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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308

1

Use of a biosynthetic intermediate to explore the chemical diversity of

pseudo-natural fungal polyketides

Teigo Asai1,*, Kento Tsukada1, Satomi Ise1, Naoki Shirata1, Makoto Hashimoto2, Isao Fujii2

Katsuya Gomi3, Kosuke Nakagawara4, Eiichi N. Kodama4 and Yoshiteru Oshima1,*

1Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-yama, Aoba-ku, Sendai,

Japan. 2School of Pharmacy, Iwate Medical University, Nishitokuta, Yahaba, Iwate, Japan. 3Graduate School of Agricultural Science, Tohoku University, Sendai, Japan. 4Graduate School of

Medicine/Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan.

*e-mail: [email protected]

Table of Contents Supplementary Figure 1 S2 General Methods S3 Experimental Procedure and Characterization data Microbial Diversification The gene cluster containing pksCH-2 S3 Dimerization to 5 and 6 S4-S8 Supplementary Figure 2 S4 Supplementary Table 1 S6 Supplementary Figure 3 and 4 S7 Supplementary Figure 5 and Table 2 S8 Oligomerization to 7, 9 and 10 S8-S10 Supplementary Table 3 S10 Dimerization to 8 and chaetophenol E S11 Chemical Diversification Supplementary Table 4 S12 Conversion of 2 under acidic condition (11, 12, 19 and 38) S12-S15 Conversion of 2 under oxidative condition (39-44) S16-S18 Conversion of 2 under basic condition (13-18 and 34-37) S19-S25 Synthesis of indole-polyketide hybrid compounds (20-33) S25-S35 Supplementary Figure 6 S28 Evaluation of anti-adenovirus activity S36 NMR Spectra The 1H and 13C NMR spectra (2, 5-44) S37-S114

Use of a biosynthetic intermediate to explore the chemical diversity of pseudo-natural fungal polyketides

© 2015 Macmillan Publishers Limited. All rights reserved



2

Supplementary Figure 1. Structural diversity of natural and pseudo-natural polyketides based on fungal NR-PKS pathways. a, Several representative polyketides based on a NR-PKS in Chaetomium indicum are listed. The draft genome sequence analysis of C. indicum and feeding experiments with 13C-labeled sodium acetate suggested that 1 is a common intermediate of the chaetophenols and their related compounds (data not shown). b, A schematic illustration of the diversity-oriented semi-synthetic process in this study and whole structures produced in the process. Compounds 5–10 and 11-44 were generated through microbial diversification and chemical diversification, respectively. The new chemical entities were classified as polyketide oligomers (5-19), indole-polyketide hybrids (20–33) and azaphilone type compounds (34–44). Entities 19 and 38 were also categorized as isoquinoline alkaloid-like compounds.

O

O

O

HOHO

OOH

OH

O

H

5 : R = Me7 : R = H8 : R = CH2CHC(CH3)2

OO

O

O

O

OH

H

R1

R1

R2

R2

OHO

OH

O

O

OH

H

O

O

OHO

HO

O

R

R

O

OHO

OH

OH

O

OH

HO

O

H

OH

HO

O

O

O

OHH

N

O

O

HO

HO

O

O

O

OH

H

O

R1R2 R3

HO

OH

O

HO OH

H

O

HO

OH

NH

HO OH

H

O

O

O

O

O

O

N O

O

HO

NHBoc

OH

O

O

O

O

O

OHO OHHO

HO

6 9 11

12 13 : R1 = OH, R2 = Me, R3 = !-H 14 : R1 = Me, R2 = OH, R3 = "-H

15 : R1 = OH, R2 = Me 16 : R1 = Me, R2 = OH

34 35 36 37

O

O

O

OH

H

17 : 5S*,6S*,7S* 18 : 5R*,6R*,7R*

ON

O

HO

O

21

NH

O OH

O

23

32

NH

HO

OH

NH

N

HO

OH

26

NHO

HO OH

33

NH

OH

O

OH

22

NH

O

OH

OH

20 : R = H24 : R = Me

OH

HOR

O

O

O

OH

HO

O

HO

OH

OOH

O

O

O

O

O

OH

HO

H

H

OH

O

O

OHO

HO

O

chaetophenol C

chaetophenol E

chaetophenol D

chaetophenol A

O

O

HO

Ospiroindicumides

1

OH

HOR

O

Aspergillus oryzae

OH

HO

O

O

Enzyme ofheterologous

host

Key equivalent of o-QMs

Multipotent intermediate

N

N OH

OH

25

27 28 29

56

7

Polyketide oligomers

Indole-polyketide hybrid compounds

Azaphylone type compounds

DIversification

Heterologousbiosynthesis

Multipotent intermediates

O

ba

N

O

OMe

OMe

HON

OHC OMe

OMe30

N

HO

O

OMe

OMeI

H

OMe

OMe

O

OH

RN

HO

OH

10

SATKS

ATPT

ACPMT

R

NR-PKSHeterologousexpression

Isolated compounds Feeding

19(Isoquinoline alkaloid-like)

38(Isoquinoline alkaloid-like)

1: R = Me3 : R = HChaetophenol A : R = CH2CHC(CH3)2

2 : R = Me4 : R = HChaetophenol B : R = CH2CHC(CH3)2

NH

O

OMe

OMe

I31

39 40 41 42 43 44

O

OH

HO

chaetophenol B

O

O

O

OO

O

O

O

OO

O

O

O

O

OO

O

O

OO

O

O

O

O

O

O

O

OO

O

O

O

O

O

O

O




3

General Methods

Analytical and preparative TLC were performed on silica gel 60 F254 (Merck) and RP-18 F254 (Merck).

Column chromatography was carried out on silica gel 60 (70–230 mesh, Merck). NMR spectra were

recorded on JEOL ECA-600. Chemical shifts for 1H and 13C NMR are given in parts per million (δ) relative

to tetramethylsilane (δH 0.00) and residual solvent signals (δC 77.0) for CDCl3, (δH 3.30) and (δC 49.0) for

CD3OD, (δH 7.19) and (δC 123.5) for py-d5, (δH 2.04) and (δC 29.8) for acetone-d6, (δH 1.93) and (δC 118.2)

for acetonitrile-d3, as internal standards. Mass spectra were measured on JEOL JMS-700 (EI-MS),

JMS-DX303 (FAB-MS), and Thermo Scientific Exactive Mass Spectrometer (ESI-MS). UV spectra were

recorded on a JASCO-V-550 spectrophotometer. IR spectra were recorded on a JASCO-FT/IR-4200

spectrometer. HPLC analysis was performed on HITACHI LaChrom Series HPLC equipped with L-7100

pump, L-7455 Diode Array Detector and D-7000 system manager.

Experimental Procedure and Characterization Data

Microbial Diversification

The gene cluster containing pksCH-2 in Chaetomium indicum

Gene Amino acid Protein homologue (origin) Identity/Similarity (%) orf1 (pksCH-2)

2655 pkeA/AN7903, polyketide synthase (Aspergillus nidulans FGSC A4)

45/62

orf2 494 Cytochrome P450 (Aspergillus flavus NRRL3357) 47/65 orf3 310 NmrA family transcriptional regulator

(Metarhizium acridum CQMa 102) 47/64

orf4 497 Aldehyde dehydrogenase (Aureobasidium melanogenum CBS 110374)

43/61

orf5 430 Tryptophan dimethylallyltransferase family protein (Metarhizium robertsii)

27/41

orf6 201 Hypothetical protein (Zymoseptoria tritici IPO323) 32/49 orf7 473 FAD dependent oxidoreductase family protein

(Metarhizium acridum CQMa 102) 36/54

orf8 409 O-Methyltransferase (Aspergillus fumigatus Af293)

29/47

orf9 575 RadR putative transcriptional regulator (Beauveria bassiana ARSEF 2860)

45/64

orf10 267 Citrinin biosynthesis oxydoreductase CtnB (Monascus purpureus)

52/66




4

Cultivation of A. oryzae (OE::pksCH-2) and isolation of 2, 5 and 6

A. oryzae (OE::pksCH-2) was cultivated in CDS (Czapek-dox Broth 3.5 g, Polypepton 1.0 g, Soluble

starch 2.0 g dissolved in per 1 L of distilled water), YM (peptone 5.0 g, yeast 3.0 g, malt extract 3.0 g, and

glucose 10.0 g dissolved in per 1 L of distilled water), and potato dextrose PDB (Potato dextrose broth 24.0 g

dissolved in per 1 L of distilled water) liquid medium under shaking conditions (180 rpm) at 30 °C.

Maltose (1 %) was added in every condition to activate amyB promoter. Each culture media were

investigated by HPLC analysis on day 2 and 5 after inoculation. In order to identified 2, 5 and 6, A. oryzae

(OE::pksCH-2) was cultivated in CDS (1.5 L) and PDB (3.6 L) (7 days). Each culture medium was extracted

with ethyl acetate, and the extract was separated by silica gel column chromatography to afford 2 (48.7 mg)

from CDS, and 5 (11.8 mg) and 6 (4.8 mg) from PDB, respectively.

Supplementary Figure 2. HPLC profiles of a each culture media with A. oryzae (OE::pksCH-2): a, CDS; b,

YM; c, PDB. The data points were collected at incubation time of 2 and 5 days. The culture medium was

extracted with EtOAc and analyzed by reversed-phase HPLC (monitored at 215 nm) on a Mightysil RP-18

column (φ 250 mm x 4.6 mm, 1.0 mL/min) with methanol and water (0-5 min: 40:60, 5-20 min: from 40:60

to 80:20, 20-25 min: from 80:20 to 100:0, 25-35 min: 0:100).

pH 4.8 pH 4.3 pH 3.7

a b c




5

Compound 1: HREIMS: m/z 222.0875 [M]+ (222.0892 calcd for C12H14O4). 1H NMR (600 MHz, CDCl3) δH

12.81 (s, 8-OH), 9.99 (s, H-10), 4.06 (s, H2-3), 2.25 (s, H3-1), 2.12 (s, H3-11), 2.12 (s, H3-12).

Compound 2: Colorless amorphous; UV (EtOH) λmax nm (log ε) 205 (4.24), 226 (4.36), 286 (4.03); IR

(KBr) νmax (cm–1) 3369, 2916, 2848, 1657, 1577, 1470, 1391, 1321, 1260, 1234, 1183, 1085, 1022;

HREIMS: m/z 206.0921 [M]+ (206.0943 calcd for C12H14O3). 1H NMR (600 MHz, acetone-d6) δH 7.11 (s,

8-OH), 6.98 (s, 6-OH), 5.75 (brs, H-3), 5.03 (s, H2-10), 2.12 (s, H3-11), 2.06 (s, H3-12), 1.87 (s, H3-1); 13C

NMR (150 MHz, acetone-d6) δC 154.9 (C-2), 153.6 (C-6), 148.8 (C-8), 129.4 (C-4), 110.3 (C-7), 109.6 (C-5),

107.2 (C-9), 99.5 (C-3), 64.7 (C-10), 19.8 (C-1), 10.7 (C-12), 9.3 (C-11).

Structure of 1 and key HMBC correlations of 2

Compound 5: Colorless amorphous; UV (EtOH) λmax nm (log ε) 205 (4.22), 283 (3.00); IR (KBr) νmax

(cm–1) 3420, 2964, 2928, 2869, 1717, 1596, 1458, 1270, 1174, 1118, 1067; HRFABMS: m/z 411.1789

[M–H]– (411.1807 calcd for C24H27O6). 1H and 13C NMR data, see Supplementary Table 1.

Compound 6: Colorless amorphous; UV (EtOH) λmax nm (log ε) 206 (4.50), 321 (4.10); IR (KBr) ν

max (cm-1) 3410, 2994, 2922, 1704, 1604, 1460, 1395, 1263, 1206, 1124, 1064; HRFABMS: m/z 413.1965

[M+H]+ (413.1964 calcd for C24H29O6). 1H and 13C NMR data, see Supplementary Table 1.

Key HMBC (red arrow), 1H-1H COSY (blue bold line) and NOE (purple arrow) correlations of 5 and 6

HO

OH

O

2

12

3

456

78 9

10

12

11

O

O

OH

HO

OH

O

1

23

4

56

78 9

10

12

11

1'

2'

3'4' 5'

6'

7'8'9'

10' 11'

12'

O

O

OH

HO

OH

OH

O

O

OHO

HO

O10

1

2 3

45

67

11

89

1'2'

3'

4'5'

12'

7'6'

11'

8'

9'

10'

O

O

O

OHHO

O

6

H

5

12H

HH

H

HH10

13

11'

11 7'

8' 9'

10'10

HO

OH

O

1

O

123

456

7

89 10

12

11




6

Supplementary Table 1. 13C (150 MHz) and 1H NMR (600 MHz) data for 5 and 6.a ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ 5 b 6 c -------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------- Position 13C 1H (multi, J in Hz) 13C 1H (multi, J in Hz) ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ 1 23.3 1.73 (3H, s) 29.3 2.12 (3H, s) 2 98.6 ------ 205.8 ------ 3 37.5 3.24 (1H, dd, 11.4, 5.5) 44.8 3.70 (1H, d, 17.4, Ha) 3.65 (1H, d, 17.4, Hb) 4 135.1 ------ 111.1 ------ 5 114.4 ------ 116.5 ------ 6 154.2 ------ 151.6 ------ 7 111.8 ------ 110.2 ------ 8 150.7 ------ 148.2 ------ 9 112.0 ------ 130.3 ------ 10 64.1 5.56 (1H, d, 15.4, Ha) 30.8 3.01 (1H, d, 17.3, Ha) 5.43 (1H, d, 15.4, Hb) 2.37 (1H, d, 17.3, Hb) 11 10.7 2.72 (3H, s) 8.4 2.14 (3H, s) 12 11.2 2.52 (3H, s) 12.1 2.10 (3H, s) 1ʹ′ 29.2 2.16 (3H, s) 20.8 2.01 (3H, s) 2ʹ′ 206.1 ------ 161.3 ------ 3ʹ′ 44.8 3.87 (1H, d, 17.3, Ha) 99.1 5.68 (1H, s) 3.80 (1H, d, 17.3, Hb) 4ʹ′ 130.0 ------ 120.2 ------ 5ʹ′ 117.7 ------ 147.8 ------ 6ʹ′ 153.5 ------ 196.6 ------ 7ʹ′ 112.0 ------ 46.3 2.82 (1H, q, 6.6) 8ʹ′ 149.5 ------ 100.4 ------ 9ʹ′ 112.8 ------ 38.4 ------ 10ʹ′ 28.9 3.09 (1H, dd, 15.4, 5.5, Ha) 66.8 4.32 (1H, dd, 11.4, 1.9, Ha) 2.83 (1H, dd, 15.4, 11.4, Hb) 4.08 (1H, d, 11.4, Hb) 11ʹ′ 10.1 2.64 (3H, s) 6.0 1.19 (3H, d, 6.6) 12ʹ′ 13.2 2.42 (3H, s) 10.1 1.83 (3H, s) OH 10.19 (1H, s) 6-OH 4.74 (1H, s) OH 10.00 (1H, s) OH 9.93 (1H, s) ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ a Assignment for all compounds were based on COSY, HMQC, HMBC experiments b in pyridine d5 c in CDCl3




7

Supplementary Figure 3. Conversion of 1 and 2 in the culture medium of A. oryzae.

Compounds 1 and 2 were added to the A. oryzae cultivating medium (PDB) with or without boiling. After 24

h of incubation, the culture medium from each condition was extracted with EtOAc and the extract was

analyzed by reversed-phase HPLC (monitored at 215 nm) on a Mightysil RP-18 column (φ 250 mm x 4.6

mm, 1.0 mL/min) with methanol and water (0-5 min: 40:60, 5-20 min: from 40:60 to 80:20, 20-25 min: from

80:20 to 100:0, 25-35 min: 0:100): a, Both 1 and 2 were converted to dimers 5 and 6 in the PDB culture on

day 3 after inoculation of A. oryzae (wild type); b, 1 was not changed in the boiled culture medium. On the

other hands, 2 was dimerized to 5 and 6 in the same condition.

Supplementary Figure 4. Putative formation of polyketide dimers from isochromene precursors through

non-enzymatic dimerization in the culture media of Cordyceps indigotica and Chaetomium indicum.

O

HO

OH O

1

O

HO

OH

2

OHO

OH

O

O

OH

H

O

O

OHO

HO

O

5 6A. oryzaein PDB

0 5 10 15 20 25 (min)

0

1.0

2.0

AU (215 nm)

1

2

5

6

0 5 10 15 20 25 (min)

0

1.0

2.0

AU (215 nm)

2

5

6

1 + A. oryzae 2 + A. oryzae

a

b

OHO

OH

ORO

O

OH

ORH

O

O

OHO

HO

O

Indigotide C : R = 4-O-b-D-glucose

OHO

OH

ORO

O

OH

ORH

Indigotide D : R = 4-O-b-D-glucose

Chaetophenol E

O

OHRO

HO

Indigotide A : R = 4-O-b-D-glucose

O

HO

OH

Chaetophenol B

Cordyceps indigotica

Chaetomium indicum

O

HO

OH O

1

O

HO

OH

2

OHO

OH

O

O

OH

H

O

O

OHO

HO

O

5 6 Boiled A. oryzaeculture medium

XXO

0 5 10 15 20 25 (min)

0

1.0

AU (215 nm)1

2.0

0 5 10 15 20 25 (min)

0

1.0

AU (215 nm)

2.0

2

5

6

1 + A. oryzae (Δ) 2 + A. oryzae (Δ)




8

Supplementary Figure 5. Confirmation of reversibility of dimerization to 5 and 6.

Dimmers 5 and 6 were incubated in the PDB culture medium (pH 3.5), respectively. After 24 h of incubation,

each culture medium was extracted with EtOAc and the analyzed reversed-phase HPLC (monitored at 215

nm) on a Mightysil RP-18 column (φ 250 mm x 4.6 mm, 1.0 mL/min) with methanol and water (0-5 min:

40:60, 5-20 min: from 40:60 to 80:20, 20-25 min: from 80:20 to 100:0, 25-35 min: 0:100): a, 5 was remained

without change in the culture medium.; b, 6 provided 2 and 5 in the culture medium, indicating that 6 was

converted to 2 through retro reaction, followed by dimerization of 2 yielded 5.

Supplementary Table 2. Conversion ration from 2 to 5 and 6, and residual ration of 2 (%).

The conversion ration from 2 to 5 and 6 under various pH conditions was investigated. The PDB culture

medium on day 3 after inoculation of A. oryzae (wild type) was filtrated. After pH value of the solution was

adjusted to 2.0, 3.0, 4.0, 5.0 and 6.0 using 1N HCl, 1 mg of 2 was fed into each medium (50 mL) and

incubated for 24 h under shaking condition (30 oC, 150 rpm). The reaction mixture was extracted with

EtOAc and analyzed by HPLC (monitored at 215 nm). The results summarized in Supplementary Table 2.

Conversion ration was calculated from peak area in HPLC chromatogram by compared to their authentic

samples.

Heterologous expression of tspks2 and cultivation of the transformant

Previously we constructed expression plasmid vector by introduction of tspks2 into fungal expression

plasmid pTAex3 possessing the α-amylase promoter (amyB) of A. oryzae. The constructed expression

plasmid pTA-tpks2 containing argB gene as selection marker was subsequently transformed into A. oryzae

M-2-3 by protoplast-PEG method for overexpression studies. Transformants selected on minimal medium. A.

oryzae (OE::tspks2) was cultivated in PDB medium (3.0 L) (30 oC, 180 rpm) for 6 days. The culture medium

(235.0 mg) was extracted by EtOAc and the extract was separated by silica gel column chromatography to

afford 4 (known, 12.2 mg), 7 (4.2 mg), 9 (3.2 mg) and 10 (2.2 mg) from PDB, respectively.

pH 2.0 3.0 4.0 5.0 6.0

5

6

2

55.4

0

0 0

0

53.9 18.5

28.1

13.7 36.2

7.3

0 0

0

17.8

5

0 5 10 15 20 25 (min)0

1.0

AU (215 nm)

2.0

0 5 10 15 20 25 (min)0

1.0

AU (215 nm)

2.0

2

5 6

a b




9

Compound 7: Colorless amorphous; UV (EtOH) λmax nm (log ε) 212 (4.63), 283 (3.64); HRESIMS: m/z

383.1502 [M–H]– (383.1489 calcd for C22H23O6); 1H NMR (600 MHz, CDCl3 (10% CD3OD)) δH 6.25 (s,

H-5ʹ′), 6.16 (s, H-5), 4.93 (d, 14.7 Hz, Ha-10), 4.81 (d, 14.7 Hz, Hb-10), 3.60 (d, 15.1 Hz, Ha-3ʹ′), 3.46 (d,

15.1 Hz, Hb-3ʹ′), 3.02 (dd, 5.1, 4.4 Hz, H-3), 2.98 (dd, 16.1, 4.4 Hz, Ha-10ʹ′), 2.86 (dd, 16.1, 5.1 Hz, Hb-10ʹ′),

2.06 (s, H3-11ʹ′), 2.03 (s, H3-11), 2.02 (s, H3-1ʹ′), 1.60 (s, H3-1); 13C NMR (150 MHz, CDCl3 (10% CD3OD))

δC 210.4 (C-2ʹ′), 154.1 (C-6), 153.5 (C-6ʹ′), 152.7 (C-8ʹ′), 150.2 (C-8), 132.3 (C-4), 129.5 (C-4ʹ′), 112.0 (C-9),

111.2 (C-7ʹ′), 110.5 (C-9ʹ′), 109.9 (C-5ʹ′), 108.3 (C-7) 104.3 (C-5), 98.5 (C-2), 60.2 (C-10), 49.4 (C-3ʹ′), 36.8

(C-3), 28.6 (C-1ʹ′), 26.1 (C-10ʹ′), 25.3 (C-1), 8.0 (C-11ʹ′), 7.6 (C-11).

Structure of 7


575.2285 [M–H]– (575.2276 calcd for C33H35O9); 1H and 13C NMR data, see Supplementary Table 3.


575.2284 [M–H]– (575.2276 calcd for C33H35O9); 1H and 13C NMR data, see Supplementary Table 3.


OHO

OH

O

O

OH

1

23

4

5

67

89

1011

1'

2'

3'4'

5'

6'

7'8'9'

10' 11'

7

H

O

O

O

HOHO HO

O

OH

O

O

OHO

OH

OH

O

OH

HO

O

9 10

O

OHO

OH

OH

O

OH

HO

O

H

O

O

O

HOHO

O

H1

24

5

67 8 911

3

1'

2'

3' 4'5'

6'

7'8'9'

10'11'

1''

2''

3''4''

5''

6''

7''11''

8''9''

10'' 10

1

2 3

456

7

11

89

1'2'

3'4'

5'6'7'8'

9'

10'

11'

1''

2''3''

4'' 5''

6''7''

11''

8''9''

10''

13

H9'

10'

11'

11HHH

10




10

Supplementary Table 3. 13C (150 MHz) and 1H NMR (600 MHz) data for 9 and 10.a ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ 9 b 10 b -------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------- Position 13C 1H (multi, J in Hz) 13C 1H (multi, J in Hz) ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ 1 22.6 1.48 (3H, s) 29.4 2.17 (3H, s) 2 98.1 ------ 208.7 ------ 3 36.5 3.03 (1H, dd, 11.7, 2.7) 47.7 3.69 (1H, d, 16.9, Ha) 3.48 (1H, d, 16.9, Hb) 4 135.2 ------ 129.5 ------ 5 115.1 ------ 110.1 6.04 (1H, s) 6 152.2 ------ 153.1 ------ 7 110.4 ------ 109.9 ------ 8 149.5 ------ 150.0 ------ 9 111.0 ------ 111.1 ------ 10 62.8 4.98 (1H, d, 15.8, Ha) 26.2 3.03 (1H, d, 15.8, Ha) 4.87 (1H, d, 15.8, Hb) 2.25 (1H, d, 15.8, Hb) 11 8.2 2.10 (3H, s) 8.3 1.90 (3H, s) 1ʹ′ 28.9 2.03 (3H, s) 21.0 2.07 (3H, s) 2ʹ′ 208.6 ------ 165.3 ------ 3ʹ′ 47.5 3.39 (2H, s) 98.9 5.92 (1H, s) 4ʹ′ 129.2 ------ 145.1 ------ 5ʹ′ 109.8 6.22 (1H, s) 122.4 ------ 6ʹ′ 153.4 ------ 201.9 ------ 7ʹ′ 111.7 ------ 49.8 ------ 8ʹ′ 150.6 ------ 98.7 ------ 9ʹ′ 111.8 ------ 39.9 3.25 (1H, dd, 12.8, 4.7) 10ʹ′ 27.4 2.36 (1H, dd, 15.7, 2.7, Ha) 66.1 4.92 (1H, dd, 10.6, 4.7, Ha) 2.27 (1H, dd, 15.7, 11.7, Hb) 4.42 (1H, dd, 12.8, 10.6, Hb) 11ʹ′ 8.3 2.11 (3H, s) 21.6 1.22 (3H, d, 7.0) 1ʹ′ʹ′ 28.8 2.02 (3H, s) 29.1 2.04 (3H, s) 2ʹ′ʹ′ 208.9 ------ 208.2 ------ 3ʹ′ʹ′ 48.5 3.69 (1H, d, 16.1, Ha) 48.4 3.54 (1H, d, 16.5, Ha) 3.60 (1H, d, 16.1, Hb) 3.41 (1H, d, 16.5, Hb) 4ʹ′ʹ′ 130.9 ------ 131.0 ------ 5ʹ′ʹ′ 109.8 6.15 (1H, s) 109.8 6.07 (1H, s) 6ʹ′ʹ′ 153.8 ------ 154.0 ------ 7ʹ′ʹ′ 111.1 ------ 112.3 ------ 8ʹ′ʹ′ 154.2 ------ 154.2 ------ 9ʹ′ʹ′ 116.4 ------ 117.5 ------ 10ʹ′ʹ′ 22.9 3.91 (1H, d, 16.1, Ha) 22.4 3.39 (1H, d, 17.3, Ha) 3.82 (1H, d, 16.1, Hb) 3.35 (1H, d, 17.3, Hb) 11ʹ′ʹ′ 8.0 1.99 (3H, s) 8.7 2.05 (3H, s) ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ a Assignment for all compounds were based on COSY, HMQC, HMBC experiments. b in CDCl3 (10% CD3OD)




11

Conversion of chaetophenol A to 8 and chaetophenol E

A. oryzae was cultivated in PDB medium (60 mL) for 3days, and then 0.6 mg of chaetophenol A was

added to the culture medium under shaking condition (30 oC, 180 rpm). After 24 h of incubation, the culture

medium was extracted with EtOAc and the extract was analyzed by reversed-phase HPLC (monitored at 215

nm) on a Mightysil RP-18 column (φ 250 mm x 4.6 mm, 1.0 mL/min) with acetonitrile and water (0-5 min:

20:80, 5-20 min: from 20:80 to 0:100, 20-30 min: 0:100). The conversion ratios of chaetophenol B (23.5%),

8 (10.7%) and chaetophenol E (14.8%) from chaetophenol A were calculated by peak area in the

chromatogram by comparing with the authentic samples. To characterize 8, the conversion experiment was

scaled up. A. oryzae was cultivated in PDB medium (150 mL x 4) for 3 days, and then 5 mg of isolated

chaetophenol A was added to the each culture medium and stirred at 30 oC (180 rpm) for 24 hours. The

culture medium was extracted with EtOAc and the extract was separated by silica gel column

chromatography to afford 8 (1.0 mg).

HPLC chromatogram of the conversion experiment of chaetophenol A in A. oryzae culture medium

and the structure of 8


519.2761 [M–H]‒ (519.2747 calcd for C32H39O6); 1H NMR (600 MHz, CDCl3) δH 5.27 (s, 6-OH), 5.08 (m,

H-13), 5.03 (m, H-13ʹ′), 5.02 (d, 15.8 Hz, Ha-10), 4.90 (d, 15.8 Hz, Hb-10), 4.61 (s, 8-OH), 3.67 (d, 17.6 Hz,

Ha-3ʹ′), 3.61 (d, 17.6 Hz, Hb-3ʹ′), 3.29 (m, H2-12), 3.24 (m, H2-12ʹ′), 2.99 (dd, 11.8, 5.5 Hz, H-3), 2.58 (dd,

15.8, 5.5 Hz, Ha-10ʹ′), 2.48 (dd, 15.8, 11.8 Hz, Hb-10ʹ′), 2.17 (s, H3-11ʹ′), 2.12 (s, H3-11), 2.09 (s, H3-1ʹ′), 1.84

(s, H3-15), 1.81 (s, H3-15ʹ′), 1.76 (s, H3-16), 1.70 (s, H3-16ʹ′), 1.56 (s, H3-1); 13C NMR (150 MHz, CDCl3) δC

206.3 (C-2ʹ′), 152.4 (C-6), 151.9 (C-6ʹ′), 148.8 (C-8ʹ′), 148.7 (C-8), 135.8 (C-14), 134.7 (C-4), 133.9 (C-14ʹ′),

128.4 (C-4ʹ′), 122.6 (C-13ʹ′), 122.1 (C-13), 118.9 (C-5ʹ′), 116.2 (C-5), 112.3 (C-9ʹ′), 111.2 (C-7ʹ′), 110.7 (C-9),

108.6 (C-7), 98.0 (C-2), 62.8 (C-10), 44.3 (C-3ʹ′), 36.8 (C-3), 29.3 (C-1ʹ′), 29.1 (C-10ʹ′), 26.2 (C-12ʹ′), 25.8

(C-15), 25.7 (C-15ʹ′), 24.4 (C-12), 22.7 (C-1), 18.1 (C-16), 18.0 (C-16ʹ′), 8.5 (C-11ʹ′), 7.9 (C-11).

0 5 10 15 20 25 (min)

0

1.0

2.0

AU (215 nm)

Chaetophenol E

Chaetophenol B

8

Chaetophenol AOHO

OH

O

O

OH

1

23

4

5

67

89

10

12

11

1'

2'

3'4' 5'

6'

7'8'9'

10' 11'

12'

13

1415

16

13'14'

15' 16'

8

H




12

Chemical Diversification

Supplementary Table 4. Acid catalyzed dimerization of 2.

Conversion of 2 to 5 and 11

To a solution of 2 (30.0 mg, 0.15 mmol) in CH3CN (1.5 ml), BF3OEt2 (1.8 µl, 0.015 mmol) was added

dropwise at room temperature. After being stirred for 30 min at the same temperature, the reaction was

quenched with water and then extracted with EtOAc. The combined organic extracts were washed with

saturated aqueous solution of brine, dried over Na2SO2, and concentrated. The residue was purified by

silica-gel column chromatography to afford 5 quantitatively. A solution of 5 (10.0 mg, 0.024 mmol) in

CH3CN (2.0 mL) was treated with excessive BF3OEt (45.8 µl, 0.38 mmol) at room temperature for 30

min. The reaction was quenched by the addition of water and the mixture was extracted using EtOAc and

the residue was purified by silica-gel column chromatography and preparative TLC to afford 11 (6.6 mg).

Hypothetical reaction mechanism to 11 from 5

OHO

OH

O

O

OH

H

OH

HO

O

2 5

Lewis acid,r.t.

OHO

OH

O

O

OH

HOH

HO

O

O

O

OHH

OH

O

OH

O

OH

O

HOHHO

O

O

O

OH

H

OH

HO

O

OH

O

OH

115

2

BF3OEt2BF3OEt2

AlCl3Zn(OTf)2

Sc(OTf)3

Ti(OiPr)4

TsOHTFA

Amount (mol %)

10101010101010100

Solvent Time (min) Yield (%)(isolated)Acid

CH2Cl2

CH2Cl2CH2Cl2CH2Cl2CH2Cl2CH2Cl2CH2Cl2

CH3CN10

106010606010

10

quant.

quant.

80quant.

061

quant.

quant.

OHO

OH

O

O

OH

H

OH

HO

O

2 5

BF3OEt2 (10 mol%),CH3CN, r.t., quant.

OH

HO

O

O

O

OHH

11

BF3OEt2 (15 eq),CH3CN, r.t., 66%




13

The 1H and 13C NMR spectra of 11 showed the two sets of signals in CDCl3 + CD3OD (10%) (ca. 3:1),

CD3CN (ca. 10:1) and CD3OD (ca. 20:1) due to the C-2 epimers. The structure determination of 11 was

established by using 1D and 2D NMR measured in CD3OD and CD3CN.


(KBr) νmax (cm-1) 3363, 3217, 3001, 2928, 1715, 1661, 1599, 1541, 1457, 1376, 1208, 1160, 1117, 1090,

1072, 1031; HRESIMS: m/z 411.1805 [M–H]– (411.1802 calcd for C24H27O6); 1H NMR (600 MHz, CD3OD)

δH 3.73 (d, 18.4 Hz, Ha-3ʹ′), 3.59 (d, 18.4 Hz, Hb-3ʹ′), 3.39 (brs, H-3), 2.86 (d, 17.9 Hz, Ha-10), 2.39 (d, 17.9

Hz, Hb-10), 2.37 (brd, 14.3 Hz, Ha-10ʹ′), 2.22 (s, H3-1ʹ′), 2.19 (s, H3-12), 2.11 (s, H3-11), 1.80 (brd, 14.3 Hz,

Hb-10ʹ′), 1.78 (s, H3-12ʹ′), 1.78 (s, H3-11ʹ′), 1.32 (s, H3-1); 13C NMR (150 MHz, CD3OD) δC 206.4 (C-2ʹ′),

189.7 (C-6ʹ′), 175.6 (C-8ʹ′), 152.7 (C-8), 151.9 (C-6), 151.3 (C-4ʹ′), 135.0 (C-5ʹ′), 133.5 (C-4), 117.7 (C-7ʹ′),

116.3 (C-5), 115.4 (C-9), 113.0 (C-7), 104.3 (C-2), 44.8 (C-3ʹ′), 41.1 (C-9ʹ′), 40.7 (C-3), 34.8 (C-10), 30.0

(C-1ʹ′), 29.1 (C-10ʹ′), 26.0 (C-1), 12.9 (C-12), 12.6 (C-12ʹ′), 9.4 (C-11), 8.0 (C-11ʹ′). 1H NMR (600 MHz,

CD3CN) δH 5.90 (OH), 5.82 (OH), 4.65 (1-OH), 3.63 (d, 18.6 Hz, Ha-3ʹ′), 3.52 (d, 18.4 Hz, Hb-3ʹ′), 3.36 (t,

3.0 Hz, H-3), 2.79 (d, 18.6 Hz, Ha-10), 2.32 (d, 18.6 Hz, Hb-10), 2.24 (dd, 12.6, 3.0 Hz, Ha-10ʹ′), 2.15 (s,

H3-1ʹ′), 2.13 (s, H3-12), 2.06 (s, H3-11), 1.76 (dd, 12.6, 3.0 Hz, Hb-10ʹ′), 1.73 (s, H3-11ʹ′), 1.72 (s, H3-12ʹ′),

1.27 (s, H3-1); 13C NMR (150 MHz, CD3CN) δC 204.9 (C-2ʹ′), 187.6 (C-6ʹ′), 172.8 (C-8ʹ′), 152.0 (C-8), 151.2

(C-6), 149.6 (C-4ʹ′), 134.6 (C-5ʹ′), 133.5 (C-4), 117.6 (C-7ʹ′), 115.1 (C-5), 114.7 (C-9), 110.5 (C-7), 103.4

(C-2), 44.4 (C-3ʹ′), 40.0 (C-9ʹ′), 39.9 (C-3), 34.7 (C-10), 30.1 (C-1ʹ′), 28.5 (C-10ʹ′), 26.0 (C-1), 12.9 (C-12),

12.5 (C-12ʹ′), 9.4 (C-11), 8.0 (C-11ʹ′).

Key HMBC (red arrow), 1H-1H COSY (blue bold line) and NOE (purple arrow) correlations of 11 in CD3OD

and CD3CN

HO

HO

O

O

O

OHH

11

OH

HO

O

O

O

OH1

23

45

6

7 8 9 1011

12

1' 2'

3'4'

5'

6'

7'8'9'

10'

1

12

11'

12'

HH

HHH H

10'

10

3'




14

Conversion of 2 to 5, 11 and 12

Compound 2 (60.0 mg) was treated with AcOH (3 mL) at reflux for 15 min. The resulting mixture was

extracted with EtOAc, washed with saturated aqueous solution of NaHCO3 and brine, dried over Na2SO4,

and concentrated. The residue was purified with column chromatography and preparative TLC to afford

12, along with 5 (>90%) and 11 (1.8 mg). Compound 12 was further purified by preparative HPLC

(monitored at 215 nm) on a Mightysil RP-18 column (φ 250 mm x 4.6 mm, 1.0 mL/min) with CH3CN and

water (0-5 min: 20:80, 5-20 min: from 20:80 to 100:0, 20-30 min: 0:100) to afford 12 (1.2 mg) in pure form.

Addition of 2-bromoethylamine hydrobromide improved the yield of 12.


(KBr) νmax (cm-1) 3419, 3210, 2924, 2853, 1685, 1653, 1594, 1558, 1541, 1507, 1457, 1376, 1354, 1214,

1173, 1107; HRESIMS: m/z 411.1806 [M–H]– (411.1802 calcd for C24H27O6); 1H NMR (600 MHz, CDCl3

(10% CD3OD)) δH 4.80 (d, 15.4 Hz, Ha-10), 4.65 (d, 15.4 Hz, Hb-10), 3.98 (s, H-3ʹ′), 2.87 (d, 11.7 Hz,

Ha-10ʹ′), 2.57 (d, 11.7 Hz, Hb-10ʹ′), 2.57 (s, H-3), 2.18 (s, H3-11ʹ′), 2.13 (s, H3-11), 2.10 (s, H3-12), 1.97 (s,

H3-12ʹ′), 1.87 (s, H3-1ʹ′), 1.46 (s, H3-1); 13C NMR (150 MHz, CDCl3 (10% CD3OD)) δC 211.3 (C-2ʹ′), 151.2

(C-6), 151.0 (C-6ʹ′), 149.3 (C-8ʹ′), 147.8 (C-8), 133.8 (C-4), 130.4 (C-4ʹ′), 116.1 (C-9ʹ′), 114.3 (C-5ʹ′), 113.3

(C-5), 112.1 (C-9), 109.7 (C-7ʹ′), 108.7 (C-7), 74.3 (C-2), 63.2 (C-3ʹ′), 60.2 (C-10), 39.8 (C-3), 29.5 (C-1ʹ′),

27.3 (C-10ʹ′), 23.0 (C-1), 11.2 (C-12ʹ′), 9.8 (C-12), 8.7 (C-11ʹ′), 8.2 (C-11).

Key HMBC (red arrow), 1H-1H COSY (blue bold line) and NOE (purple arrow) correlations of 12

HO

OH

O

HO OH

O

12

HO

OH

O

HO OH

H O

1098

711

65

42

1

3 1'2'3'

4'5'6'7'

11'

12'

8'9'

10'

H

HH

3'3

110

OHO

OH

O

O

OH

H

OH

HO

O

O

O

OHH

5 ( >90%) 11 (3%)

HO

OH

O

HO OH

H

O

12 (2%)

OH

HO

OAcOH,reflux

2




15

Conversion of 2 to 5, 19 and 38

The mixture of 2 (60.0 mg, 0.29 mmol) and NH4OAc (200 mg, 2.6 mmol) was treated with AcOH (3 mL) at

reflux for 15 min. The resulting mixture was extracted with EtOAc, washed with saturated aqueous

solution of NaHCO3 and brine, dried over Na2SO4, and concentrated. The residue was purified with

column chromatography and preparative TLC to afford 19 (4.8 mg), 38 (2.4 mg) and 5 (43.2 mg).


(KBr) νmax (cm-1) 3383, 1698, 1592, 1456, 1384, 1227, 1113, 1034; HRESIMS: m/z 412.2108 [M+H]+

(412.2118 calcd for C24H30O5N); 1H NMR (600 MHz, CD3OD) δH 4.28 (s, H-3ʹ′), 4.09 (d, 17.3 Hz, Ha-10),

4.00 (d, 17.3 Hz, Hb-10), 3.48 (t, 9.1 Hz, H-3), 3.38 (dd, 18.0, 9.1 Hz, Ha-10ʹ′), 2.34 (dd, 18.7, 9.1 Hz,

Hb-10ʹ′), 2.22 (s, H3-1ʹ′), 2.21(s, H3-12), 2.16 (s, H3-12ʹ′), 2.11 (s, H3-11ʹ′), 2.11 (s, H3-11), 1.31 (s, H3-1); 13C

NMR (150 MHz, CD3OD) δC 208.7 (C-2ʹ′), 154.2 (C-6), 153.6 (C-6ʹ′), 152.5 (C-8ʹ′), 151.1 (C-8), 135.7 (C-4),

127.6 (C-4ʹ′), 116.6 (C-5ʹ′), 115.7 (C-5), 115.1 (C-9), 113.9 (C-7ʹ′), 111.9 (C-7), 108.5 (C-9), 61.4 (C-3ʹ′), 55.5

(C-2), 39.9 (C-10), 34.7 (C-3), 31.5 (C-1ʹ′), 29.7 (C-10ʹ′), 21.0 (C-1), 12.1 (C-12ʹ′), 11.0 (C-12), 9.5 (C-11ʹ′),

9.2 (C-11).


max (cm-1) 3363, 1741, 1698, 1600, 1557, 1457, 1375, 1293, 1083, 1041; HRESIMS: m/z 234.0769 [M–H]–

(234.0761 calcd for C12H12O4N); 1H NMR (600 MHz, CDCl3) δH 9.12 (s, H-10), 7.59 (s, H-3), 2.70 (s, H3-1),

1.83 (s, H3-12), 1.72 (s, H3-11); 13C NMR (150 MHz, CDCl3) δC 208.4 (C-6), 194.8 (C-8), 166.1 (C-2), 151.9

(C-4), 149.4 (C-10), 120.6 (C-9), 120.3 (C-3), 83.3 (C-7), 74.9 (C-5), 30.2 (C-12), 26.7 (C-11), 25.1 (C-1).


O

O

NHO

HO

38

O

O

NHO

HO1

2

3

45

12

6711

89

10

11

12

HO

OH

NH

HO OH

O

19

HO

OH

NH

HO OH

H

O10

987

11

65

42

1

3 1'2'3'

4'5'6'7'

11'

12'

8'9'

10'

1'1

3

HH

H

HH10'

OHO

OH

O

O

OH

H

5 (72%)

OH

HO

O

NH4OAcAcOH, reflux

2

N

O

O

HO

HO

38 (7%)

HO

OH

NH

HO OH

H

O

19 (8%)




16

Conversion of 2 to 39-44

Compound 2 (100 mg, 0.49 mmol) was treated with silver oxide (Ag2O) (254 mg, 1.10 mmol) in CH2Cl2 (6.0

ml) at room temperature for 1 hour. The resulting solid was removed by filtration and the solution was

concentrated. The residue was purified with column chromatography, preparative TLC and preparative

HPLC to afford 39 (5.0 mg), 40 (7.1 mg), 41 (5.1 mg), 42 (2.6 mg), 43 (1.0 mg) and 44 2.1 mg). The

reaction was suppressed under argon atmosphere, indicating O2 involved in the generation of phenoxy

radical.


(KBr) νmax (cm–1) 2923, 2851, 1723, 1647, 1606, 1571, 1457, 1412, 1380, 1361, 1305, 1285, 1216, 1181,

1065; HRESIMS: m/z 431.1458 [M+Na]+ (431.1465 calcd for C24H24O6Na). 1H NMR (600 MHz, CDCl3) δH

5.79 (s, H-3, H-3ʹ′), 4.66 (d, 13.2 Hz, Ha-10, Ha-10ʹ′), 4.12 (d, 13.2 Hz, Hb-10, Hb-10ʹ′), 2.03 (s, H3-1, H3-1ʹ′),

1.87 (s, H3-12, H3-12ʹ′), 1.36 (s, H3-11, H3-11ʹ′); 13C NMR (150 MHz, CDCl3) δC 201.1 (C-8, C-8ʹ′), 192.6

(C-6, C-6ʹ′), 164.4 (C-2, C-2ʹ′), 143.1 (C-4, C-4ʹ′), 124.0 (C-5, C-5ʹ′), 99.6 (C-3, C-3ʹ′), 68.4 (C-7, C-7ʹ′), 65.9

(C-10, C-10ʹ′), 52.6 (C-9, C-9ʹ′), 21.1 (C-1, C-1ʹ′), 11.1 (C-12, C-12ʹ′), 9.9 (C-11, C-11ʹ′).


(KBr) νmax (cm–1) 2925, 1723, 1647, 1600, 1571, 1450, 1410, 1381, 1360, 1307, 1284, 1213, 1195, 1075;

HRESIMS: m/z 431.1454 [M+Na]+ (431.1465 calcd for C24H24O6Na). 1H NMR (600 MHz, CDCl3) δH 5.79

(s, H-3, H-3ʹ′), 4.66 (d, 13.2 Hz, Ha-10, Ha-10ʹ′), 4.12 (d, 13.2 Hz, Hb-10, Hb-10ʹ′), 2.03 (s, H3-1, H3-1ʹ′), 1.87

(s, H3-12, H3-12ʹ′), 1.36 (s, H3-11, H3-11ʹ′); 13C NMR (150 MHz, CDCl3) δC 201.1 (C-8, C-8ʹ′), 192.6 (C-6,

C-6ʹ′), 164.4 (C-2, C-2ʹ′), 143.1 (C-4, C-4ʹ′), 124.0 (C-5, C-5ʹ′), 99.6 (C-3, C-3ʹ′), 68.4 (C-7, C-7ʹ′), 65.9 (C-10,

C-10ʹ′), 52.6 (C-9, C-9ʹ′), 21.1 (C-1, C-1ʹ′), 11.1 (C-12, C-12ʹ′), 9.9 (C-11, C-11ʹ′).

39 (5%) 40 (7%) 41 (5%) 42 (3%)

43 (1%) 44 (2%)

O

O

O

OO

O

O

O

OO

O

O

O

O

OO

O

O

OO

O

O

O

O

O

O

O

OO

O

O

O

O

O

O

O

OH

HO

O

2

Ag2O,CH2Cl2,

r.t., 1 h




17

Key HMBC (Red arrow) and NOE (Purple arrow) correlations of 39 and 40

Compound 41: Yellow amorphous; UV (EtOH) λmax nm (log ε) 203 (4.19), 236 (3.93), 339 (4.39); IR

(KBr) νmax (cm–1) 2923, 2852, 1725, 1649, 1612, 1577, 1542, 1447, 1404, 1385, 1358, 1301, 1185, 1071,


5.82 (s, H-3), 5.49 (s, H-3ʹ′), 4.96 (d, 12.6 Hz, Ha-10ʹ′), 4.85 (d, 12.6 Hz, Hb-10ʹ′), 4.71 (d, 13.2 Hz, Ha-10),

4.20 (d, 13.2 Hz, Hb-10), 2.05 (s, H3-1), 2.03 (s, H3-1ʹ′), 1.84 (s, H3-12), 1.44 (s, H3-12ʹ′), 1.23 (s, H3-12ʹ′),

1.21 (s, H3-12); 13C NMR (150 MHz, CDCl3) δC 202.2 (C-8), 202.0 (C-6ʹ′), 191.4 (C-6), 189.0 (C-8ʹ′), 167.4

(C-2ʹ′), 163.0 (C-2), 150.0 (C-4ʹ′), 142.4 (C-4), 124.9 (C-5), 115.8 (C-9ʹ′), 100.1 (C-3), 98.8 (C-3ʹ′), 68.4 (C-7),

68.3 (C-7ʹ′), 67.2 (C-10), 64.6 (C-10ʹ′), 57.9 (C-5ʹ′), 53.7 (C-9), 21.0 (C-1), 20.5 (C-1ʹ′), 12.2 (C-12ʹ′), 11.2

(C-12), 10.4 (C-11ʹ′), 10.4 (C-11).

Compound 42: Yellow amorphous; UV (EtOH) λmax nm (log ε) 202 (4.13), 220 (4.10), 273 (3.73), 344

(4.38); IR (KBr) νmax (cm–1) 2992, 2947, 2864, 1726, 1644, 1609, 1574, 1539, 1446 1407, 1382, 1360, 1303,

1183, 1079, 1052; HRESIMS: m/z 431.1455 [M+Na]+ (431.1465 calcd for C24H24O6Na). 1H NMR (600 MHz,

CDCl3) δH 5.78 (s, H-3), 5.31 (s, H-3ʹ′), 4.98 (d, 12.6 Hz, Ha-10ʹ′), 4.83 (d, 12.6 Hz, Hb-10ʹ′), 4.61 (d, 13.2 Hz,

Ha-10), 4.13 (d, 13.2 Hz, Hb-10), 2.02 (s, H3-1), 1.99 (s, H3-1ʹ′), 1.84 (s, H3-12), 1.38 (s, H3-11ʹ′), 1.32 (s,

H3-12), 1.30 (s, H3-12ʹ′); 13C NMR (150 MHz, CDCl3) δC 202.1 (C-8), 202.1 (C-6ʹ′), 192.2 (C-6), 190.0 (C-8ʹ′),

167.4 (C-2ʹ′), 163.9 (C-2), 151.7 (C-4ʹ′), 142.5 (C-4), 124.1 (C-5), 115.0 (C-9ʹ′), 99.7 (C-3ʹ′), 98.5 (C-3), 69.2

(C-7), 68.3 (C-7ʹ′), 66.0 (C-10), 64.5 (C-10ʹ′), 57.5 (C-5ʹ′), 52.6 (C-9), 21.1 (C-1), 20.5 (C-1ʹ′), 13.1 (C-12ʹ′),

11.4 (C-11), 11.1 (C-12), 9.9 (C-11ʹ′).


39 40

OOOO

O

O

O

O

O

O

O

O

12

3

45 6

78910 11

12

1'2'3'

4'5'6'

7' 8' 9' 10'11'

12'

O

O

O

OO

O

O

O

O

O

O

O

3

11'

H

H

H

10

12

3

45 6

78910 11

12

1' 2' 3'4' 5'

6'7'8'9'

10'11'

12'

H

H

H

3

10'

HOO

OO

O

OO

O

O

O

O

O

12

3

45 6

78910 11

12

7'8'9'

11'

3'

1' 2'

4'5'

12' 6'

10'

O

O

OO

O

O

OO

O

O

O

OH

H

H

10

3

12'

3'

12

3

45 6

78910 11

12

7'8' 9'

10'

11'

3'

1'2'

4'

5' 12'6' HH

H

H

3'

11

3

11'

10

41 42




18

Compound 43: Yellow amorphous; UV (EtOH) λmax nm (log ε) 205 (4.21), 268 (3.66), 366 (4.24); IR

(KBr) νmax (cm–1) 2981, 2921, 2850, 1734, 1636, 1542, 1525, 1445, 1382, 1297, 1242, 1096, 1048, 1006;

HRESIMS: m/z 431.1456 [M+Na]+ (431.1465 calcd for C24H24O6Na). 1H NMR (600 MHz, CDCl3) δH 5.29

(s, H-3, H-3ʹ′), 4.97 (d, 12.8 Hz, Ha-10, Ha-10ʹ′), 4.78 (d, 12.8 Hz, Hb-10, Hb-10ʹ′), 1.98 (s, H3-1, H3-1ʹ′), 1.34

(s, H3-11, H3-11ʹ′), 1.29 (s, H3-12, H3-12ʹ′); 13C NMR (150 MHz, CDCl3) δC 203.0 (C-6, C-6ʹ′), 189.8 (C-8,

C-8ʹ′), 167.1 (C-2, C-2ʹ′), 151.3 (C-4, C-4ʹ′), 115.0 (C-9, C-9ʹ′), 99.7 (C-3, C-3ʹ′), 69.0 (C-7, C-7ʹ′), 64.5 (C-10,

C-10ʹ′), 57.6 (C-5, C-5ʹ′), 20.5 (C-1, C-1ʹ′), 13.1 (C-12, C-12ʹ′), 11.4 (C-11, C-11ʹ′).

Compound 44: Yellow amorphous; UV (EtOH) λmax nm (log ε) 203 (4.16), 218 (4.08), 275 (3.60), 365

(4.10); IR (KBr) νmax (cm–1) 2990, 2923, 2853, 1726, 1648, 1544, 1460, 1384, 1296, 1244, 1200, 1097, 1052,


5.28 (s, H-3, H-3ʹ′), 4.96 (d, 12.8 Hz, Ha-10, Ha-10ʹ′), 4.78 (d, 12.8 Hz, Hb-10, Hb-10ʹ′), 1.99 (s, H3-1, H3-1ʹ′),

1.41 (s, H3-12, H3-12ʹ′), 1.21 (s, H3-11, H3-11ʹ′); 13C NMR (150 MHz, CDCl3) δC 202.9 (C-6, C-6ʹ′), 188.8

(C-8, C-8ʹ′), 166.7 (C-2, C-2ʹ′), 151.0 (C-4, C-4ʹ′), 115.3 (C-9, C-9ʹ′), 99.3 (C-3, C-3ʹ′), 68.2 (C-7, C-7ʹ′), 64.5

(C-10, C-10ʹ′), 58.5 (C-5, C-5ʹ′), 20.5 (C-1, C-1ʹ′), 13.8 (C-12, C-12ʹ′), 10.5 (C-11, C-11ʹ′).


O

O

OO

O

O

O

OO

O

O

O

1'2'3'

4'5'6'7'

8' 9'

10'

11' 12'

1112

1 2

345 6 7

89

10

1112

1 2

345 6 7

89

10

12' 11'6'

1'2'3'

4'5' 7'

8'9'

10'

O

O

O

OO

O

O

O

OO

O

O

HH

11'12'

10

43 44

HH

H

10

11'

3

12'




19


A solution of 2 (20.0 mg, 0.10 mmol) and cesium carbonate (81.5 mg, 0.25 mmol) in CH3CN (2.0 ml) was

stirred at room temperature for 30 min. The resulting mixture was extracted with EtOAc, washed with

saturated aqueous solution of brine, dried over Na2SO4, and concentrated. The residue was purified with

column chromatography to afford a mixture of 34 and 35 (5.8 mg). The structure elucidation was carried

out as a mixture because it was difficult to separate each other. The ratio was calculated by the signal

ration in the 1H NMR spectrum. This reaction was suppressed under argon atmosphere suggesting that

oxygen atoms came from air.

Mixture of 34 and 35: Colorless amorphous; HRESIMS: m/z 261.0720 [M+Na]+ (261.0733 calcd for

C12H14O5Na); 1H NMR (600 MHz, CDCl3) for 34, δH 5.82 (s, H-3), 4.21 (d, 11.4 Hz, Ha-10), 3.97 (d, 11.4

Hz, Hb-10), 2.22 (s, H3-11), 2.09 (s, H3-1), 1.81 (s, H3-12); 13C NMR (150 MHz, CDCl3) for 34. δC 204.7

(C-8), 199.2 (C-6), 164.9 (C-2), 159.4 (C-4), 125.2 (C-5), 96.4 (C-3), 87.7 (C-7), 74.1 (C-9), 70.0 (C-10),

27.1 (C-11), 21.2 (C-1), 7.9 (C-12); 1H NMR (600 MHz, CDCl3) for 35, δH 5.68 (s, H-3), 5.00 (d, 12.8 Hz,

Ha-10), 4.93 (d, 12.8 Hz, Hb-10), 2.02 (s, H3-1), 1.65 (s, H3-11), 1.60 (s, H3-12); 13C NMR (150 MHz,

CDCl3) for 35, δC 208.4 (C-6), 191.7 (C-8), 168.1 (C-2), 153.3 (C-4), 110.9 (C-9), 97.5 (C-3), 87.7 (C-7),

81.7 (C-5), 64.4 (C-10), 27.4 (C-12), 27.3 (C-11), 20.4 (C-1).

Key HMBC (red arrow) and NOE (purple arrow) correlations of 34 and 35.

O

O

O

O

O

OHO OH HO

HO

3534

O

O

O

O

O

OHO OH HO

HO1

2

3

456

711

12

8 9

10

12

3

45

12

6711

89

10

11

10

11

12

O

O

OHO OH

O

O

OHO

HO

OH

HO

O

Cs2CO3,CH3CN, r.t.

2 34 (20%) 35 (5%)




20


A solution of 2 (20.0 mg, 0.10 mmol), 45 (24.1 mg, 0.10 mmol) and cesium carbonate (81.5 mg, 0.20 mmol)

in CH3CN (3.0 ml) was stirred at room temperature for 1 hour. The resulting mixture was extracted with

EtOAc, washed with saturated aqueous solution of brine, dried over Na2SO4, and concentrated. The

residue was purified with column chromatography and preparative TLC to afford 36 (2.5 mg) and 37

(2.5 mg), as well as a mixture of 34 and 35 (5.6 mg).

Hypothetical reaction mechanism to 37


(KBr) νmax (cm-1) 3364, 2979, 2930, 1715, 1604, 1520, 1478, 1449, 1391, 1369, 1294, 1243, 1160, 1050;

HRESIMS: m/z 426.1919 [M–H]‒ (426.1911 calcd for C24H28O6N); 1H NMR (600 MHz, CDCl3) δH 7.57

(brd, 8.0 Hz, H-6ʹ′), 7.40 (s, NH), 7.09 (brt, 8.0 Hz, H-5ʹ′), 6.83 (brt, 8.0 Hz, H-4ʹ′), 6.80 (brd, 8.0 Hz, H-3ʹ′),

5.51 (brs, H-3), 4.56 (d, 12.5 Hz, Ha-10), 3.66 (d, 12.5 Hz, Hb-10), 3.31 (d, 13.0 Hz, Ha-1ʹ′), 3.06 (d, 14.0 Hz,

Hb-1ʹ′), 2.52 (s, 15-OH), 1.99 (s, H3-1), 1.72 (s, H3-12), 1.68 (s, H3-11), 1.55 (s, 3 x H3-t-Bu); 13C NMR (150

MHz, CDCl3) δC 207.4 (C-8), 200.0 (C-6), 162.9 (C-2), 153.9 (NCO, Boc), 141.4 (C-4), 136.1 (C-7ʹ′), 130.4

(C-3ʹ′), 128.2 (C-2ʹ′), 127.4 (C-6ʹ′), 125.3 (C-5), 123.8 (C-6ʹ′), 123.6 (C-4ʹ′), 97.6 (C-3), 80.0 (O-C-Me3, Boc),

70.3 (C-10), 66.9 (C-9), 59.7 (C-7), 36.8 (C-1ʹ′), 28.4 (3 x Me-Boc), 27.2 (C-11), 20.8 (C-1), 10.9 (C-12).


(KBr) νmax (cm-1) 3297, 2921, 1752, 1738, 1656, 1615, 1498, 1459, 1444, 1402, 1377, 1206, 1169,

1063 ;HRESIMS: m/z 352.1186 [M–H]– (352.1179 calcd for C20H18O5N); 1H NMR (600 MHz, CDCl3) δH

8.15 (brd, 7.7 Hz H-6ʹ′), 7.23 (brd, 7.7 Hz H-3ʹ′), 7.09 (brt, 7.7 Hz, H-5ʹ′), 7.06 (brt, 7.7 Hz, H-4ʹ′), 5.61 (brs,

H-3), 4.96 (d, 12.8 Hz, Ha-10), 4.27 (d, 12.8 Hz, Hb-10), 3.55 (d, 16.5 Hz, Ha-1ʹ′), 2.91 (d, 16.5 Hz, Hb-1ʹ′),

2.07 (s, H3-1), 1.78 (s, H3-12), 1.26 (s, H3-11); 13C NMR (150 MHz, CDCl3) δC 196.7 (C-6), 160.6 (C-2),

152.1 (C-8ʹ′), 137.6 (C-4), 131.5 (C-7ʹ′), 128.8 (C-3ʹ′), 127.1 (C-5ʹ′), 124.4 (C-4ʹ′), 123.6 (C-2ʹ′), 121.5 (C-5),

117.6 (C-6ʹ′), 97.1 (C-3), 86.7 (C-8), 76.0 (C-9), 68.7 (C-10), 47.2 (C-7), 31.0 (C-1ʹ′), 21.5 (C-11), 20.7 (C-1),

11.1 (C-12).

O

HO

OH

HN BocCl

O

HO

ONHBoc

O2

O

O

ONHBoc

O

O

N O

O

HO

37

OHO

O

N OHHO

OO

2

45

O

O

OHO

HOO

O

OHO OHOH

HO

O

Cs2CO3,CH3CN, r.t.

2 34 (16%) 35 (8%)

O

O

O

O

O

N O

O

HO

NHBoc

OH

36 (6%) 37 (7%)

HN Boc

Cl

45




21


Conversion of 5 to 13, 14, 15 and 16

Compound 5 (30 mg, 0.07 mmol) was treated with cesium carbonate (115 mg, 0.35 mmol) in CH3CN (3.0

ml) at 70 oC for 3 min, then cooled to room temperature and stirred for 30 min. The resulting mixture was

extracted with EtOAc, washed with saturated aqueous solution of brine, dried over Na2SO4, and

concentrated. The residue was purified with column chromatography and preparative TLC to afford 13

(1.5 mg), 14 (0.3 mg), 15 (3.6 mg) and 16 (1.0 mg).

Hypothetical mechanism of ring contraction in the pathway to 13 and 14

OHO

OH

O

O

OH

H

OO

O

O

O

OH

H

O

HO H

13

OHO

O

O

O

OH

H

O OH

OO

O

O

O

OH

H

OHHO

OO

OO

O

OH

H

CO

OH

O

O

O

OH

H

O

HOHO O

O

O

O

OH

H

O

HO H

14

5

O

O

O

O

O

N O

O

HO

NHBoc

OH

36 37

O

O

N O

O

HO

12

3

456

711

12

89

1'2'

3'4'

5'

6' 7'

10

12

3

456

7

12

89

11

101'

2'3'

4'

5'6'

7'8'

H

H

OHO

OH

O

O

OH

H

5

OO

O

O

O

OH

H

HO

HO

O

O

O

OH

H

O

HO H

13 ( 5%) 15 (11%)

OO

O

O

O

OH

H

HO

HO

16 (3%)

O

O

O

OH

H

O

HO H

14 (1%)

Cs2CO3,CH3CN,

70 oC to r.t.




22


(KBr) νmax (cm-1) 3445, 2925, 1706, 1651, 1593, 1457, 1166, 1107, 1062, 1030; HRESIMS: m/z

399.1803 [M–H]– (399.1802 calcd for C23H27O6); 1H NMR (600 MHz, CDCl3) δH 4.72 (s, OH), 4.29 (dd,

11.7, 8.0 Hz, Ha-9), 3.77 (t, 11.7 Hz, Hb-9), 3.75 (d, 17.6 Hz, Ha-3ʹ′), 3.69 (d, 17.6 Hz, Hb-3ʹ′), 3.18 (m, H-8),

3.14 (dd, 11.4, 6.2 Hz, H-3), 2.83 (dd, 15.4, 11.4 Hz, Ha-10ʹ′), 2.52 (dd, 15.4, 6.2 Hz, Hb-10ʹ′), 2.18 (s, H3-1ʹ′),

2.16 (s, H3-11ʹ′), 2.10 (s, H3-12ʹ′), 1.78 (s, H3-11), 1.54 (s, H3-1); 13C NMR (150 MHz, CDCl3) δC 209.3 (C-6),

205.8 (C-2ʹ′), 169.3 (C-4), 151.5 (C-6ʹ′), 147.8 (C-8ʹ′), 132.9 (C-5), 129.0 (C-4ʹ′), 115.9 (C-5ʹ′), 111.1 (C-9ʹ′),

110.2 (C-7ʹ′), 98.3 (C-2), 75.0 (C-7), 63.7 (C-9), 45.0 (C-8), 44.4 (C-3ʹ′), 40.1 (C-3), 29.5 (C-1ʹ′), 24.4 (C-10ʹ′),

23.8 (C-1), 21.4 (C-10), 12.1 (C-12ʹ′), 8.4 (C-11ʹ′), 7.6 (C-11).


(KBr) νmax (cm-1) 3428, 2925, 2854, 1707, 1633, 1593, 1456, 1385, 1359, 1302, 1223, 1160, 1108, 1039,

1009; HRESIMS: m/z 399.1819 [M–H]‒ (399.1802 calcd for C23H27O6); 1H NMR (600 MHz, CDCl3) δH

4.65 (s, OH), 4.11 (dd, 10.6, 6.2 Hz, Ha-9), 3.94 (t, 10.6 Hz, Hb-9), 3.89 (d, 17.2 Hz, Ha-3ʹ′), 3.66 (d, 17.2

Hz, Hb-3ʹ′), 3.35 (d, 16.9 Hz, Ha-10), 3.05 (brd, 5.2 Hz, H-3), 2.93 (dd, 10.6, 6.2 Hz, H-8), 2.92 (dd, 16.9,

5.2 Hz, Hb-10ʹ′), 2.18 (s, H3-1ʹ′), 2.13 (s, H3-11ʹ′), 2.07 (s, H3-12ʹ′), 1.50 (s, H3-11), 1.50 (s, H3-1), 1.12 (s,

H3-10).



(KBr) νmax (cm-1) 3444, 2992, 2925, 2854, 1736, 1682, 1594, 1459, 1378, 1343, 1221, 1182, 1131, 1107,

1078, 1030; HRESIMS: m/z 467.1658 [M+Na]+ (467.1676 calcd for C24H28O8Na); 1H NMR (600 MHz,

CDCl3 (10% CD3OD)) δH 4.78 (d, 17.9 Hz, Ha-10), 4.53 (d, 17.9 Hz, Hb-10), 3.69 (d, 16.9 Hz, Ha-3ʹ′), 3.64

(d, 16.9 Hz, Hb-3ʹ′), 3.07 (dd, 16.1, 5.9 Hz, Ha-10ʹ′), 2.42 (dd, 11.0, 5.9 Hz, H-3), 2.34 (dd, 16.1, 11.0 Hz,

Hb-10ʹ′), 2.16 (s, H3-11ʹ′), 2.12 (s, H3-12ʹ′), 2.10 (s, H3-1ʹ′), 1.67 (s, H3-12), 1.63 (s, H3-11), 1.60 (s, H3-1); 13C

NMR (150 MHz, CDCl3 (10% CD3OD)) δC 208.1 (C-6), 208.1 (C-2ʹ′), 194.9 (C-8), 158.4 (C-4), 151.8 (C-6ʹ′),

147.7 (C-8ʹ′), 128.7 (C-4ʹ′), 126.9 (C-9), 116.7 (C-5ʹ′), 111.4 (C-9ʹ′), 110.9 (C-7ʹ′), 96.9 (C-2), 80.9 (C-7), 75.8

(C-5), 62.1 (C-10), 44.7 (C-3ʹ′), 36.7 (C-3), 29.1 (C-1ʹ′), 28.8 (C-10ʹ′), 26.6 (C-12), 26.6 (C-11), 22.8 (C-1),

12.3 (C-12ʹ′), 8.4 (C-11ʹ′).

O

O

O

OH

H

O

HO

HO

O

O

OH

HO

HO H

14

O

O

O

OH

O

HO

13

1

9

8

234

5

67

11

10

1'2'

3'

4'

5'6'

7'8'9'

10' 11'

12'

H

H

H

H

97

10'

3

H H




23


(KBr) νmax (cm-1) 3433, 3010, 2925, 2854, 1736, 1685, 1594, 1461, 1383, 1347, 1219, 1186, 1132, 1108,

1054, 1032; HRESIMS: m/z 467.1659 [M+Na]+ (467.1676 calcd for C24H28O8Na); 1H NMR (600 MHz,

CDCl3) δH 4.68 (d, 18.0 Hz, Ha-10), 4.64 (d, 18.0 Hz, Hb-10), 3.73 (d, 16.5 Hz, Ha-3ʹ′), 3.66 (d, 16.5 Hz,

Hb-3ʹ′), 3.07 (dd, 9.9, 5.9 Hz, H-3), 3.01 (dd, 15.4, 5.9 Hz, Ha-10ʹ′), 2.70 (dd, 15.4, 9.9 Hz, Hb-10ʹ′), 2.15 (s,

H3-12ʹ′), 2.14 (s, H3-11ʹ′), 2.13 (s, H3-1ʹ′), 1.71 (s, H3-12), 1.70 (s, H3-11), 1.47 (s, H3-1); 13C NMR (150 MHz,

CDCl3) δC 207.7 (C-6), 206.2 (C-2ʹ′), 194.3 (C-8), 157.2 (C-4), 151.7 (C-6ʹ′), 148.0 (C-8ʹ′), 128.8 (C-4ʹ′),

127.5 (C-9), 115.8 (C-5ʹ′), 110.5 (C-9ʹ′), 110.1 (C-7ʹ′), 97.1 (C-2), 82.4 (C-7), 75.0 (C-5), 61.8 (C-10), 44.8

(C-3ʹ′), 35.7 (C-3), 29.3 (C-1ʹ′), 28.5 (C-10ʹ′), 27.3 (C-12), 27.2 (C-11), 22.6 (C-1), 12.2 (C-12ʹ′), 8.3 (C-11ʹ′).



Compound 5 (20 mg, 0.05 mmol) was treated with cesium carbonate (81.5 mg, 0.25 mmol) and 45 (36.0 mg,

31.3 mg, 0.13 mmol) in CH3CN (2.0 ml) at 70 oC for 3 min, then the reaction mixture was cooled to room

temperature and stirred for 1 hour. The resulting mixture was extracted with EtOAc, washed with saturated

aqueous solution of brine, dried over Na2SO4, and concentrated. The residue was purified with column

chromatography, preparative TLC and preparative HPLC to afford 17 (1.4 mg) and 18 (0.3 mg).

OO

O

O

O

OH

H

HO

HO

15

OO

O

O

O

OH

H

HO

HO

16

OO

O

O

O

OH

HO

HO1

1'

2'

3'4' 5'

6'

7'8'9'

10' 11'

12'

2345

6

78

910

12

11

15 and 16

1 1

3 310'

55

7 7

OHO

OH

O

O

OH

H

5

Cs2CO3,CH3CN,70 oC to r.t.

HN Boc

Cl

O

O

O

OH

H

ON

O

HO

O

17 (5%)

O

O

O

OH

H

ON

O

HO

O

18 (1%)45




24

Hypothetical reaction pathway to 17 and 18


(KBr) νmax (cm-1) 3396, 2925, 2854, 1760, 1740, 1717, 1681, 1588, 1497, 1460, 1421, 1386, 1360, 1181,

1163, 1095; HRESIMS: m/z 558.2127 [M–H]– (558.2122 calcd for C32H32O8N); 1H NMR (600 MHz, CDCl3

(10% CD3OD)) δH 8.38 (brd, 8.0 Hz, H-6ʹ′ʹ′), 7.22 (brd, 8.0 Hz, H-3ʹ′ʹ′), 7.20 (brt, 8.0 Hz, H-5ʹ′ʹ′), 7.05 (brt, 8.0

Hz, H-4ʹ′ʹ′), 4.67 (d, 18.3 Hz, Ha-10), 4.39 (d, 18.3 Hz, Hb-10), 3.76 (s, H2-3ʹ′), 3.42 (d, 16.1 Hz, Ha-1ʹ′ʹ′), 3.11

(dd, 15.8, 5.9 Hz, Ha-10ʹ′), 3.04 (d, 16.1 Hz, Hb-1ʹ′ʹ′), 2.74 (dd, 11.0, 5.9 Hz, H-3), 2.34 (s, H3-1ʹ′), 2.29 (dd,

15.8, 11.0 Hz, Hb-10ʹ′), 2.14 (s, H3-11ʹ′), 2.06 (s, H3-12ʹ′), 1.80 (s, H3-12), 1.57 (s, H3-1), 1.34 (s, H3-11); 13C

NMR (150 MHz, CDCl3) δC 207.3 (C-2ʹ′), 195.7 (C-8), 151.8 (C-8ʹ′ʹ′), 151.3 (C-6ʹ′), 150.5 (C-4), 147.2 (C-8ʹ′),

132.7 (C-7ʹ′ʹ′), 129.0 (C-3ʹ′ʹ′), 128.8 (C-4ʹ′), 127.6 (C-9), 127.2 (C-5ʹ′ʹ′), 123.7 (C-4ʹ′ʹ′), 123.3 (C-2ʹ′ʹ′), 116.7

(C-5ʹ′), 116.5 (C-6ʹ′ʹ′), 110.9 (C-9ʹ′), 110.5 (C-7ʹ′), 96.3 (C-2), 88.4 (C-6), 82.4 (C-5), 62.0 (C-10), 47.2 (C-7),

44.0 (C-3ʹ′), 36.6 (C-3), 31.2 (C-1ʹ′ʹ′), 29.6 (C-1ʹ′), 29.4 (C-10ʹ′), 22.5 (C-1), 21.4 (C-11), 17.5 (C-12), 11.9

(C-12ʹ′), 8.3 (C-11ʹ′).


(KBr) νmax (cm-1) 3396, 2925, 2854, 1734, 1681, 1589, 1497, 1459, 1422, 1385, 1362, 1181, 1164, 1096;

HRESIMS: m/z 558.2134 [M–H]– (558.2122 calcd for C32H32O8N); 1H NMR (600 MHz, CDCl3 (10%

CD3OD)) δH 8.36 (brd, 8.0 Hz, H-6ʹ′ʹ′), 7.22 (brd, 8.0 Hz, H-3ʹ′ʹ′), 7.19 (brt, 8.0 Hz, H-5ʹ′ʹ′), 7.03 (brt, 8.0 Hz,

H-4ʹ′ʹ′), 4.55 (d, 18.3 Hz, Ha-10), 4.51 (d, 18.3 Hz, Hb-10), 3.73 (d, 16.1 Hz, Ha-3ʹ′), 3.66 (d, 16.1 Hz, Hb-3ʹ′),

3.45 (d, 16.1 Hz, Ha-1ʹ′ʹ′), 3.01 (d, 16.1 Hz, Hb-1ʹ′ʹ′), 2.95 (dd, 15.4, 5.5 Hz, Ha-10ʹ′), 2.77 (dd, 11.8, 5.5 Hz,

H-3), 2.47 (dd, 15.4, 11.8 Hz, Hb-10ʹ′), 2.16 (s, H3-1ʹ′), 2.15 (s, H3-11ʹ′), 2.14 (s, H3-12ʹ′), 1.84 (s, H3-12), 1.41

(s, H3-1), 1.39 (s, H3-11).

OHO

OH

O

O

OH

H

HN BocCl

OHO

O

O

O

OH

HNH

BocO

O

O

OH

H

OHN

O

HO

O

O2

OO

O

O

O

OH

HNH

BocHO

O

O

O

OH

H

ON

O

HO

O

O

17

O

O

O

OH

H

ON

O

HO

O

18

5

45




25


Synthesis of 20, 21 and 22

a) To a solution of compound 2 (20.0 mg, 0.10 mmol) and indole (23.5 mg, 0.20 mmol) in acetonitrile (1

mL), catalytic amount of BF3OEt2 (1.2 µL, 0.01 mmol) was added, and the reaction mixture was stirred at

room temperature for 30 minutes. The resulting mixture was extracted with EtOAc, washed with water,

dried over Na2SO4, and concentrated. The residue was purified with column chromatography yielding 21

(17.2 mg). b) To a solution of compound 2 (10.0 mg, 0.05 mmol) and indole (11.7 mg, 0.10 mmol) in

CH2Cl2 (0.5 mL), catalytic amount of BF3OEt2 (0.6 µL, 0.005 mmol) was added, and the reaction mixture

was stirred at room temperature for 3 minutes, yielding 20 (3.3 mg) and 21 (2.7 mg). c) Compound 21 (10.0

mg, 0.02 mmol) was dissolved in acetonitrile (0.3 mL) under argon and excess amount of BF3OEt2 (12.5 µL,

0.10 mmol) was added and the solution was stirred at room temperature for 2 hours. The solution turned to

red soon after the addition of excess amount of BF3OEt2. The resulting mixture was extracted with

EtOAc, washed with water, dried over Na2SO4, and concentrated. The residue was purified with column

chromatography and preparative TLC yielding 22 (2.6 mg) and 23 (3.4 mg). Treatment of 2 and indole

with the excess amount of BF3OEt2 also afforded to 22 and 23 in the similar yield.

O

O

O

OH

H

ON

O

HO

O

17

O

O

O

OH

H

ON

O

HO

O

18

O

O

O

OH

ON

O

HO

O

17

1

2345

67

8 910

12

11

1'2'

3'4' 5'

6'

7'8'9'

10' 11'

12'

1''2''

3''4''

5''6''

7''

8''

H H

NH

O

OH

HO

N

HO

OH

22 (c : 34%)

a) BF3OEt2 (10 mol%), r.t., CH3CN, 30 minb) BF3OEt2 (10 mol%), r.t., CH2Cl2, 3 min

2

OHN

HO

OH

23 (c : 48%)

c) BF3OEt2 (10 eq), r.t., CH3CN, 2 h.

NH

O

OH

OH

20 (a : 0%, b : 21%)

NH

HO

OH

NH

21 (a : 42%, b : 13%)

+




26

Compound 20: Yellow oil; UV (EtOH) λmax nm (log ε) 207 (4.47), 222 (4.38), 283 (3.73); IR (KBr) ν

max (cm-1) 3403, 3008, 2927, 1703, 1614, 1590, 1458, 1357, 1220, 1164, 1108; HREIMS: m/z 323.1543

[M]+ (323.1522 calcd for C20H21O3N); 1H NMR (600 MHz, CDCl3) δH 7.98 (s, NH), 7.62 (d, 8.0 Hz, H-4ʹ′),

7.35 (d, 8.0 Hz, H-7ʹ′), 7.22 (t, 8.0 Hz, H-6ʹ′), 7.14 (t, 8.0 Hz, H-5ʹ′), 6.65 (H-2ʹ′), 4.90 (s, OH), 4.71 (s, OH),

3.99 (s, H2-10), 3.82 (s, H2-3), 2.14 (s, H3-11), 2.10 (s, H3-12), 2.06 (s, H3-1); 13C NMR (150 MHz, CDCl3)

δC 206.6 (C-2), 151.2 (C-6), 151.0 (C-8), 136.7 (C-7ʹ′a), 130.6 (C-4), 127.0 (C-4ʹ′a), 122.5 (C-2ʹ′), 122.5

(C-6ʹ′), 119.6 (C-5ʹ′), 118.7 (C-4ʹ′), 117.4 (C-9), 115.0 (C-5), 113.4 (C-3ʹ′), 111.3 (C-7ʹ′), 109.6 (C-7), 45.2

(C-3), 29.5 (C-1), 23.1 (C-10), 12.3 (C-12), 8.7 (C-11).


max (cm-1) 3403, 3052, 3005, 2969, 2927, 1614, 1587, 1460, 1335, 1297, 1231, 1190, 1100, 1012; HREIMS:

m/z 422.1988 [M]+ (422.1994 calcd for C28H26O2); 1H NMR (600 MHz, acetone-d6) δH 10.10 (s, NH), 9.30 (s,

NH), 7.68 (brd, 8.0 Hz, H-4ʹ′), 7.33 (brd, 8.0 Hz, H-7ʹ′ʹ′), 7.29 (s, H-2ʹ′ʹ′), 7.08 (m, H-4ʹ′ʹ′), 7.03 (brd, 8.0 Hz,

H-7ʹ′), 6.95 (brt, 8.0 Hz, H-6ʹ′ʹ′), 6.93 (brt, 8.0 Hz, H-5ʹ′), 6.89 (brt, 8.0 Hz, H-6ʹ′), 6.66 (brt, 8.0 Hz, H-5ʹ′ʹ′),

4.67 (d, 15.4 Hz, Ha-10), 4.12 (d, 15.4 Hz, Hb-10), 3.93 (d, 14.3 Hz, Ha-3), 3.21 (d, 14.3 Hz, Hb-3), 2.17 (s,

H3-11), 2.08 (s, H3-12), 1.74 (s, H3-1); 13C NMR (150 MHz, acetone-d6) δC 151.9 (C-8), 149.7 (C-6), 141.9

(C-2ʹ′), 138.2 (C-7ʹ′ʹ′a), 136.3 (C-7ʹ′a), 136.1 (C-4), 129.0 (C-4ʹ′a), 127.3 (C-4ʹ′ʹ′a), 124.8 (C-3ʹ′ʹ′), 123.4 (C-9),

122.1 (C-2ʹ′ʹ′), 121.8 (C-5ʹ′), 121.2 (C-6ʹ′), 121.0 (C-4ʹ′ʹ′), 119.2 (C-5ʹ′ʹ′), 118.8 (C-6ʹ′ʹ′), 118.7 (C-4ʹ′), 117.0 (C-5),

112.0 (C-7ʹ′ʹ′), 111.1 (C-7ʹ′), 110.6 (C-3ʹ′), 109.7 (C-7), 42.1 (C-3), 40.7 (C-2), 26.7 (C-1), 21.1 (C-10), 13.0

(C-12), 9.9 (C-11).

Key HMBC (red arrow) and 1H-1H COSY (blue bold line) correlations of 20 and 21

Compound 22: Colorless oil; UV (EtOH) λmax nm (log ε) 208 (4.54), 229 (4.55), 285 (3.89); IR (KBr)

νmax (cm-1) 3397, 3056, 3004, 2965, 2927, 2881, 1673, 1617, 1584, 1460, 1379, 1326, 1253, 1232, 1216,

1186; HRESIMS: m/z 322.1444 [M–H]‒ (322.1438 calcd for C20H20O3N), HREIMS: m/z 323.1507 [M]+

(323.1522 calcd for C20H21O3N); 1H NMR (600 MHz, CD3OD) δH 7.56 (brd, 8.0 Hz, H-4ʹ′), 7.17 (brd, 8.0 Hz

H-7ʹ′), 6.96 (brt, 8.0 Hz, H-6ʹ′), 6.94 (brt, 8.0 Hz, H-5ʹ′), 4.17 (d, 15.9 Hz, Ha-10), 4.05 (d, 15.9 Hz, Hb-10),

3.20 (m, Ha-3), 3.12 (m, H-2), 3.09 (m, Hb-3), 2.22 (s, H3-12), 2.10 (s, H3-11), 1.34 (d, 6.2 Hz, H3-1); 13C

NMR (150 MHz, CD3OD) δC 151.8 (C-6), 149.6 (C-8), 139.9 (C-2ʹ′), 137.1 (C-4), 136.5 (C-7ʹ′a), 129.5

(C-4ʹ′a), 125.0 (C-9), 121.2 (C-6ʹ′), 119.0 (C-5ʹ′), 118.4 (C-4ʹ′), 117.3 (C-5), 112.3 (C-7), 111.1 (C-7ʹ′), 110.4

(C-3ʹ′), 35.4 (C-3), 33.6 (C-2), 21.7 (C-1), 20.8 (C-10), 12.6 (C-12), 9.9 (C-11).

NH

O

OH

OH

20

NH

HO

OH

NH

21

1

2

34 5

6

78910 11

12

2'

3'4'

5'

6'

7'

4'a

7'a

2'

3'

4'5'

6'7'

4'a

7'a

11

7

6

5

12

4

9810

1

2 3

2''

3''4''a 4''

5''

6''7''7''a




27

Compound 23: Red powder; UV (EtOH) λmax nm (log ε) 203 (4.23), 215 (4.23), 245 (4.13), 342 (4.50),

358 (4.52), 428 (3.82), 454 (3.84), 492 (3.78); IR (KBr) νmax (cm-1) 3169, 2956, 2928, 1730, 1576, 1541,

1466, 1354, 1272, 1216, 1119, 1071, 1035; HRESIMS: m/z 304.1332 [M+H]+ (304.1332 calcd for

C20H18O2N); 1H NMR (600 MHz, CD3OD) δH 9.89 (s, H-10), 8.46 (s, H-3), 8.16 (d, 8.0 Hz, H-4ʹ′), 7.68 (m,

H-7ʹ′), 7.67 (m, H-6ʹ′), 7.55 (brt, 8.0 Hz, H-5ʹ′), 2.84 (s, H3-1), 2.49 (s, H3-12), 2.32 (s, H3-11); 13C NMR (150

MHz, CD3OD) δC 165.3 (C-6), 157.7 (C-8), 150.4 (C-2ʹ′), 140.6 (C-3), 140.5 (C-7ʹ′a), 139.6 (C-10), 138.3

(C-3ʹ′), 132.2 (C-2), 130.7 (C-6ʹ′), 127.8 (C-4ʹ′a), 125.7 (C-5ʹ′), 125.5 (C-3ʹ′), 122.0 (C-9), 121.1 (C-4ʹ′), 118.6

(C-5), 118.0 (C-7), 114.4 (C-7ʹ′), 23.6 (C-1), 12.8 (C-12), 11.0 (C-11).

Key HMBC (red arrow) and 1H-1H COSY (blue bold line) correlations of 22 and 23

Hypothetical reaction scheme to 22 and 23

Confirmation of 1-hydroxy group in 22

Treatment of 22 (2.0 mg) with acetic anhydride (50 µL) in pyridine (100 µL) provided its diacetylated

derivative 22a (2.3 mg). The IR spectra of 1-hydroxyindole 22a showed broad peak around 3400 cm-1,

whereas its similar compound with indole NH group, 1,2,3,4-tetrahydrocarbazole, displayed a strong and

sharp peak at the same region, indicating the presence of a hydroxyl group in 22a.

N

HO

OH2'

3'4'

5'

6'7'

4'a7'a

11

7

6

5

12

4

9810

1

2 3

23

N

HO

OH

22

2'

3'

4'5'

6'7'

4'a7'a

11

7

6

5

12

4

9810

1

2 3

OH

OH

OH

ONH

NH

O

OH

OH NH OH

HO

OHNH

HO

OH

NH

21

NH

HO

OH

NH

22

N

HO

OH

20

2

OHNH

HO

OH

NH

H

NH

HO

OH

NH

HO

OH

H

O2

N

HO

OH

23

–H+H+




28

Compound 22a: Yellow oil; IR spectra (KBr), see Supplementary figure 6; HREIMS: m/z 407.1734 [M]+

(407.1733 calcd for C24H25O5N); 1H NMR (600 MHz, CD3OD) δH 7.45 (brd, 7.8 Hz), 7.19 (brd, 7.8 Hz),

6.99 (m), 6.97 (m), 3.93 (s, 2H), 3.29–3.18 (3H), 2.42 (s, Me (Ac)), 2.32 (s, Me (Ac)), 2.18 (s, H3-12), 1.88

(s, H3-11), 1.39 (d, 6.2 Hz, H3-1).

Structure of 22a and key NOE correlations

Supplementary Figure 6. IR spectra of 22a (left) and 1,2,3,4-tetrahydrocarbazole (right).

N

AcO

OAc

OH22a

NH

1,2,3,4-Tetrahydrocarbazole

N

AcO

OAc

OH22a

2'

3'

4'5'

6'7'

4'a7'a

117

6

5

12

4

9810

1

2 3




29

Synthesis of 24 and 25

To a solution of 2 (65 mg, 0.32 mmol) and 2-methylindole (84 mg, 0.64 mmol) in CH3CN (3 ml), BF3OEt2

(4.0 µl, 0.032 mmol) was added dropwise at room temperature and stirred for 30 min. The resulting mixture

was extracted with EtOAc, washed with water, dried over Na2SO4, and concentrated. The residue was

purified with column chromatography afforded 24 (91.4 mg). Compound 24 (100 mg) was treated with

TMS diazomethane 20% hexane solution (5 ml) and MeOH (0.5 ml) at room temperature for 2 days.

The residue was purified with column chromatography to afford 24a (32.4 mg). 24a (30.0 mg, 0.08

mmol) was treated with LHMDS 26% THF solution (154 µL, 0.20 mmol) in THF (1.5. mL) at -78 oC

and stirred for 30 min, and then the mixture was added I2 (25.4 mg, 0.10 mmol). After being slowly

warmed to room temperature, the reaction mixture was stirred for 2 hours. The mixture was added

saturated aqueous solution of Na2S2O3 and extracted with EtOAc. The combined organic extracts were

washed with brine, dried over Na2SO4, and concentrated. The residue was purified by silica-gel column

chromatography and preparative TLC to afford 25 (1.1 mg) and recovered 24a (6.4 mg, 21 %).

Compound 24: Yellow oil; UV (EtOH) λmax nm (log ε) 208 (4.53), 226 (4.51), 283 (3.87); HREIMS: m/z

337.1673 [M]+ (337.1678 calcd for C21H23O3N); IR (KBr) νmax (cm-1) 3394, 3007, 2921, 1703, 1616,

1589, 1459, 1429, 1358, 1339, 1302, 1224, 1107; 1H NMR (600 MHz, CDCl3) δH 7.82 (s, NH), 7.25 (d, 8.0

Hz, H-4ʹ′), 7.25 (d, 8.0 Hz, H-7ʹ′), 7.10 (t, 8.0 Hz, H-6ʹ′), 7.00 (t, 8.0 Hz, H-5ʹ′), 5.25 (s, 8-OH), 4.63 (6-OH),

3.97 (s, H2-10), 3.90 (s, H2-3), 2.24 (s, H3-8ʹ′), 2.12 (s, H3-12), 2.09 (s, H3-11); 13C NMR (150 MHz, CDCl3)

δC 206.7 (C-2), 151.5 (C-8), 151.1 (C-6), 135.3 (C-7ʹ′a), 132.3 (C-2ʹ′), 130.4 (C-4), 128.3 (C-4ʹ′a), 121.4

(C-6ʹ′), 119.6 (C-5ʹ′), 118.2 (C-4ʹ′), 117.4 (C-9), 114.9 (C-5), 110.3 (C-7ʹ′), 109.5 (C-7), 107.4 (C-3ʹ′), 45.4

(C-3), 29.1 (C-1), 22.9 (C-10), 12.3 (C-12), 11.7 (C-8ʹ′), 8.6 (C-11).

Key HMBC correlations of 24

Compound 24a: Colorless amorphous; HREIMS: m/z 365.2001 [M]+ (365.1991 calcd for C23H27O3N); 1H

NMR (600 MHz, CDCl3) δH 7.69 (s, NH), 7.32 (brd, 8.0 Hz), 7.20 (brd (8.0 Hz), 7.06 (brt, 8.0 Hz), 7.00 (brt,

8.0 Hz), 4.04 (s, 2H), 3.73 (s, OMe), 3.70 (s, OMe), 3.62 (s, 2H), 2.31 (s, Me), 2.04 (s, Me), 2.03 (s, Me),

1.49 (s, Me).

NH

O

OH

OH

1

2

34 5

6

78910 11

12

2'

3'4'

5'

6'7'

4'a

7'a8'

O

OH

HO

2

NH

O

OH

OH

24 2524aNH

BF3OEt2 (10 mol%),CH3CN, r.t., 86%

NH

O

OMe

OMe

TMSCH2N2,MeOH, r.t., 30%

LHMDS,THF, 0 oC,then I2, 4%

N

OMe

OMe

O




30


(KBr) νmax (cm-1) 2929, 2854, 1713, 1573, 1466, 1458, 1423, 1404, 1377, 1358, 1339, 1271, 1248, 1191,

1160, 1113, 1002; HRESIMS: m/z 386.1725 [M+Na]+ (386.1727 calcd for C23H25O3NNa), 364.1907 [M+H]+

(364.1907 calcd for C23H26O3N); 1H NMR (600 MHz, CDCl3) δH 7.58 (brd, 7.8 Hz, H-7ʹ′), 7.36 (brt, 7.8 Hz,

H-6ʹ′), 7.19 (brt, 7.8 Hz, H-5ʹ′), 7.16 (brd, 7.8 Hz, H-4ʹ′), 4.49 (s, H-3), 3.75 (s, OMe), 3.73 (s, OMe), 3.34 (d,

15.6 Hz, Ha-10), 3.17 (d, 15.6 Hz, Hb-10), 2.26 (s, H3-11), 2.14 (s, H3-8ʹ′), 1.99 (s, H3-12), 1.81 (s, H3-1); 13C

NMR (150 MHz, CDCl3) δC 205.2 (C-2), 183.2 (C-2ʹ′), 157.5 (C-6), 153.9 (C-7ʹ′a), 153.5 (C-8), 144.2 (C-4ʹ′a),

138.6 (C-4), 129.2 (C-9), 128.6 (C-6ʹ′), 126.1 (C-5ʹ′), 123.9 (C-5), 123.9 (C-7), 120.8 (C-4ʹ′), 120.2 (C-7ʹ′),

66.5 (C-3ʹ′), 65.0 (C-3), 60.2 (OMe), 60.0 (OMe), 37.5 (C-10), 30.9 (C-1), 18.0 (C-8ʹ′), 13.6 (C-12), 9.6

(C-11).

Key HMBC (red arrow) and NOE (purple arrow) correlations of 25

Synthesis of 27

To a solution of 2 (10 mg, 0.05 mmol) and 3-methylindole (13.1 mg, 0.10 mmol) in CH3CN (0.5 ml),

BF3OEt2 (0.6 µl, 0.005 mmol) was added dropwise at room temperature. After being stirred for 30 min at the

same temperature, the reaction was quenched with water and then extracted with EtOAc. The combined

organic extracts were washed with saturated aqueous solution of brine, dried over Na2SO4, and

concentrated. The residue was purified by silica-gel column chromatography to afford 27 (10.2 mg).

N

OMe

OMe

O

N

OMe

OMe

O

2'

4'5'

6'7'

4'a7'a

3'

8'

1 23

45

12

6 7 11

89

10

N

OMe

OMeO

H

HH

33

3'

4' 4'

3'1010

8' 8'

1

1

1212

O

OH

HO

2

BF3OEt2 (10 mol%),CH3CN, r.t.

NH

OH

OH

O

N OH

OHNH

26 27 (66%)




31

Synthesis of 26, 28, 29 and 30

To a solution of 2 (100 mg, 0.49 mmol) and 3-methylindole (127 mg, 0.98 mmol) in CH2Cl2 (5 ml), BF3OEt2

(6.1 µl, 0.049 mmol) was added dropwise at room temperature and stirred 3 min. The reaction was quenched

with water and then extracted with EtOAc. The combined organic extracts were washed with saturated

aqueous solution of brine, dried over Na2SO4, and concentrated. The residue was purified by silica-gel

column chromatography to afford 26 (83.0 mg). Compound 27 was also observed as a minor component

in this reaction. Compound 26 (80.0 mg) was treated with TMS diazomethane 20% hexane solution (2.5

ml) and MeOH (0.25 ml) at room temperature for 2 days, affording its dimethyl derivative 26a (67.6

mg). Compound 26a (30.0 mg, 0.14 mmol) was treated with LHMDS 26% THF solution (158 µL, 0.35

mmol) in THF (2.5 mL) at -78 oC and stirred for 30 min, and then the mixture was added I2 (20.8 mg,

0.14 mmol). After being slowly warmed to room temperature, the reaction mixture was stirred for 2

hours. The mixture was added saturated aqueous solution of Na2S2O3 and extracted with EtOAc. The

combined organic extracts were washed with brine, dried over Na2SO4, and concentrated. The residue was

purified by silica-gel column chromatography and preparative TLC to afford 28 (3.2 mg), 29 (2.4 mg)

and 30 (1.1 mg) as well as recovered 26a (7.2 mg, 24%).

Hypothetical reaction scheme to 28–30 from 26a by the intramolecular oxidative coupling

NOMe

OMe

OHC

NOMe

OMe

O

N

HO

O

OMe

OMeIH

NH

OMe

OMe

O

N

OMe

OMe

O I

NOMe

OMe

O

NOMe

OMe

O

HO

N

H2O

I

O

30 29

2826a

I

O

OH

HO

2

BF3OEt2 (10 mol%),CH2Cl2, r.t, 3 min.

NH

OH

OH

ONH

26 (51%)

27 (trace)

NOMe

OMe

OHCN

OMe

OMe

O

HO

N

HO

O

OMe

OMeI

H

30 (3%)

TMSCH2N2,MeOH, r.t.,

LHMDS,THF, -78 oC,then I2, 5%

NH

OMe

OMe

O

26a (78%)

28 (10%) 29 (8%)




32


max (cm-1) 3401, 3007, 2919, 2860, 1704, 1589, 1459, 1315, 1240, 1217, 1164, 1110; HREIMS: m/z

337.1676 [M]+ (337.1678 calcd for C21H23NO3); 1H NMR (600 MHz, CDCl3) δH 8.49 (s, NH), 7.49 (brd, 7.8

Hz, H-4ʹ′), 7.22 (brd, 7.8 Hz, H-7ʹ′), 7.10 (brt, 7.8 Hz, H-6ʹ′), 7.06 (brt, 7.8 Hz, H-5ʹ′), 3.97 (s, H2-10), 3.95 (s,

H2-3), 2.40 (s, H3-8ʹ′), 2.27 (s, H3-1), 2.09 (s, H3-12), 2.09 (s, H3-11); 13C NMR (150 MHz, CDCl3) δC 207.7

(C-2), 151.5 (C-6), 151.3 (C-8), 135.8 (C-7ʹ′a), 131.7 (C-2ʹ′a), 130.1 (C-4), 128.8 (C-4ʹ′a), 121.6 (C-6ʹ′), 119.0

(C-5ʹ′), 118.2 (C-4ʹ′), 117.0 (C-9), 114.9 (C-5), 110.8 (C-7ʹ′), 110.1 (C-7), 106.9 (C-3ʹ′), 44.8 (C-3), 30.0 (C-1),

24.1 (C-10), 12.4 (C-12), 8.6 (C-11), 8.5 (C-8ʹ′).

Compound 26a: Colorless amorphous; HREIMS: m/z 365.1988 [M]+ (365.1991 calcd for C23H27O3N); 1H

NMR (600 MHz, CDCl3) δH 8.19 (s, NH), 7.46 (brd, 8.0 Hz), 7.19 (brd (8.0 Hz), 7.06 (brt, 8.0 Hz), 7.04 (brt,

8.0 Hz), 4.00 (s, 2H), 3.80 (s, 2H), 3.70 (s, OMe), 3.65 (s, OMe), 2.36 (s, Me), 2.29 (s, Me), 2.09 (s, Me),

2.08 (s, Me).

Compound 27: Yellow oil; UV (EtOH) λmax nm (log ε) 207 (4.54), 239 (4.48), 278 (4.29), 296 (4.13); IR

(KBr) νmax (cm-1) 3359, 2921, 1637, 1512, 1457, 1393, 1310, 1260, 1202, 1183, 1106, 1082, 1033;

HREIMS: m/z 319.1557 [M]+ (319.1572 calcd for C21H21O2N); 1H NMR (600 MHz, acetone-d6) δH 7.47 (brd,

8.0 Hz, H-4ʹ′), 7.41 (brd, 8.0 Hz, H-7ʹ′), 7.21 (OH), 7.04 (brt, 8.0 Hz, H-6ʹ′), 7.00 (brt, 8.0 Hz, H-5ʹ′), 6.55 (s,

H-3), 4.73 (d, 13.2 Hz, Ha-10), 3.19 (d, 13.2 Hz, Hb-10), 2.64 (s, H3-1), 2.29 (s, H3-8ʹ′), 2.16 (s, H3-11), 2.14

(s, H3-12); 13C NMR (150 MHz, acetone-d6) δC 152.2 (C-6), 149.8 (C-8), 142.6 (C-2ʹ′), 135.6 (C-2), 135.0

(C-7ʹ′a), 132.9 (C-4), 131.7 (C-4ʹ′a), 121.3 (C-6ʹ′), 120.1 (C-5ʹ′), 119.7 (C-9), 119.0 (C-4ʹ′), 118.7 (C-3), 114.2

(C-5), 112.5 (C-7ʹ′), 112.2 (C-7), 104.3 (C-3ʹ′), 23.0 (C-10), 22.4 (C-1), 12.4 (C-12), 9.9 (C-11), 8.6 (C-8ʹ′).

Key HMBC (red arrow) and NOE (purple arrow) correlation of 26 and 27

NH

OH

OH

O

N OH

OH

26 27

4'a7'a

7'

4'

6'

5'

2'3'

1 23

4

512

67

11

89

104'a7'a

7'

4'

6'

5'

2'3'

12

3 45

12

6 7

8910

8'8'




33

Compound 28: Colorless amorphous; UV (EtOH) λ:max nm (log ε) 208 (4.55), 226 (4.54), 281 (3.89); IR

(KBr) νmax (cm-1) 3412, 2361, 2926, 2855, 1723, 1574, 1457,1405, 1382, 1356, 1323, 1262, 1237, 1187,

1107, 1085, 1064, 1023; HRESIMS: m/z 378.1712 [M–H]‒ (378.1700 calcd for C23H24O4N); 1H NMR (600

MHz, CDCl3) δH 7.54 (brd, 7.8 Hz, H-4ʹ′), 7.32 (brd, 7.8 Hz, H-7ʹ′), 7.16 (brt, 7.8 Hz, H-5ʹ′), 7.13 (brt, 7.8 Hz,

H-6ʹ′), 5.47 (s, 3-OH), 4.32 (d, 22.2 Hz, Ha-10), 4.23 (d, 22.2 Hz, Hb-10), 3.85 (s, OMe), 3.70 (s, OMe), 2.36

(s, H3-8ʹ′), 2.32 (s, H3-11), 2.27 (s, H3-12), 1.54 (s, H3-1); 13C NMR (150 MHz, CDCl3) δC 201.5 (C-2), 157.5

(C-6), 154.9 (C-8), 133.9 (C-7ʹ′a), 130.5 (C-4ʹ′a), 129.2 (C-4), 128.4 (C-2ʹ′), 126.3 (C-5), 125.8 (C-7), 122.0

(C-9), 121.3 (C-6ʹ′), 120.5 (C-5ʹ′), 117.9 (C-4ʹ′), 112.9 (C-7ʹ′), 107.5 (C-3ʹ′), 87.2 (C-3), 60.0 (OMe), 60.0

(OMe), 22.6 (C-1), 21.1 (C-10), 12.4 (C-12), 10.0 (C-11), 8.5 (C-8ʹ′).


(KBr) νmax (cm-1) 2924, 2853, 1697, 1623, 1606, 1573, 1462, 1437, 1414, 1370, 1320, 1257, 1206, 1152,

1118, 1002; HRESIMS: m/z 384.1561 [M+Na]+ (384.1570 calcd for C23H23O3NNa); 1H NMR (600 MHz,

CDCl3) δH 9.88 (s, H-1), 7.56 (s, H-3), 7.49 (brd, 7.2 Hz, H-4ʹ′), 7.15 (brt, 7.2 Hz, H-6ʹ′), 7.12 (brt, 7.2 Hz,

H-5ʹ′), 7.06 (brd, 7.2 Hz, H-7ʹ′), 4.70 (d, 14.4 Hz, Ha-10), 3.79 (s, OMe), 3.66 (s, OMe), 3.30 (d, 14.4 Hz,

Hb-10), 2.38 (s, H3-8ʹ′), 2.32 (s, H3-12), 2.27 (s, H3-11), 1.54 (s, H3-1); 13C NMR (150 MHz, CDCl3) δC 187.8

(C-1), 156.3 (C-6), 154.2 (C-8), 139.8 (C-2ʹ′), 137.1 (C-3), 134.1 (C-7ʹ′a), 133.4 (C-2), 131.0 (C-9), 130.7

(C-4ʹ′a), 128.1 (C-4), 127.3 (C-7), 126.4 (C-5), 121.7 (C-6ʹ′), 120.2 (C-5ʹ′), 118.7 (C-4ʹ′), 111.9 (C-7ʹ′), 106.8

(C-3ʹ′), 61.4 (6-OMe), 60.2 (8-OMe), 22.9 (C-10), 12.5 (C-12), 10.2 (C-11), 8.8 (C-8ʹ′).


max (cm-1) 3349, 2927, 2853, 1730, 1639, 1611, 1468, 1468, 1402, 1374, 1320, 1268, 1217, 1111, 1073,

1002; HRESIMS: m/z 528.0634 [M+Na]+ (528.0642 calcd for C23H24O4NINa); 1H NMR (600 MHz, CDCl3)

δH 7.43 (brd, 8.0 Hz, H-4ʹ′), 7.18 (brt, 8.0 Hz, H-6ʹ′), 6.89 (brt, 8.0 Hz, H-5ʹ′), 6.64 (brd, 8.0 Hz, H-7ʹ′), 4.62 (d,

1.4 Hz, H-2), 4.28 (s, 2ʹ′-OH), 4.17 (brs, H-3), 3.77 (s, 8-OMe), 3.70 (s, 6-OMe), 3.48 (d, 16.8 Hz, Ha-10),

3.10 (d, 16.8 Hz, Hb-10), 2.21 (s, H3-12), 2.21 (s, H3-11), 1.66 (s, H3-8ʹ′); 13C NMR (150 MHz, CDCl3) δC

160.6 (C-1), 157.5 (C-6), 153.4 (C-8), 147.4 (C-7ʹ′a), 138.0 (C-4), 131.4 (C-4ʹ′a), 129.9 (C-6ʹ′), 128.2 (C-9),

124.7 (C-4ʹ′), 123.9 (C-7), 123.3 (C-5), 120.2 (C-5ʹ′), 110.6 (C-7ʹ′), 93.3 (C-3ʹ′), 83.8 (C-2ʹ′), 61.7 (C-3), 60.0

(6-OMe), 59.9 (8-OMe), 43.0 (C-2), 35.2 (C-10), 22.3 (C-8ʹ′), 13.6 (C-12), 9.5 (C-11).

Key HMBC (red arrow), 1H-1H COSY (blue bold line) and NOE (purple arrow) correlation of 28–30, and

characteristic data of 30

28 29

N

O

OMe

OMe

HON

OMe

OMe

O

30

N

HO

O

OMe

OMeI

N

HO

O

OMe

OMeI H

4'a7'a

7'

4'

6'

5'

2'3'

8'

1 23

4

5 67

11

89

10 10

4'a7'a

7'

4'

6'

5'

2'3'

8'

12 3

4

512 12 11

6 7

89

H

4'a7'a

7'

4'

6'

5'

2'3'

8'10

9 8 711

6

5

432

12

3

12 12

10

8'

IR : !max 1730 cm-1

JH-2-H-3 = 1.4 Hz

NOMe

OMe

O2

12

HH




34

Conversion of 30 to 31

When NMR measurement of 30 (1.0 mg) in CD3CN (150 µL) was carried out, conversion of 30 to 31 was

observed. The reaction was monitored in situ by 1H NMR spectroscopy and completed in 30 hours at room

temperature. This conversion could not be observed in CDCl3 in 48 h.


(KBr) νmax (cm-1) 3306, 2926, 2853, 1630, 1609, 1541, 1467, 1404, 1375, 1334, 1282, 1191, 1109, 1065,

1010; HREIMS: m/z 489.0772 [M]+ (489.0801 calcd for C23H24O3NI); 1H NMR (600 MHz, CD3CN) δH

10.19 (brs, NH), 7.40 (brd, 7.8 Hz, H-4ʹ′), 7.23 (brt, 7.8 Hz, H-6ʹ′), 7.10 (brd, 7.8 Hz, H-7ʹ′), 7.03 (brt, 7.8 Hz,

H-5ʹ′), 3.99 (d, 9.5 Hz, Ha-1), 3.78 (d, 9.5 Hz, Hb-1), 3.71 (s, 2 x OMe), 3.40 (d, 14.6 Hz, Ha-10), 2.61 (d,

14.6 Hz, Hb-10), 2.23 (s, H3-11), 2.16 (s, H3-12), 1.00 (s, H3-8ʹ′); 13C NMR (150 MHz, CD3CN) δC 192.8

(C-2), 169.1 (C-2ʹ′), 158.2 (C-6), 156.8 (C-8), 145.6 (C-7ʹ′a), 137.8 (C-4ʹ′a), 134.6 (C-4), 129.2 (C-5), 124.4

(C-5), 123.7 (C-4ʹ′), 123.2 (C-6ʹ′), 122.8 (C-7), 121.6 (C-9), 111.9 (C-7ʹ′), 102.6 (C-3), 61.2 (OMe), 60.3

(OMe), 48.7 (C-3ʹ′), 32.5 (C-10), 21.5 (C-8ʹ′), 15.3 (C-12), 9.8 (C-11), 7.5 (C-1).

Key HMBC (red arrow) and NOE (purple arrow) correlations of 31

N

HO

O

OMe

OMeI

H

30

CD3CN, r.t.NH

OI

OMe

OMe

31

NHO

I

OMe

OMe

4'a7'a

7'

4'

6'

5'

2'3'

1

23

45

6

789

1011

12

8'

4'

NH O I

H H

MeO

OMe8'

3'

10

1

12




35

Synthesis of 32 and 33

To a solution of 2 (10.0 mg, 0.05 mmol) and 1,2,3,4-tetrahydrocarbazole (17.1 mg, 0.10 mmol) in CH3CN

(0.5 ml), BF3OEt2 (0.6 µl, 0.005 mmol) was added at room temperature. After being stirred for 30 min, the

reaction was quenched with water and then extracted with EtOAc. The combined organic extracts were

washed with saturated aqueous solution of brine, dried over Na2SO4, and concentrated. The residue was

purified by silica-gel column chromatography to afford 32 (2.6 mg) and 33 (13.0 mg).


max (cm-1) 3361, 3008 2932, 2858, 1699, 1612, 1591, 1462, 1356, 1222, 1122, 1049, 1001; HRESIMS: m/z

378.2038 [M+H]+ (378.2064 calcd for C24H28O3N); 1H NMR (600 MHz, CDCl3) δH 7.00 (td, 7.3, 1.1 Hz,

H-6ʹ′), 6.92 (brd, 7.3 Hz, H-4ʹ′), 6.72 (td, 7.3, 1.1 Hz, H-5ʹ′), 6.68 (brd, 7.3 Hz, H-7ʹ′), 4.57 (s, OH), 4.39 (s,

NH), 3.73 (s, H2-3), 2.94 (d, 16.2 Hz, Ha-10), 2.82 (d, 16.2 Hz, Hb-10), 2.09 (s, H3-1), 2.07 (s, H3-12), 2.01

(m, Ha-11ʹ′), 2.00 (s, H3-11), 1.77 (m, Hb-11ʹ′), 1.76 (m, Ha-8ʹ′), 1.69 (m, Ha-10ʹ′), 1.63 (m, Hb-10ʹ′), 1.51 (m,

Hb-8ʹ′), 1.48 (m, Ha-9ʹ′), 1.41 (m, Hb-9ʹ′) 1.66 (s, H3-8ʹ′); 13C NMR (150 MHz, CDCl3) δC 207.0 (C-2), 150.9

(C-8), 150.7 (C-6), 147.3 (C-7ʹ′a), 136.6 (C-4ʹ′a), 129.3 (C-4), 127.3 (C-6ʹ′), 121.3 (C-4ʹ′), 119.6 (C-5ʹ′), 114.5

(C-5), 112.2 (C-9), 110.2 (C-7), 109.8 (C-7ʹ′), 98.1 (C-2ʹ′), 45.6 (C-3ʹ′), 44.8 (C-3), 37.7 (C-8ʹ′), 31.6 (C-11ʹ′),

29.1 (C-1), 28.1 (C-10), 22.8 (C-10ʹ′), 20.6 (C-9ʹ′), 12.1 (C-12), 8.5 (C-11).


max (cm-1) 3394, 2930, 2854, 1637, 1706, 1616, 1445, 1358, 1326, 1234, 1162, 1110; HREIMS: m/z

377.1977 [M]+ (377.1991 calcd for C24H27O3N); 1H NMR (600 MHz, CDCl3) δH 7.57 (s, NH), 7.33 (d, 8.0

Hz, H-4ʹ′), 6.89 (dd, 8.0, 1.4 Hz, H-5ʹ′), 6.83 (d, 1.4 Hz, H-7ʹ′), 4.00 (s, H2-10), 3.76 (s, H2-3), 2.64 (m, H2-8ʹ′),

2.61 (m, H2-11ʹ′), 2.08 (s, H3-11), 2.07 (s, H3-12), 2.05 (s, H3-1), 1.83 (m, H2-10ʹ′ or 9ʹ′), 1.81 (m, H2-9ʹ′ or

10ʹ′); 13C NMR (150 MHz, CDCl3) δC 207.1 (C-2), 151.2 (C-8), 151.2 (C-6), 136.2 (C-7ʹ′a), 134.2 (C-2ʹ′),

131.7 (C-6ʹ′), 130.8 (C-4), 126.5 (C-4ʹ′a), 119.5 (C-5ʹ′), 118.6 (C-9), 117.9 (C-4ʹ′), 115.2 (C-5), 110.0 (C-7),

109.8 (C-3ʹ′), 109.6 (C-7ʹ′), 45.3 (C-3), 32.9 (C-10), 29.4 (C-1), 23.2 (C-11ʹ′), 23.1 (C-10ʹ′), 23.1 (C-9ʹ′), 20.9

(C-8ʹ′), 12.3 (C-12), 8.8 (C-11).

Key HMBC (red arrow), 1H-1H COSY (blue bold line) and NOE (purple arrow) correlation of 32 and 33

NH

O OH

O

NHO

OHHO

32 33

2'

4'5'

6'

7'

4'a7'a

3' 8'

9'10'

11'

1

23

45

12

6

7

11

8

9

10 2'

4'5'

6'

7'

4'a7'a

3'

8'9'

10'11'

O

OH

HO

2

BF3OEt2 (10 mol%),CH3CN, r.t.

NH

O OH

O

NHO

OHHO

32 (14%) 33 (71%)

+

NH




36

Evaluation of anti-adenovirus activities.

A human T-cell leukemia cell line, MT-2, and a human lung caricinoma cell line, A549 were grown in

RPMI1640 and DMEM media, respectively, supplemented with 10% fatal calf serum (FCS), 2 mM

L-glutamine, 100 units/mL of penicillin, and 100 µg/mL of streptomycin. Cells were incubated at 37°C in a

humidified atmosphere with 5% CO2. Human Adenovirus type 19 was propagated in A549 cells and stored

at －80°C until use. Anti-adenovirus activity was evaluated with the MTT-colorimetric method as described

previously 4 with some modifications. Breifly, MT-2 cells (2 x 104 cells/well) were mixed with 103 TCID50

(50% tissue culture infectious dose) in the presence and absence of various concentrations of the compounds

in 96well plate and incubated for 5 days. At the end of the incubation period, the cytopathic effect (CPE) was

evaluated by the MTT assay. The 50% antiviral effective concentration (EC50) was defined as the

concentration that achieved 50% protection of virus-infected cells against virus-induced destruction.

Parallelly, we determined EC50 value of cidofovir as a control (EC50 = 24 µM).

2) Kodama, E., Shigeta, S., Suzuki, T. & De Clercq, E. Application of a gastric cancer cell line (MKN-28)

for anti-adenovirus screening using the MTT method. Antiviral Res 31, 159-164 (1996).




37

OH

HO

O

1 H N

MR

(ace

tone

-d6)

of 2




38

OH

HO

O

13C

NMR(

(ace

tone

-d6)

of 2




39

1 H N

MR

(pyr

idin

e-d 5

) of 5

OHO

OH

O

O

OH

H




40

OHO

OH

O

O

OH

H

13C

NMR

(pyr

idin

e-d 5

) of 5




41

O

O

OHO

HO O 1 H N

MR

(CDC

l 3) o

f 6




42

13C

NMR

(CDC

l 3) o

f 6

O

O

OHO

HO O




43

1 H N

MR

(CDC

l 3 +

10%

CD 3

OD)

of 7

OHO

OH

O

O

OH

H




44

13C

NMR

(CDC

l 3 +

10%

CD 3

OD)

of 7

OHO

OH

O

O

OH

H




45

1 H

NM

R (C

DCl 3

+ 10

% C

D 3O

D) o

f 8

OHO

OH

O

O

OH

H




46

13C

NMR

(CDC

l 3 +

10%

CD 3

OD)

of 8

OHO

OH

O

O

OH

H




47

1 H N

MR

(CDC

l 3 +

10%

CD 3

OD)

of 9

O

OHO

OH

OH

O

OH

HO

O

H




48

13C

NMR

(CDC

l 3 +

10%

CD 3

OD)

of 9

O

OHO

OH

OH

O

OH

HO

O

H




49

1 H N

MR

(CDC

l 3 +

10%

CD 3

OD)

of 10

O

O

O

HOHO O

OH

OH

O

H




50

13C

NMR

(CDC

l 3 +

10%

CD 3

OD)

of 10

O

O

O

HOHO

OO

H

OH

O

H




51

1H N

MR

(CD 3

OD)

of 11

OH

HO

O

O

O

OH

H




52

13C

NMR

(CD 3

OD)

of 11

OH

HO

O

O

O

OH

H




53

1 H N

MR

(CDC

l 3 +

10%

CD 3

OD)

of 12

HO

OH

O

HOO

H

H

O




54

13C

NMR

(CDC

l 3 +

10%

CD 3

OD)

of 12

HO

OH

O

HOO

H

H

O




55

1H N

MR

(CDC

l 3) o

f 13

O

O

O

OH

H

OHOH




56

13

C NM

R (C

DCl 3)

of 13

O

O

O

OH

H

OHOH




57

1H N

MR

(CDC

l 3) o

f 14

O

O

O

OH

H

OHOH




58

1 H N

MR

(CDC

l 3 +

10%

CD 3

OD)

of 15

OO

O

O

O

OH

H

HO

HO




59

13C

NMR

(CDC

l 3 +

10%

CD 3

OD)

of 15

OO

O

O

O

OH

H

HO

HO




60

1 H N

MR

(CDC

l 3) o

f 16

OO

O

O

O

OH

H

HO

HO




61

13C

NMR

(CDC

l 3) o

f 16

OO

O

O

O

OH

H

HO

HO




62

1 H N

MR

(CDC

l 3) o

f 17

O

O

O

OH

H

ON

OHO

O




63

13C

NMR

(CDC

l 3) o

f 17

O

O

O

OH

H

ON

OHO

O




64

1 H N

MR

(CDC

l 3) o

f 18

O

O

O

OH

H

ON

OHO

O




65

1H N

MR

(CD 3

OD)

of 19

HO

OH

NH

HOO

H

H

O




66

13C

NMR

(CD 3

OD)

of 19

HO

OH

NH

HOO

H

H

O




67

1 H N

MR

(CDC

l 3) o

f 20

N H

OH

O

OH




68

13C

NMR

(CDC

l 3) o

f 20

N H

OH

O

OH




69

1 H

NM

R (a

ceto

ne-d

6) o

f 21

N H

HO

OH

N H




70

13C

NMR

(ace

tone

-d6)

of 21

N H

HO

OH

N H




71

1H N

MR

(CD 3

OD)

of 22

N

HO

OH

OH




72

13C

NMR

(CD 3

OD)

of 22

N

HO

OH

OH




73

1H N

MR

(CD 3

OD)

of 23

N

HO

OH




74

13C

NMR

(CD 3

OD)

of 23

N

HO

OH




75

1 H N

MR

(CDC

l 3) o

f 24

N H

OH

O

OH




76

13C

NMR

(CDC

l 3) o

f 24

N H

OH

O

OH




77

1 H N

MR

(CDC

l 3) o

f 25

N

OM

e OM

e

O




78

13C

NMR

(CDC

l 3) o

f 25

N

OM

e OM

e

O




79

1 H N

MR

(CDC

l 3) o

f 26

N H

O

OH

OH




80

13C

NM

R (C

DCl 3)

of 26

N H

O

OH

OH




81

1 H N

MR

(ace

tone

-d6)

of 27

NO

H

OH




82

13C

NMR

(ace

tone

-d6)

of 27

NO

H

OH




83

1 H N

MR

(CDC

l 3) o

f 28

N

O

OM

e

OM

e

HO




84

13C

NMR

(CDC

l 3) o

f 28

N

O

OM

e

OM

e

HO




85

1 H N

MR

(CDC

l 3) o

f 29

NO

HCO

Me

OM

e




86

13C

NM

R (C

DC

l 3) o

f 29

NO

HC

OM

e

OM

e




87

1 H N

MR

(CDC

l 3) o

f 30

NHO

O

OM

e

OM

eI

H




88

13C

NMR

(CDC

l 3) o

f 30

NHO

O

OM

e

OM

eI

H

88

13C

NMR

(CDC

l 3) o

f 30

NHO

O

OM

e

OM

eI

H




89

N HO

OM

e

OM

e

I1 H

NM

R (C

D 3CN

) of 31




90

N HO

OM

e

OM

e

I13

C NM

R (C

D 3CN

) of 31




91

N HO

OH

O

1 H N

MR

(CDC

l 3) o

f 32




92

N HO

OH

O

13C

NMR

(CDC

l 3) o

f 32




93

1 H N

MR

(CDC

l 3) o

f 33

N HO

HOO

H




94

13C

NM

R (C

DCl

3) o

f 33

N HO

HO

OH




95

1 H N

MR

(CDC

l 3) o

f 34

and

35

O

O

O

O

O

OH

OO

HH

O HO




96

13C

NMR

(CDC

l 3) o

f 34

and

35

O

O

O

O

O

OHO

OH

HO

HO




97

1 H N

MR

(CDC

l 3) o

f 36O

O

ONHBo

c

OH




98

13C

NMR

(CDC

l 3) o

f 36

O

O

ONHBo

c

OH




99

1 H N

MR

(CDC

l 3) o

f 37

O

O

NOO

HO




100

13C

NMR

(CDC

l 3) o

f 37

O

O

NOO

HO




101

1 H N

MR

(CDC

l 3) o

f 38

N

O

OHO

HO




102

13C

NMR

(CDC

l 3) o

f 38

N

O

OHO

HO




103

OOO

O O

O

1 H N

MR

(CDC

l 3) o

f 39




104

OOO

O O

O

13C

NMR

(CDC

l 3) o

f 39




105

1 H N

MR

(CDC

l 3) o

f 40

OOO

O

O

O




106

13C

NMR

(CDC

l 3) o

f 40

OOO

O

O

O




107

1 H N

MR

(CDC

l 3) o

f 41

OO

OOO

O




108

13C

NMR

(CDC

l 3) o

f 41

OO

OOO

O




109

1 H N

MR

(CDC

l 3) o

f 42

OOOO

O

O




110

13C

NMR

(CDC

l 3) o

f 42

OOOO

OO




111

1 H N

MR

(CDC

l 3) o

f 43

O

OOOO

O




112

13C

NMR

(CDC

l 3) o

f 43

O

OOOO

O




113

1 H N

MR

(CDC

l 3) o

f 44

O

OOO

O

O




114

13C

NMR

(CDC

l 3) o

f 44

O

OOO

O

O


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