Psammaplin A as a general activator of cell-based signaling assays via HDAC inhibition and studies on some bromotyrosine derivatives

Article abstract of DOI:10.1016/j.bmc.2008.10.077

The Wnt signaling pathway regulates cell growth and development in metazoans, and is therefore of interest for drug discovery. By screening a library of 5808 pre-fractionated marine extracts in a cell-based Wnt signaling assay, several signaling activator

Full text of DOI:10.1016/j.bmc.2008.10.077

Bioorganic & Medicinal Chemistry 17 (2009) 2189–2198
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Psammaplin A as a general activator of cell-based signaling assays via
HDAC inhibition and studies on some bromotyrosine derivatives
Malcolm W. B. McCulloch a, Gary S. Coombs b, Nikhil Banerjee c, Tim S. Bugni a, Kendell M. Cannon c,
Mary Kay Harper a, Charles A. Veltri a, David M. Virshup b, Chris M. Ireland a,
*
a Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA
b Duke NUS Graduate Medical School, Singapore 169547, Singapore
c Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The Wnt signaling pathway regulates cell growth and development in metazoans, and is therefore of
interest for drug discovery. By screening a library of 5808 pre-fractionated marine extracts in a cell-based
Wnt signaling assay, several signaling activators and inhibitors were observed. LCMS-based fractionation
rapidly identified an active compound from Pseudoceratina purpurea as psammaplin A, a known HDAC
inhibitor. Other HDAC inhibitors similarly activated signaling in this assay, indicating HDAC inhibitors
will be identified through many cell-based reporter assays. In a large scale analysis of P. purpurea, three
previously undescribed bromotyrosine based natural products were identified; the structure of one of
these was confirmed by synthesis. Additionally, three other derivatives of psammaplin A were prepared:
a mixed disulfide and two sulfinate esters. Finally, evidence to support a structural reassignment of
psammaplin I from a sulfone to the isomeric sulfinate ester is presented.
Received 10 April 2008
Accepted 31 October 2008
Available online 5 November 2008
Keywords:
Wnt signaling
Luciferase
HDAC
Natural product library
LCMS
Ó 2008 Elsevier Ltd. All rights reserved.
Bromotyrosine
Psammaplin
N-Methyl glutathione
Disulfide exchange
Reductive methylation
Sulfinate ester
1. Introduction
tional groups and carbon–sulfur bonds.7 Subsequently, at least
nineteen members of this group of compounds have been isolated
Cell-based drug screens are valuable tools to identify novel
compounds that block or activate signaling pathways, and it is
important to understand what classes of molecules may be de-
tected in these assays. The Wnt signaling pathway regulates cell
growth and development in metazoans. Anomalies in the regula-
tion of Wnt signaling are associated with multiple diseases includ-
ing cancer.1 Small molecule modulators of the Wnt pathway are
therefore potential therapeutics.2
Bromotyrosine derivatives are a well-known group of marine
natural products that have been isolated from numerous sponges,
most of which belong to the order Verongida. Hundreds of these
compounds are known, and they have been extensively reviewed.3
Psammaplin A (1)4–6 was the first identified member of the sub-
group of bromotyrosine derivatives that contain both oxime func-
from marine organisms.3
Herein is outlined a library screen of 5808 pre-fractionated
marine natural product extracts using a cell-based Wnt signal-
ing assay. This Wnt assay uses a cell line we termed STF3a
that was engineered to express luciferase, from an integrated
reporter construct, in response to autocrine Wnt signaling.
These cells facilitate the identification of both activators and
inhibitors of Wnt signaling. The current report focuses on
chemical investigation of bromotyrosine derivatives following
the identification of 1 as a potent activator of signaling in
the STF3a cell line. Using an LCMS-based fractionation meth-
od,8 1 was rapidly identified from an archive HP20SS fraction
of the sponge Pseudoceratina purpurea (Carter, 1880). Mechanis-
tically, 1 appears to activate signaling in the cell-based assay
through inhibition of histone deacetylases (HDACs) rather than
through a specific Wnt pathway. HDAC inhibitors in general
may score as hits in cell-based assays dependent on integrated
reporters.
* Corresponding author. Tel.:+1 801 581 8305; fax: +1 801 585 6208.
E-mail address: cireland@pharm.utah.edu (C.M. Ireland).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.077

2190
M. W. B. McCulloch et al. / Bioorg. Med. Chem. 17 (2009) 2189–2198
integrated reporter, in response to autocrine Wnt signaling. Thus,
OH
OH
OH
OH
11
this STF3a based assay will detect compounds that increase or
decrease transcription of the integrated luciferase reporter.
Using these STF3a cells, a pre-fractionated marine natural prod-
ucts library was screened. This library was generated by fraction-
ation of marine invertebrate extracts on Diaion HP20SS resin. In
the initial screen for Wnt signaling inhibitors, we noted that there
were library fractions that activated expression of the luciferase
reporter. Analysis of the data revealed 46 wells with activity
between 2- and 55-fold higher than the control (Fig. 1), along with
a number of wells that inhibited signaling. The current report
focuses on follow up investigations arising from one of these acti-
vators (well 4WA7). Full details of the STF3a cell line and assay will
be described in a separate publication.
7
Br
9 O
Br
6
Br
8
9
5 O
13
O
4
8
6
3
S
2
S
7
5
3
1
N
H
OH
N
2
H
N
N
N
O
HO
HO
1
2
OH
11
1'
2'
Br
HO
(S)
R
13
9 O
N
H
3'
8
3
6
S
O
4'
7
5
N
H
S
8'
5'
2
N
NH
2.2. Rapid identification of psammaplin A as a signaling
modulator in the STF3a cell line
HO
7'(R)
O
HN6'
10'
9'
O
Like many natural product libraries, the pre-fractionated library
used in the current screen contains mixtures of compounds. In
drug screening programs the challenges of rapid compound purifi-
cation, identification and dereplication associated with natural
product mixtures have been seen as disadvantageous vis-à-vis syn-
thetic compound libraries.12 Nonetheless, the unique chemical
space occupied by natural products provides a useful complement
to synthetic libraries.13 To circumvent challenges associated with
natural products based drug discovery a variety of different tech-
nologies have been developed.12,14,15 This report highlights an
LCMS-based fractionation method to expedite natural products
based drug discovery,8 demonstrating a rapid identification of
psammaplin A (1) from an active well identified in the library
screen.
OH
3 R=Me, 5 R=H
OH
1'
Br
O
HO
O
(S)
Me
3'
2'
N
H
S
O
4'
N
H
S
5'
N
NH
8'
HO
7'
(R)
O
HO6'
4
One of the most potent activators from the library screen
(49-fold), obtained from the sponge P. purpurea, was selected for
further analysis. Our automated LCMS fractionation protocol cou-
ples chromatography of library HP20SS fractions on a monolithic
C-18 HPLC column with the online collection of accurate mass data
on a Q-Tof mass spectrometer.8 A 1 mg sample of archive material
was subjected to automated LCMS fractionation, generating 20
fractions in a 96-well plate. The biologically active fraction, well
number six, eluted between 8 and 9 min. A number of brominated
ions were observed in the MS of this well. On the basis of the tax-
onomic identification of the source organism and an ion at m/z
662.9578 [M+H]+, the active compound was identified as psamma-
plin A (1) (calcd for C22H2579Br2N4O6S2, 662.9582). The well was
then analyzed by NMR (600 MHz, cryoprobe), and the data were
identical to those previously published for 1.16,17 Using this meth-
od the active compound was identified in less than one week.
OH
OH
11
R
Br
Br
O
13
9
O
S
O
O
S Me
O
8
3
3
6
7
5
OMe
N
H
N
H
2
2
N
N
HO
HO
6 R=H, 7 R=Br
8
In follow up chemistry studies looking for congeners of 1 three
compounds (2, 3, 4) were isolated from P. purpurea. The acid 2
has been synthesized,9 but has not been reported as a natural prod-
uct. The mixed disulfides 3 and 4 are novel. The structure of 3 was
confirmed by semi-synthesis from 1 and N-methyl glutathione.
Additionally, three other semi-synthetic derivatives of psammaplin
A (1) were prepared: the mixed disulfide 5, and the two sulfinate
esters 6 and 7. Furthermore, a structural revision is proposed for
psammaplin I10 from the sulfone 8 to the isomeric sulfinate ester 6.
2. Results and discussion
2.1. Wnt signaling assay and the library screen
In order to identify compounds that inhibit Wnt signaling at any
point in the signaling process, an assay was developed which uti-
lized a new cell line (STF3a). The STF3a cell line was generated
from the HEK293-based ‘supertopflash’ reporter cell line (STF)
developed for Wnt signaling studies by Xu et al.11 To generate
the STF3a cell line the STF cell line was modified to stably express
high levels of murine Wnt3a. The STF3a cell line constitutively
expresses high levels of luciferase, from the b-catenin-responsive
Figure 1. Wnt signaling activators identified from the library screen.

M. W. B. McCulloch et al. / Bioorg. Med. Chem. 17 (2009) 2189–2198
2191
differences to that reported for the synthetic compound, and this
was most pronounced on carbons adjacent to atoms subject to
hydrogen bonding effects. Upon acidification of 2 with HCl the 13C
chemical shifts matched the synthetic data. This suggested the
natural product was isolated as a salt, and not surprisingly that the
chemical shifts of 2 are pH dependant.
OH
Br
O
= Psub
S
N
H
N
HO
2.4.2. Mixed disulfides 3 and 4
Figure 2. Psammaplin A monomeric subunit.
The novel mixed disulfides 3 and 4 were isolated as amorphous
white NH4OAc salts,21 and exhibited UV chromophores (kmax
282 nm) cognate to psammaplin A (1). The 1H NMR spectra of 3
and 4 (Supplementary data Figs. S4 and S5) were similar, with 3
differing by one additional methylene multiplet (d 3.86). Prelimin-
ary analysis of these 1H NMR spectra suggested both compounds
were derivatives of the psammaplin A (1) monomeric subunit
(Psub, Fig. 2), with extra peptide-like functionality. Given the rela-
tively large molecular weights of 3 and 4, the comparative paucity
of material and the corollary that sensitive and time consuming
NMR experiments were necessary to obtain adequate 2D and 13C
data sets, MS and MSMS fragment analysis was relied upon heavily
in initial structural elucidation efforts.
2.3. Putative mechanism of Wnt signaling activation by
psammaplin A in the STF3a cell line: a general effect of HDAC
inhibitors
Upon considering the mechanism of Wnt signaling activation in
the STF3a cell line by psammaplin A (1), it was noted that 1 is a
known nanomolar inhibitor of HDACs.10 Therefore, other known
HDAC inhibitors were tested in the STF3a assay. Suberoylanilide
hydroxamic acid (SAHA),18 trichostatin A,19 and MS-27520 were
all found to activate Wnt signaling in a similar manner to 1 (Sup-
plementary data Figs. S1–S3). This effect is presumably caused by
a general increase in transcription activity by HDAC inhibition.
Consistent with this, 1 also activated transcription from an unre-
lated integrated reporter construct in NPSR-I107 cells.
The ESI-MS spectrum of 3 revealed a mono-brominated molec-
ular ion cluster (m/z = 652/654, [M+H]+). The approach at deriving
a molecular formula utilized a bottom-up fragment analysis of the
ESI-MSMS spectrum generated from the parent brominated cluster
(Table 1 and Supplementary data Fig. S6). This yielded a molecular
formula of C22H30N5O9BrS2 that was confirmed by high-resolution
2.4. Scale up isolation of bromotyrosine derivatives from P.
purpurea
FT-MS analysis (m/z 652.07467 [M+H]+,
D 0.015 ppm). The ESI-
MSMS spectrum showed a number of fragments providing key
structural data, including peaks supporting a disulfide derived
from Psub (Table 1, entries 2 and 3), a glycine residue (Table 1, en-
try 6) and an N-methyl group (Table 1, entry 7).
In a search for analogs of 1 and to obtain more material for
biological evaluation, a scale up extraction of P. purpurea was con-
ducted. The MeOH extract of the sponge was chromatographed on
HP20SS resin using a gradient of 100% H2O to 100% MeOH.
Psammaplin A was obtained by RP-HPLC of the 100% MeOH frac-
tion. The polar metabolites 2, 3, and 4 were isolated from the
25% MeOH fraction, by LH20 followed by RP-HPLC.
The structural elucidation of 3 was completed by analysis of
gCOSY, TOCSY, gHSQC and gHMBC data. NMR signals (Table 3) con-
sistent with cysteine, glutamate and glycine residues, along with
Psub and an N-methyl group were present. The configuration about
the oxime functionality in Psub was assigned as E based on the 13
C
2.4.1. Compound 2
shift of the benzylic carbon (d 28.5), following the literature prec-
edent.6 The N-methyl glutathione half of the disulfide was pieced
together from the amino acid residues based on the gHMBC spec-
trum; see Figure 3 for key correlations. The overall structure was
finalized by linking Psub and the N-methyl glutathione substruc-
ture, necessitating a disulfide bond between the Psub sulfur and
the cysteine sulfur. This structure was consistent with the MSMS
data (Fig. 4).
Compound 2 was obtained as an off-white amorphous solid. A
mono-brominated cluster in the (ꢀ)ESI-MS spectrum supported a
molecular formula of C9H8NO4Br (m/z 271.9558 [MꢀH]ꢀ,
D
0.0 ppm). Comparison of the NMR data observed for 2 and psamma-
plin A (1) showed analogous chemical shifts and J coupling, with the
exception that 2 lacked the CH2CH2 functionality. These data, along
with the molecular formula, suggested the simplified parent acid
structure for 2, which was supported by gHSQC, gHMBC, and gCOSY
experiments. Compound 2 has not previously been reported as a
natural product; however, 2 is known synthetically9 and its exis-
tence as a possible biosynthetic precursor of 1 has been specu-
lated.10,16 The 13C NMR data of the natural product showed some
The structural assignment of 3 was initially supported by a syn-
thesis of the analog 5 from 1 and
structure of 3 was unambiguously confirmed by its synthesis from
1 and N-methyl -glutathione (see Section 2.5). The absolute con-
figuration of each stereocenter in 3 was assigned to match that
L-glutathione. Subsequently the
L
Table 1
MSMSa analysis of 3: selected key fragments.
Entry
Fragment m/z
Assignment
Interpretation
Calcd mass (D, mmu)
1
2
3
4
5
6
7
8
177.0335
330.9753
362.9465
405.9914
509.0176
577.0367
621.0319
652.0747
[C5H9N2O3S]+
Gly-Cys derived fragment
Psammaplin A monomeric subunit (Psub
Disulfide functionality
177.0334 (0.1)
330.9752 (0.1)
+
[Psub
]
)
[PsubS]+
362.9473 (ꢀ0.8)
405.9895 (1.9)
509.0164 (1.2)
577.0426 (ꢀ5.9)b
621.0325 (ꢀ0.6)
[PsubSC2H5N]+
[PsubSC5H10N2O3]+
[MꢀGly]+
Psammaplin A monomer + a fragment of a Cys or homo-Cys derivative
Loss of N-methyl Asp derivative from [M]+
Loss of Gly from [M]+
[MꢀCH4N]+
Loss of N-methyl from [M]+
Set as lock mass, based on ESI-MS
[M+H]+ = C22H30N5O9BrS2
a
Measured on a Micromass Q-Tof mass spectrometer.
b
The error in this peak (10 ppm) is enhanced by a contribution from a smaller overlapping fragment. A minor peak observed at m/z 575.0207 is assigned as
C20H2479BrN4O7S2 (calcd 575.0270;
D
ꢀ6.3 mmu) and the 81Br isotopic ion of this fragment (calcd 577.0249) overlaps the [MꢀGly]+ fragment. An analogous signal in the
MSMS spectrum of the homolog 4 (see Table 2, entry 7), not distorted by overlapping peaks, shows a good match to the assigned formula (577.0433,
D 0.7).

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M. W. B. McCulloch et al. / Bioorg. Med. Chem. 17 (2009) 2189–2198
products that contain the N-methyl glutathione substructure. Bio-
OH
11
synthetically 3 and 4 are likely derived by disulfide exchange be-
tween 1 and oxidized N-methyl glutathione (or derivatives).
Several other mixed disulfide derivatives of 1 have been isolated
from sponges.4,10,16,17,23,24 These previously reported compounds
all contain –CH2CH2SSCH2CH2– functional groups, which contrasts
with the disulfide functionality in 3 and 4.
Br
9 O
HO
O
Me
3'
2'
N
H
8
3
S
O
7
4'
5
N
H
S
5'
2
8'
N
NH
HO
7'
HN
O
O
10'
2.5. Semi-synthesis of the mixed disulfides 3 and 5
OH
Syntheses of 3 and its de-methylated primary amine analog 5
were achieved by disulfide exchange chemistry. The latter com-
pound was readily prepared as a model compound from commer-
3
Figure 3. Selected gHMBC correlations (600 MHz) observed for 3.
cially available oxidized L-glutathione (9) and psammaplin A (1).
Based on a modification of a disulfide exchange method,25 1 and
9 were coupled in the presence of dithiothreitol (DTT) to give 5
(Scheme 1). MSMS and NMR data for 5 supported the structural
assignments of the natural products 3 and 4. The MSMS spectrum
of 5 (Supplementary data Fig. S8) displayed all the same peaks
observed in the MSMS spectrum of 3 other than those attributable
to fragments containing the N-methyl moiety (though analogous
peaks to these were observed). The NMR spectra for 3 and 5
(Supplementary data Figs. S9 and S10) were similar, with the major
difference being the chemical shift of the signals observed about
C-20 (Table 3).
of naturally occurring
between the optical rotation of the natural product 3 ([
L
-glutathione based on the close similarities
a
]
D
ꢀ21°,
MeOH), with the semi-synthetic compounds 3 ([
a]
ꢀ33°, MeOH)
D
and 5 ([
a]
ꢀ22o, MeOH).
D
The structural elucidation of 4 was achieved by reference to 3.
The molecular formula of 4 was similarly derived by bottom-up
fragment analysis of the ESI-MSMS spectrum (Table 2 and Supple-
mentary data Fig. S7). This yielded a molecular formula of
C20H27N4O8BrS2 that was confirmed by high-resolution FT-MS
analysis (m/z 595.05347 [M+H]+,
D 0.47 ppm). The molecular
While 5 was easily prepared from 1 and commercially available
9, the synthesis of 3 required access to an appropriate N-methyl
derivative of glutathione as a building block. Our approach to this
key precursor was based in part on a literature preparation of an
N-methyl glutathione containing leukotriene, which utilized a
reductive methylation strategy.26 In this direction 9 was esteri-
fied27 to give 10,28 which was then converted to the imine 11 by
condensation with 2-nitrobenzaldehyde (Scheme 2). Initial at-
tempts to convert this imine directly into the tertiary amine 12,
following a slight modification of a literature single-pot strategy
(MeOH, NaBH3CN, paraformaldehyde),29 were unsuccessful, pre-
sumably due to the low solubility of 11 in methanol. A reduction
of 11 using NaBH3CN in acetonitrile/DMSO26 successfully gave
the secondary amine 13, which was then reductively methylated
with paraformaldehyde and NaBH3CN to give 12. Subsequently,
an efficient one-pot reduction of 11 to 12 was achieved in acetoni-
trile/DMSO. In this case the thiocyanate derivative 14 was obtained
as a minor side product.
The synthesis of 3 was completed by removal of the protecting
groups on the amine and carboxylic acid functionalities, followed
by a disulfide exchange reaction (Scheme 3). First, photolytic cleav-
age of the 2-nitrobenzyl group in 12 led to N-methyl glutathione
tetra ethyl ester (15) in good yield. Compound 15 was then treated
with 5.1 equiv of LiOH (THF/H2O) to hydrolyze the ester moieties.
In the same pot, the reaction was buffered (PBS) and then DTT and
1 in MeOH were added. After reacting overnight, analytical HPLC of
this disulfide exchange reaction revealed a peak with an identical
retention time and chromophore to the 3 isolated from P. purpurea.
formula of 4 represents a difference of a glycine residue from 3.
The MSMS and NMR data (Table 3) of 4 were consistent with this
compound being a homolog of 3, lacking the glycine. The optical
rotations of 3 and 4 were similar suggesting they possess identical
absolute stereochemical configurations, as would be expected from
a biosynthetic perspective.
The mixed disulfides 3 and 4 possess some interesting struc-
tural features. We are only aware of one other report of marine
derived small molecules (two analogs) that contain the glutathione
substructure.22 Furthermore, we are unaware of any other natural
652 (M+H)+
621.032
OH
Br
O
HO
O
Me
N
H
S
O
N
H
S
N
NH
O
+2H
HO
577.043
330.975
HN
OH
O
509.016
362.947
3
Figure 4. Selected MSMS fragments for 3.
Table 2
MSMSa analysis of 4: selected key fragments.
Entry
Fragment m/z
Assignment
Interpretation
Calcd mass (D, mmu)
1
2
3
4
5
6
7
120.0202
330.9765
362.9484
405.9911
451.9975
564.0140
577.0433
[C3H6NO2S]+
Cys derived fragment
Psammaplin A monomeric subunit (Psub
Disulfide functionality
120.0119 (8.3)
330.9752 (1.3)
362.9473 (1.1)
405.9895 (1.6)
451.9949 (2.6)
564.0110 (3.0)
577.0426 (0.7)
+
[Psub
]
)
[PsubS]+
[PsubSC2H5N]+
[PsubSC3H7NO2]+
[MꢀCH4N]+
[MꢀOH]+
Psammaplin A monomer + a fragment of a Cys or homo-Cys derivative
Loss of N-methyl Asp derivative from [M]+
Loss of N-methyl from [M]+
Loss of water from [M+H]+
a
Measured on a Micromass Q-Tof mass spectrometer.

M. W. B. McCulloch et al. / Bioorg. Med. Chem. 17 (2009) 2189–2198
2193
Table 3
NMR dataa for 3, 4 and 5.
Position
3b
4c
5e
dH (m, J in Hz)
dC
dH (m, J in Hz)
dC
dH (m, J in Hz)
dC
2
2.84 (m)
38.3
2.80 (t, 6.1)
37.7
2.84 (d, 6.9)
38.7
3
3.54 (m)
39.9
3.39 (m)
38.8
3.53 (m)
39.9
4
—
—
8.02 (m)
—
—
—
5
6
7
—
—
166.0
153.5
28.5
—
—
164.1
152.3
28.2
—
—
166.1
153.2
28.8
3.78 (s)
3.68 (s)
3.78 (s)
8
9
—
130.8
134.6
110.6
153.9
116.9
130.2
—
129.3
133.4
109.5
153.1
116.9
129.7
—
130.7
134.6
110.6
153.9
117.2
130.5
7.36 (d, 2.0)
—
—
6.76 (d, 8.3)
7.06 (dd, 2.0, 8.3)
7.28 (d, 1.8)
—
—
6.86 (d, 8.0)
6.98 (dd, 1.8, 8.0)
7.35 (d, 2.0)
—
—
6.77 (d, 8.3)
7.05 (dd, 2.0, 8.3)
10
11
12
13
Glu
10
—
173.1
64.7
32.3
26.0
—
173.0d
63.0
32.3
—
174.4
55.7
—
20
3.52 (m)
2.66 (s)
2.02 (m)
2.20 (m)
2.55 (m)
—
3.28 (m)
2.48 (s)
1.94 (m)
3.66 (m)
—
2.14 (m)
N—CH3
30
26.3
28.0
40
50
32.5
175.4
2.30 (m)
—
32.6
2.54 (m)
—
33.2
175.5
175.7d
Cys
N-H
60
—
v
—
8.15 (m)
v
4.24 (m)
2.98 (m)
3.17 (m)
—
—
—
—
—
173.0
54.1
40.8
171.1d
53.7
42.8
172.4
54.3
41.3
70
4.70 (dd, 4.2, 10.0)
2.91 (dd, 10.0, 14.0)
3.26 (dd, 4.2, 14.0)
—
4.70 (dd, 4.5, 9.7)
2.93 (dd, 9.7, 14)
3.25 (dd, 4.5, 14)
—
80
N-H
Gly
90
100
—
—
—
—
173.4
42.8
—
—
—
—
—
176.2
44.6
3.82 (d, 17.0)
3.90 (d, 17.0)
1.93 (s)
3.77 (m)
NH4OAc
176.0, 22.2
1.95 (s)
172.1, 19.6
1.95 (s)
177.4, 22.2
a
Signals were assigned using a combination of gHSQC, gHMBC and gCOSY experiments.
CD3OD: 1H and 13C spectra were recorded at 600 and 150 MHz, respectively.
DMSO-d6: 1H and 13C spectra were recorded at 500 and 125 MHz, respectively.
b
c
d
e
These carbons were observed in a separate experiment; at 125 MHz in a Shigemi tube in DMSO-d6/CD3CN/H2O (5:2.5:1).
CD3OD: 1H and 13C spectra were recorded at 500 and 125 MHz, respectively.
OH
OH
HO
O
Br
O
Br
O
HO
O
NH2
NH2
O
S
S
O
S
N
H
N
H
S
2
2
NH
O
N
N
NH
O
HO
HO
HN
OH
HN
OH
O
O
1
9
5
Scheme 1. Semi-synthesis of 5. Reagents and conditions: DTT, K2CO3, THF/MeOH/PBS (pH 8.4), 22 h, 46% from 1 (unoptimized).
Purification of this material by semi-preparative HPLC gave semi-
synthetic 3, which exhibited spectroscopic data consistent with
that recorded for the natural product.
(11%) and its di-brominated analog 7 (14%) (Scheme 4). Although
yields are low and unoptimized sufficient material was obtained
for full compound characterization.
Psammaplin I is a bromotyrosine derivative reported from the
sponge P. purpurea that was assigned as the terminal methyl sul-
fone 8,10 and represents a structural isomer of the methyl sulfinate
ester 6. The 13C and 1H NMR spectra of 6 (Supplementary data Figs.
S11 and S12) are identical to those reported for psammaplin I. On
the basis of spectroscopic equivalence, the above synthesis and
additional data discussed below, we propose a structural revision
of psammaplin I from 8 to 6.
2.6. Semi-synthetic sulfinate ester derivatives of 1 and
proposed structural revision of psammaplin I
To develop structure–activity relationships and provide analogs
of psammaplin A (1), two semi-synthetic methyl sulfinate ester
derivatives of 1 were prepared by oxidative methanolysis. Follow-
ing a modified version of Brownbridge’s method,30 1 was treated
with an excess of N-bromosuccinimide (NBS) in methanol. HPLC
purification led to the isolation of the methyl sulfinate ester 6
The sulfone functionality in psammaplin I was originally as-
signed on the basis of an infrared absorbance (IR) at 1284 cmꢀ1

2194
M. W. B. McCulloch et al. / Bioorg. Med. Chem. 17 (2009) 2189–2198
1'
EtO
O
1'
EtO
O
N
1'
EtO
O
NO2
NO2
(S)
3' 2'
4'
NH2
3'
2'
3' 2'
4'
N
H
O
O
4'
O
S
8'
5'
S
8'
5'
S
8'
5'
(a)
(b)
(c-d)
NH
2
NH
2
NH
2
12
7'
(R)
O
7'
7'
HN6'
HN6'
O
HN6'
O
O
10'
9'
O
10'
9'
O
10'
9'
.2HCl
OEt
OEt
OEt
10
11
13
1'
EtO
O
1'
EtO
O
NO2
NO2
3' 2'
4'
N
Me
N
3' 2'
4'
N
Me
O
O
S
8'
5'
(b-d, one pot)
S
8'
5'
NH
NH
O
2
7'
11
7'
HN6'
O
HN6'
+
O
10'
9'
O
10'
9'
OEt
OEt
12
14
Scheme 2. Reductive alkylation reactions. Reagents and conditions: (a) NaOH (2.1 equiv), 2-nitrobenzaldehdyde (2.2 equiv), MeOH/H2O, overnight, 83%; (b) NaBH3CN, ACN/
DMSO, AcOH(cat), 3–16 h, 13 71%; (c) paraformaldehyde (excess), ACN/DMSO, 8–16 h; (d) NaBH3CN, AcOH(cat), overnight, 12 66–84%, 14 6%.
1'
1'
EtO
O
EtO
O
OH
NO2
Br
O
HO
O
3' 2'
4'
3' 2'
4'
N
Me
NH
Me
Me
O
O
N
H
S
8'
S
8'
5'
5'
S
O
(a)
(b-c)
NH
O
NH
O
N
H
S
2
2
7'
7'
N
NH
O
HN6'
10'
9'
HN6'
10'
9'
HO
O
O
HN
OH
O
OEt
OEt
12
15
3
Scheme 3. Photolysis of 12 and the semi-synthesis of 3. Reagents and conditions: (a) 0.5 mmol in ACN, h
(pH 8.4), MeOH, 1 (6 equiv), DTT, overnight, 38% over two steps.
m 360 nm, 2 h, 91%; (b) LiOH (5.1 equiv), THF/H2O, overnight; (c) PBS
and the 13C NMR shift of the methyl group at dc 54.1.10,31 Most
anol; comparable peaks of significant intensity are not observed
from sulfone isomers.35 Additional evidence supporting the struc-
tural assignment of 6 as a methyl sulfinate ester was obtained by
FT-ESI-MSMS analysis (Fig. 5). The molecular ion from 6 was frag-
mented and formed a base peak at m/z 347, resulting from a loss
known psammaplin analogs reportedly exhibit IR absorbances in
the range 1255–1290 cmꢀ1 10,16,32
;
for example, psammaplin E
(16) and F (17) both show peaks at 1287 cmꢀ1 10
.
Thus, the IR
absorbance at 1284 cmꢀ1 in psammaplin I can not be reliably
attributed to a sulfone moiety. Also, the 13C NMR shift at dc 54.1
is not consistent with a terminal methyl sulfone substructure, such
methyl carbons are usually observed in the range of dc 37–45.33 We
conducted DFT calculations34 modeling the 13C NMR shifts of 8 and
6 (Table 4). These showed a much better fit to the observed data for
the sulfinate ester structure.
of methanol (D 32.0264, CH3OH calcd 32.0262). This fragmentation
pattern would not be expected for the sulfone 8; rather a peak aris-
ing from a loss of the terminal methyl group would be expected.
3. Conclusions
In conclusion, we screened a pre-fractionated marine natural
product library in a luciferase based Wnt signaling assay. This assay
utilized the STF3a cell line, which constitutively expressed lucifer-
ase in response to autocrine Wnt signaling. Several activators and
inhibitors of signaling were identified from the library. Psammaplin
A (1), a known HDAC inhibitor from P. purpurea, was rapidly iden-
tified as an activator of the reporter construct in the STF3a cell line;
other known HDAC inhibitors elicited similar activations of signal-
ing. In follow up chemical studies three new bromotyrosine based
natural products (2, 3 and 4) were identified. The structures of 3
and 4 were supported by a synthesis of the former compound and
of 5. Finally, a structural reassignment of psammaplin I from the
sulfone 8 to the isomeric sulfinate ester 6, supported by synthetic
and spectroscopic studies, was presented.
OH
Br
O
O
S
S
R
N
H
N
H
N
O
HO
16 R=NH2, 17 R=OH
Methyl sulfinate esters and their isomeric terminal methyl sulf-
ones can be distinguished by mass spectrometry. Methyl sulfinate
esters fragment to give intense peaks resulting from a loss of meth-

M. W. B. McCulloch et al. / Bioorg. Med. Chem. 17 (2009) 2189–2198
2195
OH
OH
OH
Br
O
Br
Br
O
Br
O
O
S
O
S
+
S
N
H
OMe
OMe
N
H
N
H
2
N
N
N
HO
HO
HO
1
6
7
Scheme 4. Oxidative methanolysis of 1. Reagents and conditions: NBS (2.4 equiv), MeOH, 0 °C ? rt, 40 min, 6 11%, 7 14% (unoptimized).
ear gradient from 10% to 100% over 16 min and held at 100% ACN
for 4.5 min, returning to starting conditions over 0.5 min (total run
time 22.0 min). A solvent delay was set on the mass spectrometer
for 1.8 min to allow the DMSO slug to elute prior to acquiring MS
data. At 2 min, the fraction collector began collecting one min frac-
tions for a total of 20 fractions. The active fraction was number six
and eluted between 8 and 9 min. MS gave [M+H]+ m/z 662.9578,
calcd for psammaplin A (1) C22H2579Br2N4O6S2, 662.9582. Leucine
enkephalin was used for lock mass and set at m/z 556.2771 for
the [M+H]+.
Table 4
Calculated NMR shifts for 6 and 8 compared to observed data.
Position
Synthetic product
6
8
Observed dC
Calcd dC
D
d
Calcd dC
Dd
CH3
C-2
C-3
55.7
57.4
33.5
53.3
59.9
34.8
ꢀ2.4
2.5
1.3
43.8
58.9
38.4
ꢀ11.9
1.5
4.9
4. Experimental
4.3. HDAC inhibitor effect on Wnt signaling
4.1. General procedures
HDAC inhibitors were tested by plating 20,000 STF3a cells/well
Optical rotations were measured on a Perkin-Elmer Model 343
Digital Polarimeter. Infrared spectra were recorded on NaCl plates,
using a JASCO FTIR-420 spectrophotometer. UV spectra were ac-
quired on a Hewlett–Packard 8452A spectrophotometer. ESI-MS
spectra were obtained using a Q-Tof Micromass spectrometer.
FT-MS spectra were obtained using a ThermoFinnigan LTQ-FT mass
spectrometer. NMR spectra were obtained either on a Varian Mer-
cury spectrometer operating at 400 and 100 MHz for 1H and 13C,
respectively, or on Varian INOVA spectrometers operating at 500/
600 and 125/150 MHz for 1H and 13C, respectively. Chemical shifts
are reported in ppm and were referenced to residual solvent sig-
nals: CD3OD (dH 3.30; dC 49.15), DMSO-d6 (dH 2.50; dC 40.3), CD3CN
(dH 1.94; dC 1.32).
in 96-well tissue culture plates pretreated with poly-
Plates were incubated overnight at 37 °C in 5% CO2. Just prior to
adding compounds, media was replaced with 200 L of fresh med-
ia containing HDAC inhibitors in a range from 30 M to 10 nM and
incubated overnight. Cells were washed once with 200 L of cold
PBS and then lysed in 20 L Cell Culture Lysis Reagent (Promega)
and complete protease inhibitor cocktail without EDTA (Roche).
Lysates were mixed with 60 L of 0.15 mg/mL -luciferin in
L-lysine.
l
l
l
l
l
D
25 mM gly–gly, pH 7.8, 15 mM MgSO4, and 4 mM EGTA. Lumines-
cence data were obtained using a Veritas microplate luminometer
(Turner Biosystems).
After luminescence data were collected, lysates were mixed
with 60 lL of substrate solution for a coupled assay to detect lac-
tate dehydrogenase activity. Light absorbance at 490 nm was read
every 10 s for 5 min, and slopes were plotted and used for normal-
ization of luminescence data. The substrate solution was made by
combining three separate component solutions. Component one:
4.2. LCMS-based analysis of library hit 4WA7
The preliminary LCMS fractionation of the active HP20SS frac-
tion (4WA7 = FJ04-4-36-F2), utilized a sample of archive material
(200 lL, 5.0 mg/mL). The HPLC method started with 10% ACN/
316 mM sodium
L-lactate in 10 mM Tris, pH 8.5; component two:
40 mM iodonitrotetrazolium chloride in DMSO; component three:
H2O (flow rate 1.5 mL/min) and held for one min, followed by a lin-
10 U/mL diaphorase, 325 lg/mL BSA, 12 mg/mL sucrose, and 3 mg/
mL NAD+ in PBS. Components one and three were frozen in 20 mL
aliquots at ꢀ20 °C. Component two was frozen in 2 mL aliquots at
ꢀ20 °C and was diluted in 18 mL of PBS before mixing with compo-
nents one and three for use.
+
H+
OH
Br
O
O
S
4.4. Collection, extraction and identification of P. purpurea
OMe
N
H
N
HO
The sponge P. purpurea (Carter, 1880) was collected by SCUBA
off the coast of Fiji in 2004 (S 17° 00.0640, E 178° 37.7050). A vou-
cher specimen (FJ04-4-36) is retained at the University of Utah.
A portion of the frozen sponge (300 g, wet weight) was broken
into pieces and extracted with MeOH (2ꢁ 700 mL), which was fil-
tered and concentrated in vacuo. The combined vacuum dried
extracts (15 g) were adsorbed onto diaion HP20SS resin (5 g) in a
minimal volume of MeOH. The resulting HP20SS slurry was con-
centrated in vacuo and then loaded on the top of a packed HP20SS
column (25 g resin, length 15 cm, radius 1.5 cm) pre-equilibrated
with H2O. The column was eluted with a gradient of H2O/MeOH
(four steps 100:0 ? 75:25 ? 50:50 ? 25:75 ? 0:100) followed
by 100% IPA. The 100% MeOH fraction contained fairly pure
FT-MSMS
Δ 32.0264
CH3OH Calcd 32.0262
OH
Br
+
O
S
O
N
H
N
HO
Figure 5. FT-MSMS of sulfinate ester 6 showed a loss of methanol.

2196
M. W. B. McCulloch et al. / Bioorg. Med. Chem. 17 (2009) 2189–2198
psammaplin A (1, 2.5 g), a sample of which was further purified by
RP-HPLC (Phenomenex, phenylhexyl, 5
8.07 (d, 8.0 Hz, ArH), 7.98 (d, 7.8 Hz, ArH), 7.82 (m, ArH), 7.74 m,
ArH), 4.60 (m, H-7), 4.15 (m, H-2), 4.12 (q, 7 Hz, CH2CH3), 4.05
(q, 7 Hz, CH2CH3), 3.80 (d, 5.7 Hz, H-10), 3.08 (dd, 4.5, 13.6 Hz,
H-8a), 2.80 (dd, 10, 13.6 Hz, H-8b), 2.17 (m, H-4) 2.16, 1.99 (m,
H-3 a & b), 1.20 (t, 7 Hz, CH2CH3), 1.17 (t, 7 Hz, CH2CH3). 13C
NMR (100 MHz, DMSO-d6 d ppm: 172.4 (C-5), 171.6 (C-1), 171.4
(C-6), 170.2 (C-9), 161.0 (imine-CH), 149.6 (ArC), 134.5 (ArC),
132.5 (ArC), 130.7 (ArC), 130.4 (ArC), 125.1 (ArC), 71.7 (C-2), 61.4
(CH2CH3), 61.2 (CH2CH3), 52.2 (C-7), 41.6 (C-10), 40.9 (C-8), 32.0
(C-4), 29.2 (C-3), 14.8 (2ꢁ CH3).
l
m, 250 ꢁ 108 mm, 4 mL/
min) using isocratic MeOH/H2O (65:35, retention time 14.5 min).
The 25% MeOH fraction off the HP20SS column was subjected to
gel permeation chromatography on a Sephadex LH-20 column
(length 30 cm, radius 1.5 cm, 100% MeOH) yielding 14 sub-frac-
tions. The seventh LH-20 sub-fraction (7 mg) was purified by RP-
HPLC (Phenomenex, C-18, 5
an isocratic mixture (4:21) of ACN and aqueous NH4OAc buffer
(0.1 M), to give (1.5 mg, retention time 21.5 min) and
(1.0 mg, retention time 23.8 min) as amorphous white solids.
lm, 250 ꢁ 10 mm, 4 mL/min) using
3
4
The eighth LH-20 sub-fraction (14 mg) was also purified by
RP-HPLC. The material was first processed on a C-18 column (Phe-
4.6. Synthesis of the N-(2-nitrobenzyl) amine 13
nomenex, 5
l
m, 250 ꢁ 10 mm, 4 mL/min) using an isocratic mix-
To a solution of the imine 11 (122 mg, 123
DMSO (3:1, 4 mL) was added NaBH3CN (61 mg, 970
l
mol) in acetonitrile/
mol) fol-
ture (27:73) of ACN and aqueous AcOH (0.1%) to give impure 2
(ꢂ2 mg, retention time 11.5 min). Reprocessing of the material
l
lowed by AcOH (6 drops). After stirring for 3 h saturated NaHCO3
solution (50 mL) and methylene chloride (150 mL) were added.
The mixture was extracted and separated. The aqueous phase
was then re-extracted with methylene chloride (150 mL). The com-
bined organic phases were dried (MgSO4) and concentrated in
vacuo. The resulting crude material was purified by gel permeation
chromatography on LH-20 (30 cm, 90 mL, 100% MeOH) to give 13
on a phenylhexyl column (Phenomenex, 5
lm, 250 ꢁ 10 mm,
4 mL/min) using an isocratic mixture of MeOH/H2O (12:88) yielded
a salt of 2 (ꢂ1 mg, retention time 7.7 min) as an amorphous off-
white solid. The salt of 2 was dissolved in H2O and acidified with
HCl (1 M, 40 lL), before being extracted with EtOAc (5 mL). The or-
ganic phase was concentrated in vacuo to yield 2 (<1 mg).
(87 mg, 87.6 lmol, 71%) as an amorphous off-white solid.
4.4.1. Compound 3
(+) ESI-MS m/z: 652 ([M+H]+, 79Br), 654 ([M+H]+, 81Br). (+)
FT-MS m/z: found 652.07467 (C22H31N5O9S2Br [M+H]+ calcd
652.07466). ESI-MSMS: Table 1, and Supplementary data Fig. S6.
4.6.1. Compound 13
(+) ESI-MS m/z: 995.3516 (C42H59N8O16S2 [M+H]+ calcd
995.3490). [
a]
ꢀ61° (MeOH). 1H NMR (400 MHz, CD3OD d ppm:
D
UV (MeOH) kmax (log
e
): 212 (4.36), 282 (3.75). [
a]
D ꢀ21° (MeOH).
7.91 (1H, d, 6.6 Hz, Ar-H), 7.63 (2 H, m, ArH), 7.47 (1 H, m ArH),
4.88 (m, H-7), 4.15 (q, 7 Hz, CH2CH3), 4.09 (q, 7 Hz, CH2CH3), 3.98
(d, 8.3 Hz, NHCH2), 3.93 (d, 2.5 Hz, H-10), 3.27 (m, H-2), 3.19 (dd,
4.7, 14.5 Hz, H-8a), 2.92 (dd, 9.2, 14.5 Hz, H-8b), 2.40 (t, 7.1 Hz,
H-4), 2.01–1.82 (m, H-3), 1.23 (t, 7 Hz, CH2CH3). 13C NMR
(125 MHz, CD3OD d ppm: 176.3 (C-1), 175.5 (C-5), 173.2 (C-6),
171.2 (C-9), 150.9 (ArC), 136.3 (ArC), 134.4 (ArC), 132.8 (ArC),
129.6 (ArC), 125.8 (ArC), 62.5 (CH2CH3), 62.2 (CH2CH3), 61.8 (C-
2), 53.8 (C-7), 49.7 (NHCH2), 42.3 (C-10), 42.0 (C-8), 33.2 (C-4),
30.0 (C-3), 14.7 (CH3), 14.6 (CH3).
IR
m
(cmꢀ1): 2800–3600, 1654, 1539. 1H NMR: see Table 3. 13C
NMR: see Table 3.
4.4.2. Compound 4
(+) ESI-MS m/z: 595 ([M+H]+, 79Br), 597 ([M+H]+, 81Br). (+)
FT-MS m/z: found 595.05347 (C20H28N4O8S2Br [M+H]+ calcd
595.05319). ESI-MSMS: Table 2, and Supplementary data Fig. S7.
[a
]
D
ꢀ26° (MeOH). 1H NMR: see Table 3. 13C NMR: see Table 3.
4.5. Synthesis of the imine 11
4.7. Synthesis of the tertiary amine 12 and the thiocyanate
derivative 14
The imine 11 was prepared by the following two step process.
Anhydrous HCl gas (420 mmol) was bubbled into an ice cooled
and stirred suspension of 9 (Acros, 1.18 g, 1.9 mmol) in dry EtOH
(25 mL) over 40 min. The resulting solution was sealed and stored
at 4 °C for 3 days. The solution was then concentrated in vacuo to
afford 10 (1.54 g, 1.9 mmol, quant.).
4.7.1. Synthesis of compound 12 from 13
A solution of the amine 13 (76.5 mg, 77
dehyde (Acros, 40 mg, 1.3 mmol) in acetonitrile/DMSO (3:1, 4 mL)
was stirred for 8 h. NaBH3CN (35 mg, 557 mol) and AcOH (6
lmol) and paraformal-
l
NaOH solution (2.13 mL, 1.0 M) was added to an ice cooled stir-
red solution of 10 (850 mg, 1.07 mmol) in MeOH/H2O (2:10,
12 mL), followed by the addition of 2-nitrobenzaldehyde (Acros,
331 mg, 2.19 mmol). After stirring at room temperature overnight
the resulting white suspension was collected by filtration (washing
with ice cold water) and dried in vacuo to yield 11 (873 mg,
0.9 mmol, 83%) as an amorphous white solid.
drops) were then added, and the solution was left to stir overnight.
Saturated NaHCO3 solution (50 mL) and methylene chloride
(25 mL) were then added and the mixture extracted and separated.
The aqueous phase was re-extracted with methylene chloride
(25 mL). The combined organic phases were dried (MgSO4) and
concentrated in vacuo. The resulting crude material was purified
by gel permeation chromatography on LH-20 (30 cm, 90 mL,
100% MeOH) to give 12 (51.5 mg, 51.8 lmol, 66%) as an oil.
4.5.1. Compound 10
(+) ESI-MS m/z 725 [M+H]+.
[
a]
ꢀ77° (MeOH).1H NMR
4.7.2. Synthesis of compounds 12 and 14 from 11
D
(400 MHz, CD3OD d ppm: 4.76 (dd, 4.5, 9.4 Hz, H-7), 4.30 (q, 7 Hz,
CH2CH3), 4.17 (q, 7 Hz, CH2CH3), 4.13 (m, H-2), 3.95 (d, 2.1 Hz,
H-10), 3.26 (dd, 4.1, 13.8 Hz, H-8a), 2.97 (dd, 13.8, 9.4 Hz, H-8b),
2.59 (t, 7.6 Hz, H-4), 2.25 (m, H-3), 1.33 (t, 7 Hz, CH2CH3), 1.26 (t,
7 Hz, CH2CH3). 13C NMR (100 MHz, CD3OD d ppm: 174.5, 173.1,
171.2, 170.3, 64.0, 62.5, 54.0, 53.8, 42.3, 41.5, 32.4, 27.1, 14.64, 14.57.
To a solution of the imine 11 (773 mg, 0.78 mmol) in acetonitrile/
DMSO (3:1, 12 mL) was added NaBH3CN (105 mg, 1.67 mmol) fol-
lowed by AcOH (6 drops). After stirring overnight paraformaldehyde
(Acros, 200 mg, 6.7 mmol) was added. After another nine hours
additionalNaBH3CN (400 mg, 6.4 mmol) was added and the solution
was again stirred overnight. Saturated NaHCO3 solution (75 mL) and
methylene chloride (150 mL) were then added. The mixture was
separated, and the aqueous phase re-extracted with methylene
chloride (150 mL). The combined organic phases were dried
(MgSO4) and concentrated in vacuo. The resulting crude material
was purified by gel permeation chromatography on LH-20 (30 cm,
4.5.2. Compound 11
(+) ESI-MS m/z: 991.3189 (C42H55N8O16S2 [M+H]+ calcd
995.3177). 1H NMR (500 MHz, DMSO-d6 d ppm: 8.65 (s, imine-
CH), 8.38 (t, 6.0 Hz, glycine-NH), 8.24 (d, 8.5 Hz, cysteine-NH),

M. W. B. McCulloch et al. / Bioorg. Med. Chem. 17 (2009) 2189–2198
2197
90 mL, 100% MeOH) to give 12 (672 mg, 0.65 mmol, 84%) as an
immobile oil, along with 14 (48 mg, 6%) as an amorphous solid.
overnight, at room temperature, the reaction was concentrated in
vacuo and the crude material purified by RP-HPLC (Phenomenex,
C-18, 5
(19:81) of ACN and aqueous NH4OAc buffer (0.1 M), to give 3
(4.7 mg, 7.2 mol, 38% based on 15; retention time 12.7 min).
l
m, 250 ꢁ 10 mm, 4 mL/min) using an isocratic mixture
4.7.3. Compound 12
(+) ESI-MS m/z: 1023.3806 (C44H63N8O16S2 [M+H]+ calcd
l
1023.3803). [
a]
ꢀ74° (MeOH). 1H NMR (500 MHz, CD3OD d
D
ppm: 7.80 (1H, m, ArH), 7.61 (2H, m, ArH), 7.45 (1H, m, ArH),
4.86 (dd, 5.1, 9.2 Hz, H-7), 4.18 (m, CH2CH3), 4.05, 3.96 (both d,
14.5 Hz, NHCH2), 3.93 (d, 2.5 Hz, H-10), 3.32 (m, H-2), 3.19 (dd,
5.1, 14.0 Hz, H-8a), 2.94 (dd, 9.2, 14.0 Hz, H-8b), 2.31 (m, H-4),
2.17 (s, N-CH3), 1.97 (m, H-3), 1.29 (t, 7 Hz, CH2CH3), 1.23 (t,
7 Hz, CH2CH3). 13C NMR (125 MHz, CD3OD d ppm: 175.7 (C-1),
173.4 (C-5), 173.2 (C-6), 171.2 (C-9), 151.5 (ArC), 135.5 (ArC),
133.9 (ArC), 132.6 (ArC), 129.6 (ArC), 125.6 (ArC), 66.6, (C-2), 62.5
(CH2CH3), 61.6 (CH2CH3), 57.6 (NHCH2), 53.9 (C-7), 42.3 (C-10),
42.1 (C-8), 37.0 (N-CH3), 33.3 (C-4), 26.5 (C-3), 15.0 (CH2CH3),
14.6 (CH2CH3).
4.10. Synthesis of compound 5
A solution of psammaplin A (1, 37
l
mol), oxidized
L-glutathione
(Acros, 68 lmol), K2CO3 (90 mg, excess) and DTT (38 lmol) in
THF:MeOH (1:6, 7 mL) and phosphate buffer (3 mL, pH 8.4,
12 mM phosphates) was stirred at room temperature for 22 h.
The reaction was then concentrated in vacuo and the crude mate-
rial purified by RP-HPLC (Phenomenex, C-18, 5
using an isocratic mixture (19:81) of ACN and aqueous NH4OAc
buffer (0.1 M), to give 5 (11 mg, 17 mol, 46%, based on 1, unop-
l
m, 250 ꢁ 10 mm)
l
timized, retention time 11.6 min) as an off-white amorphous solid.
4.7.4. Compound 14
4.10.1. Compound 5
ESI-MS m/z: 538.1978 (C23H32N5O8S [M+H]+ calcd 538.1972).
(ꢀ) ESI-MS m/z: found 638.0592 (C21H29N5O9S2Br [M+H]+ calcd
[
a]
D
ꢀ27° (MeOH). IR
m
(cmꢀ1): 2156 sharp (C„N stretch). 1H
638.0590). ESI-MSMS: Supplementary data Fig. S8. [
a
]
D
ꢀ22°
NMR (400 MHz, CD3OD d ppm: 7.81 (1H, m, ArH), 7.61 (2H, m,
ArH), 7.47 (1H, m, ArH), 4.81 (dd, 5.0, 7.7 Hz, H-7), 4.18 (m,
CH2CH3), 4.08, 3.97 (both d, 14.5 Hz, NHCH2), 3.94 (d, 2.5 Hz, H-
10), 3.50 (dd, 5.0, 13.5 Hz, H-8a), 3.33 (m, H-2), 3.29 (dd, 7.7,
13.5 Hz, H-8b), 2.31 (m, H-4), 2.18 (s, N-CH3), 1.99 (m, H-3), 1.31
(t, 7 Hz, CH2CH3), 1.25 (t, 7 Hz, CH2CH3). 13C NMR (100 MHz,
CD3OD d ppm: 175.9 (C-1), 173.4 (C-5), 171.6 (C-6), 171.2 (C-9),
151.5 (ArC), 135.5 (ArC), 133.9 (ArC), 132.6 (ArC), 129.6 (ArC),
125.6 (ArC), 113.6 (C„N), 66.6, (C-2), 62.5 (CH2CH3), 61.6
(CH2CH3), 57.7 (NHCH2), 53.9 (C-7), 42.3 (C-10), 36.8 (N-CH3),
36.4 (C-8), 33.2 (C-4), 26.2 (C-3), 15.0 (CH2CH3), 14.6 (CH2CH3).
(MeOH). IR
m
(cmꢀ1): 2800–3600, 1653, 1539. 1H NMR: see Table
3. 13C NMR: see Table 3.
4.11. Oxidative methanolysis of psammaplin A: synthesis of 6
and 7
To an ice cooled, stirred solution of psammaplin A (1, 4 mg,
6 lmol) in MeOH (10 mL) was added NBS (2.6 mg, 14.6 lmol), in
MeOH (2.6 mL), over a period of 2 min. After 40 min the flask
was allowed to warm to room temperature, and after another
40 min the reaction mixture was partitioned between methylene
chloride (50 mL) and water (75 mL). The organic phase was sepa-
rated and the aqueous phase twice more extracted with methylene
chloride (2ꢁ 50 mL). The combined methylene chloride extracts
were concentrated in vacuo to give a crude mixture of two major
products. HPLC purification of this material (Phenomenex, phenyl-
4.8. Synthesis of oxidized glutathione N-methyl amine,
tetraethyl ester (15)
The amine 12 (28.5 mg, 27 lmol) was photolyzed in six batches.
Samples dissolved in acetonitrile (concentration of ꢂ0.5 mmol/L)
were placed in pear shaped Kontes flasks disposed ꢂ10 cm away
from a 360 nm lamp. No cooling was necessary and each sample
was irradiated for 2 h. The combined photolyzed samples were
concentrated in vacuo to give an orange gum. Purification by gel
permeation chromatography on LH-20 (30 cm, 90 mL, 100% MeOH)
hexyl, 5
the methyl sulfinate esters 6 (0.5 mg, 1.3
retention time 6.8 min) and 7 (0.8 mg, 1.7
l
m, 250 ꢁ 10 mm, 4 mL/min, MeOH/H2O [60:40]) yielded
mol, 11%, unoptimized,
mol, 14%, retention
l
l
time 10.5 min) as colorless amorphous solids.
4.11.1. Compound 6
yielded 15 (19 mg, 25
l
mol, 91%) as a semi-solid.
(+) ESI-MS m/z: found 400.9785 (C12H15BrN2O5SNa [M+Na] calcd
400.9783). (+) FT-MS m/z: found 378.9963 (C12H16BrN2O5S [M+H]+
4.8.1. Compound 15
calcd 378.9963). IR m
(cmꢀ1): 3000–3600, 1655, 1540, 1421, 1282,
(+) ESI-MS m/z: 753.3165 (C30H53N6O12S2 [M+H]+ calcd
1101, 982. 1H NMR (500 MHz, CD3OD d ppm: 7.35 (d, 2.0 Hz, H-9),
7.06 (dd, 2.0, 8.0 Hz, H-13), 6.75 (d, 2.0 Hz, H-12), 3.78 (s, H-7),
3.75 (s, OCH3), 3.62 (dt, 2.0, 6.5 Hz, H-3), 2.89-3.05 (m, H-2). 13C
NMR (125 MHz, CD3OD d ppm: 165.9 (C-5), 153.8 (C-11), 153.0
(C-6), 134.5 (C-9), 130.6 (C-8), 130.4 (C-13), 117.0 (C-12), 110.5 (C-
10), 57.4 (C-2), 55.7 (OCH3), 33.5 (C-3), 28.8 (C-7).
753.3163). [
a
]
ꢀ34° (MeOH). 1H NMR (500 MHz, CD3OD d ppm:
D
4.82 (dd, 4.5, 9.5 Hz, H-7), 4.20 (q, 7 Hz, CH2CH3), 4.17 (q, 7 Hz,
CH2CH3), 3.95 (d, 3.0 Hz, H-10), 3.27 (dd, 6.5, 13.5 Hz, H-2), 3.22
(dd, 4.5, 14.0 Hz, H-8a), 2.92 (dd, 9.5, 14.0 Hz, H-8b), 2.36 (m, H-
4), 2.34 (N-CH3), 1.97 (m, H-3), 1.28 (t, 7 Hz, CH2CH3), 1.25 (t,
13
7 Hz, CH2CH3). C NMR (125 MHz, CD3OD d ppm: 175.3 (C-5),*
175.2 (C-1),* 173.2 (C-6), 171.2 (C-9), 63.5 (C-2), 62.5 (CH2CH3),
62.3 (CH2CH3), 53.8 (C-7), 42.3 (C-10), 41.8 (C-8), 34.4 (N-CH3),
33.0 (C-4), 29.1 (C-3), 14.8 (CH2CH3), 14.6 (CH2CH3). * Interchange-
able assignments.
4.11.2. Compound 7
(+) ESI-MS m/z: found 478.8883 (C12H14Br2N2O5S [M+Na] calcd
478.8888). 1H NMR (500 MHz, CD3OD d ppm: 7.38 (s, H-9), 3.78 (s,
H-7), 3.76 (s, OCH3), 3.64 (dt, 2.0, 6.0 Hz, H-3), 2.90-3.04 (m, H-2).
13C NMR (125 MHz, CD3OD d ppm: 165.8 (C-5), 152.6 (C-6), 151.1
(C-11), 134.1 (C-9), 132.2 (C-8), 112.2 (C-10), 57.4 (C-2), 55.7
(OCH3), 33.6 (C-3), 28.5 (C-7).
4.9. Synthesis of the natural product 3
To a vigorously stirred solution of 15 (14 mg, 18.6
lmol) in THF/
H2O (1:1, 3 mL) was added aqueous LiOH (95 mol from a 1.0 M
l
4.12. DFT calculations on structures 6 and 8
solution). The solution was stored at 4 °C overnight and then stirred
4 h at room temperature. Phosphate buffer (4 mL, pH 8.4, 12 mM
For each structure, a low energy conformer was identified by a
Monte Carlo search using Ghemical 2.10. The low energy conforma-
tions were imported to Gaussian03,36 and each structure was sub-
phosphates) was then added followed by DTT (2 mg, 13
lmol) and
psammaplin A (1, 75 mg, 113 mol) in MeOH (10 mL). After stirring
l

2198
M. W. B. McCulloch et al. / Bioorg. Med. Chem. 17 (2009) 2189–2198
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Acknowledgments
The authors thank Dr. Chad Nelson and Dr. Krishna Parsawar,
fromtheUniversityof Utah, MassSpectrometryandProteomicsCore
Facility, for recording the FT-MS spectra. An allocation of computer
timefromtheCenterfor HighPerformanceComputingattheUniver-
sity of Utah is gratefully acknowledged. The computational re-
sources for this project have been provided by the National
Institutes of Health (Grant # NCRR 1 S10 RR17214-01) on the Arches
Metacluster, administered by the University of Utah Center for High
Performance Computing. The following NIH and NSF grants funded
NMR instrumentation: RR14768, RR06262, RR13030, DBI-
0002806. This research was also supported by The Huntsman Cancer
Institute, a University of Utah Seed Grant: Pharmacologic regulation
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