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AMinorDiterpenoidwithaNew6/5/7/3FusedringSkeletonfrom

Euphorbiamicractina
Ye Tian, Qinglan Guo, Wendong Xu, Chenggen Zhu, Yongchun Yang, Jiangong Shi*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese
Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

S1
Contents

No. Content Page


1 Experimental Section S3
2 Figure S1. The optimized conformer of 1. S4
3 Figure S2. (a) The experimental CD (black) and the calculated ECD (blue, blue-shifted by 11.5) spectra of 1 S4
and the enantiomer (1, red) after blue-shifted by 11.5. (b) The experimental (black) and calculated (red,
blue-shifted by 11.5) UV spectra of 1.
4 Figure S3. The IR spectrum of compound 1. S5
5 Figure S4. The (+)-ESIMS of compound 1. S6
6 Figure S5. The ()-ESIMS of compound 1. S7
7 Figure S6. The (+)-HRESIMS of compound 1. S8
8 The (+)-HRESIMS tabulated data of compound 1. S9
9 Figure S7. The 1H NMR spectrum of compound 1 in (CD3)2CO (500 MHz). S10
10 Figure S8. The 1H NMR spectrum of compound 1 in CD3OD (600 MHz). S11
11 Figure S9. The 13C NMR spectrum of compound 1 in (CD3)2CO (125 MHz) S12
12 Figure S10. The DEPT spectrum of compound 1 in (CD3)2CO (125 MHz) S13
13 Figure S11. The 1H-1H gCOSY spectrum of compound 1 in (CD3)2CO (600 MHz for 1H) S14
1 1 1
14 Figure S12. The regionally enlarged H- H gCOSY spectrum 1 of compound 1 in (CD3)2CO (600 MHz for H) S15
1 1 1
15 Figure S13. The regionally enlarged H- H gCOSY spectrum 2 of compound 1 in (CD3)2CO (600 MHz for H) S16
1 1 1
16 Figure S14. The regionally enlarged H- H gCOSY spectrum 3 of compound 1 in (CD3)2CO (600 MHz for H) S17
17 Figure S15. The gHSQC spectrum of compound 1 in (CD3)2CO (600 MHz for 1H) S18
18 Figure S16. The gHMBC spectrum of compound 1 in (CD3)2CO (600 MHz for 1H) S19
19 Figure S17. The regionally enlarged gHMBC spectrum 1 of compound 1 in (CD3)2CO (600 MHz for 1H) S20
1
20 Figure S18. The regionally enlarged gHMBC spectrum 2 of compound 1 in (CD3)2CO (600 MHz for H) S21
1
21 Figure S19. The regionally enlarged gHMBC spectrum 3 of compound 1 in (CD3)2CO (600 MHz for H) S22
1
22 Figure S20. The regionally enlarged gHMBC spectrum 4 of compound 1 in (CD3)2CO (600 MHz for H) S23
23 Figure S21. The regionally enlarged gHMBC spectrum 5 of compound 1 in (CD3)2CO (600 MHz for 1H) S24
1
24 Figure S22. The regionally enlarged gHMBC spectrum 6 of compound 1 in (CD3)2CO (600 MHz for H) S25
1
25 Figure S23. The regionally enlarged gHMBC spectrum 7 of compound 1 in (CD3)2CO (600 MHz for H) S26
1
26 Figure S24. The regionally enlarged gHMBC spectrum 8 of compound 1 in (CD3)2CO (600 MHz for H) S27
27 Figure S25. The NOE difference spectrum 1 of compound 1 in (CD3)2CO (600 MHz) S28
28 Figure S26. The NOE difference spectrum 2 of compound 1 in (CD3)2CO (600 MHz) S29
29 Figure S27. The NOE difference spectrum 3 of compound 1 in (CD3)2CO (600 MHz) S30
30 Figure S28. The CD (upper curve) and UV (bottom curve) spectra of compound 1 in MeOH S31

S2
Experimental Section
General Experimental Procedures. Optical rotations were measured on a Rudolph Research Autopol III automatic polarimeter
and UV spectra were obtained on a Cary 300 spectrometer. CD spectra were recorded on a JASCO J-815 CD spectrometer. IR
spectra were recorded on a Nicolet 5700 FT-IR microscope instrument (FT-IR microscope transmission). NMR spectra were
obtained at 600 MHz for 1H and 150 MHz for 13C, respectively, on an Inova 600 MHz spectrometer with solvent peaks being used
as references. ESIMS and HRESIMS data were measured with a Q-Trap LC/MS/MS (Turbo ionspray source) spectrometer.
Column chromatography was performed using silica gel (200-300 mesh, Qingdao Marine Chemical Inc., China), Sephadex LH-20
(Pharmacia Biotech AB, Uppsala Sweden) and MCI gel (CHP20P). Preparative thin-layer chromatography was performed using
high-performance silica gel preparative TLC plates (HSGF254, glass precoated, Yantai Jiangyou Silica Gel Development Co., Ltd.,
Yantai, Peoples Republic of China). High-performance liquid chromatography (HPLC) was performed on a Waters 600, using a
Waters 2487 dual absorbance detector and a Prevail (250 10 mm i.d.) C18 column (5 m). TLC was carried out on precoated
glass silica gel GF254 plates. Spots were directly visualized under UV light or by spraying with 5% H2SO4 in 95% EtOH, followed
by heating. Unless otherwise noted, all chemicals were commercially available and used without further purification.
Plant Material. The roots of Euphorbia micractina were collected at Zhang County, Gansu Province, Peoples Republic of
China in September 2002. The plant was identified by Associate Professor Lin Ma (Institute of Materia Medica, Beijing). A
voucher specimen (no. 02086) was deposited at the Herbarium of the Department of Medicinal Plants, Institute of Materia Medica.
Extraction and Isolation. The air-dried root of Euphorbia micractina (11.2 kg) was powdered and extracted with 30 L of 95%
EtOH at room temperature for 3 48 h. The EtOH extract was evaporated under reduced pressure to yield a dark brown residue
(1092 g). The residue was suspended in H2O (1500 mL) and then partitioned with EtOAc (4 1000 mL). After removing the
solvent, the EtOAc extract (323.5g) was subjected to CC over silica gel, eluting with a gradient of increasing acetone
concentrations (0100%) in petroleum ether, to afford 20 fractions (A1A20) on the basis of TLC analysis. Fraction A11 (10.1 g)
was separated by normal-phase silica gel CC, eluting with a gradient of acetone (1050%) in petroleum ether, to give fraction
A11-1A11-12. Subsequent fractionation of fraction A11-8 (1.3 g) by flash chromatography over RP silica gel, eluting with a
gradient of EtOH (095%) in H2O, afforded fractions A11-8-1A11-8-6. Fraction A11-8-3 (80 mg), which showed activity against
HIV-1 replication with an inhibition rate of 32% at 10 mg/ml, was chromatographed over Sephadex LH-20, eluting with petroleum
ether-CHCl3-MeOH (5:5:1), and then purified by RP HPLC, using MeOH-H2O (60:40) as the mobile phase, to yield 1 (1.2 mg).
ECD and UV Calculations of 1. Conformational analysis of 1 was performed by using the MMFF94 molecular mechanics force
field via the MOE software package.1 The molecule of 1 showed one lowest energy conformer whose relative energy within 2
kcal/mol (Figure S1). The conformer was further optimized at the B3LYP/6-31g(d, p) level in the gas phase. The energies,
oscillator strengths, and rotational strengths of the first 30 electronic excitations were calculated using the TDDFT methodology at
the B3LYP/6-311G (2d, 2p) level. ECD spectrum of the conformer was simulated using a Gaussian function with a
half-bandwidth of 0.33 eV. The corresponding theoretical ECD spectrum of the enantiomer 1 was depicted by inverting that of 1.
All quantum computations were performed using Gaussian 03 program package,2 on an IBM cluster machine located at the High
Performance Computing Center of Peking Union Medical College. In the 200-400 nm region, the theoretically calculated ECD and
UV spectra of 1 were in good agreement with the experimental ECD and UV spectra (Figure S2). This supported the assignment of
the absolute configuration for 1.

(1) MOE 2009.10, Chemical Computing Group Inc., www.chemcomp.com.


(2) Gaussian03, Gaussian, Inc., www.gaussian.com.
S3
Figure S1. The optimized conformer of 1.

(a) (b)

Figure S2. (a) The experimental CD (black) and the calculated ECD (blue, blue-shifted by 11.5) spectra of 1 and the enantiomer (1,
red) after blue-shifted by 11.5. (b) The experimental (black) and calculated (red, blue-shifted by 11.5) UV spectra of 1.

S4
Figure S3. The IR spectrum of compound 1.

S5
Figure S4. The (+)-ESIMS of compound 1.

S6
Figure S5. The ()-ESIMS of compound 1.

S7
Figure S6. The (+)-HRESIMS of compound 1.

S8
The tabulated (+)-HRESIMS data of compound 1.

S9
(CD3) 2CO

H2O

Figure S7. The 1H NMR spectrum of compound 1 in (CD3)2CO (500 MHz).

S10
H2O
CD3OD

Figure S8. The 1H NMR spectrum of compound 1 in CD3OD (600 MHz).

S11
(CD3) 2CO
(CD3) 2CO

Figure S9. The 13C NMR spectrum of compound 1 in (CD3)2CO (125 MHz).

S12
Figure S10. The DEPT spectrum of compound 1 in (CD3)2CO (125 MHz).

S13
(CD3) 2CO

H2O

Figure S11. The 1H-1H gCOSY spectrum of compound 1 in (CD3)2CO (600 MHz for 1H).

S14
H-11H-12 H-8aH-9 H-8bH-9

H-2H3-16

H-7aH-7b
H-7bH-8b
H-7bH-8b
19
10
11 18
HO O H-1bH-2 H-7bH-8b
OH 9
20
1 15 13
H-1aH-1b H-8aH-8b
3 5 7
16
5' 17
O OH
1'
7'
O 1
3'

Figure S12. The regionally enlarged 1H-1H gCOSY spectrum 1 of compound 1 in (CD3)2CO (600 MHz for 1H).

S15
H-3H-4 H-4H-5

H-5OH-5
19
10
11 18
HO O
OH 9
20
1 15 13
3 5 7
16
5' 17
O OH
1'
7'
O 1
3'

Figure S13. The regionally enlarged 1H-1H gCOSY spectrum 2 of compound 1 in (CD3)2CO (600 MHz for 1H).

S16
H-2H-3 H-3H-4
H-5 H-6 H-4 H-5

Figure S14. The regionally enlarged 1H-1H gCOSY spectrum 1 of compound 1 in (CD3)2CO (600 MHz for 1H).

S17
Figure S15. The gHSQC spectrum of compound 1 in (CD3)2CO (600 MHz for 1H).

S18
Figure S16. The gHMBC spectrum of compound 1 in (CD3)2CO (600 MHz for 1H).

S19
H3-16 C-3

H3-17 C-5

H3-20 C-14 H3-20 C-12

O
HO OH
20
15
1
3 5 7
16 17 H-7b C-5
O OH

O 1

Figure S17. The regionally enlarged gHMBC spectrum 1 of compound 1 in (CD3)2CO (600 MHz for 1H).

S20
H3-16 C-1
H3-17 C-13 H3-17 C-6

H3-20 C-13 H3-20 C-6

O
HO OH H-7b C-6
13 20
1

16 17
O OH

O 1
H-7a C-13 H-7a C-6

Figure S18. The regionally enlarged gHMBC spectrum 2 of compound 1 in (CD3)2CO (600 MHz for 1H).

S21
H-11 C-9

H3-18/H3-19 C-9 H3-18/H3-19 C-10


H3-16 C-2 H3-18/H3-19 C-11

H3-17 C-7 H3-18 C-19


H3-19 C-18

H-7b C-9 H-7b C-8

H-7a C-9 H-7a C-8


H-8a C-9
H-8a C-11

Figure S19. The regionally enlarged gHMBC spectrum 3 of compound 1 in (CD3)2CO (600 MHz for 1H).

S22
OH-14 C-15 OH-14 C-14

H-5 C-3

O
HO OH
1 15
5
3
O OH

O 1

H-3 C-14

Figure S20. The regionally enlarged gHMBC spectrum 4 of compound 1 in (CD3)2CO (600 MHz for 1H).

S23
H-12 C-9/C-11

H-12 C-20

OH-14 C-4/C-13

H-5 C-4 H-5 C-17

HO O 12
OH 11 9
20
1
14
5
17
3
O OH

O 1

H-3 C-1 H-3 C-16

Figure S21. The regionally enlarged gHMBC spectrum 5 of compound 1 in (CD3)2CO (600 MHz for 1H).

S24
H-1a C-2

HO O
OH 11 9
20
1 13

5
3
O OH

O 1

H-12 C-9/C-11 H-12 C-20

Figure S22. The regionally enlarged gHMBC spectrum 6 of compound 1 in (CD3)2CO (600 MHz for 1H).

S25
H-1b C-15 H-1b C-14 H-1b C-3

H-1a C-15 H-1a C-3


O
HO 15 OH
1
5
16 3
O OH
H-4 C-14 H-4 C-3/C-5
O 1
H-4 C-15

Figure S23. The regionally enlarged gHMBC spectrum 7 of compound 1 in (CD3)2CO (600 MHz for 1H).

S26
H-3 C-7

H-3,H-5C-1

H-3C-5
H-3/ H-5C-4
H-5 C-3
H-4C-2,C-6

H-2,H-6 C-7
H-2,H-6 C-4
H-2C-6
H-6 C-2

Figure S24. The regionally enlarged gHMBC spectrum 8 of compound 1 in (CD3)2CO (600 MHz for 1H).

S27
OH-14 H3-20
OH-15 OH-5 H2O

H-12

H-5 H-8

OH-15
H2O

H-3 H-4
H-2 H3-16

19 18
H2O (CD3) 2CO
16 10
11
1
15
9
3
13 20

5
7
17

Figure S25. The NOE difference spectrum 1 of compound 1 in (CD3)2CO (600 MHz).

S28
H-9/11
H3-20 H3-18

H3-20

H-4 H3-17

H3-17
OH-14 H-9/11
H3-20

H-5
H-12 H3-19

19 18

16 10
11
1
15
9
3
13 20

5
7
17

Figure S26. The NOE difference spectrum 2 of compound 1 in (CD3)2CO (600 MHz).

S29
H3-17
H-3

OH-14 H-4
OH-5 H-2

19 18

16 10
11
1
15
9
3
13 20

5
7
17

Figure S27. The NOE difference spectrum 3 of compound 1 in (CD3)2CO (600 MHz).

S30
Figure S28. The CD (upper curve) and UV (bottom curve) spectra of compound 1 in MeOH, with the 3D structure viewed from the
para position of benzene ring and a projection of the six-membered ring moiety in the benzoate sectors.

S31