three new phloroglucinol derivatives from hypericum scabrum

NOTE

Three new phloroglucinol derivatives from Hypericum scabrum

Jie Ma, Teng-Fei Ji, Jian-Bo Yang, Ai-Guo Wang and Ya-Lun Su*

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute ofMateria Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,

Beijing 100050, China

(Received 27 February 2012; final version received 26 March 2012)

A chemical investigation on the aerial parts of Hypericum scabrum L. resulted in theisolation of three new phloroglucinol derivatives, hyperscabrins A (1), B (2), and C (3),together with one known compound, (2R,3R,4S,6R)-3-methyl-4,6-di(3-methyl-2-butenyl)-2-(2-methyl-1-oxopropyl)-3-(4-methyl-3-pentenyl)-cyclohexanone (4). Thestructures were elucidated by means of spectroscopic methods, including MS, IR, 1DNMR, and 2D NMR, and the absolute configurations of chiral centers in thesephloroglucinol derivatives were determined for the first time by studying their circulardichroism spectra.

Keywords: Guttiferae; Hypericum scabrum; phloroglucinol

1. Introduction

Hypericum scabrum L. is a herbaceous

perennial plant, belonging to the family

Guttiferae, which only distributes in

Xinjiang Uygur Autonomous Region,

China. So far, only a few papers reported

the chemical constituents and pharmaco-

logical activities of this medicinal plant.

Liu et al. [1] reported that the ethanolic

extract exhibited anti-bacterial activities

against Staphylococcus aureus, Escheri-

chia coli, and Pseudomonas aeruginosa. In

the course of searching bioactive com-

ponents from Chinese medicinal plants, a

chemical investigation on the ethanolic

extract of the aerial parts ofH. scabrumwas

conducted. This resulted in the isolation of

four phloroglucinol derivatives (1–4)

including three new prenylated phloroglu-

cinols (1–3), together with one known

phloroglucinol (4) (Figure 1). The known

phloroglucinol was previously reported as

(2R,3R,4S,6S)-3-methyl-4,6-di(3-methyl-

2-butenyl)-2-(2-methyl-1-oxopropyl)-3-

(4-methyl-3-pentenyl)-cyclohexanone [2],

however, the configuration of C-6 should

be 6R instead of 6S according to the

nomenclature of organic chemistry [3]. The

structures of new compounds are eluci-

dated as (2 )-(2S,4S,6R)-3,3-dimethyl-4,6-

di(3-methyl-2-butenyl)-2-(2-methyl-1-

oxopropyl)-cyclohexanone (1), (þ )-



(4-methyl-3-pentenyl)-cyclohexanone (2),

and (þ )-(2R,3R,4S,6S)-3-methyl-4,6-di(3-

methyl-2-butenyl)-2-(2-methyl-1-oxobu-

tyl)-3-(4-methyl-3-pentenyl)-cyclohexa-

none (3) by means of spectroscopic

analysis and chromatographic methods.

Herein, we report the isolation and

structural elucidation of new compounds,

together with the absolute configuration of

the known compound.

ISSN 1028-6020 print/ISSN 1477-2213 online

q 2012 Taylor & Francis

http://dx.doi.org/10.1080/10286020.2012.680445

http://www.tandfonline.com

*Corresponding author. Email: [email protected]

Journal of Asian Natural Products Research

Vol. 14, No. 5, May 2012, 508–514

http://dx.doi.org/10.1080/10286020.2012.680445

http://www.tandfonline.com

2. Results and discussion

Compound 1 was isolated as a colorless

oil, which showed a red spot on thin layer

chromatography (TLC) plates when

sprayed with anisaldehyde–sulfuric acid

reagent. The HR-ESI-MS of 1 exhibited a

pseudo-molecular ion at m/z 333.2781

[M þ H]þ, consistent with a molecular

formula of C22H36O2. The IR spectrum

showed an absorption peak at 1693 cm21,

indicative of the presence of carbonyl

groups. The 1H NMR spectrum of 1

showed the presence of four methyl groups

[d 1.52 (s), 1.52 (s), 1.60 (s), and 1.65 (s)]

and two vinylic protons [d 4.97 (t,

J ¼ 7.2 Hz) and 5.09 (t, J ¼ 7.2 Hz)],

suggesting the presence of two prenyl

side chains. The presence of one isopropyl

group was indicated by two methyl

doublets [d 0.98 (d, J ¼ 7.2Hz) and 0.96

(d, J ¼ 7.6Hz)] and a related proton signal

[d 2.61 (m)]. The NMR data analysis of

1 indicated that it has the same

planar structure as garcinielliptone N [4].

The difference from them was the

stereochemistry at C-4 and C-6. By

extensive comparison, the NMR data of 1

with that of garcinielliptone N, the

chemical shift values of H-4 and H-6

were shifted toward downfield by Dd 0.81

and 0.30 ppm, and those of C-4 and C-6

were shifted toward upfield by Dd 8.20 and

3.40 ppm, respectively. The HMBC corre-

lations from H-2 to C-4, C-6, and C-11,

H2-13 to C-3 and C-5, and H2-18 to C-1

and C-5 (Figure 2) further confirmed the

structure of 1, which was established as

shown in Figure 1.

The relative configuration at C-2, C-4,

and C-6 was assigned on the basis of

NOESY correlations of H-2/H-5b, H-

2/H3-11, H3-11/H-5b, H-4/H-6, H-5a/H-

4, and H-5a/H-6 (Figure 3). Thus, prenyl

groups at C-4 and C-6 oriented to the same

side, while 1-oxo-2-methylpropyl group at

C-2 was on the opposite side.

The absolute configuration of 1 was

determined by circular dichroism (CD)

method. Compound 1 contains a cyclohex-

anonemoiety, and this prompts us to study its

CD spectrum by applying the octant rule [5].

OO1

35

7 9

1011

1213

151617

18

20

21

22

1

4

OO

OO

Garcinielliptone N

O O

R

2 R = CH3

3 R = CH2CH3

345

8

9

11

12

14

1617

18

202122

2325

26

27

1

10

10 28

Figure 1. Structures of compounds 1–4.

Journal of Asian Natural Products Research 509

According to this rule, a negative cotton

effect at 318 nm was correlated with the

conformation as shown in Figure 1. There-

fore, the structure of compound 1 was

elucidated as (2)-(2S,4S,6R)-3, 3-dimethyl-

4,6-di(3-methyl-2-butenyl)-2-(2-methyl-1-

oxopropyl)-cyclohexanone (Figure 1),

named hyperscabrin A.

Compound 2 was also obtained as a

viscous colorless oil. It exhibited a quasi-

molecular ion peak at m/z 401.3450

[M þ H]þ in HR-ESI-MS, consistent with

a molecular formula of C27H44O2. IR, UV,

and 1H NMR spectra of 2 were almost

identical with those of (2R,3R,4S,6R)-3-

methyl-4,6-di(3-methyl-2-butenyl)-2-(2-

methyl-1-oxopropyl)-3-(4-methyl-3-pente-

nyl)-cyclohexanone (4). Extensive analysis

of 2D NMR spectra (HSQC and HMBC)

revealed that compound 2 shared the same

planar structure as 4 (Figure 1). The relative

configuration at all chiral centres of 2 was

determined by interpretation of NOESY

correlations. In addition, significant NOE

enhancements (Figure 3) were observed

between H2-23 and H-2, and between H-4

and H2-23, as well as between H-6 and H-

5b. So it was determined that the configur-

ation ofC-6 in 2was different from that in 4.

The absolute configuration of com-

pound 2was also elucidated by CDmethod

according to the octant rule. In the CD

spectrum, it showed the positive sign at

300.5 nm (Figure 4). Hence, the structure of

compound 2 was assigned as (þ )-



(4-methyl-3-pentenyl)-cyclohexanone,

named hyperscabrin B.

The molecular formula of compound 3

was determined as C28H46O2, as inferred

from the pseudo-molecular ion peak at m/z

415.3601 [M þ H]þ by positive HR-ESI-

MS, which was 14 amu more than that of

compound 2. The 13C NMR spectrum of 3

was almost identical with that of 2 except for

the appearance of an additional methylene

carbon in 3. Meanwhile, in the 1H NMR

spectrum of 3, a methyl triplet [d 0.88 (3H, t,

J ¼ 7.6Hz, H3-28)] replaced the doublet

methyl in 2. So it is possible that one methyl

group in 2was replaced by an ethyl group in

3.And thiswasconfirmedby the cross-peaks

between H3-28 and H2-10 (d 1.65, 1.24, m),

which in turn correlatedwithH3-9 (d 1.03, d,

O H

HH

H

H

1

O6

54

32

13

O O

H

H

H

H

H

2

2

11

12

184

5

6

23

O O

H

H

H

H

H

3

11

4

2

6

23

12

23

5

Figure 3. Significant NOESY correlations for 1–3.

O O

1

O OO O

32

1028

9

Figure 2. Key HMBC (arrows) and gCOSY (thick lines, 3) correlations of compounds 1–3.

J. Ma et al.510

J ¼ 6.8Hz) in the 1H–1H COSY spectrum

(Figure 2). Similar NOESY correlations,

together with the coupling constants in 3,

secured the assignment of a same relative

configuration as 2 at all chiral centers except

for C-8. Likewise, a positive cotton effect

revealed 3 also shared a same absolute

configuration as previously mentioned

(Figure 4). Hence, the structure of 3 was

determined as (þ)-(2R,3R,4S,6S)-3-methyl-

4,6-di(3-methyl-2-butenyl)-2-(2-methyl-1-

oxobutyl)-3-(4-methyl-3-pentenyl)-cyclo-

hexanone, named hyperscabrin C.

The structure of compound 4 was

identified as (þ )-(2R,3R,4S,6R)-3-methyl-

4,6-di(3-methyl-2-butenyl)-2-(2-methyl-

1-oxopropyl)-3-(4-methyl-3-pentenyl)-

cyclohexanone by comparison of its

spectroscopic (MS and NMR) data with

those reported in literature [2]. In addition,

the positive sign at 298 nm was observed

in the CD spectrum (Figure 4). Thus, the

absolute stereochemistry of compound 4

was established for the first time.

3. Experimental

3.1 General experimental procedures

Optical rotations were measured on a

JASCO P2000 polarimeter (JASCO Inc.,

Tokyo, Japan) using CH2Cl2 as solvent.

UV spectra were determined with a

JASCO V650 spectrophotometer (JASCO

Inc.). IR spectra were carried out on a

Nicolet 5700 using FT-IR Microscope

Transmission (Thermo Nicolet Inc., Wal-

tham, MA, USA). 1D NMR, HSQC, and

HMBC spectra were recorded on SYS-

600, Mercury-400, and Mercury-300 spec-

trophotometers (Varian Inc., Palo Alto,

CA, USA) with trimethylsilane (TMS) as

an internal standard. HR-ESI-MS and ESI-

MS were performed on an Agilent 1100

LC/MSD Trap-SL mass spectrometer

(Agilent Technologies Ltd, Santa Clara,

CA, USA). Silica gel (160–200 mesh and

200–300 mesh; Qingdao Marine Chemi-

cal Factory, Qingdao, China) and sinica

gel H were used for column chromatog-

raphy and silica gel GF-254 (Qingdao

Marine Chemical Factory) was used for

TLC. HPLC experiments were carried out

on a preparative YMC-Pack ODS-A

column (10mm, 250mm £ 20mm; YMC,

Kyoto, Japan) equipped with a Shimadzu

SPD-20A UV spectrophotometric detector

(Shimadzu, Kyoto, Japan).

3.2 Plant material

The aerial parts of H. scabrum were

collected from Wusun Mountain, Xinjiang

Uygur Autonomous Region, China, in

August 2010, and identified by Prof. Jin Li

(Xinjiang Normal University). A voucher

specimen (No. ID-S-2370) was deposited

4

2

0

–2

–4

–5220 250 350

4

3

2

1

Wavelength [nm]

Mol

.CD

300

Figure 4. CD spectra for 1–4.


in the herbarium of the Institute of

Materia Medica, Chinese Academy of

Medical Sciences, Peking Union Medical

College.

3.3 Extraction and isolation

Air-dried aerial parts of H. scabrum

(80 kg) were extracted with 95% EtOH

under reflux. After evaporation of the

solvents under vacuum, the residue

(,11 kg) was suspended in water and

then partitioned with petroleum ether,

ethyl acetate, and n-BuOH successively.

Part of the petroleum ether fraction

(1.2 kg) was chromatographed over three

same silica gel columns (160–200 mesh,

10 £ 45 cm, 500g) in reduced pressure,

eluted by petroleum ether–ethyl acetate

(100:1–50:1–20:1–9:1–4:1, V/V) to give

fractions of 1–60. Fractions 1–4 (392 g,

named Fraction A) were subjected to

column chromatography over silica gel

(160–200 mesh, 10 £ 100 cm, 2100g),

eluting with petroleum ether–Et2O (20:1,

V/V), to give 10 fractions A-0–A-9.

Fraction A-2 (65 g) was further separated

by silica gel H column chromatography

[eluted with petroleum ether–CH2Cl2(1:1), petroleum ether–Et2O (20:1)]

repeatedly to give compounds 1 (4mg)

and 2 (31mg). Fraction A-3 (51 g) was

purified on silical gel H column [eluted

with petroleum ether–Et2O (40:1)] to give

compound 4 (550 mg). Compound 3

(10 mg) was obtained by preparative

HPLC eluted with 100% CH3CN (flow

rate, 6mlmin21; l ¼ 210 nm; t ¼ 48min)

from fraction A-3.

3.3.1 Hyperscabrin A (1)

Viscous colorless oil; ½a�20D 2 85.1 (c 0.06,

CH2Cl2); UV (CH2Cl2) lmax nm: 284 (log

e 3.15); IR (KBr) nmax: 2964, 1693, 1464,

1378, and 1095 cm21; for 1H and 13C

NMR spectral data, see Table 1; HR-ESI-

MS: m/z 333.2781 [M þ H]þ (calcd for

C22H37O2, 333.2788).

3.3.2 Hyperscabrin B (2)

Viscous colorless oil; ½a�20D þ 27.6 (c 0.08,


e 3.05) and 265 (log e 3.01); IR (KBr)

nmax: 2968, 2952, 1726, 1701, 1450, and

1380 cm21; for 1H and 13C NMR spectral

data, see Table 2; HR-ESI-MS: m/z

401.3450 [M þ H]þ (calcd for C27H45O2,

401.3456).

3.3.3 Hyperscabrin C (3)

Viscous colorless oil; ½a�20D þ 22.4 (c 0.10,


e 3.05) and 268 (log e 3.01); IR (KBr)

nmax: 2972, 2933, 1722, 1708, 1462, and

Table 1. 1H and 13C NMR spectral data for 1(600MHz for 1H NMR and 150MHz for 13CNMR, CDCl3, J in Hz, d in ppm).

1

Position C H (J, Hz)

1 208.02 76.5 3.52 (1H, s)3 42.94 40.0 2.35 (1H, tt, J ¼ 11.4 and

3.6)5 34.4 2.03 (1H, m, H-5a)

0.99 (1H, m, H-5b)6 47.5 2.65 (1H, m)7 210.48 43.8 2.61 (1H, m)9 17.8 0.96 (3H, d, J ¼ 7.6)10 17.3 0.98 (3H, d, J ¼ 7.2)11 25.9 0.93 (3H, s)12 21.9 0.74 (3H, s)13 28.0 2.03 (1H, m, H-13a)

1.55 (1H, m, H-13b)14 123.6 5.09 (1H, t, J ¼ 7.2)15 132.416 25.8 1.65 (3H, s)17 17.8 1.52 (3H, s)18 27.5 2.25 (1H, dt, J ¼ 15.0 and

6.6, H-18a)1.85 (1H, dt, J ¼ 15.0,7.2, H-18b)

19 121.8 4.97 (1H, t, J ¼ 7.2)20 132.921 25.8 1.60 (3H, s)22 17.8 1.52 (3H, s)

J. Ma et al.512

1380 cm21; for 1H and 13C NMR spectral

data, see Table 2; HR-ESI-MS: m/z

415.3601 [M þ H]þ (calcd for C28H47O2,

415.3606).

3.4 Determination of absoluteconfiguration of compounds 1–4using the CD method

CH2Cl2 was obtained from Beijing Chemi-

cal Works (Beijing, China) and dried

according to the common procedures.

According to the common procedure, test

solutionswere prepared using 0.60mgml21

of compound 1, 0.80 mgml21 of 2,

0.97mgml21 of 3, and 1.00mgml21 of 4.

At first, the blank solvent CD spectrum was

recorded and then sample solutions were

monitored for three times. The sign of the

diagnostic band between 295 and 325 nm is

correlated to the absolute configuration of

the cyclohexanone. The inherent CD of the

solvent CH2Cl2 was subtracted. In the CD

spectrum, the value of the diagnostic band at

318 nm of compound 1was24.0, the value

of the diagnostic band at 300.5 nm of 2 was

3.5, the value of the diagnostic band at

Table 2. 1H and 13C NMR spectral data for 2 and 3 (300MHz for 1H NMR and 100MHz for 13CNMR, CDCl3, J in Hz, d in ppm).

2 3

Position C H (J, Hz) C H (J, Hz)

1 212.5 212.42 64.2 3.95 (1H, s) 64.6 3.93 (1H, s)3 45.3 45.54 38.0 1.88 (1H, m) 38.0 1.86 (1H, m)5 31.3 1.80 (1H, m, H-5a)

1.62 (1H, m, H-5b)31.3 1.78 (1H, m, H-5a)

1.63 (1H, m, H-5b)6 49.8 2.47 (1H, m) 49.8 2.48 (1H, m)7 211.4 210.88 42.7 2.46 (1H, m) 49.7 2.30 (1H, m)9 18.4 1.03 (3H, d, J ¼ 7.2) 14.6 1.03 (3H, d, J ¼ 6.8)10 17.7 1.05 (3H, d, J ¼ 7.2) 25.3 1.65 (1H, m, H-10a)

1.24 (1H, m, H-10b)11 17.7 1.05 (3H, s) 17.7 1.05 (3H, s)12 37.3 1.46 (2H, m) 37.4 1.46 (2H, m)13 22.0 2.06 (1H, m, H-13a)

1.82 (1H, m, H-13b)22.0 2.05 (1H, m, H-13a)

1.84 (1H, m, H-13b)14 123.6 5.00 (1H, td, J ¼ 7.2 and 1.2) 123.6 4.99 (1H, t, J ¼ 7.2)15 131.8 131.816 25.7 1.67 (3H, s) 25.9 1.67 (3H, s)17 17.7 1.59 (3H, s) 17.7 1.60 (3H, s)18 26.8 2.11 (1H, dd, J ¼ 13.8, 3.6, H-18a)

1.77 (1H, m, H-18b)26.8 2.10 (1H, m, H-18a)

1.75 (1H, m, H-18b)19 123.1 5.07 (1H, t, J ¼ 6.6) 123.1 5.06 (1H, t, J ¼ 8.8)20 132.8 132.821 25.9 1.72 (3H, s) 25.8 1.72 (3H, s)22 18.0 1.61 (3H, s) 17.7 1.60 (3H, s)23 30.6 2.40 (1H, m, H-23a)

2.27 (1H, m, H-23b)30.5 2.40 (1H, m, H-23a)

2.24 (1H, m, H-23b)24 120.9 5.04 (1H, t, J ¼ 7.8) 121.0 5.04 (1H, t, J ¼ 7.2)25 134.0 133.926 25.8 1.71 (3H, s) 25.7 1.71 (3H, s)27 18.0 1.67 (3H, s) 18.0 1.67 (3H, s)28 11.5 0.88 (1H, t, J ¼ 7.6)


300.5 nm of 3 was 3.1, the value at 298 nm

of 4 was 3.1, respectively.

Acknowledgments

The authors acknowledge the Department ofInstrumental Analysis, Institute of MateriaMedica, Chinese Academy of MedicalSciences, and Peking Union Medical Collegefor all spectral analysis. We are all greatlyindebted to Prof. Yi-Kang Si for her help in thedetermination of absolute configuration and toProf. Jin Li of Xinjiang Normal University forthe identification of plant.

References

[1] S.H. Liu, G.S. Jiang, Y.N. Wang, F. Chen,and J.K. AHai, J. Med. Pharm. Chin.Minor. 21, 131 (2005).

[2] M.D. Shan, L.H. Hu, and Z.L. Chen, J. Nat.Prod. 64, 127 (2001).

[3] X.T. Liang, IUPAC Nomenclature ofOrganic Chemistry Section A, B, C, D, E, Fand H, 1979 Edition, et al., (Science Press,Beijing, 1987), p. 615.

[4] J.R. Weng, L.T. Tsao, J.P. Wang, W.R.Wu, and C.N. Lin, J. Nat. Prod. 67, 1796(2004).

[5] D.N. Kirk, Tetrahedron 42, 777 (1986).

J. Ma et al.514

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three new phloroglucinol derivatives from hypericum scabrum

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