Bromotyrosine alkaloids from the Australian marine sponge Pseudoceratina verrucosa

Article abstract of DOI:10.1021/np300648d

Two new bromotyrosine alkaloids, pseudoceralidinone A (1) and aplysamine 7 (2), along with three known compounds were isolated from the Australian sponge Pseudoceratina verrucosa. Their structures were characterized by NMR and MS data and the synthetic ro

Full text of DOI:10.1021/np300648d

Article
pubs.acs.org/jnp
Bromotyrosine Alkaloids from the Australian Marine Sponge
Pseudoceratina verrucosa
Trong D. Tran, Ngoc B. Pham, Gregory Fechner, John N. A. Hooper, and Ronald J. Quinn*
Eskitis Institute, Griffith University, Brisbane, Queensland 4111, Australia
S
* Supporting Information
ABSTRACT: Two new bromotyrosine alkaloids, pseudocer-
alidinone A (1) and aplysamine 7 (2), along with three known
compounds were isolated from the Australian sponge
Pseudoceratina verrucosa. Their structures were characterized
by NMR and MS data and the synthetic route. Their
cytotoxicity was evaluated against cancer cell lines (HeLa
and PC3) and a noncancer cell line (NFF).
new alkaloids, 1 and 2, in TFA salt forms, along with three
known bromotyrosine derivatives, 3−5.
he chemistry of sponges of the order Verongida has been
Tinvestigated for the past three decades, with hundreds of
bromotyrosine alkaloids being isolated.1 Over 90% of the 280
reported bromotyrosines are derived from four sponge families:
Aplysinidae, Aplysinellidae, Ianthellidae, and Pseudoceratini-
dae.2 Bromotyrosines, which have been considered as distinct
chemotaxonomic markers for the verongida sponges, are
further classified into six categories including simple bromotyr-
osine derivatives, oximes, bastadins, spirocyclohexadienylisox-
azolines, spirooxepinisoxazolines, and other structural classes
(geodiamolides, jaspamides, polyandrocarpamides, polycitones,
polycitrins, and chelonins).3 These marine alkaloids are well-
known for possessing a wide range of bioactivities including
histamine H2 and H3 and adrenergic α1D and α2A receptor
antagonists,4,5 anti-human immunodeficiency virus 1 (HIV-1),6
antiangiogenic,7 antibacterial,8,9 antifouling,10 isoprenylcysteine
carboxyl methyltransferase11,12 and mycothiol-S-conjugate
amidase11,12 inhibitory, cytotoxic,13 and anticancer14−16 activ-
ities.
In the course of our continuing studies on marine natural
products, the Australian sponge Pseudoceratina verrucosa was
found to contain two new bromotyrosine alkaloids, pseudocer-
alidinone A (1) and aplysamine 7 (2), together with three
known compounds, aerophobin 217 (3), fistularin 218 (4), and
fistularin 314,18 (5). Herein the isolation and structure
elucidation of the two new compounds (1 and 2) are described.
Cytotoxicities of all isolated compounds toward human cancer
cell lines (HeLa and PC3) and a noncancer cell line (NFF) are
also reported.
Pseudoceralidinone A (1) was purified as a colorless,
amorphous solid. The (+)-LRESIMS spectrum of 1 displayed
an isotopic cluster of ions [M + H]+ at m/z 421, 423, and 425
in the ratio 1:2:1, indicating the presence of two bromines. A
pseudomolecular ion peak in the (+)-HRESIMS spectrum at
m/z 420.9746 allowed the molecular formula C14H18Br2N2O3
to be assigned to 1. Analysis of the 13C NMR and HSQCAD
spectra indicated the molecule contained five quaternary
carbons, three methines, four methylenes, and two methyl
groups (Table 1). The 1H NMR and COSY spectra
demonstrated the presence of two spin systems, −CH−
CH2−NH− (moiety a, Figure 1) and (CH3)2−NH−CH2−
CH2−CH2− (moiety c, Figure 1). A two-proton singlet signal
RESULTS AND DISCUSSION
■
The freeze-dried P. verrucosa sponge was sequentially extracted
with n-hexane, CH2Cl2, and MeOH. The CH2Cl2/MeOH
extracts were then combined and chromatographed using C18
bonded silica HPLC (MeOH/H2O/0.1% TFA) to yield two
Received: September 20, 2012
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Table 1. NMR Data for Pseudoceralidinone A (1) in DMSO-
d6 at 30 °C
Table 2. NMR Data for Aplysamine 7 (2) in DMSO-d6 at 30
°C
a
a
position
δC, type
δH (J in Hz)
gCOSY
gHMBC
position
δC, type
δH (J in Hz) gCOSY
ROESY
gHMBC
3, 4, 6, 7
2
3
4
158.2, C
1
2
3
4
5
6
130.3, C
7.75, s
47.0, CH2 Ha 3.85, m;
Hb 3.36, t (9.0)
5.59, t (7.8)
4
2, 4, 5
132.9, CH
110.2, C
7.37, d (1.8)
6
7
3, 4b, 5;
3, 4a, 5
2, 5, 6;
2, 5, 6
153.7, C
5
74.4, CH
4a, 4b
2, 4, 6, 7,
11
112.5, CH
129.1, CH
6.99, d (8.4)
6
O-CH3
7
1, 3, 4
7.12, dd
(8.4, 1.8)
2, 5
2, 4, 5, 7
6
139.0, C
130.4, CH
117.8, C
152.0, C
7, 11
8, 10
9
7.71, s
5, 8, 9, 10
7
27.6, CH2 3.71, s
2, 6, N- 1, 2, 6, 8, 9
OH
8
151.3, C
163.0, C
13
70.3, CH2 4.04, t (6.0)
24.8, CH2 2.18, m
54.3, CH2 3.36, m
14
9, 14, 15
13, 15
9
14
13, 15
14, 16
10
11
7.79, t (6.0) 11
11, 12
9, 11
15
13, 14, N-
CH3
46.1, CH2 3.39, m;
3.27, m
10, 12;
10, 12
9, 12, 13;
9, 12, 13
16
9.63, brs
15, N-CH3
16
12
69.4, CH
4.67, t (6.0) 11
10, 14,
18
11, 13, 14,
18
N-CH3
42.3, CH3 2.84, d (4.2)
15
13
143.0, C
a
Referenced to residual DMSO-d6 at δC 39.52 ppm and δH 2.50 ppm.
14, 18
130.4, CH
7.56, s
11, 12
12, 15, 16,
17
15, 17
16
117.0, C
150.8, C
20
70.1, CH2 3.99, t (6.0) 21
22
16, 21, 22
20, 22
21
24.8, CH2 2.16, m
20, 22
b
22
54.3, CH2 3.35
21, 23
20, 21, N-
CH3
23
9.43, brs
22, N-
CH3
Figure 1. Partial structures a, b, and c (in bold) and key HMBC
correlations of 1.
O-CH3
N-OH
N-CH3
56.1, CH3 3.80, s
11.94, s
5
4
2, 6, 7
22
8
42.4, CH3 2.84, d (4.2) 16
22
at δH 7.71 ppm correlating with carbons at δC 117.8, 139.0, and
152.0 ppm was assigned to two symmetrical aromatic protons
in a 3,5-dibromotyrosine skeleton (moiety b, Figure 1).15 A 5-
substituted oxazolidinone ring (moiety a) was deduced from
the system −CH−CH2−NH− due to the HMBC correlations
from H-3 (δH 7.75 ppm), H-4 (δH 3.36 and 3.85 ppm), and H-
5 (δH 5.59 ppm) to the same relatively upfield resonance C-2
(δC 158.2 ppm). HMBC correlations from H-5 to C-7 (δC
130.4 ppm) and C-11 (δC 130.4 ppm) and from H-4 to C-6 (δC
139.0 ppm) indicated the connection of moieties a and b at C-5
and C-6 (Figure 1). The HMBC correlation from H-13 (δH
4.04 ppm) to C-9 (δC 152.0 ppm) supported the assemblage of
moieties b and c at O-12 (Figure 1). Pseudoceralidinone A was
elucidated as 1.
Aplysamine 7 (2) was obtained as a colorless, amorphous
solid. The (+)-LRESIMS spectrum of 2 exhibited an isotopic
cluster of ions [M + H]+ at m/z 664, 666, 668, and 670 in the
ratio 1:3:3:1, indicating the presence of three bromine atoms.
The molecular formula of 2 was determined to be
C23H28Br3N3O5 by a pseudomolecular ion peak in the
(+)-HRESIMS spectrum at m/z 663.9662. The 1H NMR
spectrum of 2 indicated the presence of a 1,3,4-trisubstituted
aromatic ring containing H-2 (δH 7.37, d, 1.8 Hz), H-5 (δH
6.99, d, 8.4 Hz), and H-6 (δH 7.12, dd, 8.4 and 1.8 Hz) (Table
2). Two substituents, a methoxy group (δH 3.80, δC 56.1 ppm)
and a bromine atom, were assigned to positions 4 and 3 of the
aromatic ring by HMBC correlations. The upfield chemical
shift of C-3 (δC 110.2 ppm) is due to the shielding effect of the
bromine atom on the chemical shift of an aromatic carbon.3
HMBC correlations from H-7 (δH 3.71 ppm) to aromatic
carbons (δC 112.5, 130.3, and 132.9 ppm) and two downfield
quaternary carbons (δC 151.3 and 163.0 ppm) indicated the
a
Referenced to residual DMSO-d6 at δC 39.52 ppm and δH 2.50 ppm.
b
Obscured by H2O peak.
isolated methylene carbon C-7 to be a β-carbon in a 3-(3-
bromo-4-methoxyphenyl)-2-(hydroxyimino)propanoic acid res-
idue (moiety a, Figure 2), which was also found in the
Figure 2. Partial structures a, b, and c (in bold) and key HMBC
correlations of 2.
psammaplin and purpuramine skeletons.3 This assignment was
supported by an HMBC correlation of a hydroxy signal in an
oxime group (δH 11.94 ppm) to C-8 (δC 151.3 ppm). The
upfield resonance of C-7 at δC 27.6 ppm suggested an E
configuration for the oxime functionality, as the corresponding
value for a Z-geometry would be >35 ppm.16,19,20 COSY and
HMBC correlations also led to the establishment of 1-
substituted-2-aminoethoxy and 3,5-dibromo-4-(3-
dimethylamino)propyloxyphenyl residues (moieties b and c,
Figure 2). The HMBC correlation from an exchangeable NH
proton (δH 7.79 ppm) to a carbonyl carbon (δC 163.0 ppm)
established the formation of an amide bond between moieties a
and b (Figure 2). HMBC correlations from H-11 (δH 3.27 and
3.39 ppm) to C-13 (δC 143.0 ppm) and from H-12 (δH 4.67
ppm) to C-14 and C-18 (δC 130.4 ppm) confirmed the
B
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Scheme 1. Modification of Pseudoceralidinone A (1) to Determine the Absolute Configuration
Figure 3. ΔδSR values for MTPA derivatives (7 and 8) and their configurational correlation model.
Scheme 2. Total Synthesis of Oxime-Protected Aplysamine 7 (17)
Figure 4. Stereochemical structures of two enantiomers (17a and 17b) separated from 17.
connectivity of b and c (Figure 2). Thus, aplysamine 7 was
assigned as 2.
CD spectra have been recorded for 1 and 2; however no
characteristic curves were observed. Here we used a synthetic
route to determine the absolute configurations for C-5 of 1 and
C-12 of 2.
hydrochloric acid using microwave-assisted conditions at 140
°C for 10 min, followed by Boc-protection in aqueous sodium
bicarbonate for 30 min at room temperature (rt) to produce 6.
The hydrolysis conditions should not change the configurations
at C-5.21,22 It was noticed that the specific rotation of 6 ([α]25
D
+3.1 (c 0.1, MeOH)) had the same sign compared with that of
The absolute configuration of 1 was examined by Mosher’s
1 ([α]25 +3.5 (c 0.1, MeOH)). Mosher’s analysis was then
D
analysis (Scheme 1). Compound 1 was hydrolyzed with 6 N
performed on 6 to determine its absolute configuration. An S
C
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configuration was deduced due to the ΔδSR values obtained by
subtracting the chemical shift (δR) of a proton of the (R)-
MTPA ester (8) from that (δS) of the (S)-MTPA ester (7)
(Figure 3). This result led to the assignment of the S
configuration for 1.
aqueous EtOH at reflux for 40 min (95%).39 The O-benzyl
product 20 was formed with a yield of 90% using KOH in dry
DMF or DMSO at rt for 2 h.40 Other basic conditions with
Na2CO3, K2CO3, or CsCO3 gave no or very low yield.
Treatment of 17 with benzyl bromide using KOH in DMF at rt
monitored by LC/MS showed that the alcohol group was not
benzylated. Therefore, KOH in DMF was chosen for the
conversion of 2 to 21.
The absolute configuration of 2 was addressed by a total
synthesis (Scheme 2). The alpha-protected 3-bromo-4-
methoxyphenylpyruvic acid oxime (12) was prepared from O-
methyl-L-tyrosine (9). Compound 9 was converted to 3-bromo-
4-methoxyphenylpyruvic acid (11) via an azlactone in three
steps.23 Compound 11 was treated with O-benzylhydroxyl-
amine in EtOH at reflux for 4 h24 to give 12 with a yield of
64%. A racemate of commercially available octopamine (13)
was chosen as a starting material for synthesis of 16.
Dibromination of 13 with bromine in hydrochloric acid at rt
gave the 3,5-dibromo derivative, which was then protected by
(Boc)2O under basic conditions to give 14 with an overall yield
of 80% for the two steps. O-Alkylation of 14 with N-(3-
chloropropyl)-N,N-dimethylamine hydrochloride using potas-
sium carbonate and potassium iodide in (Me)2CO/MeCN
(1:1) at reflux25 produced 15 (50%). Removal of the Boc group
in 15 was achieved with 50% trifluoroacetic acid (TFA) in
CH2Cl2 to obtain 16 (90%). This compound was coupled with
12 using EDCI/HOBt24,26 to give 17 with a yield of 60%.
The racemic mixture of 17 was subjected to a chiral-phase
HPLC column and resolved into two enantiomers. The
absolute configurations of these two stereoisomers were then
assigned by Mosher’s ester analysis. The results indicated that
17a with a specific rotation of +5.2 (c 0.08, MeOH) was
determined to have an S configuration, and its stereoisomer
(17b), with a specific rotation of −6.7 (c 0.08, MeOH), had an
R configuration (Figure 4).
Attempts at hydrogenolysis of the O-benzyl oxime-protecting
group on 17a and 17b to determine the absolute configuration
of 2 were unsuccessful. Hydrogenolysis with palladium black in
AcOH/dioxane (1:1)27 or Pd/C in EtOH28 led to decom-
position. Attempted deprotection using HBr/AcOH (1:3)29
resulted in decomposition; neat TFA30 or TFA/toluene (1:1)31
resulted in no conversion. The difficulty of deprotection of the
O-benzyl group was also reported in a synthetic route to
purealidin, a bromotyrosine natural product.32 Since we could
not deprotect the O-benzyl group of 17a (S configuration) and
17b (R configuration), we converted the natural product 2 to
the oxime-protected compound (21) and used the specific
rotation to assign the configuration for 21 and thus 2. Oxime
ethers can be made under anhydrous conditions using strong
bases such as NaH33,34 or sodium alkoxides.35,36 However,
strong bases could also benzylate the oxygen of the alcohol
group C-12 in 2.37,38 Therefore, model reactions were
conducted to determine the least basic conditions to O-
benzylate an oxime (Scheme 3).
Reaction between 2 and benzyl bromide using KOH in DMF
at rt for 1 h was successful in selectively benzylating the oxime
(21) with a yield of 18% (Scheme 4). Compounds 21 and 17a
Scheme 4. Synthesis of O-Benzylaplysamine 7 (21)
displayed the same retention time by chiral-phase HPLC at
14.4 min, while 17b had a retention time of 15.3 min, and the
measured specific rotation of 21 of +5.8 (c 0.04, MeOH)
confirmed an S configuration for 21. Therefore, the S
configuration was assigned for 2.
The known compounds (3−5) were characterized by
comparison of their spectroscopic data (UV, NMR, and MS)
and specific rotations with the values in the literature.17,18
The cytotoxicities of the isolated compounds were evaluated
against three cell lines, cervical adenocarcinoma cells (HeLa),
prostate adenocarcinoma cells (PC3), and human neonatal
foreskin fibroblast cells (noncancer cells, NFF) (Table 4). The
new compound aplysamine 7 (2) showed potency against PC3
cells with an IC50 of 4.9 μM and was inactive (IC50 > 10 μM)
against HeLa cells and NFF cells, while pseudoceralidinone A
(1) and the other known bromotyrosines (3−5) displayed no
cytotoxicity against all three cell lines.
In summary, two new marine metabolites, pseudoceralidi-
none A (1) and aplysamine 7 (2), were isolated from the
Australian sponge P. verrucosa. Their absolute configurations
were determined by spectroscopic analysis and chemical
synthesis. Compound 2 showed cytotoxic activity against the
human prostate cancer cell line PC3.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a JASCO P-1020 polarimeter (10 cm cell). UV and IR
spectra were recorded on a CAMSPEC M501 UV/vis spectropho-
tometer and Bruker Tensor 27 FT-IR spectrometer, respectively.
NMR spectra were recorded at 30 °C on Varian Inova 600 and 500
1
MHz spectrometers. The H and 13C chemical shifts were referenced
to the DMSO-d6 solvent peaks at δH 2.50 and δC 39.52 ppm. Standard
parameters were used for the 2D NMR spectra obtained, which
included gCOSY, HSQCAD (1JCH = 140 Hz), gHMBC (nJCH = 8.3
Hz), and ROESY. Low-resolution mass spectra were acquired using a
Mariner TOF mass spectrometer (Applied Biosystems Pty Ltd.).
High-resolution mass measurement was acquired on a Bruker
Daltonics Apex III 4.7e Fourier transform mass spectrometer, fitted
with an Apollo API source. A Betasil C18 column (5 μm, 21.2 × 150
mm) and Hypersil BDS C18 column (5 μm, 10 × 250 mm) were used
for semipreparative HPLC. A Phenomenex Luna C18 column (3 μm,
4.6 × 50 mm) was used for LC/MS controlled by MassLynx 4.1
software. All solvents used for extraction and chromatography were
Omnisolv HPLC grade, and the H2O used was Millipore Milli-Q PF
Model oxime 19 was made from acetophenone (18) and
hydroxylamine in the presence of potassium acetate in 80%
Scheme 3. Protection of the O-Benzyl Group in the Model
System
D
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extracts were combined, and solvents were then evaporated to yield a
yellow residue (1.5 g). This extract was preadsorbed onto C18 (1.0 g)
and packed dry into a small cartridge, which was connected to a C18
preparative HPLC column (5 μm, 21.2 × 150 mm). A linear gradient
from 100% H2O (0.1% TFA) to 100% MeOH (0.1% TFA) was
performed over 60 min at a flow rate of 9 mL/min, and 60 fractions
(1.0 min each) were collected. Fistularin 3 (5, 40.0 mg, 0.8% dry wt)
was obtained from fractions 44−48. Fractions 23−37 were combined
and loaded onto a C18 preparative HPLC column (5 μm, 21.2 × 150
mm) with a 60 min linear gradient from 20% MeOH (0.1% TFA)−
80% H2O (0.1% TFA) to 90% MeOH (0.1% TFA)−10% H2O (0.1%
TFA). A new bromotyrosine, pseudoceralidinone A (1, 12 mg, 0.24%
dry wt), and two known compounds, aerophobin 2 (3, 18 mg, 0.36%
dry wt) and fistularin 2 (4, 1.5 mg, 0.03%), were isolated respectively.
Further purification for fractions 37 to 39 on the same reversed-phase
HPLC column with a linear gradient from 20% MeOH (0.1% TFA)−
80% H2O (0.1% TFA) to 80% MeOH (0.1% TFA)−20% H2O (0.1%
TFA) in 60 min yielded the new aplysamine 7 (2, 4.5 mg, 0.09% dry
wt).
Table 3. NMR Data for Compounds 2 and 17 in DMSO-d6 at
30 °C
a
2
17
position
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
1
2
3
4
5
6
130.3, C
129.6, C
132.9, CH
110.2, C
7.37, d (1.8)
133.1, CH
110.3, C
7.36, m
153.7, C
154.0, C
112.5, CH
129.1, CH
6.99, d (8.4)
112.6, CH
129.2, CH
6.97, d (8.4)
7.08, d (8.4)
7.12, dd (8.4,
1.8)
7
8
9
27.6, CH2 3.71, s
151.3, C
28.6, CH2 3.72, s
152.2, C
163.0, C
162.2, C
10 (NH)
11
7.79, t (6.0)
7.96, t (6.0)
46.1, CH2 3.39, m;
3.27, m
46.3, CH2 3.36, m;
3.27, m
Pseudoceralidinone A (1): colorless, amorphous solid; [α]25 +3.5
D
12
69.4, CH
143.0, C
130.4, CH
117.0, C
150.8, C
4.67, t (6.0)
69.4, CH
143.1, C
130.4, CH
117.2, C
150.8, C
4.67, t (6.0)
(c 0.1, MeOH); UV (MeOH) λmax (log ε) 257 (3.4), 362 (2.7) nm; IR
(film) νmax 3444, 1748, 1682, 1203 cm−1; 1H (600 MHz) and 13C (150
MHz) NMR data are summarized in Table 1; (+)-HRESIMS m/z
420.9746 [M + H]+ (calcd for [C14H1979Br2N2O3]+, 420.9757, Δ −2.6
ppm).
13
14, 18
15, 17
16
7.56, s
7.57, s
Aplysamine 7 (2): colorless, amorphous solid; [α]24 +8.1 (c 0.08,
20
70.1, CH2 3.99, t (6.0)
24.8, CH2 2.16, m
70.2, CH2 3.98, t (6.0)
24.8, CH2 2.16, m
54.4, CH2 3.35, m
9.47, brs
D
21
MeOH); UV (MeOH) λmax (log ε) 282 (3.5) nm; IR (film) νmax 3405,
1
1673, 1204 cm−1; H (600 MHz) and 13C (150 MHz) NMR data are
b
22
54.3, CH2 3.35
summarized in Table 2; (+)-HRESIMS m/z 663.9662 [M + H]+
23 (NH)
O-CH3
N-CH3
O-CH2-Ph
Ph (OBn)
9.43, brs
56.1, CH3 3.80, s
(calcd for [C23H2979Br3N3O5]+, 663.9652, Δ 1.5 ppm).
56.2, CH3 3.80, s
42.4, CH3 2.84, d (4.2)
76.6, CH2 5.25, s
136.8, C
Compound 6. Compound 1 (4.6 mg, 0.011 mmol) was hydrolyzed
in 200 μL of 6 N HCl using microwave-assisted conditions at 140 °C
for 10 min. The solvent was removed under reduced pressure. The
residue was then basified with aqueous saturated NaHCO3 (1 mL)
followed by Boc-protection with Boc2O (3 mg, 0.014 mmol) for 30
min at rt. The crude product was concentrated in vacuo and separated
42.4, CH3 2.84, d (4.2)
128.0, CH
128.5, CH
128.1, CH
7.33, d (7.2)
7.36, m
7.36, m
by RP-HPLC to obtain 6 as a colorless, amorphous solid (2.5 mg, 46%
1
yield in two step): [α]25 +3.1 (c 0.1, MeOH); H NMR (600 MHz,
N-OH
11.94, s
D
a
DMSO-d6) δ 7.51 (2H, s, H-7 and H-11), 6.67 (1H, t, J = 5.4 Hz, NH-
3), 4.54 (1H, t, J = 6.0, H-5), 3.95 (2H, t, J = 6.6 Hz, H-13), 3.08 (2H,
m, H-4), 2.41 (2H, t, J = 6.6 Hz, H-15), 2.14 (6H, s, 2 × NCH3), 1.91
(2H, pentet, J = 6.6 Hz, H-14), 1.32 (9H, s, 3 × CH3-Boc); 13C NMR
(150 MHz, DMSO-d6, obtained from correlations observed in
HSQCAD and gHMBC spectra) δ 155.7 (C, C-2), 151.5 (C, C-9),
143.2 (C, C-6), 130.3 (CH, C-7 and C-11), 117.3 (C, C-8 and C-10),
77.7 (C, C-Boc), 71.5 (CH2, C-13), 69.8 (CH, C-5), 55.4 (CH2, C-
15), 47.0 (CH2, C-4), 44.9 (CH3, 2 × NCH3), 27.8 (CH3, 3 × CH3-
Boc), 27.4 (CH2, C-14); (+)-LRESIMS m/z 495, 497, 499 (in the
ratio 1:2:1).
Referenced to residual DMSO-d6 at δC 39.52 ppm and δH 2.50 ppm.
Obscured by H2O peak.
b
Table 4. Cytotoxicity Evaluation for Compounds 1−5
a
IC50 SD (μM) or % inhibition SD
compound
HeLa
PC3
NFF
1
2
3
4
5
44 14%
19 5.2
35 6.7%
4.9 1.8
29 2.5%
16 7.2
20 4.7%
40 3.4
66 4.8%
25 8.1%
Preparation of MTPA Esters (7 and 8). (R)- or (S)-MTPA-Cl (2
μL, 0.01 mmol) was added to 6 (0.5 mg, 0.001 mmol) in anhydrous
pyridine (150 μL) and stirred at rt. Reactions were monitored by LC/
MS and stopped after 24 h. An aliquot was then dried under dry
nitrogen. The residue was partitioned with the solvent system H2O/
CH2Cl2 (1:1). The CH2Cl2 fraction was evaporated to dryness to yield
73 12%
68 3.4%
46 14%
60 20%
48 12%
vincristine
sulfate
0.0358 0.0035
0.0153 0.0011
0.0169 0.0032
a
Each IC50 or % inhibition at 100 μM was determined as the mean
SD of two independent experiments with triplicate determinations for
each concentration.
1
Mosher’s esters 7 and 8. The H and COSY NMR experiments were
performed on the Mosher’s esters (7 and 8) to obtain the δS and δR
values, which were used to determine the absolute configuration at
position C-5.
filtered. A Phenomenex chiral-phase column, Lux 5 μm, Amylose-2,
4.6 × 250 mm, was used for separation of compounds 17a and 17b
and confirmation of the absolute configuration of compound 21.
Animal Material. A specimen of P. verrucosa was collected at the
depth of 9.4 m, at Hook Reef Lagoon, Queensland, Australia, in 1995.
It was identified as Pseudoceratina verrucosa Bergquist (phylum
Porifera, class Demospongiae, order Verongida, family Aplysinellidae).
A voucher specimen (QMG315237) has been deposited at the
Queensland Museum, South Brisbane, Queensland, Australia.
1
(S)-MTPA ester of 6 (7): H NMR (500 MHz, DMSO-d6) δ 7.62
(2H, s, H-7 and H-11), 7.49 (2H, m, MTPA-ArH), 7.46 (3H, m,
MTPA-ArH), 7.13 (1H, t, J = 5.5 Hz, NH-3), 5.96 (1H, t, J = 6.0 Hz,
H-5), 4.01 (2H, t, J = 6.0 Hz, H-13), 3.49 (3H, s, MTPA-OCH3), 3.45
(1H, m, H-4a), 3.32 (1H, m, H-4b), 3.31 (2H, m, H-15), 2.79 (6H, s,
2 × NCH3), 2.24 (2H, m, H-14), 1.32 (9H, s, Boc-H); (+)-LRESIMS
m/z 711, 713, 715 (in the ratio 1:2:1).
1
(R)-MTPA ester of 6 (8): H NMR (500 MHz, DMSO-d6) δ 7.63
Extraction and Isolation. A freeze-dried sample of P. verrucosa (5
g) was extracted exhaustively with hexane (250 mL), CH2Cl2 (250
mL), and MeOH (2 × 250 mL), respectively. The CH2Cl2 and MeOH
(2H, s, H-7 and H-11), 7.50 (2H, m, MTPA-ArH), 7.46 (3H, m,
MTPA-ArH), 7.12 (1H, t, J = 5.5 Hz, NH-3), 5.95 (1H, t, J = 6.0 Hz,
H-5), 4.01 (2H, t, J = 6.0 Hz, H-13), 3.49 (3H, s, MTPA-OCH3), 3.44
E
dx.doi.org/10.1021/np300648d | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(1H, m, H-4a), 3.30 (1H, m, H-4b), 3.33 (2H, m, H-15), 2.83 (6H, s,
2 × NCH3), 2.24 (2H, m, H-14), 1.32 (9H, s, Boc-H); (+)-LRESIMS
m/z 711, 713, 715 (in the ratio 1:2:1).
13C NMR (125 MHz, DMSO-d6) δ 155.5 (C), 151.3 (C), 142.7 (C),
130.4 (2 × CH), 117.0 (2 × C), 77.6 (C), 71.7 (CH2), 70.0 (CH),
55.6 (CH2), 47.3 (CH2), 45.1 (2 × NCH3), 28.1 (3 × CH3), 27.7
(CH2); (+)-LRESIMS m/z 495, 497, 499 (in the ratio 1:2:1).
( )-2-Amino-1-(3,5-dibromo-4-(3-(dimethylamino)propoxy)-
phenyl)ethanol (16). Compound 15 (158 mg, 0.3 mmol) was taken in
2 mL of CH2Cl2/TFA (1:1) and stirred for 30 min. After the solvent
was removed, the crude product was purified by RP-HPLC to obtain
free amine 16 (114 mg, 90%): IR (film) νmax 3408, 3033, 2739, 1676,
3-Bromo-O-methyl-L-tyrosine (10). To a cooled solution (5 °C) of
O-methyl-L-tyrosine (9, 261 mg, 1.3 mmol) in glacial acetic acid (8.0
mL, 0.14 mol) was added Br2 (0.12 mL, 2.3 mmol), and the mixture
stirred for 3 h at rt. The reaction was then quenched with saturated
Na2S2O3 solution, and the solvent was removed under vacuum. The
reaction mass was extracted with EtOAc. The organic layer was dried
prior to being purified by RP-HPLC to give 10 as a colorless,
amorphous solid (285 mg, 80%): IR (film) νmax 3411, 1625, 1255,
1
1202, 1133 cm−1; H NMR (600 MHz, DMSO-d6) δ 8.10 (2H, brs),
7.68 (2H, s), 4.82 (1H, dd, J = 9.0, 3.0 Hz), 4.00 (2H, t, J = 6.6 Hz),
3.36 (2H, t, J = 7.2 Hz), 3.10 (1H, d, J = 12.0 Hz), 2.89 (1H, t, J = 11.6
Hz), 2.84 (6H, s), 2.19 (2H, m); 13C NMR (150 MHz, DMSO-d6) δ
151.4 (C), 141.4 (C), 130.5 (2 × CH), 117.6 (2 × C), 70.3 (CH2),
67.6 (CH), 54.3 (CH2), 45.1 (CH2), 42.3 (2 × NCH3), 24.8 (CH2);
(+)-LRESIMS m/z 395, 397, 399 (in the ratio 1:2:1).
1
1024 cm−1; H NMR (600 MHz, DMSO-d6) δ 8.31 (3H, brs), 7.47
(1H, d, J = 1.8 Hz), 7.23 (1H, dd, J = 9.0, 1.8 Hz), 7.07 (1H, d, J = 8.4
Hz), 4.17 (1H, brs), 3.83 (3H, s), 3.05 (2H, m); 13C NMR (150 MHz,
DMSO-d6) δ 170.3 (C), 154.7 (C), 133.8 (CH), 130.1 (CH), 128.5
(C), 112.7 (CH), 110.6 (C), 56.2 (OCH3), 53.1 (CH), 34.5 (CH2);
(+)-LRESIMS m/z 274, 276 (in the ratio 1:1).
( )-2-(Benzyloxyimino)-3-(3-bromo-4-methoxyphenyl)-N-(2-(3,5-
dibromo-4-(3-(dimethylamino)propoxy)phenyl)-2-hydroxyethyl)-
propanamide (17). To a solution of 16 (30 mg, 0.076 mmol) in dry
dimethylformamide (DMF, 3.5 mL) was added N-hydroxybenzo-
triazole (HOBt, 20 mg, 0.15 mmol), and the reaction mixture was
stirred for 15 min at rt. The reaction mixture was then cooled to 0 °C,
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDCI, 28.7 mg,
0.15 mmol) was added, and stirring continued for 30 min at 0 °C. To
this mixture was then added compound 12 (28 mg, 0.076 mmol), and
the mixture was stirred for 2 h at rt. The crude product was
concentrated in vacuo and separated by RP-HPLC (MeOH, H2O, 0.1%
TFA) to yield a racemic mixture of 17 (34 mg, 60%): IR (film) νmax
3395, 2940, 1673, 1496, 1204 cm−1; 1H NMR (600 MHz, DMSO-d6)
δ 9.47 (1H, brs), 7.96 (1H, t, J = 6.0 Hz), 7.57 (2H, s), 7.36 (3H, m),
7.33 (2H, d, J = 7.2 Hz), 7.08 (1H, d, J = 8.4 Hz), 6.97 (1H, d, J = 8.4
Hz), 5.25 (2H, s), 4.67 (1H, t, J = 6.0 Hz), 3.98 (2H, t, J = 6.0 Hz),
3.80 (3H, s), 3.72 (2H, s), 3.36 (1H, m), 3.35 (2H, m), 3.27 (1H, m),
2.84 (6H, d, J = 4.2 Hz), 2.16 (2H, m); 13C NMR (150 MHz, DMSO-
d6) δ 162.2 (C), 154.0 (C), 152.2 (C), 150.8 (C), 143.1 (C), 136.8
(C), 133.1 (CH), 130.4 (2 × CH), 129.6 (C), 129.2 (CH), 128.5 (2 ×
CH), 128.1 (CH), 128.0 (2 × CH), 117.2 (2 × C), 112.6 (CH), 110.3
(C), 76.6 (CH2), 70.2 (CH2), 69.4 (CH), 56.2 (OCH3), 54.4 (CH2),
46.3 (CH2), 42.4 (2 × NCH3), 28.6 (CH2), 24.8 (CH2);
(+)-LRESIMS m/z 754, 756, 758, 760 (in the ratio 1:3:3:1).
Isolation of Two Enantiomers from a Synthetic Racemic Oxime-
Protected Aplysamine 7 Mixture (17). Compound 17 was further
purified by HPLC on a chiral-phase HPLC column (Phenomenex, Lux
5 μm, Amylose-2, 4.6 × 250 mm) with an isocratic condition of 30%
MeCN (0.1% TFA)−70% H2O (0.1% TFA) in 15 min at a flow rate of
0.8 mL/min. Two enantiomers (17a and 17b) eluted at 7.5 and 9.8
min, respectively.
3-Bromo-4-methoxyphenylpyruvic acid (11). A solution of 10
(161 mg, 0.6 mmol) in (CF3CO)2O (3.0 mL, 21.2 mmol) was heated
at 90 °C for 18 h. The solvent was removed under reduced pressure.
The residue was again dissolved in 70% aqueous TFA and allowed to
stand for 16 h at rt. The product was chromatographed on RP-HPLC
to yield 11 (74 mg, 46%): IR (film) νmax 3410, 1650, 1254, 1054 cm−1;
1H NMR (600 MHz, DMSO-d6) δ 9.28 (1H, brs), 8.10 (1H, d, J = 1.8
Hz), 7.66 (1H, dd, J = 8.4, 1.8 Hz), 7.10 (1H, d, J = 8.4 Hz), 3.85 (3H,
s); 13C NMR (150 MHz, DMSO-d6) δ 166.2 (C), 154.3 (C), 141.1
(C), 133.2 (CH), 130.2 (CH), 129.2 (C), 112.5 (CH), 110.5 (C),
108.2 (CH), 56.2 (OCH3); (−)-LRESIMS m/z 271, 273 (in the ratio
1:1).
2-(Benzyloxyimino)-3-(3-bromo-4-methoxyphenyl)propanoic
acid (12). To a solution of 11 (33 mg, 0.12 mmol) in EtOH (2 mL)
was added O-benzylhydroxylamine (56 mg, 0.35 mmol), and the
mixture refluxed for 4 h. The crude product was purified by RP-HPLC
to afford 12 (29 mg, 64%): IR (film) νmax 3420, 2940, 1625, 1599,
1541, 1397, 1255, 1022, 806 cm−1; 1H NMR (600 MHz, DMSO-d6) δ
7.42 (1H, d, J = 1.8 Hz); 7.32 (2H, d, J = 7.8 Hz), 7.29 (1H, d, J = 7.2
Hz), 7.25 (2H, d, J = 7.2 Hz), 7.19 (1H, dd, J = 8.4, 1.8 Hz), 6.96 (1H,
d, J = 8.4 Hz), 5.05 (2H, s), 3.79 (3H, s), 3.72 (2H, s); 13C NMR (150
MHz, DMSO-d6) δ 164.7 (C), 158.2 (C), 153.5 (C), 138.2 (C), 133.1
(CH), 131.5 (C), 129.6 (CH), 128.2 (2 × CH), 127.54 (2 × CH),
127.50 (CH), 112.3 (CH), 110.0 (C), 74.8 (CH2), 56.2 (OCH3), 30.8
(CH2); (+)-LRESIMS m/z 378, 380 (in the ratio 1:1).
( )-tert-Butyl-2-(3,5-dibromo-4-hydroxyphenyl)-2-hydroxyethyl-
carbamate (14). To a solution of racemic octopamine (13, 384 mg,
2.0 mmol) in distilled H2O (3 mL) was added 6 N HCl (3 mL), and
the mixture was cooled to 5 °C. Bromine (0.35 mL, 6.8 mmol) was
then injected into the stirred solution. The reaction was performed in
1 h and then dried in vacuo. It was then basified with NaOH 10% (5
mL), and Boc2O (500 mg, 2.2 mmol) was added with stirring at rt for
30 min. The solvent was removed in vacuo, and the residue was
subjected to RP-HPLC to give 14 (654 mg, 80% yield in two steps):
IR (film) νmax 3411, 2977, 2932, 1693, 1468, 1250, 1170, 1055 cm−1;
1H NMR (600 MHz, DMSO-d6) δ 7.10 (2H, s), 6.64 (1H, t, J = 5.4
Hz), 4.29 (1H, dd, J = 7.2, 5.4 Hz), 3.00 (1H, m), 2.92 (1H, m), 1.36
(9H, s); 13C NMR (150 MHz, DMSO-d6) δ 160.8 (C), 155.7 (C),
128.8 (2 × CH), 123.9 (C), 114.4 (2 × C), 77.5 (C), 70.7 (CH), 48.3
(CH2), 28.3 (3 × CH3); (+)-LRESIMS m/z 410, 412, 414 (in the ratio
1:2:1).
( )-tert-Butyl-2-(3,5-dibromo-4-(3-(dimethylamino)propoxy)-
phenyl)-2-hydroxyethyl carbamate (15). To a racemic mixture of
compound 14 (327 mg, 0.8 mmol), K2CO3 (0.90 g, 6.5 mmol), and KI
(1.08 g, 6.5 mmol) in dry Me2CO/MeCN (1:1, 5.0 mL) was added 3-
dimethylamino-1-propyl chloride hydrochloride (406 mg, 2.5 mmol),
and the mixture was refluxed for 16 h. The solvent was evaporated in
vacuo, and crude product was extracted with EtOAc. The organic layer
was concentrated and purified by RP-HPLC to obtain 15 (198 mg,
50%): IR (film) νmax 3336, 2974, 2823, 1698, 1456, 1253, 1168, 1042
cm−1; 1H NMR (500 MHz, DMSO-d6) δ 7.52 (2H, s), 6.76 (1H, brs),
4.55 (1H, t, J = 6.0 Hz), 3.95 (2H, t, J = 6.5 Hz), 3.08 (2H, t, J = 6.5
Hz), 2.41 (2H, t, J = 7.0 Hz), 2.14 (6H, s), 1.91 (2H, m), 1.32 (9H, s);
Compound 17a: colorless, amorphous solid; [α]24 +5.2 (c 0.08,
D
1
MeOH); H NMR (600 MHz, DMSO-d6) δ 9.56 (1H, brs, NH-23),
7.96 (1H, t, J = 6.0 Hz, NH-10), 7.57 (2H, s, H-14 and H-18), 7.36
(3H, m, H-2 and H-Ph), 7.33 (3H, d, J = 7.2 Hz, H-Ph), 7.08 (1H, d, J
= 8.4 Hz, H-6), 6.97 (1H, d, J = 8.4 Hz, H-5), 5.25 (2H, s, OCH2-Ph),
4.67 (1H, t, J = 6.0 Hz, H-12), 3.98 (2H, t, J = 6.0 Hz, H-20), 3.80
(3H, s, OCH3), 3.72 (2H, s, H-7), 3.36 (1H, m, H-11a), 3.35 (2H, m,
H-22), 3.27 (1H, m, H-11b), 2.84 (6H, d, J = 4.2 Hz, 2 × NCH3), 2.16
(2H, m, H-21); (+)-LRESIMS m/z 754, 756, 758, 760 (in the ratio
1:3:3:1); (+) HRESIMS m/z 754.0086 [M + H]+ (calcd for
[C30H3579Br3N3O5]+, 754.0121, Δ −4.6 ppm).
Compound 17b: colorless, amorphous solid; [α]24 −6.7 (c 0.08,
D
1
MeOH); H NMR (600 MHz, DMSO-d6) δ 9.55 (1H, brs, NH-23),
7.96 (1H, t, J = 6.0 Hz, NH-10), 7.57 (2H, s, H-14 and H-18), 7.36
(3H, m, H-2 and H-Ph), 7.33 (3H, d, J = 7.2 Hz, H-Ph), 7.08 (1H, d, J
= 8.4 Hz, H-6), 6.97 (1H, d, J = 8.4 Hz, H-5), 5.25 (2H, s, OCH2-Ph),
4.67 (1H, t, J = 6.0 Hz, H-12), 3.98 (2H, t, J = 6.0 Hz, H-20), 3.80
(3H, s, OCH3), 3.72 (2H, s, H-7), 3.36 (1H, m, H-11a), 3.35 (2H, m,
H-22), 3.27 (1H, m, H-11b), 2.84 (6H, d, J = 4.2 Hz, 2 × NCH3), 2.16
(2H, m, H-21); (+)-LRESIMS m/z 754, 756, 758, 760 (in the ratio
1:3:3:1); (+) HRESIMS m/z 754.0090 [M + H]+ (calcd for
[C30H3579Br3N3O5]+, 754.0121, Δ −4.1 ppm).
F
dx.doi.org/10.1021/np300648d | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Preparation of MTPA Esters (17a and 17b). The absolute
configuration at C-12 of compounds 17a and 17b was assigned using
the same procedure as that for compound 6.
HCOOH) in 25 min and isocratic at 100% MeOH (0.1% HCOOH)
for the next 5 min at a flow rate of 0.8 mL/min. Compound 21 eluted
at 14.4 min. Using the same procedure as for compound 17, the two
enantiomers 17a and 17b were separated with retention times of 14.4
and 15.3 min, respectively.
1
(S)-MTPA ester of 17a (22a): H NMR (600 MHz, DMSO-d6) δ
7.99 (1H, t, J = 6.0 Hz, NH-10), 7.54 (2H, s, H-14 and H-18), 5.259
(2H, s, OCH2-Ph), 4.715 (1H, t, J = 6.0 Hz, H-12), 3.26 (1H, m, H-
11), 2.79 (6H, s, 2 × NCH3); (+)-LRESIMS m/z 970, 972, 974, 976
(in the ratio 1:3:3:1).
Cytotoxicity Assay. Human neonatal foreskin fibroblast (non-
cancer cells, NFF) was grown in DMEM media supplemented with
10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/
mL streptomycin. Cervical adenocarcinoma cells (HeLa) and prostate
adenocarcinoma cells (PC3) were grown in RPMI media supple-
mented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL
streptomycin. Cells were grown under 5% CO2 in a humidified
environment at 37 °C. Forty-five microliters of media containing 1000
cells was added to a 384-well microtiter plate (Falcon black clear 384
TC microtiter plates). Plates were incubated overnight at 37 °C, 5%
CO2, and 80% humidity to allow cells to adhere. Stock concentrations
of pure compounds were prepared at 10 mM in 100% DMSO.
Compounds were then diluted 1 in 10 in media. Five microliters of
diluted compound was added to the cells to give a total volume of 50
μL. The final compound concentration range tested was 100 μM to 3
nM (final DMSO concentration of 1%). Each concentration in media
was tested in triplicate. Vincristine sulfate was used as a positive
control. Cells and compounds were then incubated for 72 h at 37 °C,
5% CO2, and 80% humidity. Cell proliferation was measured with the
addition of 10 μL of a 60% Alamar blue solution in media to each well
of the microtiter plate to give a final concentration of 10% Alamar
blue. The plates were incubated at 37 °C, 5% CO2, and 80% humidity
within 24 h. The fluorescence of each well was read at excitation 535
nm and emission 590 nm on a Victor II Wallac plate reader
(PerkinElmer). Ten-point concentration response curves were then
analyzed using nonlinear regression, and IC50 values were determined
by using GraphPad Prism 5 software.
1
(R)-MTPA ester of 17a (23a): H NMR (600 MHz, DMSO-d6) δ
7.97 (1H, t, J = 6.0 Hz, NH-10), 7.55 (2H, s, H-14 and H-18), 5.254
(2H, s, OCH2-Ph), 4.710 (1H, t, J = 6.0 Hz, H-12), 3.25 (1H, m, H-
11), 2.80 (6H, s, 2 × NCH3); (+)-LRESIMS m/z 970, 972, 974, 976
(in the ratio 1:3:3:1).
1
(S)-MTPA ester of 17b (22b): H NMR (600 MHz, DMSO-d6) δ
7.97 (1H, t, J = 6.0 Hz, NH-10), 7.543 (2H, s, H-14 and H-18), 5.257
(2H, s, OCH2-Ph), 4.71 (1H, t, J = 6.0 Hz, H-12), 3.24 (1H, m, H-11),
2.80 (6H, s, 2 × NCH3); (+)-LRESIMS m/z 970, 972, 974, 976 (in
the ratio 1:3:3:1).
1
(R)-MTPA ester of 17b (23b): H NMR (600 MHz, DMSO-d6) δ
7.98 (1H, t, J = 6.0 Hz, NH-10), 7.535 (2H, s, H-14 and H-18), 5.261
(2H, s, OCH2-Ph), 4.71 (1H, t, J = 6.0 Hz, H-12), 3.25 (1H, m, H-11),
2.79 (6H, s, 2 × NCH3); (+)-LRESIMS m/z 970, 972, 974, 976 (in
the ratio 1:3:3:1).
(E)-Acetophenone oxime (19): A suspension of acetophenone (18)
(200 μL, 1.7 mmol), hydroxylamine hydrochloride (419 mg, 6 mmol),
and KOAc (410 mg, 4.2 mmol) in 80% aqueous EtOH (35.0 mL) was
heated gently to reflux for 40 min. Following cooling to rt, the solvent
was removed in vacuo and the residue purified by flash chromatog-
raphy (9:1 hexane/EtOAc eluent) to provide 19 (218 mg, 95% yield):
IR (film) νmax 3220, 2920, 1447, 1305, 1008, 928, 764 cm−1; 1H NMR
(500 MHz, CDCl3) δ 9.51 (1H, brs), 7.65 (2H, m), 7.41 (3H, m),
2.33 (3H, s); 13C NMR (125 MHz, CDCl3) δ 156.1 (C), 136.7 (C),
129.4 (C), 128.7 (2 × CH), 126.2 (2 × CH), 12.5 (CH3);
(+)-LRESIMS m/z 136.
ASSOCIATED CONTENT
■
(E)-Acetophenone O-benzyl oxime (20). Benzyl bromide (2.1 μL,
0.0178 mmol) was added to the solution of 19 (2 mg, 0.0148 mmol)
and KOH (40 mg) in dry DMF (600 μL). The reaction mixture was
stirred for 2 h at rt. Brine (1 mL) was then added to the reaction
followed by extraction with EtOAc (6 mL). The organic layer was
washed three times with brine (0.5 mL), and the solvent was removed
under dry nitrogen. The residue was purified by flash chromatography
(100% hexane eluent) to provide 20 (3 mg, 90% yield): IR (film) νmax
S
* Supporting Information
1H and 13C NMR spectra for 1, 2, and other synthetic
compounds and also a photograph of the sponge Pseudoceratina
verrucosa are available free of charge via the Internet at http://
pubs.acs.org.
1
2929, 1455, 1378, 1304, 1001, 919, 772 cm−1; H NMR (500 MHz,
AUTHOR INFORMATION
■
CDCl3) δ 7.65 (2H, m), 7.43 (2H, d, J = 7.5 Hz), 7.36 (5H, m), 7.31
(1H, m), 5.25 (2H, s), 2.28 (3H, s); 13C NMR (125 MHz, CDCl3) δ
155.1 (C), 138.3 (C), 136.8 (C), 129.2 (CH), 128.5 (4 × CH), 128.3
(2 × CH), 127.9 (CH), 126.2 (2 × CH), 76.3 (CH2), 13.0 (CH3);
(+)-LRESIMS m/z 226.
Corresponding Author
*Tel: +61-7-3735-6009. Fax: +61-7-3735-6001. E-mail: r.
quinn@griffith.edu.au.
Notes
O-Benzylaplysamine 7 (21). Benzyl bromide (0.64 μL, 0.0054
mmol) was added to the solution of 2 (3 mg, 0.0045 mmol) and KOH
(40 mg) in dry DMF (600 μL). The reaction mixture was stirred for 1
h at rt and then stopped by adding 1 mL of brine followed by
extraction with EtOAc (6 mL). The organic layer was washed three
times with brine (0.5 mL), and the solvent was then removed under
dry nitrogen. The residue was purified by RP-HPLC (MeOH, H2O) to
yield compound 21 (0.6 mg, 18% yield) in a neutral form: [α]26D +7.2
(c 0.04, MeOH, neutral form); [α]26D +5.8 (c 0.04, MeOH, TFA salt);
1H NMR (600 MHz, DMSO-d6) δ 7.90 (1H, t, J = 6.0 Hz, NH-10),
7.54 (2H, s, H-14 and H-18), 7.33−7.36 (6H, m, H-2 and H-Ph), 7.06
(1H, dd, J = 8.4, 1.8 Hz, H-6), 6.97 (1H, d, J = 8.4 Hz, H-5), 5.71 (1H,
d, J = 4.8 Hz, 12-OH), 5.25 (2H, s, OCH2-Ph), 4.67 (1H, dd, J = 6.0,
5.4 Hz, H-12), 3.95 (2H, t, J = 6.0 Hz, H-20), 3.80 (3H, s, OCH3),
3.72 (2H, s, H-7), H-11 and H-22 obscured by H2O peak, 2.15 (6H, s,
2 × NCH3), 1.91 (2H, m, H-21); (+)-LRESIMS m/z 754, 756, 758,
760 (in the ratio 1:3:3:1); (+)-HRESIMS m/z 754.0151 [M + H]+
(calcd for [C30H3579Br3N3O5]+, 754.0121, Δ 4.0 ppm).
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
One of the authors (T. D.T.) acknowledges Education Australia
Ltd. for the provision of the “EAL Postgraduate Research
Student Mobility Scholarships”. We thank Dr. H. T. Vu for
acquiring the HRESIMS measurement and Ms. B. Aldred for
provision of the cell lines. We also thank the reviewers of this
paper for valuable contributions to the assignment of the
absolute configuration.
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