Antioxidative bromoindole derivatives from the mid-intestinal gland of the muricid gastropod Drupella fragum

Article abstract of DOI:10.1021/np980097r

Three new bromoindoles, 6-bromo-5-hydroxyindole (1), 6-bromo-4,5- dihydroxyindole (2), and 6-bromo-4,7-dihydroxyindole (3), were isolated from the midintestinal gland of the muricid gastropod Drupella fragum. The structures of 2 and 3 were elucidated mainly by NMR spectroscopic analyses of their acetyl derivatives, whereas the structure of 1 was established by spectroscopic methods and total synthesis. Antioxidative activity for compounds 1-3 was evaluated by the POV method, and compound 1 was found to have as strong an antioxidative potency as BHT.

Full text of DOI:10.1021/np980097r

J . Nat. Prod. 1998, 61, 1043-1045
1043
An tioxid a tive Br om oin d ole Der iva tives fr om th e Mid -In testin a l Gla n d of th e
Mu r icid Ga str op od Dr u pella fr a gu m
,
†
†
†
‡
‡
Masamitsu Ochi,* Kumi Kataoka, Saori Ariki, Chie Iwatsuki, Mitsuaki Kodama, and
,
‡
Yoshiyasu Fukuyama*
Department of Chemistry, Faculty of Science, Kochi University, Kochi 780-8072, J apan, and
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, J apan
Received March 16, 1998
Three new bromoindoles, 6-bromo-5-hydroxyindole (1), 6-bromo-4,5-dihydroxyindole (2), and
6
-bromo-4,7-dihydroxyindole (3), were isolated from the midintestinal gland of the muricid
gastropod Drupella fragum. The structures of 2 and 3 were elucidated mainly by NMR
spectroscopic analyses of their acetyl derivatives, whereas the structure of 1 was established
by spectroscopic methods and total synthesis. Antioxidative activity for compounds 1-3 was
evaluated by the POV method, and compound 1 was found to have as strong an antioxidative
potency as BHT.
The muricid gastropod Drupella fragum (Muricacea)
is well known to be a predator on Madreporaria corals,
and a population explosion of the gastropod may cause
widespread destruction of corals, resulting in an ecologi-
cal imbalance.1 No effective way for exterminating
muricid predation on corals has been proposed to data.
Chemical studies on the midintestinal gland of D.
fragum may allow the isolation of metabolites from
previously uninvestigated Madreporaria corals and may
also lead to the discovery of new biologically active
Ta ble 1. NMR Spectra Data of Compounds 1, 2a , and 3a in
CDCl3 (400 MHz for H NMR, 100 MHz for C NMR)
1
13
1
2a
3a
position
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
,2
2
3
7.15 (t, 2.9) 126.0 7.36 (d, 3.7) 127.5 7.42 (d, 3.7) 126.7
6.42 (d, 2.9) 102.4 6.55 (d, 3.7) 105.3 6.49 (d, 3.7) 105.7
3a
4
5
6
7
7a
OH
128.8
105.7
145.9
106.3
113.8 7.38 (s)
131.5
126.4
140.6
134.2 8.69 (s)
113.9
121.0
124.5
136.4
118.5
113.8
135.4
134.2
7.48 (s)
7.24 (s)
5.19 (s)
3
-5
129.1
substances.
D. fragum was collected from the shallow waters
along the Ohtsuki coast, Kochi, J apan. The midintes-
tinal gland of D. fragum was extracted with methanol
and the methanol extract was partitioned sequentially
between water and hexane, dichloromethane, and n-
butanol. Compound 1 was isolated from the dichlo-
romethane soluble portion, whereas compounds 2 and
OAc Me
2.37 (s)
2.42 (s)
2.60 (s)
20.9 2.380 (s)
24.9 2.382 (s)
2.63 (s)
167.0
68.6
68.7
20.4
20.5
23.8
167.7
167.9
168.4
OAc CdO
1
1
Ta ble 2. Antioxidative Activities of Compounds 1-3
Evaluated by the POV Method
3
were obtained from the n-butanol layer.
a
b
sample
POVs /POVc (mequiv/kg)
1
2
3
0.036
0.194
0.106
0.062
0.031
R-tocopherol
BHTc
a
Peroxide value with the sample. b Peroxide value without the
sample (control). c Butylated hydroxytolune.
formula, C8H6BrON, consistent with six unsaturation
units, was secured by HREIMS measurements at m/z
2
3
13 and 211. Absorption bands in the IR (3570 and
520 cm ) and UV (218, 282, 296, and 312 nm) spectra
-1
suggested that 1 was a hydroxyindole derivative. The
1
3
C NMR data of 1 (Table 1) indicated that all the
2
Compound 1 was obtained as a colorless amorphous
carbons were sp and comprised four methines and four
quaternary carbons. The H NMR spectrum contained
1
solid. Its EIMS exhibited molecular ion peaks at m/z
13 and 211 in a 1:1 ratio, suggesting the presence of
one bromine atom in the molecule. The molecular
2
two aromatic singlet signals at δH 7.24 and 7.48 and a
set of doublet signals at δH 6.42 and 7.15 (J ) 2.9 Hz)
assignable to H-3 and H-2 on the indole nucleus.
Treatment of 1 with NaH-MeI in DMF yielded the
dimethyl derivative 1a [δH 3.75 (3H, s, NMe), 3.92 (3H,
s, OMe)], confirming the presence of a hydroxy group
*
To whom correspondence should be addressed. Fax: +81-886-55-
3
051. E-mail: fukuyama@ph.bunri-u.ac.jp.
†
Kochi University.
‡
Tokushima Bunri University.
S0163-3864(98)00097-4 CCC: $15.00
© 1998 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/03/1998

1
044 J ournal of Natural Products, 1998, Vol. 61, No. 8
Notes
Sch em e 1a
dole. On the other hand, the sole singlet proton signal
at δH 8.69 in 3a was assigned to H-5 by the observation
of its HMBC correlation to the C-3a resonance at δC
1
24.50, but not to the C-7a resonance at δC 134.18.
Thus, 3a was formulated as N-acetoxy-6-bromo-4,7-
diacetoxyindole. Compounds 2 and 3 were thus as-
signed as 6-bromo-4,5-dihydroxyindole and 6-bromo-4,7-
dihydroxyindole, respectively.
The bromoindoles 1-3 isolated from D. fragum were
tested for antioxidant activity by the peroxide value
a
Key: (a) pyrrolidine, Me2NH(OMe)2, DMF, 100 °C; (b) TiCl3,
8
(POV) method. Among compounds 1-3, 1 exhibited
NH4OAc, DMF, rt; (c) NaH, DMF, MeI 0 °C f rt, 47%.
the most potent antioxidative activity, higher than that
of R-tocopherol and almost equal to that of BHT. It
should be noted that the promising antioxidant 1 is
stable under the isolation procedures and the conditions
of spectral measurements, while the same is not true
for compounds 2 and 3.
Exp er im en ta l Section
1
13
F igu r e 1. Significant H- C couplings observed for 2a and
a in HMBC (8.1 Hz) experiments.
Gen er al Exper im en tal P r ocedu r es. Melting points
were determined with a Mitamura hot-stage microscope
and are uncorrected. IR and UV spectra were obtained
using a J ASCO FTIR-5300 and a J ASCO UVIDEC 670
spectrophotometer, respectively. NMR spectra were
recorded on a J EOL GX-400 and a Varian Unity-200,
with TMS as the internal standard. The HMBC,
HMQC, and ROESY were run on a J EOL at 400 MHz.
MS spectra were measured with a J EOL J MS-D300 and
a J EOL J MS HX-100. HRMS were recorded on a J MS
HX-100. HPLC was carried out on a Waters Model
6000A with detection at 254 nm. Si gel 60 (Merck) and
Wako gel C-300 (Wako) were used for column chroma-
tography. Analytical TLC were carried out on silica gel
plates (Kieselgel 60, Merck) and visualized by 5% CeSO4
in H2SO4 followed by heating.
Collection , Extr a ction , a n d Isola tion . The gas-
tropod D. fragum was collected by scuba in November
1992, from Ohtsuki coast, Kochi, J apan. The gastropod
was freeze-dried upon arrival and kept frozen until
extraction. A voucher specimen has been stored at the
Chemistry Department of Kochi University. After the
samples (26.7 kg) were defrosted, the midintestinal
gland (1.5 kg) of D. fragum was blended with MeOH,
and after filtration the crude extract (20 L) was evapo-
rated under vacuum to yield a residue (250 g) that was
partitioned between hexane and H2O. The aqueous
portion was extracted with CH2Cl2, and then the aque-
ous layer was extracted with n-BuOH. The CH2Cl2
extract was concentrated to give a residue (6.2 g), which
was loaded onto a size-exclusion column (Sephadex LH-
20) and eluted with MeOH to yield 10 fractions. Frac-
tion 9 (313 mg) was purified successively by column
chromatography on Si gel (60 g) with Me2CO-hexane
(3:7) and HPLC [TSK-GEL LS-410K] with MeOH-H2O
(3:7) to give 6-bromo-5-hydroxyindole (1) (47 mg). The
n-BuOH extract (12 g) was subjected to Sephadex LH-
20 column chromatography eluted with MeOH, yielding
seven fractions. Fraction 3 (100 mg) was chromato-
graphed over a TOYOPEARL HW-40 with MeOH to
yield 6-bromo-4,5-dihydroxyindole (2), and fraction 5
(112 mg) was purified by column chromatography on
Sephadex LH-20 with MeOH to give 6-bromo-4,7-
dihydroxyindole (3).
3
and an indole moiety. Although the locations of the
bromine atom and the hydroxy group were deduced from
comparison with the spectral data of the previously
known eudistomin C to be on the C-6 and C-5 positions,
respectively, the need to confirm the proposed structure
6
prompted us to synthesize the dimethyl derivative 1a
(Scheme 1).
5
-Acetoxy-4-bromo-2-nitrotoluene (4) was condensed
with dimethylformamide dimethyl acetal in the pres-
ence of pyrrolidine followed by reduction of the nitro
group into the amine with titanous chloride in a 4 M
ammonium acetate buffer to give 5-bromo-6-methoxy-
7
indole (5). Treatment of 5 with MeI-NaH in DMF
furnished the N-methylindole in 47% yield, which was
identical in all respects with 1a derived from 1. Thus,
the structure of 1 was established as 5-bromo-6-hy-
droxyindole.
Compounds 2 and 3 gradually decomposed during
NMR measurements, and the IR spectra indicated the
presence of a hydroxy group. The stable triacetyl
derivatives 2a and 3a were thus prepared from 2 and 3
by acetylation. The same molecular formula, C14H12-
BrO5N, for 2a and 3a was established by HREIMS
measurements at m/z 353, indicating again the presence
of one bromine atom. Their UV absorption, analogous
to that of 1, suggested the presence of an indole nucleus
1
(
see the Experimental Section). The H NMR spectrum
of 2a (Table 1) showed a singlet aromatic signal at δH
7
6
.38 and a set of signals coupled to each other at δH
.55 (J ) 3.2 Hz, H-3) and 7.36 (J ) 3.2 Hz, H-2) in
addition to three acetoxyl signals at δH 2.37, 2.42, and
.60. The NMR data of 3a (Table 1) were similar to
those of 2a . These spectral data indicated that 2a and
a are regioisomers in regard to the two hydroxy groups
2
3
on the benzene ring of the indole nucleus. The positions
of the acetoxy groups for 2a and 3a were clarified by
HMBC experiments, and the results are summarized
in Figure 1. The singlet carbon resonances in the
spectrum of 2a at δC 126.4 and 129.1, which were
assigned to C-3a and C-7a by HMBC correlation from
H-2 and H-3, respectively, showed further long-range
couplings to the singlet proton signal at δH 7.38. The
sole aromatic proton was thus placed on the C-7
position, and 2a is N-acetoxy-6-bromo-4,5-diacetoxyin-
6-Br om o-5-h yd r oxyin d ole (1): colorless amorphous
solid; IR (CHCl3) 5570, 3520, 1580, 1460, 1355, 1220,

Notes
J ournal of Natural Products, 1998, Vol. 61, No. 8 1045
1
1
6
150, 990 cm- ; UV λmax (EtOH) 312 (ꢀ 4480), 296 (ꢀ
argon atmosphere and heated at 100-110 °C for 2.5 h.
The red solution was cooled to room temperature, and
a solution of 4 M ammonium acetate (2.5 mL) in DMF
(2.5 mL) was added dropwise. The red solution was
stirred for 25 min while 20% (w/v) TiCl3 (2.8 mL) was
added dropwise. The resulting gray suspension was
extracted with Et2O, washed with H2O and brine, and
then dried. The solvent was removed in vacuo to give
the crude residue, which was chromatographed on Si
gel with hexanes-EtOAc (6:1) to yield compound 5 (42
mg, 25%) as an oil: IR (neat) 3397, 2963, 1740, 1308,
1
13
300), 282 (ꢀ 7800), 218 (ꢀ 24 300) nm; H and C NMR,
see Table 1; EIMS m/z 213 (99), 211 (100); HREIMS m/z
12.9623 [M ] (calcd for C8H6 BrON, 212.9612),
10.9633 [M ] (calcd for C8H6 BrON, 210.9632).
-Br om o-4,5-d ih yd r oxyin d ole (2): colorless amor-
phous solid; IR (neat) 3430, 1630, 1590, 1500, 1260 cm
H NMR (400 MHz, CD3OD) δ 6.55 (1H, br s), 7.07 (1H,
br s), 7.11 (1H, br s).
-Br om o-4,7-d ih yd r oxyin d ole (3): colorless amor-
phous solid; IR (neat) 3420, 1630, 1580, 1480, 1350, 1270
cm ; H NMR (400 MHz, CD3OD) δ 6.50 (1H, d, J )
.1 Hz), 7.07 (1H, d, J ) 3.1 Hz), 7.15 (1H, br s).
Meth yla tion of 1 (1a ). To a suspension of NaH (50%
+
81
2
2
+
79
6
-1
;
1
6
-
1
1
-1
1
1209, 1155, 1040 cm ; H NMR (200 MHz, CDCl3) δ
3.92 (3H, s, OMe), 6.48 (1H, d, J ) 2.4 Hz, H-3), 7.13
(1H, s, H-4), 7.17 (1H, t, J ) 2.4 Hz, H-2), 7.59 (1H, s,
H-7), 8.03 (1H, br d, J ) 2.4 Hz, NH); EIMS m/z 227
3
dispersion in oil, 8 mg) in DMF (1 mL) was added a
solution of compound 1 (10 mg) in DMF (0.2 mL) at 0
+
(100), 225 (99); HREIMS m/z 224.9803 [M ] (calcd for
°
C under an argon atmosphere, and the reaction mix-
C9H8BrON, 224.9790).
ture was allowed to stir at room temperature for 1 h.
MeI (0.6 mL) was added to this solution. After being
stirred for 1 h, the reaction mixture was diluted with
Et2O, washed with H2O and brine, and then dried. The
solvent was removed in vacuo to give the crude oil,
which was chromatographed on Si gel with EtOAc to
yield dimethyl derivative 1a (5 mg) as colorless prisms:
mp 113-114 °C; IR (KBr) 3441, 1630, 1483, 1244, 1154,
N-Meth yl-6-br om o-5-m eth oxyin d ole (1a ). To a
suspension of NaH (50% dispersion in oil, 4 mg) in DMF
(0.5 mL) was added a solution of compound 1 (4.0 mg)
at 0 °C under an argon atmosphere, and the reaction
mixture was allowed to stir at room temperature for 1
h. MeI (0.05 mL) was added to this solution. After
being stirred for 1 h, the reaction mixture was diluted
with Et2O, washed with H2O and brine, and then dried.
The solvent was removed in vacuo to give the crude oil,
which was chromatographed on Si gel with EtOAc to
-
1
1
1
042 cm ; H NMR (400 MHz, CDCl3) δ 3.75 (3H, s,
OMe), 3.92 (3H, s, OMe), 6.38 (1H, dd, J ) 3.2. 0.7 Hz,
H-3), 7.02 (1H, d, J ) 3.2 Hz, H-2), 7.17 (1H, s, H-7),
1
yield 6-bromo-5-methoxyindole (2.0 mg), the H NMR,
IR and MS spectra of which were identical with those
of dimethyl derivative 1a converted from 1.
7
.52 (1H, d, J ) 0.7 Hz, H-4); EIMS m/z 241 (100), 239
+
79
(99); HREIMS m/z 238.9941[M ] (calcd for C10H10 Br
ON, 238.9946).
Bioa ssa y. POV (peroxide value) was measured ac-
cording to the standard method recommended by the
J apanese Society of Fat and Oil.
8
Aceta te d er iva tive of 2 (2a ). Compound 2 (9.7 mg)
was acetylated with Ac2O (0.1 mL) + pyridine (0.1 mL),
affording triacetate 2a (5.7 mg) as colorless prisms: mp
Ack n ow led gm en t. We thank Mr. S. Takoka and
Mrs. I. Okamoto of the Instrument Analysis Center for
Pharmaceutical Sciences, Tokushima Bunri University,
for MS and NMR measurements, and X-ray analyses.
-
1
1
56-156.5 °C; IR (Nujol) 1760, 1470, 1370, 1200 cm
UV λmax (EtOH) 308 (ꢀ 9000), 298 (ꢀ 8000), 270 (ꢀ
8 000), 263 (ꢀ 19 000), 236 (ꢀ 38 000), 208 (ꢀ 36 000);
;
1
1
13
H and C NMR, see Table 1; HREIMS m/z 352.9882
+
[M ] (calcd for C14H12BrO5N, 352.9899).
Refer en ces a n d Notes
Aceta te Der iva tive of 3 (3a ). Compound 3 (10 mg)
(
1) Boucher, L. M. Bull. Mar. Sci. 1986, 38, 9-11.
(2) Moyer, J . T.; Emerson, W. K.; Ross, M. Nautilus 1982, 96, 69-
2.
was acetylated with Ac2O (0.1 mL) + pyridine (0.1 mL),
affording triacetate 3a (2 mg). 3a : colorless needles;
mp 142.5-143.5 °C; IR (Nujol) 1770, 1720, 1400, 1380,
8
(3) Ochi, M.; Tatsukawa, A.; Kotsuki, H.; Fukuyama, Y.; Shibata,
K. Chem. Lett. 1994, 89-92.
-
1
(4) Ochi, M.; Yamada, K.; Kotuski, H.; Shibata, K. Chem. Lett. 1991,
1
2
3
3
200 cm ; UV λmax (EtOH) 304 (ꢀ 9300), 293 (ꢀ 8900),
74 (ꢀ 17 200), 268 (ꢀ 16 800), 240 (ꢀ 30 800), 204 (ꢀ
4
27-430.
(
5) Ochi, M.; Yamada, K.; Shirase, K.; Kotsuki, H.; Shibata, K.
1
13
2 000); H and C NMR, see Table 1; HREIMS m/z
Heterocycles 1991, 32, 28-21.
+
(6) Lake, R. J .; Blunt, J . W.; Munro, M. H. G. Aust. J . Chem. 1989,
52.9872 [M ] (calcd for C14H12BrO5N, 352.9899).
4
2, 1201-1206.
7) Ayer, W. A.; Ma, Y.; Miao, S. Tetrahedron 1992, 48, 2919-2924.
(8) Nose, M.; Fujino, N. Nippon Shokuhin Kogyo Gakkasihi 1982,
9, 507-512 and references therein.
6
-Br om o-5-m eth oxyin d ole (5). A solution of 5-ac-
(
etoxy-4-bromo-2-nitrotoluene (200 mg, 0.73 mmol), di-
methylformamide dimethyl acetal (0.3 mL), and pyrro-
lidine (0.1 mL) was stirred in DMF (2 mL) under an
2
NP980097R