introduction microtropis fokienensis dunn 1) (celastraceae) is a small shrub that grows in high...

IntroductionIntroductionMicrotropis fokienensis Microtropis fokienensis DunnDunn1)1)(Celastraceae) is a small shrub that grows in high altitude forests throughout southern China a(Celastraceae) is a small shrub that grows in high altitude forests throughout southern China and Taiwan. Various dihydroagarofuranoid sesquiterpenes, sesquiterpene alkaloids, and triterpenes are widely distributed in nd Taiwan. Various dihydroagarofuranoid sesquiterpenes, sesquiterpene alkaloids, and triterpenes are widely distributed in plants of the family Celastraceae. Many of these compounds exhibit insecticidal, anti-inflammatory, anti-AIDS, and antitumoplants of the family Celastraceae. Many of these compounds exhibit insecticidal, anti-inflammatory, anti-AIDS, and antitumor activities. In our continuing studies on the antitubercular constituents of Formosan plants, over 400 species have been screer activities. In our continuing studies on the antitubercular constituents of Formosan plants, over 400 species have been screened for ned for in vitroin vitro antituberculosis activity to date, and antituberculosis activity to date, and Microtropis fokienensisMicrotropis fokienensis has been found to be one of the active species. In has been found to be one of the active species. In our previous studiesour previous studies2)2), we have reported four new dihydroagarofuranoid sesquiterpenes, fokienagarofuran A (10), fokienagar, we have reported four new dihydroagarofuranoid sesquiterpenes, fokienagarofuran A (10), fokienagarofuran B (11), fokienagarofuran C (12), and fokienagarofuran D (13) from the stem of this plant. Continuing investigation of ofuran B (11), fokienagarofuran C (12), and fokienagarofuran D (13) from the stem of this plant. Continuing investigation of the EtOAc-soluble fraction of the stem of the EtOAc-soluble fraction of the stem of M. fokienensisM. fokienensis led to the isolation of a new led to the isolation of a new -dihydroagarofuranoid sesquiterpene, -dihydroagarofuranoid sesquiterpene, 8-acetoxymutangin (1), along with eight known compounds, mutangin (2), vanillin (3), 8-acetoxymutangin (1), along with eight known compounds, mutangin (2), vanillin (3), pp-hydroxy-benzaldehyde (4), syringic -hydroxy-benzaldehyde (4), syringic acid (5), 2,6-dimethoxy-acid (5), 2,6-dimethoxy-pp-benzoquinone (6), -benzoquinone (6), -sitosterol (7), and a mixture of 1-tetracosanol (8) and 1-hexacosanol (9). The st-sitosterol (7), and a mixture of 1-tetracosanol (8) and 1-hexacosanol (9). The structures of this new compound was determined through spectral analyses. The structural elucidation of this new compound aructures of this new compound was determined through spectral analyses. The structural elucidation of this new compound and antitubercular activities of all isolates will be discussed in this symposium.nd antitubercular activities of all isolates will be discussed in this symposium.

AcknowledgementAcknowledgement This work was supported by a grant from the National Science Council of the This work was supported by a grant from the National Science Council of the Republic of China (NSC 93-2320-B-127-003).Republic of China (NSC 93-2320-B-127-003).

Results and DiscussionResults and DiscussionThe antituberculosis effects of the isolated compounds from the stems of The antituberculosis effects of the isolated compounds from the stems of M. fokienensis M. fokienensis were tested were tested in vitroin vitro against against Mycobacterium tuberculosisMycobacterium tuberculosis 90-2 90-221387. The antituberculosis activity data are shown in 21387. The antituberculosis activity data are shown in TableTable. The clinicall. The clinically used antituberculosis agent, ethambutol, was used as the positive control.y used antituberculosis agent, ethambutol, was used as the positive control. From the results of our antituberculosis tests, the following conclusions ca From the results of our antituberculosis tests, the following conclusions can be drawn regarding these isolated compounds: (a) Among the n be drawn regarding these isolated compounds: (a) Among the -dihydro-dihydroagarofuranoid sesquiterpene analogues (1 and 2), compound 1 with an 8-aagarofuranoid sesquiterpene analogues (1 and 2), compound 1 with an 8-acetoxy moiety exhibited stronger antituberculosis activity than 2 against cetoxy moiety exhibited stronger antituberculosis activity than 2 against M. tuberculosisM. tuberculosis 90-221387. (b) 8-Acetoxymutangin (1) is the most effective 90-221387. (b) 8-Acetoxymutangin (1) is the most effective among the isolated compounds, with an MIC of 10.0 among the isolated compounds, with an MIC of 10.0 g/ml against g/ml against M. tubeM. tuberculosisrculosis 90-221387. (c) The compounds 3–5, 7, 8, and 9 showed no antitube 90-221387. (c) The compounds 3–5, 7, 8, and 9 showed no antituberculosis activities. (d) Agents acting specifically against rculosis activities. (d) Agents acting specifically against M. tuberculosisM. tuberculosis ha have been less well characterized than other antimicrobial drugs. However, ive been less well characterized than other antimicrobial drugs. However, it is thought that several of them owe their activity to selective effects on tht is thought that several of them owe their activity to selective effects on the unique structure of the mycobacterial envelope. The positive control, ethe unique structure of the mycobacterial envelope. The positive control, ethambutol, inhibits arabinosyl transferases. These enzymes bring about the ambutol, inhibits arabinosyl transferases. These enzymes bring about the polymerization of arabinose to form arabinan, a polysaccharide componepolymerization of arabinose to form arabinan, a polysaccharide component of the core polymers of the mycobacterial cell wall. The structures of 1 nt of the core polymers of the mycobacterial cell wall. The structures of 1 and 2 with and 2 with -dihydroagarofuranoid sesquiter-pene skeleton are quite diffe-dihydroagarofuranoid sesquiter-pene skeleton are quite different from those of known antituberculosis agents, rent from those of known antituberculosis agents, e.g.e.g. ethambutol, isoniazi ethambutol, isoniazid, and artemisinin. Thus, the detail action mechanisms of 1 and 2 need furd, and artemisinin. Thus, the detail action mechanisms of 1 and 2 need further experiments to elucilate.ther experiments to elucilate.

ReferenceReference1.Lu SY, Yang YP. Celastaceae in 1.Lu SY, Yang YP. Celastaceae in Flora of TaiwanFlora of Taiwan; 2en ed.; Editorial commi-t; 2en ed.; Editorial commi-t

tee of the Flora of Taiwan, Taipei, Taiwan, 1993; Vol. 3, 640-660.tee of the Flora of Taiwan, Taipei, Taiwan, 1993; Vol. 3, 640-660.2.Chen JJ, Chou TH, Duh CY, Chen IS. Cytotoxic Dihydroagarofuranoid Sesq2.Chen JJ, Chou TH, Duh CY, Chen IS. Cytotoxic Dihydroagarofuranoid Sesq

uiterpenes from the Stem of uiterpenes from the Stem of Microtropis fokienensis. J. Nat. Prod. Microtropis fokienensis. J. Nat. Prod. 2006, 69, 62006, 69, 685-688.85-688.

8-Acetoxymutangin (1)8-Acetoxymutangin (1)Amorphous powderAmorphous powder HR-ESI-MS: CHR-ESI-MS: C

3535HH4040OO1111, found: 659.2701 [M+H], found: 659.2701 [M+H]++, calcd: 659.2703, calcd: 659.2703

ESI-MSESI-MS m/z m/z (%): (%): 659 ([M+H]659 ([M+H]++, 100). , 100). IRIRννmaxmax(KBr) cm(KBr) cm-1-1: : 1748 (C=O), 1723 (C=O).1748 (C=O), 1723 (C=O).

UV UV maxmax(MeOH) nm (log (MeOH) nm (log εε): ): 230 (4.14), 274 (3.15), 281 (3.08).230 (4.14), 274 (3.15), 281 (3.08).

CD (MeOH): CD (MeOH): 236.7 (236.7 ( －－ 13.9), 220.8 (13.9), 220.8 ( ＋＋ 5.33).5.33).[[aa] ] DD

2525 + 85.4+ 85.4°° ( (cc 0.12, CHCl 0.12, CHCl33).).

Key wordsKey wordsMicrotropis fokienensisMicrotropis fokienensis, , CelastraceaeCelastraceae, stem, , stem, dihydroagarofuranoid sesqui-dihydroagarofuranoid sesqui-terpenesterpenes, 8-acetoxymutangin, 8-acetoxymutangin, , antitubercular activity.antitubercular activity.

1313C-NMR date (CDClC-NMR date (CDCl33,100MHz) of 1,100MHz) of 1

11H-NMR date (CDClH-NMR date (CDCl33,400MHz) of 1,400MHz) of 1 Significant NOESY correlations of 1Significant NOESY correlations of 1

Significant HMBC correlations of 1Significant HMBC correlations of 1

11H-NMR date (CDClH-NMR date (CDCl33,400MHz) of 10,400MHz) of 10


Antitubercular effects of compounds isolated from the stems of Antitubercular effects of compounds isolated from the stems of Microtropis fokieMicrotropis fokienensisnensis against against Mycobacterium tuberculosisMycobacterium tuberculosis 90-221387 90-221387

CompoundCompound MIC MIC [[g mLg mL-1-1]]

8-Acetoxymutangin A (1)8-Acetoxymutangin A (1) 1010Mutangin A (2)Mutangin A (2) 3535Vanillin (3)Vanillin (3) > 100> 100pp-Hydroxybenzaldehyde (4)-Hydroxybenzaldehyde (4) 9090Syringic acid (5)Syringic acid (5) 65652,6-Dimethoxy-2,6-Dimethoxy-pp-benzoquinone (6)-benzoquinone (6) 5757-Sitosterol (7)-Sitosterol (7) > 100> 100Mixture of 1-tetracosanol (8) and 1-hexacosanol (9)Mixture of 1-tetracosanol (8) and 1-hexacosanol (9) > 100> 100

EthambutolEthambutolaa 6.256.25

a Ethambutol was used as a positive control.

Fokienagarofuran A (10)Fokienagarofuran A (10)Amorphous powderAmorphous powder HR-FAB-MS: CHR-FAB-MS: C

4040HH4343OO1111, found: 699.2802 [M+H], found: 699.2802 [M+H]++, calcd: 699.2806, calcd: 699.2806

FAB-MSFAB-MS m/z m/z (%): (%): 699 ([M+H]699 ([M+H]++, 28). , 28). IRIRννmaxmax(KBr) cm(KBr) cm-1-1: : 1746 (C=O), 1716 (C=O).1746 (C=O), 1716 (C=O).


[[aa] ] DD2525 + 32.4+ 32.4°° ( (cc 0.15, CHCl 0.15, CHCl

33).). Fokienagarofuran B (11)Fokienagarofuran B (11)Amorphous powderAmorphous powder HR-ESI-MS: CHR-ESI-MS: C

4040HH4343OO1111, found: 601.2417 [M+Na], found: 601.2417 [M+Na]++, calcd: 601.2413, calcd: 601.2413

ESIESI-MS -MS m/z m/z (%): 601(%): 601 ([M+Na] ([M+Na]++, 100). , 100). IRIRννmaxmax(KBr) cm(KBr) cm-1-1: : 1725 (C=O).1725 (C=O).


[[aa] ] DD2525 + + 46.846.8°° ( (cc 0.12, CHCl 0.12, CHCl

33).).

Fokienagarofuran C (12)Fokienagarofuran C (12) Amorphous powderAmorphous powder HR-FAB-MS: HR-FAB-MS: CC3838HH4747OO1111, found: 679.3122 [M+H ], found: 679.3122 [M+H ]++ calcd: 679.3119 calcd: 679.3119

FAB-MS FAB-MS m/zm/z (%): 679 ([M+H] (%): 679 ([M+H]++, 100)., 100). IRIRννmaxmax(KBr) cm(KBr) cm-1-1: : 1720 (C=O).1720 (C=O).


[[aa] ] DD2525 + 34.5+ 34.5°° ( (cc 0.18, CHCl 0.18, CHCl

33).). Fokienagarofuran D (13)Fokienagarofuran D (13)Amorphous powderAmorphous powder HR-ESI-MS: CHR-ESI-MS: C

4545HH4444OO1111, found: 783.2784 [M+Na], found: 783.2784 [M+Na]++, calcd: 783.2781, calcd: 783.2781

ESIESI-MS -MS m/z m/z (%): 783(%): 783 ([M+Na] ([M+Na]++, 100). , 100). IRIRννmaxmax(KBr) cm(KBr) cm-1-1: : 1749 (C=O), 1721 (C=O).1749 (C=O), 1721 (C=O).


[[aa] ] DD2525 + + 35.435.4°° ( (cc 0.13, CHCl 0.13, CHCl

33).).







AcO OBzOBz

OAcO

AcO R1

2

34

56

7

89

10

11

12

13

14

15

15

14

13

12

11

10

9

8

7

65

43

2

1

BzO

OBz

O

OAc

AcO

R

AcO

8-acetoxymutangin (1)8-acetoxymutangin (1)** R = OAc R = OAc

mutangin (2) R = Hmutangin (2) R = H

12

34

56

7

89

10

11

12

13

14

15

O

O OOO

R1

R2

R3 1010** R R11 = OBz, R = OBz, R22 = OAc, R = OAc, R33 = OBz = OBz

1111** R R11 = H, R = H, R22 = OAc, R = OAc, R33 = OBz = OBz

1212** R R11 = OBz, R = OBz, R22 = OAC, R = OAC, R33 = isobutanoyloxy = isobutanoyloxy

1313** R R11 = R = R2 2 = R= R33 = OBz = OBz

OH

R3

CO R1

R2

12

34

5

6

3 R3 R11 = H, R = H, R22 = H, R = H, R33 = OCH3 = OCH3

4 R4 R11 = R = R22 = R = R33 = H = H

5 R5 R11 = OH, R = OH, R22 = R = R33 = OCH3 = OCH3

O

OCH3H3CO

O

66

HO

77CHCH33(CH(CH22)nCH)nCH22OHOH

1-Teteracosanol (8) n = 22 1-Teteracosanol (8) n = 22 1-Hexacosanol (9) n = 241-Hexacosanol (9) n = 24

1*

2 ~ 910 ~ 13

H2O (Fr.C, 85 g)

n-BuOH (Fr.B, 168 g)

H2O : n-BuOH (1:1)

EtOAc (Fr.A, 38 g)

H2O

EtOAc : H2O (1:1)

MeOH extract(315 g)

MeOH

Dried Stems (3.2 kg) of Microtropis fokienensis

**New compoundNew compound★★New compound reported beforeNew compound reported before

J = 3.2

J = 3.2J = 15.0, 6.8, 3.6

J = 15.0

6"

5"4"

3"

2"1"

6'

5'4'

3'

2'1'

12

34

5

6

7

89

10

11

12

13

14

15

OOO

OO OOO

O

O

O

O

O

7.60 (t, J = 7.5)

7.51 (t, J = 7.5)

8.31 (d, J = 7.5)

7.59 (t, J = 7.5)

7.45 (t, J = 7.5)

8.02 (d, J = 7.5)

1.83 s

2.06 s

2.13 s

1.59 s

5.33 d, J = 13.04.97 d, J = 13.0

1.28 d, J = 7.5

1.43 s

1.46 s

5.70 s

5.32 d

2.50 d6.29 s

2.46-2.52 m

2.55 ddd

1.82 br. d

5.65 q J = 3.6

5.79 d, J = 3.6

21.0

21.3

21.4

20.4166.6

165.3

169.7

169.9

170.0

169.5

133.3

128.7

129.6

129.1

133.4

128.3

130.0

129.8

66.0

18.030.6

26.0

82.5

53.5

72.2

70.3

53.2

74.9

89.533.530.9

69.5

71.6

6"

5"4"

3"

2"1"

6'

5'4'

3'

2'1'

12

34

5

6

7

89

10

11

12

13

14

15

OOO

OO OOO

O

O

O

O

O

133.4

133.2

130.1

30.4

26.082.7

48.8

34.8

69.4

53.4

89.2

71.570.3

78.4

65.7

18.1

33.030.9

169.4

20.4

21.3

169.8

172.8

166.1

165.3

129.3

129.7

129.8

129.8

130.1

128.3 128.3

128.7

128.7

OOO

OO OOO

O

O

O43.6

22.522.5

25.6

6"

5"4"

3"

2"1"

12

34

5

6

7

89

10

11 12

13

14

151'

2'3'

4'5'

6'

J = 7.5J = 7.5 J = 16.0, 6.5, 3.4

J = 16.0

J = 4.0

J = 7.5

J = 7.5

J = 8.0

J = 7.4

J = 12.4

8.12 d7.50 t

7.50 t

8.06 d

7.45 t

7.58 t

7.58 t

7.45 t

8.06 d

8.12 d

1.52 s

1.97 br d2.57 ddd 2.45 m

1.24 d

5.87 q

5.84 d

4.48 d5.29 d

5.47 d

5.95 s

2.57 ddd, J = 15.6, 7.4 3.62.24 m

2.30 dd, J = 3.6, 2.8

2.10 s

1.44 s

1.48 sO

OO

OO OOO

O

O

O

J = 3.4

1.04 d1.05 dJ = 6.8J = 6.4

2.24 m2.41 d, J = 5.22.43 d, J = 6.0

1.51 s

1.47 s

2.10

2.41 m

2.29 dd, J = 16.3, 2.82.70 ddd, J = 16.3, 7.5, 3.8

6.13 s

5.56 d

5.42 d4.95 d

5.91 d

5.92 m

1.33 d

2.51 m2.62 ddd2.03 d

1.55 s

OOO

OO OOO

O

O

O

8.22 d

8.15 d

8.07 d

7.46 t

7.60 t

7.58 t

7.46 t

8.07 d

7.52 t

7.52 t8.15 d

8.22 d

7.54 t 7.54 t

7.63 t

J = 12.5

J = 7.0

J = 7.5

J = 7.5

J = 7.5

J = 4.5

J = 16.0

J = 16.0, 6.5, 3.3

J = 7.5

J = 7.5J = 7.5

J = 7.5

6"

5"4"

3"

2"1"

6'5'

4'

3'2'

1'15

14

13

1211

109

8

7

6

5

4

3

21

1"'2"'

3"'4"'

5"'

6"'

133.4

133.3

133.2

130.1129.7

OOO

OO OOO

O

O

O

30.4

26.082.8

48.7

34.7

69.2

53.5

89.2

71.670.3

78.7

66.4

18.3

33.130.9

169.4

20.4

21.3

169.9

166.8

166.1

165.2

129.1

129.8

130.0

129.7

129.8

129.8

130.1

128.3 128.3128.6

128.7

128.6

128.7

2.31 m1.52 m

J = 14.0, 4.0 1.99 dd1.64 m

2.40 m

J = 8.01.07 d

1.53 s

J = 12.5, 4.55.64 dd

1.47 s

1.44 s

2.35 m

2.25 dd, J = 16.0, 3.02.59 ddd, J = 16.0, 7.0, 3.0

J = 7.05.51d

J = 12.3

4.99 d4.72 d

7.63 t

7.53 t 7.53 t7.58 t

7.45 t 7.45 t8.22 d8.22 d8.06 d 8.06 d

6.01s

2.10 s

OOO

OO OOO

O J = 7.5

J = 7.5

J = 7.5

J = 7.5

J = 7.5

J = 7.5

12

3

4

5

6

7

8

910

11

12

13

14

15

1'

2'

3'4'

5'

6'1"

2"

3"4"

5"

6"

J = 3.6, 2.8

J = 15.2, 2.4

7.58 t

7.46 t 7.46 t

J = 7.68.03 d 8.03 d

6""

5""3""

2""1""

4""

6"'

5"'4"'

3"'

2"'1"'

12

3

4

5

6

7

89

10

11

12

1314

151'

2'3'

4'5'

6'

1"2"

3"4"

5"

6"

J = 7.6

J = 7.6

J = 4.5

J = 7.6

J = 7.6

J = 7.6

J = 7.2

J = 12.8

7.65 t

7.58 t

8.14 d7.50 t

7.50 t

7.58 t

7.59 t

7.46 t

8.08 d

8.14d

8.27d

OOO

OO OOO

O

O

O1.56 s

2.07 dd2.70 m 2.69 m

1.34 d

5.95 m

5.95 d

5.00 d5.42 d

5.56 d

6.36 s

2.79 ddd, J = 16.2, 7.2, 3.62.34 dd, J = 16.2, 2.8

2.57 dd

1.52 s

1.53 s129.7

129.1

129.9

129.7

128.7

128.7128.7

128.7

128.7

129.5 129.5

129.8

129.8

129.8

128.3

130.2

133.3

133.4

133.2

133.5

165.4

166.8

165.3

166.1

169.5

20.5

79.3

71.6

70.3

31.0 33.5

18.4

34.8

30.6

26.0

82.9

89.4

69.266.4

53.6

48.8

OOO

OO OOO

O

O

O

6"

5"4"

3"

2"1"

6'5'

4'

3'2'

1'15

1413

12

11

109

8

7

6

5

4

3

21

1"'2"'

3"'4"'

5"'

6"'

4""

1""2""

3"" 5""

6""

130.0130.0129.8 129.8 128.3128.3

128.6128.6133.4

133.4

78.9

65.6

21.3

166.7

129.3

129.6

165.3

53.4

48.6

69.6

34.4

82.5

30.4

26.0

170.0

89.5

73.6

169.8

20.8

OOO

OO OOO

O

16.7

26.4 33.5

22.2

6"

5"4"

3"

2"1"

6'

5'4'

3'

2'1'

12

34

5

6

7

89

10

11

12

13

14

15

OOO

OO OOO

O

O

O

O

O

6"

5"4"

3"

2"1"

6'

5'4'

3'

2'1'

15

14

13

12

11

109

8

7

6

54

3

21

OOO

OO OOO

O

O

O

O

O

Tsung-Hsien Chou a, Ih-Sheng Chen a, Chien-Fang Peng b, Lin-Yang Cheng c, Ya-Chih Chang c, Jih-Jung Chen c,*

a Graduate Institute of Pharmaceutical Sciences, Kaohsiung Medical University, Kaohsiung, 807, Taiwan b Faculty of Biomedical Laboratory Sciences, College of Health Science, Kaohsiung Medical University, Kaohsiung 807, Taiwan

c Graduate Institute of Pharmaceutical Technology & Department of Pharmacy, Tajen University, Pingtung 907, Taiwan

Tsung-Hsien Chou a, Ih-Sheng Chen a, Chien-Fang Peng b, Lin-Yang Cheng c, Ya-Chih Chang c, Jih-Jung Chen c,*

a Graduate Institute of Pharmaceutical Sciences, Kaohsiung Medical University, Kaohsiung, 807, Taiwan b Faculty of Biomedical Laboratory Sciences, College of Health Science, Kaohsiung Medical University, Kaohsiung 807, Taiwan

c Graduate Institute of Pharmaceutical Technology & Department of Pharmacy, Tajen University, Pingtung 907, Taiwan

A New Antituberculosis Dihydroagarofuranoid SesquiterpeA New Antituberculosis Dihydroagarofuranoid Sesquiterpe

ne from the Stems of Microtropis fokienensisne from the Stems of Microtropis fokienensis

A New Antituberculosis Dihydroagarofuranoid SesquiterpeA New Antituberculosis Dihydroagarofuranoid Sesquiterpe

ne from the Stems of Microtropis fokienensisne from the Stems of Microtropis fokienensis

introduction microtropis fokienensis dunn 1) (celastraceae) is a small shrub that grows in high...

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