 0.56 mol, 84%) as an off-white solid: mp
80ꢀ82 ꢀC (lit.10 78ꢀ80 ꢀC); 1H NMR (DMSO, 400 MHz) δ
10.99 (1H, s), 9.99 (1H, s), 7.37 (5H, m), 6.61 (1H, m), 6.54
(1H, d), 5.15 (2H, s).
2-(50-Benzyloxy-20-hydroxymethyl)phenoxy-1-(20-benzyl-
oxy-40-methoxy-methy-lenoxy)phenylethanone (18). To a
solution of salicyl alcohol 15 (107.5 g, 0.46 mol) and R-iodo
ketone 17 (115.7 g, 0.28 mol) in Me2CO (1.4 L) was added
K2CO3 (46.32 g, 0.34 mol). The reaction mixture was stirred at
reflux for 18ꢀ20 h. Reaction progress was followed by TLC and
1H NMR. After completion, solvents were evaporated under
vacuum, and the residue was dissolved in EtOAc/H2O (1:1)
mixture. The organic layer was washed with 0.1 M HCl, saturated
NaHCO3, H2O and brine, dried over anhyd Na2SO4, and evapo-
rated to dryness under vacuum. The solid residue was recrys-
tallized from EtOAc/hexane (700 mL, 1:1) to obtain 18 (107.8 g,
0.21 mol, 72%) as a white solid: mp 122ꢀ124 ꢀC [lit.10 115ꢀ
118 ꢀC]; TLC Rf = 0.3 [EtOAc/hexane (1:2)]; 1H NMR
((CD3)2CO, 400 MHz) δ 7.87 (1H, d, J = 8.8 Hz), 7.64 (2H,
d, J = 7.6 Hz), 7.41 (6H, m), 7.33 (1H, m), 7.24 (2H, m),
6.93 (1H, d, J = 2 Hz), 6.77 (1H, dd, J = 2, 8.8 Hz), 6.56 (1H, dd,
J = 2.4, 8.4 Hz), 6.26 (1H, m), 5.32 (2H, s), 5.31 (2H, s), 5.22
(2H, s), 4.57 (2H, d, J = 6.4 Hz), 4.11 (1H, t, J = 6.4 Hz), 3.45
(3H, s).
4-Benzyloxysalicyl alcohol (15). To a solution of the ben-
zaldehyde 15a (127.5 g, 0.52 mol) in MeOH (1 L) at 0 ꢀC,
NaBH4 (16 g, 0.42 mol) was added. The reaction mixture was
stirred for 18 h at rt. Reaction progress was monitored by TLC.
Upon completion, the reaction mixture was reduced to one-
fourth volume and was treated with 0.5 N aq H2SO4 to adjust pH
to about 6ꢀ7 followed by addition of H2O (1 L). Upon vigorous
stirring white precipitate formed. After filtration, the precipitate
was lyophilized, washed using a chilled toluene (∼ꢀ50 ꢀC) and
dried under vacuum to obtain 15 (89 g, 0.38 mol, 73%) as an off-
white solid: mp 86ꢀ88 ꢀC (lit.10 88ꢀ90 ꢀC); 1H NMR (DMSO,
400 MHz) δ 9.33 (1H, s), 7.35 (5H, m,) 7.08 (1H, d, J = 8.4 Hz),
6.42 (2H, m), 5.01 (2H, s), 4.80 (1H, m), 4.39 (2H, s).
2-Benzyloxy-4-methoxymethylenoxyacetophenone (16a).
To a solution of dimethoxymethanal (150.5 g, 1.97 mol, 175 mL)
and Zn(OAc)2 (43.88 mg, 0.24 mmol) in EtOAc (350 mL),
CH3COCl (154.7 g, 1.97 mol) was added over 2ꢀ3 h. The reac-
tion mixture was stirred for an additional 2ꢀ3 h and then cooled
to 0 ꢀC. A solution of 16 (200.0 g, 1.31 mol) in EtOAc (615 mL)
was then added slowly, followed by dropwise addition of Hunig’s
base (211.6 g, 1.63 mol). The reaction mixture was stirred for 18
h while being monitored by TLC. After completion, H2O
(300 mL) was added and the mixture further stirred for 1 h.
The organic phase was washed with 1 M NaOH, brine and dried
over anhyd Na2SO4. Solvents were evaporated under vacuum to
obtain a yellowish oily residue which was utilized directly in the
next step.
20,7-Dibenzyloxy-40-(methoxymethylenoxy)isoflav-3-ene
(8a). To a suspension of 18 (30.1 g, 58.5 mmol) in anhyd
CH3CN (1 L), PPh3 HBr (20.1 g, 58.4 mmol) was added in five
3
portions of ∼4.0 g each at intervals of ∼15ꢀ20 min. By the last
addition, the reaction mixture became a clear solution. The
progress of the reaction was checked by TLC (CH2Cl2:MeOH
[15/1] as developing system). The reaction was complete in
1ꢀ2 h. Solvents were then evaporated to dryness under vacuum
at rt to obtain an off-white residue which was used in the next step
without further purification.
To a solution of product from the previous step in anhyd
MeOH (1.5 L), potassium tert-butoxide (13.1 g, 0.12 mol)
was added with stirring. The reaction mixture was refluxed for
18ꢀ24 h. Reaction progress was monitored by TLC. After
completion, the mixture was cooled to rt and filtered. The
precipitate was dissolved in CH2Cl2. The organic layer was
washed with H2O and dried over anhyd Na2SO4. After filtration,
the solvents were evaporated under vacuum to obtain 8a (21.4 g,
44.5 mmol, 76% over two steps) as an off-white solid: mp
126 ꢀ131 ꢀC (lit.10 115ꢀ118 ꢀC) ; TLC Rf 0.39 (hexanes/
EtOAc [5:1]); 1H NMR ((CD3)2CO, 600 MHz) δ 7.42 (10H,
m), 7.28 (1H, d, J = 8.4 Hz), 7.02 (1H, d, J = 8.4 Hz), 6.81 (1H, d,
J = 2.4 Hz), 6.69 (1H, dd, J = 2.4, 8.4 Hz), 6.61 (1H, s) 6.57 (1H,
dd, J = 2.4, 8.4 Hz), 6.47 (1H, d, J = 2.4 Hz).15 (2H, s), 5.10 (2H,
s), 4.94 (2H, d, J = 1.2 Hz), 0.97 (9H, s), 0.19 (6H, s);13C NMR
((CD3)2CO, 150 MHz) δ 160.4, 159.2, 158.1, 155.6, 138.2,
137.7, 129.98, 129.91, 129.3, 129.2, 128.8, 128.7, 128.5, 128.34,
128.30, 122.5, 121.8, 118.0, 108.9, 108.8, 102.9, 102.4, 95.0, 90.1,
71.0, 70.4, 68.9, 56.0, 3.37, 3.35, ꢀ11.4.
To a solution of the above product (128.3 g) in Me2CO
(1.3 L), BnBr (168.6 g, 0.98 mol) and K2CO3 (96.74 g) were
added under a flow of N2. The reaction mixture was stirred under
reflux while progress was monitored by TLC. After completion,
the mixture was filtered and solvents were evaporated under
vacuum. The product was recrystallized from MeOH to obtain
16a (249.0 g, 0.97 mol, 74% in two steps) as off-white crystals:
mp 71ꢀ73 ꢀC (lit.10 70ꢀ71 ꢀC); TLC Rf = 0.63 [EtOAc/
1
hexanes (1:2)]; H NMR (CDCl3, 600 MHz) δ 7.82 (1H, d,
J = 8.4 Hz), 7.40 (5H, m), 6.68 (2H, m), 5.19 (2H, s), 5.14
(2H, s), 3.47 (3H, s), 2.55 (3H, s).
1-(20-Benzyloxy-40-methoxymethylenoxy)phenyl-2-iodo-
ethanone (17). To a solution of acetophenone 16a (124.26 g,
0.43 mol) in anhyd CH2Cl2 (280 mL) and anhyd MeOH (1.7 L),
Selectfluor (100 g, 0.26 mol) was added followed by elemental I2
(49.88 g, 0.22 mol) under a flow of N2. The reaction mixture was
stirred for 24 h. Reaction progress was monitored by TLC and 1H
NMR. After completion, the mixture was filtered and the preci-
pitate was washed extensively with CH2Cl2. The combined organic
phase was evaporated under vacuum at 25ꢀ30 ꢀC. The residue was
again dissolved in CH2Cl2. The organic layer was washed with
freshly prepared Na2S2O3 solution (10% w/v), dried over anhyd
Na2SO4 and evaporated under vacuum. The residue was recrys-
tallized from MeOH to obtain 17 (140.1 g, 0.34 mol, 79%) as
yellowish crystals: mp 76ꢀ78 ꢀC (lit.10 66ꢀ68 ꢀC); TLC Rf =
20,7-Dibenzyloxy-40-(tert-butyldimethylsilyloxy)isoflav-3-
ene (8). To a solution of 20,7-dibenzyloxy-40-(methoxymethalenoxy)-
isoflav-3-ene 8a (19.6 g, 40.8 mmol) in anhyd CH2Cl2 (200 mL)
PPh3 HBr (17.6 g, 51.2 mmol) was added. The reaction mixture was
3
stirred at rt for 1ꢀ2 h while followed by TLC (EtOAc/hexanes 1:2).
After completion, Et3N (7.8 g, 75.6 mmol, 10 mL) and TBDMS-Cl
(7.6 g, 50.4 mmol) were added. The reaction was stirred at rt for
12ꢀ15 h. After completion, solvents were evaporated under vacuum
at 30 ꢀC. The solid residue was dissolved in CH2Cl2 (2 L), and oven-
dried silica (340 g, dried overnight at 120 ꢀC in oven and cooled in a
desiccator) and a small amount of TFA were added; the mixture was
gently stirred until complete disappearance of PPh3 (TLC). After
filtration, the filtrates were passed through a pad of silica. The solvents
1
0.65 [EtOAc/hexanes (1:2)]; H NMR (CDCl3, 600 MHz)
δ 7.88 (1H, d, J = 9.6 Hz), 7.49 (2H, m), 7.41 (3H, m), 6.71
(2H, m), 5.20 (2H, s), 5.17 (2H, s), 4.40 (2H, s), 3.48 (3H, s).
1160
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Organic Process Research & Development
ARTICLE
were evaporated under vacuum, and the residue was recrystallized
from CH2Cl2/MeOH (1:5) to obtain 8 (15.9 g, 28.6 mmol, 70%) as
white crystals: mp 104ꢀ106 ꢀC [lit.10 106ꢀ107 ꢀC]; TLC Rf = 0.42
[EtOAc/hexanes (1:2)]; 1H NMR ((CD3)2CO, 600 MHz) δ 7.40
(10H, m), 7.25 (1H, d, J = 8.4 Hz), 7.02 (1H, d, J = 8.4 Hz), 6.62
(2H, m), 6.57 (1H, dd, J = 2.4, 8.4 Hz), 6.51 (1H, dd, J= 2.4, 8.4 Hz),
6.47 (1H, d, J = 2.4 Hz), 5.15 (2H, s), 5.10 (2H, s), 4.94 (2H, d,
J = 1.2 Hz), 0.97 (9H, s), 0.19 (6H, s);13C NMR ((CD3)2CO,
150 MHz) δ 160.4, 158.1, 157.5, 155.6, 138.2, 137.9, 130.0, 129.8,
129.3, 129.2, 128.7, 128.57, 128.52, 128.3, 128.29, 128.28, 121.8,
118.1, 113.1, 108.9, 105.9, 102.9, 90.1, 70.9, 70.4, 68.9, 25.9, ꢀ4.3,
ꢀ11.4; Anal. (%) calcd for C17H18O7, C 76.30, H 6.95, found, C
76.02, H 7.09.
ꢀ4.39, ꢀ4.40, ꢀ11.45, ꢀ11.46; Anal. (%) calcd for C21H26O5Si,
C 65.26, H 6.78, found, C 65.75, H 6.76.
9-(tert-Butyldimethylsilyloxy)glyceollin I (21). To a mixture
of 6a-hydroxypterocarpan 20 (3.07 g, 7.94 mmol) in anhyd
p-xylene (40 mL) were added 1,1-diethoxy-3-methyl-2-butene
(2.6 g, 16.4 mmol, 3.1 mL) and 3-picoline (0.2 g, 2.1 mmol,
0.3 mL) successively under a flow of N2. The reaction flask was fitted
with a distillation assembly and was stirred at 125 ꢀC (internal temp.
120 ꢀC). Progress of the reaction was followed by TLC. After
completion, the reaction mixture was directly applied to a column
and was purified by gravity column chromatography using a step
gradient [first hexanes/CH2Cl2 (2:1), 450 mL then was changed to
hexanes/CH2Cl2/EtOAc (20: 10:1) to obtain 21 (2.2 g, 4.8 mmol,
60%) as off-white solid: mp 69ꢀ75 ꢀC; TLC Rf 0.57 [EtOAc/
hexanes (1:2)]; 1H NMR ((CD3)2CO, 600 MHz) δ 7.28 (1H, d,
J = 8.4 Hz), 7.24 (1H, d, J = 8.4 Hz), 6.57 (1H, d, J = 10.2 Hz),
6.46 (1H, m), 6.28 (1H, d, J = 2.4 Hz), 5.66 (1H, d, J = 10.2 Hz),
5.28 (1H, s), 5.09 (1H, s,), 4.20 (1H, d, J = 11.4 Hz), 4.08 (1H, d,
J = 11.4 Hz), 1.39 (3H, s), 1.35 (3H, s), 0.97 (9H, s), 0.20 (6H, s);
Anal. (%) calcd for C21H26O5Si, C 68.99, H 7.13, found, C 68.91,
H 7.27.
(+)-40-tert-Butyldimethylsilyloxy-20,7-(dibenzyloxy)isoflavan-
3,4-diol (19). To a solution of chiral ligand (DHQD)2PHAL
(15.6 g, 20.0 mmol) in CH2Cl2 (80 mL), OsO4 (5 g, 20.0 mmol)
was added. After stirring at ꢀ20 ꢀC for 1 h, a solution of 8 (10 g,
18.1 mmol) in CH2Cl2 (80 mL) was slowly added over 10ꢀ
15 min, and the mixture was stirred at ꢀ20 ꢀC for 18ꢀ20 h.
Reaction progress was monitored by TLC. After completion, the
reaction was allowed to warm to rt, and 10% sodium sulfite
(100 mL, pH ∼9.0) and 10% sodium bisulfite (100 mL, pH ∼4)
solution was added. After stirring at rt for 2 h, a mixture of THF/
EtOAc (1:4, 1 L) was added to the reaction mixture and further
stirred at 55 ꢀC (external oil bath temp) for an additional 3ꢀ4 h.
The reaction mixture was cooled to rt and filtered. The aq phase
was extracted with EtOAc. The combined organic phase was
washed wit 0.1 M HCl and brine and dried over anhyd Na2SO4
and evaporated under vacuum. The product was recrystallized
from EtOAc/hexanes to obtain 19 (10.2 g, 17.3 mmol, 95%,
(ꢀ)-Glyceollin I (1). To a solution of 21 (1 g, 2.2 mmol) in
CH2Cl2 (30 mL) were added Et3N 3HF (30 mmol) and excess
3
pyridine (45 mmol). The reaction mixture was stirred at rt for
6 h. Progress was followed by TLC. After completion, solvents
were evaporated under vacuum at 20 ꢀC and directly applied to a
flash column (silica ∼20 g) using ‘dry sample’ loading techniques
[EtOAc/hexanes (1:1)]. The eluting fractions were collected,
solvents were removed under vacuum, and the resulting residue
was lyophilized after dissolving in a minimal amount of MeOH
to obtain 1 (0.67 g, 1.97 mmol, 90%) as pinkish-white solid: mp
>98% ee) as a white solid: mp 75ꢀ77 ꢀC; [R]25 +6.7 (c 1.6,
D
95ꢀ101 ꢀC; [R]25 ꢀ202.6 (c 0.15, EtOAc); TLC Rf = 0.33
MeOH); TLC Rf = 0.28 [EtOAc/hexanes (1:3)]; Chiral HPLC
RT = 10.35 min [Standard racemate RT = 10.38 and 15.31 min],
mobile phase was 2-propanol/hexanes (25:75) at 1.0 mL/min;
1H NMR ((CD3)2CO, 600 MHz) δ 7.59 (1H, dd, J = 2.4. 8.4
Hz), 7.39 (11H, m), 6.58 (2H, m), 6.49 (1H, d, J = 2.4 8.4 Hz),
6.38 (1H, d, J = 2.4 Hz), 5.52 (1H, d, J = 6.6 Hz), 5.20 (2H, s),
5.07 (2H, s), 4.73 (1H, d, J = 11.4 Hz), 4.26 (1H, m), 4.21
(1H, m), 4.02 (1H, d, J = 11.4 Hz), 0.96 (9H, s), 0.17 (6H, s); 13C
NMR ((CD3)2CO, 100 MHz) δ 159.9, 157.4, 157.2, 155.7,
138.4, 137.8, 130.7, 130.1, 129.3, 129.2, 128.6, 128.6, 128.4,
128.2, 128.1, 123.5, 118.3, 112.4, 108.7, 106.1, 102.1, 72.0, 70.8,
7.2, 67.6, 67.4, 25.9, 18.6, ꢀ4.3; Anal. (%) calcd for C35H40O6Si,
C 71.89, H 6.89, found, C 71.83, H 6.92.
D
(MeOH/CH2Cl2/hexanes (1:10:10)); Chiral HPLC RT =
11.75 min [standard racemate RT = 11.74 and 13.51 min],
mobile phase was 2-propanol/hexanes (10:90) at 1.5 mL/min;
1H NMR (CD3OD, 600 MHz) δ 7.21 (1H, d, J = 8.4 Hz), 7.16
(1H, d, J = 8.0 Hz), 6.60 (1H, d, J = 10 Hz), 6.46 (1H, d, J = 8.4
Hz), 6.40 (1H, dd, J = 2.0, 8.4 Hz), 6.22 (1H, d, J = 2 Hz),
5.62 (1H, d, J = 10 Hz), 5.16 (1H, s), 4.16 (1H, d, J = 11.6 Hz),
3.93 (1H, d, J = 11.2 Hz), 1.38 (3H, s), 1.35 (3H, s); Anal.
(%) calcd for C20H18O5 0.1H2O, C 70.62, H 5.39, found, C
3
70.35, H 5.65.
Chiral NMR Shift Reagent Studies.26. Europium(III) tris-
[3-(heptafluoro-propylhydroxy-methylene)-l-camphorate was de-
ployed in 20% mole ratio by dissolving it in the same deuterated
solvent as for the routine NMR sample, adding the solution directly
to the latter, and then rerunning the NMR spectrum.
(ꢀ)-9-(tert-Butyldimethylsilyloxy)glycinol (20). To a solu-
tion of 19 (5.01 g, 8.5 mmol) in anhydrous EtOH (110 mL) at
0 ꢀC, 10% Pd/C (1.01 g) was added. The mixture was stirred at rt
for 4 h under hydrogen atmosphere (35 psi). Progress was
followed by TLC. Prolonged reaction times can cause losses in
overall yield. After completion, the reaction mixture was passed
through a pad of Celite which was then washed with EtOH (3 ꢁ
50 mL). The combined solvents were evaporated under vacuum
to obtain 20 (3.27 g, 8.5 mmol, 100%) as an off-white powder:
mp 196ꢀ198 ꢀC; [R]25D ꢀ209.5 (c 0.3, MeOH); TLC Rf = 0.41
Chiral Column Chromatography.11,45 In addition to the
method delineated in Table 2, a chiral Cyclobond column from
ASTEC Inc. can be used in a Waters HPLC equipped with a
model 2659 separation module, a quaternary pump, a degasser, an
auto sampler/injector (syringe volume =100 μL), a column oven
and a model 2996 photodiode array detector. The mobilephase in
this case used a gradient solvent system having water, acetonitrile,
and methanol in the following percentages with time: 60, 0, 40 at
the start; 45, 0, 55 at 30 min; and 60, 10, 30 at 48 min, after which
the system was stepped back to start conditions and flushed for 12
min. Temperature was 35 ꢀC, flow rate was 0.5 mL/min, and the
detector was set at 254 nm. Retention times (min) were: 52.7 for
authentic GLY I standard from stressed soybean; 53.2 for
synthesized material; 49.4 for synthesized unnatural (+) material;
and 49.5 + 53.3 for synthesized racemic material.
1
[MeOH/CH2Cl2/hexanes (1:10:10)]; H NMR ((CD3)2CO,
600 MHz) δ 8.57 (1H, s), 7.31 (1H, d, J = 8.4 Hz), 7.26 (1H, d,
J = 7.8 Hz), 6.56 (1H, dd, J = 8.4 Hz, J = 2.4 Hz), 6.46 (1H, dd, J =
2.4, 8.4 Hz), 6.33 (1H, d, J = 2.4 Hz), 6.27 (1H, d, J = 1.8 Hz),
5.28 (1H, s), 5.03 (1H, s), 4.13 (1H, d, J = 11.4 Hz), 4.01 (1H, d,
J = 11.4 Hz), 0.97 (9H, s), 0.20 (6H, s); 13C NMR ((CD3)2CO,
150 MHz) δ 161.6, 159.6, 158.6, 157.0, 133.1, 125.0, 123.6,
113.3, 113.0, 110.7, 103.7, 103.2, 90.1, 85.8, 76.6, 70.5, 25.9,
1161
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Organic Process Research & Development
ARTICLE
’ ASSOCIATED CONTENT
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Supporting Information. Experimental details for synthesis
b
of chiral ligand ((DHQD)2PHAL); chiral HPLC chromatograms
of 1, its racemate, and opposite enantiomer; NMR chiral shift
reagent data pertaining to the enantiomeric diols. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
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Corresponding Author
*Telephone: (419) 530-2167. Fax: (419) 530-7946. E-mail:
paul.erhardt@utoledo.edu.
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’ ACKNOWLEDGMENT
This work was supported by grants from the USDA ARS
Southern Regional Research Center and the Ohio Soybean
Council.
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