Briaexcavatolides A–J, new diterpenes from the gorgonian briareum excavatum

TETRAHEDRON Tetrahedron 55 (1999) 14555-14564

Pergamon

Briaexcavatolides A-J, New Diterpenes From The Gorgonian Briareum excavatum Jyh-Horng

Sheu, *,a Ping-Jyun Sung,a Jui-Hsin Su,a Hsiao-Yu Liu,a Chang-Yih Duh,a and Michael Y. Chiangb

“Department of Marine Resources, National Sun Yat-Sen University, Kaohsiung, 804, Taiwan bDepartment of Chemistry, National Sun Yat-Sen University, Kaohsiung, 804, Taiwan Received

Abstract:

Ten new briarane-type

excavatum.

The structures

established

by spectroscopic

briaexcavatolide

8 September

diterpenes

of these

have been isolated

secondary

and chemical

B (2), was further confirmed

15October 1999

1999; accepted

metabolites,

methods.

from the gorgonian

named

The structure,

by a single-crystal

octocoral

briaexcavatolides including

Briareum

A-J (l-lo),

were

the relative configuration

X-ray structure analysis. Cytotoxicity

of

of these

toward various cancer cell lines also is described.@ 1999 Elsevier Science Ltd. All rights reserved.

compounds

K~YwM%: Brtareum e~cnu~fun~. bnarane diterpene, briaexcavatolide,

gorgonian, cytotoxicity

Introduction Gorgonian active

octocorals

and structurally

(phylum Cnidaria, interesting

invertebrates,

the gorgonian

investigation.

The previous

excavatum A-2,2-5

order Gorgonacea)

terpenoids.1

Briareum

studies on the chemical

constituents

highly oxygenated

a y-lactone in a bicycle [8,4,0]

Besides,

briaexcavatolide l-10

from the further investigation E (5) was isolated

were elucidated

by extensive

marine

has been the subject of an

of the West Pacific Ocean gorgonian briarane-type

diterpenes,

system. Briarane diterpenes

B.

excavatolides

continue to attract our

toward various cancer cell lines.2a,

In this paper. we report the isolation of the additional nine new briarane-type

metabolites

of the Taiwanese

Nutting (family Briareidae)

attention as several briaranes have been shown to exhibit cytotoxicity

D and F-J (l-4 and 6-lo),

as rich sources of biological

As part of our investigations

excavafum

have led to the isolation of twenty-six

which containing

are recognized

diterpenes,

brlaexcavatolides

6-11 A-

of the organic extract of the gorgonian B. excavatum.

for the first time as a natural spectral analyses and chemical

with the physical and spectral data from other known briarane-type

product. methods

The structures

of

and by comparison

compounds.

Results and Discussion Specimens *Author

were frozen immediately to whom correspondence

after collection and subsequently should be addressed.

freeze-dried.

Tel.: 886-7-5252000

5255020. E-mail: [email protected]

0040-4020/99/$ - see front matter 0 1999 Elsevier Science Ltd. All rights reserved. PII: SOO40-4020(99)0093 I -X

The minced

organisms

ext. 5030. Fax: 886-7-

J.-H. Shea et al. /Tetrahedron

14556

were extracted absorbent

successively

with EtOAc,

and the extract

(SiO2 gel) to give the ten new metabolites,

;@I’

55 (1999) 14555-14564

was fractionated

extensively

A-J (l-10).

briaexcavatolides

2$qoq

1

using normal-phase

see Experimental

Section.

o$2cH3

0

0 3: R=Ac

A (1) was obtained

Briaexcavatolide C26H320lo.

The IR spectrum

ester carbonyl

the presence

Its HRFABMS

of a carbonyl

groups (umax 1740 cm-I) and an cc&unsaturated

was confirmed

by an UV absorption

shifts (Table

1) associated

COSY, HETCOR,

and NOESY).

were determined

From the *H-tH

established

the molecular

group of y-lactone

ketone carbonyl

(vmax 1700 cm-l).

long-range

by a series of 2D NMR experiments of 1, it was possible

COSY spectrum

correlations

observed

in an HMBC experiment,

established

was confirmed

the connectivities

ring which is fused to the ten-membered

10, was elucidated

between

by the key HMBC correlations

and C-20; and H- 11 and C- 1, C-9. The ring juncture between

correlations

between

framework

of 1. The relative

groups

constant

stereochemistry

between

H3-18 when H-7 was P-oriented

the p-orientation

by HRFABMS.

of 2 showed the presence )), and ester carbonyls room temperature

of molecular

of a hydroxyl

(u max 1734 cm-t)

conformers

interconversion

to the point

and

are situated

the NOE correlations

of H-10

on the same face of the structure

and

at C- 1. The signal of H3- 18 showed

of H3-18. Also, H-9 was found to exhibit correlations close to H-7 and

on the o! face. Based on the above observations,

were elucidated

2 contained

the

unambiguously. formula of C2sH3sOlu

ten degrees of unsaturation.

The IR absorptions

a carbonyl group of y-lactone (qnax 1790 cm-

in the structure of 2. The tH and l3C NMR spectra of 2 measured at revealed

of 2. As increase

mostly broad signals, of temperature

where the NMR spectrometer

and 13C NMR spectra of 2 were measured

the molecular constants

of the C-13/C- 14 double bond was indicated by a 10.4

group (u ,,,ax 3572 cm-l),

in CDCI3 or acetone-de

interconverting

coupling

B (2) was isolated as a white solid. The molecular

Thus, metabolite

also revealed

with the HMBC

established

tH-tH

models, H-9 was found to be reasonably

and H-9 was placed

the relative stereochemistry

The new briarane briaexcavatolide was established

from vicinal

since the C- 15 methyl is the B-substituent

with H3-20, indicating

structure of 1, including

of 1 was deduced

that these four protons

with H-7 and H3-18. From consideration

at C- 1 from the key

the HMBC correlations

H-13 (6 6.00) and H- 14 (6 6.48). Furthermore,

as the o! protons

between

ring at C-l and C-

to C-2, C-3, and C-9. These data, together

(Figure 1). The cis geometry

with H-2, H-3, and H-l 1 indicated NOE correlation

C- 15 methyl group was positioned

H-9 and C- 17; H3- 18 and C-8, C- 17, and C- 19, unambiguously

from a NOESY experiment

were assigned

attached

from C- 1

H-2 and C- 14; H-9 and C- 11; H- 10 and C- 12, C- 14.

H3-15 and C-l, C-2, C-10, and C-14. Furthermore,

of three acetoxyl

Hz coupling

(IH-tH

to establish

by the HMBC correlations

H3-16 and C-4, C-5, and C-6. The cyclohexenone

the positions

The latter

from H-2 to H2-4; H-6 to H-7; H-9 to H-l 1; and H-13 to H-14. These data. together with

to C- 14. A vinyl methyl group attached at the C-5 position

correlations

formula

(unuix 1786 cm-l),

of 1 and all of the 1H and 13C chemical

at 222 nm. The gross structure

with the molecule

HMBC,

the proton sequences the lH-t3C

as a white powder.

of 1 showed

revealing

was expected

the existence to speed

up the rate of

could record an “average” conformation,

at elevated temperature

(70 “C) in pyridine-d5

of slowly

both tH

(Table 1). Fortunately,

J.-H. Sherr et al. /Tetrahedron

Table 1. The 1H and 13C NMR Chemical 1 CA-I no. 1 2 3

4

13cb

‘HO 5.28 br s 4.76 br s

2.02 br d (156)g 3.41 dd (15.6; 4.8)

33.3 (t)

5 ; 8 1: 11 12 13 14 :; 17

5.28 d (7.6) 5.34 d (7.6) 5.13 d (10.4) 3.23 dd $l$m4.4) 6.00 d (10.4) 6.48 d (10.4) 0.99 s 1.66 s

139.3 123.0 73.6 69.1 66.7 38.6

(s) (d) (d) (s) (d) (d)

41.3 (s) 200.9 (d) 126.1 (d) 155.2 (d) 17.0 (cl)

1.51 s :98 20 acetate methyls

;:.l;;

(11.2)

2.20 s 2.25 s

ester carbonyls

171.1 14.2 21.2 21.7 22.6 169.0 169.8 170.4

Shifts of Diterpenes l-4 compound 2 13cd

‘HC

43.0 (s)h 81.5 (d) 71.9 (d)

(s) (q) (q) (q) (q) (s) (s) (s)

55 (1999) 14555-14564

5.04 br 1.91 br (13.2) 2.52 br (13.2) 2.02 m 2.69 br (10.0)

s d

46.4 (s) 80.1 (d) 32.3 (t)

d 26.3 (t) d

5.65 d (9.6) 6.02 d (9.6) 6.29 d (4.8) 3.14 br s

145.8 120.2 74.9 71.0 68.6 42.5

(s) (d) (d) (s) (d) (d)

72.9 (s) 74.1 (d) 120.7 (d)

5.35 d (5.6) 5.97 dd (10.0: 5.6) 5.70 d (ld.0) 142.2 (d) 1.54 s 20.2 (4) 1.98 s 26::; 2,’ 1.78 s 10.4 (9) 170.1 (s) 1.69 s 28.1 tq) 2.21 s 21.1 (4) 2.25 s 21.6 (q) 169.3 (s) 171.8 (s)

3

‘HC 5.05 br s 19Obrd (13.2) 2.49 br d (13.2) 1.97 m 2.68 br d (10.0)

5.62 d (9.6) 6.03 d (9.6) 65;

14557

clr(i.8)

4 13cd

‘He

‘30 46.3 (s) 79.3 (d) 27.4 (t)

46.5 (s) 80.2 (d) 3.90 br s 32.3 (t) 1.6Om 2.15 m 26.5 (t) 1.91 br t (13.8) 3.01 m 145.9 120.6 74.8 71.1 42.7 69.0

25.2 (t)

(s) (d) 5.26 d (8.7) (d) 5.70 d (8.7) (s) (d) 3.93 3.45 ddd(11.2)

(11.2: 4.8) 72.9 (s) 2.61 m 74.8 (d) 121.0 (d) 5.86 d (10.5)

5.32 d (5.6) 5.93 dd (10.0; 5.6) 5.64 d (10.0) 142.4 (d) 1.50 s 20.3 (q) 1.95 s 21.2 (q) 63.3 (s) 1.74 s 10.7 (4) ., 170.3 (s) 1.65 s 28.2 (q) 2.05 s 21.3 (4) 2.19 s 21.4 (9) 2.26 s 21.9 (9) 169.6 (s) 170.2 (s) 172.1 (s)

6.54 d (10.5) 1.50 s 1.67 s

145.4 122.1 74.2 71.6 67.5 39.7

(s) (d) (d) (s) (d) (d)

41.7 (d) 202.4 (s) 126.1 (d) 157.4 (d) 19.2 (9) ‘53d$, 9.5 (q) 173.1 (q) 14.4 (q)

1.56 s 1.14 d (7.5)

n-butyrate

172.6 (s) 1.00 t (7.2) 13.7 (q) 1.75 m 18.8 (t) 2.38 t (7.2) 36.7 (t) %pectra recorded at 400 MHz in CDC13 at 25 “C. blOO MHz in CDC13 at 25 “C. C400 MHz in pyridine-ds at 70 “C. dlOO MHz in pyridine-d5 at 70 “C. e300 MHz in acetone-& at 25 “C. 05 MHz in aCetOne-&j at 25 “C. g J values (in Hz) in parentheses. ‘iMultiplicity deduced by DEPT and indicating by usual symbols. The values are ppm downfield from TMS. it was found that at this temperature assigned by the assistance

the signals

for each proton and carbon were sharpened

was deduced from the signals of two carbons at 6 120.2 (d) and 145.8 (s), and a disubstituted from the signals of carbons at 6 120.7 (d) and 142.2 (d). An 8,17-epoxide two quatemary

oxygenated

of 2, four carbonyl resonances

and 172.6 (s), and further confirmed

acetate methyls

was confirmed

olefin

olefin was found from the signals of

carbons at 6 63.2 (s) and 71.0 (s), and from the chemical shift of H3-18 (6 1.78,3H,

s). In the 13C NMR spectrum

above observations,

and could be

of DEPT and 2D NMR spectra. From the above spectral data, a trisubstituted

diterpene

were observed

the presence

were appeared at 6 169.3 (s), 170.1 (s), 171.8 (s),

of a y-lactone and three other ester groups in 2. Based on the

2 was found to be a tetracyclic

compound.

(6 2.21, 3H, s; 2.25, 3H, s). The additional

In the 1H NMR spectrum

of 2, two

acyl group was found to be an n-

J.-H. Shea et al. /Tetrahedron

14558

hutyryloxyl

55

group based on the tH NMR studies, including

(I 999) 14555-14564

an tH-tH

COSY experiment,

(s) was correlated

with the signals of the methylene

of 2. and was consequently experiment

assigned

of 2. it was possible

protons of the n-butyrate

as the carbon atom of the Jr-butyrate

to establish

the separate spin systems

at 6 2.38 in the HMBC spectrum carbonyl.

observed

Furthermore,

the rr-butyrate

carbon

unambiguously. confirms

in the HMBC experiment

(6 173.6) of the fr-butyrate.

with the tH-t3C

the briarane-type of 2, su,,ODested .

ester was positioned

at C-12 from the connectivity

By above spectral

analyses,

The X-ray structure of 2 (Figure 2) also demonstrates

the relative, not the absolute configuration

From the tH-tH

COSY

that map out the proton sequences

H-2 to HZ-~; H-6 to H-7: H-9 to H-10; and H-12 to H-14. These data, together correlations carbonyl

which revealed seven

protons (6 1.OO. 3H. t, J = 7.2 Hz: I .75, 2H. m; 2.38, 2H. t, J = 7.2 Hz). The carbon signal at 6 172.6

contiguous

molecular between

the structure

from

long-range

framework

of 2.

H-12 (6 5.35) and

of 2 were determined

the location of the rr-butyrate at C-12 and

of 2.

010

08

2 Cl6

0 Figure 1. Selective

NOE Correlations

of 1

Figure 2. A computer-generated ORTEP plot of 2 showing relative configuration. Hydrogen atoms have been omitted for calrity. Briaexcavatolide

C (3). was isolated

the basis of HRFABMS. 1786 cm-t.

Its IR spectrum

and ester carbonyls

temperature

and the sharpened

as an amorphous exhibited

at 1734 cm-t.

The broad NMR signals

NMR (tH and t3C) signals of diterpene

found to be very close to those of 2. Furthermore, with 2, it was revealed

solid and had the molecular

a broad OH stretch at 3528 cm-t,

that the signals

by comparison

corresponding

acetyl derivative Diterpene spectrum

of 4 showed absorptions

I784 cm-t),

spectrum, spectrum

and a$-unsaturated

D), C2oH2606 that indicated

at room

at 70 “C were

of the tH and t3C NMR spectral data of 3 group in 2 were replaced

by an

C (3) was found to be the 12-0-debutyryl-12-0-

ketone (urnax 1690 cm-t).

at 224 nm, the presence

and the presence

by HRFABMS, the presence

of signals

of a mutually

coupled

that the spectral

was isolated

of two different

as a white powder.

carbonyl

The latter structural

types:

The IR

y-lactone (urnax

feature was confirmed

by an

at 6 202.4 (s), 126.1 (d), and 157.4 (d) in the t3C NMR pair of doublet

of 4 at S 5.86 (H-13) and 6.54 (H-14) corresponding

was observed

in pyridine-ds

at

of 2.

4 (briaexcavatolide

UV absorption

carbonyl

(t H and t3C) measured

3 measured

to the n-butyryloxyl

acetoxyl group in 3. Based on the above data, briaexcavatolide

formula C26H34Dto on a y-lactone

signals

(J = 10.5 Hz) in the tH NMR

to the a and p olefinic

data (I H and t3C NMR) of 4 were similar

protons,

respectively.

to those of a known diterpene,

It

J.-H. Sheu et al. /Tetrahedron

briareolide

I (11).t2

However,

replaced by hydroxyl observations,

55 (1999) 14555-14564

it was found that the acyloxyl

groups at C-2 and C-9 of compound

11 were

the related spectral data of 4 with those of 11. Based on the above

groups by comparing

briaexcavatolide

14559

D (4) was assigned as the 2-0-debutyryl-9-0-deacetyl

derivative

of briareolide

I

(11).

R*o$&C1

J$+$Cl

5 : R’ = R2 = AC

0

Ho*C,

8:R=H 9:R=Ac

6 : R’ = H, R2 = n-PrCO 7:R’=R2=H

0

10

0

12 : R’ = H, R2 = AC Briaexcavatolide CzeH330t cm-‘),

E (5) was isolated

1C1, for this compound.

a carbonyl

resonances

as a white powder.

The IR spectrum

group of y-lactone

HRFABMS

established

of 5 showed absorptions

(2),,,ax 1781 cm-l),

the molecular

of a hydroxyl

and ester carbonyls

formula.

group (urnax 3484

(urnax 1740 cm-t).

Carbonyl

in the t3C NMR spectrum of 5 (Table 3) at 6 168.8 (s), 169.0 (s). 169.5 (s), and 174.0 (s) confirmed

the presence

of a y-lactone

methyls were observed optical rotation) diterpenes

and three other esters in 5. In the tH NMR spectrum

at 6 2.10 (3H, s), 2.14 (3H, s), and 2.22 (3H, s). Comparison

and spectral

acetylation

of the physical (mp and

(IR, MS, ‘H, and 13C NMR) data found for 5 with those of other briarane-type

reported in the literatures

was isolated previously

of 5 (Table 2). three acetate

indicated that diterpene

from an Okinawa

gorgonian

of 12 in the same work. however,

5 is the acetate derivative

coral Briareum

of brianolide

sp. 13 Diterpene

(12). which

5 has been obtained

this is the first time that 5 (briaexcavatolide

by

E) was isolated from

natural source. The new briarane diterpenes of C26H3501uCl

briaexcavatolide

F (6) and briaexcavatolide

and C22H2909C1, respectively,

as determined

G (7) had the molecular

by HRFABMS.

It was found that the spectra1

data (IR, IH, and 13C NMR) of 6 and 7 were very similar to those of brianolide j3C NMR spectra (Tables 2 and 3) revealed disappeared,

that the signals corresponding

and were replaced by those of an n-butyryloxyl

the HMBC experiment found to be correlated

carbonyl.

hydroxyl group was positioned

at C-12 in 7. Furthermore,

confirmed

by HRFABMS. diterpene

E (5) by comparison

showed

and 12-0-deacetyl

at 228 nm suggested

assigned as the

at C-12 in 6 and the

of 7 gave a less polar product, which was of the physical

derivatives

as a white solid. The molecular

A strong UV absorption

8. The IR spectrum

hydroxyl, y-lactone, exocyclic

H (8) was obtained

and spectral

the structures

data, and

of 6 and 7 were

of 12, respectively.

formula C24H3 109Cl, was established

the presence

bands at 3396, 1778, and 1727 cm-l,

of a conjugated consistent

diene system in

with the presence

and ester carbonyl groups. In the 1% NMR spectrum of 8 (Table 3) a disubstituted

olefins were deduced

In

with H-12 (6 5.17) was

ester should be positioned

acetylation

7. On the basis of the above observations,

found to be the 12-0-deacetyl-12-0-n-butyryl Briaexcavatolide

and a hydroxyl groups in 6 and 7. respectively.

Thus, the n-butyrate

with briaexcavatolide

the structure of diterpene

the 1H and

group in 12 were

protons at 6 2.30, and was consequently

carbon atom of the n-butyrate

found to be identical

(12).13 However,

to an acetoxyl

of 6, the carbon signal at 8 173.0 (s) which showed a correlation with the signal of the methylene

formulas

of

and an

from the signals of four carbons at 6 118.3 (t), 126.1 (d), 135.1 (d), and 137.0

J.-H. Sheu et al. /Tetrahedron

I4560

(s). An 13,14-epoxide

group was confirmed

and from the chemical NMR spectrum the presence

from the two tertiary oxygenated

of a carbon resonance

resonances

appeared at 6 170.1 (s), 170.2 (s), and 174.4 (s) and confirmed

of y-lactone

and two other ester groups. In the tH NMR of 8, two acetate The cis geometry

methyls (6 2.15, 3H, s; 2.17, 3H, s) were observed.

suggested represented

carbons at 6 58.0 (d) and 62.5 (d),

shifts of H-13 (6 3.23, d, J = 3.6 Hz) and H-14 (6 3.16, d, J = 3.6 Hz). From the t3C

of 8, three carbonyl

by a 12.0 Hz coupling

55 (1999) 14555-14564

constant

that metabolite

between

8 is probably

by formula 8. The molecular

of the C-3/C-4 double bond was indicated

H-3 (6 5.83) and H-4 (6 5.84). Based on the above findings, a deoxygenated

derivative

of 12 and should possess

structure of 8 was further confirmed

it was

a structure

by 2D NMR experiments

as

(‘H-*H

COSY. HMBC. and NOESY. see fieures 3 and 4).

-

‘H-‘H

-

HMBC

COSY

Figure 4. Selective

Figure 3. ‘H-‘H COSY and HMBC Correlations for 8 Table 2. The tH NMR Chemical H 2 3 4 6 ; 10

f: 13 14 15 16 17 :: acetate methyls n-butyrate

Shifts of Diterpenes

4.30 3.98 (9.0; 4.29

6b d (9.0) dd 3.9) d (3.9)

5.37 5.04 5.34 1.75 (8,8; 2.24 4.61

d (3.2) d (3.2) d (8.8) dd 2.4) m d (4.8)

5.83 5.73 5.80 2.43 (8.7; 3.04 5.17

d (3.3) d (3.3) d (8.7) dd 1.8) m d (5.1)

5.86 d (3.6) 5.73 d (3.6) ;:;; d&9.0)

3.13 2.89 1.22 6.03 6.12 2.45 1.17 1.04 2.10 2.14 2.22

d d s d d q d d s s s

3.77 3.48 1.50 5.57 5.99 3.20 1.41 1.34 2.32

d d s d d q d d s

3.80 d 3.7 1 d 1.57 s 5.59 d 6.04d 3.18 q 1.53 d 1.42 d 2.30 s

(3.2) (3.2) (2.4) (2.4) (7.2) (7.2) (7.2)

(3.9) (3.9) (1.5) (1.5) (6.9) (6.9) (6.9)

of 8

S-10 compound

5a 5.13 d (9.2)d 3.40dd ’ (9.2; 4.0) 3.62 d (4.0)

4.29 4.01 (9.0; 4.28

NOE Correlations

76 d (9.0) dd 3.9) d (3.9)

(9.0; 1.8) 3.05 m 4.33 d (5.4) (3.9) (3.9) (1.5) (1.5) (7.2) (7.2) (6.9)

5.15 r(6.4) 5.83 dd (12.0; 6.4) 5.84 d (12.0)

6.13 r(9.2) 5.62 dd (12.0; 9.2) 5.90 d (12.0)

5.20 4.95 5.24 1.93 (7.6; 2.13 4.60

d (4.0) d (4.0) d (7.6) dd 2.0) m d (4.8)

5.22 4.94 5.25 2.02 (7.6; 2.17 4.62

3.23 3.16 1.10 5.58 5.93 2.35 1.15 1.06 2.15 2.17

d d s d d q d d s s

3.16 d 2.98 d 1.17 s 6.01 d 6.29 d 2.34 q 1.15 d 1.04 d 2.04 s 2.10s 2.17 s

(3.6) (3.6) (2.8) (2.8) (7.2) (7.2) (7.2)

d (2.8) d (2.8) d (7.6) dd 2.8) m d (4.4) (3.2) (3.2) (2.4) (2.4) (7.2) (7.2) (6.8)

1oC 3.62 d (3.3) 5.17 d (3.3) 1.99 2.86 5.20 4.92 5.22 1.95

m m d (3.0) d (3.0) d (4.2) d (4.2)

1.99 4.27 (6.9; 3.20 2.92 1.21 6.03 6.06 2.36 1.15 1.07 2.15 2.17

m dd 3.6) d (3.6) d (3.6) s d (1.8) d (1.8) q (7.2) d (7.2) d (7.2) s s

0.85 t (7.5) 1.59 m 2.30 t (7.5) aSpectra recorded at 400 MHz in WC13 at 25 “C. b3OO MHz in pyridine-dg at 25 “C. C300 MHz in CDC13 at 25 “C. dJ values (in Hz) in parentheses. The values are ppm downfield from TMS.

J.-H. Shell et al. /Tetrahedron

Briaexcavatolide

I (9) had the molecular

55 (1999) 1455.5-14564

formula C26H33OtuCl. as determined

that the NMR spectra ()H and t3C) of 9 were very similar to those of 8. However, which were observed

14561

by HRFABMS.

It was found

the 1H NMR spectrum of 9

showed the presence

of three acetate methyls

at 6 2.04 (3H, s), 2.10 (3H, s), and 2.17

(3H, s). The positions

of the three acetoxyl groups at C-2, C-9, and C-12 were confirmed

by the connectivities

between the three methine protons at 6 6.13 (H-2). 5.25 (H-9). and 4.62 (H- 12) and the ester carbonyls at 169.7 (s), 170.0 (s), and 170.1 (s), respectively, (9) was established

Table 3. The 13C NMR Chemical position C-l c-2 c-3 c-4 c-5

C-6 c-7 C-8 c-9 c-10 c-11 c-12 c-13 c-14 c-15 C-16 c-17 C-18 c-19 c-20 acetate methyls ester carbonyls

in the HMBC spectrum of 9. Thus, the structure of briaexcavatolide

as the 2-acetyl derivative

5a 38.3 (s)d 74.9 (d) 60.0 (d) 57.1 (d) 133.4 (s) 61.1 (d) 76.5 (d) 83.9 (s) 69.3 (d) 37.4 (d) 35.9 (d) 71.7 (d) 56.6 (d) 61.4 (d) 16.5 (q) 120.9 (t) 45.5 (d) 6.2 (q) 174.0 (s) 9.7 (9) 21.0 (9) 21.0 (4) 22.0 (4) 168.8 (s) 169.0 (s) 169.5 (s)

I

of 8.

Shifts of Diterpenes

5-10

66 40.0 (s) 73.3 (d) 64.3 (d) 58.8 (d) 138.7 (s) 61.8 (d) 79.8 (d) 84.5 (s) 70.6 (d) 38.0 (d) 37.5 (d) 73.8 (d) 64.2 (d) 58.3 (d) 17.0 (q) 117.5 (t) 45.8 (d) 7.1 (cl) 175.9 (s) 10.7 (q) 22.4 (4)

compound 76 40.9 (s) 73.3 (d) 64.4 (d) 58.7 (d) 138.8 (s) 61.8 (d) 79.8 (d) 84.8 (s) 70.9 (d) 38.6 (d) 40.0 (d) 70.9 (d) 64.4 (d) 60.8 (d) 17.2 (q) 117.3 (t) 45.8 (d) 7.1 (q) 175.9 (s) 10.6 (q) 22.3 (9)

171.6 (s)

171.6 (s)

80 41.3 (s) 71.7 (d) 135.1 (d) 126.1 (d) 137.0 (s) 62.4 (d) 77.7 (d) 83.9 (s) 69.9 (d) 37.6 (d) 37.2 (d) 72.6 (d) 58.0 (d) 62.5 (d) 15.1 (9) 118.3 (t) 45.1 (d) 6.3 (4) 174.4 (s) 9.5 (4) 21.1 (q) 21.1 (4) 170.1 (s) 170.2 (s)

9a 40.6 (s) 75.2 (d) 130.8 (d) 128.0 (d) 136.0 (s) 62.4 (d) 77.7 (d) 83.9 (s) 69.8 (d) 37.6 (d) 37.1 (d) 72.2 (d) 57.3 (d) 62.0 (d) 15.9 (9) 119.4 (t) 45.1 (d) 6.3 (q) 174.3 (s) 9.4 (4) 21.0 (4) 21.0 (4) 22.0 (9) 169.7 (s) 170.0 (s) 170.1 (s)

1oC 40.2 70.6 64.7 35.4 144.3 74.5 77.0 84.4 71.4 37.5 42.0 70.1 59.3 63.3 17.6 121.2 43.7 6.9 174.5 8.6 21.2 22.0

(s) (d) (d) (d) (s) (d) (d) (s) (d) (d) (d) (d) (d) (d) (q) (t) (d) (q) (s) (4) (4) (4)

169.5 (s) 173.1 (s)

173.0 (S) CH3 14.1 (q) CH;? 19.2 (t) CH2 36.6 (t) aSpectra recorded at 100 MHz in CDC13 at 25 “C. b75 MHz in pyridine-dg at 25 “C. c75 MHz in CDC13 at 25 “C. dhrlultiplicity deduced by DEPT and indicating by usual symbols. The values are in ppm downfield from TMS n-butyrate

Briaexcavatolide J (10)had the molecular formula C24H330toCl as determined by HRFABMS. The IR spectrum of 10 showed the presence of hydroxyl groups (vrriax 3424 cm-t), carbonyl group of y-lactone (urnax

1780 cm-t), and ester carbonyls (U,,,ax 1733 cm-t). The gross structure of 10 and all of the tH and t3C chemical

shifts associated

with the molecule

were also determined

by the assistance

of DEPT and 2D NMR

experiments (tH--‘H COSY, HETCOR, HMBC, and NOESY). From the above spectral data, an exocyclic olefin was deduced from the signals of two carbons at 6 121.2 (t) and 144.3 (s). An 13,14-epoxide group was confirmed from the signals of two oxygenated methine carbons at 6 59.3 (d) and 63.3 (d), and from the chemical shifts of H-13 (6 3.20, d, J = 3.6 Hz) and H-14 (6 2.92, d, J = 3.6 Hz). In the t3C NMR spectrum of 10, three carbonyl resonances appeared at 6 169.5 (s), 173.1 (s), and 174.5 (s), and confirmed the presence of a

J.-H. Sheu et al. /Tetrahedron

14562

55 (1999) 14555-14564

y-lactone and two other ester groups in 10. In the 1H NMR spectrum of 10, two acetate methyls (6 2.15, 3H. s; 2.17, 3H, s) were also observed. sequences

From the IH-IH

COSY spectrum,

it was possible

to establish

the protons

from H-2 to Hz-4; H-6 to H-7; and H-9 to H-14. The positions of the two acetoxyl groups at the C-3

and C-9 were confirmed connectivities

between

respectively,

by the IH NMR chemical the two methine

in the HMBC spectrum

experiment

protons

shifts of H-3 (6 5.17) and H-9 (6 5.22) and by the

and the ester carbonyls

at 6 169.5 (s) and 173.1 (d),

of 10 was established

of 10. The relative stereochemistry

10 were established.

(Figure 5). On the basis of the above analyses, the structure of diterpene

noted that the briarane-type

diterpenes

The absolute configuration

of brianolide

organism.2

it is reasonable

which was found to be identical Therefore,

(12) has been determined

on biogenetic

as 12. The acetylation

briaexcavatolides

NOE Correlations

A-J (l-10)

12.13 As briaexcavatolides

configurations

It is

known possess three hydroxyl groups in structures are rarely found.3

Figure 5. Selective

by structure

by a NOESY

grounds

were isolated

previously

by X-ray analysis, as shown

along with brianolide

to assume that diterpenes

of briaexcavatolide

with briaexcavatolide

A-J were assumed

of 10

l-10

G (7) and brianolide

have the same absolute

(12) gave the same compound

E (5). and further confirmed

to possess

the absolute

from the same

the above assumption.

configurations

as represented

by

l-10.

structures

The cytotoxicity

l-10 against the growth of P-388, KB. A549, and HT-29 tumor cells

of the new diterpenes

1,3,4,5.

were studied, and the results showed that diterpenes the above cells. Compound

2 exhibited

1.3 and 1.5 pg/mL. respectively,

significant

and compound

and 7-10 were found to be not cytotoxic toward

cytotoxicity 6 exhibited

against P-388 and KB tumor cells with EDso of significant

cytotoxicity

against A549 tumor cells

with EDso of 1.3 pg/mL.t4 Experimental General

Experimental

spectrophotometer.

Procedures.

Ultraviolet

The NMR spectra were recorded

Section

spectra

and 75 MHz for 13C or on a Bruker

AMX-400

respectively,

using TMS as internal

in CDC13 or pyridine-d5

general experimental

procedures

Collection, Extraction,

FT-NMR

and was stored in a freezer

followed

and Separation. until extraction.

National Sun Yat-Sen University the standard procedures

chromatographed l-10. Diterpene

on a Hitachi FT-NMR

U-3210

UV

at 300 MHz for tH

at 400 MHz for IH and 100 MHz for 13C, standard,

unless otherwise

indicated.

Other

followed those reported previously.2,3 B. excavatum

The organism

using scuba at South Bay, Kenting, located in the southernmost

Resources,

were recorded

on a Varian VXR-30015

A voucher (specimen

(3.6 kg wt) was collected

by hand

tip of Taiwan in July 1995, at depths of 4-5 m

specimen

is stored

at the Department

no. KTSC- 103). The extraction

of Marine

and isolation schemes

that have been reported by our group previously.233 The organic extract was

on SiO2 gel CC, using hexanes and EtOAc mixtures of increasing 1 was eluted with hexanes-EtOAc

(5: l), 2 with hexanes-EtOAc

polarity, yielded diterpenes (5: l-4: 1). 3 with hexanes-

J.-H. Sheu et al. /Tetrahedron

EtOAc (4: 1). 5 with hexanes-EtOAc 8 with hexanes-EtOAc hexanes-EtOAc

(4: l-7:2),

55 (1999)

14555-14564

9 with hexanes-EtOAc

(2: l), 6 with hexanes-EtOAc

(2: l-3:2).

14563

(3: 1), 4 with hexanes-EtOAc

7 with hexanes-EtOAc

(3:2-l:

(3: l-2: l),

I), 10 with

( 1: 1).

Briaexcavatolide

A (1): white solid (4.1 mg); mp 85-87 “C; [a]25~ -10”

h max 222 nm (E 5524); IR (KBr) U,ax

1786, 1740, and 1700 cm- 1; I H and 13C NMR data, see Table 1;

FABMS m/z 505 [ 11.0, (M + H>+]; HRFABMS Briaexcavatolide

(c 0.1, CHC13); UV (CHCl3)

m/z 505.207 1 (calcd for C26H33010 505.2064).

B (2): white powder (60.1 mg); mp 219-221 “C; [cz]~~D -111” (c 1.0, CHCl3); IR (KBr)

urnax 3572, 1790, and 1734 cm- ‘: IH and 13C NMR data, See Table 1: FABMS

m/z 535 [0.5. (M + H)+];

HRFABMS m/z 535.2573 (calcd for C28H39010,535.2544). Briaexcavatolide

C (3): white powder (9.8 mg); mp 226-228

Umax 3528, 1786, and 1734 cm-l; HRFABMS

“C; [a125~ -96” (c 0.4, CHC13); IR (KBr)

‘H and I 3C NMR data, See Table

m/z 507 [2. (M + H)+];

m/z 507.2235 (calcd for C26H35010,507.2220).

Briaexcavatolide

D (4): white powder (5.6 mg); mp 217-219 “C; [a]25~ -14” (c 0.2, CHC13): UV (CHC13)

h max 224 nm (E 6074); IR (KBr) urnax 3330, 1784, and 1690 cm-l; FABMS m/z 363 10.3, (M + H)+l; HRFABMS Briaexcavatolide

3400, 1765. and 1745 cm-l;

‘H and 13C NMR data. see Tables 2 and 3; FABMS m/z 545 [0.4, (M + H + m/z 543.1987 (calcd for C26H36010C1, 543.1987).

G (7): white powder (6.0 mg): mp 233-235 “C: [cx]~~D -18” (c 0.2, MeOH): IR (KBr)

Umax 3371, 1768, and 1740 cm-l;

IH and 13C NMR data, see Tables 2 and 3; FABMS m/z 475 [0.2, (M + H +

2)+], 473 IO.4, (M + HI+]; HRFABMS Briaexcavatolide

m/z 557.1795 (calcd for C26H3401 lC1, 557.1780).

F (6): white powder (9.7 mg); mp 184-186 “C; [(r]25~ -21” (c 0.1, MeOH); IR (KBr)

2)+], 543 [O-2, (M + H)+]; HRFABMS Briaexcavatolide

363.18 11 (calcd for C2oH2706.363.1800).

IH and 13C NMR data, see Tables 2 and 3; FABMS m/z 559 [0.2, (M + H +

2)+], 557 [0.4, (M + H)+]; HRFABMS Briaexcavatolide

&z

IH and 13C NMR data, See Table 1;

E (5): white powder (2.2 mg); mp 171-173 “C; [a]26~ -27°C~ 0.1, MeOH); IR (KBr)

Umax 3484, 1781, and 1740 cm-l;

U,a,

1; FABMS

m/z 473.1577 (calcd for C22H3009Cl. 473.1570).

H (8): white solid (3.5 mg); mp 201-203 “C; [cx]~~D-254”

h max 228 nm (E 5426); IR (KBr) urnax 3396, 1778, and 1727 cm-l;

(c 0.1. CHC13); UV (CHC13)

IH and l3C NMR data, see Tables 2 and 3;

FABMS m/z 501 t0.6, (M + H + 2)+], 499 [ 1.4, (M + H)+]; HRFABMS

m/z 499.1727 (calcd for C24&209Cl,

499.1726). Briaexcavatolide Lax

I (9): white solid (2.7 mg); mp 224-226 “C; [cx]~~D -53”

226 nm (E 5568); IR (KBr) u,,,

3422,

1781,

and 1734 cm-l;

(c 0.1. CHC13); UV (CHC13)

1H and 13C NMR data, See Tables 2 and 3;

FABMS nJz 543 [0.3, (M + H + 2)+], 541 t0.8, (M + H)+]; HRFABMS

r& 541.1841 (calcd for C2&I3401oCl,

541.1831). Briaexcavatolide

J (10): white powder (2.5 mg); mp 183-185 “C; [a&)

Umax 3424, 1780, and 1733 cm-‘;

2)+], 517 [0.8, (M + H)+]; HRFABMS Acetylation

of Briaexcavatolide

m/z 517.1837 (calcd for C24H3401&1,517.1831). G (7): Briaexcavatolide G (7) (2.0 mg) was stirred with 1 mL of A~20 in

1 mL of pyridine for 48 h at room temperature. by column chromatography

-26” (c 0.1, CHC13); IR (KBr)

IH and 13C NMR data, see Tables 2 and 3; FABMS m/z 519 [O.4, (M + H +

After evaporation

of excess reagent, the residue was separated

on SiO2 gel to give pure briaexcavatolide

E (5) (hexanes

: EtOAc = 7 : 2; 1.9 mg,

8 1%); physical and spectral data were in full agreement with those of the natural product 5. Cytotoxicity

Testing.

Illinois at Chicago;

KB and P-388 cells were kindly provided by Prof. J. M. Pezzuto,

A549 and HT-29 cells were purchased

from the American

University

Type Culture Collection.

of The

J.-H. Sheu et al. /Tetrahedron

14564

cytotoxic

activities

of tested compounds

55 (1999) 14555-14564

against the above four cancer cells were assayed with a modification

of the MTT [3-(4,5-dimethylthiazo1-2-yl)-2,5-diphenyltetrazolium assays were carried out according Single-Crystal

to the procedure

X-ray Crystallography

described

bromide]

of 2. 17 Suitable colorless

= 14.785 (5), b = 21.551 (5), c = 8.773 (4) A,V= 2795 (1) A3,Z= data were

reflections -squares

measured

were collected. procedure.

converged

on Rigaku

The structure

The nonhydrogen

AFC6S

was solved atoms

system, space group P212121 with a

4, Dcatc = 1.268 g/cm3, h (MoKa)

diffractometer by dirtect

were given

method. Cytotoxicity

prisms of 2 were otabined from a solution

in EtOH. The crystal (0.33 X 0.50 X 0.50 mm) belongs to the monoclinic A. Intensity

calorimetric

previously.t5,16

up to 28 of 47.1”.

method

anisotropic

to a final R = 0.091, R, = 0.057 for 132 1 observed

and refined

thermal

reflections

= 0.71069

All 2407 unique

by a full-matrix

parameters.

least

The refinement

[I > 3.00 a (I)] and 153 variable

parameters. Acknowledgment.

We gratefully

Science Council of the Republic

acknowledge

the generous

financial

support

of this work by National

of China (Contract no. NSC-88-2113-M-110-011)

awarded to J.-H. Sheu.

References and Notes 1. Faulkner, D. J. Nur. Prod. Rep. 1999. 16, 155, and previous reports in this series. 2. Sheu, J.-H.; Sung, P.-J.; Cheng, M.-C.; Liu, H.-Y.: Fang, L.-S.; Duh, C.-Y.; Chiang. M. Y. J. Nut. Prod. 1998, 61, 602. 3. Sung, P.-J.; Su, J.-H.; Wang, G.-H.; Lin, S.-F.; Duh, C.-Y.; Sheu, J.-H. J. Nat. Prod. 1999,62,457. 4. Sheu, J.-H.; Sung, P.-J.; Su, J.-H.; Wang, G.-H.; Duh, C.-Y.; Shen, Y.-C.; Chiang, M. Y.; Chen, I.-T. J. Nat. Prod. 1999, (in press). 5. Neve, J. E.; McCool,

B. J.; Bowden,

B. F. Aust. J. Chem. 1999,52, 359.

6. Sheu, J.-H.: Sung. P.-J.: Huang, L.-H.: Lee, S.-F.: Wu, T.; Chang, B.-Y.; Duh, C-Y.; Fang, I..&:

Soong,

K.; Lee, T.-J. J. Nat. Prod. 1996,59,935. 7. Coval, S. J.; Cross, S.; Bernardinelli, 8. Natori. T.; Kawai, H.; Fusetani, 9. Bloor. S. J.; Schmitz, 10. Schmitz,

G.; Jefford, C. W. J. Nat. Prod. 1988, 51, 98 1.

N. Tetrahedron Lett. 1990,31, 689.

F. J.; Hossain, M. B.; van der Helm, D. J. Org. Chem. 1992,.57, 1205.

F. J.; Schulz, M. M.; Siripitayananon,

J.; Hossain,

M. B.; van der Helm, D. J. Nut. Prod. 1993,

56, 1339. 11. Rodriguez,

J.: Nieto. R. M.; JimCnez, C. J. Nut. Prod. 1998,61,

12. Pordesimo,

E. 0.; Schmitz,

F. J.; Ciereszko,

3 13.

L. S.; Hossain, M. B.; van der Helm, D. J. Org. Chem. 1991,

56.2344. 13. Kobayashi, J.; Cheng, J.-F.; Nakamura, Jacobs, R. S.; Kato. Y.: Brinen, 14. For significant Greenberg,

activity

H.; Ohizumi,

L. S.; Clardy,

of pure compounds,

N. H.; MacDonald,

Y.; Tomotake,

J. Experientia

1991,47,

Y.; Matsuzaki,

an EDso value of I 4.0 pg/mL

M. M.; Schumacher,

T.; Grace, K. J. S.;

501. is required.

Geran, R. I.;

A. M.; Abbott, B. J. Cancer Chemother. Rep. 1972,

3, 1. 15. Mosmann,

T. J. Immunol. Methods

1983,65,55.

16. Sheu, J.-H.; Liaw, C.-C.; Duh, C.-Y. J. Nat. Prod. 1995,58, 17. Atomic coordinates Centre,

for this structure have been deposited

12 Union Road, Cambridge

CB2 lEZ, UK.

1521.

with the Cambridge

Crystallographic

Data