Psoracorylifols A–E, five novel compounds with activity against Helicobacter pylori from seeds of Psoralea corylifolia

Tetrahedron 62 (2006) 2569–2575

Psoracorylifols A–E, five novel compounds with activity against Helicobacter pylori from seeds of Psoralea corylifolia Sheng Yin, Cheng-Qi Fan, Lei Dong and Jian-Min Yue* State Key Laboratory of Drug Research, Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai, 201203, People’s Republic of China Received 26 August 2005; revised 15 December 2005; accepted 16 December 2005 Available online 19 January 2006

Abstract—Five novel compounds, psoracorylifols A–E (1–5) with important activity against Helicobacter pylori have been isolated from a well-known traditional Chinese medicine (TCM), the seeds of Psoralea corylifolia. The structures of compounds 1–5, including their absolute configurations, were established on the basis of spectral methods and biogenetic reason. The structure of 1 was confirmed by a single-crystal X-ray diffraction. Psoracorylifols D and E (4 and 5) represent an unprecedented carbon skeleton. The biogenetic origin of psoracorylifols A–E (1–5) was also postulated. q 2005 Elsevier Ltd. All rights reserved.

1. Introduction Five natural Helicobacter pylori inhibitors, psoracorylifols A–E (1–5) were isolated from the seeds of Psoralea corylifolia L. (Fabaceae), which is a well-known traditional Chinese medicine (TCM), and has been applied to cure gynecological bleeding, vitiligo and psoriasis.1 A series of compounds2,3 isolated from this TCM showed important biological activities, such as antibacterial,2a,b,4 antiplatelet,5a DNA polymerase and topoisomerase II inhibition.5b H. pylori infection is closely associated with gastritis, peptic ulcer, and gastric cancer, and eradication of the infection is now recommended as the primary therapy for patients with peptic ulcer disease.6 Psoracorylifols A–E (1–5) showed important inhibitory activity against two strains of H. pylori (SS1 and ATCC 43504) at the level of MICs of 12.5–25 mg/mL, especially against H. pylori-ATCC 43504, a drug resistant strain with the MIC of 128 mg/mL to resist metronidazole. Metronidazole is a main ingredient for the combination therapies of H. pylori infection. However, metronidazole resistant H. pylori has been reported worldwide and drug resistance has now become one of major reasons for the failure of antimicrobial therapies.6 The structures of psoracorylifols A–E (1–5) were elucidated by spectral methods. Their absolute configurations were proposed on the basis of biogenetic reason and demonstrated by CD spectra. The structure of 1 was confirmed by a Keywords: Psoracorylifols A–E; Anti Helicobacter pylori; Structure elucidation; Biosynthesis; Natural products. * Corresponding author. Tel./fax: C86 21 50806718; e-mail: [email protected] 0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2005.12.041

single-crystal X-ray diffraction. The origin of psoracorylifols A–E (1–5) could be biogenetically traced back to (S)-(C)-bakuchiol (6), a coexisting major compound. We describe herein the isolation, structure elucidation and biosynthetic ways of these antimicrobial compounds. OH 18 H 7

H 3

1

8

O HO

16

9

17

H H O

11

HO

12 13

5 14

O HO

O

15

O

2

1 H 1

3

HO

H H

10

H

7

15 14 5

18

8 13

4

H

16 17 9

H

10 11

HO

12

O

3

H

O

H 5

2. Results and discussion Psoracorylifol A (1) showed the molecular formula of C18H24O3 as deduced by HREIMS. The IR absorptions indicated the presence of hydroxyls (3473 and 3265 cmK1) and aromatic ring (1616 and 1518 cmK1). From its 1H and 13 C NMR data (Tables 1 and 2), six typical aromatic carbons for a 1,4-substituted benzene ring were easily distinguished, the remaining twelve carbons were attributable to the functionalities of two methyls, four methylenes (two olefinic), four methines (one olefinic and three oxygenated) and two quaternary carbons (one olefinic), indicating existence of two terminal double bonds. The structural assignment of 1 was fully achieved by interpretation of 2D NMR including HMQC, HMBC and NOESY (Fig. 1a)

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Table 1. 1H NMR data of compounds 1–5 (CD3OD)a Protons

1

2

3

4

5

2/6 3/5 7 8 10a 10b 11a 11b 12 13 14

7.12 (d, 8.4) 6.70 (d, 8.4) 4.63 (d, 5.3) 3.38 (d, 5.3) 1.62 (d, 7.5)b 1.60 (d, 7.5)b 1.98 (m) 1.85 (m) 4.40 (t, 4.5)

7.12 (d, 8.5) 6.67 (d, 8.5) 5.04 (br s) 3.83 (br s) 1.63 (m) 1.76 (m) 1.72 (m) 1.52 (m)

7.12 (d, 8.5) 6.70 (d, 8.5) 4.93 (br s) 3.88 (br s) 2.02b 1.46 (m) 1.66 (m) 1.81 (m)

7.17 (dd, 0.8, 8.6) 6.72 (d, 8.6) 5.23 (br s) 2.09 (br s) 1.99 (dd, 6.7, 14.4) 1.58 (m) 1.83 (m) 1.66 (m) 3.84 (d, 4.2)

7.12 (d, 8.3) 6.70 (d, 8.3) 4.93 (br s) 2.05 (br s) 1.98b 1.47 (m) 1.80 (m) 1.99b 3.88 (d, 4.3)

1.95 (m) 0.94 (3H, d, 6.9)

2.02b 1.02 (3H, d, 7.0)

1.13 (3H, s)

0.76 (3H, s)

0.93 (3H, d, 6.9) 6.03 (dd, 10.9, 17.7) 4.99 (dd, 1.1, 10.9) 5.06 (dd, 1.1, 17.7) 0.98 (3H, s)

1.07 (3H, d, 7.0) 5.85 (dd, 11.0, 17.6) 5.15 (dd, 1.0, 11.0) 5.08 (dd, 1.0, 17.6) 1.18 (3H, s)

0.73 (3H, s) 6.10 (dd, 10.9, 17.8) 4.93 (dd, 0.8, 10.9) 5.05 (d, 17.8) 1.08 (3H, s)

1.29 (3H, s) 6.00 (dd, 10.9, 17.6) 4.98 (d, 10.9) 4.95 (d, 17.6) 1.31 (3H, s)

a 4.89 (br s) b 4.91 (br s) 1.55 (3H, s) 6.40 (dd, 11.0, 17.7) 4.97 (dd, 1.5, 17.7) 5.02 (dd, 1.5, 11.0) 0.84 (3H, s)

15 16 17a 17b 18 a b

Data were recorded at 400 MHz, chemical shifts are in ppm, and the coupling constant J is in Hz (in parentheses). Signals were overlapped in the same vertical column.

Table 2. 13C NMR data of compounds 1–5 (CD3OD)a Carbons

1

2

3

4

5

1 2/6 3/5 4 7 8 9 10 11 12 13 14 15 16 17 18

135.6 130.0 116.0 158.2 74.5 83.6 40.0 35.7 24.5 76.9 145.9 113.3 20.4 144.7 112.7 25.3

135.3 129.0 116.3 158.3 79.6 90.5 40.0 30.1 27.7 113.5 37.0 18.1 18.7 145.5 113.0 24.1

135.2 129.0 116.3 158.3 80.2 89.7 40.2 28.4 27.3 113.9 37.0 18.1 18.7 146.2 114.6 23.4

138.0 126.6 116.2 157.0 81.9 60.9 42.5 28.3 27.7 85.8 44.6 25.3 30.0 151.2 109.5 32.9

138.1 126.7 116.2 157.0 83.9 60.2 42.9 29.4 27.3 85.8 44.6 30.9 24.9 153.4 111.1 29.3

a

Data were recorded at 100 MHz, chemical shifts (d) are in ppm.

spectra, and was finally confirmed by a single-crystal X-ray diffraction (Fig. 1b). Psoracorylifol B (2) gave the molecular formula C18H24O3 as determined by HREIMS. IR implied the existence of hydroxyl (3332 cmK1) and aromatic ring (1616 and 1518 cmK1). Analysis of its 1H and 13C NMR data (Tables 1 and 2) has led to identification of all the functionalities. One benzene ring and a terminal double bond accounted for five degrees of unsaturation, according to the molecular

formula, two additional rings in 2 were required. HMQC and 1 H–1H COSY spectra revealed three structural fragments of –CH]CH2, –CH2CH2– and –CH(CH3)2. The linkage of structural fragments, quaternary carbons, oxygen atoms and other functional groups were furnished by HMBC spectrum (Fig. 2) to outline the skeleton of 2. In the HMBC, H-7 correlated with C-1 and C-2/6 to attach the benzene ring to the C-7; H-8 correlated with C-7, and H-7 correlated with C9 to tentatively link C-7 and C-9 to C-8 (although C-7 and C8 are bearing protons, the correlation between H-7 and H-8 was not observed in the 1H–1H COSY due to the unfavorable dihedral angle between the two protons); H-10, H-16 and H18 showed correlations with the quaternary carbon C-9 to locate C-10, C-16 and C-18 at C-9; H2-11 and H-13 correlated with the quaternary carbon C-12 at d 113.5 to connect the whole carbon backbone together; both H-7 and H-8 at the oxygenated carbons correlating with C-12 strongly, indicated that C-7, C-8 and C-12 were connected via a ketal group. The presence of one ketal group was supported by the quaternary carbon at d 113.5 (C-12) and two tertiary carbon signals at 79.6 (C-7) and 90.5 (C-8).7,8,10 The relative configuration and the conformation of 2 were mainly assigned by NOESY spectrum (Fig. 3). The H-16 correlated with H-11a, indicating that H-11a and vinyl moiety taking axial bonds were at the same side of the sixmembered ring, and were arbitrarily fixed as a-orientation. For the compounds with a 6,8-dioxabicyclo[3,2,1]octane

Figure 1. (a) Selected NOESY correlations of 1; (b) single-crystal X-ray structure of 1.

S. Yin et al. / Tetrahedron 62 (2006) 2569–2575

HO

HO

4

4

2 18 6

17

8

16

7

O

10

O

17

8

7

16

14 10 12

11

15

and

C) and 1H–1H COSY

Figure 3. Key NOESY correlations of 2 and 3 (

6

18

O

13

2

2

15

11 14

Figure 2. Selected HMBC (H

2571

3

4 and

5

of 2–5.

).

core,7–10 like 2, the six-membered ring always takes a chair conformation, and the five-membered ring occupying two axial bonds at the six-membered ring should be envelope conformation. Accordingly, C-7 and its bonding oxygen atom in the five-membered ring might take the two axial bonds at C-8 and C-12 to form the bottom of the envelope, and was definitely assigned as in b-orientation. H-7 correlating with both Me-18 and H-10b showed that the 4-hydroxyphenyl was on the opposite side toward C-10. Psoracorylifol C (3) was determined to share a common planar structure with that of 2 by HREIMS and 2D NMR spectra (Fig. 2, Tables 1 and 2). The relative stereochemistry of 3 was also established by NOESY (Fig. 3). The Me-18 correlating with H-11b indicated that both them took the axial bonds, and were randomly designated as b-configuration. CH-7 and its bonding oxygen atom in the fivemembered ring occupying two axial bonds at C-8 and C-12 were accordingly assigned as a-configured. H-7 correlated with H-10a allowed us to locate the 4-hydroxyphenyl on the opposite direction toward the C-10. In a similar way as that in 2, the solution conformation of 3 was also determined as a chair conformation for the six-membered ring and envelope conformer for the five-membered ring. Psoracorylifols D (4) and E (5) were stereoisomers with a new carbon skeleton, as established by HREIMS and spectral analysis, especially by the strategic application of combined 2D NMR spectra (Fig. 2, Tables 1 and 2). The correlation between H-7 and H-8 in 4 was not observed in the 1H–1H COSY due to the unfavorable dihedral angle between the two protons.7–10 In the HMBC of 4, H-7 correlated with C-1 and C-2/6 enabled us to attach 4-hydroxyphenyl to the C-7. The HMBC correlations of H-7 with C-8 and C-9, and H-8 with C-9 indicated a linkage of C-7, C-8 and C-9. The Me-18, H-16 and H2-10 were all

correlated with the quaternary carbon C-9 to attach Me-18, vinyl group and C-10 to C-9. The C-8, C-14 and C-15 were located to another quaternary carbon C-13 by HMBC correlations of H-8, Me-14 and Me-15 with C-13. The strong HMBC correlation between H-12 and C-7 established the linkage of a 7,12-epoxy. Even though the HMBC correlation between H-12 and C-13 was not observed in 4, the correlations of H-11b/C-13, H-14/C-12 and H-15/C-12 could still direct a connection of C-12 and C-13, which was the only possibility after the other linkages were settled. The relative configuration and conformation of 4 were established by NOESY (Fig. 4). The NOESY correlation between Me-14 and H-16 indicated that Me-14 and vinyl moiety might take the axial bonds, and were designated as a-configuration. CH-7 and its bonding oxygen formed the bottom of furan envelope might occupy the two axial bonds at C-8 and C-12, respectively, and were accordingly b-oriented. The H-7 correlating with both Me-18 and H-10b (d 1.58, m) showed that 4-hydroxyphenyl group was far away from the C-10. The relative stereochemistry of 5 was fixed by using NOESY and NOE difference spectra (Fig. 4). In the NOESY, H-7 correlating with H-10a showed that the 4-hydroxyphenyl was far away from the six-membered ring, and H-10a occupying the axial bond was randomly put in a-oriented; the Me-15 correlated with H-8 and H-11b, indicating that they were at the same side and were b-oriented; both Me-18 and H-16 showed correlations with H-8, and the correlation of Me-15 and Me-18 showed uncertainty as they were nearly overlapped. The NOE difference spectra of 5 were thus performed to assign the relative configuration of Me-18, in which, the interactions of Me-15 (after irradiation) with Me-14 and Me-18, and the interactions of Me-18 (after irradiation) with Me-15 and

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Figure 4. Key NOESY correlations of 4 and 5 (

). NOE difference of 5 (irradiated H

enhanced H).

(9S)-(C)-bakuchiol (6),2b a coexisting major compound (up to 3–6% in the seeds). After oxidation, 6 could be transformed into an intermediate i, which would then undergo rearrangements to give two key intermediates ii and iii. The ii could be transformed to 1 by an acid induced intramolecular rearrangement. The intermediate iii had two isomers 2iii and 3iii (at the epoxy), which could be

H-10b indicated that the Me-18 taking axial bond was b-configuration. These results also indicated that the cyclohexane ring took a chair conformation, and the furan ring was envelope.7–10 The origin of psoracorylifols A–E (1–5) could be rationalized biogenetically (Scheme 1), and traced back to 16 17 9

8

10

7 11

Ar

[O]

O

12

13 14

O

Ar

O

i

15

O

Ar

iii

(9S)-(+)-bakuchiol (6)

H

Ar

Ar

H

HO H

O

O

OH

H

+

ii

2iii

Ar

H

O

-H

-H

Ar

H

O

H H O

H

3iii

-H

H O

O

H

H O

Ar HO

1

-H Ar

iv

H

v

H

Ar

O H

H

H O

H

H

H

H

+

H

Ar

H

O H

4

OH

Ar =

OH

Ar

O H

H

O

3

2

[O]

6

Ar

Ar

H

4v

Scheme 1. Biogenetic pathways proposed for psoracorylifols A–E (1–5).

5v

H

Ar O

5

S. Yin et al. / Tetrahedron 62 (2006) 2569–2575

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Table 3. Absolute configuration and Cotton effects of compounds 1–5 Compounds

1 2 3 4 5

Chiral centers

Cotton effects

C-7

C-8

C-9

C-12

270 nm

227 nm

S R S S R

S R S S R

S S S S S

S R S R S

C K C C K

K C K K C

Figure 5. CD and UV spectra of psoracorylifols A–E (1–5).

transformed to 2 and 3, respectively, by intramolecular cyclizations triggered by an acidic catalysis. The iv produced by epoxidation of 6 would be transformed to the key intermediate v via an acid inducing rearrangement. The key intermediate v (12-hydroxyisobakuchiol11), a mixture of two isomers 4v and 5v, was also isolated in the current research. The intermediates 4v and 5v could be transformed to 4 and 5, respectively, also by an acid catalyzed intramolecular cyclization. The absolute configurations of psoracorylifols A–E (1–5) (Table 3) were proposed on the basis of biogenetic reason and demonstrated by CD spectra. The origin of 1–5 was proposed to be (9S)-(C)-bakuchiol (6), whose absolute configuration was determined by a total synthesis,2b and a hypothesis was thus made that the 9S-configuration were retained at the biosynthetic procedures of 1–5. Accordingly, the absolute configuration of 1–5 could be predicted by referring to (9S)-(C)-bakuchiol (6). Observation of CD spectra of 1–5 (Fig. 5), the patterns of Cotton effects corresponding to the UV absorptions around 270 and 227 nm seemed to be closely related with the chiral centers of C-7 and C-8.12 Although it was not determined whether C-7/ and C-8 were corresponded to these Cotton effects, compounds 1, 3 and 4 proposed as 7S,8S-configurations showed positive chirality (positive first Cotton effect around 270 nm and negative second Cotton effect centered at 227 nm); while compounds 2 and 5 with 7R,8Rconfigurations gave negative chirality (Cotton effects are totally reversed by comparing with those of 1, 3 and 4). The CD spectra of 1–5 showed good consistency with the absolute structures predicted on the biogenetic reason. Psoracorylifols A–E (1–5) were tested on the antimicrobial assay against H. pylori (Hp) in vitro, and metronidazole was

used as the positive control (Table 4). All the tested compounds showed significant inhibitory activity against two strains of H. pylori (SS1 and ATCC 43504) at the level of MICs of 12.5–25 mg/mL, the latter one was a drug (metronidazole) resistant Hp strain.6 It is remarkable that psoracorylifols A–E (1–5) are more stronger (5–10 times) than metronidazole, a critical ingredient for combination therapies of H. pylori infection, against H. pylori ATCC 43504. Table 4. Inhibitory activities of compounds 1–5 against H. pylori Samples

MICs (mg/mL) Hp ATCC 43504 Hp SS1

1 2 3 4 5 Metronidazole

25 12.5 12.5 12.5 25 128

25 12.5 12.5 12.5 25 0.5

3. Experimental 3.1. General experimental procedures Melting points were measured with an SGWX-4 apparatus and uncorrected. Optical rotation was determined on a Perkin-Elmer 341 polarimeter, and CD was obtained on a Jasco 810 spectrometer. UV was measured on Varian Cary 300 BIO spectrometer. IR spectra were recorded on a Perkin-Elmer 577 spectrometer. NMR spectra were measured on a Bruker AM-400 spectrometer with TMS as internal standard. EIMS (70 eV) were carried out on a

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Finnigan MAT 95 mass spectrometer. Semi-preparative HPLC was performed on a Waters 515 pump with a Waters 2487 detector (254 nm), and a YMC-Pack ODS-A column (250!10 mm, S-5 mm, 12 nm) was used. 3.2. Plant material The seeds of P. corylifolia were harvested in September 1999 from Anhui Province of China, and authenticated by Dr. Y. Xiang of Shanghai Institute of Materia Medica, where a voucher specimen has been deposited (Accession number Pc-1999-1Y). 3.3. Extraction and isolation The dried seeds powder of P. corylifolia (2.0 kg) was extracted with 95% EtOH to give 592 g crude, and part of which (392 g) was suspended in 2.5 L water and then partitioned with petroleum ether and ethyl acetate successively to give petroleum ether soluble fraction PE (143 g) and ethyl acetate soluble fraction EA (120 g). EA (100 g) was subjected to silica gel column chromatography (CC) eluted with an increasingly gradient of acetone in petroleum ether to obtain nine fractions Frs 1–9 according to TLC monitor. Frs 1–4 (total 46 g) mainly contained bakuchiol (6) (Scheme 1). Fr 5 (4.5 g) was further separated on a silica gel CC eluted with CHCl3 to afford subfractions Frs 5a–5e. Fr 5c was purified on a silica gel column eluted with petroleum ether–EtOAc (6/1) to offer 1 (31 mg, recrystallized in acetone). Fr 5a (0.72 g) was treated on a Sephadex LH-20 column eluted with EtOH to obtain a major mixture, which was then separated on a silica gel CC eluted with petroleum ether–EtOAc (15/1–3/1) to give two major gum-like mixtures Frs 5a-1 and 5a-2, each of them was purified by preparative HPLC with the mobile phase of 60% CH3CN in water (at flow rate of 3 mL/min), Fr 5a-1 yielded compounds 2 (12.3 mg) and 3 (10.2 mg), and Fr 5a-2 gave compounds 4 (7.5 mg) and 5 (5.4 mg). 3.3.1. Psoracorylifol A (1). Colorless needles from MeOH; mp 152–154 8C; [a]20 D C39.5 (c 1.30, MeOH); UV (MeOH): lmax (log 3)Z226 (4.00), 277 (3.19) nm; IR (KBr): nmaxZ3473, 3265, 1616, 1518, 1452 cmK1; 1H and 13 C NMR: see Tables 1 and 2; EIMS (70 eV): m/z (%): 288 [M]C (2), 270 (13), 189 (10), 165 (34), 123 (100), 107 (26); HREIMS: m/z 288.1729 [M]C (calcd for C18H24O3: 288.1725). 3.3.2. Psoracorylifol B (2). Colorless gum; [a]20 D 0 (c 0.61, MeOH); UV (MeOH): lmax (log 3)Z227 (4.06), 277 (3.23) nm; IR (KBr disc): nmaxZ3332, 1616, 1518, 1448 cmK1; 1H and 13C NMR: see Tables 1 and 2; EIMS (70 eV): m/z (%): 288 [M]C (3), 200 (34), 185 (100), 172 (9), 153 (9), 136 (16), 123 (10), 107 (29); HREIMS: m/z 288.1717 [M]C (calcd for C18H24O3: 288.1725). 3.3.3. Psoracorylifol C (3). Colorless gum; [a]20 D K30.7 (c 0.39, MeOH); UV (MeOH): lmax (log 3)Z227 (4.09), 277 (3.19) nm; IR (KBr): nmaxZ3357, 1612, 1516, 1443 cmK1; 1 H and 13C NMR: see Tables 1 and 2; EIMS (70 eV): m/z (%): 288 [M]C (3), 200 (32), 185 (100), 172 (9), 153 (19), 136 (21), 123 (8), 107 (38); HREIMS: m/z 288.1722 [M]C (calcd for C18H24O3: 288.1725).

3.3.4. Psoracorylifol D (4). Colorless gum; [a]20 D C21.0 (c 0.22, MeOH); UV (MeOH) lmax (log 3)Z226 (3.95), 280 (3.17) 363 (3.14), 381 (3.10) nm; IR (KBr): nmaxZ3394, 1614, 1514, 1469 cmK1; 1H and 13C NMR: see Tables 1 and 2; EIMS (70 eV): m/z (%): 272 [M]C (8), 150 (30), 135 (78), 121 (25), 107 (100); HREIMS: m/z 272.1780 [M]C (calcd for C18H24O2: 272.1776). 3.3.5. Psoracorylifol E (5). Colorless gum; [a]20 D K69.0 (c 0.16, MeOH); UV (MeOH): lmax (log 3)Z226 (3.86), 279 (3.12) nm; IR (film): nmaxZ3346, 1614, 1514, 1444 cmK1; 1 H and 13C NMR: see Tables 1 and 2; EIMS (70 eV): m/z (%): 272 [M]C (18), 257 (18), 187 (76), 149 (100), 135 (44), 121 (94), 107 (70); HREIMS: m/z 272.1767 [M]C (calcd for C18H24O2: 272.1776). 3.4. X-ray crystallographic analysis of 1 Single-crystals suitable for X-ray analysis were obtained by recrystallization from methanol. All measurements were made on a Rigaku AFC7R four circle diffractometer employing graphite monochromated Mo Ka radiation (lZ ˚ ) at 293 K and operating in the f–u scan mode. 0.71073 A Crystal data: C18H24O3, (Mr 288.17), monoclinic, space ˚ , bZ6.162(8) A ˚ , cZ group P2(1), aZ10.896(15) A 3 ˚ ˚ 11.850(16) A, bZ98.899(2)8, VZ786.03(18) A , ZZ2, D calcdZ1.22 g/cm 3, F(000)Z312 and m(Mo Ka)Z 0.081 mmK1. The structure was solved by direct methods (SHELXS-97) and refined with full-matrix least-squares calculations on F2 using SHELXL-97. Copies of the data can be obtained, free of charge, on application to CCDC (CCDC deposition number: 275109), 12 Union Road, Cambridge CB2 1EZ, UK [fax: 44 1223 336033 or e-mail: [email protected]]. 3.5. In vitro test of anti-H. pylori activity by agar dilution method6 A series of agar plates were prepared with the base of Campylobacter selective agar (Merck) containing 5% of fetal bovine serum, and various concentrations of two-fold diluted samples (1–5) were dispersed into the culture medium. To the well-prepared agar plates, H. pylori (Hp SS1 or ATCC 43504 strain) cells suspended in saline at the density of 108 CFU/mL were inoculated and incubated at 37 8C for 96 h under an atmosphere of 5% O2, 10% CO2 and 85% N2. The blank controls (H. pylori cultures in the agar plates, no test samples were dispersed) and the positive controls (H. pylori cultures in the agar plates dispersed with various concentrations of two-fold diluted metronidazole) were incubated under the same condition. The MIC was defined as the lowest concentration of test samples, at which the visible growth was completely inhibited. All measurements were repeated three times under the same condition.

Acknowledgements National Natural Science Foundation of China (30025044), Foundation from the Ministry of Science and Technology of China (2002CB512807), and Shanghai Municipal Scientific

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Foundation (03DZ19529; 04XD14019) are gratefully acknowledged.

4.

References and notes 5. 1. Ou, M. Chinese-English Manual of Common-used in Traditional Chinese Medicine; Guangdong Scientific and Technologic: Guangzhou, 1992; p 535. 2. (a) Mehta, G.; Nayak, U. R.; Dev, S. Tetrahedron 1973, 29, 1119–1125. (b) Prakasa Rao, A. S. C.; Bhalla, V. K.; Nayak, U. R.; Dev, S. Tetrahedron 1973, 29, 1127–1130. (c) Khanna, P. L.; Seshadri, T. R. Curr. Sci. 1971, 40, 505. 3. (a) Bhalla, V. K.; Nayak, U. R.; Dev, S. Tetrahedron Lett. 1968, 20, 2401–2406. (b) Jain, A. C.; Gupta, G. K.; Rao, P. R. Indian J. Chem. 1974, 12, 659–660. (c) Bajwa, B. S.; Khanna, P. L.; Seshadri, T. R. Indian J. Chem. 1974, 12, 15–19. (d) Zhu, D. Y.; Chen, Z. X.; Zhou, B. N.; Liu, J. S.; Huang, B. S.; Xie, Y. Y.; Zeng, G. F. Yaoxue Xuebao 1979, 14, 605–611. (e) Suri,

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