Biomimetic syntheses and antiproliferative activities of racemic, natural (-), and unnnatural (+) glyceollin i

ARTICLE

pubs.acs.org/jmc

Biomimetic Syntheses and Antiproliferative Activities of Racemic,

Natural (ꢀ), and Unnnatural (þ) Glyceollin I

Rahul S. Khupse,^‡Jeﬀrey G. Sarver,^†Jill A. Trendel,^†Nicole R. Bearss,^†Michael D. Reese,^†Thomas E. Wiese,^§

Stephen M. Boue,^||Matthew E. Burow,^{^}Thomas E. Cleveland,^||Deepak Bhatnagar,^||and Paul W. Erhardt*^,†

^†Center for Drug Design and Development, Department of Medicinal and Biological Chemistry, College of Pharmacy, The University of

Toledo, 2801 West Bancroft Street, Toledo, Ohio 43606, United States

^‡University of Findlay, College of Pharmacy, Findlay, Ohio 45840, United States

^§Division of Basic Pharmaceutical Sciences, College of Pharmacy, Xavier University of Louisiana, New Orleans, Louisiana 70125,

United States

Southern Regional Research Center, Agricultural Research Station, United States Department of Agriculture, New Orleans,

Louisiana 70124, United States

^{^}Department of Hematology and Medical Oncology, School of Medicine, and Center for Bioenvironmental Research,

Tulane University, New Orleans, Louisiana 70112, United States

S Supporting Information

b

ABSTRACT: A 14-step biomimetic synthetic route to glyceollin I (1.5% overall yield) was developed

and deployed to produce the natural enantiomeric form in soy, its unnatural stereoisomer, and a racemic

mixture. Enantiomeric excess was assessed by asymmetric NMR shift reagents and chiral HPLC.

Antiproliferative eﬀects were measured in human breast, ovarian, and prostate cancer cell lines, with all

three chiral forms exhibiting growth inhibition (GI) in the low to mid μM range for all cells. The natural

enantiomer, and in some cases the racemate, gave signiﬁcantly greater GI than the unnatural

stereoisomer for estrogen receptor positive (ER^þ) versus ER^ꢀbreast/ovarian cell lines as well as for

androgen receptor positive (AR^þ) versus AR^ꢀprostate cancer cells. Surprisingly, diﬀerences between ER^þand ER^ꢀcell lines were

not altered by media estrogen conditions. These results suggest the antiproliferative mechanism of glyceollin I stereoisomers may be

more complicated than strictly ER interactions.

’ INTRODUCTION

of hurdles, we have adopted a strategy to examine promising

dietary supplements as their individual nutraceutical⁵compo-

nents while applying the same level of rigor during preclinical

development that is typically aﬀorded to the closely regulated

process of drug discovery. Upon credentializing their potential

utility, such components would then be ideally positioned for

clinical validation within personalized treatment paradigms.⁹Ulti-

mately, these well-deﬁned studies can be coupled with subsequent

preclinical evaluations directed toward evolving the optimal natural

product mixtures and administration protocols that would best

serve the more complex matrices’ deployment as cancer preventa-

tive dietary supplements tailored for speciﬁc patient populations.

Our collaborating laboratories have been undertaking these

investigations by systematically examining the possibility for

stress-induced fortiﬁcation⁵of beneﬁcial anticancer principles

within legumes that are staples of the U.S. agricultural industry.¹⁰

The common soybean, Glycine max, can be considered to be a

credible functional food that has well-established beneﬁcial

eﬀects on the cardiovascular system. Its natural matrices and

several of its puriﬁed constituents have also undergone studies

Interest in the potential for chemoprevention of cancer¹has

grown signiﬁcantly over the past two decades.^2,3Drawing from

rapidly increasing knowledge about prognostic biomarkers, it

may soon be possible to prevent or delay the onset of certain

cancers in well-deﬁned, high-risk populations as well as to reduce

the recurrence of cancer in selected patients undergoing re-

mission.⁴Within this context, the use of dietary supplements

becomes particularly appealing as long-term, prophylactic treat-

ments can be conveniently accommodated within a “normal”

diet. To develop herbs and functional foods⁵as credible dietary

supplements, deﬁnitive chemical ﬁngerprinting⁶and systematic

biological characterizations of both the natural matrices and their

relevant constituents represent critical steps toward credentializing⁷

a new treatment paradigm prior to clinical validation⁷and for

assuring quality control (QC) of the marketed products. Because

of the normal variation associated with the complex blend of

natural materials,⁶and the sometimes tedious methods that may

be required to purify their phytochemical constituents, these

steps can be diﬃcult to achieve. In addition, long-standing issues

about the reliability of cancer prevention models to predict clinical

outcomes⁸further complicates the development of naturally de-

rived products for this type of indication. Given this combination

Received: December 27, 2010

Published: April 22, 2011

r

2011 American Chemical Society

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Figure 1. Selected structures. Genistein 1 and daidzein 2 are common isoﬂavones produced by soybean grown under normal conditions. The glyceollins, such

as GLY I 3, GLY II 4, and GLY III 5, are phytoalexins produced by soybean in response to speciﬁc stress conditions. All of the GLYs are derived from the

phytochemical intermediate glycinol 6. Structure 7 depicts the typical pterocarpan ring system and speciﬁcally denotes the “6a” position. Structures 8 and 9 are

(þ) pisatin and (þ) variabilin, respectively; these compounds are 6a-hydroxypterocarpan natural products which have been previously synthesized by other

investigators. Structure 10 was originally thought to be that of GLY I (see text for details). Structure 11 is an appropriately substituted isoﬂav-3-ene system that

serves as a key intermediate for both our former total synthesis and the present biomimetic synthesis of the GLY I stereoisomers. Structure 12 is lespedezol A₁,

which we previously prepared and use herein as a model system to explore alternative synthetic entries toward GLY I (see text for details).

for potential use in cancer treatment or prevention. None of

these studies, however, have examined the potential anticancer

beneﬁts that may become enhanced when soy is subjected to

speciﬁc stress conditions. Of particular note in soy are the glyceollins

(GLYs), phytoalexins that belong to the pterocarpan class of

natural products.¹¹Phytoalexins are secondary metabolites thought

to be produced as a defense mechanism when plants become

stressed.¹²Because the phytoalexins are typically present in very

low amounts for select periods of time, they represent an ad-

ditional challenge toward potential development as either a nutra-

ceutical or a dietary supplement. For example, the GLYs can be

elicited in trace amounts and isolated as a complex mixture only

when soybean plants are subjected to speciﬁc types of stress such

as when soy cotyledons are infected with Aspergillus.¹³Unlike the

abundant isoﬂavonoids genistein and daidzein (1 and 2 in

Figure 1) which are normally present in soy,¹⁴we have shown

that the GLYs exhibit marked antiestrogenic activity in some

tissues¹⁵as well as demonstrating unique anticancer properties¹⁶

and promise for use as selective estrogen receptor modulators

(SERMs).^17,18To date, there have been three GLYs separately

isolated from natural sources (3ꢀ5 in Figure 1), for which GLY I,

3, is the most prevalent and appears to be the most interesting

family member in terms of exhibiting promising anticancer properties

by virtue of its antiestrogenic activities.¹⁹Most recently, the GLYs’

close phytochemical precursor, glycinol (6 in Figure 1), also has

been isolated and studied for these types of eﬀects.²⁰Likely owing to

its display of two hydroxyl groups in a spatial arrangement that is

similar to that for estradiol,²¹6 exhibits potent estrogen receptor

agonist activity that prompts its interest as a potential SERM.²⁰

As part of our broad investigation to systematically examine

the practical beneﬁts of stressing soy for eventual use as a cancer-

preventing dietary supplement, and to potentially develop one or

more of the family members as distinct anticancer nutraceutical

agents, we have undertaken medicinal chemistry studies directed

toward synthesizing and rigorously evaluating each of the in-

dividual GLYs.^22ꢀ24Herein, we report our biomimetic synthesis

of GLY I and its characterization as an antiproliferative agent within a

panel of human, hormone-related cancer cell lines. In addition, the

racemate and GLY I’s opposite enantiomer were prepared to probe

the biological consequences resulting from the distinct stereochem-

istry that is exquisitely bestowed to this intriguing family of phytoalexin

natural products by virtue of their unique 6a-position hydroxyl group.

’ CHEMISTRY

While the pterocarpans, as represented by 7 in Figure 1, are

the second largest group of natural isoﬂavonoids,²⁵their 6a-

hydroxy-substituted family has only a few members. The ﬁrst

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Scheme 1. Previous^23cTotal Synthesis of GLY I Utilizing a Nonbiomimetic Wittig Reaction (Steps (a) and (b)) to Obtain the Key

Intermediate Isoﬂav-3-ene 11 (after Adjustment of Protecting Groups)^a

^aReagents and conditions: (a) PPh₃HBr, CH₃CN, room temp; (b) t-BuONa, methanol, reﬂux, 78% (over two steps); (c) OsO₄, (DHQD)₂PHAL,

3

CH₂Cl₂, ꢀ78 ꢀC; (d) PdꢀC(10%), H₂, acetone, 80%; (e) polymeric base, ethanol, 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) N(Et)₃3HF, CH₃CN, ꢀ20 to 4 ꢀC, 77%.

3

6a-hydroxypterocarpan, (þ)-pisatin, was isolated in 1960.²⁶

Owing in part to the lengthy, multistep routes needed to prepare

these contiguous ring systems while maintaining stereocontrol,

only four members of this family have been synthesized to date:

pisatin,²⁷variabilin,²⁸and more recently in our laboratories GLY

key isoﬂav-3-ene intermediate 11, which was then elaborated

to 3 in six steps.^23c

In addition to being amenable to the production of analogues

for future SAR studies, we felt it was important to devise a

biomimetic synthetic strategy so that we could also provide

analytical standards that might be useful for tracking the chal-

cone-, isoﬂavone-, and pterocarpan-related biochemical inter-

mediates that are traversed during stress-induced biosynthesis of

the GLYs. The biomimetic approach described herein reserves

construction of the isoprenyl-related ring system until the end,

such that late-stage divergent chemistry can allow for both GLY I

and GLY II analogues to be obtained from the same overall route.

Introduction of the 6a-hydroxy-group, either within the context

of the cis-fused ring system or as a prelude to the latter’s con-

struction, represents one of the most intricate steps leading to

these natural products.³⁴Previous strategies have typically uti-

lized the isoﬂav-3-ene from which dihydroxylation and closure to

the dihydrobenzofuran produces the natural and more stable cis-

fused, multiring system.³⁵Alternatively, as shown in retrosyn-

thetic Scheme 2, we imagined that assembly of the GLYs’ central

skeleton might be accomplished by either of two routes. Both

proceed from the same isoﬂavone and subsequently take advan-

tage of what we perceived to be very accessible alkene inter-

mediates for potential insertion of the 6a-hyroxy-group. The diol

I

^23cand glycinol²⁴(structures 8, 9, 3, and6in Figure 1, respectively).

GLY I was ﬁrst isolated in 1971 by Keen et al. from soybean

hypocotyls inoculated with phytophthora which served as a stress

factor to elicit the requisite phytochemical pathway.²⁹However,

they assigned the structure incorrectly and thus initially mis-

named it as part of the phaseollin family. When Burden et al. later

obtained 6a-hydroxyphaseollin, 10, as a metabolite from phaseollin,³⁰

the spectroscopic properties did not match those given by Keen

et al. To address the descrepancy, Burden et al. isolated the soybean

phytoalexin and determined its correct structure by parallel degra-

dation and mass spectroscopic studies of both compounds

wherein each can be shown to have a distinct and characteristic

retro-DielsꢀAlder fragmentation pattern.³¹In 1976, Lyne

et al. isolated all of the GLYs from stressed soybean and

provided deﬁnitive NMR data for the revised structure of

GLY I along with the other two family members.³²Subse-

quently, Keen et al. renamed the new phytoalexin framework as

“glyceollin.”³³In our prior total synthesis of GLY I (Scheme 1),

we took advantage of an internal Wittig reaction to form the

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Scheme 2. Retrosynthetic Approaches to the 6a-Hydroxypterocarpan System Required for GLY I

Scheme 3. Biomimetic Route to the Key Isoﬂav-3-ene Intermediate 11^a

^aReagents and conditions: (a) BnBr, K₂CO₃, CH₃CN, reﬂux, 10 h, 88%; (b) MOMCl, K₂CO₃, acetone, room temp, 24 h, 70%; (c) BnBr, K₂CO₃,

CH₃CN, reﬂux, 4 h, 80%; (d) piperidine, methanol, 60 ꢀC, 6 h, 80%; (e) acetic anhydride, N(Et)₃, room temp, 92%; (f) Tl(NO₃)₃, 3H₂O, methanol:

TMOF (1:1), room temp, 8 h; (g) 10% HCl, THF, reﬂux, 6 h, 68% (over 2 steps); (h) TBDMSCl, N(Et)₃, CH₂Cl₂, 90%; (i) (1) LiBH₄, THF, 0 ꢀC, 6 h,

(2) 10% HCl, reﬂux, 2 h, 45% (over 2 steps).

route is similar to the literature,^35,36while the other imagines

adding a water molecule across the double-bond of a pterocar-

pene analogous to what has been reported for indene.³⁷For this

second route, however, we did not observe any indication of a

successful hydration reaction after preparing lespedazol A₁,

12,^23band using it as a model pterocarpene that we subjected

to these types of conditions. Details for the reactions associated

with our unsuccessful initial strategy can be found in the

Supporting Information. The dihydroxylation approach was

pursued next and proved to be successful. For this, we adopted

the biomimetic route shown in Scheme 3 to produce the key

isoﬂav-3-ene intermediate 11. Interestingly, while this route

begins with the same two starting materials that were used during

our former total syntheses (Scheme 1), these initial building

blocks are now destined to become reversed within the ﬁnal

product.

In Scheme 3, a ClaisenꢀSchmidt condensation³⁸produced

chalcone 19 after regioselective protection of the ketone and

aldehyde partners 17 and 18, respectively. We found that the use

of piperidine in anhydrous methanol³⁹is advantageous to avoid

concomitant cyclization of the chalcone to the ﬂavone.^22,23,40

Conversions of chalcones to isoﬂavones are typically accom-

plished through an acetal intermediate 21 by using a thallium-

mediated oxidative rearrangement.⁴¹We also attempted this

conversion via a newer method that employs Kosher’s reagent.⁴²

Yields for the latter were low (10ꢀ15%), however, when

compared to the thallium-mediated approach (68%). The nature

of the protecting groups inﬂuences this rearrangement where

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Table 1. NMR Shift (PPM) for Enantiomeric Diols and Their

Diastereomeric Complexes with Chiral Shift Reagent (CSR)

(the Molar Ratio of Diol:CSR was 5:1)

compd

(þ) diol

ArꢀH8

H4

H2 equatorial

H2 axial

6.59

6.61

6.62

6.59

6.61

6.62

5.51

5.75

5.94

5.52

5.78

5.91

4.74

4.88

5.0

4.03

4.25

4.45

4.03

4.28

4.41

(þ) diol (þ) CSR

(þ) diol (ꢀ) CSR

(ꢀ) diol

4.74

4.90

4.85

(ꢀ) diol (ꢀ) CSR

(ꢀ) diol (þ) CSR

ring “B” must migrate in preference to ring “A”.⁴³Because

electron withdrawing groups decrease the migratory aptitude,

we deployed Bn- and MOM-ether protecting groups for the

aldehyde partner 18 which becomes ring “B” in 19. Although the

ketone partner’s unprotected ortho-hydroxyl group, now present

on phenyl ring “A”, is needed for the subsequent ring closure, we

found that formation of acetal 21 required that this hydroxyl

groupbetransiently protected. Use ofanacetyl group(toform20)

became ideal because its electron withdrawing nature de-

creases the competing migratory aptitude of ring “A” while being

labile enough to be lost under conditions envisioned for the

subsequent ring closure.⁴¹Addition of trimethyl orthoformate

led to a cleaner reaction and improved the yield of 21, perhaps by

removing water from the coordination sphere of thallium and

promoting its formation of a chelate with 20. When acidic

conditions were deployed to form isoﬂavone 22, the acetyl was

lost and eﬃcient ring closure was observed. These conditions

also removed the MOM protecting group on ring “B”, which we

felt was additionally advantageous.^23cSeeking a labile protecting

group conducive to later steps in the overall synthesis, we chose

TBDMS because it is stable to basic conditions and to hydro-

genation while being subject to removal under neutral conditions.^23c

Reprotection of 22 with TBDMS to aﬀord 23 was accomplished

by standard methods.⁴⁴The ﬁnal reaction leading to the key

isoﬂav-3-ene intermediate 11 involved lithium borohydride re-

duction of isoﬂavone 23 to an isoﬂavan-4-ol which undergoes

1,2-dehydration upon treatment with aqueous HCl to provide

the desired product.⁴⁵Because 11 is labile under these condi-

tions, it is critical to remove all traces of acid during the workup.

Even with such care, however, isolated yields for this ﬁnal step

can still vary from as much as 45% to as little as 20%.

Figure 2. Glyceollin I chiral HPLC ﬁngerprinting. Chiral column.

Gradient solvent system utilizing water:methanol:acetonitrile (see Ex-

perimental Section for details).

technology for stereospeciﬁc introductions of the 6a-hydroxy

group,⁴⁶(ii) generation of a quinone-methide to pull the

dihydrobenzofuran ring together in its desired cis-orientation,⁴⁷

and (iii) use of a modiﬁed aldol condensation to form the ﬁnal

isoprenyl-ring system in a manner that predominately provides

the regiochemistry desired for GLY I.⁴⁸These steps were conducted

in parallel for each enatiomer of 13 after reﬁning the chemistry

for the individual steps by ﬁrst deploying the less precious

racemic material as a model system. Absolute stereochemistry

and ee were assessed at selected intermediate steps by using

circular dichroism (CD) and NMR chiral shift reagent studies.^23c

The remaining steps in the overall synthesis involved: (i)

construction of the dihydrobenzofuran ring while simultaneously

establishing the 6a-hydroxy group in a stereospeciﬁc manner,

(ii) elaboration of the isoprenyl-containing ring systems, and

(iii) ﬁnal deprotection. All of this chemistry was accomplished

according to our previously published total syntheses for which

these latter steps are explicitly shown in Scheme 1. Highlights

include: (i) deployment of the Sharpless asymmetric dihydroxylation

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Figure 3. GI eﬀects of standards and diﬀerent enantiomeric forms of GLY I on the ER^þMCF7 breast cancer cell line (solid circles, solid line), the ER^ꢀ

MCF12A breast cell line (open squares, dashed line), and the ER^ꢀNCI/ADR-RES ovarian cancer cell line (open triangles, dotted line), all cultured in

low E2 media. Values are the average of eight sets of duplicate well measurements for each condition. Symbols indicate the value for MCF7 cells has a

statistically signiﬁcant diﬀerence (p < 0.05) versus MCF12A cells (A) or NCI-ADR-RES cells (N). For the GLY results, symbols further indicate the

value for MCF7 cells has a statistically signiﬁcant diﬀerence versus corresponding measurement for the ꢀ, þ, or racemic (R) forms.

The overall yield to GLY I from the biomimetic route is ca. 1.4%,

which is somewhat less than the 3.4% previously achieved via our

nonbiomimetic route wherein a Wittig reaction was instead

utilized to form the key isoﬂav-3-ene system 11.

during the routine NMR experiments performed upon comple-

tion of step c in Scheme 1 so as to be able to quickly assess the

status of enantioselectivity for this key dihydroxylation reaction

with about (5% precision. Assessments for the latter are particularly

relevant because these diol intermediates appear to have inher-

ently low optical rotations. The NMR shift reagent data are

summarized in Table 1. Similarly, a chiral column chromatogra-

phy method proved to be very convenient for even more pre-

cisely determining the ee of the ﬁnal product materials. Repre-

sentative chromatograms from these types of assessments are

shown in Figure 2 wherein it can be seen that a nearly baseline

separation of the two enantiomers was achieved.

The proton NMR spectra of our synthesized GLYs resemble

the data reported for the materials obtained from nature, all of

which bear cis stereochemistry.³²Most conspicuous are the

protons at C-6, which appear as two separate doublets with the

C-6 equatorial proton appearing downﬁeld compared to the C-6

axial proton and wherein W coupling occurs with the C-11a and

C-6 equatorial proton, an event not possible for the trans system.

Both COSY and NOSEY spectra also conﬁrm this relationship

between the C-6 equatorial and C-11a protons. Absolute stere-

ochemistry was conﬁrmed by CD⁴⁹and chiral HPLC^23cstudies,

which deployed an authentic sample of (ꢀ)-(6aS,11aS)-GLY I

obtained from nature as a standard. High-resolution mass spectro-

scopic data were in agreement with theoretical values for each

enantiomer, as well as for racemic GLY I, and correctly supported

a common molecular formula of C₂₀H₁₈O₅. Speciﬁc analytical

details are conveyed in the Experimental Section and Supporting

Information, the latter also providing NMR spectra, CD spectra,

and chiral column chromatograms. Noteworthy for future QC

purposes is the convenience of deploying chiral shift reagents

’ BIOLOGICAL RESULTS AND DISCUSSION

The cell growth inhibition (GI) activity of several drugs and

selected natural product standards, along with the GLY I stereo-

isomers, were assessed in a panel of human cell lines that are

relevant to hormone-related cancers, namely breast, ovarian, and

prostate. Experiments in 96-well format were adapted from our

previous methods using breast⁵⁰and prostate⁵¹cancer cells so as

to allow for a range of hormone conditions to be studied across a

range of test agent concentrations.

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Figure 4. GI eﬀects of standards and diﬀerent enantiomeric forms of GLY I on the ER^þMCF7 breast cancer cells cultured in low E2 (solid circles, solid

line), intermediate E2 (open squares, dashed line), and high E2 (open triangles, dotted line). Values are the average of eight sets of duplicate well

measurements for each condition. Symbols indicate the value for low E2 media has a statistically signiﬁcant diﬀerence (p < 0.05) versus intermediate (I)

or high E2 media (E).

Results From Human Breast and Ovarian Cell Lines. GI

activities were measured on the ER^þMCF7 breast cancer cell

line, the ER^ꢀMCF12A immortalized normal breast epithelial

cell line, and the ER^ꢀNCI/ADR-RES ovarian cancer cell line,

wherein the ER^þand the ER^ꢀdesignations reflect the presence

or absence of functional estrogen receptors, respectively. Three

different types of media conditions having specified levels of

estradiol (E2) were used for each cell line: low E2 media (0.003

nM E2, 0.01 nM testosterone), intermediate E2 media (0.6 nM

E2, 0.7 nM testosterone), and high E2 media (100 nM E2, 0.7

nM testosterone). A full graphical summary of the data from all

three cell types in each media condition is provided in the Support-

ing Information. Selected subsets of these results that illustrate the

overall findings are provided in Figures 3 and 4 and Table 2.

The results from all three cell lines at low E2 (Figure 3) clearly

showed diﬀerences in the GI eﬀects of the test agents on ER^þ

versus ER^ꢀcell lines. Statistically signiﬁcant diﬀerences (p < 0.05)

between the ER^þcell line and the two ER^ꢀcell lines are indicated

in these graphs by italic letters A and N. In line with previous

literature, the pure antiestrogenic drug fulvestrant signiﬁcantly

reduced cell growth of the ER^þcells while having little or no

impact on the growth of either of the ER^ꢀcell lines.⁵²4-Hydro-

xytamoxifen, the active metabolite of tamoxifen,⁵³speciﬁcally

inhibited only the ER^þcell line at 10^ꢀ8M to 10^ꢀ6M, with both

ER^þand ER^ꢀcell lines being aﬀected at higher concentrations.

Tamoxifen itself showed a less ER-speciﬁc eﬀect on growth but

still yielded signiﬁcant diﬀerences in response of ER^þand ER^ꢀ

cells at intermediate concentrations. In agreement with previous

ﬁndings that genistein can have diﬀerential estrogenic eﬀects at

diﬀerent concentrations,⁵⁴in our experiments this common soy

natural product appeared to have some inhibitory eﬀect on ER^þ

cell growth at the lowest concentration while increasing estro-

gen-responsive cell growth at higher concentrations, although

few of the diﬀerences between cell lines are statistically signiﬁ-

cant. The GLY I enantiomers and racemic mixture aﬀected the

growth of all three cell lines at higher concentrations. While the

natural (ꢀ) enantiomer and the racemic mixture showed statis-

tically signiﬁcant diﬀerences between ER^þand ER^ꢀcells at 10^ꢀ5

M, the unnatural (þ) enantiomer only caused diﬀerences at the

highest concentration. Statistically signiﬁcant diﬀerences between

the eﬀects of (ꢀ), (þ), and racemic GLY I on MCF7 cells are

also indicated by symbols on the graphs. All three GLY forms

yielded signiﬁcantly diﬀerent levels of MCF7 GI at 10^ꢀ5M.

Natural GLY I had the largest eﬀect, the unnatural enantiomer

had the least eﬀect, and the racemic mixture was in-between with

signiﬁcantly diﬀerent values versus each of the other forms.

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GI results for the ER^þMCF7 cell line at each of the three E2

media conditions are graphically summarized in Figure 4. In this

series of graphs, statistically signiﬁcant diﬀerences between the low

E2 media versus the intermediate and high E2 media conditions are

indicated by the bold letters I and E, respectively. Once again,

fulvestrant provided the most consistent diﬀerences, with GI eﬀects

decreasing systematically with increasing E2 levels and signiﬁcant

diﬀerences between the low E2 media and the two elevated E2 levels

at all concentrations tested. 4-Hydroxytamoxifen showed signiﬁcant

diﬀerences between low E2 and both elevated E2 levels at 10^ꢀ8Mto

10^ꢀ6M, a diﬀerence between the low and highest E2 levels at 10^ꢀ5

M, and no diﬀerence between E2 levels at 10^ꢀ4M. Tamoxifen only

showed signiﬁcant diﬀerences at intermediate agent levels,

and the diﬀerences were only signiﬁcant between the low and high

E2 levels. The apparent low concentration GI eﬀects and high

concentration growth enhancement eﬀects of genistein seem to be

muted by increasing E2 levels, although diﬀerences were once again

not generally statistically signiﬁcant. Interestingly, none of the GLY I

forms showed any diﬀerences in MCF7 growth inhibition with

changing levels of E2, nor were there any signiﬁcant diﬀerences

between the eﬀects of the diﬀerent enantiomeric forms at any

of the tested agent concentrations.

Another means of comparing GI eﬀects is to consider speciﬁc

GI₅₀values, the latter representing the estimated concentration

of test agent that causes a 50% reduction in cell growth versus

vehicle-only control wells. GI₅₀values for each cell line in each

type of media with each test agent are summarized in Table 2.

Results are recorded as log₁₀(GI₅₀) values to make the table

more legible and to account for our experience that GI₅₀data

generally follows a log-normal distribution pattern.^50,51Statisti-

cally signiﬁcant diﬀerences between log₁₀(GI₅₀) values have

again been indicated by italic letters A or N for ER^þMCF7

comparisons versus ER^ꢀMCF12A and NCI-ADR-RES cell lines,

respectively, and by bold letters L, I, or E for statistical sig-

niﬁcance versus low, intermediate, and high E2 levels, respec-

tively. Fulvestrant’s GI was less than 50% for all concentrations

(GI₅₀> 10^ꢀ4M) across all cell lines and conditions except ER^þ

MCF7 cells in low E2 media for which inhibition is above 50% for

all concentrations (GI₅₀< 10^ꢀ8M). This resulted in fulvestrant’s

log₁₀(GI₅₀) values being below ꢀ8.0 for this lone condition, or

above ꢀ4.0 for all other conditions, with the MCF7 cells in low

E2 media being statistically diﬀerent from all of the other

conditions and no further statistical analysis being possible.

Tamoxifen and 4-hydroxytamoxifen log₁₀(GI₅₀) values showed

statistical diﬀerences between ER^þMCF7 cells and both ER^ꢀ

cells at low and intermediate E2 levels, and consistent diﬀerences

between MCF7 results at high E2 levels versus both low and

intermediate E2. Genistein gave consistent statistical diﬀerences

between ER^þand ER^ꢀcell lines but no signiﬁcant diﬀerences

between ER^þMCF7 cells at diﬀerent E2 levels. Tamoxifen,

4-hydroxytamoxifen, and genistein all occasionally exhibited

statistically signiﬁcant diﬀerences between E2 levels for one or

both ER^ꢀcell lines, but none of these diﬀerences showed a

consistent relationship between E2 level and eﬀect (i.e., none

have E > I > L or L > I > E). Conversely, results for the diﬀerent

forms of GLY I were highly consistent in showing no signiﬁcant

diﬀerences between E2 levels for any cell line. There were,

however, signiﬁcant diﬀerences for each GLY I form at all E2

levels between ER^þMCF7 cells and both ER^ꢀcell lines.

Statistically signiﬁcant diﬀerences between (ꢀ), (þ), and

racemic GLY I are indicated by ꢀ, þ, and R symbols in

Table 2, respectively. There were no signiﬁcant diﬀerences

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Figure 5. GI eﬀects of standards and diﬀerent enantiomeric forms of GLY I on the AR^þLNCaP prostate cancer cell line (solid circles, solid line) and

AR^ꢀPC3 (open squares, dashed line) and DU145 (open triangles, dotted line) prostate cancer cell lines. Values are the average of eight sets of duplicate

well measurements for each condition. Symbols indicate the value for LNCaP cells has a statistically signiﬁcant diﬀerence (p < 0.05) versus DU145 cells

(D) or PC3 cells (P). For the GLY results, symbols further indicate the value for LNCaP cells has a statistically signiﬁcant diﬀerence versus corresponding

measurement for the ꢀ, þ, or racemic (R) forms.

between GLY I forms for either of the ER^ꢀcell lines, but

MCF7 cells had signiﬁcant diﬀerences between the natural

and both other forms at low and intermediate E2 levels, while

the (ꢀ) and (þ) forms remained signiﬁcantly diﬀerent for

high E2 conditions.

differences in GI between the ARþ and ARꢀ cell lines at the

highest concentration, while the (ꢀ) and racemic forms also

showed significant differences between cell lines at the 10^ꢀ5M

level. The (ꢀ) and racemic forms also showed significant

differences versus the (þ) form at both 10^ꢀ6M and 10^ꢀ5

M

Results From Human Prostate Cell Lines. The GI effects of

the same standards and GLY I forms also were evaluated in media

containing 0.6 nM E2 and 0.7 nM testosterone across a panel of

human prostate cancer cell lines. The panel included AR^þ

LNCaP cells and AR^ꢀPC3 and DU145 cells wherein the AR^þ

and AR^ꢀdesignations reflect the presence or absence of func-

tional androgen receptors, respectively. These GI results are

summarized graphically in Figure 5, with statistically significant

differences between LNCaP cells versus PC3 and DU145 cells

indicated by italic letters P and D, respectively. As with the

ERþ cell line, fulvestrant displayed a highly selective growth

inhibitory effect on the AR^þLNCaP cells, with significant differ-

ences versus both AR^ꢀcell lines at the three highest concentra-

tions. Tamoxifen and 4-hydroxytamoxifen exhibited only small

and occasionally significant differences between cell lines, while

genistein showed no significant differences between cell lines.

Alternatively, each of the GLY I forms yielded significant

levels, as similarly indicated on these graphs by the ꢀ, þ, and R

symbols. Using the same symbols to indicate statistically sig-

nificant differences, Table 3 shows that the log₁₀(GI₅₀) values

for the prostate cancer cell lines exhibited little or no cell line

differences for tamoxifen, 4-hydroxytamoxifen, or genistein but

consistent significant differences between AR^þand AR^ꢀcell

lines for each of the GLY I forms. LNCaP cells also showed

significant differences in GI₅₀values for GLY I’s natural and

racemic forms versus the unnatural form. PC3 and DU145 cell

lines exhibited smaller differences between the GLY I forms,

albeit some of the differences remained statistically significant.

Discussion. Tamoxifen, 4-hydroxytamoxifen, and fulvestrant

yielded GI effects that are dependent on ER expression and E2 level,

as would be expected for agents acting primarily through antagonistic

ER interactions. Genistein appeared to exhibit weak antagonistic

effects at low concentrations and weak agonistic effects at high

concentrations, in agreement with previously reported studies.⁵⁵

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Table 3. Measured log₁₀(GI₅₀) Values (n = 8, GI₅₀in Molar

Concentration) of Standards and Enantiomeric Forms of GLY

I for the AR^þLNCaP and the AR^ꢀPC3 and DU145 Prostate

Cell Lines^a

In addition to antagonizing estrogen- and androgen-

mediated signaling pathways, other anticancer mechanisms

for genistein detailed in the literature include antioxidant

properties, cell cycle arrest, increased apoptosis, and inhibi-

tion of angiogenesis and metastasis.^14,16c,55cOur preliminary

mechanistic studies¹⁶with a mixture of GLY I, II, and III

showed increased G0/G1 cell cycle arrest in LNCaP cells

analogous to genistein induced changes, but in PC3 cells the GLY

mix appeared to cause S phase arrest while genistein increased

G2/M arrest. Also unlike genistein, the GLY mix did not

appear to exhibit signiﬁcant eﬀects on apoptotic events or

caspase activation in LNCaP cells.¹⁶The antioxidant proper-

ties of GLY I (þ, ꢀ, and racemic) as well as GLY mix have

been measured in our laboratory and have been found to be

similar to genistein (data not shown). Further testing of GLY I

eﬀects on angiogenesis, metastasis, and signaling cascades will

be needed to complete the comparison of genistein and GLY

anticancer activities.

As stereochemical probes per se, the GLY I racemate and

enantiomers appear to represent the ﬁrst phytoestrogen-related

anticancer or SERM natural products to be undergoing studies in

this manner.^19bThe closest work in this regard would be that for

equol, which is an asymmetric, gut-bacteria-derived human

metabolite of the common soy natural product daidzein. Shown

to be formed as its (S)-enantiomer,⁶⁰stereochemical studies pre-

viously have demonstrated diﬀerential interactions between the

two possible equol enantiomers for the ERs.⁶¹Alternatively,

several studies have investigated the stereochemistry associated

with common synthetic SERM compounds. For example, asym-

metric derivatives of diethylstilbestrol,⁶²dichlorodiphenyltri-

chloroethane,⁶³raloxifene,⁶⁴and diarylpropionitrile⁶⁵have all

shown diﬀerences in their biological activities that may be related

to preferential binding of one of the enantiomers to ERs. Our

prior studies involving speciﬁc characterization of interactions

with the ERs suggest that while both of GLY I’s enantiomers can

bind with similar aﬃnities to both receptor subtypes, natural GLY I

exhibits only antagonistic actions, whereas (þ) GLY I can serve

as a weak agonist. Additionally, each GLY I enantiomer was

found to “induce unique gene expression proﬁles in a PCR array

panel of genes commonly altered in breast cancer.”^19bThe data

reported herein implies that the overall antiproliferative proper-

ties exhibited by the GLY I stereoisomers may be derived from a

more complicated mechanistic picture than can be explained by

relying upon just the classical types of interactions with ERs. This

further prompts the need for additional investigations of this

interesting compound that are directed toward delineating its

principal mechanism(s) of action.

log₁₀[GI₅₀(M)] ( 95% CI

agent

tamoxifen

PC3

DU145

LNCaP

ꢀ4.96 ( 0.05 ꢀ4.86 ( 0.04 ꢀ5.00 ( 0.04 D

4-hydroxytamoxifen ꢀ5.12 ( 0.09 ꢀ4.97 ( 0.05 ꢀ5.08 ( 0.09

fulvestrant

genistein

(. ꢀ4.0)

(ꢀ3.4)

ꢀ4.37 ( 0.11 ꢀ4.30 ( 0.21 ꢀ4.41 ( 0.13

Gly I (ꢀ)

Gly I (þ)

Gly I (rac)

ꢀ4.51 ( 0.03 ꢀ4.44 ( 0.13 ꢀ4.94 ( 0.07

þ

PD þ

ꢀ4.32 ( 0.05 ꢀ4.24 ( 0.10 ꢀ4.56 ( 0.11

ꢀR PD ꢀR

ꢀ4.49 ( 0.09 ꢀ4.53 ( 0.10 ꢀ4.89 ( 0.05

PD þ

ꢀ

þ

^aLower values indicate greater activity. Values in parentheses have been

estimated by extrapolation beyond the measured range of concentra-

tions. Italic symbols below an entry indicate the value for LNCaP cells

has a statistically signiﬁcant diﬀerence (p < 0.05) versus PC3 cells (P) or

DU145 cells (D) in the same media. For the GLY results, symbols below

the value further indicate a statistically signiﬁcant diﬀerence versus

corresponding measurement for the ꢀ, þ, or racemic (R) forms in

the same media for a given cell line.

The natural and racemic forms of GLY I, and to a lesser extent

the unnatural form, displayed larger GI effects on ER^þversus

ER^ꢀcells in low E2 media, as again expected based on the

antiestrogenic activities reported for GLY mixtures and GLY I

from some of our previous studies.^15d,19aSurprisingly, how-

ever, all three GLY I forms exhibited substantial GI effects on

ER^ꢀcell lines, at concentrations that are not all that much

greater than those inhibiting ER^þcell growth. Further, the

GI effects of the different forms of GLY I appeared to be

completely unaffected by changes in E2 levels from 0.003 nM

E2 (near the low end of the normal range for postmenopausal

women⁵⁶) to 100 nM E2 (nearly 2 orders of magnitude above

the high end of the normal range for premenopausal

women⁵⁶). These results suggest that the primary mechan-

ism for the GI effects observed for the GLY I forms in the

present studies may not be through interactions with ERs.

The experiments with AR^þand AR^ꢀprostate cancer cell lines

indicate that GLY I, particularly its natural and racemic forms,

had a greater GI eﬀect on AR^þLNCaP cells than the AR^ꢀPC3

and DU145 cell lines. Fulvestrant signiﬁcantly and selectively

inhibited the growth of AR^þLNCaP cells, while tamoxifen,

4-hydroxytamoxifen, and genistein displayed little or no diﬀer-

ence in eﬀect on AR^þversus AR^ꢀcell lines. The GI eﬀects of

fulvestrant and GLY I could potentially be due to antiestrogenic

eﬀects against ERβ, which is reported to be the more dominant

form of ER expressed in prostate cancer cells⁵⁷(whereas ERR is

generally the dominant form expressed in breast cancer cells⁵⁸).

Alternatively, these agents could be interacting with the AR

expressed in LNCaP cells, which is known to have a mutation

giving it relatively promiscuous steroid-binding characteristics.⁵⁹

Further testing will be necessary to elucidate whether the LNCaP

GI activity is related to ERβ, a promiscuous AR, or possibly a

previously unrecognized AR-related mechanism.

’ CONCLUSION

A biomimetic synthetic route to GLY I has been developed. In

addition to being useful for the elaboration of analogues that can

contribute to SAR, this method provides practical access to

analytical standards that may be used for QC purposes when

GLY I is obtained naturally from stressed soy bean. For the latter,

asymmetric NMR shift reagents and chiral HPLC have been

shown to be very practical methods for preliminary and ﬁnal

assessments of ee. Both enantiomers, as well as the GLY I

racemate, were studied in antiproliferation studies using

hormone-related human cancer cell lines. Within the breast and

ovarian cell culture panel, all three GLY I forms exhibit sub-

stantial GI eﬀects on ER^ꢀcell lines, at concentrations that are not

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ARTICLE

all that much greater than those inhibiting ER^þcell growth.

Within the prostate cell culture panel, GLY I, particularly its

natural and racemic forms, have a greater GI eﬀect on AR^þ

LNCaP cells than the AR^ꢀPC3 and DU145 cell lines. Thus,

although not pronounced, there do appear to be some diﬀerences

between the GLY I enantiomers in their overall GI proﬁles. Perhaps

more importantly, however, the composite of these studies

suggest that the overall antiproliferative properties exhibited by

the GLY I stereoisomers may be derived from a more compli-

cated mechanistic picture than can be explained by relying upon

just the classical types of interactions with ERs. This, in turn,

further prompts the need for additional investigations of this

interesting natural product that are directed toward delineating

its principal mechanism(s) of action, as well as toward conﬁrming

its promise as a potential anticancer and cancer preventative agent.

δ 203.9, 165.4, 164.6, 136.9, 134, 129.2, 128.8, 128.5, 114.6, 108.5, 102.4,

70.3, 27.3.

2-Benzyloxy-4-methoxymethyloxy-benzaldehyde (18). Oven-dried

potassium carbonate (1.38 g, 10 mmol) was added to an ice-cooled

solution of 2,4-dihydroxy-benzaldehyde (1.38 g, 10 mmol) in 10 mL of

acetone. MOMCl (1.54 mL, 20 mmol) was added dropwise, and the

mixture was stirred at 0 ꢀC for 1 h, after which it was gradually allowed to

come to room temp. The reaction was further stirred for 24 h at room

temp and then poured into ice water with vigorous stirring. A buff-

colored solid precipitated. The solid was filtered, dried, and recrystal-

lized from MeOH:DCM (ca. 30:5 mL) to obtain 1.31 g (72%) of

2-hydroxy-4-methoxymethyloxy-benzaldehyde as a white solid: mp

52ꢀ53 ꢀC [lit.⁶⁷50ꢀ51 ꢀC]. TLC R_f0.43 in hexanes:EtOAc (2:1).

¹H NMR (400 MHz, CDCl₃) δ 11.38 (s, 1H, OH), 9.74 (s, 1H, CHO),

7.45 (d, 1H, J = 8.4 Hz, ArꢀH6), 6.65 (dd, 1H, ²J = 8.4 Hz, ³J = 2 Hz,

ArꢀH5), 6.60 (d, 1H, ³J = 2 Hz), 5.24 (s, 2H, O-CH₂-O), 3.48 (s, 3H,

OCH₃). ¹³C NMR (100 MHz, CDCl₃) δ 194.8, 164.5, 164.4, 135.6,

116.2, 109.3, 103.6, 94.3, 56.7.

’ EXPERIMENTAL SECTION

Oven-dried potassium carbonate (0.272 g, 2 mmol) was added to a

solution of 2-hydroxy-4-methoxymethy-benzaldehyde (0.182 g, 1 mmol) in

10 mL of acetonitrile, and the mixture was stirred for 1 h at room temp.

Benzylbromide (0.24 mL, 2 mmol) was added, and the mixture was

reﬂuxed for 10 h. After disappearance of 2-hydroxy-4-methoxymethy-

benzaldehyde (TLC), the solvent was evaporated. The residue was

chromatographed over silica using hexanes:EtOAc (5:1). The organic

fractions were evaporated to obtain 0.218 g (80%) of 18 as a yellow oil.

TLC R_f0.27 hexanes:EtoAc (5:1). ¹H NMR (400 MHz, CDCl₃) δ 10.4

(s, 1H, CHO), 7.82 (d, 1H, J = 8.4 Hz, ArꢀH6), 7.46ꢀ7.39 (m, 5H,

C₆H₅), 6.7ꢀ6.69 (m, 2H, ArꢀH3/ArꢀH5), 5.21 (s, 2H, OꢀCH₂-O),

5.16 (s, 2H, PhCH₂), 3.48 (s, 3H, OCH₃). ¹³C NMR (100 MHz,

CDCl₃) δ 188.6, 163.9, 162.9, 136.1, 130.5, 128.9, 128.5, 127.6, 120.2,

108.8, 101, 94.4, 70.7. Anal. (C₁₆H₁₆O₄) C, H, O.

Chemistry. General Methods. Chemical reactions were conducted

under nitrogen in anhydrous solvents unless stated otherwise. Reagents

obtained from commercial suppliers were used without further purifica-

tion. Acetone was dried over 4 Å molecular sieves. Tetrahydrofuran

(THF) was distilled under nitrogen over sodium-benzophenone. Thin-

layer chromatography (TLC) was done on 250 μ fluorescent plates and

visualized by using UV light or iodine vapor. Normal phase flash column

chromatography was performed using silica gel (200ꢀ425 mesh 60 Å

pore size) and ACS grade solvents. Melting points (mps) are uncor-

rected. Nuclear magnetic resonance (NMR) spectra were recorded on

either a 600 or 400 MHz instrument. Peak locations were referenced

using either tetramethyl silane (TMS) or residual nondeuterated solvent

as an internal standard. Proton coupling constants (J values) are expressed

in hertz using the following designations: s (singlet), d (doublet), br s

(broad singlet), m (multiplet), and q (quartet). In some cases, over-

lapping signals occurred in the ¹³C NMR spectra. Circular dichroism

(CD) studies were performed on an AVIV CD spectrometer for which

access was provided by Bowling Green State University, Ohio. High

resolution mass spectroscopy (HR MS) was conducted on a Micromass

LCT electrospray mass spectrometer via service provided by the Mass

Spectroscopy and Proteomics Facility within The Ohio State University,

Ohio. Chiral high performance liquid chromatography (HPLC) experi-

ments are described below and gave acceptable results (peak area purity

>98%). Combustion analyses were performed by Atlantic Microlab, Inc.

(Norcross, GA) and gave satisfactory results (C, H, O within 0.4% of

calculated values). In all cases, spectroscopic data are in agreement with

known compounds and assigned structures. Structural integrity and

sample purity for test compounds were established by the composite of

TLC, NMR, CD, HR MS, chiral HPLC, and in some cases additionally

included combustion analyses.

4-Benzyloxy-2-hydroxy-acetophenone (17). Oven-dried potassium

carbonate (1.65 g, 12 mmol) was added to a solution of 2,4-dihydroxy-

acetopheneone (1.52 g, 10 mmol) in 10 mL of acetonitrile, and the

mixture was stirred for 1 h at room temp. Benzylbromide (1.3 mL, 11 mmol)

was added, and the mixture was refluxed for 10 h. After disappearance of

2,4-dihydroxy-acetopheneone (TLC), the solvent was evaporated to

one-third volume and poured into ice water with vigorous stirring. A

pinkishꢀwhite solid precipitated. The solid was recrystallized from

methanol (ca. 20 mL) to obtain 2.12 g (88%) of 17 as a white product:

mp 109ꢀ110 ꢀC [lit.⁶⁶108ꢀ109 ꢀC]. TLC R_f0.47 hexanes:EtOAc

(5:1). ¹H NMR (400 MHz, DMSO-d₆) δ 12.56 (s, 1H, OH), 7.79 (d,

1H, J = 8.8 Hz, ArꢀH6), 7.38ꢀ7.28 (m, 5H, C₆H₅), 6.54 (dd, 1H, ²J =

8.8 Hz, ³J = 2.4 Hz, ArꢀH5), 6.5 (d, 1H, ³J = 2.4 Hz, ArꢀH3), 5.13 (s,

2H, OꢀCH₂), 2.5 (s, 3H, CH₃CO). ¹³C NMR (100 MHz, DMSO-d₆)

2-Benzyloxy-4-methoxymethyloxy-4⁰-benzyloxy-2⁰-hydroxy-chalcone

(19). A mixture of 2-benzyloxy-4-methoxymethyloxy-benzaldehyde (2.72 g,

10 mmol) and 4-benzyloxy-2-hydroxy-acetophenone (2.42 g, 10 mmol)

in 50 mL of anhydrous methanol was refluxed in the presence of

piperidine (1 mL, 10 mmol) for ca. 4 h, after which it was cooled to

room temp. A yellow solid precipitated. The solid was filtered and

washed with 100 mL of methanol. The filtrate was reduced to ca. 50 mL

and refluxed for 2 h, after which it provided additional product upon

cooling in the refrigerator. The combined yield of yellow solid 19 was

4.16 g (84%): mp 147ꢀ148 ꢀC. TLC R_f0.19 in hexanes:EtOAc (5:1).

¹H NMR (400 MHz, DMSO-d₆) δ 8.01 (d, 1H, J = 15.2, CHd), 7.84

(d, 1H, J = 15.2, CHd), 7.89 (d, 1H, J = 8.8 Hz, ArꢀH6⁰), 7.73 (d, 1H,

J = 9.2 Hz, ArꢀH6), 7.56ꢀ7.36 (m, 10H, 2 ꢁ C₆H₆), 6.89 (d, 1H, J = 2.4

Hz, ArꢀH3⁰), 6.73 (dd, 1H, ²J = 8.8 Hz, ³J = 2.4 Hz, ArꢀH5⁰), 6.58 (d,

3

2

3

1H, J = 2.8 Hz, ArꢀH3), 6.48 (dd, 1H, J = 9.2 Hz, J = 2.4 Hz,

ArꢀH5), 5.29 (s, 2H, OꢀCH₂), 5.20 (s, 4H, PhꢀCH₂), 3.40 (s, 3H,

OCH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 192.6, 166.3, 166.4, 61.2,

160, 140.3, 137.1, 136.9, 132.8, 132.5, 129.4, 129.2, 129, 128.8, 128.6,

119.4, 117.6, 114.6, 109, 108.5, 102.6, 102.1, 94.5, 70.9, 70.4, 56.6. Anal.

(C₃₁H₂₈O₆) C, H, O.

2-Benzyloxy-4-methoxymethyloxy-4⁰-benzyloxy-2⁰-acetoxy-chalcone

(20). To a solution of 2-benzyloxy-4-methoxymethyloxy-4⁰-benzyloxy-

2⁰-hydroxy-chalcone (0.496 g, 1 mmol) in 10 mL of acetic anhydride was

added triethylamine (0.25 mL, 2 mmol) and the solution heated at 60 ꢀC

for ca. 6 h. After disappearance of starting material (TLC), the hot

reaction mixture was poured into iceꢀwater:methanol (1:1) ca. 50 mL.

A solid precipitated upon vigorous stirring. The solid was filtered, dried,

and recrystallized from methanol (ca. 15 mL) to obtain 0.495 g (92%) of

20 as a white solid; mp 94ꢀ96 ꢀC. TLC R_f0.21 in hexanes:EtOAc (3:1).

¹H NMR (400 MHz, DMSO-d₆) δ 7.78ꢀ7.75 (m, 2H), 7.70 (d, 1H, J =

8.8 Hz, ArꢀH6⁰), 7.49ꢀ7.36 (m, 10 þ 1H, 2 ꢁ C₆H₆þ ArꢀH8), 6.98

2

3

(dd, 1H, J = 8.4 Hz, J = 2.4 Hz, ArꢀH5⁰), 6.90 (d, 1H, J = 2.4 Hz,

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2

ArꢀH3⁰), 6.85 (d, 1H, J = 2 Hz, ArꢀH3), 6.70 (dd, 1H, J = 8.4 Hz,

³J = 2.4 Hz, ArꢀH5), 5.26 (s, 2H, OꢀCH₂), 5.20 (s, 4H, OꢀCH₂), 3.39

(s, 3H, OCH₃), 2.18 (s, 3H, CH₃CO). ¹³C NMR (100 MHz, DMSO-

d₆) δ 189,169.5, 162.5, 160.9, 159.6, 151.4, 139.5, 137.2, 136.9, 132.3,

131.7, 129.3, 129.2, 128.8, 128.6, 128.6, 125.2, 123.3, 117.6, 113, 110.8,

109.1, 102.2, 94.5, 70.7, 70.5, 56.6, 21.5. Anal. (C₃₃H₃₀O₇) C, H, O.

2⁰-7-Dibenzyloxy-4⁰-hydroxy-isoflavone (22). 2-Benzyloxy-4-meth-

oxymethyloxy-4⁰-benzyloxy-2⁰-acetoxy-chalcone (0.538 g, 1 mmol) was

dissolved in 2 mL of methylene chloride and 25 mL of methanol:

trimethyl orthoformate (1:1) was added, followed by addition of thallium

nitrate-trihydrate (0.666 g, 1.5 mmol). After stirring the suspension for

2 h at room temp, it became a clear solution with white thallium(I)

nitrate precipitating. After stirring for an additional 4 h, the TLC showed

complete disappearance of chalcone 20. Solvents were evaporated to

reduce the volume to ca. one-third, and then 20 mL of DCM was added,

followed by the addition of 0.2 g of sodium bisulfite. After stirring for 1 h,

the entire solid was filtered, solvent evaporated, and the resulting residue

passed through a short column of silica with hexanes:EtOAc (10:1) as

eluant. The solvents were evaporated to obtain 0.48 g (80%) of the acetal

21 as a yellowish oil which was used directly in the next step without

further purification. Crude acetal (ca. 0.48 g) was dissolved in 10 mL of

methanol and refluxed with 2 mL of 1N HCl for about 12 h. After

disappearance of the acetal (TLC), the reaction mixture was poured into

ice water (100 mL). A yellowishꢀwhite solid precipitated. The pre-

cipitate was filtered, dried, and recrystallized from MeOH:DCM

(ca. 15:2 mL) to obtain 0.306 g (68%) of 22 as a white powder having

a yellow tinge; mp 116ꢀ117 ꢀC. TLC R_f0.25 hexanes:EtOAc (1:1). ¹H

NMR (400 MHz, DMSO-d₆) δ 9.59 (s, 1H, OH), 8.22 (s, 1H, OCHd),

8.02 (d, 1H, J = 8.8 Hz, ArꢀH5), 7.50ꢀ7.23 (m, 10 þ 1H, 2 ꢁ C₆H₅þ

ArꢀH8), 7.14 (dd, 1H, ²J = 9.2 Hz, ³J = 2.4 Hz, ArꢀH6), 7.05 (d, 1H,

J = 8 Hz, ArH-6⁰), 6.5 (d, 1H, J = 1.6 Hz, ArꢀH3⁰), 6.41 (dd, 1H, ²J = 8.4

Hz, ArꢀH5⁰), 5.27 (s, 2H, PhꢀCH₂), 5.02 (s, 2H, PhꢀCH₂). ¹³C NMR

(100 MHz, DMSO-d₆) δ 75.3, 163.2, 159.5, 158.1, 158, 154.8, 138,

136.8, 132.7, 129.2, 128.9, 128.8, 128.7, 128.2, 127.7, 127.6, 123, 118.4,

115.8, 112.5, 108, 102.3, 101, 70.7, 70. Anal. (C₂₉H₂₂O₅) C, H, O.

2⁰,7-Dibenzyloxy-4⁰-(t-butyldimethylsilyloxy)-isoflavone (23). To a

solution of 2⁰,7-dibenzyloxy-4⁰-hydroxy-isoflavone (0.450 g, 1 mmol) in

10 mL of DCM was added triethylamine (0.25 mL, 2 mmol) and

TBDMSCl (0.225 g, 1.5 mmol). The reaction mixture was stirred at

room temperature for ca. 6 h. After disappearance of starting material

(TLC), the reaction was quenched with 1N HCl (ca. 2 mL). The

mixture was extracted with DCM:water (2 ꢁ 10 mL:10 mL). The

organic layer was dried over sodium sulfate and evaporated to give

0.507 g (90%) of 23 as a fluffy, white solid; mp 132ꢀ133 ꢀC. TLC R_f

0.89 in hexanes:EtOAc (1:1). ¹H NMR (400 MHz, DMSO-d₆) δ 8.28

(s, 1H, OCHd), 8.03 (d, 1H, J = 8.8 Hz, ArꢀH5), 7.50ꢀ7.13 (m, 10 þ

3H, C₆H₅, ArꢀH6, H6⁰, H8), 6.53ꢀ6.48 (m, 2H, ArꢀH3⁰/ArꢀH5⁰),

5.27 (s, 2H, PhꢀCH₂), 5.08 (s, 2H, PhCH₂), 0.93 {s, 9H, Si (CH₃)₃},

0.16 {s, 6H, Si (CH₃)₂}. ¹³C NMR (100 MHz, DMSO-d₆) δ 175.1,

163.3, 158.1, 157.9, 157, 155, 137.9, 136.8, 132.7, 129.2, 128.9, 128.8,

128.6, 128.1, 127.7, 127.6, 122.7, 118.4, 115.9, 115.3, 112.1, 105.9, 102.4,

using DCM:hexanes (1:1). The organic fractions were evaporated to

provide 0.247 g (45%) of 11 as a white solid; mp 105ꢀ107 ꢀC. TLC R_f

0.75 in DCM:hexanes (2:1). ¹H NMR (400 MHz, DMSO-d₆) δ

7.46ꢀ7.32 (m, 10H, 2 ꢁ C₆H₅), 7.21 (d, 1H, J = 8.4 Hz, ArꢀH5),

7.02 (d, 1H, J = 8.4 Hz), 6.63 (s, 1H, CHd), 6.58ꢀ6.55 (m, 2H,

ArꢀH3⁰/H5⁰), 6.49ꢀ6.46 (m, 2H, ArꢀH6/8), 5.11 (s, 2H, PhꢀCH₂),

5.07 (s, 2H, PhꢀCH₂), 4.89 (s, 2H, OCH₂), 0.93 {s, 9H, Si (CH₃)₃},

0.17 {s, 6H, Si (CH₃)₂}. ¹³C NMR (100 MHz, DMSO-d₆) δ 199.6,

159.7, 157.5, 156.8, 154.7, 137.7, 137.5, 129.7, 129.2, 129.1, 128.6, 128.5,

128.4, 128.3, 128.1, 121.5, 121.4, 117.5, 112.8, 108.9, 105.9, 102.8, 70.5,

69.9, 68.3, 26.3, 18.7. Anal.(C₃₅H₃₈O₄Si 0.25H₂O) C, H.

3

(() 4⁰-t-Butyl Dimethylsilyloxy-2⁰,7-(dibenzyloxy)isoflavan-3,4-diol

(() (13). 2⁰,7-Dibenzyloxy-4⁰-t-butyldimethylsilyloxy-isoflav-3-ene (0.537 g,

1 mmol) was dissolved in 10 mL of acetone, and 1 mL of water was

added. Methane sulfonamide (0.095 g, 1 mmol) and 4-methylmorpho-

line-4-oxide (NMO) (0.140 g, 1.2 mmol) were then added, and the

resulting mixture stirred at 0 ꢀC for 30 min. Osmium tetroxide solution

in water (1 mL = 0.004 mmol) was added slowly, upon which the

reaction turned brownishꢀyellow. The reaction was gradually allowed

to come to room temp and stirred for 24 h. After disappearance of the

reactant (TLC), the reaction was quenched with 1 g of sodium bisulfate

followed by evaporation of acetone. The residue was extracted with

EtOAc:water (2 ꢁ 10 mL:10 mL). The organic layers were combined,

dried over sodium sulfate, filtered, and evaporated. The residue was

chromatographed over silica with hexanes:EtOAc (5:1). The organic

fractions were evaporated to provide 0.525 g (90%) of 13 as a fluffy,

white solid; mp 110ꢀ112 ꢀC. TLC R_f0.25 in hexanes:EtOAc (5:1). ¹H

NMR (400 MHz, acetone-d₆) δ 7.46 (d, 1H, J = 8.4 Hz, ArꢀH5),

7.46ꢀ7.28 (m, 10 þ 1H, 2 ꢁ C₆H₅þ ArꢀH6⁰), 6.58ꢀ6.56 (m, 2H,

ArꢀH8/ArꢀH5⁰), 6.48 (q, 1H, J = 8.4 Hz, J = 1.8 Hz, ArꢀH6), 6.37 (d,

1H, J = 2.8 Hz, ArꢀH3⁰), 5.51 (d, 1H, J = 7.2, H4), 5.19 (s, 2H, PhCH₂),

5.06 (s, 2H, PhCH₂), 4.72 (d, 1H, J = 11.4 Hz, H-2 equiv), 4.23 (d, 1H,

J = 7.24, 4-OH), 4.19 (s, 1H, 3-OH), 4.02 (d, 1H, J = 11.4 Hz, H2 ax),

0.96 {s, 9H, Si (CH₃)₃}, 0.17 {s, 6H, Si (CH₃)₂}. ¹³C NMR (100 MHz,

acetone-d₆) δ 160.6, 158.2, 157.9, 156.4, 139.1, 138.6, 131.5, 130.8,

130.1, 129.9, 129.3, 129.1, 128.9, 128.8, 124.2, 119.1, 113.2, 109.4, 106.9,

102.8, 72.7, 71.6, 70.9, 68.3, 26.6, 19.3. Anal. (C₃₅H₄₀O₆Si) C, H.

(þ) 4⁰-t-Butyl Dimethylsilyloxy-2⁰,7-(dibenzyloxy)isoflavan-3,4-diol

(þ) (13). Prepared according to previously published procedure.^23c

White solid: 0.385 g (66%). TLC R_f0.25 in hexanes:EtOAc (5:1). ¹H

NMR (400 MHz, acetone-d₆) δ 7.46 (d, 1H, J = 8.4 Hz, ArꢀH5),

7.46ꢀ7.28 (m, 10 þ 1H, 2 ꢁ C₆H₅þ ArꢀH6⁰), 6.58ꢀ6.56 (m, 2H,

ArꢀH8/ArꢀH5⁰), 6.48 (q, 1H, J = 8.4 Hz, J = 1.8 Hz, ArꢀH6), 6.37 (d,

1H, J = 2.8 Hz, ArꢀH3⁰), 5.51 (d, 1H, J = 7.2, H4), 5.19 (s, 2H, PhCH₂),

5.06 (s, 2H, PhCH₂), 4.72 (d, 1H, J = 11.4 Hz, H-2 equiv), 4.23 (d, 1H,

J = 7.24, 4-OH), 4.19 (s, 1H, 3-OH), 4.02 (d, 1H, J = 11.4 Hz, H2 ax),

0.96 {s, 9H, Si (CH₃)₃}, 0.17 {s, 6H, Si (CH₃)₂}.

(ꢀ) 4⁰-t-Butyl Dimethylsilyloxy-2⁰,7-(dibenzyloxy)isoflavan-3,4-diol

(ꢀ) (13). Prepared according to previously published procedure.^23c

White solid: 0.410 g (70%). TLC R_f0.25 in hexanes:EtOAc (5:1). ¹H

NMR (400 MHz, acetone-d₆) δ 7.46 (d, 1H, J = 8.4 Hz, ArꢀH5),

7.46ꢀ7.28 (m, 10 þ 1H, 2 5ꢁ C₆H₅þ ArꢀH6⁰), 6.58ꢀ6.56 (m, 2H,

ArꢀH8/ArꢀH5⁰), 6.48 (q, 1H, J = 8.4 Hz, J = 1.8 Hz, ArꢀH6), 6.37 (d,

1H, J = 2.8 Hz, ArꢀH3⁰), 5.51 (d, 1H, J = 7.2, H4), 5.19 (s, 2H, PhCH₂),

5.06 (s, 2H, PhCH₂), 4.72 (d, 1H, J = 11.4 Hz, H-2 equiv), 4.23 (d, 1H,

J = 7.24, 4-OH), 4.19 (s, 1H, 3-OH), 4.02 (d, 1H, J = 11.4 Hz, H2 ax),

0.96 {s, 9H, Si (CH₃)₃}, 0.17 {s, 6H, Si (CH₃)₂}.

70.8, 70.1, 26.2, 18.6. Anal. (C₃₅H₃₆O₅Si 0.25H₂O) C, H.

3

2⁰,7-Dibenzyloxy-4⁰-(t-butyldimethylsilyloxy)-isoflav-3-ene (11). To

an ice-cooled solution of 2⁰,7-dibenzyloxy-4⁰-(t-butyldimethylsilyloxy)-

isoflavone (0.564 g, 1 mmol) in 10 mL of THF was added 1.6 M lithium

borohydride solution in THF (3 mmol) dropwise. The reaction mixture

was allowed to come to room temp gradually and then stirred for ca. 2 h.

After disappearance of starting material (TLC), the reaction was

quenched with 1N HCl (pH 4). The reaction was further stirred at

room temp followed by refluxing at 60 ꢀC for ca. 2 h. The reaction

mixture was extracted with DCM:water (2 ꢁ 15 mL:15 mL). The

organic layers were combined and washed with 1N NaHCO₃

(2 ꢁ 15 mL), dried over sodium sulfate, and evaporated at 20 ꢀC to

obtain an oily residue. The residue was chromatographed over silica

(() 4⁰-t-Butyldimethylsilyloxy-2⁰,7-dihydroxyisoflavan-3,4-diol (()

(14). To a solution of 4⁰-t-butyldimethylsilyloxy-2⁰,7-dibenzyloxyisofla-

van-3,4-diol (0.584 g, 1 mmol) in 6 mL of acetone was added 10% PdꢀC

(0.1 g) in 4 mL of cold acetone. This reaction mixture was hydrogenated

at room temperature under 1 atm (15 psi) of H₂pressure for ca. 4 h. After

the disappearance of starting material (TLC), the catalyst was filtered

through a pad of Celite. The Celite pad was washed with 10 ꢁ 2 mL of

3517

dx.doi.org/10.1021/jm101619e |J. Med. Chem. 2011, 54, 3506–3523

Journal of Medicinal Chemistry

ARTICLE

1

methanol. The solution was filtered once more through filter paper and

evaporated to obtain a white solid with red tinge: 0.360 g (89%); mp

decomposes at 142 ꢀC. TLC R_f0.23 in hexanes:EtOAc (2:1). ¹H NMR

(400 MHz, acetone-d₆) δ 7.20 (d, 1H, J = 8.4 Hz, ArꢀH5), 7.17 (d, 1H,

J = 8.4 Hz, ArꢀH6⁰), 6.42 (dd, 1H, ²J = 8.4 Hz, ³J = 2.4 Hz, ArꢀH6),

6.34 (d, 1H, J = 2.4 Hz, ArꢀH8), 6.28 (dd, 1H, ²J = 8.4 Hz, ³J = 2.4 Hz,

ArꢀH5⁰), 6.26 (d, 1H, J = 2.4 Hz), 5.09 (s, 1H, H4), 4.35 (d, 1H, J = 11.4

Hz, H2 equiv), 4.13 (d, 1H, J = 11.4 Hz, 4-OH), 4.19 (s, 1H, 3-OH), 4.02

(d, 1H, J = 11.4 Hz, H2 ax), 0.96 {s, 9H, Si (CH₃)₃}, 0.18 {s, 6H, Si

(CH₃)₂}. ¹³C NMR (100 MHz, acetone-d₆) δ 159.5, 150.8, 157.7,

155.9, 132.0, 129.0, 120.5, 116.7, 112.3, 110, 109.7, 103.4, 73.9, 70.1,

69.1, 26.5, 19.2. Anal. (C₂₁H₂₈O₆Si) C, H.

(60%). TLC R_f0.38 in hexanes:EtOAc (2:1). H NMR (400 MHz,

acetone-d₆) δ 8.55 (s.1H, ꢀOH), 7.30 (d, 1H, J = 8.4 Hz, ArꢀH1), 7.25

(d, 1H, J = 8.4, ArꢀH7), 6.55 (dd, 1H, ²J = 8.4 Hz, ³J = 2.4 Hz, ArꢀH-2),

6.45 (dd, 1H, ²J = 8.4 Hz, ³J = 2.4 Hz, ArꢀH8), 6.3 (d, 1H, J = 2.4 Hz,

ArH-4), 6.26 (d, 1H, J = 2.4 Hz, Ar H-10), 5.26 (s, 1H, 11a-H), 5.01 (s,

1H, 6aꢀOH), 4.14 (d, 1H, J = 12 Hz, H6 equiv), 4.02 (d, 1H, J = 12 Hz,

H6 ax), 0.96 {s, 9H, Si (CH₃)₃}, 0.19 {s, 6H, Si (CH₃)₂}.

(() 9-(t-Butyldimethylsilyloxy) Glyceollin I (() (16). To a solution of

9-(t-butyl-dimethylsilyloxy)-glycinol (0.040 g, 0.1 mmol) in 5 mL of

xylene was added 1,1-diethoxy-3-methyl-2-butene (0.2 mmol in 0.2 mL

of xylene) and 3-picoline (0.025 mmol in 0.3 mL xylene). The reaction

mixture was refluxed at 130 ꢀC for 18 h. After disappearance of reactant

(TLC), the solvent was evaporated and the residue was chromato-

graphed over silica using DCM as eluant. The organic fractions were

evaporated to provide two yellowish oils: a major isomer I and a minor

isomer II in ca. 5:1 ratio. Major yellow oil: 0.022 g (50%). TLC R_f0.46 in

toluene:methanol (10:1). ¹H NMR (400 MHz, acetone-d₆) δ 7.27 (d,

1H, J = 8.4 Hz, ArꢀH1), 7.24 (d, 1H, J = 8.4 Hz, ArꢀH7), 6.57 (d, 1H,

J = 9.6 Hz, H12), 6.48ꢀ6.45 (m, 2H, ArꢀH7/ArꢀH8), 6.3 (d, 1H, Ar-

10H), 5.65 (d, 1H, J = 10.2 Hz, H13), 5.27 (s, 1H, 11a-H), 5.09 (s, 1H,

6aꢀOH), 4.20 (d, 1H, J = 11.4 Hz, H6 equiv), 4.08 (d, 1H, J = 11.4 Hz,

H6 ax), 1.38,1.35 (2 s, 2 ꢁ 3H, H15/H16), 0.96 {s, 9H, Si (CH₃)₃}, 0.19

{s, 6H, Si (CH₃)₂}. ¹³C NMR (100 MHz, acetone-d₆) δ 162.3, 159.3,

155.4, 152.2, 132.7, 130.7, 125.6, 124, 117.6, 114.7, 114.1, 111.7, 111.2,

103.9, 86.5, 77.3, 77.2, 71.5, 28.6, 26.6, 19.3. HRMS calcd (M^þþ Na)

475.1917, found 475.1923.

(þ) 4⁰-t-Butyldimethylsilyloxy-2⁰,7-dihydroxyisoflavan-3,4-diol (þ)

(14). Prepared according to previously published procedure.^23cWhite

solid with red tinge: 0.364 g (90%); mp decomposes at 142 ꢀC. TLC R_f

0.23 in hexanes:EtOAc (2:1). ¹H NMR (400 MHz, acetone-d₆) δ 7.20

(d, 1H, J = 8.4 Hz, ArꢀH5), 7.17 (d, 1H, J = 8.4 Hz, ArꢀH6⁰), 6.42 (dd,

1H, ²J = 8.4 Hz, ³J = 2.4 Hz, ArꢀH6), 6.34 (d, 1H, J = 2.4 Hz, ArꢀH8),

6.28 (dd, 1H, ²J = 8.4 Hz, ³J = 2.4 Hz, ArꢀH5⁰), 6.26 (d, 1H, J = 2.4 Hz),

5.09 (s, 1H, H4), 4.35 (d, 1H, J = 11.4 Hz, H2 equiv), 4.13 (d, 1H, J =

11.4 Hz, 4-OH), 4.19 (s, 1H, 3-OH), 4.02 (d, 1H, J = 11.4 Hz, H2 ax),

0.96 {s, 9H, Si (CH₃)₃}, 0.18 {s, 6H, Si (CH₃)₂}.

(ꢀ) 4⁰-t-Butyldimethylsilyloxy-2⁰,7-dihydroxyisoflavan-3,4-diol (ꢀ)

(14). Prepared according to previously published procedure.^23cWhite

solid with red tinge: 0.34 g (84%); mp decomposes at 144 ꢀC. TLC R_f

0.23 in hexanes: EtOAc (2:1). ¹H NMR (400 MHz, acetone-d₆) δ 7.20

(d, 1H, J = 8.4 Hz, ArꢀH5), 7.17 (d, 1H, J = 8.4 Hz, ArꢀH6⁰), 6.42 (dd,

1H, ²J = 8.4 Hz, ³J = 2.4 Hz, ArꢀH6), 6.34 (d, 1H, J = 2.4 Hz, ArꢀH8),

6.28 (dd, 1H, ²J = 8.4 Hz, ³J = 2.4 Hz, ArꢀH5⁰), 6.26 (d, 1H, J = 2.4 Hz),

5.09 (s, 1H, H4), 4.35 (d, 1H, J = 11.4 Hz, H2 equiv), 4.13 (d, 1H,

J = 11.4 Hz, 4-OH), 4.19 (s, 1H, 3-OH), 4.02 (d, 1H, J = 11.4 Hz, H2 ax),

0.96 {s, 9H, Si (CH₃)₃}, 0.18 {s, 6H, Si (CH₃)₂}.

(ꢀ) 9-(t-Butyldimethylsilyloxy) Glyceollin I (ꢀ) (16). Prepared

according to previously published procedure.^23cMajor yellow oil:

1

0.027 g (61%). TLC R_f0.46 in toluene:methanol (10:1). H NMR

(400 MHz, acetone-d₆) δ 7.27 (d, 1H, J = 8.4 Hz, ArꢀH1), 7.24 (d, 1H,

J = 8.4 Hz, ArꢀH7), 6.57 (d, 1H, J = 9.6 Hz, H12), 6.48ꢀ6.45 (m, 2H,

ArꢀH7/ArꢀH8), 6.3 (d, 1H, Ar-10H), 5.65 (d, 1H, J = 10.2 Hz, H13),

5.27 (s, 1H, 11a-H), 5.09 (s, 1H, 6aꢀOH), 4.20 (d, 1H, J = 11.4 Hz, H6

equiv), 4.08 (d, 1H, J = 11.4 Hz, H6 ax), 1.38ꢀ1.35 (2 ꢁ s, 2 ꢁ 3H,

H15/H16), 0.96 {s, 9H, Si (CH₃)₃}, 0.19 {s, 6H, Si (CH₃)₂}.

The other material obtained during this reaction, (ꢀ) 9-(t-butyldi-

methylsilyloxy)glyceollin II (14b), eluted as an inseparable mixture with

additional side products. No attempts were made to further isolate this

isomer.

(() 9-(t-Butyl Dimethyl Silyloxy)-glycinol (() (15). To a solution of

4⁰-t-butyldimethylsilyloxy-2⁰,7-dihydroxyisoflavan-3,4-diol (0.041 g, 0.1

mmol) in 20 mL of anhydrous ethanol was added polymer-bound

1,3,4,6,7,8-hexahydro-2H-pyrimido(1,2-a)pyrimidine (4 mg, 0.01 mmol)

and 4 Å molecular sieves (0.1 g). The reaction mixture was refluxed at

80 ꢀC for 6 h with continuous distillation of the ethanolꢀwater azeotrope.

After disappearance of reactant (TLC), the molecular sieves and polymeric

base were filtered. The filtrate was dried over sodium sulfate, filtered, and

solvent evaporated at 20 ꢀC. The residue was chromatographed over

silica using hexanes:EtOAc (4:1). The organic fractions were evaporated

at 20 ꢀC to provide a pinkishꢀwhite solid: 0.023 g (60%); mp

(þ) 9-(t-Butyldimethylsilyloxy) Glyceollin I (þ) (16). Prepared

according to previously published procedure.^23cMajor yellow oil:

1

0.025 g (57%). TLC R_f0.46 in toluene:methanol (10:1). H NMR

(400 MHz, acetone-d₆) δ 7.27 (d, 1H, J = 8.4 Hz, ArꢀH1), 7.24 (d, 1H,

J = 8.4 Hz, ArꢀH7), 6.57 (d, 1H, J = 9.6 Hz, H12), 6.48ꢀ6.45 (m, 2H,

ArꢀH7/ArꢀH8), 6.3 (d, 1H, Ar-10H), 5.65 (d, 1H, J = 10.2 Hz, H13),

5.27 (s, 1H, 11a-H), 5.09 (s, 1H, 6aꢀOH), 4.20 (d, 1H, J = 11.4 Hz, H6

equiv), 4.08 (d, 1H, J = 11.4 Hz, H6 ax), 1.38ꢀ1.35 (2ꢁ s, 2 ꢁ 3H, H15/

H16), 0.96 {s, 9H, Si (CH₃)₃}, 0.19 {s, 6H, Si (CH₃)₂}.

The other material obtained during this reaction, (þ)9-(t-butyldi-

methylsilyloxy)glyceollin II (14b), eluted as an inseparable mixture with

additional side products. No attempts were made to further isolate this

isomer.

1

197ꢀ199 ꢀC. TLC R_f0.38 in hexanes:EtOAc (2:1). H NMR (400

MHz, acetone-d₆) δ 8.55 (s.1H, ꢀOH), 7.30 (d, 1H, J = 8.4 Hz,

ArꢀH1), 7.24 (d, 1H, J = 8.4, ArꢀH7), 6.55 (dd, 1H, ²J = 8.4 Hz, ³J = 2.4

Hz, ArꢀH-2), 6.45 (dd, 1H, ²J = 8.4 Hz, ³J = 2.4 Hz, ArꢀH8), 6.3 (d,

1H, J = 2.4 Hz, ArH-4), 6.26 (d, 1H, J = 2.4 Hz, Ar H-10), 5.26 (s, 1H,

11a-H), 5.01 (s, 1H, 6aꢀOH), 4.14 (d, 1H, J = 12 Hz, H6 equiv), 4.02

(d, 1H, J = 12 Hz, H6 ax), 0.96 {s, 9H, Si (CH₃)₃}, 0.19 {s, 6H, Si

(CH₃)₂}. ¹³C NMR (100 MHz, acetone-d₆) δ 162.4, 160.3, 159.3,

157.7, 133.8, 125.6, 124.3, 114.1, 113.8, 111.4, 104.5, 103.9, 86.6, 77.4,

71.3, 26.6, 19.3. Anal. (C₂₁H₂₆O₅Si) C, H.

General Procedure for Deprotection of the TBDMS Group. 9-(t-

Butyldimethyl-silyloxy)-glyceollin I (0.043 g, 0.1 mmol) was dissolved

in 1 mL of acetonitrile, and the solution was cooled to ꢀ20 ꢀC.

(ꢀ) 9-(t-Butyl Dimethyl Silyloxy)-glycinol (ꢀ) (15). Prepared accord-

ing to previously published procedure.^23cPinkishꢀwhite solid: 0.025 g

1

(64%). TLC R_f0.38 in hexanes:EtOAc (2:1). H NMR (400 MHz,

N(Et)₃3HF in acetonitrile (1.2 mL, 0.12 mmol) was added and the

3

acetone-d₆) δ 8.55 (s.1H, ꢀOH), 7.30 (d, 1H, J = 8.4 Hz, ArꢀH1), 7.25

(d, 1H, J = 8.4, ArꢀH7), 6.55 (dd, 1H, ²J = 8.4 Hz, ³J = 2.4 Hz, ArꢀH-2),

6.45 (dd, 1H, ²J = 8.4 Hz, ³J = 2.4 Hz, ArꢀH8), 6.3 (d, 1H, J = 2.4 Hz,

ArH-4), 6.26 (d, 1H, J = 2.4 Hz, Ar H-10), 5.26 (s, 1H, 11a-H), 5.01 (s,

1H, 6aꢀOH), 4.14 (d, 1H, J = 12 Hz, H6 equiv), 4.02 (d, 1H, J = 12 Hz,

H6 ax), 0.96 {s, 9H, Si (CH₃)₃}, 0.19 {s, 6H, Si (CH₃)₂}.

mixture stirred for 8 h at 4 ꢀC. After disappearance of reactant (TLC), the

pH was adjusted to 7ꢀ8 by addition of triethylamine and the mixture filtered

through a silica column using DCM:MeOH (10:1). Evaporation of the

solvent at 20 ꢀC provided a brownish oily residue which was chromato-

graphed over silica using hexanes:DCM:methanol (10:10:1). The organic

fractions were evaporated at 20 ꢀC to provide the solids specified below.

(() Glyceollin I (() (3). White solid with red tinge: 0.023 g (69%).

TLC R_f0.22 in hexanes:DCM:methanol (10:10:1). ¹H NMR (400 MHz,

(þ) 9-(t-Butyl Dimethyl Silyloxy)-glycinol (þ) (15). Prepared accord-

ing to previously published procedure.^23cPinkishꢀwhite solid: 0.023 g

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Journal of Medicinal Chemistry

ARTICLE

methanol-d₄) δ 7.20 (d, 1H, J = 8.4 Hz, ArꢀH1), 7.07 (d, 1H, J = 8.4 Hz,

ArꢀH7), 6.59 (d, 1H, J = 10.2 Hz, H12), 6.46 (d, 1H, J = 8.4 Hz,

ArꢀH2), 6.39 (dd, 1H, ²J = 8.4 Hz, ³J = 2.4 Hz, ArꢀH8), 6.22 (d, 1H, J =

2.4 Hz, ArꢀH10), 5.61 (d, 1H, J = 10.2 Hz, H13), 5.16 (s, 1H, 11a-H),

4.90 (s, 1H, 6aꢀOH), 4.16 (d, 1H, J = 11.4 Hz, H6 equiv), 3.93 (d, 1H,

J = 11.4 Hz, H6 ax), 1.38,1.37 (2 s, 2 ꢁ 3H, H15/H16). ¹³C NMR (100

MHz, methanol d₄) δ 162.3, 161.3, 155.4, 151.9, 132.4, 130.5, 125.3,

121.3, 117.7, 114.3, 111.7, 111.4, 109.5, 99.1, 86.1, 77.3, 77.2, 71.3,

28.2. HRMS calcd (M^þþ Na) 361.1052, found 361.1052. Anal.

of DMEM and F-12 Ham media containing 2.5 mM ^L-glutamine and

15 mM HEPES, supplemented with 0.2% sodium bicarbonate, 10 mg/mL

insulin, 500 μg/L hydrocortisone, 20 μg/L epidermal growth factor,

100 μg/L cholera toxin, 5% fetal bovine serum, and 50 μg/mL gentamicin.⁷³

All cells were cultured at 37 ꢀC in a 5% CO₂/100% humidity environment.

Growth Inhibition Assays. Cell growth inhibition assays were per-

formed by a sulforhodamine B staining method described previously.^50,51,74

Cell loading densities yielding exponential growth over the 48 h

exposure period with final optical density (OD) values less than 2.0

were determined in preliminary tests. Three media conditions were

employed during testing. For intermediate E2 level tests (0.6 nM E2, 0.7 nM

testosterone), all cell lines were loaded in maintenance media and test

agents were added in 5%FBS/5%NS RPMI maintenance media, using

the following cell loading densities: DU145 2300 cells per well (cpw),

PC3 1300 cpw, LNCaP 3600 cpw, MCF12A 1300 cpw, MCF7 2000

cpw, and NCI-ADR-RES 3500 cpw. For tests with MCF7, NCI-ADR-

RES, and MCF12A cell lines in high E2 conditions, cell loading was

carried out as described for the intermediate E2 conditions, but agents

were loaded in 5%FBS/5%NS media with added E2 to give a final media

concentration during the exposure period of 100 nM E2. For low E2

tests (0.003 nM E2, 0.01 nM testosterone) with MCF7, NCI-ADR-RES,

and MCF12A cell lines, MCF7 and NCI-ADR-RES cells were precul-

tured for at least 4 days in RPMI with 5% FBS (same as RPMI

maintenance media but without added NS), after which these two cell

lines were loaded in this 5% FBS media while MCF12A cells were loaded

in their normal maintenance media at cell densities of MCF12A 1600

cpw, MCF7 3500 cpw, and NCI-ADR-RES 6000 cpw, and test agents

were loaded in the 5% FBS RPMI media for all three cell lines. For each

E2 condition tested, the percent of control cell growth was determined

for each cell line at each agent concentration by comparing OD readings

to those of matched vehicle-only control cells (0.25% DMSO). Agent

concentrations causing 50% growth inhibition (GI₅₀) versus vehicle-

only control cells were estimated by a Hill equation fit of the data. A log-

normal distribution provided the best approximation for the GI₅₀

measurements, so statistical analyses were performed using log₁₀

(GI₅₀) values. All results have been listed as mean (95% confidence

interval (CI). Statistically significant differences were determined by

analysis of variance (ANOVA) using a Bonferroni posthoc analysis for

pairwise comparisons, with p < 0.05 used as the criteria for statistical

significance performed using SYSTAT 12 (SYSTAT Software Inc., San

Jose, CA).

(C₂₀H₁₈O₅0.25H₂O) C, H.

3

Glyceollin I (3). White solid with red tinge: 0.026 g (77%). TLC R_f

0.22 in hexanes:DCM:methanol (10:10:1). Chiral HPLC showed an

essentially one peak chromatogram. ¹H NMR (400 MHz, methanol-d₄)

δ 7.20 (d, 1H, J = 8.4 Hz, ArꢀH1), 7.07 (d, 1H, J = 8.4 Hz, ArꢀH7), 6.50

(d, 1H, J = 10.2 Hz, H12), 6.37 (d.1H, J = 8.4 Hz, ArꢀH7), 6.30 (dd, 1H,

²J = 8.4 Hz, ³J = 2.4 Hz, ArꢀH8), 6.13 (d, 1H, J = 2.4 Hz, ArꢀH7), 5.52

(d, 1H, J = 10.2 Hz, H13), 5.06 (s, 1H, 11a-H), 4.81 (s, 1H, 6aꢀOH),

4.07 (d, 1H, J = 11.4 Hz, H6 equiv), 3.93 (d, 1H, J = 11.4 Hz, H 6ax),

1.38ꢀ1.35 (2 s, 2 ꢁ 3H, H15/H16). HRMS calcd [M^þþ Na]

361.1052; found 361.1059. Anal. (C₂₀H₁₈O₅0.25H₂O) C, H.

3

(þ) Glyceollin I (3). White solid with yellow tinge: 0.025 g (75%).

TLC R_f0.22 in hexanes:DCM:methanol (10:10:1). Chiral HPLC

1

showed an essentially one peak chromatogram. H NMR (400 MHz,

methanol-d₄) δ 7.21ꢀ7.19 (d, 1H, J = 8.4 Hz, ArꢀH1), 7.07 (d, 1H,

J = 8.4 Hz, ArꢀH7), 6.51ꢀ6.49 (d, 1H, J = 10.2 Hz, H12), 6.38ꢀ6.36

(d.1H, J = 8.4 Hz, ArꢀH7), 6.31ꢀ6.29 (dd, 1H, ²J = 8.4 Hz, ³J = 2.4 Hz,

ArꢀH8), 6.13 (d, 1H, J = 2.4 Hz, ArꢀH7), 5.53ꢀ5.51 (d, 1H, J = 10.2

Hz, H13), 5.06 (s, 1H, 11a-H), 4.81 (s, 1H, 6aꢀOH), 4.08ꢀ4.06 (d, 1H,

J = 11.4 Hz, H6 equiv), 3.94ꢀ3.92 (d, 1H, J = 11.4 Hz, H6 ax),

1.38ꢀ1.35 (2 s, 2 ꢁ 3H, H15/H16). HRMS calcd [M^þþ Na]

361.1052; found 361.1059.

Chiral Column Chromatography.^24,68A chiral Cyclobond

column from ASTEC Inc. was 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 mobile phase 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

(þ) material, and 49.5 þ 53.3 for synthesized racemic material.

Chiral NMR Shift Reagent Studies.⁶⁹Europium(III) tris-

[3-(heptafluoro-propylhydroxy-methylene)-^L-camphorate was deployed

in 20% mole ratio by dissolving it in the same deuterated solvent as

the routine NMR sample, adding the solution directly to the latter and

then rerunning the NMR spectrum. Results for the key diol intermediate

are summarized in Table 1.

’ ASSOCIATED CONTENT

S

Supporting Information. Historical structural assign-

b

ment; phytochemical pathway; attempted addition of water

across the pterocarpene “6aꢀ11a” double-bond; NMR and CD

studies with selected spectra; chiral HPLC studies with selected

chromatograms; HR MS and combustion analyses data; biolo-

gical data; NMR spectra for new intermediates and ﬁnal test

compounds. This material is available free of charge via the

Internet at http://pubs.acs.org.

Biology. Cell Lines. Androgen receptor negative (AR^ꢀ) human

prostate cancer (PC) cell lines DU145 and PC3,⁷⁰the estrogen receptor

positive (ER^þ) human breast cancer cell line MCF7,⁷¹and the estrogen

receptor negative (ER^ꢀ) human ovarian cancer cell line NCI-ADR-

RES⁷²were generously provided by the National Cancer Institute

Division of Cancer Treatment and Diagnosis Tumor Repository. The

androgen receptor positive (AR^þ) human PC cell line LNCaP⁷⁰and

ER^ꢀimmortalized “normal” human breast cell line MCF12A⁷³were

obtained from the American Type Culture Collection (ATCC).

Cell Culture. All cell lines except MCF12A were routinely maintained

in RPMI-1640 media containing 2.0 mM ^L-glutamine supplemented

with 20 mM HEPES, 0.2% sodium bicarbonate, 5% fetal bovine serum

(FBS), 5% NuSerum (NS) IV (BD Biosciences, Bedford, MA), and

50 μg/mL gentamicin. MCF12A cells were maintained in a 1:1 mixture

’ AUTHOR INFORMATION

Corresponding Author

*Phone: 419-530-2167. Fax: 419-530-1994. E-mail: paul.erhardt

@utoledo.edu.

’ ACKNOWLEDGMENT

This work was supported by grants from the USDA (ARS

SRRC) and the Ohio Soybean Council. We thank Dr. Ogawa and

Mr. Fei at Bowling Green State University for their assistance

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Journal of Medicinal Chemistry

ARTICLE

during the CD studies. MCF7, NCI/Adr-Res. DU145, and PC3

cell lines were provided by the NCI Division of Cancer Treat-

ment and Diagnosis (DCTD) Tumor Repository.

selected progeny. J. Chromatogr., A 2001, 915, 61ꢀ74 (with subsequent

erratum noted 2003, 989, 317).

(7) The following deﬁnitions, as meant for use herein, have been

adapted from analogies to the development of small molecule pharma-

ceutical agents wherein the attrition rates observed during drug devel-

opment candidates derived from new mechanistic pathways thought to

represent novel therapeutic targets, and likewise for their accompanying

“drug wannabe” compounds, is so high that success in preclinical models

is now regarded by many as being only a credentialization of such

pathways and their associated compounds, rather than as a validation.

Actual validation is then reserved for success observed in clinical studies.

For discussions of this topic, see:(a) Erhardt, P. W.; Proudfoot, J. R.

Drug Discovery: Historical Perspective, Current Status, and Outlook. In

Comprehensive Medicinal Chemistry II; Taylor, J. B., Triggle, D. J.,

Kennewell, P. D., Eds.; Elsevier, Ltd.: Oxford, U.K, 2007; pp 29ꢀ96.

(b) Erhardt, P. W. Drug Discovery. In Pharmacology—Principles and

Practice; Hacker, M., Bachmann, K., Messer, W., Eds.; Elsevier, Ltd.:

Oxford, 2009; pp 475ꢀ560.

’ ABBREVIATIONS USED

GI, growth inhibitory; ER, estrogen receptor; AR, androgen

receptor; QC, quality control; GLYs, glyceollins; SERMs-selec-

tive estrogen receptor modulatorsCD, circular dichroism; E2,

estradiol; THF, tetrahydrofuran; TLC, thin-layer chromatogra-

phy; mps, melting points; TMS, tetramethyl silane; HR MS, high

resolution mass spectroscopy

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