Supporting Information

Biomimetic Syntheses and Antiproliferative Activities of Racemic, Natural (-), and Unnnatural (+) 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, The University of Toledo College of Pharmacy; University of Findlay College of Pharmacy; Division of Basic Pharmaceutical Sciences, College of Pharmacy, Xavier University of Louisiana; Southern Regional Research Center, Agricultural Research Station, United States Department of Agriculture; and, Department of Hematology and Medical Oncology, School of Medicine, and Center for Bioenvironmental Research, Tulane University

* To whom correspondence should be addressed. Phone: 419-530-2167. Fax: 419-530-1994. E-mail: [email protected] †

University of Findlay College of Pharmacy.

$

University of Toledo College of Pharmacy.

§

Xavier University of Louisiana.

‡

United States Department of Agriculture.

¶

Tulane University.

Supporting Information (i) Historical structural assignment. (ii) Phytochemical pathway. (iii) Attempted addition of water across the pterocarpene ‘6a,11a’ double-bond. (iv) NMR and CD studies with selected spectra. (v) Chiral HPLC studies with selected chromatograms. (vi) HR MS and combustion analysis data. (vii) Biological data. (viii) NMR spectra for new intermediates and final compounds (separately located in accompanying file). S1

`

(i) Original and corrected structural assignments for the glyceollins The pterocarpans are the second largest group of natural isoflavonoids. The first 6ahydroxypterocarpan, (+)-pisatin, was isolated in 1960. However, owing in part to the lengthy, multistep routes needed to prepare these contiguous ring systems, only two members of this class have been synthesized to date, namely the pisatins for which both enantiomers were obtained (1) and the variabilins for which all four stereoisomers were obtained. (2) The natural forms of these compounds are shown below. O

O

O

O

OH

OH

(+)-Pisatin O

(+)-Variabilin O OH

O

Glyceollin I was first isolated in 1971 by Keen et al. from soybean hypocotyls inoculated with phytophthora which serves as a stress factor to elicit the requisite phytochemical pathway.(3) However, they assigned the structure incorrectly and thus misnamed it as part of the phaseollin family. The latter is shown below (left) along with glyceollin I’s correct structure (right). HO

O

6a OH

O

O OH

O (-)-6a-Hydroxyphaseollin

O

(-)-Glyceollin I

O OH

In 1974 when Burden et al. later obtained 6a-hydroxyphaseollin as a metabolite of phaseollin,(4) the spectral properties did not match those given by Keen et al. In particular, for glyceollin I a λ max (EtOH) was observed at 286 nm with shoulders at 291, 306 and 318 nm, while 6a-hydroxyphaseollin showed absorption maxima at 280 nm with shoulders at 286 and 314 nm. Also, the TLC Rf values did not match. To address these discrepancies, Burden et al. isolated the soybean phytoalexin and determined its correct structure to be that shown above by parallel degradation studies of both compounds. These key degradation studies are shown below in Scheme 1.

S2

O

HO

O

O OH

OH 11aH O

Glyceollin I

6a-Hydroxyphaseollin

O O

OH

Formic Acid

O

Formic Acid HO

O

O

O

O

O

OH

Hydrogenolysis Pd/C

Mass-spectral analysis

O

Hydrogenolysis Pd/C

HO

O

Mass-spectral analysis

O

HO

HO HO

HO m/e 136

OH

Isoflavan A

OH

Isoflavan B

O O

m/e 204

Scheme 1. Structural elucidation of glyceollin I by comparison to 6a-hydroxyphaseollin. The correct structure of the new phytoalexin differs from the 6a-hydroxyphaseollin by the opposite locations of their dimethyl chromene ring and phenolic hydroxyl group. To confirm the correct structure, Burden et al. dehydrated both compounds with formic acid to yield the 6a-11a dehydropterocarpans followed by hydrogenolysis using palladium on carbon to yield the respective hexahydro derivatives. The structures of these hexahydro derivatives were identical to isoflavans A and B as shown in Scheme 1. Interestingly, the mass-spectral analysis of each compound showed a well-known and distinct pattern arising from a retro-Diels-Alder fragmentation.(5) The isoflavan B from phaseollin showed a base peak at m/e 204 while in the spectra of the isoflavan A from glyceollin, this peak was replaced by one at m/e 136. The NMR data of the compound isolated by Burden et al. concurred with the initial NMR data previously provided by Keen et al. In 1977 Keen et al. renamed the new phytoalexin as glyceollin.(6) Meanwhile, in 1976 Lyne et al. also isolated the glyceollins from soybean and provided complete NMR data of the revised structures for three of the four glyceollins.(7) The NMR structural data provided for the glyceollins is summarized in Figure 1 and Table 1 where deuterated acetone was used as solvent on a 100 MHz instrument to obtain the proton NMR spectra of the three major glyceollins. (7) S3

15 13 12 16

15

A O

O B

C

2

11a

1

H

I

6

6a

H H OH

O

E

O OH

16

12

7

D

4

O

1

O

II

8

OH

10

OH 15

13

H

O

O

O

HO

OH 16

OH

12

1

O

III

IV OH

O

OH

Figure 1. Structures and numbering of the glyceollin family members (I, II and III) along with the phytochemical intermediate (IV) that ultimately leads to GLY I. Table 1. Proton NMR of glyceollins (shifts in ppm).(7) Proton H-1 H-2 H-4 H-6 H-6’ H-7 H-8 H-10 H-11a H-12 H-13 H-15 H-16

Glyceollin I δ (ppm) J (Hz) 7.24 d 8.5 6.47 d 8.5 ----4.12 d 12 4.32 d 12 7.22 d 8 6.43 q 8;2 6.25 d 2 5.27 s --6.53 d 10 5.65 d 1.36 s 1.39 s

10 -----

Glyceollin II δ (ppm) J (Hz) 7.15 s ------6.21 s --4.04 d 12 4.13 d 12 7.21 d 8 6.43 q 8;2 6.25 d 2 5.25 s --6.41 d 10 5.65 d 1.37 s 1.40 s

10 -----

Glyceollin III δ (ppm) J (Hz) 7.27 s ------6.27 s --4.05 d 11 4.15 d 11 7.23 d 8 6.46 q 8;2 6.26 d 2 5.30 s --3.05 ABX 16,8 3.42 16,8 5.26 8 4.91 s --1.77 s ---

The mass spectrum of glyceollin I has likewise been fully delineated.(3) Glyceollin I gives a parent ion at m/e 338, in accordance with an empirical formula C20H18O5. Other major peaks were found S4

at m/e 323 (M - CH3, Base Peak), 320 (M-H2O), 305 (M - CH3 &H2O), 293 (M-CH3-CO) and 277 (MCH3&H2O &CO).

(ii) Glyceollins’ phytochemical pathway The glyceollins are one of the phytoalexin(10) end-products from stress-induced biosynthesis in various soy plant parts, such as in roots upon attack by cyst root nematodes,(11) or in seeds suffering from fungal infections.(12) Scheme 2 depicts the biosynthetic pathway to glyceollins I, II and III wherein it can be observed that several of the steps involve oxidative enzymes from the plant cytochrome P-450 family.(13)

Scheme 2. Biosynthesis of glyceollins. Reactions involve the following sequence of plant cytochrome P450 enzymes : a) L-Phenylalanine ammonia-lyase or PAL; b) Cinnamate 4-hydroxylase; c) Chalcone S5

synthase; d) Isoflavone synthase; e) Daidzein 2’-hydroxylase; f) 3,9-Dihydroxypetrocarpan 6ahydroxylase; g) Dimethylallyltransferase.

(iii) Attempts to add water to the pterocarpene ‘6a,11a’ double-bond Although speculative, one potential route to the 6a-hydroxypterocarpans involves adding the equivalent of a water molecule across the double-bond of a pterocarpene system. This approach is illustrated below in Scheme 3. Boron reagents have been deployed for these types of conversions across a wide range of alkenes under mild reaction conditions.(14) In particular, we noted that catechol borane can promote an appropriate addition across the conjugated 2,3-double bond of indene when catalyzed by rhodium.(15) RO

O

RO

O

H2O

OH

6a

O

O OR'

OR'

Scheme 3. Potential route to 6a-hydroxypteocarpans from pterocarpenes. To initially assess the feasibility of the borane chemistry, a model reaction was undertaken wherein the eventual 3-position oxygen atom in GLY I or II was instead substituted with a simple methyl-group so as to take advantage of the established pterocarpene natural product known as ‘lespedezol A1’(Scheme 4). We have previously obtained the latter in ca. 33% yield by a practical synthesis involving four steps from the appropriately substituted chalcone.(16) While we were able to repeat an insertion of a water molecule for the indene case, we did not observe any of such reaction when either lespedazol A1 or the even simpler benzofuran system were used as models and subjected to various of these types of conditions. Although the indene double-bond is highly conjugated, it is not fully aromatic like the benzofuran system that is embedded within the pterocarpene nucleus. It is likely that the inherent stability of the latter serves as a larger hurdle in this attempted application. We are continuing to explore this route by other strategies. OH

1) Catecholborane Rhodium catalys 2) H2O2, NaOH

Indene

O

1-Indanol

O

O

O OH

6a

O

Lespedezol A1

O OH

OH

6a-hydroxylespedezol A1 OH

O

Benzofuran

O

3-hydroxybenzofuran

Scheme 4. Successful addition of water across indene but not for lespedazol A1 or benzofuran. S6

(iv) NMR and CD studies with selected spectra Natural Stereochemistry of the 6a-hydroxy group There are two asymmetric centers in glyceollin, namely at positions 6a and 11a. It has been shown by computational studies that the cis ring junction is energetically favored over the trans.(8) The majority of the known natural pterocarpans have a cis ring junction and are levorotatory.(9) Ferreira et al., has synthesized a trans pterocarpan.(2) They observed that the C-11a proton is axially oriented in the trans isomer and this results in its conspicuous shielding (δ = 1 ppm) as compared to the cis isomer. In the glyceollin NMR spectra, such shielding was absent indicating that there is a cis ring junction. The diagnostic protons at the C-6 position appear as two separate doublets with the C-6 equatorial proton appearing downfield compared to the C-6 axial proton. The cis ring junction for the glyceollins is also confirmed by the W coupling between the C-11a proton and the lower field C-6 equatorial proton. The negative optical rotation of the glyceollins also suggests an S,S configuration at the 6a,11a ring junction by analogy to all of the other natural Pterocarpans.(9) Circular Dichroism (CD) and Optical Rotatory Dispersion (ORD) studies also corroborate the absolute configuration at these stereocenters. The ORD curves of the glyceollins show a large negative trough in the region of 240 nm which is consistent with other pterocarpans having the same absolute configuration at the 6a-11a ring junction.(17) The CD features of the pterocarpans have been used historically for determining their absolute configuration. The CD of pterocarpans is characterized by two bands, namely a high energy/low wavelength (220-240 nm) 1La and a low energy/high wavelength (260-310 nm) 1Lb band, contributed by the chroman ring and benzofuran ring chromophores. According to Antus et al.(18) and Slade et al.,(9) the negative Cotton effect in the 220-240 nm range and positive Cotton effect in the 260-310 nm range confirms the (6aS,11aS) configuration at the junction of the chroman and benzofuran rings within the cis 6a-hydroxy pterocarpans. These assignments are further shown in Table 2. Table 3 provides actual data for several of the natural pterocarpans. The optical rotation of the 6a-hydroxypterocarpans is solely determined by the absolute configurations at the 6a and 11a ring carbons. Thus, in the 6ahydroxypterocarpan family, all the levorotatory compounds can be associated with a (6aS,11aS)-cis configuration and all the dextrorotatory compounds with a (6aR,11aR)-cis configuration. Standard projection

Compound

Newman projection and torsional angle

H 7a

4

O

3 2

11a

1

6

5

H

HC

H H 6'

6a

Sign of La band CD

1

P

Positive

Negative

M

Negative

Negative

6a

O

Sign of Lb band CD

1

O

OH

11

7

O

6 8

11

10

Helicity

9

C

O 1a

C

H H

Table 2. Circular dichroism: helicity and Cotton effects of pterocarpans.(18)

S7

Table 3. CD data for natural pterocarpans. Adapted from Antus et al.(18a) R2

6

O

R1

H H 6' OH

2 1

H

R3

O 10

R4

S8

New chiral shift reagent NMR studies for diols We deployed lanthanide shift reagents to establish the optical purity of diols 13. We observed no doubling of the peaks in the NMR spectra for the enantiopure diols, in contrast to the NMR spectrum of the racemate. The racemate diol forms diastereomeric complexes with europium chiral shift reagents, namely europium(III) tris[3-(heptafluoropropylhydroxymethylene)-l-camphorate], which can be clearly observed as separate NMR resonances. Thus, NMR spectra of each stereoisomer indicated the presence of only one enantiomer. Successful asymmetric dihydroxylation was achieved using dihydroqunidine (DHQD) ligand for synthesis of dextrorotatory (+) diol needed for natural (-) GLY I whereas dihydroquinine (DHQ) ligand was used for the synthesis of levororotatory (-) diol. The effect of chiral shift reagents was not observed on the C-3 and C-4 hydroxy NMR shifts due to a broadening effect. However, their effect was conspicuously seen on the NMR shifts of the C-4 proton and the C-8 aromatic proton, suggesting the proximity of europium agent to both of these protons. The complex of (+) diol and (+) chiral shift reagent europium(III) tris[3-(heptafluoropropylhydroxymethylene)-d-camphorate] is an enantiomer to the complex of (-) diol and (-) chiral shift reagent europium(III) tris[3(heptafluoropropylhydroxymethylene)-l-camphorate], and this pair appropriately provides identical NMR spectra. However, the complex of (+) diol and (+) chiral shift reagent europium(III) tris[3(heptafluoropropylhydroxymethylene)-d-camphorate] is in a diastereomeric relation with the complex of (-) diol and (+) chiral shift reagent europium(III) tris[3-(heptafluoropropylhydroxymethylene)-dcamphorate], and this pair thus provides different NMR spectra. These observations support the opposite stereochemistry of the diols for each enantiomer. This situation is depicted in Figures 2 and 3. The most relevant NMR shift data is summarized below in Table 4 8

BnO

H

O

1 2

4

6 5

3

H OH

HO H

OBn

Table 4. NMR shift (PPM) for enantiomeric diols and their diastereomeric complexes with chiral shift reagent (CSR). The molar ratio of diol:CSR is 5:1. Compound (+) Diol (+) Diol (+) CSR (+) Diol (-) CSR (-) Diol (-) Diol (-) CSR (-) Diol (+) CSR

Ar-H 8 6.59 6.61 6.62 6.59 6.61 6.62

H4 5.51 5.75 5.94 5.52 5.78 5.91

H 2 equatorial 4.74 4.88 5.0 4.74 4.90 4.85

H 2 axial 4.03 4.25 4.45 4.03 4.28 4.41 S9

NMR spectrum before addition of chiral shift reagent:

NMR spectrum after addition of (+) chiral shift reagent:

Figure 2. Chiral Shift Reagent NMR studies for (+) Diol.

S10

8

H H TBDMSO

OBn

1

2

3

OBn OH

O H 4

5

OH

(-) Diol ( 3R 4S ) NMR spectrum before addition of chiral shift reagent:

NMR spectrum after addition of (+) chiral shift reagent:

Figure 3. Chiral Shift Reagent NMR studies for (-) Diol.

S11

CD studies of Diols The absolute stereochemical assignments for the diols were made by relying upon prior CD studies. T.G van Aardt et al. describe the diol intermediate for the synthesis of the unnatural variabilin in which the CD spectra for this enantiomer shows a negative Cotton effect in the region of 220-250 nm, and then it shows a positive Cotton effect in the region of 270-290 nm.(2) Similar types of Cotton effects were observed by Mori and Kisida for the diol intermediates synthesized on route to unnatural pisatin.(1) They also used the CD spectra to establish the absolute stereochemistry of the diol intermediate. The CD spectra of the diol intermediate for synthesis of unnatural (-) pisatin, our (+) diol intermediate for synthesis of natural (-) glyceollin I, and our (-) diol intermediate for synthesis of unnatural (+) glyceollin I, are compared in Figures 4 and 5

Figure 4. Diol needed for (-) Pisatin. Copied from reference.(1)

Figure 5. CD spectra of diols prepared herein for natural (-) glyceollin I (left panel) and for unnatural (+) glyceollin (right panel).

S12

NMR and CD studies of TBDMS protected glycinols The absolute stereochemistry and cis ring junction of the benzofuran and chromene ring systems were confirmed by NMR spectroscopic methods combined with CD analysis. As indicated, T.G. van Aardt et al. previously synthesized the cis and trans pterocarpan skeletons and characterized the ring junction by using extensive NMR studies.(2) The NMR shift of the C-11a proton is clearly diagnostic for the cis versus trans isomers. In the trans isomer, the C-11a proton is oriented axial relative to both aromatic rings. This relationship causes an up-field shift of about 1 ppm for the C-11a proton in the trans isomer compared to the cis isomer.(2) The NMR spectra of the synthesized glycinol derivative showed a C-11a proton at about 5.2 ppm which correlates with the shift reported for the cis isomer of variabilin by T.G. van Aardt et al., and for the cis isomer of pisatin as separately reported by Mori et al.(1) It has been previously established that the natural glyceollins have a cis ring junction between rings C and D. The proton NMR spectra of the glyceollins show the C-6 protons as two separate doublets, namely a downfield equatorial alpha-proton, and an upfield axial beta-proton. The COSY spectra of our glycinol derivative showed the same correlation between the C-11a proton and the downfield equatorial proton, the C-6 due to W coupling (Figure 6). This effect would not be possible in the trans isomer. 6

HO

H H 6'

O 6a

2 1

OH 7

H

11a

O

8 10

OTBDMS

Figure 6. NMR COSY relationship between the equatorial C-6 and C-11a proton. These assignments were further supported by CD studies. The CD spectra of the cis and trans isomers of other 6a-hydroxy-pterocarpans have been previously reported. Depending upon the observed Cotton effects, the configuration can be similarly assigned for our closely analogous system. The assignment of absolute configuration for other 6a-hydroxy-pterocarpan structures and the Cotton effects observed in their CD spectra have been summarized by Slade et al.(9) These are shown in Table 5 and Figure 7. The measured CD spectra of our synthesized glycinol derivative match with those for the cis fused 6a-hydroxy pterocarpans. Our results are shown in Figure 8. Table 5. Correlation of absolute configuration and cotton effects for 6a-hydroxy pterocarpans. Copied from reference.(9)

S13

HO

O

HO

H H

O H

O

H

O

OR (-)-(6aR,11aR)-cis HO

HO

O

O OH

OH H

H O OR

(-)-(6aS,11aS)-cis

OR

(+)-(6aS,11aS)-cis

O OR

(+)-(6aR,11aR)-cis

Figure 7. Stereochemistry and absolute configuration of cis and trans pterocarpans accompanied by their CD spectra. Adapted from reference.(9). 40.000

30.000

CD spectra of TBDMS protected Glycinols

20.000

10.000

Δε

Series2

0.000

Series1

-10.000

-20.000

-30.000

-40.000 223

231

239

247

255

263

271

279

287

295

303

311

319

Wavelength (nm)

- - - - Series1 (-) (6a-S,11a-S) Glycinol TBDMS. ____ Series2 (+) (6a-R,11a-R) Glycinol TBDMS. Figure 8. Measured CD spectra of glycinol derivatives synthesized herein.

S14

Final Assignment of Structure for the Synthesized Glyceollins The CD spectral data is in accord with all of the previous literature, as well as with our prior data for the asymmetric intermediates. The CD spectra for the glyceollin enantiomers are shown in Figure 9. 20

10

32 9

29 9

26 9

23 9

0

-10

Δε

O

O

-20

OH -30

O OH

-40

(-) Glyceollin I -50

-60

Wavelength (nm )

60

50

O

O

40

OH O 30

(+) Glyceollin I

OH

Δε 20

10

32 9

29 9

26 9

23 9

0

-10

Wavelength(nm)

-20

Figure 9. CD spectra of (-)-glyceollin and (+)-glyceollin.

S15

(v) Chiral HPLC studies with selected chromatograms Method: Chiral CyclobondTM (ASTEC) column; Temperature 35 oC; Flow rate 0.5 mL/minutes; Gradient solvent system having water:methanol:acetonitrile as shown below. Time (minutes) 0 30 31 48 49 60

% Water 60 45 60 60 60 60

% CH3CN 0 0 1 10 0 0

% MeOH 40 55 39 30 40 40

Compound

Retention Time (Minutes)

(±) Synthetic Glyceollin I (-) Natural Glyceollin I

Peak one 49.5 Peak two 53.3 52.7

(-) Synthetic Glyceollin I

53.2

(+) Synthetic Glyceollin I

49.4

Peak areas indicated > 98% purity for all synthesized glyceollin I materials and also > 98% ee for each of the synthesized enantiomers.

S16

Representative HPLC Chromatograms: Natural (-) glyceollin I 0.12

0.10

AU

0.08

0.06

0.04

0.02

0.00 0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00 Minutes

40.00

45.00

50.00

55.00

60.00

65.00

70.00

Synthetic racemic mixture ( + ) glyceollin 0.040 0.035 0.030

AU

0.025 0.020 0.015 0.010 0.005 0.000 0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00 Minutes

40.00

45.00

50.00

55.00

60.00

65.00

70.00

50.00

55.00

60.00

65.00

70.00

50.00

55.00

60.00

65.00

70.00

Synthetic (-) glyceollin 0.050

AU

0.040

0.030

0.020

0.010

0.000 0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00 Minutes

40.00

45.00

Synthetic (+) glyceollin 0.050

AU

0.040

0.030

0.020

0.010

0.000 0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00 Minutes

40.00

45.00

Figure 10. Glyceollin I chiral HPLC fingerprinting. Column: CyclobondTM (astec). Gradient solvent system utilizing water:methanol:acetonitrile (see preceding table).

S17

Synthetic racemic mixture ( + ) glyceollin 0.040 0.035 0.030

AU

0.025 0.020 0.015 0.010 0.005 0.000 0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00 Minutes

40.00

45.00

50.00

55.00

60.00

65.00

70.00

65.00

70.00

Synthetic (-) glyceollin spiked with natural (-) glyceollin 0.080 0.070 0.060

AU

0.050 0.040 0.030 0.020 0.010 0.000 0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00 Minutes

40.00

45.00

50.00

55.00

60.00

Synthetic (+) glyceollin spiked with natural (-) glyceollin 0.050

0.040

AU

0.030

0.020

0.010

0.000 0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00 Minutes

40.00

45.00

50.00

55.00

60.00

65.00

Figure 11. Chiral HPLC spiking studies. Column: CyclobondTM (astec). Gradient solvent system utilizing water:methanol:acetonitrile (see preceding table).

S18

(vi) HR MS and combustion analysis data The high resolution mass spectral (HS MS) data for the glyceollins are in accord with the empirical formula C20H18O5. This information along with combustion analysis data for several synthetic intermediates is summarized in Tables 6a and 6b. Table 6a. High resolution mass spectral data for the final glyceollin test compounds.

(+) Glyceollin I

Theoretical Mass (amu) [M+ + Na] 361.1052

Observed Mass (amu) [M+ + Na] 361.1052

(-) Glyceollin I

361.1052

361.1059

+ 1.9

(+) Glyceollin I

361.1052

361.1065

+ 3.6

Compound

Table 6b. Combustion analysis data. Compound (# from text) Formula C

Theoretical H O

Δ PPM 0

C

Founda H O

18

C16H16O4

70.57

5.92

23.50

70.35

5.95

23.72

19

C31H28O6

74.98

5.68

19.33

75.01

5.69

19.27

20

C33H30O7

73.60

5.61

20.81

73.86

5.62

20.79

22

C29H22O5 • 0.25 H2O

76.61

4.99

18.00

76.69

4.76

17.68

23

C35H36O5Si • 0.25 H2O 73.78

6.42

-

73.85

6.38

-

11

C35H38O4Si • 0.25 H2O 75.71

6.99

-

76.00

6.70

-

(±) 13

C35H40O6Si

71.89

6.89

-

71.72

6.81

-

(±) 14

C21H28O6Si

62.35

6.98

-

62.38

7.01

-

(±) 15

C21H26O5Si

65.26

6.78

-

65.10

6.67

-

70.06

5.44

-

70.32

5.42

-

(±) 3 [(±)GlyI] C20H18O5 • 0.25 H2O

(-) 3 [GlyI] C20H18O5 • 0.25 H2O 70.06 5.44 69.96 5.32 a Oxygen analyses were requested only when silicon (Si) was not present (because of its noted interference with accurate determinations); and when larger amounts of sample were available to allow for such.

S19

(vii) Biological data Figure 12 provides a complete graphical summary of cell growth inhibition activity of standards and different enantiomeric forms of glyceollin I on the ER+ MCF7 breast cancer cell line, the ER− MCF12A immortalized normal breast epithelial cell line, and the ER− NCI/ADR-RES ovarian cancer cell line for each media condition tested. Figure 13 illustrates the growth of the ER+ MCF7 breast cancer cell line, the ER− MCF12A breast cell line, and the ER− NCI/ADR-RES ovarian cancer cell line in vehicle-only control wells over 48 hr exposure period for each media condition tested. Table 7 offers a comparison of measured log10(GI50) values (GI50 in molar concentration) of standards in 5%FBS media compared to the values reported by the NCI Development Therapeutics Program for the same experimental methods and conditions.

S20

5%FBS

5%FBS/5%NS

100 80 60 40 20 0

Tamoxifen

-40 -9 10

10

-8

-7

10

-6

10

-5

10

10

-4

100 80 60 40 20 0 -20

Tamoxifen

-40 -9 10

-3

10

10

-8

Concentration (M)

10

80 60 40 20 0 -20

4-Hydroxytamoxifen 10

-8

-7

10

-6

10

-5

10

10

-4

10

80 60 40 20 0 -20

4-Hydroxytamoxifen

10

10

-8

-7

10

-6

10

-5

10

10

-4

10

40 20 0

Fulvestrant -7

-6

10

-5

10

10

-4

80 60 40 20 0 -20

Fulvestrant

-40 -9 10

-3

10

60 40 20 0

Genistein -8

-7

10

-6

10

-5

10

10

-4

10

-8

Percent of Control Growth (%)

80 60 40 20 0

-40 -9 10

-6

10

-5

10

10

-4

Gly I (-) 10

-8

-7

10

-6

10

-5

10

Concentration (M)

10

-4

-3

10

-4

10

-3

10

-4

10

80 60 40 20 0 -20

4-Hydroxytamoxifen 10

-8

-7

10

-6

10

-5

10

-3

60 40 20 0 -20

Fulvestrant

-40 -9 10

-3

10

10

-8

80 60 40 20 0 -20

Genistein 10

-8

-7

10

-6

10

-5

10

10

-4

-5

10

10

-4

-3

10

80 60 40 20 0 -20

Genistein 10

-8

-7

10

-6

10

-5

10

10

-4

-3

10

Concentration (M) 120

80 60 40 20 0

-40 -9 10

-6

10

100

-40 -9 10

-3

10

100

-20

-7

10

Concentration (M)

120

100

10

100

Concentration (M)

120

-5

10

120

Concentration (M)

-20

-7

10

100

-40 -9 10

-3

10

-6

10

80



80

-7

10

100

120

100

10

-8

Concentration (M)

120

-40 -9 10

10

120

100

Concentration (M)

-20

Tamoxifen

Concentration (M) Percent of Control Growth (%)


60

10

0 -20

Concentration (M)

80

-8

20

-40 -9 10

-3

120

10

40

Concentration (M)

100

-40 -9 10

-3

100

-40 -9 10

60

-40 -9 10

-3




100

120


10

-4

80

120

Concentration (M)


10

-5

120

-40 -9 10


10

-6

100

Concentration (M)

120

-20

-7


-20

120


120



120

5%FBS/5%NS + 100nM E2

Gly I (-) 10

-8

-7

10

-6

10

-5

10

Concentration (M)

10

-4

-3

10

100 80 60 40 20 0 -20 -40 -9 10

Gly I (-) 10

-8

-7

10

-6

10

-5

10

10

-4

-3

10

Concentration (M)

S21

100 80 60 40 20 0

Gly I (+)

-40 -9 10

10

-8

-7

10

-6

10

-5

10

10

-4

100 80 60 40 20 0 -20

Gly I (+)

-40 -9 10

-3

10

10

-8

Concentration (M)

-5

10

10

-4

100 80 60 40 20 0

Gly I (rac)

-40 -9 10

10

-8

-7

10

-6

10

-5

10

Concentration (M)

60 40 20 0 -20

Gly I (+)

-40 -9 10

-3

10

10

-8

10

-4

-3

10

-6

10

-5

10

10

-4

-3

10

120

100 80 60 40 20 0 -20

-7

10

Concentration (M)

120



-6

10

80

Concentration (M)

120

-20

-7

10

100


-20

120


120



120

Gly I (rac)

-40 -9 10

10

-8

-7

10

-6

10

-5

10

Concentration (M)

10

-4

-3

10

100 80 60 40 20 0 -20

Gly I (rac)

-40 -9 10

10

-8

-7

10

-6

10

-5

10

10

-4

-3

10

Concentration (M)

Figure 12. Growth inhibitory effects of standards and different enantiomeric forms of glyceollin I on the ER+ MCF7 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) for all media conditions tested. Values are the average of eight sets of duplicate well measurements for each condition.

S22

Vehicle-Only Control Growth over 48 hr Exposure Period (%)

700 600 500 400 300 200 100 0 MCF7

MCF12A

NCI-ADR-RES

Cell Line

Figure 13. Growth of ER+ MCF7 breast cancer cell line, the ER− MCF12A breast cell line (lighter gray bars), and the ER− NCI/ADR-RES ovarian cancer cell line (darker gray bars) in vehicle-only control wells over 48 hr exposure period in media supplemented with 5%FBS (low E2, white bars, n = 52 for each cell line), 5%FBS/5%NS (intermediate E2, light gray bars, n = 50 for each cell line), and 5%FBS/5%NS + 100 nM E2 (high E2, darker gray bars, n = 32 for each cell line).

S23

Table 7. Comparison of measured log10(GI50) values (GI50 in molar concentration) of standards in 5%FBS media compared to the values reported by the NCI Development Therapeutics Program (http://dtp.nci.nih.gov/dtpstandard/cancerscreeningdata/index.jsp) for the same experimental methods and conditions. log10[GI50 (M)] MCF7 Agent

Measured

NCI-ADR-RES NCI DTP

Measured

PC3

NCI DTP

Measured

DU145 NCI DTP

Measured

NCI DTP

n

Ave ± 95%CI

n

Ave

n

Ave ± 95%CI

n

Ave

n

Ave ± 95%CI

n

Ave

n

Ave ± 95%CI

n

Ave

Paclitaxel

8

-8.69 ± 0.31

30

-8.55

8

-5.77 ± 0.22

30

-5.54

8

-8.15 ± 0.08

30

-8.41

8

-8.53 ± 0.06

30

-8.35

Docetaxel

8

-8.71 ± 0.05

2

-9.39

8

-6.05 ± 0.13

5

-6.17

8

-8.29 ± 0.04

2

-8.24

8

-8.50 ± 0.11

5

< -8.0

Vinblastine

8

-9.04 ± 0.07

117

-9.35

8

-6.80 ± 0.07

117

-6.94

8

-8.96 ± 0.08

117

-9.39

2

-9.25 ± 0.07

117

-9.40

Topotecan

8

-7.47 ± 0.22

8

-7.83

8

-6.74 ± 0.11

8

-6.71

8

-6.01 ± 0.15

8

-7.11

8

-7.61 ± 0.03

8

-7.77

Tamoxifen

8

−5.64 ± 0.06

12

-5.79

8

−5.24 ± 0.08

12

-5.28

8

-5.24 ± 0.07

12

-5.44

8

-4.92 ± 0.05

12

-5.16

Fulvestrant

8

< -8.0

2

< -8.0

8

> -4.0

2

> -4.0

8

> -4.0

2

> -4.0

8

> -4.0

2

> -4.0

Genistein

8

−4.21 ± 0.06

1

> -4.0

8

−4.49 ± 0.09

1

> -4.0

8

-4.40 ± 0.09

1

-4.03

8

-4.39 ± 0.05

1

> -4.0

Lower values indicate greater activity.

S24

References for Supporting Information Section (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11)

(12) (13)

(14)

(15)

Mori, K.; Kisida, H. Syntheses of pterocarpans. II. Synthesis of both the enantiomers of pisatin. Liebigs Ann. Chem. 1989, 35-39. van Aardt, T. G.; van Rensburg, H.; Ferreira, D. Synthesis of isoflavonoids. Enantiopure cis- and trans-6a-hydroxypterocarpans and a racemic transpterocarpan. Tetrahedron 2001, 57, 7113-7126. Sims, J. J.; Keen, N. T.; Honwad, V. K. Hydroxyphaseollin, and induced antifungal compound from soybeans. Phytochemistry 1972, 11, 827-828. Burden, R. S.; Bailey, J. A. Structure of the phytoalexin from soybean. Phytochemistry 1975, 14, 1389-1390. Pelter, A.; Stainton, P. Mass spectra of oxygen heterocycles. I. 4-Hydroxy-3phenylcoumarins isoflavonols. J. Heterocycl. Chem 1965, 2, 256-261. Partridge, J. E.; Keen, N. T. Soybean phytoalexins: rates of synthesis are not regulated by activation of initial enzymes in flavonoid biosynthesis. Phytopathology 1977, 67, 50-55. Lyne, R. L.; Mulheirn, L. J.; Leworthy, D. P. New pterocarpinoid phytoalexins of soybean. J. Chem. Soc., Chem. Commun.1976, 497-498. Schoening, A.; Friedrichsen, W. The stereochemistry of pterocarpanoids. A theoretical study. Zeitschrift fuer Naturforschung, B: Chemical Sciences 1989, 44, 975-982. Slade, D.; Ferreira, D.; Marais, J. P. J. Circular dichroism, a powerful tool for the assessment of absolute configuration of flavonoids. Phytochemistry 2005, 66, 21772215. Hammerschmidt, R. Phytoalexins: what have we learned after 60 years? Ann. Rev. Phytopath. 1999, 37, 285-306. Faghihi, J.; Jiang, X.; Vierling, R.; Goldman, S.; Sharfstein, S.; Sarver, J.; Erhardt, P. Reproducibility of the high-performance liquid chromatographic fingerprints obtained from two soybean cultivars and a selected progeny. J. Chromatogr. A 2001, 915, 61-74 with subsequent correction noted 2003, 989, 317. Boue, S. M.; Carter, C. H.; Ehrlich, K. C; Cleveland, T. E. Induction of the Soybean Phytoalexins Coumestrol and Glyceollin by Aspergillus J. Ag. Food Chem. 2000, 48, 2167-2172. a)Kochs, G.; Grisebach, H. Phytoalexin synthesis in soybean:purification and reconstitution of cytochrome P450 3,9-dihydroxypterocarpan 6a-hydroxylase and separation from cytochrome P450 cinnamate 4-hydroxylase Arch. Biochem. and Biophy. 1988, 263, 191-198 b) Welle, R.; Grisebach H. Induction of phytoalexin synthesis in soybean: enzymic cyclization of prenylated pterocarpans to glyceollin isomers. Arch. Biochem. and Biophy. 1988, 263, 191-198. a) Brown, H. C.; Vara Prasad, J. V. N. Hydroboration of heterocyclic olefins - a versatile route for the synthesis of both racemic and optically active heterocyclic compounds Heterocycles 1987, 25, 641-657. b) Brown H. C. New reagents for hydroboration and for synthesis via boranes Aldrichimica Acta 1974, 7, 43-52. Doucet, H.; Fernandez, E.; Layzell, T. P.; Brown, J. M. The scope of catalytic asymmetric hydroboration /oxidation with rhodium complexes of 1, 1'-(2-

S25

diarylphosphino-1-naphthyl)isoquinolines Chemistry-A European Journal 1999, 5, 1320-1330. (16) Khupse, R. S.; Erhardt, P. W. Practical synthesis of lespedezol A1 J. Nat. Prod. 2008, 71, 275-277. (17) Pelter, Andrew; Amenechi, P. I. Isoflavonoid and pterocarpinoid extractives of Lonchocarpus laxiflorus J. Chem. Soc. [Section] C: Organic 1969, 6, 887-896. (18) a) Kiss, L.; Kurtan, T.; Antus, S.; Benyei, A. Chiroptical properties and synthesis of enantiopure cis and trans pterocarpan skeleton. Chirality 2003, 15, 558-563 b) Kiss, Lorand; Kurtan, Tibor; Antus, Sandor; Benyei, Attila. Chiroptical properties and synthesis of enantiopure cis and trans pterocarpan skeleton. Chirality (2003), 15(6), 558-563.

S26

Biomimetic Syntheses and Antiproliferative Activities of Racemic, Natural (-), and Unnnatural (+) 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, The University of Toledo College of Pharmacy; University of Findlay College of Pharmacy; Division of Basic Pharmaceutical Sciences, College of Pharmacy, Xavier University of Louisiana; Southern Regional Research Center, Agricultural Research Station, United States Department of Agriculture; and, Department of Hematology and Medical Oncology, School of Medicine, and Center for Bioenvironmental Research, Tulane University

* To whom correspondence should be addressed. Phone: 419-530-2167. Fax: 419-5301994. E-mail: [email protected] †

University of Findlay College of Pharmacy.

$

University of Toledo College of Pharmacy.

§

Xavier University of Louisiana.

‡

United States Department of Agriculture.

¶

Tulane University.

S27

(vii) NMR spectra for new intermediates and final compounds 1) 1H, COSY, 13C NMR Spectra of compound 17………………………………………3 2) 1H, COSY, 13C NMR Spectra of compound 18……………………………………...6 3) 1H, COSY, 13C NMR Spectra of compound 19…………………………………..….9 4) 1H, COSY, 13C NMR Spectra of compound 20…………………………………..…12 5) 1H, COSY, 13C NMR Spectra of compound 22………………………………….….15 6) 1H, COSY, 13C NMR Spectra of compound 23…………………………………..…18 7) 1H, COSY, 13C NMR Spectra of compound 11…………………………………..….21 8) 1H, COSY, 13C NMR Spectra of compound 13……………………………….…….24 9) 1H, COSY, 13C NMR Spectra of compound 14………………………………..…...27 10) 1H, COSY, 13C NMR Spectra of compound 15…………………………………...30 11) 1H, COSY, 13C NMR Spectra of compound 16…………………………………...33 12) 1H, COSY, 13C NMR Spectra of compound 3……………………………………...36

2 S28

17

S29

S30

S31

18

S32

S33

S34

19

S35

S36

S37

20

S38

S39

S40

22

S41

S42

S43

23

S44

S45

S46

11

S47

S48

S49

13

S50

S51

S52

14

S53

14

S54

14

S55

15

S56

S57

S58

16

S59

S60

S61

3

36 S62

37 S63

38 S64