Micromide and Guamamide: Cytotoxic Alkaloids from a Species of the Marine Cyanobacterium Symploca

Article abstract of DOI:10.1021/np030215x

Two new cytotoxins have been isolated from a species of marine cyanobacterium belonging to the genus Symploca that was collected in Guam. These new compounds, micromide (1) and guamamide (2), were accompanied by the known lipopeptides apramides A (3), B (4), and G (5). The planar structures of both alkaloids were elucidated by standard 2D NMR techniques, and the configurations of the amino acid-derived units in 1 were determined by chiral HPLC. The stereochemistry of the β-methoxyhexanoic acid in I was determined by derivatization with methyl D-mandelate, after acid hydrolysis, and comparison with synthetic standards.

Full text of DOI:10.1021/np030215x

J . Nat. Prod. 2004, 67, 49-53
49
Micr om id e a n d Gu a m a m id e: Cytotoxic Alk a loid s fr om a Sp ecies of th e Ma r in e
Cya n oba cter iu m Sym ploca
†
†
,†
‡,§
Philip G. Williams, Wesley Y. Yoshida, Richard E. Moore,* and Valerie J . Paul
Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii 96822, and University of Guam Marine
Laboratory, UOG Station, Mangilao, Guam 96913
Received May 14, 2003
Two new cytotoxins have been isolated from a species of marine cyanobacterium belonging to the genus
Symploca that was collected in Guam. These new compounds, micromide (1) and guamamide (2), were
accompanied by the known lipopeptides apramides A (3), B (4), and G (5). The planar structures of both
alkaloids were elucidated by standard 2D NMR techniques, and the configurations of the amino acid-
derived units in 1 were determined by chiral HPLC. The stereochemistry of the â-methoxyhexanoic acid
in 1 was determined by derivatization with methyl D-mandelate, after acid hydrolysis, and comparison
with synthetic standards.
Historically cyanobacterial extracts have been known for
their toxic properties, but more recent chemical investiga-
tions have revealed that they are a promising group of
organisms from which to isolate a wide variety of biologi-
cally active natural products.1 In particular, marine cy-
Resu lts a n d Discu ssion
The lipophilic extract of VP727 was separated by suc-
cessive gel permeation and repeated reversed-phase chro-
matography to afford 1 in 0.12% yield, based on the crude
extract.
2
anobacteria have yielded a diverse array of cytotoxins,
3
4
5
immunosuppressives, antibiotics, ichthyotoxins, tumor
6
7-9
promoters, and proteinase inhibitors.
Our efforts in recent years have focused on screening
marine cyanobacteria10 for natural products8 that are
effective against multi-drug-resistant solid tumors. Of the
extracts screened in 2001, two displayed significant selec-
tivity for solid-tumor cell lines when compared to the
leukemia and control cell lines. Here we report the results
of the bioassay-guided fractionation of one of these solid-
tumor selective extracts11 that has led to the isolation and
structure determination of two new alkaloids, micromide
(
1) and guamamide (2), along with three known lipopep-
1
2
tides, apramides A (3), B (4), and G (5). Micromide (1)
and guamamide (2) displayed IC50 values against KB cells
at 260 and 1200 nM, respectively.
The initial spectral data of the amorphous powder
-
1
showed IR and UV/vis absorptions at 1680 cm and 225
nm that suggested 1 was a peptide containing a hetero-
aromatic ring. HRMS (MALDI) established the ele-
mental composition of the optically active compound as
C
49
H
73
N
7
3
O
7
S, despite the presence of only 44 carbon signals
in the C NMR spectrum. Distinctive carbon resonances
at δ 136.8, 135.9, 129.3, 128.5, 128.4, 126.9, and 126.6
suggested the presence of two monosubstituted phenyl
1
C
1
3
rings and brought the total carbon count from the C NMR
spectrum into agreement with the mass spectrometry data.
This also accounted for eight of the 17 double-bond
equivalents implied by the molecular formula. On the basis
2
of the number of sp carbons and their chemical shifts, the
remaining degrees of unsaturation could be ascribed to
seven carbonyl equivalents, one carbon-carbon double
bond, and one more ring system.
*
To whom correspondence should be addressed. Tel: (808) 956-7232.
Examination of the spectral data recorded in CDCl
3
(Table 1) established a series of partial structures. In
Fax: (808) 956-5908. E-mail: moore@gold.chem.hawaii.edu.
†
University of Hawaii at Manoa.
University of Guam Marine Laboratory.
Present address: Smithsonian Marine Station, Fort Pierce, FL 34949.
‡
conjunction with the UV/vis and MS data, a pair of doublets
§
1
at δ
H
7.64 and 7.26 in the HSQC spectrum with
J
C,H
1
0.1021/np030215x CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/19/2003

5
0
J ournal of Natural Products, 2004, Vol. 67, No. 1
Williams et al.
Ta ble 1. NMR Spectral Data for 1 in CDCl3
1
1
HMBCb
C/H no.
δH(J in Hz)
7.64, d (3.3)
δCa
H- H COSY
N-Me-Gly-thiazole
1
2
3
4
142.0, d
120.1, d
165.6, s
48.9, t
2
1
7.26, d (3.3)
4
5
4.88, d (-15.1)
4u
4
.38, d (-15.1)
5
6
7
8
2.56, s
34.8, q
169.8, s
53.8, d
35.1, t
4
N-Me-Phe
4, 5, 7
8, 15
7
5.34, dd (10.4, 4.2)
8
7
3.11, dd (-13.1, 10.4)
2
.22, dd (-13.1, 4.2)
9
1
1
1
1
136.8, s
129.3, d
128.5, d
126.9, d
30.8, q
171.4, s
57.0, d
33.3, d
23.9, t
8, 11/13
8, 14/10
12, 13/11
0/14
1/13
2
7.02, d (7.7)
7.22, t (7.7)
7.13, t (7.7)
2.94, s
5
7
N-Me-Ile
16
7, 15, 17
21, 22
17
1
1
1
7
8
9
5.22, d (10.9)
2.19, m
1.24, m
18
17, 19, 21
18, 20
18, 20
19
20, 21
1
.00, m
2
2
2
0
1
2
0.87, t (6.6)
0.84, d (6.0)
3.24, s
10.9, q
15.4, q
30.3, q
171.3, s
54.0, d
18
17
17
22, 24
26, 27
Val
23
2
2
2
2
2
4
4.77, dd (8.3, 4.1)
6.95, d (8.3)
1.88, m
0.93, d (6.9)
0.81, d (6.8)
24-NH, 25
24
24, 26, 27
25
25
4-NH
5
6
7
30.5, d
19.9, q
16.9, q
170.0, s
62.4, d
25.0, d
19.8, q
18.7, q
30.7, q
171.6, s
50.1, d
24, 26, 27
24, 27
24, 26
24-NH, 24, 29
31, 32, 33
31, 32
32
30, 31
29
33, 35
36
N-Me-Val
28
2
3
3
3
3
9
0
1
2
3
4.65, d (11.2)
2.34, m
1.01, d (6.4)
0.84, d (5.9)
2.90, s
30
29, 31, 32
30
30
Phe
34
3
3
3
5
5.19, ddd (8.0, 7.2, 6.0)
6.82, d (8.0)
36
5-NH
6
3.22, dd (-13.5, 6.0)
36.9, t
35, 36u
36d
2
.80, dd (-13.5, 7.2)
3
3
3
4
7
135.9, s
129.3, d
128.4, d
126.6, d
170.5, s
40.7, t
36, 39/41
36, 40
8/42
9/41
0
6.83, d (7.9)
7.12, t (7.9)
7.17, t (7.9)
3
-methoxyhexanoic acid
43
35-NH, 44, 45
45
44, 49
4
4
4
4
5
6
2.30, d (6.1)
3.45, m
1.40, m
45
44, 46
45
77.4, d
35.4, t
44, 45, 48
1
.28, m
4
4
4
7
8
9
1.27, m
0.86, t (6.6)
3.27, s
18.3, t
14.1, q
56.6, q
48
47
48
45
a
Multiplicity deduced by HSQC. b Protons showing long-range correlation with indicated carbon.
correlations to sp2 carbon signals at δ
suggested the presence of a thiazole ring. HMBC correla-
tions to a carbon at δ 165.6 from a pair of diastereotopic
methylene protons (H-4) expanded this fragment into a
thiazole-glycine unit. A second fragment was constructed
142.0 and 120.1
and C-16, and from H-22 to C-17 and C-23. HMBC
correlations to the secondary amide proton signals provided
the remaining connectivites needed to establish unambigu-
ously the planar structure depicted.
C
C
The absolute stereochemistry of the units in 1 was
determined by a combination of chiral and RP-HPLC.
Ozonolysis and acid hydrolysis liberated the amino acid-
derived centers in 1 that were subsequently analyzed by
chiral HPLC. This established the stereocenters derived
from N-Me-L-Ile, N-Me-L-Phe, N-Me-D-Val, L-Val, and
L-Phe. The configuration of the final stereocenter (C-45)
H
starting from an oxygenated methine at δ 3.45. A series
3
of J C,H correlations from this proton (H-45) to C-43, C-44,
C-46, and C-49 revealed the majority of this unit. Further
HMBC cross-peaks from a methyl triplet (H-48) to C-47
and C-46 established this last unusual moiety as 3-meth-
oxyhexanoic acid. The remaining fragments, assembled
from COSY and HMBC correlations, were one isoleucine,
two valine, and two phenylalanine units. The sequence of
1
3
was determined by comparison with synthetic standards.
Using a chiral column, racemic methyl 3-hydroxyhexanoate
was resolved and the enantiomers were identified based
on comparison of their optical rotations with the reported
values.14 In the successful procedure (Figure 1), the hy-
1
was determined through HMBC correlations. Two frag-
ments, (Thz-N-Me-Gly)-(N-Me-Phe)-(N-Me-Ile)-Val (C-1
through C-27) and (N-Me-Val)-Phe (C-28 through C-40),
were constructed by HMBC correlations from the N-
methylamide proton signals. To be specific, cross-peaks
were observed from H-5 to C-4 and C-6, from H-15 to C-7
1
5
droxyester was methylated in petroleum ether with
dimethyl sulfate, potassium hydroxide, and a catalytic
amount of triethylamine, before saponification of the ester.

Cytotoxic Alkaloids from Symploca
J ournal of Natural Products, 2004, Vol. 67, No. 1 51
Ta ble 2. NMR Spectral Data for 2 in CDCl3
a
1
1
HMBCb
C/H no.
δ
H
(J in Hz)
δ
C
H- H COSY
1
2
3
4
172.6, s
38.6, t
67.1, d
44.5, t
2, 22
3
2.50, d (4.3)
4.14, m
3
2, 4
3, 4, NH
d
2, 4
2
d
3.62, ddd (-11.6,
7
.4, 4.4)
3
.08, ddd (-11.6,
4
4
d
, NH
7
.4, 4.4)
NH
5
6
7
8
6.17, t (4.4)
F igu r e 1. Synthesis of methyl D-mandelate derivatives.
173.1, s
42.4, d
74.1, d
69.5, d
14.4, q
173.3, s
34.4, t
24.8, t
24.0, t
u
4 , NH, 7, 23
2.55, p (7.1)
7, 23
6, 8
7, 9
8
7, 23
6, 9, 8, 23
6, 7, 9
7, 8
8, 11
12
5.25, dd (7.1, 4.4)
5.00, qd (6.5, 4.4)
1.23, d (6.5)
Unfortunately, attempts to resolve these methoxyacids (7)
failed on the OD or OJ chiral columns that were available
to us. In the end, separation was achieved of the methyl
D-mandelate derivatives (8) by C18 HPLC. The remaining
quantity of 1 was therefore hydrolyzed with 6 N HCl at
9
10
1
1
1
1
1
2
3
4
2.26, t (7.4)
1.58, m
1.20, m
1.20, m
1.20, m
1.20, m
1.20, m
1.20, m
1.20, m
12
11, 13
12
11
1
18 °C for 24 h and the residue exhaustively extracted with
c
29.6, t
29.6, tc
29.5, tc
CH Cl . Subsequent derivatization of this residue with
2
2
15
1
1
1
1
6
7
8
9
methyl D-mandelate, followed by purification and analysis
by C18 HPLC, established the configuration of C-45 as R.
Thus the absolute stereochemistry of 1 is 7S,17S,18S,24S,-
c
29.3, t
29.3, tc
31.9, t
21
21
2
9R,35S,45R.
20
21
1.21, m
0.87, t (6.9)
3.72, s
22.6, t
21
20
Examination of other fractions from the Sephadex LH-
0 separation of the crude extract led to the isolation of
14.1, q
52.0, q
13.5, q
170.3, s
20.8, q
2
2
2
2
2
3
4
5
2
1.17, d (7.1)
6
6, 7
7, 25
guamamide (2). This optically active amorphous powder
[R]21
+6°) was less cytotoxic than 1, with an IC50 value
against KB cells of 1200 nM. High-resolution FABMS
(
D
2.11, s
a
Multiplicity deduced by HSQC. b Protons showing long-range
established the molecular formula of 2 as C25
H
45NO
8
based
correlation with indicated carbon. c These carbons may be inter-
+
on a pseudo-molecular ion peak at 488.3225 ([M + H]
∆
changed.
0
.2 mDa) and revealed four degrees of unsaturation. These
1
3
could be accounted for by the C NMR spectrum, which
along with three oxygenated methine signals (δ 74.1, 69.5,
and 67.1) contained four carbonyl signals (δ 173.3, 173.1,
72.6, and 170.3). From the proton NMR spectrum, it was
apparent that 2 contained one acetate (δ 2.11), one
secondary amide (δ 6.17), one methoxy (δ 3.72), and two
methyl doublet proton signals (δ 1.23 and 1.17). These
fragments were expanded with the aid of the 2D NMR data
C
C
1
H
H
H
H
F igu r e 2. ∆δ (δ
R S
- δ ) values in ppm for the MPA esters of 2.
(
Table 2) to provide the gross structure depicted for 2.
Only the configuration of C-3 in 2 was established. After
7
.26, δ 77.00). FABMS and HRFABMS were recorded in the
C
positive mode on a VG ZAB2SE spectrometer, and the MALDI
spectra were recorded on a DE-STR. HPLC separations were
performed on a Beckman 110B apparatus coupled to an
Applied Biosystems 759A absorbance detector. The chiral OD
and OJ silica columns, derivatized with cellulose tris(3,5-
dimethylphenyl carbamate) and cellulose tris(4-methylben-
zoate), respectively, were purchased from Chiral Technologies,
Inc. Racemic methyl 3-hydroxyhexanoate was purchased from
CTC Organics. HMBC experiments were optimized for J CH
7
derivatization with R-methoxyphenylacetic acid (MPA) the
difference in the δ values of the (R)-MPA and (S)-MPA
adducts (Figure 2) indicated C-3 possessed an R absolute
configuration.
Micromide and guamamide are part of an ever-increas-
ing class of polyketide-derived compounds that have been
isolated from marine cyanobacteria. The most common of
these metabolites are the malyngamides, characterized by
a (-)-7-methoxy-4(E)-enoic acid moiety of varying length,
Η
n
)
Hz. All synthetic yields are unoptimized.
Biologica l Ma ter ia l. The cyanobacterium VP727 was
appended through an amide linkage to a functionalized
cyclohexane ring.16 Micromide (1) contains structural
collected in the spring of 2001 in Guam at Fingers Reef. A
voucher is maintained in formalin at the Smithsonian Marine
Station, Fort Pierce, FL.
features common to many cyanobacterial metabolites,
including N-methylated amino acids, a D-amino acid, and
a modified cysteine unit in the form of a thiazole ring. It
should be noted that despite the structural similarity
between micromide and the apramides, the IC50 of the
former compound against KB cells was an order of mag-
nitude greater than the latter series.
Extr a ction a n d Isola tion . The cyanobacterium was ex-
haustively extracted with 4:1 CH
3
2 2
CN-CH Cl to afford 1.23
g of lipophilic extract. After initial partitioning between hexane
and 80% aqueous MeOH, the organic residue from a second
partitioning with n-BuOH and water was loaded onto a
Sephadex LH-20 column (360 × 20 mm) and eluted with 5%
3
MeOH in CHCl . The bioactive fraction that eluted between
Exp er im en ta l Section
120 and 140 mL was further purified by C18 chromatography,
and the two fractions that eluted with 50 and 70% CH CN
were combined. Separation by RP-HPLC [Ultracarb ODS, 250
10 mm, 3 mL/min, detection at 254 nm] afforded apramide
A (t 22 min, 1.3 mg), apramide B (t 15 min, 1.0 mg), and a
mixture of apramide G and micromide (t 41 min). This latter
3
Gen er a l Exp er im en ta l P r oced u r es. The optical rotations
were measured on a J asco-DIP-700 polarimeter at the sodium
D line (589 nm). The UV spectra were determined on a
Hewlett-Packard 8453 spectrophotometer, and the IR spectra
were recorded on a Perkin-Elmer 1600 FTIR instrument as a
film on a NaCl disk. The NMR spectra of 1 and 2 were recorded
×
R
R
R
fraction was further purified by a linear gradient of 80 to 100%
aqueous MeOH [YMC-AQ ODS column, 250 × 10 mm, 3 mL/
in CDCl
3
on a Varian Inova 500 operating at 500 and 125 MHz
R
min, PDA detection] to afford the former (t 14.2 min) and 1
using the residual solvent signal as the internal reference (δ
H
R
(t 15.9 min) in 1.8 and 1.7 mg, respectively. The Sephadex

5
2
J ournal of Natural Products, 2004, Vol. 67, No. 1
Williams et al.
fraction eluting between 180 and 200 mL was purified by RP-
HPLC with a linear gradient of 65 to 75% aqueous CH CN
over 30 min [YMC-AQ ODS, 250 × 10 mm, 3 mL/min, PDA
detection] to afford 1.7 mg of 2 (t 23 min).
Micr om id e (1): amorphous powder; [R]
MeOH); UV (MeOH) λmax (log ꢀ) 201 (4.40), 225 (3.80), 274
a stream of N
2
, the residue was dissolved in water and
3
exhaustively extracted with CH
combined and dried over MgSO
This residue was then dissolved in 0.1 mL of CH
this were added 1 crystal of DMAP, 1.2 mg of methyl
D-mandelate in 0.5 mL of CH Cl , and 0.02 mL of 1 M DCC.
After stirring for 3 h the mixture was purified by HPLC
[Bondclone Si, 250 × 10 mm, a linear gradient of 1 to 15%
i-PrOH in hexane, 2 mL/min, PDA detection]. The fraction
eluting between 9 and 10 min was subsequently repurified by
HPLC [Phenosphere Silica, 150 × 4.6 mm, 0.5% i-PrOH in
hexane, 0.75 mL/min, PDA detection].
HP LC An a lysis of (Meth yl D-Ma n d elyl) 3-Meth oxyh ex-
a n oa te. The derivatized hydrolyzate of 1 was analyzed by
HPLC [Phenosphere Silica, 150 × 4.6 mm, 0.5% i-PrOH in
hexane, 0.75 mL/min, PDA detection] to establish the config-
uration of C-45 of 1 by comparison with the synthetic stan-
dards (SR)-8 and (R)-8. The derivatized hydrolyzate was found
to contain (methyl D-mandelyl) 3R-methoxyhexanoate (27.3
min), which was verified by co-injection of the appropriate
standard. The retention times of the synthetic standards under
the same HPLC conditions were 24.1 and 27.3 min for (S)-
and (R)-8, respectively.
2
Cl
, and the solvent was removed.
Cl , and to
2
. The organic extracts were
4
R
2
2
2
1
D
-28° (c 0.5,
2
2
-1 1
(
2.40) nm; IR (film) νmax 3311, 1680, 1462, 1379 cm ; H NMR,
1
3
1
1
C NMR, H- H COSY, and HMBC data, see Table 1; MALDI
+
+
m/z [M + Na] 926.5; HRMALDI m/z [M + Na] 926.5155
calcd for C49 SNa 926.5184).
Gu a m a m id e (2): amorphous powder: [R]
MeOH); UV (MeOH) λmax (log ꢀ) 201 (3.71) nm; IR (film) νmax
(
73 7 7
H N O
2
1
D
+6° (c 0.4,
-
1
1
13
3
382, 1739, 1651, 1371, 1233, 1095 cm ; H NMR, C NMR,
H- H COSY, and HMBC data, see Table 2; FAB m/z [M +
1
1
+
+
Na] 510; HRFAB m/z [M + H] 488.3225 (calcd for C25
NO 488.3223).
Ozon olysis a n d Acid Hyd r olysis of 1. Ozone was bubbled
through a solution of 0.3 mg of 1 in 3 mL of CH Cl for 15 min
46
H -
8
2
2
until a blue color persisted. The solvent was then removed and
the residue hydrolyzed with 6 N HCl for 24 h at 118 °C. After
removal of the acid under a stream of N
dissolved in 10% aqueous methanol and passed over a small
18 column. The components of this mixture were then
analyzed by chiral HPLC [Column Chirex Phase 3126 (D), 250
4.6 mm, Phenomenex, flow rate 0.8 mL/min, detection at
54 nm]. The retention times (t , min) of the standards with
(5:95) were N-Me-L-Val (14.0), N-Me-
2
, the residue was
C
MP A Der iva tives of 2: (R)-MP A-2. To 0.5 mg of 2 in 0.3
×
mL of CH Cl2 were added 1 crystal of DMAP, 4 mg of (R)-
2
2
R
methoxyphenylacetic acid (MPA), and 4 mg of 1-[3-(dimethy-
lamino)propyl]-3-ethylcarbodiimide hydrochloride. The solu-
tion was stirred at room temperature overnight and then
quenched with cold 1 N HCl (0.4 mL). After partitioning
between ethyl acetate and water, the organic layer was dried
CH
3
CN-2 mM CuSO
4
D-Val (19.2), L-Val (20.3), D-Val (27.1), N-Me-L-allo-Ile (27.0),
N-Me-L-Ile (28.1), N-Me-D-Ile (42.0), and N-Me-D-allo-Ile (42.1).
The retention times of the standards with CH
3
CN-2 mM
CuSO (15:85) were N-Me-L-Phe (33.4), N-Me-D-Phe (36.2),
4
over MgSO . The residue was dissolved in 20% EtOAc in
4
L-Phe (41.0), and D-Phe (42.5). The retention times of the
components of the hydrolyzate were N-Me-D-Val (19.2), L-Val
hexanes and applied to a prepacked silica column (500 mg).
(R)-MPA-2 eluted in the 40% EtOAc in hexane fraction (4 mL)
after 2 mL of hexane and 2 mL of 25% EtOAc had passed
(20.3), N-Me-L-Ile (28.1), N-Me-L-Phe (33.4), and L-Phe (41.0),
1
as confirmed by co-injection.
through the column. H NMR (CDCl , 500 MHz): δ (position,
3
H
Ch ir a l Sep a r a tion of Meth yl 3-Hyd r oxyh exa n oa te. A
racemic mixture of methyl 3-hydroxyhexanoate was separated
by chiral HPLC [OD gold, 250 × 10 mm, 15% i-PrOH in
hexane, 1 mL/min, PDA detection] to afford methyl (R)- and
multiplicity, J in Hz) 7.47-7.37 (Ph-MPA), 5.29 (NH, t, J )
6.2), 5.31 (H-3, ddt, J ) 6.2, 3.7, 1.5), 5.15 (H-7, dd, J ) 7.1,
5.6), 4.93 (H-8, qd, J ) 6.6, 5.6), 4.78 (R-MPA, s), 3.65 (H-22,
s), 3.45 (H-4 , ddd, J ) -13.9, 6.2, 3.7), 3.25 (H-4 , dt, J )
L
H
2
3
(
-
S)-3-hydroxyhexanoate at 21.4 and 27.6 min with [R]
D
of
-13.9, 6.2), 3.42 (OMe-MPA, s), 2.60 (H-2, d, J ) 1.5), 2.26
(H-11, t, J ) 7.5), 2.18 (H-6, p, J ) 7.1), 2.08 (H-25, s), 1.51
(H-12, m), 1.28 (H-13 to H-20, m), 1.15 (H-9, d, J ) 6.6), 0.98
(H-23, d, J ) 7.1), 0.88 (H-21, t, J ) 6.9).
1
5
22° and +23° (c 0.8, MeOH), respectively.
Ra cem ic a n d (R)-3-Meth oxyh exa n oic Acid (6). To 0.5
mL of racemic methyl 3-hydroxyhexanoate in 20 mL of
petroleum ether (bp 40-60 °C) were added KOH (4 pellets),
(
S)-MP A-2. This derivative was prepared in the same
1
4 µL of triethylamine, and 0.39 mL of dimethyl sulfate. The
manner as (R)-MPA-2 except using (S)-methoxyphenylacetic
solution was allowed to stir at room temperature for 5 h before
removal of the solvent under a stream of N . To this mixture
1
acid. H NMR (CDCl
3
H
, 500 MHz): δ (position, multiplicity, J
2
in Hz) 7.44-7.32 (Ph-MPA), 6.01 (NH, t, J ) 6.4), 5.31 (H-3,
were then added 5 mL of 0.5 N NaOH and 5 mL of MeOH,
and the solution was stirred overnight. The mixture was then
acidified to pH 2 with 1 N HCl and extracted into diethyl ether.
The organic layer was dried with MgSO and concentrated in
4
vacuo to afford racemic 6 in approximately 80% yield. Treat-
ddt, J ) 6.4, 4.4, 1.2), 5.24 (H-7, dd, J ) 7.1, 5.1), 4.98 (H-8,
qd, J ) 6.6, 5.1), 4.79 (R-MPA, s), 3.55 (H-4
L
, ddd, J ) -13.4,
6
3
2
1
0
.4, 4.4), 3.47 (H-4 , dt, J ) -13.4, 6.4), 3.42 (OMe-MPA, s),
H
.40 (H-22, s), 2.53 (H-2, d, J ) 1.2), 2.43 (H-6, p, J ) 7.1),
.27 (H-11, t, J ) 7.4), 2.10 (H-25, s), 1.51 (H-12, m), 1.28 (H-
3 to H-20, m), 1.20 (H-9, d, J ) 6.6), 1.10 (H-23, d, J ) 7.1),
.88 (H-21, t, J ) 6.5).
ment of methyl (R)-3-hydroxyhexanoate in a similar manner
1
provided (R)-6. H NMR of (R)-6 (CDCl
(
3
, 300 MHz):
δ
H
integration, multiplicity, J in Hz) 3.64 (1H, m), 3.39 (3H, s),
2
0
.53 (2H, d, J ) 5.9), 1.61 (1H, m), 1.49 (1H, m), 1.40 (2H, m),
Ack n ow led gm en t. We thank the National Cancer Insti-
.93 (3H, t, J ) 7.3).
tute for providing support through R01 grant CA12623 and
NCNPDDG grant CA53001. The upgrades to the spectrom-
eters used in this research were funded by grants from
the CRIF Program of the National Science Foundation
CHE9974921), the Air Force Office of Scientific Research
F49620-01-1-0524), and the Elise Pardee Foundation. The
cyanobacterium was collected by J . Starmer and identified
with the assistance of E. Cruz-Rivera. G. Tien, University of
Hawaii, Department of Chemistry, carried out the bioassays.
The UCR Mass Spectrometry Facility, Department of Chem-
istry, University of California, performed mass spectral analy-
ses at Riverside. We thank M. A. Tius and T. K. Hemsheidt,
University of Hawaii Chemistry Department, for the use of
the Chiral OD and the Phenosphere C18 columns.
Ma n d ela te Der iva tives (8). To 10 mg of 6 in 0.2 mL of
Cl were added 2 mg of DMAP, 12 mg of methyl D-
2 2
CH
mandelate, and 70 µL of 1 M N,N′-dicyclohexylcarbodiimide
DCC) in CH Cl . The mixture was stirred at room tempera-
(
2
2
(
(
ture for 3 h before filtration and evaporation. The crude
residue was purified by silica chromatography eluting with a
mixture of 6:1 hexane-i-PrOH. Evaporation of the solvent
afforded a mixture of (methyl D-mandelyl) (R)- and (S)-3-
methoxyhexanoate [(R)- and (S)-8] in 85% yield. (R)-6 was
treated in the same manner to afford enantiomerically pure
1
(R)-8. H NMR of (R)-8 (CDCl
3
H
, 300 MHz): δ (integration,
multiplicity, J in Hz) 7.46 (5H, m), 5.95 (1H, s), 3.72 (3H, s),
.70 (1H, m), 3.34 (3H, s), 2.67 (1H, dd, J ) -15.3, 7.3), 2.57
1H, dd, J ) -15.3, 5.6), 1.6-1.4 (4H, m), 0.88 (3H, t, J )
.5).
Acid Hyd r olysis a n d Ma n d ela te Der iva tiza tion of 1.
3
(
6
Refer en ces a n d Notes
To 0.5 mg of 1 was added 0.3 mL of 6 N HCl, and the solution
(1) Burja, A. M.; Banaigs, B.; Abou-Mansour, E.; Burgess, J . G.; Wright,
was refluxed at 118 °C for 24 h. After removal of the acid under
P. C. Tetrahedron 2001, 57, 9347-9377.

Cytotoxic Alkaloids from Symploca
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(
2) Nogle, L. M.; Gerwick, W. H. Org. Lett. 2002, 4, 1095-1098.
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000, 63, 1106-1112.
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(
13) Attempts to demethylate 1 with BBr
3
failed to liberate the secondary
1
995, 117, 8281-8282.
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(
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(
(
14) Lemieux, R. U.; Giguere, J . Can. J . Chem. 1951, 29, 678-690.
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NP030215X