Stictamides A-C, MMP12 inhibitors containing 4-amino-3-hydroxy-5- phenylpentanoic acid subunits

Article abstract of DOI:10.1021/jo200241h

An extensive study of the secondary metabolites produced by a new Sticta sp. of lichen has led to the isolation of three new compounds containing the 4-amino-3-hydroxy-5-phenylpentanoic acid residue (Ahppa). The structures of stictamides A-C (1-3) were as

Full text of DOI:10.1021/jo200241h

FEATURED ARTICLE
pubs.acs.org/joc
Stictamides AꢀC, MMP12 Inhibitors Containing
4-Amino-3-hydroxy-5-phenylpentanoic Acid Subunits
Zhibin Liang,† Analia Sorribas,† Florian J. Sulzmaier,^ Jorge I. Jimꢀenez,|| Xin Wang,‡ Thomas Sauvage,§
Wesley Y. Yoshida,† Guangyi Wang,‡ Joe W. Ramos,^ and Philip G. Williams*,†,^
†Departments of Chemistry, ‡Oceanography, and §Botany, University of Hawaii at Manoa, Honolulu, Hawaii 96822, United States
^University of Hawaii Cancer Center, 651 Ilalo Street, Honolulu, Hawaii 96813, United States
AgraQuest, Inc., 1540 Drew Avenue, Davis, California 95618, United States
S Supporting Information
b
ABSTRACT: An extensive study of the secondary metabolites
produced by a new Sticta sp. of lichen has led to the isolation of
three new compounds containing the 4-amino-3-hydroxy-5-
phenylpentanoic acid residue (Ahppa). The structures of stic-
tamides AꢀC (1ꢀ3) were assigned by 2D NMR spectroscopic
and chemical methods. Due to extensive epimerization of the
Ahppa residue observed after acid hydrolysis, the configuration
of this unit was deduced through conversion of 1 to an
appropriate derivative and application of our recently developed
statine NMR database. Evaluation of stictamide A against a
panel of disease-relevant proteases showed that it inhibited MMP12 at 2.3 μM and significantly reduced invasion in the human
glioma cell line U87MG. Docking studies suggest that stictamide A inhibits MMP12 by a non-zinc-binding mechanism.
’ INTRODUCTION
Statine-like amino acids are a well-known motif in natural
products. Originally identified as a residue in the classic protease
inhibitor pepstatin,1 the biosynthesis of these γ-amino-β-hydro-
xy acids involves the condensation of a protogenic amino acid
with malonate via an oxidative decarboxylation reaction.2 The
unit has attracted considerable interest as natural products
containing this moiety have been reported to display a wide
spectrum of biological activities ranging from anticancer3 to the
inhibition of P-glycoprotein efflux pumps.4 As statine moieties
function as peptide isosteres, being transition state mimics of
peptide hydrolysis,5 they are common design elements in
protease inhibitors.6 Incorporation of this residue can significantly
improve the pharmacokinetic properties and potency of that class of
compounds. As part of a general screening program for protease
inhibitors, an extract derived from a new Sticta sp. (Order Peltiger-
ales: Family Lobariaceae) of lichen was identified which showed
protease inhibition in our initial screens. Fractionation of the extract
has now led to the identification of three statine-containing peptides,
stictamides AꢀC (1ꢀ3). We report here their isolation and
structure determination. In addition, we report the protease profil-
ing and evaluation of the effect of 1 on invasion of U87-MG, a
human glioblastoma cell line, and its viability.
was obtained from the mass spectrum, which showed a molecular
ion peak at m/z 759.4082 [M]þ. On the basis of HR-ESI-
TOFMS data, the molecular formula was defined as
C41H55N6O8, which indicated that 1 contained 18 double bond
equivalents and a positive charge. In total, 35 carbon resonances
were observed in the 13C NMR and multiplicity-edited HSQC
spectra, suggesting that elements of symmetry were present in
the compound. On the basis of the number of sp2 carbons and
their chemical shifts, these symmetry elements were assigned to
one mono- and two disubstituted phenyl rings. The 41 carbons
required by the molecular formula could therefore be ascribed to
11 quaternary, 18 methine, 10 methylene, and 2 methyl carbons.
’ RESULTS AND DISCUSSION
Stictamide A (1) was isolated as an optically active light yellow
powder ([R]D ꢀ0.2 (c 0.2, MeOH)). The molecular weight of 1
Received: February 1, 2011
Published: April 18, 2011
r
2011 American Chemical Society
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remaining nitrogen atoms, on the basis of HMBC correlations
observed from both H-25 and H-10 to C-26. Two functional
groups were still possible for this sp2 carbon (C-26) that
would result in 1 containing either an N0-prenylarginine or an
N0-prenylcitrulline unit, with a carboxylic acid or primary amide,
respectively, at C-1. These two possible structures could not be
conclusively distinguished via the IR spectrum nor could they be
distinguished via a chemical shift argument.
In the end, this required the treatment of a 500 μg sample of 1
with trimethylsilyldiazomethane to determine whether C-1 was a
carboxylic acid or a primary amide. The progress of the reaction
was monitored by LC-MS analysis (Figure 2), which after an
hour indicated a complex mixture of mono-, di-, and trimethy-
lated products that slowly coalesced over the next 2.5 h into one
major product (4) corresponding to the trimethylated derivative
(m/z 801.4547 [M]þ). To confirm that methylation occurred
exclusively at the phenolic and carboxylic acid oxygens, the
product was isolated and the proton spectrum recorded. The
1H NMR spectrum of the isolated product was essentially
identical to the starting material except for two additional
methoxy singlets at δH 3.65 and δH 3.73 integrating to 3 and
6, respectively (see Supporting Information Figure S12). Anal-
ysis of the HMBC spectrum of this derivative indicated that
the two methyl signals at δH 3.73 showed clear correlations to
O-arylcarbons at approximately δC 159.0, while the methoxy
signal at δH 3.65 clearly correlated to a carbonyl carbon at δC
173.0 (see Supporting Information Figure S13), establishing
conclusively that C-1 in 1 was a carboxylic acid and unit C
contained a guanidine rather than a carbamate functional group.
Analysis of the MS/MS fragmentation pattern provided addi-
tional data to support this assignment (vide infra).
Figure 1. Initial fragments assembled from analyses of the 2D
NMR data.
These could be further assigned as five carbonꢀheteroatom
double bonds (δC-1 178.9; δC-10 172.5; δC-21 174.3; δC-26 157.3;
δC-27 175.9) and 10 carbonꢀcarbon double bonds that ac-
counted for 15 of the total 18 degrees of unsaturation implied
by the molecular formula. This indicated that 1 contained
three rings.
Analysis of the spectroscopic data established the units that
constituted 1. Briefly, HMBC correlations from a pair of diaster-
eotopic benzylic protons (δH-3 3.13 and 2.86) to a para-
substituted aromatic ring, a downfield methine (δC-2 57.8),
and a carbonyl carbon (δC-1 178.9) established the presence of
a tyrosine moiety (fragment A). A second unit (fragment B)
could be established beginning with the carbinol proton signal
(H-12) at δH 4.08. This proton resonance displayed TOCSY
correlations to a methine proton signal at δH 4.10 (H-13) and
showed COSY cross peaks to methylene proton signals at δH-11
2.39 and 2.29 that, in conjunction with HMBC correlations from
H-12 to C-11, from H-14 to C-13, and from H-14 to C-15,
indicated this fragment was 4-amino-3-hydroxy-5-phenylpenta-
noic acid (Ahppa; fragment B). One-dimensional TOCSY
experiments defined a continuous four-carbon spin system
composed of three methylenes and one methine (C-22 to
C-25) that was built into fragment C. The downfield chemical
shift of the carbons at each end (δC-22 54.5; δC-25 41.9)
indicated that these carbons were adjacent to nitrogen atoms,
while the HMBC correlation from the proton attached to C-22
to the carbonyl carbon C-21 indicated that fragment C was
ornithine. Fragment D evidently contained a second para-
substituted phenyl ring, based on the characteristic AB system
Detailed LC-MS analyses of the initial column fractions led to
the identification of two analogues. Subsequent isolation and
characterization of the more polar metabolite 2 indicated that 1
and 2 possessed similar structures, although a comparison of the
high-resolution ESI-TOF data established that 2 (C36H47N6O8)
was 68 amu smaller than 1. This mass difference was easily
explained after the observation that the two allylic methyl
1
resonances in the H NMR spectrum of 1 were missing in the
spectrum of 2, which indicated that the isoprenyl moiety was
absent in 2. Analysis of the 2D NMR spectra (see Table S2 in
Supporting Information) confirmed this conclusion. The third
compound, stictamide C (3), was present only in a quantity
sufficient for characterization by 1H NMR and HRMS spectro-
metry. These data suggested that 3 was a methoxy derivative of 1,
based on the 14 amu difference in mass, while the chemical shift
of this methoxy group noted in the 1H NMR spectrum suggested
a methyl ester rather than a phenolic ether. This conclusion
1
(δH-32 6.99 d, 8.4 Hz; δH-33 6.68 d, 8.4 Hz) visible in the H
NMR spectrum. This spin system could be attached on the
basis of a series of HMBC correlations to the terminal end of
an n-butanoic acid unit, thus defining the 4-(4-hydroxyphe-
nyl)butanoic acid residue (fragment D). The final unit (frag-
ment E) contained two methyl singlets with proton chemical
shifts indicative of attachment to a sp2 carbon. HMBC correla-
tions from these methyl groups to each other and to two sp2
carbons, which also were coupled to a methylene carbon,
defined this unit as a prenyl moiety.
HMBC correlations provided the final carbonꢀcarbon con-
nectivities. Fragment B (Ahppa) was linked to the nitrogen atom
of fragment A (Figure 1) by an HMBC correlation from H-2 to
C-10. Fragment C was located between the amino terminus of
Ahppa and C-27 of fragment D based on HMBC correlations
from H-13 to C-21 and H-22 to C-27. Finally, the last remaining
carbon C-26 at δC 157.3 was linked to fragments C and E via the
1
was supported by a comparison of the H NMR spectrum
(Figure S11) with that of the trimethyl derivative 4, prepared
above, for which the methyl ester could be ascribed to the singlet
at δH 3.64 through HMBC correlations. Additional support for
this assignment was obtained from a comparison of the MS
fragmentation patterns of 1 and 3 (Figure 3). Key fragmentations
that established the position of methylation included a common
m/z 536 ion derived from a retro-aldol fragmentation of the
Ahppa unit and a y1þ ion in 1 that was shifted from m/z 182.3 by
14 amu to m/z 196.1 in 3. Taken together, these fragments
indicated that the C-terminal tyrosine had been methylated in 3.
Finally, identification of an ion at m/z 147.0 corresponding to
[y1-NH3-CH3OH]þ conclusively established the methyl ester
due to its loss of MeOH, which is typical of this functional group.
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Figure 2. Methylation of 1 to distinguish a possible C-terminal carboxylic acid from a primary amide. LC-MS chromatogram of (A) starting material,
(B) 15 min time point, (C) 1 h time point, and (D) 3.5 h time point.
Figure 4. (A) L-FDLA and (B) DL-FDLA Ahppa extract-ion chromato-
grams of the derivatized hydrolysate of 2.
the advanced Marfey’s technique.8 These experiments estab-
lished that 2 contained D-Arg and L-Tyr. The identification of the
latter was complicated by the predominance of O-aryl tyrosine
products, where the diastereomeric derivatives were poorly
resolved. The expected N-aryl tyrosine derivatives were even-
tually detected by extract-ion analysis of the LC-MS chromato-
Figure 3. MS fragmentation patterns of (A) 1 and (B) 3.
grams and then confirmed through comparison with retention
times and molecular weights of the appropriate standards.
Methoxyaryl units, in contrast, tend to extrude carbon monoxide,
which was not observed.
To determine the absolute configurations of the amino acid
constituents, the advanced Marfey method was applied to 2.
The acid hydrolysate (0.5 mg, 6 N HCl, 118 ꢀC, 10 h) of 2 was
divided in two portions and derivatized with L- and DL-mixtures
of (1-fluoro-2,4-dinitrophenyl)-5-leucinamide (FDLA)7 as per
Assigning the configuration of the vicinal centers in the Ahppa
moiety via Marfey’s analysis proved more challenging for multi-
ple reasons. Initial experiments with synthetic standards9 demon-
strated that, while Marfey’s derivatives could be prepared,
distinguishing the 3S,4S and 3R,4S epimeric derivatives would
be difficult under the standard solvent systems due to near
identical retention times. Repeated attempts to optimize this
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Figure 5. Preparation of the chloroform-soluble derivative 6 and its 3JH,H values used to establish the configuration of the intact Ahppa unit.
separation by varying the gradient or pH failed to resolve these
standards. Unfortunately, analysis of the derivatized L-FDLA
hydrolysate indicated that 2 contained one of these unresolved
diastereomers (Figure 4A). While this established the 4S config-
uration, the relative stereochemistry still needed to be deduced.
In the end, this was accomplished by “racemization” of the
hydrolysate through derivatization with DL-FDLA and analyzing
for D-FDLA-3S,4S- and D-FDLA-3R,4S-Ahppa. This analysis
revealed that peaks corresponding to both C-3 epimers were
present in the hydrolysate, although the 3S,4S diastereomer
predominated by a ratio of approximately 2:1. Clearly, C-3, the
β-alcohol of the Ahppa unit, undergoes epimerization at this
center under the hydrolysis conditions. A similar phenomenon
was noted by Sakai and Rinehart10 during the structural char-
acterization of the isostatine unit contained in didemnin B, which
necessitated synthesis of that compound to clarify the issue. In
our case, assuming the ratio of products in the hydrolysate does
not simply represent a thermodynamic mixture, the configura-
tion of the Ahppa unit therefore would be 3S,4S.
Although the preceding results established the stereochemis-
try of the hydrolyzed Ahppa unit, they did not conclusively
establish the stereochemistry of this unit in the natural product
due to the possibility of inversion at C-3 during hydrolysis. We
recently proposed a simple method to assign the relative config-
uration of statine derivatives (5-alkyl-4-amino-3-hydroxybuta-
noic acids) to address this question.9 This method allows
configurational assignment of statine-type units within complex
natural products without degradation, as the syn and anti
diastereomers can be distinguished using a combination of
chemical shift and coupling constant information derived from
the R-methylene ABX system. The method was validated
using 73 examples, ranging in structural complexity from
simple statine-type units to cyclic depsipeptides, such as taman-
darin B,11 demonstrating the scope and limitations of the
methodology.
the derivatization of the guanidino amine was so slow that
hydrolysis of the methyl ester competed, under a variety of
conditions, to yield a mixture of products. Given this liability,
compound 1 was therefore converted to the secondary amide 5
through coupling with benzylamine, and then the phenolic
groups were methylated (Figure 5). Treatment of this derivative
with Fmoc-OSu cleanly afforded the desired product 6 without
competing hydrolysis. Careful analysis of the proton spectrum of
this derivative, 6, recorded in CDCl3, revealed the correct
diagnostic pattern for the methylene protons of the ABX system
of the Ahppa unit. In this case, the downfield methylene
proton resonating at δH 2.33 was a doublet of doublets with a
3
large vicinal JH,H value of 8.3 Hz, while the upfield signal (δH
2.24) had the small vicinal proton coupling (5.1 Hz). These
results confirmed the anti stereochemical configuration of the
Ahppa unit.
The Ahppa unit has been found in a rather limited number of
compounds. It is a component of the cyanobacterial metabolites,
tasiamide B13 and hapalosin,4 and of some protease inhibitors
isolated from Streptomyces spp.14 The stictamides represent the
first reported example of this structural motif in a lichen
secondary metabolite.
Given the presence of a γ-amino-β-hydroxy acid moiety in 1,
which is a common design element in protease inhibitors,
stictamide A (1) was evaluated for selectivity against a series of
18 proteases (Table 2). Compound 1 did not inhibit the serine
proteases trypsin, chymotrypsin, or elastase in standard chemi-
luminescent assays at concentrations up to 100 μM.15 It was
inactive (IC50 >100 μM) against the Alzheimer’s relevant
proteases BACE1 (β-secretase), ADAM10 (R-secretase), and
TACE (potential γ-secretase) and against angiotensin convert-
ing enzyme 1 (ACE1), caspase 3, ADAM9, and calpain. Com-
pound 1 did inhibit matrix metalloproteinase-12 (MMP12) with
an IC50 value of 2.3 μM (Ki = 4.9 μM), while related MMPs
(2 and 9) were inhibited only at significantly higher concentra-
tions (37 and 65 μM).
Matrix metalloproteinases (MMPs) are zinc-dependent en-
dopeptidases that mediate extracellular matrix degradation and
thereby are involved in many physiological and pathological
processes including angiogenesis and tumor invasion. That
MMPs are required to initiate tumor cell invasion into the
surrounding matrix has been the underlying principle in the
study of cancer cell invasion for a number of years.16 Despite
One of the principle limitations of the method is the require-
ment for CDCl3 solubility, presumably to facilitate hydrogen
bonding between the various heteroatoms in this unit. As com-
pounds 1ꢀ3 are insoluble in CDCl3, we sought to prepare a
more lipophilic derivative. Treatment of 1 with an excess of
trimethylsilyldiazomethane,12 as described above, yielded 4, but
subsequent derivatization with Fmoc-OSu failed to provide the
desired derivative. While the methylation proceeded smoothly,
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Table 1. NMR Spectroscopic Data for Stictamide A (1) at 500 MHz in MeOH-d4
residue
Tyr
position
δC, mult.
δH (J in Hz)
COSY
HMBC
TOCSYa
1
178.9, qC
57.8, CH
38.2, CH2
H-2, H-3a, H-3b
H-3a, H-3b
H-2, H-5
2
3
4.44, dd (8.5, 4.3)
3.13, dd (14.0, 4.3)
2.86, dd (14.0, 8.5)
H-3a, H-3b, NH(1) a
H-2
H-6
4
130.5, qC
131.4, CH
116.0, CH
156.9, qC
172.5, qC
41.4, CH2
H-2, H-6
5 and 9
6 and 8
7
7.06, d (8.5)
6.66, d (8.5)
H-3a, H-3b
H-5, H-6
Ahppa
10
H-2, H-11a, H-11b, H-12
H-12
11
2.39, dd (14.1, 7.0)
2.29, dd (14.1, 6.8)
4.08, m
H-12
H-12
12
13
14
70.1, CH
56.8, CH
36.7, CH2
H-11a, H-11b, H-14b
H-11a, H-11b, H-14a, H-14b
H-16
H-11a, H-11b, H-13, H-14a, H-14b
H-11a, H-11b, H-12, H-14a, H-14b
4.10, m
2.95, dd (13.7, 4.5)
2.84, dd (13.7, 10.5)
H-13
H-13
15
140.1, qC
130.4, CH
129.3, CH
127.2, CH
174.3, qC
54.5, CH
30.2, CH2
H-14a, H-14b, H-17
H-14a, H-14b, H-18
16 and 20
7.22, d (7.4)
7.19, t (7.4)
7.11, t (7.4)
H-17
H-17
17 and 19
18
21
22
23
H-16
Arg
H-13, H-22
H-23a
4.19, dd (7.6, 6.2)
1.60, m
H-23a, H-23b, H-24a, H-24b, H-25
H-22, H-24
H-25
H-22, H-24b, H-25
1.42, m
24
26.1, CH2
1.40, m
H-22, H-23a, H-25
1.31, m
25
41.9, CH2
157.3, qC
40.5, CH2
119.6, CH
138.7, qC
25.8, CH3
18.0, CH3
175.9, qC
36.4, CH2
29.0, CH2
35.5, CH2
133.8, qC
130.4, CH
116.1, CH
156.5, qC
3.07, t (5.9)
26
10
20
30
40
50
H-25, H-10
N-prenyl
3.76, d (7.1)
5.24, t (7.1)
H-20, H-40, H-50
H-20
H-10, H-40, H-50
H-10, H-40, H-50
H-20, H-50
H-10, H-40, H-50
1.74, s
1.69, s
H-20
H-20
H-20, H-40
HO-Ph-Bu
27
H-22, H-28, H-29
H-29, H-30
H-28, H-30
H-28, H-29, H-32
H-29, H-30, H-33
H-30
28
2.23, t (7.6)
1.83, p (7.6)
2.52, t (7.6)
29
H-28, H-30
H-33
30
H-28, H-29
31
32 and 36
33 and 35
34
6.99, d (8.4)
6.68, d (8.4)
H-32
H-32, H-33
NH(1)a
7.75, d (8.1)
a Protons observed in MeOH-d3.
significant research, the timing of this process, how it is regulated
in the tumor microenvironment, and how it varies with cell type
still remain major unanswered questions in cancer biology.17 The
lack of more detailed mechanisms of action for the full range of
MMPs in the tumor microenvironment, and of the other
physiological roles of MMPs, undoubtedly contributed to the
lack of efficacy observed in the clinical trials of broad spectrum
MMP inhibitors.18 In the past few years, there has been a
resurgence of interest in these targets as they have been
discovered to modulate a diverse array of biochemical processes
in cancer, including growth signals, tumor angiogenesis, apop-
tosis, neoplastic progression, invasion, and metastasis.17 In
particular, MMP12, secreted macrophage metalloelastase, has
recently been shown to promote glioma invasion and to be
upregulated in gliomas by the matrix glycoprotein tenascin C.19 It
is also proinflammatory, as MMP12 overexpression has been
implicated in emphysema,20 osteoarthritis,21 atherosclerosis,22
and chronic obstructive pulmonary disease.23
Matrix metalloproteinases have attracted considerable interest
as targets for antiangiogenic agents, although it is now recognized
that isozyme selective inhibitors are needed to exploit these
targets effectively.17 Inhibitors of MMP2 and MMP9, based
primarily on hydroxamate zinc-binding motifs, were evaluated
in clinical trials, although none displayed the requisite safety and
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Table 2. Protease Screening of 1
target protease
IC50 (μM)
inactive
ADAM9
MMP12
2.3
11
20
26
37
49
65
84
cathepsin E
cathepsin D
DDP IV
ADAM10
ACE1
calpain
MMP2
caspase 3
TACE
cathepsin B
MMP9
trypsin
caspase 9
chymotrypsin
elastase
BACE1
Figure 7. Effects of stictamide A (1) on U78-MG cells after 20 h. (A)
Cell viability (5 μM). (B) Cell invasion (5 μM). The fluorescence
emission (RFU) is proportional to the number of invasive cells. Error
bars indicate the standard error of the mean.
mation, angiogenesis, cancer cell growth, cell death, and cell
invasion.32,33 In particular, MMP2, MMP12, cathepsin B, and
cathepsin D are known to contribute to glioma cell invasion.32ꢀ37
As such, we examined the effect of 1 on invasion of a glioblastoma
derived cell line (U87-MG) through a three-dimensional extra-
cellular matrix (Matrigel) in an in vitro transwell assay. At a
concentration of 5 μM, cell invasion was significantly affected in
the treated cells (Figure 7B). To exclude any possible cytotoxic
effects of 1 on U87-MG cells, we examined cell viability after
incubation with 5 μM (Figure 7A) or 50 μM (data not shown) of
stictamide A. Compared to control samples that were incubated
with the DMSO vehicle, 1 did not significantly alter U87-MG
viability at either concentration (Figure 7A). Therefore, at a
concentration that predominantly impairs MMP12 function in
enzymatic systems, 1 significantly reduces invasion of U87-MG
cells in vitro.
Figure 6. Docked structure of 1 with MMP12.
efficacy profiles required for phase III approval,18 which suggests
a more complex interplay of factors than previously realized.
Carboxylate inhibitors of MMPs have also been reported that
bind to the zinc ion,24 which, given the presence of this
functionality in 1, suggested a possible mechanism by which 1
was inhibiting MMP12.
The interaction of 1 with MMP12 was further examined by
docking with AutoDock Vina 1.1.1,25 using the X-ray structure of
MMP12(PDB code 1RMZ).26 Watermolecules within the crystal
structure were removed, while hydrogens attached to electroneg-
ative atoms and Gasteiger charges were added using AutoDock-
Tools. Optimized zinc parameters were applied to improve the
accuracy of the docking.27 Ligands, which were optimized for their
energy and geometry using MM2 and AM1 force fields, were
docked into this protein structure. Using these parameters, ligand
NNGH was successfully docked into MMP12, with a rmsd of
0.84 Å compared to its original crystal structure. Stictamide A (1)
was then docked as the carboxylate and the guanidinium zwitter-
ion to reflect a physiologically relevant state (Figure 6). These
results suggest that 1 and MMP12 interact via a non-zinc-binding
mode, rather than the more common carboxylateꢀzinc interac-
tion. Non-zinc-binding inhibitors of MMP12 have been pre-
viously reported that exploit the deep hydrophobic S10 pocket
for both potency and selectivity, including those with carboxylate
termini.28 In the case of 1, the 4-(4-hydroxyphenyl)butanoic acid
residue is situated within this critical pocket potentially stabilized
by interactions with Phe237, Phe248, and Val 235. Given the low
homology of S10 pockets in MMPs, optimizing interactions within
this unit will be crucial for improving selectivity and potency.29ꢀ31
As noted above, 1 inhibited several proteases that can affect
the tumor microenvironment leading to changes in inflam-
’ EXPERIMENTAL PROCEDURES
Collection and Identification. The sample designated 122899-
SOLE-5 was collected on the river banks of “RIO SOLE” in the
community of Guaca, near David City, Chiriqui, Panama, on December
28, 1999. A NCBI BLAST analysis of the ITS sequence of this strain
showed the highest identity to the lichen genus Sticta. The multiple
sequences available for this genus on Genbank were downloaded and
aligned in MEGA 4. A sequence of the closely related genus Lobaria
(Lobaria linita AB239702) was also included. Ambiguous parts of the
alignment were trimmed, leaving 388 bp for sequence clustering using
neighbor joining (maximum likelihood composite model with a 1000
bootstrap and pairwise deletion option). Branch support >70% is not
displayed on the resulting tree. The labels for the polyphyletic species
were also not included for tree clarity, as they represent taxonomic
discrepancies between researchers.
The analysis demonstrated that the Sticta species investigated lay on a
long branch and was well separated from other members of the genus
that have been characterized using molecular methods to date. A
voucher specimen has been stored at the UH Manoa, Department of
Chemistry, and is available upon request. Sequences obtained from the
specimen have been deposited in GenBank under accession numbers
HQ149042ꢀHQ149045.
Extraction and Isolation. A portion of the freeze-dried sample
was exhaustively extracted with MTBE/MeOH (2:8) to yield 9.6 g of
crude extract, which after trituration with MeOH afforded 3.7 g of a
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FEATURED ARTICLE
desalted extract. This mixture was separated on a C8 flash column (25 g)
eluting with a step gradient of 25, 50, 75, and 100% MeOH (ꢁ2) in H2O
to provide five fractions (AꢀE) of 2.8 g, 87 mg, 78 mg, 203 mg, and 33
mg, respectively. LC-MS analyses of these fractions revealed that three of
them contained a series of compounds with molecular weights between
600 and 800 amu. Separation of fraction C (78 mg) by reversed-phase
HPLC [Onyx C18, 100 ꢁ 10 mm, a linear gradient over 35 min from 5 to
100% MeCN in H2O with 0.1% formic acid added to both solvents, flow
rate 5 mL/min, PDA and ELSD detection] afforded stictamide A (1)
(tR 13.7 min, 2.5 mg). Separation of fraction B (87 mg) by RP-HPLC
[Onyx C18, 100 ꢁ 10 mm, a linear gradient over 15 min from 15 to 50 %
MeCN in H2O then 50 to 100% over 10 min with 0.1 % formic acid
added to both solvents, flow rate 5 mL/min, PDA detection] afforded
stictamide A (tR 9.6 min, 2.0 mg, 0.047 % total yield) and stictamide B
(2) (tR 7.4 min, 3.1 mg, 0.032 % yield). Separation of fraction D (203
mg) using the same conditions as for fraction C reported above, yielded
impure stictamide C (tR 15.6 min), which was repurified by RP-HPLC
[Luna C8, 150 ꢁ 4.6 mm, a linear gradient from 5 to 50% aqueous
MeCN with 0.1% formic acid in both solvents over 30 min, flow rate
0.7 mL/min, PDA] to yield stictamide C (3) (tR 29.5 min, 0.2 mg,
0.002% yield).
Benzylamidation of 1 (5). A sample containing 2 mg of EDCI
(13 μmol), 2 mg of HOBt (15 μmol), and 1.4 mg of 1 (1.9 μmol) was
dissolved in 500 μL of CH2Cl2 and cooled in an ice bath under a N2
atmosphere. To this solution was added 10 μL of benzylamine
(90 μmol). A second and third aliquot of EDCI (32 μmol each time)
were added on days 2 and 3, while the reaction stirred at room
temperature. The reaction was quenched after a total of 7 days by the
addition of 1 N HCl and residue partitioned between H2O and EtOAc.
The final product was purified from the organic residue by RP-HPLC
[Luna C8, 150 ꢁ 4.6 mm, a linear gradient over 40 min using 20ꢀ80%
aqueous MeCN with 0.1% formic acid in both solvents, flow rate
0.7 mL/min, PDA detection] to afford 1.3 mg of 5 (83% yield). 5:
Light yellow amorphous powder; [R]22 0.4 (c 0.2, MeOH); UV
D
(MeOH) λmax (log ε) 225 (7.4), 278 (5.7) nm; IR (CaF2) νmax 3395,
2965, 2931, 2820, 1639, 1593, 1515, 1454, 1385, 1353, 1085 cmꢀ1; 1H
NMR (500 MHz, MeOH-d4) δH 1.30 (m, 1H, H-24b), 1.35 (m, 1H,
H-24a), 1.45 (m, 1H, H-23b), 1.60 (m, 1H, H-23a), 1.70 (s, 3H, H-50),
1.75 (s, 3H, H-40), 1.83 (p, J = 7.6 Hz, 2H, H-29), 2.18 (t, J = 7.6 Hz, 2H,
H-28), 2.32 (dd, J = 14.4, 7.2 Hz, 1H, H-11b), 2.40 (dd, J = 14.4, 7.2 Hz,
1H, H-11a), 2.48 (t, J = 7.6 Hz, 2H, H-30), 2.77 (dd, J = 13.0, 6.7 Hz, 1H,
H-14b), 2.79 (m, 1H, H-3b), 2.86 (dd, J = 13.0, 5.5 Hz, 1H, H-14a), 2.96
(m, 2H, H-25), 3.02 (m, 1H, H-3a), 3.76 (d, J = 6.8 Hz, 2H, H-10), 4.05
(td, J = 7.2, 1.4 Hz, 1H, H-12), 4.09 (m, 1H, H-22), 4.11 (m, 1H, H-13),
4.32 (d, J = 4.6 Hz, 2H, H-60), 4.47 (dd, J = 8.2, 5.9 Hz, 1H, H-2), 5.24 (t,
J = 6.8 Hz, 1H, H-20), 6.58 (d, J = 8.3 Hz, 2H, H-6, H-8), 6.59 (d, J = 8.3
Hz, 2H, H-33, H-35), 6.87 (d, J = 8.3 Hz, 2H, H-32, H-36), 6.92 (d, J =
8.3 Hz, 2H, H-5, H-9), 7.11 (m, 1H, H-18), 7.12 (m, 2H, H-16, H-20),
7.17 (m, 3H, H-17, H-19, H-100), 7.18 (m, 2H, H-80, H-120), 7.26 (t, J =
7.4 Hz, 2H, H-90, H-110); HRESI-TOFMS m/z [M]þ 848.4704 [calcd
for C48H62N7O7þ, 848.4711, 0.8 ppm error].
Stictamide A (1): Light yellow amorphous powder; [R]22 ꢀ0.2
D
(c 0.2, MeOH), UV (MeOH) λmax (log ε) 219 (8.9), 276 (6.8) nm; IR
(CaF2) νmax 3399, 2924, 2856, 1629, 1449, 1383, 1260, 1088 cmꢀ1; see
Table 1 for tabulated spectral data; HRESI-TOFMS m/z [M]þ
759.4082 [calcd for C41H55N6O8þ, 759.4081, ꢀ0.1 ppm error].
Stictamide B (2): Light yellow amorphous powder; [R]22 ꢀ0.5
D
(c 0.2, MeOH); UV (MeOH) λmax (log ε) 219 (9.3), 276 (7.5) nm; IR
(CaF2) νmax 3275, 2830, 2361, 1591, 1514, 1362, 1245 cmꢀ1; see Table
S2 in the Supporting Information for tabulated spectral data; HRESI-
TOFMS m/z [M]þ 691.3457 [calcd for C36H47N6O8þ, 691.3455, ꢀ0.2
ppm error].
Methylation and Fmoc Protection (6). A 300 μL aliquot of 0.4 M
TMSCHN2 in toluene (120 μmol) was added to a solution of 1.3 mg of 5
(1.5 μmol) dissolved in 200 μL of methanol. After stirring for 4 h at
room temperature, the solvent was removed and the crude reaction
mixture was purified by RP-HPLC [Luna C8, 150 ꢁ 4.6 mm, a linear
gradient over 40 min using 20ꢀ80% aqueous MeCN with 0.1% formic
acid in both solvents, flow rate 0.7 mL/min, PDA detection] to afford
the dimethyl product in 90% yield (1.2 mg). This product (1.4 μmol)
was dissolved in 9% Na2CO3 (150 μL) and dioxane (150 μL). After
stirring in an ice bath for 15 min, 50 μL of a 0.28 M solution of Fmoc-
OSu (14 μmol) was added to the reactants and the mixture was stirred at
0 ꢀC for an additional 30 min before warming to room temperature. Two
additional aliquots of 0.28 M Fmoc-OSu (each 14 μmol in 50 μL) were
added on days 2 and 4. On day 6, the solvent was removed in vacuo and
then the residue was partitioned between EtOAc and H2O. The organic
residue was purified by RP-HPLC [Luna C8, 150 ꢁ 4.6 mm, a linear
gradient over 40 min using 20ꢀ80% aqueous MeCN with 0.1% formic
acid in both solvents, flow rate 0.7 mL/min, PDA detection] to afford 6
Stictamide C (3): Light yellow amorphous powder; [R]22D 0.1 (c 0.2,
MeOH); UV (MeOH) λmax (log ε) 219 (7.5), 276 (6.4) nm; IR (CaF2)
νmax 3404, 2951, 2925, 2853, 1628, 1454, 1385, 1353, 1318, 1088 cmꢀ1
;
1H NMR (500 MHz, MeOH-d4) δH 1.31ꢀ1.60 (m, 4H, H-23a, H-23b,
H-24a, H-24b), 1.70 (s, 3H, H-50), 1.75 (s, 3H, H-40), 1.84 (p, J = 7.3 Hz,
2H, H-29), 2.15 (t, J = 7.3 Hz, 2H, H-28), 2.23 (dd, J = 14.0, 7.0 Hz, 1H,
H-11b), 2.35 (dd, J = 14.0, 7.0 Hz, 1H, H-11a), 2.51 (t, J = 7.3 Hz, 2H,
H-30), 2.87 (dd, J = 13.9, 9.0 Hz, 1H, H-14b), 2.92 (dd, J = 14.3, 6.5 Hz,
1H, H-3b), 2.99 (dd, J = 13.9, 5.7 Hz, 1H, H-14a), 3.04 (br, 2H, H-25),
3.18 (dd, J = 14.3, 4.6 Hz, 1H, H-3a), 3.64 (s, 3H, H-60), 3.77 (d, J = 6.2
Hz, 2H, H-10), 4.00 (dt, J = 7.0, 1.5 Hz, 1H, H-12), 4.19 (dd, J = 6.0, 4.8
Hz, 1H, H-22), 4.24 (ddd, J = 9.0, 5.7, 1.5 Hz, 1H, H-13), 4.54 (dd, J =
6.5, 4.6 Hz, 1H, H-2), 5.25 (t, J = 6.2 Hz, 1H, H-20), 6.67 (d, J = 8.1 Hz,
2H, H-6, H-8), 6.69 (d, J = 7.8 Hz, 2H, H-33, H-35), 6.97 (d, J = 7.8 Hz,
2H, H-32, H-36), 7.00 (d, J = 8.1 Hz, 2H, H-5, H-9), 7.14 (m, 1H, H-18),
7.22 (m, 2H, H-17, H-19), 7.24 (m, 2H, H-16, H-20); HRESI-TOFMS
m/z [M]þ 773.4243 [calcd for C42H57N6O8þ, 773.4238, ꢀ0.6 ppm error].
Trimethyl Stictamide A (4). A 150 μL aliquot of 0.2 M TMSCHN2 in
toluene (30 μmol) was added to a solution of 0.5 mg of 1 (0.7 μmol) in
100 μL of methanol. After stirring for 3 h at room temperature, another
100 μL of 0.2 M TMSCHN2 (20 μmol) was added and the solution
stirred for an additional 1 h before the solvent was removed under a
vacuum to afford the crude reaction mixture. The final product was
purified by RP-HPLC [Luna C8, 150 ꢁ 4.6 mm, a linear gradient over 40
min using 20ꢀ80% aqueous MeCN with 0.1% formic acid in both
solvents, flow rate 0.7 mL/min, PDA detection] to afford 4 (tR 19.5 min,
0.4 mg, 76% yield). 4: Light yellow amorphous powder; [R]22D ꢀ0.4 (c
0.2, MeOH); UV (MeOH) λmax (log ε) 225 (7.9), 276 (6.1) nm; IR
(CaF2) νmax 3442, 2957, 2926, 2851, 1638, 1596, 1513, 1450, 1385,
1353, 1319, 1247, 1085 cmꢀ1; see Table S3 in the Supporting Informa-
tion for tabulated spectral data; HRESI-TOFMS m/z [M]þ 801.4547
[calcd for C44H61N6O8þ, 801.4551, 0.5 ppm error].
(1.2 mg, 80% yield). 6: White amorphous powder; [R]22 0.5 (c 0.2,
D
MeOH); UV (MeOH) λmax (log ε) 219 (8.8), 228 (8.6), 265 (7.8), 300
(4.3) nm; IR (CaF2) νmax 3440, 2952, 2923, 2852, 1593, 1513, 1450,
1383, 1352, 1318, 1247, 1085 cmꢀ1; see Table S4 in the Supporting
Information for tabulated spectral data; HRESI-TOFMS m/z [M þ
H]þ 1098.5667 [calcd for C65H76N7O9þ, 1098.5705, 3.5 ppm error],
m/z [M þ Na]þ 1120.5503 [calcd for C65H75N7O9Naþ, 1120.5524, 1.9
ppm error].
Advanced Marfey Analysis. A 500 μg sample of 2 was hydro-
lyzed at 118 ꢀC for 10 h with 200 μL of 6 N HCl. The hydrolysate was
passed over a small C8 column and the resulting eluant divided into two
portions before drying. Each portion was derivatized by adding 100 μL
of H2O, 50 μL of 1 M sodium bicarbonate, and 50 μL of 6 mg/mL of
either L- or DL-FDLA in acetone. After stirring for 3 min at 81 ꢀC, the
reaction was quenched by the addition of 50 μL of 1 N HCl and then
diluted with 150 μL of acetonitrile. The resulting L- and DL-FDLA
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FEATURED ARTICLE
derivatives were analyzed by ESI-LCMS-TOF [Luna C8, 150 ꢁ 4.6 mm,
a linear gradient with 30ꢀ50% aqueous MeCN with 0.01% TFA in both
solvents over 50 min, flow rate 0.7 mL/min], and the retention times
were compared with those of standards. The retention times of the
DL-FDLA-derivatized (3S,4S)-Ahppa standards were 32.6 and 45.7 min,
while the retention times of the DL-FDLA-derivatized (3R,4S)-Ahppa
standards were 32.2 and 44.2 min. The retention times of the L-FDLA-
derivatized amino acids from the hydrolysate were D-Arg (11.3 min),
L-Tyr (27.1 min), and (3S,4S)-Ahppa (32.4 min). The retention times of
the D-FDLA-derivatized amino acids from the DL-FDLA-derivatized
hydrolysate were D-Arg (14.4 min), L-Tyr (30.1 min), and (3S,4S)-
Ahppa (45.7 min).
’ ASSOCIATED CONTENT
S
Supporting Information. Tabulated NMR data for 1, 2,
b
4, and 6 along with H, 13C, gCOSY, gHMBC, and gHSQC
NMR spectra for 1, 2, and 6; 1H and gHMBC spectra for 4 and 5,
and ESI-LC-MS-TOF chromatograms of amino acid FDLA
derivatives. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 808-956-5720. Fax: 808-956-5908. E-mail: philipwi@
hawaii.edu.
Cell Invasion Assay. ThinCert tissue culture inserts (8.0 μm pore
size) were coated overnight at 4 ꢀC with 10 μg/mL human plasma
fibronectin. The inside of the cell culture inserts was then coated with
Matrigel Basement Membrane Matrix (growth factor reduced) accord-
ing to the manufacturer’s protocol. The matrix was supplemented with
5 or 50 μM of 1 or DMSO control. On the day of the assay, U87-MG
cells were trypsinized and a total of 1 million cells were plated on the
coated tissue culture inserts in D-MEM that had been supplemented
with non-essential amino acids. Inserts were placed in the wells of a
24-well plate filled with D-MEM supplemented with non-essential
amino acids and 10 ng/mL EGF. Cells were incubated for 20 h at
37 ꢀC, 5% CO2. Cell culture medium was then removed from the wells,
replaced with D-MEM containing 8 μM calcein-AM, and incubated for
45 min at 37 ꢀC under 5% CO2. Then, the culture medium was removed
from the tissue culture inserts, and the inserts were transferred to a
freshly prepared well containing 0.05% trypsin-EDTA. After a 10 min
incubation at 37 ꢀC under 5% CO2 with sporadic agitation, the inserts
were discarded. The trypsin-EDTA solution then contained the de-
tached invasive cells. Invasion was quantified by measuring the fluores-
cence emission of calcein-AM at 520 nm upon excitation at 485 nm in
black 96-well plates with a fluorescence plate reader. Experiments were
done in duplicate, and fluorescence emissions were measured in
triplicate. All data are shown as mean plus standard error of the mean
(SEM). Statistical significance was calculated using the Student’s t test.
The p values of <0.05 were considered to be statistically significant.
Cell Viability Assay. U87-MG cells (104 per well) were incubated
with 1 at a concentration of 5 or 50 μM in a 96-well plate. Control
samples were incubated with the carrier DMSO in an equivalent volume.
After an incubation time of 20 h, cell viability was measured using the
alamarBlue cell viability reagent according to the manufacturer’s proto-
col. Cell viability was determined by measuring the fluorescence
emission intensity at 590 nm upon excitation at 560 nm with a
fluorescence plate reader. Samples were prepared in quintuplicate, but
the experiment was repeated once. All data are shown as mean plus
standard error of the mean (SEM). Statistical significance was calculated
using the Student’s t test. The p values of <0.05 were considered to be
statistically significant.
’ ACKNOWLEDGMENT
This work was funded by grants from the Alzheimer’s Associa-
tion (NIRG-08-90880), Victoria S. and Bradley L. Geist Founda-
tion, the Alzheimer’s Drug Discovery Foundation (281204), the
National Institute of Aging (1R21AG032405), and NIGMS
(R01GM088266 to J.R.). Funds for the upgrades of the NMR
instrumentation were provided by the CRIF program of the
National Science Foundation (CH E9974921) and the Elsa
Pardee Foundation. The purchase of the Agilent LC-MS was
funded by Grant W911NF-04-1-0344 from the Department of
Defense. We thank A. Preciado for the phenylalanine-statine
standards, F.D. Horgen and T. Vansach for help with acquisition
of the LC-MS spectra, and B. Rubio for the serine protease data.
’ REFERENCES
(1) Morishima, H.; Takita, T.; Aoyagi, T.; Takeuchi, T.; Umezawa,
H. J. Antibiot. 1970, 25, 263–265.
(2) Morishima, H.; Sawa, T.; Takita, T.; Aoyagi, T.; Takeuchi, T.;
Umezawa, H. J. Antibiot. 1974, 27, 267–273.
(3) Rinehart, K.; Gloer, J.; Hughes, R.; Renis, H.; McGovren, J.;
Swynenberg, E.; Stringfellow, D.; Kuentzel, S.; Li, L. Science 1981,
212, 933–935.
(4) Stratmann, K.; Burgoyne, D. L.; Moore, R. E.; Patterson,
G. M. L.; Smith, C. D. J. Org. Chem. 1994, 59, 7219–7226.
(5) Marciniszyn, J.; Hartsuck, J. A.; Tang, J. J. Biol. Chem. 1976,
251, 7088–7094.
(6) Fehrentz, J. A.; Chomier, B.; Bignon, E.; Venaud, S.; Chermann,
J. C.; Nisato, D. Biochem. Biophys. Res. Commun. 1992, 188, 865–872.
(7) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591–596.
(8) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K.-I. Anal. Chem.
1997, 69, 5146–5151.
(9) Preciado, A.; Williams, P. G. J. Org. Chem. 2008, 73, 9228–9234.
(10) Rinehart, K. L.; Sakai, R.; Kishore, V.; Sullins, D. W.; Li, K. M.
J. Org. Chem. 1992, 57, 3007–3013.
(11) Vervoort, H.; Fenical, W.; Epifanio, R. de A. J. Org. Chem. 2000,
65, 782–792.
(12) K€uhnel, E.; Laffan, D. D. P.; Lloyd-Jones, G. C.; Martínez del
Campo, T.; Shepperson, I. R.; Slaughter, J. L. Angew. Chem., Int. Ed.
2007, 46, 7075–7078.
(13) Williams, P. G.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J. J. Nat.
Prod. 2003, 66, 1006–1009.
(14) Sato, T.; Shibazaki, M.; Yamaguchi, H.; Abe, K. J. Antibiot. 1994,
47, 588–590.
(15) Gunasekera, S. P.; Miller, M. W.; Kwan, J. C.; Luesch, H.; Paul,
Molecular Docking. Compound 1 was docked with MMP12 using
the X-ray structure of MMP12 (PDB code 1RMZ)26 and AutoDock
Vina 1.1.1.25 Water molecules within the crystal structure were removed
and polar hydrogens and Gasteiger charges were added using Auto-
DockTools. Optimized zinc parameters were applied to improve dock-
ing accuracy.27 Ligands were optimized for their energy and geometry
using MM2 and AM1 force fields before docking. In the structures of the
ligands, all bonds were treated as rotatable except for the aromatic,
alkenyl, amide, and guanidino bonds. Stictamide A (1) was docked as the
carboxylate and the guanidinium zwitterion to reflect a physiologically
relevant state. The dimensions of the grid map were 26 ꢁ 24 ꢁ 26 points
with a grid-point spacing of 1 Å. The center of the grid was set to
coincide with the catalytic zinc atom. Docking proceeded with an
exhaustiveness value of 600 and a maximum output of 100 structures.
V. J. J. Nat. Prod. 2010, 73, 459–462.
(16) Liotta, L. A.; Tryggvason, K.; Garbisa, S.; Hart, I.; Foltz, C. M.;
Shafie, S. Nature 1980, 284, 67–68.
(17) Kessenbrock, K.; Plaks, V.; Werb, Z. Cell 2010, 141, 52–67.
3642
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The Journal of Organic Chemistry
FEATURED ARTICLE
(18) Coussens, L. M.; Fingleton, B.; Matrisian, L. M. Science 2002,
295, 2387–2392.
(19) Sarkar, S.; Nuttall, R. K.; Liu, S.; Edwards, D. R.; Yong, V. W.
Cancer Res. 2006, 66, 11771–11780.
(20) Houghton, A. M.; Quintero, P. A.; Perkins, D. L.; Kobayashi,
D. K.; Kelley, D. G.; Marconcini, L. A.; Mecham, R. P.; Senior, R. M.;
Shapiro, S. D. J. Clin. Invest. 2006, 116, 753–759.
(21) Liu, M.; Sun, H.; Wang, X.; Koike, T.; Mishima, H.; Ikeda, K.;
Watanabe, T.; Ochiai, N.; Fan, J. Arthritis Rheum. 2004, 50, 3112–3117.
(22) Halpert, I.; Sires, U. I.; Roby, J. D.; Potter-Perigo, S.; Wight,
T. N.; Shapiro, S. D.; Welgus, H. G.; Wickline, S. A.; Parks, W. C. Proc.
Natl. Acad. Sci. U.S.A. 1996, 93, 9748–9753.
(23) Shapiro, S. D.; Kobayashi, D. K.; Ley, T. J. J. Biol. Chem. 1993,
268, 23824–23829.
(24) Li, W.; Li, J.; Wu, Y.; Rancati, F.; Vallese, S.; Raveglia, L.; Wu, J.;
Hotchandani, R.; Fuller, N.; Cunningham, K.; Morgan, P.; Fish, S.;
Krykbaev, R.; Xu, X.; Tam, S.; Goldman, S. J.; Abraham, W.; Williams,
C.; Sypek, J.; Mansour, T. S. J. Med. Chem. 2009, 52, 5408–5419.
(25) Trott, O.; Olson, A. J. J. Comput. Chem. 2010, 31, 455–461.
(26) Bertini, I.; Calderone, V.; Cosenza, M.; Fragai, M.; Lee, Y.-M.;
Luchinat, C.; Mangani, S.; Terni, B.; Turano, P. Proc. Natl. Acad. Sci. U.S.
A. 2005, 102, 5334–5339.
(27) Hu, X.; Shelver, W. H. J. Mol. Graphics Modell. 2003,
22, 115–126.
(28) Dublanchet, A.-C.; Ducrot, P.; Andrianjara, C.; O’Gara, M.;
Morales, R.; Compꢁere, D.; Denis, A.; Blais, S.; Cluzeau, P.; Courtꢀe, K.;
Hamon, J.; Moreau, F.; Prunet, M.-L.; Tertre, A. Bioorg. Med. Chem. Lett.
2005, 15, 3787–3790.
(29) Pirard, B.; Matter, H. J. Med. Chem. 2006, 49, 51–69.
(30) Morales, R.; Perrier, S.; Florent, J.-M.; Beltra, J.; Dufour, S.; De
Mendez, I.; Manceau, P.; Tertre, A.; Moreau, F.; Compere, D.; Dublan-
chet, A.-C.; O’Gara, M. J. Mol. Biol. 2004, 341, 1063–1076.
(31) Bertini, I.; Calderone, V.; Fragai, M.; Luchinat, C.; Maletta, M.;
Yeo, K. J. Angew. Chem., Int. Ed. 2006, 45, 7952–7955.
(32) Gabelloni, P.; Da Pozzo, E.; Bendinelli, S.; Costa, B.; Nuti, E.;
Casalini, F.; Orlandini, E.; Da Settimo, F.; Rossello, A.; Martini, C.
Neuroscience 2010, 2, 514–522.
(33) Rao, J. S. Nat. Rev. Cancer 2003, 3, 489–501.
(34) Uhm, J. H.; Dooley, N. P.; Villemure, J. G.; Yong, V. W. Clin.
Exp. Metastasis 1996, 14, 421–433.
(35) Abe, T.; Mori, T.; Kohno, K.; Seiki, M.; Hayakawa, T.; Welgus,
H. G.; Hori, S.; Kuwano, M. Clin. Exp. Metastasis 1994, 12, 296–304.
(36) Sarkar, S.; Nuttall, R. K.; Liu, S.; Edwards, D. R.; Yong, V. W.
Cancer Res. 2006, 66, 11771–11780.
(37) Sivaparvathi, M.; Sawaya, R.; Chintala, S. K.; Go, Y.; Gokaslan,
Z. L.; Rao, J. S. Neurosci. Lett. 1996, 208, 171–174.
3643
dx.doi.org/10.1021/jo200241h |J. Org. Chem. 2011, 76, 3635–3643