Oxazinins from toxic mussels: isolation of a novel oxazinin and reassignment of the C-2 configuration of oxazinin-1 and -2 on the basis of synthetic models

Article abstract of DOI:10.1016/j.tet.2006.05.070

The analysis of a batch of toxic mussels (Mytilus galloprovincialis) from the Northern Adriatic Sea led to the isolation of a novel oxazinin, oxazinin-4. Its structure including the relative stereochemistry has been elucidated through extensive NMR analys

Full text of DOI:10.1016/j.tet.2006.05.070

Tetrahedron 62 (2006) 7738–7743
Oxazinins from toxic mussels: isolation of a novel oxazinin and
reassignment of the C-2 configuration of oxazinin-1 and -2
on the basis of synthetic models
a
a
a
Patrizia Ciminiello,a, Carmela Dell’Aversano, Ernesto Fattorusso, Martino Forino,
Silvana Magno,a Federico Umberto Santelia,a Vassilios I. Moutsos,b
Emmanuel N. Pitsinosb and Elias A. Couladourosb,c
*
aDipartimento di Chimica delle Sostanze Naturali, Universitaꢀ degli Studi di Napoli ‘Federico II’, Via D. Montesano 49,
80131 Napoli, Italy
bNatural Product Synthesis and Bioorganic Chemistry Laboratory, Institute of Physical Chemistry, NCSR ‘Demokritos’,
PO Box 60228, GR 153 10 Ag. Paraskevi, Greece
cChemistry Laboratories, Agricultural University of Athens, Iera Odos 75, GR 118 55 Athens, Greece
Received 9 March 2006; revised 9 May 2006; accepted 25 May 2006
Available online 21 June 2006
Abstract—The analysis of a batch of toxic mussels (Mytilus galloprovincialis) from the Northern Adriatic Sea led to the isolation of a novel
oxazinin, oxazinin-4. Its structure including the relative stereochemistry has been elucidated through extensive NMR analysis. A synthetic
route to oxazinins has been crucial in establishing the absolute stereochemistry of oxazinin-4 and for reassigning the absolute C-2 configu-
ration of oxazinin-1 and -2 previously isolated from toxic shellfish and stereostructurally characterized.
Ó 2006 Elsevier Ltd. All rights reserved.
OH
7
1. Introduction
H
3 N
4
O
5
5'
4'
6'
7'
2''
1''
During our continuous analysis on toxic shellfish from the
Northern Adriatic Sea,1 we have isolated and fully character-
ized several cytotoxic compounds2–4 in addition to typical
marine biotoxins. These cytotoxic compounds appear to be
of great interest not only for their quite unique structural fea-
tures, but also because they cause human seafood intoxica-
tion. In 2001, we reported the structure and the relative
stereochemistry of three novel compounds oxazinin-1, -2,
and -3 of which oxazinin-1 has emerged as a cytotoxic
molecule.5
3'
3''
4''
O
O
2
6
1
2'
HN
6''
1'
5''
Oxazinin-1
7''
8''
OH
CN
H
N
O
O
HN
OH
Oxazinin-2
Figure 1. Previously reported stereostructures of oxazinin-1 and -2.5
Subsequently, we have assigned the absolute stereochemis-
try of both oxazinin-1 and -26 by applying the Riguera’s
method (Fig. 1).7 Recently, Couladouros et al. reported an
effective synthesis of oxazinin-1, -2, and -3, which has
allowed the absolute stereochemistry of these compounds
to be determined (Fig. 2).8
In the present paper we report the structure of a novel dia-
stereoisomer of oxazinin-1 (oxazinin-4) isolated from toxic
mussels. The successive elucidation of the relative stereo-
structure of oxazinin-4 in combination with the isolation
of a further amount of oxazinin-1 gave us the chance to
H
O
N
S
S
O
OH
Keywords: Oxazinins; Adriatic shellfish; Natural products; Synthetic
models; Mytilus galloprovincialis; NMR spectroscopy.
* Corresponding author. Tel.: +39 081 678507; fax: +39 081 678552;
e-mail: ciminiel@unina.it
HN
Figure 2. Stereostructure of oxazinin-3.
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.05.070

P. Ciminiello et al. / Tetrahedron 62 (2006) 7738–7743
7739
reassess and correct the relative configuration of both oxazi-
nin-1 and -2. A synthetic study allowed us to definitively
confirm the C-2 configuration of oxazinin-1 and -2 and
unambiguously assign the absolute stereochemistry of
oxazinin-4.
Since the structure of oxazinin-4 was coincident with that re-
ported for oxazinin-1 only a diastereoisomeric relationship
between the two compounds could explain the significant
differences in their chromatographic and NMR properties
(Tables 1 and 2). A ROESY spectrum was indispensable
for assigning the relative configuration of oxazinin-4. As-
suming that this compound presented a preferential chair-
like conformation in analogy with oxazinin-1,5–6 an intense
2. Results and discussion
A toxic batch of mussels (Mytilus galloprovincialis) was col-
lected along the Emilia Romagna coasts, Italy in March 2002
during a period of high toxicity. Oxazinin-4 was successfully
isolated according to the previous reported procedure.5 The
chromatographic behavior, the preliminary NMR data, and
the mass spectrum of this novel compound (Table 2 and Sec-
tion 3) indicated a similar structure as oxazinin-1. Such
a consideration eased us to individuate the presence of
both an indole ring substituted at C-3 and a para-disubsti-
tuted phenyl ring linked to a –OCH2CH2CN moiety. Run-
ning COSY, HOHAHA, and ROESY spectra were crucial
to determine the spin system including C-5, C-6, C-7, and
H-N-4. A strong IR absorption band at 1661 cmꢀ1 along
with a carbonyl carbon resonating at d 169.4 ppm pointed
out an amide functionality. Finally, correlations emerging
from a HMBC experiment (Fig. 3) allowed to establish the
planar structure of oxazinin-4.
Table 2. 13C and 1H NMR spectroscopic dataa of oxazinin-4 and 9b
(CD3CN)
Position
Oxazinin-4
Compound 9b
dC (ppm) dH (ppm) J (Hz) dC (ppm) dH (ppm) J (Hz)
2
3
75.3
169.4
5.48
75.6
170.7
5.43
4
5
6
6.61
3.93
4.79
3.36
3.49
9.28
7.37
6.66
3.88
4.69
3.31
3.44
9.26
7.33
60.8
77.6
62.0
60.7
78.1
62.0
9.7
8.2
9.9
7a
7b
10
20
126.8
112.8
126.8
120.0
118.4
120.1
112.4
137.9
132.0
130.4
116.0
160.4
64.2
126.2
112.0
126.9
120.3
30
30a
40
7.42
7.12
7.20
7.72
7.41
7.08
7.16
7.71
8.2
50
7.5, 8.2 120.5
7.5, 8.2 122.8
7.7, 8.2
7.7, 8.2
8.2
60
700
8.2
112.4
137.6
129.7
129.8
116.1
158.0
700a
100
OH
H
7
O
N
5
200–600
3 –500
400
7.41
6.98
8.4
8.4
7.25
6.79
8.2
8.2
2"
2 O 6
3'
1"
2'
700
800
4.21
2.87
6.0
6.0
HN
O
19.6
118.9
CN
7-OH
CN
3.05
3.05
a
Assignments are based on HMQC and HMBC experiments.
Figure 3. HMBC correlations for oxazinin-4.
Table 1. 13C and 1H NMR spectroscopic data of oxazinin-1, -2,5 and 9aa (CD3CN)
Position
Oxazinin-1
dH (ppm)
5.59
Oxazinin-2
Compound 9a
dH (ppm)
5.57
dC (ppm)
J (Hz)
dC (ppm)
dH (ppm)
J (Hz)
dC (ppm)
J (Hz)
2
3
72.9
170.1
72.8
170.1
5.57
72.8
170.1
4
5
6
6.70
3.71
4.61
3.26
3.43
9.40
7.29
6.64
3.70
4.56
3.26
3.42
9.37
7.28
6.64
3.70
4.56
3.26
3.42
9.37
7.28
59.9
71.1
62.0
59.9
71.2
62.1
59.9
71.2
62.1
9.2
9.2
9.2
7a
7b
10
20
126.1
112.3
127.5
120.1
120.3
122.8
112.4
137.4
131.8
130.0
115.5
159.0
63.9
126.1
112.4
127.5
120.1
120.3
122.8
112.4
137.4
130.0
129.9
116.0
157.9
126.1
112.4
127.5
120.1
120.3
122.8
112.4
137.4
130.0
129.9
116.0
157.9
30
30a
40
7.58
6.99
7.13
7.43
8.2
7.58
7.00
7.14
7.42
8.2
7.58
7.00
7.14
7.42
8.2
50
7.5, 8.2
7.5, 8.0
8.0
7.7, 8.2
7.7, 7.9
7.9
7.7, 8.2
7.7, 7.9
7.9
60
700
700a
100
200–600
3 –500
400
7.20
6.88
8.4
8.4
7.08
6.73
8.4
8.4
7.08
6.73
8.4
8.4
700
800
4.14
2.82
6.0
6.0
19.0
118.9
CN
7-OH
3.12
3.10
3.10
a
Assignments are based on HMQC and HMBC experiments.

7740
P. Ciminiello et al. / Tetrahedron 62 (2006) 7738–7743
ROE correlation between H-2 and H-6 was a clear clue of
their cis-relationship, while the high coupling constant be-
tween H-5 and H-6 suggested their trans-orientation (Fig. 4).
protecting group switched to the more stable tert-butyl-
diphenylsilyl ether 6. Subsequent reduction of the keto func-
tionality with NaBH4 provided diol 7, as a 1:1 mixture of
diastereomers, thus setting the stage for the crucial morpho-
linone ring-forming step. Treatment of a solution of diol 7 in
refluxing acetonitrile with PPTS afforded C-2 epimers 8a
and 8b. Finally, debenzylation and silyl deprotection gave
phenols 9a and 9b.
OH
H
N
H
O
2
6
O
H
H
HN 2'
O
Once isolated, compounds 9a and 9b were fully investigated
by NMR. Basically, a difference in their ROESY spectra was
observed: a strong correlation peak between H-2 and H-6
was present in the spectrum of 9b, while it was missing in
the spectrum of 9a (Fig. 5).
CN
Figure 4. ROE correlations for oxazinin-4.
These data surprisingly pointed to the same relative stereo-
chemistry reported for oxazinin-1. In order to clarify such
an ambiguity, we decided to re-examine the ROE correla-
tions of oxazinin-1. This time we did not detect any correla-
tion between H-2 and H-6 in oxazinin-1 indicating an actual
trans-orientation of the two protons. Since this time we could
employ a more powerful NMR instrument, such as
a 700 MHz instead of the 500 MHz, and rely on a larger
availability of pure oxazinin-1 isolated along with oxazi-
nin-4, we can confidently conclude that the H-2/H-6 ROE
correlation described for oxazinin-1 in our precedent paper
was actually an artifact possibly due to the low signal–noise
ratio of the former experiment.
OH
H
O
N
2
6
O
H
H
HN
OH
9b
Figure 5. ROE correlation in compound 9b indicating the cis-relationship of
H-2 and H-6.
A comparison of NMR data and optical rotation collected on
both the synthetic compounds with those recorded for the
natural molecules (Tables 1 and 2) led us to unambiguously
correlate 9a to oxazinin-2. This was crucial for confirming
the absolute stereochemistry previously assigned at C-5
and C-6, and for re-examining and properly reassigning
the stereochemistry at C-2 as R (Fig. 6). Once established
the overlapping of oxazinin-2 with 9a, we could extend
our analysis to oxazinin-1. In fact, the difference between
oxazinin-1 and 9a/oxazinin-2 is restricted to the presence
of the –OCH2CH2CN segment, which does not affect signifi-
cantly the optical rotation. As a consequence, the absolute
stereochemistry at C-5 and C-6 of oxazinin-1 was confirmed
and the stereochemistry at C-2 was reassigned again as R
(Fig. 6). Similarly, the absolute stereochemistry of oxazi-
nin-4 was achieved on the basis of the parallelism of the
In order to unambiguously prove the stereochemistry of
oxazinin-1 and -4, we undertook the synthesis set up by Cou-
ladouros et al. (Scheme 1). Thus, the prerequisite amine 3
was obtained by protective group manipulation of the
known9 tyrosine derivative 1. Subsequent coupling with
3-indoleglyoxylic acid 410 afforded amide 5. A further
OBn
OBn
O
c
OH
O
N
H
OR
HO
OTHP
NH2
3
O
O
NH
4
1: R = Ac
a,b
2: R = THP
d
94%
OH
O
OH
OR
O
OH
OR
H
N
H
N
g
OH
H
O
98%
N
OBn
N
H
OBn
O
N
R
H
7: R = TBDPS
50%
5: R = THP
R
e,f
h
6: R = TBDPS
O R
Oxazinin-1
OTBDPS
OH
H
N
H
HN
O
O
O
O
N
i,j
76%
O
8a
O
9a
OH
HN
HN
HN
HN
CN
OBn
OTBDPS
OH
H
N
O
+
R
OH
H
N
H
N
R
O
i,j
77%
R
O
O
8b
O
9b
HN
HN
OH
OBn
OH
Oxazinin-2
OH
Scheme 1. Preparation of morpholinones 9a and 9b. Reagents and condi-
tions: (a) 3 N LiOH(aq)/THF/MeOH (1:10:10 v/v), 0 ꢁC, 10 min; (b) DHP,
PPTS, CH2Cl2, reflux, 1 h, 93% for two steps; (c) 3% KOH(aq)/toluene
(1:1 v/v), reflux; (d) 4, EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2/
DMF (14:1 v/v), 0 ꢁC/2 h, rt/22 h, 94% for two steps; (e) TsOH,
MeOH, rt, 20 min, 98%; (f) TBDPSCl, 2,6-lutidine, 55 ꢁC, 24 h, 95%; (g)
NaBH4, MeOH/THF (1:1 v/v), 0 ꢁC/rt, 30 min, 98%; (h) PPTS,
CH3CN, reflux, 2 h, 50% combined yield; (i) H2, Pd(OH)2/C, EtOAc/
EtOH (4:1 v/v), rt, 8 h; (j) 1.0 M TBAF, THF, rt, 30 min; 9a 76% for two
steps, 9b 77% for two steps.
H
N
O
R
S
O R
Oxazinin-4
O
CN
Figure 6. Absolute stereochemistry of oxazinin-1, -2, and -4.

P. Ciminiello et al. / Tetrahedron 62 (2006) 7738–7743
7741
optical rotation and NMR properties of oxazinin-4 with
those of compound 9b (Table 2; Fig. 6).
from 60% to 100% of methanol. The fraction eluted with
90% of methanol was successively separated on a Toyopearl
HW-40 SF column with 100% methanol as an eluent. The
fraction containing oxazinins was first purified on reverse
phase HPLC eluted with CH3CN/H2O/CH3OH 15:50:35
and then on a silica gel HPLC column using EtOAc/
CH3OH 95:5 as an eluent to afford 1.2 mg of pure oxazi-
nin-4.
In conclusion, a novel oxazinin has been isolated and
stereostructurally characterized; and a synthetic route to
oxazinin-2 has given the opportunity to correct the absolute
stereochemistry of oxazinin-1 and -2.
3. Experimental
3.1. General
3.2.1. Oxazinin-4. [a]2D5 +60.0 (c 0.2, MeOH). nmax (KBr)
3478, 3342, 3186, 2930, 2262, 1661, 1623 cmꢀ1. ESI posi-
tive ion mode MS: m/z 391.9 [M+H]+ and m/z 413.7
[M+Na]+. HRMS (ESI positive ion mode): [M+H]+, found
392.1627. C22H22N3O4 requires 392.1610. 1H and 13C
NMR spectroscopic data (CD3CN) are reported in Table 1.
1H–1H COSY correlations: H-5/H-6; H-5/H2-7; H-5/7-OH;
H-10/H-20; H-40/H-50; H-50/H-60; H-60/H-70; H-200(600)/
H-300(500); H2700/H2800. HMBC correlations: H-2/C-3,C-20,
C-30,C-30a,C-6; H-6/C-2,C-100,C-200(600); H-20/C-30,C-30a,
C-700a; H-40/C-30,C-60,C-70a; H-50/C-30a,C-70; H-60/
C-5 ,C-70a; H-70/C-50,C-30a; H-200(600)/C-6,C-400,C-600(200);
H-300(500)/C-100,C-400,C-500(300); H-700/CN; H-800/C-700,CN.
NMR spectra were measured on a Varian Unity Inova700
spectrometer and the solvent was used as an internal stan-
dard (CD3CN: dH 1.94; dC 1.3 and 118.2). ESI positive ion
mode spectra were obtained on a API-2000 triple quadrupole
mass spectrometer equipped with a turbo ion spray source
(Applied biosystem, Thornhill, ON, Canada). Optical rota-
tions were measured on a Perkin–Elmer 192 polarimeter in
methanol solution, using a sodium lamp at 589 nm. NMR
and MS experiments were performed at ‘Centro di Servizi
ꢀ
Interdipartimentale di Analisi Strumentale’, Universita degli
Studi di Napoli Federico II.
3.3. Synthetic studies
Medium-pressure liquid chromatography (MPLC) was per-
formed on a Buchi 861 apparatus equipped with Develosil
ODS and Toyopearl HW-40 SF columns. HPLC separations
were performed on a Varian apparatus equipped with Waters
490 MS UV and RI-3 index detectors and Luna 5u C18 and
Luna 5u Silica columns. UV detector was set at 230 nm;
TLC was performed on silica gel 60 plates (Merck, pre-
coated), with EtOAc/MeOH (95:5) as a mobile phase; the
oxazinins were detected by heating the plates after spraying
with 50% sulfuric acid. All reactions were carried out under
a dry argon atmosphere with anhydrous, freshly distilled sol-
vents under anhydrous conditions unless otherwise noted.
All reactions were magnetically stirred with Teflon stir
bars, and temperatures were measured externally. Reactions
requiring anhydrous conditions were carried out in oven
dried (120 ꢁC, 24 h) or flame dried (vacuum<0.5 Torr)
glassware. Yields refer to chromatographically and spectro-
scopically (1H NMR) obtained homogeneous materials.
E. Merck silica gel (60, particle size 0.040–0.063 mm) was
used for flash column chromatography.
3.3.1. THP ether 2. Acetate 1 (1.5 g, 4.39 mmol) was dis-
solved in a mixture of THF/MeOH (40:40 mL), the solution
was cooled to 0 ꢁC, and aqueous LiOH (3 N, 4 mL) was
added slowly. After 10 min the solution was neutralized
with a 1 N aqueous HCl and extracted with EtOAc
(3ꢂ50 mL). The combined organic extracts were washed
with brine (2ꢂ30 mL), dried over Na2SO4, and concentrated
under reduced pressure. To a stirred solution of the above
crude product (1.3 g) in dichloromethane (80 mL), 3,4-di-
hydro-2H-pyran (8 mL) was added at ambient temperature,
followed by a catalytic amount of pyridinium p-toluenesul-
fonate (20 mg) and the mixture was refluxed at 80 ꢁC. After
1 h the reaction mixture was poured into water (50 mL) and
extracted with dichloromethane (2ꢂ50 mL). The combined
organic extracts were washed with brine (2ꢂ50 mL), dried
over Na2SO4, and concentrated under reduced pressure.
The residue was purified by flash column chromatography
(30% EtOAc in hexane) affording 1.56 g (4.07 mmol) of
the tetrahydropyranyl ether 2 (93% combined yield for two
steps) as colorless amorphous solid. Rf¼0.38 (60% EtOAc
in hexane); [a]2D5 +62 (c 0.30, Acetone); nmax (KBr) 3284,
3.2. Collection and extraction
1758, 1614, 1514 cmꢀ1 1H NMR (500 MHz, CDCl3):
.
d 7.44–7.24 (m, 14H, ArH), 7.00 (d, J¼8.7 Hz, 4H, ArH),
6.37 (d, J¼3.7 Hz, 1H, NHCO), 6.30 (d, J¼5.7 Hz, 1H,
NHCO), 5.21 (m, 2H, ArCHOCO), 5.08 (s, 4H, OCH2Ph),
4.65 (br s, 2H, OCHO), 3.97–3.80 (m, 6H, CH2OTHP+
CH2CHHO), 3.63–3.50 (m, 4H, CHNH+CH2CHHO),
1.85–1.54 (m, 12H, CH2CH2CH2); 13C NMR (125 MHz,
CDCl3): d 159.2, 159.1, 158.9, 136.6, 130.6, 130.5, 128.5,
128.5, 128.4, 128.2, 128.0, 127.4, 127.3, 127.2, 115.1,
99.4, 99.1, 80.1, 80.0, 70.0, 69.0, 68.8, 62.4, 62.3, 60.2,
59.9, 30.3, 30.2, 25.2, 25.1, 19.2, 19.1. HRMS (ESI positive
ion mode): [M+H]+, found 384.1828. C22H26NO5 requires
384.1811.
Toxic mussels M. galloprovincialis were collected along the
coasts of Cesenatico (Adriatic Sea) in March 2002 at 3 m
depth, which corresponds to the upper levels of mussel
farm in this area. Reference specimens were deposited at
the Dipartimento di Chimica delle Sostanze Naturali,
Napoli, Italy. After collection, the mussels were stored at
ꢀ20 ꢁC until extraction. The digestive glands (5000 g of
dry weight after extraction) were removed, homogenized
with a Waring blender and extracted with CH3CN/H2O
8:2—0.1% HCOOH twice at room temperature. The com-
bined extracts, after filtration, were concentrated in vacuo
to give a residue, which was dissolved in CH3CN/H2O 2:1
and partitioned with CH2Cl2. The dichloromethane layer
was concentrated and then chromatographed by MPLC on
a Develosil ODS column using a solvent gradient system
3.3.2. Amide 5. An aqueous solution of 3% KOH (40 mL)
was added to a stirred solution of cyclic carbamate 2
(1.5 g, 3.91 mmol) in toluene (40 mL) and the mixture was

7742
P. Ciminiello et al. / Tetrahedron 62 (2006) 7738–7743
warmed to reflux for 24 h. After cooling to ambient temper-
ature the reaction mixture was poured into a saturated aque-
ous ammonium chloride (30 mL) neutralized with a 1 N
aqueous HCl (pH¼8) and extracted with EtOAc (3ꢂ
100 mL). The combined organic extracts were washed with
brine (2ꢂ50 mL), dried over Na2SO4, and concentrated un-
der reduced pressure. To a stirred solution of the crude amine
3 (1.4 g, 3.91 mmol) and 3-indoleglyoxylic acid 4 (874 mg,
4.62 mmol) in a mixture of dichloromethane (500 mL) and
DMF (36 mL) were added at 0 ꢁC N,N-diisopropylethyl-
amine (867 mL, 4.98 mmol), 1-hydroxy-benzotriazole
(720 mg, 5.33 mmol), and 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride (954 mg, 4.98 mmol).
The temperature was maintained at 0 ꢁC for 2 h, and then
the mixture was warmed gradually to ambient temperature.
After 22 h the reaction mixture was poured into water
(200 mL) and extracted with dichloromethane (2ꢂ
200 mL). The combined organic extracts were washed with
brine (2ꢂ200 mL), dried over Na2SO4, and concentrated
under reduced pressure; the residue was purified by flash
column chromatography (50% EtOAc in hexane) affording
1.95 g (3.68 mmol) of the amide 5 (94% yield) as colorless
oil. Rf¼0.30 (50% EtOAc in hexanes); [a]2D5 ꢀ80 (c 0.80,
CHCl3); nmax (NaCl) 3393, 3284, 1743, 1683, 1633, 1500,
122.7, 115.2, 113.1, 72.0, 70.3, 62.8, 57.9; HRMS (ESI
positive ion mode): [M+H]+, found 445.1777. C26H25N2O5
requires 445.1763.
3.3.4. Amide 6-protection. The above diol (1.1 g,
2.47 mmol) was dissolved in 2,6-lutidine (4 mL) and TPSCl
(1.3 mL, 4.95 mmol) was added at ambient temperature. The
mixture was warmed to 55 ꢁC and stirred for 24 h. The reac-
tion mixture was poured into saturated aqueous ammonium
chloride (50 mL) and extracted with EtOAc (3ꢂ50 mL). The
combined organic extracts were washed with saturated aque-
ous copper sulfate (2ꢂ30 mL) brine (30 mL), dried over
Na2SO4, and concentrated under reduced pressure. The resi-
due was purified by flash column chromatography (40%
EtOAc in hexane) affording 1.60 g (2.35 mmol) of the amide
6 (95% yield) as colorless oil. Rf¼0.60 (50% EtOAc in hex-
ane); [a]2D5 ꢀ29.0 (c 0.48, Acetone); nmax (NaCl) 3398, 1739,
1
1681, 1641, 1635, 1510, 1430 cmꢀ1. H NMR (500 MHz,
CD3CN): d 10.2 (br s, 1H, ArNH), 8.83 (d, J¼3.2 Hz, 1H,
ArH), 8.33 (m, 1H, ArH), 7.86 (d, J¼9.3 Hz, 1H, ArH),
7.75–7.62 (m, 5H, ArH), 7.55 (m, 1H, ArH), 7.46–7.26
(m, 14H, ArH+CONH), 6.92 (d, J¼8.6 Hz, 2H, ArH), 5.05
(br s, 3H, OCH2Ph+CHOH), 4.23 (br s, 1H, OH), 4.18
(m, 1H, CHNH), 3.80 (m, 1H, CHHOTBDPS), 3.67 (m,
1H, CHHOTBDPS), 1.01 (s, 9H, (CH3)3C); 13C NMR
(125 MHz, CDCl3): d 139.8, 136.4, 136.4, 135.6, 130.9,
130.4, 129.4, 129.3, 128.8, 128.6, 128.5, 124.8, 123.9,
122.7, 115.4, 113.3, 72.2, 70.5, 64.4, 57.7, 27.2, 27.0;
HRMS (ESI positive ion mode): [M+H]+, found 683.2920.
C42H43N2O5Si requires 683.2941.
1
1455 cmꢀ1. H NMR (500 MHz, CDCl3): d 9.08 (br s, 2H,
ArNH), 8.92 (s, 2H, ArH), 8.43 (d, J¼7.6 Hz, 2H, ArH),
8.29 (d, J¼8.8 Hz, 1H, ArH), 8.14 (d, J¼8.8 Hz, 1H, ArH),
7.44–7.24 (m, 20H, ArH+NHCO), 6.94 (d, J¼7.2 Hz, 4H,
ArH), 5.10 (m, 2H, CHOH), 5.02 (s, 4H, OCH2Ph), 4.64
(br s, 1H, OCHO), 4.57 (br s, 1H, OCHO), 4.31 (m, 2H,
CHNH), 3.98–3.79+3.69–3.53 (m, 4H+m, 6H, CH2OTHP+
CH2CH2O+OH), 1.78–1.50 (m, 12H, CH2CH2CH2); 13C
NMR (125 MHz, CDCl3): d 180.7, 180.3, 163.0, 162.8,
158.4, 138.2, 138.2, 135.7, 133.3, 128.5, 128.3, 127.9,
127.5, 126.6, 124.1, 124.1, 123.4, 123.3, 122.4, 114.8,
111.6, 99.8, 99.5, 74.3, 73.7, 70.0, 68.4, 68.0, 63.2, 62.5,
55.2, 54.7, 30.7, 30.3, 29.7, 25.2, 25.2, 19.9, 19.4; HRMS
(ESI positive ion mode): [M+H]+, found 529.2329.
C31H33N2O6 requires 529.2338.
3.3.5. Diol 7. Sodium borohydride (56 mg, 1.5 mmol) was
added in small portions to a stirred solution of amide 6
(500 mg, 0.73 mmol) in a mixture of MeOH (5 mL) and
THF (5 mL) at 0 ꢁC. The reaction was warmed to ambient
temperature and after 30 min saturated aqueous ammonium
chloride (10 mL) was carefully added. The mixture was
extracted with EtOAc (3ꢂ30 mL); the combined organic
extracts were washed with brine (20 mL), dried over
Na2SO4, and concentrated under reduced pressure. The
residue was purified by flash column chromatography
(80% EtOAc in hexane) affording 490 mg (0.72 mmol) of
the diol 7 (98% yield) as amorphous white solid. Rf¼0.15,
0.22 (50% EtOAc in hexane); nmax (KBr) 3394, 1729,
3.3.3. Amide 6-deprotection. To a stirred solution of amide
5 (1.95 g, 3.68 mmol) in MeOH (100 mL) was added a cata-
lytic amount of p-toluenesulfonic acid monohydrate (30 mg)
at ambient temperature. After 20 min the acid was quenched
with a saturated aqueous NaHCO3 (2 mL), the reaction mix-
ture was poured into water (100 mL) and extracted with
EtOAc (4ꢂ100 mL). The combined organic extracts were
washed with brine (100 mL), dried over Na2SO4, and con-
centrated under reduced pressure; the residue was purified
by flash column chromatography (50–80% EtOAc in hex-
ane) affording 1.60 g (3.60 mmol) of the corresponding
diol (98% yield) as amorphous white solid. Rf¼0.33 (80%
EtOAc in hexane); [a]2D5 ꢀ112 (c 0.98, Acetone); nmax
(KBr) 3395, 3286, 1736, 1684, 1618, 1509, 1431 cmꢀ1. 1H
NMR (500 MHz, Acetone-d6): d 11.3 (br s, 1H, ArNH),
8.99 (d, J¼3.1 Hz, 1H, ArH), 8.38 (m, 1H, ArH), 8.03 (m,
1H, ArH), 7.56 (m, 1H, ArH), 7.46–7.26 (m, 8H, ArH),
6.96 (d, J¼8.7 Hz, 2H, ArH), 5.17 (m, 1H, CHOH), 5.05
(s, 2H, OCH2Ph), 4.87 (m, 1H, NHCO), 4.27 (m, 1H, OH),
4.17 (m, 1H, CHNH), 3.84 (m, 1H, CHHOH), 3.74 (m,
1H, CHHOH), 2.99 (s, 1H, OH); 13C NMR (125 MHz,
CDCl3): d 181.6, 163.3, 159.0, 139.7, 138.4, 137.2, 136.1,
129.2, 128.5, 128.4, 128.1, 127.7, 124.4, 124.4, 123.5,
1664, 1516, 1462 cmꢀ1 1H NMR (500 MHz, CD3CN):
.
d 9.25 (br s, 1H, ArNH), 9.21 (br s, 1H, ArNH), 7.67–7.58
(m, 6H, ArH), 7.54–7.29 (m, 30H, ArH), 7.27–7.18 (m,
3H, ArH), 7.15–6.86 (m, 8H, ArH), 6.75 (d, J¼8.5 Hz,
2H, ArH), 6.63 (br s, 1H, NHCO), 5.22 (br s, 1H,
COCHOH), 5.20 (s, 1H, COCHOH), 5.09 (br s, 1H,
CHCHOH), 5.05 (s, 2H, OCH2Ph), 5.03 (s, 2H, OCH2Ph),
4.97 (br s, 1H, CHCHOH), 4.09 (br m, 4H, CHNH+OH),
3.90–3.80 (m, 2H, OH), 3.78–3.57 (m, 4H, CH2OTBDPS),
1.05 (s, 18H, (CH3)3C); HRMS (ESI positive ion mode):
[M+H]+, found 685.3117. C42H45N2O5Si requires 685.3097.
3.3.6. Morpholinones 8a and 8b. A catalytic amount of
pyridinium p-toluenesulfonate (10 mg) was added to a stirred
solution of diol 7 (490 mg, 0.72 mmol) in acetonitrile
(100 mL) at ambient temperature and the mixture was
warmed to 80 ꢁC. After completion of the reaction (2 h)
half of the volume of the solvent was removed under reduced
pressure and the rest was poured into water (30 mL) and

P. Ciminiello et al. / Tetrahedron 62 (2006) 7738–7743
7743
extracted with EtOAc (3ꢂ30 mL). The combined organic
extracts were washed with brine (30 mL), dried over
Na2SO4, and concentrated under reduced pressure. The
residue was purified by flash column chromatography
(30% EtOAc in hexane) affording, in order of elution, mor-
pholinones 8a (122 mg, 0.183 mmol) and 8b (120 mg,
0.180 mmol) (50% combined yield) as colorless oils.
morpholinone 9a (0.034 mmol, 76% yield for two steps)
or 11.7 mg of morpholinone 9b (0.035 mmol, 77% yield
for two steps) as colorless oils.
3.3.7.1. Compound 9a. Rf¼0.27 (50% acetone in di-
chloromethane); [a]D25 +132.5 (c 0.2, MeOH); nmax (NaCl)
3274, 1663, 1618, 1520, 1460 cmꢀ1. 1H and 13C NMR spec-
troscopic data (CD3CN) are reported in Table 1. HRMS (ESI
positive ion mode): [M+H]+, found 339.1361. C19H19N2O4
requires 339.1345.
3.3.6.1. Compound 8a. Rf¼0.46 (50% EtOAc in hex-
ane); [a]2D5 +36.9 (c 0.80, MeOH); nmax (NaCl) 3398,
1
3289, 1666, 1612, 1513, 1454 cmꢀ1. H NMR (500 MHz,
CD3CN): d 9.35 (br s, 1H, ArNH), 7.58 (m, 3H, ArH),
7.52 (d, J¼6.8 Hz, 2H, ArH), 7.48–7.22 (m, 13H, ArH),
7.17 (t, J¼7.4 Hz, 1H, ArH), 7.06 (d, J¼8.6 Hz, 2H,
ArH), 6.97 (t, J¼7.4 Hz, 1H, ArH), 6.82 (d, J¼8.6 Hz, 2H,
ArH), 6.64 (br s, 1H, NHCO), 5.59 (s, 1H, COCHO), 5.03
(s, 2H, OCH2Ph), 4.73 (d, J¼9.2 Hz, 1H, CHCHO), 3.83
(m, 1H, CHNHCO), 3.56 (dd, J¼11.0, 2.9 Hz, 1H,
CHHOTPS), 3.38 (dd, J¼11.0, 4.7 Hz, 1H, CHHOTPS),
1.03 (s, 9H, (CH3)3C); 13C NMR (125 MHz, CDCl3):
d 170.7, 159.8, 136.5, 136.4, 130.9, 130.9, 130.1, 129.5,
129.3, 128.9, 128.8, 128.8, 128.6, 127.5, 126.1, 122.9,
120.4, 120.3, 115.6, 112.5, 112.2, 73.4, 70.9, 70.6, 64.1,
59.6, 27.3, 19.8; HRMS (ESI positive ion mode): [M+H]+,
found 667.3018. C42H43N2O4Si requires 667.2992.
3.3.7.2. Compound 9b. Rf¼0.43 (50% acetone in di-
chloromethane); [a]2D5 +73.2 (c 0.6, MeOH); nmax (NaCl)
3314, 1656, 1616, 1518, 1453 cmꢀ1. 1H and 13C NMR spec-
troscopic data (CD3CN) are reported in Table 2. HRMS (ESI
positive ion mode): [M+H]+, found 339.1350. C19H19N2O4
requires 339.1345.
Acknowledgements
This work is a result of research supported by BIOTOX
marin 2005 and PRIN 2004 ‘Algal toxins contaminating
shellfish in Italian coastal waters: monitoring, isolation and
structural characterization’.
3.3.6.2. Compound 8b. Rf¼0.20 (50% EtOAc in hex-
ane); [a]2D5 +71.7 (c 1.69, MeOH); nmax (NaCl) 3396,
Supplementary data
1
3283, 1676, 1613, 1514, 1460 cmꢀ1. H NMR (500 MHz,
Supplementary data associated with this article can be found
in the online version, at doi:10.1016/j.tet.2006.05.070.
CD3CN): d 9.28 (br s, 1H, ArNH), 7.68 (d, J¼7.8 Hz, 1H,
ArH), 7.61 (m, 3H, ArH), 7.56 (t, J¼6.4 Hz, 1H, ArH),
7.47–7.28 (m, 13H, ArH), 7.21 (d, J¼8.5 Hz, 2H, ArH),
7.16 (t, J¼7.4 Hz, 1H, ArH), 7.09 (t, J¼7.4 Hz, 1H,
ArH), 6.87 (d, J¼8.5 Hz, 2H, ArH), 6.72 (br s, 1H, NHCO),
5.46 (s, 1H, COCHO), 5.04 (s, 2H, OCH2Ph), 4.78 (d,
J¼9.7 Hz, 1H, CHCHO), 3.96 (m, 1H, CHNHCO), 3.53 (d,
J¼4.3 Hz, 2H, CH2OTPS), 1.06 (s, 9H, (CH3)3C); 13C
NMR (125 MHz, CDCl3): d 171.3, 160.5, 137.1, 137.0,
131.6, 131.6, 130.7, 130.4, 130.1, 129.5, 129.2, 127.7,
126.9, 123.4, 121.2, 121.0, 116.4, 113.1, 78.5, 76.3, 71.3,
64.8, 61.0, 27.9, 20.4; HRMS (ESI positive ion mode):
[M+H]+, found 667.2971. C42H43N2O4Si requires 667.2992.
References and notes
1. Ciminiello, P.; Dell’Aversano, C.; Fattorusso, E.; Forino, M.;
Magno, S. Pure Appl. Chem. 2003, 75, 325–336 and literature
cited there.
2. Ciminiello, P.; Fattorusso, E.; Forino, M.; Di Rosa, M.; Ianaro,
A.; Poletti, R. J. Org. Chem. 2001, 66, 578–582.
3. Ciminiello, P.; Dell’Aversano, C.; Fattorusso, E.; Forino, M.;
Magno, S.; Di Rosa, M.; Ianaro, A.; Poletti, R. J. Am. Chem.
Soc. 2002, 124, 13114–13120.
4. Ciminiello, P.; Dell’Aversano, C.; Fattorusso, E.; Forino, M.;
Magno, S.; Di Meglio, P.; Ianaro, A.; Poletti, R. Tetrahedron
2004, 60, 7093–7098.
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Magno, S.; Di Rosa, M.; Ianaro, A. Eur. J. Org. Chem. 2001,
49–53.
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3.3.7. General procedure for the preparation of 9a and
9b. To a solution of 8a or 8b (30 mg, 0.045 mmol) in a mix-
ture of EtOAc/EtOH (25 mL, 4:1 v/v) at ambient tempera-
ture was added a catalytic amount of Pd(OH)2/C (10 mg)
and the mixture was stirred under a hydrogen atmosphere
for 8 h. The reaction mixture was filtered through Celite
and concentrated under reduced pressure to give the corre-
sponding free phenol as a white amorphous solid. A solution
of the above phenol in THF (1 mL) was treated for 30 min at
ambient temperature with 1.0 M solution of TBAF in THF
(50 mL). The reaction mixture was poured into saturated
aqueous ammonium chloride (5 mL) and extracted with
EtOAc (3ꢂ5 mL). The combined organic phases were
washed with water (3 mL), brine (3 mL), dried over
Na2SO4, and concentrated under reduced pressure. The
residue was purified by flash column chromatography
(40% acetone in dichloromethane) affording 11.6 mg of
10. Shaw, K. N. F.; McMillan, A.; Gudmundson, A. G.; Armstrong,
M. D. J. Org. Chem. 1958, 23, 1171–1178.