Novel bromotyrosine derivatives that inhibit growth of the fish pathogenic bacterium Aeromonas hydrophila, from a marine sponge Hexadella sp.

Article abstract of DOI:10.1021/jo048203j

(Chemical Equation Presented) Three new bromotyrosine derivatives, 11-N-methylmoloka′iamine (1), 11-N-cyano-11-N-methylmoloka′iamine (2), and kuchinoenamine (3), were isolated as antibacterial constituents from a marine sponge Hexadella sp. Their structur

Full text of DOI:10.1021/jo048203j

we found that the hydrophilic extract of a marine sponge
Hexadella sp. collected in southern Japan exhibited
moderate antibacterial activity against the bacterium
Aeromonas hydrophila, which causes hemorrhagic sep-
ticemia in fish.14 Bioassay-guided fractionation afforded
three new bromotyrosine derivatives, 11-N-methylmolo-
ka′iamine (1), 11-N-cyano-11-N-methylmoloka′iamine (2),
and kuchinoenamine (3). This paper describes the isola-
tion, structure elucidation, and antimicrobial activities
of these compounds.
The MeOH extract of the frozen sponges (1.0 kg) were
partitioned between Et2O and H2O. The aqueous layer
was further partitioned between n-BuOH and H2O. The
n-BuOH layer was sequencially separated by ODS flash
chromatography, gel filtration, centrifugal partition chro-
matography, and reversed-phase HPLC to afford 11-N-
methylmoloka′iamine (1), 11-N-cyano-11-N-methylmolo-
ka′iamine (2), and kuchinoenamine (3) in yields of 638.2,
1.0, and 13.2 mg, respectively.
Novel Bromotyrosine Derivatives That
Inhibit Growth of the Fish Pathogenic
Bacterium Aeromonas hydrophila, from a
Marine Sponge Hexadella sp.1
Shigeki Matsunaga,† Hirotsugu Kobayashi,†
Rob W. M. van Soest,‡ and Nobuhiro Fusetani*,†
Laboratory of Aquatic Natural Products Chemistry,
Graduate School of Agricultural and Life Sciences, The
University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan,
and Institute for Systematics and Ecology, University of
Amsterdam, 1090 GT Amsterdam, The Netherlands
anobu@mail.ecc.u-tokyo.ac.jp
Received October 12, 2004
Three new bromotyrosine derivatives, 11-N-methyl-
moloka′iamine (1), 11-N-cyano-11-N-methylmoloka′iamine
(2), and kuchinoenamine (3), were isolated as antibacterial
constituents from a marine sponge Hexadella sp. Their
structures were elucidated on the basis of spectral and
chemical methods. They exhibited moderate antibacterial
activity against the fish pathogenic bacterium Aeromonas
hydrophila.
Marine sponges of the order Verongida have proved
to be a rich source of bromotyrosine-derived metabolites2
which show a wide range of biological activities including
anti-HIV,3-5 antibacterial,6,7 antifouling,6,7 cytotoxicity,6-10
toxicity to the crab,11 Na,K-ATPase inhibitory,12 and
histamine H3 antagonistic.13 In our search for biologically
active compounds from Japanese marine invertebrates,
11-N-Methylmoloka′iamine (1) had a molecular for-
mula of C12H18N2OBr2 as analyzed by HRFABMS. Inter-
pretation of the 1H NMR spectrum together with HMQC
data led to the presence of an N-methyl (δH 2.67, δC 33.7),
two unfunctionalized methylenes (δH 2.16, δC 29.2; δH
2.90, δC 31.8), two nitrogenous methylenes (δH 3.26, δC
38.8; δH 3.19, δC 50.7), an oxygenated methylene (δH 4.08,
δC 71.7), and an aromatic signal [δH 7.53 (2H), δC134.5
(2C)]. In addition, four nonprotonated sp2 carbon signals
[δC 119.3 (2C), 153.1, and 137.4] were observed in the
13C NMR spectrum. The COSY spectrum revealed spin
systems for N-CH2-CH2-CH2-O and CH2-CH2-N
units, thereby indicating 1 to be an N-methyl derivative
of moloka′iamine (7).15 11-N-Methylation was deduced on
† The University of Tokyo.
‡ University of Amsterdam.
(1) Bioactive Marine Metabolites. 130. Part 129: Hamada, T.;
Matsunaga, S.; Yano, G.; Fusetani, N. J. Am. Chem. Soc., 2005, 127,
110-118.
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Prinsep, M. R. Nat. Prod. Rep. 2003, 20, 1-48 and the previous reviews
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1609-1612.
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P.; Scheuer, P. J.; Hamann, M. T. J. Nat. Prod. 2000, 63, 501-503.
(6) Tsukamoto, S.; Kato, H.; Hirota, H.; Fusetani, N. J. Org. Chem.
1996, 61, 2936-2937.
(7) Tsukamoto, S.; Kato, H.; Hirota, H.; Fusetani, N. Tetrahedron
1996, 52, 8181-8186.
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Experimentia 1990, 47, 299-300.
(11) Fusetani, N.; Masuda, Y.; Nakao, Y.; Matsunaga, S.; van Soest,
R. W. M. Tetrahedron 2001, 57, 7507-7511.
(9) Kobayashi, J.; Honma, K.; Sasaki, T.; Tsuda, M. Chem. Pharm.
Bull. 1995, 43, 403-407.
(12) Nakamura, H.; Wu, H.; Kobayashi, J.; Nakamura, Y.; Ohizumi,
Y. Tetrahedron Lett. 1985, 37, 4517-4520.
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Patel, M.; Coval, S. J. J. Nat. Prod. 1994, 57, 175-177.
10.1021/jo048203j CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/28/2005
J. Org. Chem. 2005, 70, 1893-1896
1893

SCHEME 1a
a Reagents and conditions: (a) N-carbethoxyphthalimide, Na2CO3, THF-H2O (8:2), rt, 30 min; (b) CNBr, NaHCO3, EtOH, rt, 12 h; (c)
H2NNH2‚H2O, EtOH, rt, 15 h.
TABLE 1. 1H and 13C NMR Spectral Data in CD3OD at
the basis of an HMBC correlation between the N-methyl
proton and C-11.
600/150 MHz for Kuchinoenamine (3)
position
δH (mult, J in Hz) 3
δC
3
HMBC
C2, 3, 1′
11-N-Cyano-11-N-methylmoloka′iamine (2) had
a
1
3.86 (m, br)
2.19 (s, br)
4.06 (m, br)
49.4
31.3
71.3
153.3
119.5
134.4
137.1
31.8
50.9
33.7
157.6
111.4
205.0
48.7
51.1
201.6
51.1
50.7
205.3
59.5
molecular formula of C13H17N3OBr2 as established by
2
HRFABMS, indicating that a hydrogen in 1 was replaced
3
1
by a CN unit. In fact, the H NMR spectrum measured
4
in CD3OD exhibited the same sets of signals as observed
in 1. HMBC cross-peaks were observed between N-Me
and C-11 and between N-Me and a carbon at δ 118.8
which was assigned as a cyano group. This was in
agreement with an IR band at 2210 cm-1. Other NMR
data were superimposable on those of 1. Therefore, 2 was
assigned to the 11-N cyanamide of 1. To confirm this
assignment an 11-N cyano derivative of 1 was prepared
as outlined in Scheme 1. Compound 1 was converted to
the phthalimide 4,16 which was treated with CNBr to give
the cyanamide 5.17 Removal of the phthalimide group of
5 with hydrazine18 afforded 2 which was indistinguish-
5, 9
6, 8
7
7.55 (s)
C5, 6, 7, 8, 9, 10
10
11
12
1′
2.95 (t, 6.9)
3.24 (t, 6.9)
2.72 (s)
C6, 7, 8, 11
C7, 10, 12
C11
7.89 (s)
C1, 2′ 3′
2′
3′
4′
3.38 (dd, 5.8, 8.9)
3.49 (dd, 4.8, 8.9)
C2′, 3′, 5′, 6′, 7′, 8′
C3′, 4′, 6′, 7′, 9′, 10′
5′
6′
7′
3.12 (s, br)
4.64 (d, 4.2)
8′
C4′, 7′, 9′
1
9′
able from the natural product in the H NMR and FAB
10′
11′
3.10 (d, 4.2)
4.53 (s)
C4′, 5′, 7′, 9′, 11′
C4′, 7′, 10′
mass spectra. To the best of our knowledge, 2 is the first
marine natural product encompassing a cyanamide moi-
ety.2 As to natural products containing a cyanamide
moiety, only one compound was reported from a mush-
room.19
79.1
Kuchinoenamine (3) exhibited a cluster of ion peaks
in a ratio of 1:3:3:1 at m/z 647, 649, 651, and 653 in
FABMS, indicating the presence of three bromine atoms.
The molecular formula of C23H25O5N2Br3 was determined
1
on the basis of HRFABMS and NMR data. The H and
13C NMR spectra (Table 1) readily indicated the presence
FIGURE 1. Structures of units A and B of 3.
(14) Roberts, R. J. In Bacterial Disease of Fish; Inglis, V., Roberts,
R. J., Bromage, N. R., Eds.; Blackwell Sci. Publ.: Oxford, 1993; pp
143-155.
of fragment A in 3 (Figure 1). The remaining elements
of C11H8O4Br were composed of a polarized trisubstituted
olefin (δH 7.89, δC 157.6; δC 111.4), six methines (δH 3.10,
δC 59.5; δH 3.12, δC 51.1; δH 3.38, δC 48.7; δH 3.49, δC 51.1;
δH 4.53, δC 79.1; δH 4.64, δC 50.7), and three ketones (δC
201.6, 205.0, and 205.3), thereby suggesting the presence
of three rings. Some 1H and 13C NMR signals were either
broadened or doubled, indicating the presence of confor-
mational equilibrium in the molecule. Interpretation of
(15) Hamann, M. T.; Scheuer, P. J. J. Org. Chem. 1993, 58, 6565-
6569.
(16) McArthur, C. R.; Worster, P. M.; Okon, A. U. Synth. Commun.
1983, 13, 311-318.
(17) Snider, B. B.; O’Hare, S. M. Tetrahedron Lett. 2001, 42, 2455-
2358.
(18) Sasaki, T.; Minamoto, K.; Itoh, H. J. Org. Chem. 1978, 43,
2320-2325.
(19) Gasco, A.; Serafino, A.; Mortarini, V.; Menziani, E.; Bianco, M.
A.; Scurti, J. C. Tetrahedron Lett. 1974, 38, 3431-3432.
1894 J. Org. Chem., Vol. 70, No. 5, 2005

kuchinoenamine (3) had a unique tricyclo[5.2.1.02,6]-
decane skeleton attached to 1 via an enamine linkage
and is biogenetically related to psammaplysin E (8)4 and
waiana′enamines A (9), and B (10);21 the tricyclo[5.2.1.02,6]-
decane moiety may be formed by a [4 + 2] cycloaddition
of a cyclopenta-2,4-dienol and an N-substituted 2-ami-
nomethylenecyclopent-4-ene-1,3-dione followed by bro-
mohydrin formation and oxidation.
SCHEME 2
the COSY and HOHAHA spectra led to the six continu-
ous methine system (C-8′, C-7′, C-4′, C-5′, C-10′, and
C-11′). A slow exchange of H-8′ signal in CD3OD solution
allowed to link a ketone (C-9′) to C-8′ which must be
brominated as judged from its chemical shift values
(δH 4.64, δC 50.7). HMBC cross-peaks, H-5′/C-9′ and
H-10′/C-9′, connected C-9′ and C-10′, while an HMBC
cross-peak H-11′/C-4′ led to the connectivity of C-11′ and
C-7′. HMBC cross-peaks H-4′/C-3′, H-4′/C-6′, H-5′/C-3′,
and H-5′/C-6′ were consistent with that C-4′ and C-5′
were adjacent to a keto group, respectively. An insertion
of the polarized trisubstituted olefin between the keto
groups at C-3′ and C-6′ was implied by an HMBC cross-
peak H-1′/C-3′, thereby completing the gross struc-
ture of the unit B. Units A and B were connected be-
tween 1-N and C-1′ on the basis of an HMBC cross-peak
H2-1/C-1′.
To confirm this structure, we attempted to reduce the
C-9′ keto group. Due to its alkaline-labile property, 3 was
decomposed upon treatment with NaBH4. However, a
clean reduction proceeded when treated with Na(OAc)3BH
in MeOH to afford a single isomer of diol 6. COSY cross-
peaks between H-8′ and H-9′ placed an oxymethine
between C-8′ and C-10′ in 6. The doubled NMR signals
were accounted for by the equilibria as shown in Scheme
2. We were not able to infer which one dominates.
The relative stereochemistry of the tricyclic portion was
derived from the 1H-1H coupling constants and NOESY
data. A coupling constant of 8.9 Hz between H-4′ and H-5′
suggested that they were both exo, which was supported
by coupling constants JH-4′, H-7′ ) 4.8 Hz and JH-5′, H-10′
) 5.8 Hz. A coupling constant of 4.2 Hz between H-7′
and H-8′ revealed that H-8′ was exo.20 A NOESY correla-
tion between H-8′ and H-11′ showed the orientation of
the C-11′ hydroxyl group. As to the C-9′ stereochemistry
in 6, a large coupling constant between H-8′ and H-9′
indicated that H-9′ was also exo (Figure 2).
Compounds 1-3 showed antibacterial activity in disk
agar diffusion assay against the fish pathogenic bacte-
rium Aeromonas hydrophila (100 µg/ diameter 6.5 mm
disk; 1, 7.5 mm zone of inhibition; 2, 8 mm; 3, 7 mm).
Two bromotyrosine derivatives related to aerothionin
and bis-indole alkaloids have so far been reported from
marine sponges of the genus Hexadella.22,23 Compounds
1 and 2 are N-substituted derivatives of moloka′iamine
(7) from an unidentified genus of verongid sponge, while
Experimental Section
Animal Material. The sponge specimens were collected by
hand using scuba at a depth of 10 m off Kuchinoerabu-jima
Island in the Satsunan Islands (30° 27.22′ N; 130° 11.43′ E).
They were immediately frozen and preserved at -20 °C until
extraction. The sponge was identified as Hexadella sp. (family
Ianthellidae, order Verongida): a massive sponge with irregular
surface, which is in places heavily encrusted by other organisms,
and shows some irregularly distributed openings. Color is shiny
gray, turning dark brown in alcohol. In cross section the sponge
is shown to be a thin sheet of 3-5 mm thickness consolidating
thick masses of debris, causing the irregular surface. At the
microscopic level, the surface shows characteristic striation
patterns known for Hexadella, and the debris is seen to be
enveloped in collagen, without forming fibers, showing that the
sponge is using the debris for support. This phenomenon is
atypical for Hexadella, but has also been described for the North
Atlantic deep-water species Hexadella detritifera. Currently we
do not consider these sponges conspecific on account of large
differences in geographic distance and habitat. No Hexadella
species have been described from Japan. The voucher is incor-
porated in the Zoological Museum of Amsterdam under reg. no.
17063
Extraction and Isolation. The frozen sponges (1.0 kg wet
weight) were extracted three times with MeOH, and the
combined extracts were concentrated and partitioned between
water and Et2O. The aqueous layer was further extracted with
n-BuOH. The n-BuOH fraction was separated by ODS flash
chromatography with aqueous MeOH. The fraction eluted with
(20) Stevens, V. R.; Gaeta, F. C. A.; Lawrence, D. S. J. Am. Chem.
Soc. 1983, 105, 7713-7719.
(21) Lacy, C.; Scheuer, P. J. J. Nat. Prod. 2000, 63, 119-121.
(22) Morris, S. A.; Andersen, R. J. Can. J. Chem. 1989, 67, 677-
681.
(23) Morris, S. A.; Andersen, R. J. Tetrahedron 1990, 46, 715-720.
J. Org. Chem, Vol. 70, No. 5, 2005 1895

FIGURE 2. Relative stereochemistry of 3 and 6.
H2O was gel-filtered on Sephadex G-10 with H2O. The active
fraction was further separated by two rounds of HPLC on a
phenylhexyl column, first using a gradient elution of 0-50%
MeCN containing 0.05% TFA and then isocratic elution with 9%
MeCN containing 0.05% TFA to afford compound 1 (638.2 mg).
The fractions eluted with 20% and 50% MeOH from ODS flash
chromatography were combined and gel-filtered on Sephadex
LH-20 with MeOH/H2O (50:50). The active fractions were
separated by centrifugal partition chromatography [rotation,
1700 rpm; mobile phase, n-BuOH/MeOH/H2O (4:1:5); flow rate,
3 mL/min; ascending mode] to afford 35 fractions (15 mL each)
and additional 16 fractions by a reversed elution. The active
fractions eluted between 135 and 210 mL were separated by
ODS HPLC (gradient elution of 0-80% MeOH containing 0.05%
TFA) followed by reversed-phase HPLC on a C30 column (23%
MeCN containing 0.05% TFA) to afford compound 3 (13.2 mg).
Another fraction from the second ODS HPLC was finally purified
by reversed-phase HPLC on a phenylhexyl column (25% MeCN
containing 0.05% TFA) to furnish compound 2 (1.0 mg).
11-N-Methylmoloka′iamine (1): white powder; UV (MeOH)
λmax 277 (ꢀ 340), 284 nm (330); IR νmax (film) 3850, 3732, 3356,
2943, 2832, 1682, 1455, 1117, 1033 cm-1; HRFABMS m/z
366.9831 (M + H)+ (calcd for C12H19ON279Br81Br, ∆ -1.3 mmu);
1H NMR (600 MHz, CD3OD) δ 7.53 (s, 2H, H-6, 8), δ 4.08 (t, J
) 5.8 Hz, 2H, H-3), δ 3.26 (t, J ) 7.5 Hz, 2H, H-1), δ 3.19 (t, J
) 7.7 Hz, 2H, H-11), δ 2.90 (t, J ) 7.7 Hz, 2H, H-10), δ 2.67 (s,
3H, H-12), δ 2.16 (quint, J ) 5.8, 7.5 Hz, 2H, H-2); 13C NMR
(150 MHz, CD3OD) δ 153.1 (C-4), δ 137.4 (C-7), δ 134.5 (C-6, 8),
δ 119.3 (C-5, 9), δ 71.7 (C-3), δ 50.7 (C-11), δ 38.8 (C-1), δ 33.7
(C-12), δ 31.8 (C-10), δ 29.2 (C-2).
4. The product was used in the next reaction without purifica-
tion. 4: 1H NMR (600 MHz, CD3OD) δ 7.85 (dd, J ) 5.0, 3.1 Hz,
2H), δ 7.81 (dd, J ) 5.0, 3.1 Hz, 2H), δ 7.53 (s, 2H), δ 4.07 (t, J
) 6.2 Hz, 2H), δ 3.95 (t, J ) 7.3 Hz, 2H), δ 3.22 (t, J ) 7.7 Hz,
2H), δ 2.92 (t, J ) 7.7 Hz, 2H), δ 2.70 (s, 3H), δ 2.25 (quint, J )
6.2, 7.3 Hz, 2H).
Preparation of Cyanamide 5. To a mixture of 4 prepared
as described above and NaHCO3 (42.0 mg, 0.5 mmol) in EtOH
(2.5 mL) was added dropwise a solution of CNBr (53.0 mg, 0.5
mmol) in EtOH (2.5 mL) at rt. After stirring for 12 h at rt, the
reaction mixture was evaporated, suspended in H2O, and
extracted with EtOAc to give 5: 1H NMR (600 MHz, CD3OD) δ
7.85 (dd, J ) 5.4, 3.1 Hz, 2H), δ 7.80 (dd, J ) 5.4, 3.1 Hz, 2H),
δ 7.51 (s, 2H), δ 4.07 (t, J ) 6.2 Hz, 2H), δ 3.96 (t, J ) 7.3 Hz,
2H), δ 3.26 (t, J ) 7.1 Hz, 2H), δ 2.86 (t, J ) 7.1 Hz, 2H), δ 2.84
(s, 3H), δ 2.24 (tt, J ) 6.2, 7.3 Hz, 2H).
Preparation of 2. A solution of hydrazine monohydrate (32.0
mg, 1.0 mmol) in EtOH (2.0 mL) was added dropwisely to a
solution of 5 in EtOH (5.0 mL) at rt. The reaction mixture was
stirred for 15 h at rt and evaporated. The residue was purified
by reversed-phase HPLC on a phenylhexyl column (25% MeCN
containing 0.05% TFA) to afford 2 (19.5 mg, 31.4%) whose 1H
NMR and FAB mass spectra were indistinguishable from those
of the natural product.
Preparation of the Diol 6. A solution of 3 (5.0 mg, 7.7 µmol)
in MeOH (1 mL) was added to Na(OAc)3BH (31 mg, 146 µmol)
at rt. The mixture was stirred for 1 h at rt and evaporated. The
residue was purified by ODS HPLC (gradient elution of 5-35%
MeCN containing 0.05% TFA) to afford 6 (1.5 mg, 30.0%) and
unreacted 3 (3.5 mg). 6: 1H NMR (600 MHz, CD3OD) δ 7.86
(br, 1H), δ 7.53 (s, 2H), δ 4.42 (br, 1H), δ 4.20 (s, 1H), δ 4.11 (br,
1H), δ 4.07 (br, 2H), δ 3.85 (br, 2H), δ 3.22 (t, 6.9 Hz, 2H), δ
3.22 (overlapped, 1H), δ 3.12 (br, 1H), δ 2.92 (t, 6.9 Hz, 2H), δ
11-N-Cyano-11-N-methylmoloka′iamine (2): colorless solid;
UV (MeOH) λmax 277 (ꢀ 440), 284 nm (440); IR νmax (film) 3418,
2210, 1652, 1457, 1203, 1140 cm-1; HRFABMS m/z 391.9788
(M + H)+ (calcd for C13H18ON379Br81Br, ∆ -0.8 mmu); 1H NMR
(600 MHz, CD3OD) δ 7.56 (s, 2H, H-6, 8), δ 4.13 (t, J ) 5.8 Hz,
2H, H-3), δ 3.31 (t, J ) 7.1 Hz, 2H, H-1), δ 3.27 (t, J ) 6.9 Hz,
2H, H-11), δ 2.89 (t, J ) 6.9 Hz, 2H, H-10), δ 2.86 (s, 3H, H-12),
δ 2.20 (quint, J ) 5.8, 7.1 Hz, 2H, H-2); 13C NMR (150 MHz,
CD3OD) δ 152.6 (C-4), δ 139.2 (C-7), δ 134.5 (C-6, 8), δ 119.0
(C-5, 9), δ 118.8 (N-CN), δ 71.6 (C-3), δ 54.6 (C-11), δ 39.1
(C-12), δ 38.9 (C-1), δ 33.1 (C-10), δ 29.0 (C-2).
2.84 (br, 1H), δ 2.80 (br, 1H), δ 2.70 (s, 3H), δ 2.20 (br, 2H); 13
C
NMR (HSQC, CD3OD) δ 156.0 (C-1′), δ 133.0 (C-6, 8), δ 78.5
(C-11′), δ 69.9 (C-3), δ 67.3 (C-9′), δ 52.5 (C-7′), δ 52.2 (C-8′), δ
51.5 (C-10′), δ 49.2 (C-11), δ 48.2 (C-4′), δ 48.0 (C-5′), δ 47.7
(C-1), δ 33.2 (C-12), δ 30.6 (C-10), δ 30.1 (C-2).
Acknowledgment. Thanks are due to the crew of
R/V Toyoshio-maru for assistance in collecting the
sponge. This work was partly supported by a Grant-in-
Aid from the Japan Society for the Promotion of Science.
Kuchinoenamine (3): colorless solid; [R]21 +21 (c 0.05,
D
MeOH); UV (MeOH) λmax 244 (ꢀ 8620), 310 nm (12730); IR νmax
(film) 3440, 1757, 1730, 1682, 1614, 1556, 1471, 1257, 1203,
1136, 721 cm-1; HRFABMS m/z 650.9357 (M + H)+ (calcd for
C
23H26O5N279Br81Br2, ∆ +0.6 mmu); 1H and 13C NMR data, see
Supporting Information Available: General experimen-
tal procedure; 1H NMR, 13C NMR, COSY, HMBC, and
HMQC spectra of 1-3 in CD3OD; HOHAHA spectrum of 3 in
CD3OD; NOESY spectrum of 3 in CD3OH. This material is
available free of charge via the Internet at http://pubs.acs.org.
Table 1.
Preparation of Phthalimide 4. A solution of N-carboeth-
oxyphthalimide (65.7 mg, 0.3 mmol) in THF/H2O (4:1, 2.0 mL)
was added to a solution of compound 1 (59.6 mg, 0.1 mmol) and
Na2CO3 (63.6 mg, 0.6 mmol) in THF/H2O (4:1, 2.0 mL) at rt.
The mixture was stirred at rt for 30 min to afford phtahlimide
JO048203J
1896 J. Org. Chem., Vol. 70, No. 5, 2005