Supplementary materials

Chemical and Pharmaceutical Bulletin

Chemical Constituents from the Leaves of Pachyrhizus erosus Collected in Vietnam

Tran Thi Minh, Ho Khanh Toan, Hoang Thi Lan Anh, Tran Thu Huong, Do Thi Thao, and
Vu Dinh Hoang

© 2021 The Pharmaceutical Society of Japan

Content Pages
Acid hydrolysis and sugar identification S3
Cytotoxic activity assay S3
Figure S1. HR-ESI mass spectrum of 1 S4
Figure S2. 1H-NMR (500 MHz, CD3OD) spectrum of 1 S5
Figure S3. 1H-NMR (500 MHz, CD3OD) spectrum of 1 (extension) S5
Figure S4. 13C-NMR (125 MHz, CD3OD) spectrum of 1 S6
Figure S5. DEPT 90 and 135 (125 MHz, CD3OD) spectrum of 1 S7
Figure S6. HSQC (500 MHz, CD3OD) spectrum of 1 S8
Figure S7. COSY (500 MHz, CD3OD) spectrum of 1 S9
Figure S8. HMBC (500 MHz, CD3OD) spectrum of 1 S10
Figure S9. NOESY (500 MHz, CD3OD) spectrum of 1 S11
Figure S10. HR-ESI mass spectrum of 9 S12
Figure S11. 1H-NMR (CD3OD, 500 MHz) spectrum of 9 S13
Figure S12. 13C-NMR (CD3OD, 125 MHz) spectrum of 9 S14
Figure S13. HSQC (CD3OD, 500 MHz) spectrum of 9 S15
Figure S14. COSY (CD3OD, 500 MHz) spectrum of 9 S16
Figure S15. HMBC (CD3OD, 500 MHz) spectrum of 9 S17
Figure S16. NOESY (CD3OD, 500 MHz) spectrum of 9 S18
Figure S17. NOESY (CD3OD, 500 MHz) spectrum of 9 (extension) S19
Figure S18. 1H-NMR (500 MHz, CD3OD) spectrum of 7 S20
Figure S19. 13C-NMR (125 MHz, CD3OD) spectrum of 7 S21
Figure S20. 1H-NMR (500 MHz, DMSO-d6) spectrum of 8 S22
Figure S21. 13C-NMR (125 MHz, DMSO-d6) spectrum of 8 S23
Table S1. 1H (500 MHz) and 13C (125 MHz) NMR data for compounds 1, 1a and 2 S24
Table S2. 13C NMR (125 MHz) data for compounds 3-6 S25
Table S3. 1H (500 MHz) and 13C (125 MHz) NMR data for compounds 9 and 10 in S26
CD3OD
Table S4. 13C NMR data for compounds 11-15 S27

S2
Acid hydrolysis and sugar identification Compound 9 (4.0 mg) was refluxed with 2 N

methanolic HCl (5 mL) for 2 h. After neutralization with 1 N sodium carbonate, the reaction

mixture was extracted with CHCl3 to remove the aglycone. In the aqueous layer, D-glucose

was identified by the optical rotation and TLC comparison with an authentic sample [Rf 0.17,

EtOAc-MeOH-H2O (4 : 1 : 0.1, v/v), [α ]25

D +45.7].

Cytotoxic activity assay Cytotoxic activity against LU-1 (lung carcinoma), MCF7 (breast

carcinoma), and HepG2 (hepatocellular carcinoma) were determined using the

sulforhodamine B assay.32) All tumor cell lines were supplied by Professor J. M. Pezzuto

(Long-Island University, US) and Professor Jeanette Maier (Milan University, Italia).

Ellipticine was used as a positive control. The cancer cells (3 × 104 cells/mL), suspended in

180 µL/well of DMEM medium containing 10% Fetal Bovine Serum (FBS), L-glutamine (2

mM), sodium pyruvat, NaHCO3, penicillin (100 UI/mL), streptomycin (100 UI/mL), and

trypsin-EDTA (0.05%), were seeded onto a 96-well culture plate at 37oC in air/CO2 (95:5),

various concentrations of the test solution (20 µL) were added. After incubation for 72 h, the

cell layers were fixed with 20% (w/v) trichloroacetic acid (TCA), washed three times with

acetic acid, and stained for 30 min with sulforhodamine B reagent (SRB) at 37oC. The cell

density after sample treatment was then measured optical density (OD) at 540 nm using an

ELISA Plate Reader (Biotek). The control sample without sample treatment (Day0), was

incubated for 1 h, and fixed with TCA 20%. The negative reference sample was treated with

10% DMSO. All tests were performed in triplicate. Cell viability was measured and the IC50

value was calculated by TableCurve 2Dv4 software. [Hughes J. P., Rees S., Kalindjian S. B.,

Philpott K. L., British J. Pharm., 162, 1239-1249 (2011)]

ODsample − ODDay 0
Cell viability (%) = ×100(%)
ODnegative reference −ODDay 0

Inhibition (%) = 100 – Cell viability (%)

S3
Figure S1. HR-ESI mass spectrum of 1

S4
 Figure S2. 1H-NMR (500 MHz, CD3OD) spectrum of 1

1
Figure S3. H-NMR (500 MHz, CD3OD) spectrum of 1 (extension)

S5
Figure S4. 13C-NMR (125 MHz, CD3OD) spectrum of 1

S6
Figure S5. DEPT 90 and 135 (125 MHz, CD3OD) spectrum of 1

S7
Figure S6. HSQC (500 MHz, CD3OD) spectrum of 1

S8
Figure S7. COSY (500 MHz, CD3OD) spectrum of compound 1

S9
Figure S8. HMBC (500 MHz, CD3OD) spectrum of 1

S10
Figure S9. NOESY (500 MHz, CD3OD) spectrum of 1

S11
Figure S10. HR-ESI mass spectrum of 9

S12
Figure S11. 1H-NMR (CD3OD, 500 MHz) spectrum of 9

S13
Figure S12. 13C-NMR (CD3OD, 125 MHz) spectrum of 9

S14
Figure S13. HSQC (CD3OD, 500 MHz) spectrum of 9

S15
Figure S14. COSY (CD3OD, 500 MHz) spectrum of 9

S16
Figure S15. HMBC (CD3OD, 500 MHz) spectrum of 9

S17
Figure S16. NOESY (CD3OD, 500 MHz) spectrum of 9

S18
Figure S17. NOESY (CD3OD, 500 MHz) spectrum of 9 (extension)

S19
1,4-methano-3-benzo[d]oxepin-2(1H)-one (7): 1H NMR (500 MHz, CD3OD): δH 7.72 (1H, d,
J = 8.0 Hz, H-6), 7.37 (1H, d, J = 8.0, H-9), 7.19 (1H, s, H-5), 7.12 (1H, t, J = 8.0 Hz, H-7),
7.05 (1H, t, J = 8.0 Hz, H-8), 3.79 (1H, dd, J = 4.0, 9.0 Hz, H-1), 3.48 (1H, dd, J = 4.0, 15.0
Hz, H-10a), 3.10 (1H, dd, J = 9.0, 15.0 Hz, H-10b); 13C NMR (125 MHz, CD3OD): δC 176.5
(C-2), 138.3 (C-5a), 128.6 (C-9a), 124.9 (C-5), 122.6 (C-7), 120.0 (C-8), 119.4 (C-6), 112.3
(C-9), 110.4 (C-4), 57.1 (C-1), 29.6 (C-10).

Figure S18. 1H-NMR (500 MHz, CD3OD) spectrum of 7

S20
Figure S19. 13C-NMR (125 MHz, CD3OD) spectrum of 7

S21
3-(2’,3’-dihydroxy-4’-hydroxymethyl-tetrahydrofuran-1’-yl)pyridine-4,5-diol (8): 1H NMR
(500 MHz, DMSO-d6): δH 8.34 (1H, s, H-2), 8.13 (1H, s, H-6), 7.32 (2H, br s, 4-OH, 5-OH),
5.87 (1H, d, J = 6.0 Hz, H-1’), 5.42 (2H, br s, 2’-OH, 5’-OH), 5.17 (1H, br s, 3’-OH), 4.60
(1H, br s, H-2’), 4.14 (1H, br s, H-3’), 3.96 (1H, q, J = 3.5 Hz, H-4’), 3.68 (1H, br d, J = 12.0
Hz, H-5’a), 3.55 (1H, m, H-5’b); 13C NMR (125 MHz, DMSO-d6): δC 156.1 (C-5), 152.3 (C-
6), 149.0 (C-4), 139.9 (C-2), 119.3 (C-3), 87.9 (C-1’), 85.9 (C-4’), 73.4 (C-2’), 70.6 (C-3’),
61.6 (C-5’).

Figure S20. 1H-NMR (500 MHz, DMSO-d6) spectrum of 8

S22
Figure S21. 13C-NMR (125 MHz, DMSO-d6) spectrum of 8

S23
 5' 3
HO 4' 4 OH O OH HO OH
O 6'
α
2 O
3' ′
5" 3" β 5
2" 1' 1
1" 2' β 6

4"
OH O OH O OH O
1 1α 2

Table S1. 1H (500 MHz) and 13C (125 MHz) NMR data for compounds 1, 1a, and 2
1a 1ab 2c
Position
δC
δH (J in Hz) δC δH (J in Hz) δC
1 - 127.9 (C) 127.5 - 127.8
2,6 131.8 (CH)
7.65 (2H, d, J = 8.5) 131.9 7.76 (2H, d, J = 8.7) 130.5
3,5 116.9 (CH)
6.87 (2H, d, J = 8.5) 116.8 6.94 (2H, d, J = 8.7) 116.0
4 - 161.5 (C) 161.1 - 158.1
1’ - 114.5 (C) 115.2 - 114.2
2’ - 164.0 (C) 164.1 - 163.9
3’ - 111.1 (C) 112.7 - 114.1
4’ - 165.3 (C) 164.6 - 161.5
5’ 108.1 (CH)
6.51 (1H, d, J = 9.0) 103.1 6.69 (1H, d, J = 8.8) 107.7
6’ 131.5 (CH)
7.96 (1H, d, J = 9.0) 131.7 8.23 (1H, d, J = 8.8) 129.2
α 118.4 (CH)
7.66 (1H, d, J = 16.0) 118.2 7.82 (1H, d, J = 15.5) 118.1
β 145.6 (CH)
7.83 (1H, d, J = 16.0) 145.5 7.87 (1H, d, J = 15.5) 144.0
β' - 193.7 (C) 193.4 - 192.2
1” 33.3 (CH2)
4.09 (2H, s) 32.7 4.08 (2H, s) 21.8
2” - 201.9 (C) 198.3 - 121.2
3” - 145.5 (C) 145.1 - 135.8
4” 125.6 (CH2)
6.24 (1H, br s) 124.6 5.86 (1H, s) 18.0
5.88 (1H, br s) 6.21 (1H, s)
5” 17.9 (CH3) 1.91 (3H, s) 17.9 1.85 (3H, s) 25.8
4’-OCH3 - - 56.4 3.89 (3H, s)
a b c
Recorded in CD3OD, in CD3COCD3, in CDCl3

Xanthoangelol K (1a)29) [Li J., Gao L., Meng F., Tang C., Zhang R., Li J., Luo C., Li J., Zhao
W., Bioorg. and Med. Chem. Lett., 25, 2028-2032 (2015).].

S24
 HO
HO OH

HO O 4' OH
HO 4' OH 1"
8 8 3'
HO 9 O HO O HO O 2
2
1" O O 3' 1' R
2"
7
3
HO 2
1'
7
4" 7
6 10 OH 4
3" 5 4 1' 4 5
1"
OH O 5 OH O
5" 4' OH OH O R=H
5
R = OH
3 4 6

Table S2. 13C NMR (125 MHz) data for compounds 3-6
Position 3a 4b 5c 6b
2 152.6 80.7 164.2 166.6
3 123.5 44.1 102.6 103.6
4 180.9 198.5 182.3 184.1
5 159.7 164.6 160.6 162.7
6 110.2 98.0 98.3 99.4
7 161.4 167.0 162.8 164.7
8 94.0 96.9 104.7 105.1
9 156.2 165.1 156.2 158.2
10 106.0 104.9 104.2 105.7
1’ 123.2 130.9 121.8 124.0
2’ 130.4 129.1 129.1 115.0
3’ 115.6 116.3 116.1 147.0
4’ 155.9 159.1 161.3 151.0
5’ 115.6 116.3 116.1 116.7
6’ 130.4 129.1 129.1 120.9
1” 21.5 101.3 73.5 75.3
2” 121.0 74.6 71.1 72.8
3” 136.1 78.2 78.8 80.3
4” 25.8 71.1 70.7 72.3
5” 17.9 77.8 81.9 82.9
6” 62.3 61.5 63.2
a b c
Recorded in CDCl3, in CD3OD, in DMSO-d6

S25
 OH
O
12 11 O 1' OHOH OGlc
7
HO
1 9
2 6 8 10
3 5
4 13
HO HO
9 10

Table S3. 1H (500 MHz) and 13C (125 MHz) NMR data for compounds 9 and 10 in CD3OD
Position 9 10
δC δH mult. (J in Hz) δC δH mult. (J in Hz)
1 34.3 - 35.9 -
2 47.3 1.68 dt (2.5, 14.5) 51.1 1.72 ddd (2.0, 4.0, 12.0)
1.38 dd (3.0, 14.5) 1.13 dd (10.0, 12.0)
3 68.1 4.06 m 67.3 3.74 m
4 42.8 1.83 m 45.5 2.00 m
1.22 m 0.90 m
5 27.3 1.88 m 32.1 1.56 m
6 59.5 1.41 t (10.0) 58.6 1.34 t (10.0)
7 133.8 5.46 dd (10.0, 15.5) 133.2 5.37 dd (10.0, 15.5)
8 135.7 5.54 dd (6.5, 15.5) 136.4 5.56 dd (6.5, 15.5)
9 78.0 4.37 m 77.9 4.37 m
10 21.4 1.31 d (6.5) 21.4 1.30 d (6.5)
11 24.0 1.07 s 21.7 0.89 s
12 33.1 0.86 s 32.2 0.92 s
13 21.7 0.81 d (6.5) 21.8 0.84 d (6.5)
1’ 102.2 4.38 d (8.0) 102.2 4.38 d (8.0)
2’ 75.4 3.22 m 75.4 3.21 m
3’ 78.1 3.36 m 78.1 3.35 m
4’ 71.4 3.36 m 71.4 3.35 m
5’ 77.9 3.22 m 77.9 3.21 m
6’ 62.5 3.82 dd (3.0, 12.0) 62.5 3.82 dd (2.5, 12.0)
3.71 dd (4.5, 12.0) 3.69 dd (5.0, 12.0)

S26
Table S4. 13C NMR data for compounds 11-15
Position 11a 12a 13b 14b 15b
1 39.4 42.6 43.9 39.3 42.3
2 45.3 51.4 52.4 56.6 50.6
3 66.7 212.0 214.9 213.9 201.2
4 39.3 45.1 46.1 50.0 127.1
5 34.1 36.4 37.7 34.8 167.2
6 76.9 76.9 78.0 57.8 79.9
7 133.0 131.6 133.9 131.5 131.4
8 134.4 135.2 134.9 137.6 135.2
9 68.5 68.2 77.7 77.6 77.3
10 23.9 23.8 21.4 21.4 21.2
11 25.1 24.5 25.3 21.3 24.6
12 24.5 24.4 24.9 31.2 23.4
13 15.8 15.9 16.4 21.7 19.6
1’ 102.6 102.3 102.6
2’ 75.3 75.3 75.1
3’ 78.1 78.1 78.0
4’ 71.5 71.4 71.5
5’ 77.9 77.9 77.9
6’ 62.7 62.6 62.7
a b
Recorded in CDCl3 (150 MHz), in CD3OD (125 MHz)

S27