Antineoplastic agents. 600. from the south pacific ocean to the silstatins

Article abstract of DOI:10.1021/np501004h

The recent advances in the development of antibody and other drug conjugates for targeted cancer treatment have further increased the need for powerful cancer cell growth inhibitors. Toward that objective we have extended our earlier discovery of the remarkable anticancer bacillistatins 1 and 2 from Bacillus silvestris to SAR and other structural modifications such as availability of a free hydroxy group for antibody-drug conjugate (ADC) and other prodrug linkage. That direction has resulted in seven structural modifications designated silstatins 1-8 (7a, 8a, 8b, 14a, 15a, 15b, 18a, and 18b), where the exceptional cancer cell growth inhibition of some of them are in the range GI₅₀ 10^-3-10^-4 μM/mL. Silstatin 7 (18a) was converted to a glucuronic conjugate (28) that displayed an impressive reduction in toxicity during transport.

Full text of DOI:10.1021/np501004h

Article
pubs.acs.org/jnp
Antineoplastic Agents. 600. From the South Pacific Ocean to the
Silstatins
George R. Pettit,* Pablo M. Arce, Jean-Charles Chapuis, and Christian B. Macdonald
Department of Chemistry and Biochemistry, Arizona State University, P.O. Box 871604, Tempe, Arizona 85287-1604, United States
S
* Supporting Information
ABSTRACT: The recent advances in the development of
antibody and other drug conjugates for targeted cancer
treatment have further increased the need for powerful cancer
cell growth inhibitors. Toward that objective we have extended
our earlier discovery of the remarkable anticancer bacillistatins
1 and 2 from Bacillus silvestris to SAR and other structural
modifications such as availability of a free hydroxy group for
antibody−drug conjugate (ADC) and other prodrug linkage.
That direction has resulted in seven structural modifications
designated silstatins 1−8 (7a, 8a, 8b, 14a, 15a, 15b, 18a, and
18b), where the exceptional cancer cell growth inhibition of
some of them are in the range GI50 10−3−10−4 μM/mL.
Silstatin 7 (18a) was converted to a glucuronic conjugate (28) that displayed an impressive reduction in toxicity during transport.
n 2009 we reported the isolation and structures of two very
potent cancer cell growth inhibitors, cyclodepsipeptides
I
designated bacillistatins 1 and 21,2 from Bacillus silvestris carried
by a South Pacific (Chile) crab. Subsequently, we completed
the total synthesis of bacillistatin 2.2 Meanwhile, the promise of
marine-derived microorganisms as productive sources of new
anticancer and antiproliferative drugs continues to expand.
Recent examples include discoveries of marine microorganisms
containing small-molecule cancer cell growth inhibitors,3a−h
antibiotics from bacteria,4a−f antibiotics from fungi,5a−f various
inhibitors from cyanobacteria,6a−c and the increasing potential
of deep-sea microorganisms.7a−d Structurally, the bacillistatins
are similar to valinomycin, a well-known antibiotic and
cytotoxic cyclodepsipeptide that acts as a carrier-type potassium
ionophore. Another cyclic depsipeptide that resembles the
structures of valinomycin and bacillistatins is cereulide, a toxin
isolated in 1995 from Bacillus cereus.8
The cancer cell growth inhibition values for these molecules
are on the order of 10−4−10−5 μg/mL, which makes them good
candidates as therapeutic agents for cancer treatment, especially
when linked to monoclonal antibodies. That is a powerful
technique to reduce side effects and increase selectivity as well
as other targeting systems that allow the release of the drug,
from a nontoxic precursor (prodrug), in the solid tumor
microenvironment or inside the cancer cells. One of the major
obstacles for this approach with the bacillistatins is the lack of
easily derivatizable groups (e.g., −NH2, −SH, −OH, −COOH,
etc.) necessary for conjugation of these K+ ionophores with
antibodies/carriers.
overall lipophilicity of the modified bacillistatins containing a
polar group (hydroxy) can lead to compounds with high cancer
cell growth inhibition. Also it can provide useful bacillistatin
conjugates such as the glucuronide prodrug of structure 18a.
Therefore, we undertook the synthesis and biological
evaluation of a series of structural modifications of the
bacillistatins containing hydroxy groups that could serve as
coupling handles. Subsequent results showed that managing the
Special Issue: Special Issue in Honor of William Fenical
Received: December 12, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/np501004h
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Scheme 1. Synthesis of Silstatins 1, 2, and 3 (7a, 8a, and 8b)
and one with the D configuration in alternating positions. The L
amino acid was used as the N terminus of each fragment to
produce each fragment sequence as L-(amino acid)-D-(α-
hydroxy acid)-D-Val-L-lactic acid.
The first series of new structures (7a, 8a, and 8b, named
silstatins 1, 2, and 3, respectively) was synthesized as shown in
Scheme 1. First, compound 1 was prepared by coupling benzyl
lactate with Boc-D-Val-OH using dicyclohexylcarbodiimide
(DCC) in the presence of 4-dimethylaminopyridine (DMAP)
in high yield. Compound 1 was N-deprotected using
RESULTS AND DISCUSSION
■
The structure of bacillistatin 1 consists of six amino acids (three
with L configuration and three with D configuration) and six α-
hydroxy acids (three with L configuration and three with D
configuration). These 12 units are symmetrically arranged along
the structure, thus forming three principal fragments (with four
units each) that are attached head to tail and cyclized. Using
this analysis we proceeded to synthesize fragments containing
four units that consisted of two amino acids, one with L and one
with the D configuration, and two α-hydroxy acids, one with L
B
DOI: 10.1021/np501004h
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Scheme 2. Synthesis of Silstatins 4, 5, and 6 (14a, 15a, and 15b)
trifluoroacetic acid (TFA) and next coupled to (R)-2-
hydroxyisocaproic acid (Hica) using N-(3-(dimethylamino)-
propyl)-N′-ethylcarbodiimide hydrochloride (EDCI), 1-hy-
droxybenzotriazole (HOBt), and triethylamine (TEA) to afford
alcohol 2 in high yield. Next, compounds 3a, 3b, and 3c were
prepared by coupling alcohol 2 with the corresponding
protected amino acid in high yields using DCC and DMAP.
Compounds 3a, 3b, and 3c were subjected to hydrogenolysis to
obtain compounds 4a, 4b, and 4c in quantitative yields and
were used as obtained. Next, the fragments were attached head
to tail before cyclization. Compound 3a was N-deprotected
using 2,2′,2″-triaminotriethylamine (TAEA) and then coupled
C
DOI: 10.1021/np501004h
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Scheme 3. Synthesis of silstatins 7 and 8 (18a and 18b)
to 4a using N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)-
uronium hexafluorophosphate (HBTU) and TEA to afford
compound 5 in high yield. Compound 5 was treated in the
same way as for 3a and then coupled to the corresponding
fragment (4a, 4b, and 4c) to afford compounds 6a, 6b, and 6c.
The Fmoc and benzyl protecting groups on 6a, 6b, and 6c were
removed in one step using Pd(OH)2-on-carbon and hydrogen.
The crude intermediates were cyclized using bromotripyrroli-
dinophosphonium hexafluorophosphate (PyBroP) and TEA to
afford compounds 7a, 7b, and 7c in low yields. Compounds 7b
and 7c were next treated with TFA to remove the tert-butyl
protecting group from threonine and tyrosine side chains,
obtaining compounds 8a and 8b in high yields, respectively.
For the synthesis of silstatins 4, 5, and 6 (14a, 15a, and 15b)
(Scheme 2), the (R)-2-hydroxyisocaproic acid was replaced
with (R)-2-hydroxy-3-cyclohexylpropionic acid (Hcha), which
was prepared from D-cyclohexylalanine following a literature
procedure.9 The strategy and the series of reactions were the
same as for the preparation of 7a, 8a, and 8b, obtaining similar
yields in each step.
was treated with 4-(methylamino)butyric acid and potassium
carbonate to obtain compound 26, which was partially
separated and then coupled to compound 18a using DCC
and DMAP to afford compound 27 in moderate yield over two
steps. Using 4-(methylamino)butyric acid as a spacer allowed
us to link the drug to the glucuronic acid moiety through a
carbamate and ester bond, which are more stable than a
benzylic carbonate that would have formed otherwise.13 The
allyl protecting groups of compound 27 were finally
deprotected using Pd(PPh3)4 in the presence of triethylammo-
nium formate formed in situ by mixing TEA and formic acid to
afford glucuronide prodrug 28.
The mechanism by which the drug is liberated follows the
same pattern of glucuronide prodrugs already published
(Scheme 5).14 Briefly, the glucuronic acid is cleaved by the
action of β-glucuronidase, and then the nitrobenzyl group
cleaves, forming carbon dioxide and 2-nitroquinone methide,
which reacts with H2O to form 4-hydroxy-3-nitrobenzyl
alcohol. The 4-(methylamino)butyric ester formed self-cleaves
to release the drug (18a) and N-methylpyrrolidone.14c
The last SAR series consisted of a combination of fragments
used in the synthesis of the previous analogues (vide supra).
Intermediate 5 was N-deprotected using TAEA and then
coupled to 11b and 11c using HBTU and TEA to afford
compounds 16a and 16b in good yields. Compounds 16a and
16b were deprotected and cyclized using the same reagents as
for the previous analogues to obtain 17a and 17b. Silstatins 7
and 8 (18a and 18b) were prepared by TFA treatment of 17a
and 17b in high yields (Scheme 3).
Compound 18a was later coupled to glucuronic acid through
a self-immolative linker (Scheme 4). The starting material,
methyl 1-bromo-2,3,4-tri-O-acetyl-α-D-glucuronate, was pre-
pared from glucuronolactone following Bollenback et al.’s
strategy.10 Compound 20 was prepared according to Duimstra
et al.’s procedure.11 Compound 25 was synthesized following
Grinda et al.’s strategy starting from crude 20.12 Compound 25
The growth inhibition properties of the silstatins and some
intermediates was evaluated against a minipanel of cancer cell
lines (Table 1). An overall observation pointed to small
modifications on the structure of the bacillistatin, while keeping
the overall lipophilicity as shown for silstatin 1 (7a), which did
not affect its high potency. However, the introduction of polar
groups (hydroxy) somewhat decreased activity, as shown for
silstatins 2 and 3 (8a and 8b). We observed that the only
difference between valinomycin and bacillistatins 1 and 2 is an
α-hydroxy acid that repeats three times along the structure. The
structure of valinomycin contains (R)-2-hydroxyisovaleric acid,
and the bacillistatins 1 and 2 contain (R)-2-hydroxyisocaproic
acid and (2R,3S)-2-hydroxy-3-methylpentanoic acid, which are
slightly more lipophilic than the 2-hydroxyisovaleric acid
present in valinomycin. Following the same logic we introduced
an even more lipophilic residue on the same position. However,
D
DOI: 10.1021/np501004h
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Scheme 4. Synthesis of Glucuronide Prodrug 28
introduction of an α-hydroxy acid with a more lipophilic side
chain in place of the three (R)-2-hydroxyisocaproic acid units in
silstatin 1 caused a marked reduction of cancer cell growth
inhibition capability, as seen in silstatins 4 and 6 (14a and 15b).
In contrast, silstatin 5 (15a) showed very high inhibitory
activity.
interchanged with (2R)-2-hydroxy-3-cyclohexylpropionic acid,
thereby rescuing the activity as cancer cell growth inhibitors of
the structures containing a hydroxy group, as shown by
silstatins 7 and 8 (18a and 18b), although without reaching the
activity shown by silstatin 1 (7a) or the bacillistatins.
The position where the hydroxy group is introduced plays a
big role in the cancer cell growth inhibition. Recently, it was
observed that introduction of a hydroxy group on one of the L-
In order to maximize the activity displayed versus lip-
ophilicity, only one (R)-2-hydroxyisocaproic acid residue was
E
DOI: 10.1021/np501004h
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Scheme 5. Drug (18a) Liberation
Table 1. Growth Inhibition of Human Cancer Cell Lines (GI50 μg/mL [nM])
a
cell line
compound
BXPC-3
MCF-7
SF-268
NCI-H460
KM20L2
DU-145
bacillistatin 11
bacillistatin 21
silstatin 1 (7a)
7c
0.00095 [0.82]
0.00034 [0.29]
0.0008 [0.69]
0.030 [24]
0.0040 [3.5]
0.0050 [4.1]
0.5 [392]
0.00061 [0.53]
0.00031 [0.27]
0.00011 [0.095]
0.0042 [3.3]
0.0022 [1.9]
0.0016 [1.3]
0.18 [141]
0.00045 [0.39]
0.0018 [1.6]
0.00021 [0.18]
0.0025 [2.0]
0.0031 [2.7]
0.0040 [3.3]
0.081 [63.6]
0.075 [56.3]
>1 [>717]
0.0023 [1.2]
0.00045 [0.39]
0.0007 [0.61]
0.029 [23]
0.0032 [2.8]
0.0037 [3.0]
0.4 [314]
0.00087 [0.75]
0.00026 [0.23]
0.00016 [0.14]
0.0048 [3.8]
0.0033 [2.9]
0.0011 [0.90]
0.18 [141]
0.0015 [1.3]
0.00086 [0.75]
0.00042 [0.36]
0.011 [8.6]
0.0060 [5.2]
0.0051 [4.2]
0.22 [173]
0.12 [90]
silstatin 2 (8a)
silstatin 3 (8b)
silstatin 4 (14a)
14b
0.18 [135]
>1 [>717]
0.03 [24]
0.075 [56.3]
>1 [>717]
0.32 [240]
>1 [>717]
0.021 [16]
0.32 [239]
0.009 [7.2]
0.12 [91]
0.08 [60]
14c
>1 [>717]
>1 [>717]
silstatin 5 (15a)
silstatin 6 (15b)
17a
0.0031 [2.4]
0.12 [89.7]
0.0044 [3.4]
0.05 [37.4]
0.004 [3.2]
0.06 [46]
0.0044 [3.4]
0.09 [67.3]
0.0024 [1.9]
0.052 [40]
0.005 [3.9]
0.22 [164]
0.007 [5.6]
0.04 [30]
0.53 [396]
0.023 [18]
0.09 [68]
0.001 [0.79]
0.032 [24]
17b
silstatin 7 (18a)
silstatin 8 (18b)
28
0.0038 [3.2]
0.006 [4.8]
0.20 [120]
0.0014 [1.5]
0.0011 [0.88]
0.031 [18.6]
0.0031 [2.6]
0.003 [2.4]
0.05 [30.0]
0.0032 [3.3]
0.004 [3.2]
0.07 [42.0]
0.0015 [1.7]
0.0021 [1.7]
0.04 [24.0]
0.003 [2.5]
0.0054 [4.3]
0.13 [78.1]
a
Cancer cell lines in order: pancreas (BXPC-3); breast (MCF-7); CNS (SF-268); lung (NCI-H460); colon (KM20L2); prostate (DU-145).
valine or D-valine residues of valinomycin did not prominently
affect its activity. However, when the hydroxy group was
introduced on the (R)-2-hydroxyisovaleric acid, the activity as
cancer cell growth inhibitor was decreased dramatically.15 For
that reason we introduced a threonine or a tyrosine residue in
place of one of the L-valine residues present in bacillistatin 1
and bacillistatin 2. Another aspect that might be important, and
needs to be assessed, is whether the position of the (2R)-2-
hydroxy-3-cyclohexylpropionic acid residue needs to be
adjacent to the hydroxy-containing residue (compounds 18a
and 18b) or not. Future analogues in which the D-valine residue
is replaced with a hydroxy-containing residue would also be of
interest.
The structural characteristics of valinomycin provide the
capacity to act as a potassium ion carrier. Valinomycin is
lipophilic enough to solubilize in the lipid membrane and polar
enough to reach the surface of the membrane in order to
coordinate with the potassium ion.16 The structural resem-
blance of the bacillistatins to valinomycin is such that it is
believed that bacillistatins have the ability to transport
potassium ions through the membranes in the same manner
as valinomycin. Thus, the absolute configuration, the order of
the residues (D and L configurations), and the overall
lipophilicity along the structure are vital for high activity.
To demonstrate the utility of the new silstatins’ feasibility for
forming prodrugs and linkers to monoclonals, prodrug 28 was
F
DOI: 10.1021/np501004h
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
obtained in the Arizona State University CLAS High Resolution Mass
Spectrometry Laboratory.
synthesized and evaluated against the same minipanel of cancer
cell lines (Table 1). The use of glucuronide prodrugs has been
validated by in vivo studies, showing superior therapeutic
efficacy compared to the parent drug due to a difference in
levels of β-glucuronidase in tissues. More importantly, β-
glucuronidase is also observed in the tumor microenviron-
ment.17 Importantly, Papot and colleagues have completed a
detailed study developing a glucuronide prodrug derived from
the powerful anticancer drug auristatin E as its des-methyl
counterpart. The conclusion was that the in vivo data for that
combination had promise for selective treatment of cancer.17d
In turn, 28 proved to be a prodrug that should have greatly
reduced toxicity during transport to the tumor, where a
glucuronidase would release silstatin 7. Evidence of that
expected result was obtained as follows. The cancer cell growth
inhibition activity for prodrug 28 was found to be decreased
16−52 times with respect to the parent silstatin 7. Also,
prodrug 28 was tested against two normal cell lines and
compared to the parent drug (18a) (Table 2), and, as
The reactions were carried out under an atmosphere of nitrogen
unless specified otherwise. Column chromatography was conducted
using silica gel (E. Merck 60 Å, 230−400 mesh), applying a low-
pressure stream of nitrogen. Analytical thin-layer chromatography
separations were carried out on glass plates coated with silica gel
(Analtech, GHLF uniplates). The TLC chromatograms were
visualized using UV (short wave) lamp irradiation or by immersing
the plates in ceric ammonium molybdate (CAM) staining solution
followed by heating with a heat gun. Reagents and anhydrous solvents
were purchased from Sigma−Aldrich Chemical Co. and Alfa-Aesar Inc.
and were used as received. Degussa type E101 NE/W was employed
for 20% palladium hydroxide on carbon.
Boc-D-Val-Lac-OBn (1). To a stirred solution containing Boc-D-Val
(1.00 g, 4.60 mmol) and benzyl L-lactate (638 mg, 3.54 mmol) in 20
mL of anhydrous dichloromethane (DCM) at 0 °C was added DMAP
(86 mg, 0.71 mmol) followed by DCC (840 mg, 4.07 mmol). The
reaction mixture was stirred at 23 °C for 4 h. The mixture was filtered
to remove most of the dicyclohexylurea (DCU), and the filtrate was
diluted with 80 mL of DCM. The organic solution was washed with 50
mL of 0.3 N HCl, 50 mL of saturated aqueous NaHCO3, and 50 mL of
brine. The organic solution was dried over MgSO4 and concentrated
under reduced pressure. The residue was dissolved in 50 mL of cold
CH3CN, and the formed precipitate was filtered. The filtrate was
Table 2. Growth Inhibition of Human Normal Cell Lines
(GI50 μg/mL [nM])
a
concentrated under reduced pressure to afford 1 as a colorless oil:
cell line
1
yield 97% (1.32 g, 3.47 mmol); [α]24 −3.43 (c 0.18, EtOAc); H
D
compound
MCF-10A
CRL-2221
NMR (CDCl3, 400 MHz) δ 7.25 (5H, m), 5.06 (4H, m), 4.22 (1H,
m), 2.11 (1H, m), 1.42 (3H, d, J = 7.2 Hz), 1.37 (9H, s), 0.90 (3H, d, J
= 6.8 Hz), and 0.83 (3H, d, J = 6.8 Hz); 13C NMR (CDCl3, 101 MHz)
δ 171.3, 169.9, 155.4, 135.1, 128.4, 128.5, 128.0, 79.5, 69.0, 66.9, 58.5,
31.0, 28.2, 18.8, 17.4, and 16.8; HRMS (APCI) m/z 380.2067 [M +
H]+ (calcd for C20H30NO6, 380.2073).
silstatin 7 (18a)
0.004 [3.35]
0.25 [150]
0.004 [3.35]
0.05 [30.0]
28
a
Breast (MCF-10A); prostate (CRL-2221).
D-Hica-D-Val-Lac-OBn (2). To a stirred solution containing ester 1
(4.12 g, 10.9 mmol) in 60 mL of anhydrous DCM was added TFA (15
mL). The reaction mixture was stirred at 23 °C for 5 h and then
concentrated under reduced pressure. The residue was dissolved in
140 mL of DCM and washed with two 100 mL portions of saturated
aqueous NaHCO3 and 100 mL of brine. The organic solution was
dried over MgSO4 and concentrated under reduced pressure. The
residue was dissolved in 60 mL of anhydrous DCM and cooled to 0
°C. Next, (R)-2-hydroxyisocaproic acid (1.44 g, 10.9 mmol) was added
followed by HOBt (2.22 g, 16.4 mmol), TEA (2.27 mL, 1.66 g, 16.4
mmol), and EDCI hydrochloride (3.14 g, 16.4 mmol). The reaction
mixture was stirred at 23 °C for 18 h. The solution was diluted with
140 mL of DCM and washed with 100 mL of 0.3 N HCl, 100 mL of
saturated aqueous NaHCO3, and 100 mL of brine. The organic
solution was dried over MgSO4 and concentrated under reduced
pressure. The residue was separated by chromatography on a silica gel
column. Elution with 1:2 EtOAc/hexanes gave 2 as a colorless oil:
presumed, the prodrug 28 normal cell growth inhibition activity
was decreased 62.5 and 12.5 times with respect to the parent
silstatin 7 drug (18a) in the MCF-10A and CRL-2221 cell lines,
respectively.
The quite high activity of prodrug 28 displayed in cancer and
normal cell lines might be attributed to the fact that the
prodrug could be crossing the cell membrane and releasing the
drug (18a). Recently, a doxorubicin glucuronide prodrug
capable of binding albumin through a maleamide moiety was
synthesized, and the effects of this capability led to a relatively
nontoxic prodrug with higher accumulation in the tumor (more
selective) as compared to the glucuronide prodrug without the
maleamide moiety.18
CONCLUSION
■
In summary, a series of bacillistatin structural modifications
have been synthesized that can be bonded to linkers for
transport to the cancer targets. In general, we observed an
overall lipophilicity range at which these carrier-type potassium
ionophores perform better as antiproliferative agents. Illus-
trative is the hydroxy linker site of bacillistatin modification
18a, which can now be linked to other and more efficient
targeting moieties especially monoclonal antibodies.
yield 86% (3.68 g, 9.35 mmol); TLC Rf 0.40 (1:2 EtOAc/hexanes);
1
[α]24 +12.8 (c 0.18, EtOAc); H NMR (CDCl3, 400 MHz) δ 7.33
D
(5H, m), 6.93 (1H, d, J = 9.2 Hz), 5.14 (3H, m), 4.64 (1H, dd, J = 9.2
Hz, 4.8 Hz), 4.14 (1H, m), 2.66 (1H, d, J = 4.8 Hz), 2.21 (1H, m),
1.84 (1H, m), 1.65 (1H, m), 1.53 (4H, m), and 0.93 (12H, m); 13C
NMR (CDCl3, 101 MHz) δ 174.4, 171.2, 170.2, 135.3, 128.8, 128.6,
128.3, 71.1, 69.5, 67.3, 56.7, 44.0, 31.4, 24.7, 23.6, 21.5, 19.1, 17.7, and
17.1; HRMS (APCI) m/z 394.2239 [M + H]+ (calcd for C21H32NO6,
394.2230).
Fmoc-Val-D-Hica-D-Val-Lac-OBn (3a). To a stirred solution
containing unit 2 (3.58 g, 9.10 mmol), Fmoc-Val (3.70 g, 10.9
mmol), and DMAP (334 mg, 2.73 mmol) in 100 mL of anhydrous
DCM at 0 °C was added DCC (2.06 g, 10.0 mmol). The reaction
mixture was stirred at 23 °C for 2 h. The solution was filtered, and the
filtrate was concentrated under reduced pressure. The crude product
was separated by chromatography on a silica gel column. Elution with
1:4 EtOAc/hexanes gave 3a as a colorless solid: yield 77% (5.0 g, 6.98
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points are un-
corrected and were determined with a Fisher−Johns melting point
apparatus. Optical rotations were measured by use of a Perkin−Elmer
241 polarimeter, and the [α]D values are given in 10−1 deg cm2 g−1. 1H
and 13 C NMR spectra were recorded on a Varian Unity INOVA 400
1
instrument with deuterated solvents; H NMR chemical shifts were
mmol); TLC Rf 0.40 (1:4 EtOAc/hexanes); mp 43−47 °C; [α]24
recorded relative to residual CHCl3 at 7.26 ppm or MeOH 3.31 ppm;
13C NMR chemical shifts were reported relative to residual CHCl3 at
77.16 ppm or MeOH at 49.00 ppm. High-resolution mass spectra were
D
+6.59 (c 0.43, EtOAc); 1H NMR (CDCl3, 400 MHz) δ 7.74 (2H, d, J
= 7.2 Hz), 7.58 (2H, d, J = 7.6 Hz), 7.39 (2H, m), 7.33 (7H, m), 6.76
G
DOI: 10.1021/np501004h
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(1H, d, J = 8.4 Hz), 5.39 (1H, d, J = 8.8 Hz), 5.24 (1H, m), 5.09 (3H,
m), 4.55 (1H, m), 4.41 (1H, m), 4.25 (3H, m), 2.28 (1H, m), 2.15
(1H, m), 1.74 (3H, m), 1.42 (3H, d, J = 6.8 Hz), and 0.95 (18H, m);
13C NMR (CDCl3, 101 MHz) δ 171.6, 170.6, 170.1, 170.0, 156.5,
144.0, 143.8, 141.4, 135.3, 128.6, 128.4, 128.2, 127.8, 127.1, 125.2,
120.1, 120.0, 73.6, 69.3, 67.4, 67.1, 59.8, 57.4, 47.2, 40.8, 30.8, 30.7,
24.5, 23.3, 21.5, 19.2, 19.1, 18.0 and 16.8; HRMS (APCI) m/z
715.3586 [M + H]+ (calcd for C41H51N2O9, 715.3594).
Hz), and 0.95 (36H, m); 13C NMR (CDCl3, 101 MHz) δ 171.99,
171.04, 170.91, 170.85, 170.52, 170.41, 170.28, 170.21, 156.89, 143.87,
143.62, 141.28, 135.29, 128.48, 128.24, 128.12, 127.72, 127.02, 125.02,
119.97, 73.15, 73.07, 70.43, 69.12, 67.46, 66.92, 60.40, 59.02, 57.80,
47.03, 40.68, 40.44, 34.64, 31.56, 30.38, 30.08, 29.72, 29.42, 25.25,
24.41, 23.19, 22.62, 21.42, 21.37, 19.17, 19.06, 19.01, 18.89, 18.79,
18.55, 18.32, 17.42, and 16.75; HRMS (APCI) m/z 1099.586 [M +
H]+ (calcd for C60H82N4O15, 1099.585).
Fmoc-[Val-D-Hica-D-Val-Lac]3-OBn (6a). By means of the strategy
followed for the preparation and purification of 5, unit 5 (345 mg, 0.31
mmol) was deprotected with 2,2′,2″-triaminotriethylamine (467 μL,
453 mg, 3.10 mmol) in 10 mL of DCM and then coupled to unit 4a
(194 mg, 0.31 mmol) using HBTU (235 mg, 0.62 mmol) and TEA
(43 μL, 31 mg, 0.42 mmol) in 5 mL of anhydrous DMF. After
separation by column chromatography 6a was obtained as a colorless
Fmoc-Thr(tBu)-D-Hica-D-Val-Lac-OBn (3b). Using the strategy
followed for the preparation and purification of 3a, unit 2 (492 mg,
1.25 mmol) was coupled to Fmoc-Thr(tBu) (596 mg, 1.50 mmol)
using DMAP (46 mg, 0.38 mmol) and DCC (285 mg, 1.38 mmol) in
15 mL of anhydrous DCM. After separation by column chromatog-
raphy 3b was obtained as a colorless solid. Yield: 83% (800 mg, 1.04
mmol); TLC Rf 0.40 (1:3 EtOAc/hexanes); mp 40−44 °C; [α]24
D
+10.8 (c 0.42, EtOAc); 1H NMR (CDCl3, 400 MHz) δ 7.74 (2H, d, J
= 7.6 Hz), 7.58 (2H, t, J = 7.2 Hz), 7.38 (2H, t, J = 7.6 Hz), 7.29 (7H,
m), 6.77 (1H, d, J = 8.4 Hz), 5.69 (1H, d, J = 8.4 Hz), 5.18 (1H, m),
5.09 (3H, m), 4.55 (1H, dd, J = 8.8 Hz, 5.6 Hz), 4.35 (3H, m), 4.21
(2H, m), 2.27 (1H, m), 1.79 (3H, m), 1.45 (3H, d, J = 6.8 Hz), 1.23
(3H, d, J = 6.4 Hz), 1.18 (9H, s) and 0.95 (12H, m); 13C NMR
(CDCl3, 101 MHz) δ 170.51, 170.50, 169.99, 169.72, 156.57, 144.03,
143.75, 141.32, 135.22, 128.60, 128.43, 128.18, 127.75, 127.10, 125.23,
120.01, 74.44, 73.87, 69.28, 67.42, 67.13, 67.07, 60.06, 57.23, 47.16,
40.89, 30.95, 28.55, 24.32, 23.08, 21.92, 20.53, 18.99, 17.93, and 16.85;
HRMS (APCI) m/z 773.4015 [M + H]+ (calcd for C44H57N2O10,
773.4013).
solid: yield 62% (284 mg, 0.19 mmol); TLC Rf 0.35 (1:2 EtOAc/
1
hexanes); mp 59−63 °C; [α]24 +3.56 (c 0.23, EtOH); H NMR
D
(CDCl3, 400 MHz) δ 7.74 (2H, d, J = 7.6 Hz), 7.66 (2H, m), 7.58
(2H, dd, J = 14.8 Hz, 7.2 Hz), 7.51 (1H, d, J = 6.0 Hz), 7.39 (3H, m),
7.33 (8H, m), 5.74 (1H, d, J = 7.2 Hz), 5.34 (1H, m), 5.20 (4H, m),
5.10 (3H, m), 4.38 (3H, m), 4.22 (1H, t, J = 7.2 Hz), 4.13 (2H, m),
4.04 (1H, t, J = 6.4 Hz), 3.95 (2H, m), 2.27 (5H, m), 1.99 (1H, m),
1.74 (9H, m), 1.41 (9H, d, J = 6.8 Hz), and 0.95 (54H, m); 13C NMR
(CDCl3, 101 MHz) δ 172.54, 171.69, 171.55, 171.40, 170.96, 170.70,
170.68, 170.59, 170.58, 170.41, 170.38, 170.36, 157.20, 144.16, 143.78,
141.44, 135.49, 128.60, 128.35, 127.85, 127.19, 125.25, 120.10, 73.35,
73.10, 72.68, 70.54, 70.17, 69.21, 67.64, 67.04, 60.77, 60.04, 59.82,
59.66, 59.08, 58.16, 47.19, 40.94, 40.59, 40.51, 34.79, 31.71, 30.42,
30.08, 29.77, 29.47, 29.29, 29.18, 25.40, 24.57, 24.53, 24.50, 23.38,
23.35, 22.77, 21.68, 21.49, 21.40, 19.48, 19.35, 19.30, 19.26, 19.19,
19.16, 19.06, 18.88, 18.64, 17.56, 17.35, and 16.91; HRFTMS (ESI)
m/z 1505.788 [M + Na]+ (calcd for C79H114N6O21Na, 1505.793).
Fmoc-Thr(tBu)-D-Hica-D-Val-Lac-[Val-D-Hica-D-Val-Lac]2-OBn
(6b). With the procedure followed for the preparation and purification
of 5, unit 5 (300 mg, 0.27 mmol) was deprotected with 2,2′,2″-
triaminotriethylamine (407 μL, 395 mg, 2.70 mmol) in 12 mL of
DCM and then coupled to unit 4b (184 mg, 0.27 mmol) using HBTU
(205 mg, 0.54 mmol) and TEA (37 μL, 27 mg, 0.27 mmol) in 5 mL of
anhydrous DMF. After separation by column chromatography 6b was
obtained as a colorless solid: yield 74% (308 mg, 0.20 mmol); TLC Rf
Fmoc-Tyr(tBu)-D-Hica-D-Val-Lac-OBn (3c). Using the procedure
followed for the preparation and purification of 3a, unit 2 (492 mg,
1.25 mmol) was coupled to Fmoc-Tyr(tBu) (584 mg, 1.27 mmol)
using DMAP (39 mg, 0.32 mmol) and DCC (241 mg, 1.17 mmol) in
15 mL of anhydrous DCM. After separation by column chromatog-
raphy 3c was obtained as a colorless solid: yield 94% (830 mg, 0.99
mmol); TLC Rf 0.40 (1:3 EtOAc/hexanes); mp 45−50 °C; [α]24
D
+6.04 (c 0.27, EtOAc); 1H NMR (CDCl3, 400 MHz) δ 7.74 (2H, d, J
= 7.6 Hz), 7.55 (2H, m), 7.38 (2H, t, J = 7.6 Hz), 7.26 (7H, m), 7.08
(2H, d, J = 8.4 Hz), 6.95 (1H, d, J = 8.8 Hz), 6.90 (2H, d, J = 8.4 Hz),
5.55 (1H, d, J = 7.2 Hz), 5.22 (1H, m), 5.07 (3H, m), 4.55 (2H, m),
4.35 (1H, m), 4.28 (1H, m), 4.15 (1H, t, J = 7.2 Hz), 3.13 (1H, m),
3.04 (1H, m), 2.31 (1H, m), 1.71 (2H, m), 1.50 (1H, m), 1.45 (3H, d,
J = 7.2 Hz), 1.31 (9H, s), 0.97 (6H, m), and 0.87 (6H, m); 13C NMR
(CDCl3, 101 MHz) δ 171.19, 170.51, 170.11, 170.06, 156.01, 154.68,
143.81, 143.67, 141.26, 135.18, 130.17, 129.66, 128.54, 128.36, 128.11,
127.74, 127.05, 125.07, 124.12, 119.97, 78.34, 73.66, 69.16, 67.31,
67.01, 57.40, 55.67, 47.01, 40.70, 36.93, 30.58, 28.84, 24.37, 23.14,
21.55, 19.05, 18.10, and 16.79; HRMS (APCI) m/z 835.4176 [M +
H]+ (calcd for C49H59N2O10, 835.4169).
0.35 (1:2 EtOAc/hexanes); mp 62−65 °C; [α]24 +13.0 (c 0.10,
D
EtOH); 1H NMR (CDCl3, 400 MHz) δ 7.73 (2H, d, J = 7.6 Hz), 7.70
(1H, d, J = 6.0 Hz), 7.56 (4H, m), 7.51 (2H, t, J = 7.6 Hz), 7.33 (8H,
m), 7.17 (1H, d, J = 6.4 Hz), 5.68 (1H, d, J = 7.2 Hz), 5.23 (5H, m),
5.10 (3H, m), 4.35 (3H, m), 4.22 (1H, t, J = 7.2 Hz), 4.04 (6H, m),
2.27 (5H, m), 1.74 (9H, m), 1.41 (9H, m), 1.22 (3H, d, J = 5.6 Hz),
1.18 (9H, s), and 0.95 (48H, m); 13C NMR (CDCl3, 101 MHz) δ
171.34, 171.27, 171.25, 170.89, 170.83, 170.81, 170.63, 170.59, 170.45,
170.44, 170.29, 170.27, 156.80, 143.84, 143.58, 141.25, 135.35, 128.46,
128.21, 128.14, 127.72, 127.04, 125.05, 119.98, 74.62, 73.22, 72.99,
72.54, 70.19, 70.08, 69.07, 67.50, 66.88, 66.83, 60.19, 59.91, 59.80,
59.46, 59.26, 58.05, 47.03, 40.79, 40.33, 34.62, 30.23, 29.65, 29.57,
29.33, 28.51, 28.46, 25.23, 24.36, 24.33, 24.21, 23.22, 23.02, 21.71,
21.40, 21.33, 20.54, 19.34, 19.22, 19.19, 19.15, 19.11, 19.09, 19.04,
18.97, 18.93, 18.52, 17.25, 17.12, and 16.74; HRFTMS (ESI) m/z
1563.833 [M + Na]+ (calcd for C82H120N6O22Na, 1563.835).
Fmoc-[Val-D-Hica-D-Val-Lac]2-OBn (5). To a stirred solution
containing compound 3a (300 mg, 0.42 mmol) in 10 mL of
anhydrous DCM was added 2,2′,2″-triaminotriethylamine (633 μL,
614 mg, 4.20 mmol). The reaction mixture was stirred at 23 °C for 2 h.
The solution was diluted with 10 mL of DCM and washed with two 10
mL portions of phosphate buffer (25.3 g of K2HPO4/12.3 g of
KH2PO4 in 250 mL of H2O) and 10 mL of brine. The organic solution
was dried over MgSO4 and concentrated under reduced pressure. The
residue was dissolved in 5 mL of anhydrous DMF, and unit 4a (262
mg, 0.42 mmol) was added followed by TEA (58 μL, 42 mg, 0.42
mmol) and HBTU (319 mg, 0.84 mmol). The solution was stirred at
23 °C for 16 h, then diluted with 50 mL of EtOAc, and washed with 50
mL of brine. The organic solution was dried over MgSO4 and
concentrated under reduced pressure. The mixture was separated by
chromatography on a silica gel column. Elution with 1:4 EtOAc/
hexanes gave 5 as a colorless solid: yield 83% (384 mg, 0.35 mmol);
Fmoc-Tyr(tBu)-D-Hica-D-Val-Lac-[Val-D-Hica-D-Val-Lac]2-OBn
(6c). Employing the strategy followed for the preparation and
purification of 5, unit 5 (630 mg, 0.57 mmol) was deprotected with
2,2′,2″-triaminotriethylamine (850 μL, 834 mg, 5.70 mmol) in 20 mL
of DCM and then coupled to unit 4c (425 mg, 0.57 mmol) using
HBTU (434 mg, 1.14 mmol) and TEA (79 μL, 58 mg, 0.57 mmol) in
5 mL of anhydrous DMF. After separation by column chromatography
6c was obtained as a colorless solid: yield 81% (744 mg, 0.46 mmol);
TLC Rf 0.20 (1:4 EtOAc/hexanes); mp 59−64 °C; [α]24 +6.27 (c
D
TLC Rf 0.35 (1:2 EtOAc/hexanes); mp 62−65 °C; [α]24 +5.71 (c
1
0.26, EtOAc); H NMR (CDCl3, 400 MHz) δ 7.74 (2H, d, J = 7.2
D
0.28, EtOH); 1H NMR (CDCl3, 400 MHz) δ 7.78 (1H, d, J = 7.6 Hz),
7.74 (2H, d, J = 7.6 Hz), 7.67 (1H, m), 7.62 (1H, d, J = 6.4 Hz), 7.52
(2H, m), 7.44 (1H, m), 7.37 (3H, m), 7.30 (7H, m), 7.06 (2H, d, J =
8.0 Hz), 6.89 (2H, d, J = 8.4 Hz), 5.73 (1H, d, J = 6.4 Hz), 5.18 (8H,
m), 4.35 (4H, m), 4.19 (1H, t, J = 6.8 Hz), 4.06 (3H, m), 3.95 (1H, t, J
Hz), 7.58 (2H, dd, J = 13.2 Hz, 7.6 Hz), 7.39 (3H, m), 7.33 (7H, m),
7.20 (1H, d, J = 7.2 Hz), 7.16 (1H, d, J = 8.8 Hz), 5.51 (1H, d, J = 7.6
Hz), 5.27 (2H, m), 5.23 (1H, m), 5.09 (3H, m), 4.42 (2H, m), 4.33
(1H, m), 4.20 (2H, m), 4.11 (1H, t, J = 7.6 Hz), 4.04 (1H, t, J = 7.6
Hz), 2.27 (3H, m), 2.15 (1H, m), 1.74 (6H, m), 1.41 (6H, d, J = 6.4
H
DOI: 10.1021/np501004h
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
= 6.4 Hz), 3.03 (2H, m), 2.25 (5H, m), 1.74 (8H, m), 1.59 (1H, m),
1.41 (9H, m), 1.30 (9H, s), and 0.95 (48H, m); 13C NMR (CDCl3,
101 MHz) δ 171.92, 171.59, 171.45, 171.42, 171.21, 170.97, 170.88,
170.72, 170.64, 170.53, 170.44, 170.43, 156.72, 154.89, 144.01, 143.74,
141.36, 135.51, 130.70, 129.76, 128.61, 128.35, 128.31, 127.89, 127.17,
125.21, 124.24, 120.13, 80.72, 73.45, 73.14, 72.74, 70.46, 70.28, 69.26,
67.68, 67.05, 59.95, 59.92, 59.70, 59.24, 58.24, 56.19, 47.14, 40.93,
40.52, 36.61, 36.59, 30.40, 29.83, 29.79, 29.55, 29.47, 29.38, 29.26,
29.19, 28.99, 24.53, 24.51, 24.34, 23.39, 23.23, 21.65, 21.49, 19.46,
19.43, 19.31, 19.30, 19.27, 19.21, 19.12, 19.09, 18.68, 17.49, 17.44 and
16.91; HRFTMS (ESI) m/z 1625.844 [M + Na]+ (calcd for
C87H122N6O22Na, 1625.850).
Cyclo-[Val-D-Hica-D-Val-Lac]3 (7a, silstatin 1). To a stirred solution
containing depsipeptide 6a (250 mg, 0.17 mmol) in 5 mL of 4:1
EtOAc/MeOH was added 20% palladium hydroxide-on-carbon (125
mg). The mixture was stirred under a hydrogen atmosphere (1 atm) at
23 °C for 7 h and then filtered through Celite. The filtrate was
concentrated under reduced pressure, and the residue was dissolved in
125 mL of anhydrous DCM. TEA (24 μL, 17 mg, 0.17 mmol) was
added followed by PyBroP (238 mg, 0.51 mmol), and the reaction
mixture was stirred at 23 °C for 24 h. The solution was filtered
through a silica gel plug, and the filtrate was concentrated under
reduced pressure. The residue was separated by chromatography on a
silica gel column. Elution with 1:4 EtOAc/hexanes gave 7a (silstatin 1)
dd, J = 9.6 Hz, 7.6 Hz), 3.95 (1H, dd, J = 9.6 Hz, 7.2 Hz), 3.89 (2H,
dd, J = 10 Hz, 6.0 Hz), 3.10 (2H, m), 2.23 (5H, m), 1.74 (7H, m),
1.55 (2H, t, J = 6.8 Hz), 1.42 (6H, d, J = 6.8 Hz), 1.36 (3H, d, J = 6.8
Hz), 1.30 (9H, s), 1.05 (18H, m), 0.92 (24H, m), 0.75 (3H, d, J = 6.8
Hz), and 0.71 (3H, d, J = 6.4 Hz); 13C NMR (CDCl3, 101 MHz) δ
172.49, 172.37, 172.10, 172.00, 171.71, 171.69, 171.53, 171.43, 170.59,
170.52, 170.27, 170.03, 154.31, 131.01, 129.66, 123.91, 78.11, 73.61,
73.18, 70.71, 70.50, 70.07, 60.41, 60.37, 59.39, 59.28, 58.39, 54.57,
40.73, 40.63, 40.61, 40.48, 35.27, 30.32, 29.65, 28.80, 28.58, 28.53,
28.46, 28.40, 24.45, 24.16, 23.71, 23.31, 22.88, 21.80, 21.24, 21.08,
19.73, 19.46, 19.35, 19.33, 19.17, 19.14, 19.11, 19.06, 19.04, 18.84,
17.19, 17.09, and 16.81; HRMS (APCI) m/z 1273.744 [M + H]+
(calcd for C65H105N6O19, 1273.743).
Cyclo-{Thr-D-Hica-D-Val-Lac-[Val-D-Hica-D-Val-Lac]2} (8a, silstatin
2). To a stirred solution containing cyclodepsipeptide 7b (70 mg, 58
μmol) in 1 mL of anhydrous DCM was added TFA (250 μL). The
reaction mixture was stirred at 23 °C for 2 h. The solution was
concentrated under reduced pressure, and the residue was separated
by chromatography on a silica gel column. Elution with 1:3 EtOAc/
hexanes gave 8a (silstatin 2) as a colorless solid: yield 94% (63 mg, 55
μmol); TLC Rf 0.25 (1:3 EtOAc/hexanes); mp 75−78 °C; [α]24
D
+25.9 (c 0.19, EtOH); 1H NMR (CDCl3, 400 MHz) δ 7.99 (1H, d, J =
7.2 Hz), 7.91 (1H, d, J = 7.2 Hz), 7.81 (3H, m), 7.57 (1H, d, J = 6.4
Hz), 5.28 (2H, m), 5.17 (3H, m), 5.05 (1H, m), 4.87 (1H, br s), 4.14
(1H, t, J = 9.2 Hz), 3.95 (6H, m), 2.23 (4H, m), 2.12 (1H, m), 1.74
(9H, m), 1.41 (9H, m), 1.20 (3H, d, J = 5.6 Hz), 1.10 (18H, m), and
0.90 (30H, m); 13C NMR (CDCl3, 101 MHz) δ 173.51, 172.82,
172.39, 172.32, 172.20, 171.92, 171.53, 171.20, 171.00, 170.92, 170.29,
169.97, 74.49, 73.29, 73.16, 70.76, 70.70, 70.42, 66.19, 60.84, 60.47,
60.14, 59.89, 59.60, 58.52, 40.84, 40.70, 40.62, 28.72, 28.67, 28.55,
28.49, 28.21, 24.67, 24.57, 23.49, 23.37, 23.35, 23.33, 21.53, 21.36,
21.35, 19.70, 19.60, 19.53, 19.48, 19.47, 19.46, 19.45, 19.41, 19.37,
19.35, 18.98, 17.53, 17.10, and 17.03; HRMS (APCI) m/z 1155.664
[M + H]+ (calcd for C56H95N6O19, 1155.665).
as a colorless solid: yield 28% (54 mg, 47 μmol); TLC Rf 0.42 (1:4
1
EtOAc/hexanes); mp 137−138 °C; [α]24 +30.9 (c 0.11, EtOH); H
D
NMR (CDCl3, 400 MHz) δ 7.78 (3H, d, J = 7.6 Hz), 7.73 (3H, d, J =
6.4 Hz), 5.21 (3H, m), 5.13 (3H, m), 4.06 (3H, dd, J = 9.6 Hz, 8.0
Hz), 3.93 (3H, dd, J = 9.6 Hz, 6.4 Hz), 2.23 (6H, m), 1.74 (9H, m),
1.44 (9H, d, J = 7.2 Hz), 1.04 (18H, m), and 0.95 (36H, m); 13C
NMR (CDCl3, 101 MHz) δ 172.4, 172.0, 171.6, 170.4, 73.5, 70.6, 60.3,
59.1, 40.8, 28.7, 28.5, 24.6, 23.4, 21.4, 19.8, 19.6, 19.3, 19.2, and 17.2;
HRMS (APCI) m/z 1153.685 [M + H]+ (calcd for C57H97N6O18,
1153.686).
Cyclo-{Thr(tBu)-D-Hica-D-Val-Lac-[Val-D-Hica-D-Val-Lac]2} (7b). By
the method followed for the preparation and purification of 7a,
depsipeptide 6b (300 mg, 0.20 mmol) was deprotected using 20%
palladium hydroxide-on-carbon (300 mg) in 10 mL of 4:1 EtOAc/
MeOH and then cyclized with PyBroP (373 mg, 0.80 mmol) and TEA
(111 μL, 81 mg, 0.80 mmol) in 200 mL of anhydrous DCM. After
separation by column chromatography 7b was obtained as a colorless
Cyclo-{Tyr-D-Hica-D-Val-Lac-[Val-D-Hica-D-Val-Lac]2} (8b, silstatin
3). Using the strategy followed for the preparation and purification of
8a, cyclodepsipeptide 7c (7 mg, 5.5 μmol) was deprotected with TFA
(250 μL) in 1 mL of anhydrous DCM. After separation by column
chromatography 8b (silstatin 3) was obtained as a colorless solid: yield
75% (5 mg, 4.1 μmol); TLC Rf 0.25 (1:3 EtOAc/hexanes); mp 65−68
1
°C; [α]24 +35.0 (c 0.10, EtOAc); H NMR (CDCl3, 400 MHz) δ
D
solid: yield 33% (81 mg, 67 μmol); TLC Rf 0.5 (1:3 EtOAc/hexanes);
7.77 (6H, m), 7.04 (2H, d, J = 8.4 Hz), 6.73 (2H, d, J = 8.4 Hz), 5.20
(5H, m), 4.96 (1H, t, J = 6.0 Hz), 4.43 (1H, q, J = 7.6 Hz), 4.10 (3H,
m), 3.95 (2H, m), 3.10 (2H, m), 2.23 (5H, m), 1.65 (9H, m), 1.42
(6H, d, J = 6.8 Hz), 1.36 (3H, d, J = 7.2 Hz), 1.04 (18H, m), 0.92
(24H, m), 0.71 (3H, d, J = 6.4 Hz), and 0.71 (3H, d, J = 6.8 Hz); 13C
NMR (CDCl3, 101 MHz) δ 172.50, 172.38, 172.23, 172.04, 171.98,
171.94, 171.81, 171.53, 171.43, 170.36, 170.28, 170.20, 155.19, 130.57,
127.82, 115.78, 73.90, 73.58, 73.43, 70.73, 70.64, 60.57, 60.22, 59.01,
58.81, 58.80, 55.29, 40.94, 40.80, 40.68, 35.44, 29.85, 29.81, 29.08,
28.79, 28.73, 28.63, 24.68, 24.64, 24.28, 23.49, 23.20, 22.84, 21.66,
21.41, 21.31, 19.83, 19.71, 19.59, 19.55, 19.47, 19.38, 19.37, 19.12,
18.98, 18.83, 17.42, 17.18, and 17.04; HRMS (APCI) m/z 1217.681
[M + H]+ (calcd for C61H97N6O19, 1217.681).
D-Hcha-D-Val-Lac-OBn (9). Using the strategy followed for the
preparation and purification of 2, unit 1 (2.52 g, 6.64 mmol) was
deprotected with TFA (7.5 mL) in 30 mL of anhydrous DCM and
then coupled to (2R)-2-hydroxy-3-cyclohexylpropionic acid (1.23 g,
7.14 mmol) using HOBt (1.35 g, 9.96 mmol), TEA (1.38 mL, 1.10 g,
9.96 mmol), and EDCI hydrochloride (1.91 g, 9.96 mmol) in 30 mL
of anhydrous DCM. After separation by column chromatography 9
was obtained as a colorless oil (solidifies upon standing): yield 63%
(1.80 g, 4.15 mmol); TLC Rf 0.30 (1:3 EtOAc/hexanes); mp 55−57
°C; [α]24D +19.1 (c 0.32, EtOH); 1H NMR (CDCl3, 400 MHz) δ 7.34
(5H, m), 6.90 (1H, d, J = 9.2 Hz), 5.18 (2H, m), 4.64 (1H, dd, J = 9.2
Hz, 4.8 Hz), 4.14 (1H, m), 2.62 (1H, br s), 2.21 (1H, m), 1.78 (1H, d,
J = 12.8 Hz), 1.66 (5H, m), 1.50 (5H, m), 1.20 (3H, m), and 0.93
(8H, m); 13C NMR (CDCl3, 101 MHz) δ 174.5, 171.1, 170.1, 135.3,
128.8, 128.6, 128.3, 70.5, 69.5, 67.3, 56.7, 42.7, 34.3, 34.1, 32.3, 31.4,
1
mp 75−78 °C; [α]24 +23.3 (c 0.06, EtOH); H NMR (CDCl3, 400
D
MHz) δ 7.83 (1H, d, J = 8.0 Hz), 7.74 (1H, d, J = 7.2 Hz), 7.66 (1H,
d, J = 8.4 Hz), 7.49 (1H, d, J = 8.0 Hz), 7.45 (1H, d, J = 5.6 Hz), 7.37
(1H, d, J = 4.8 Hz), 5.20 (6H, m), 4.29 (1H, t, J = 8.4 Hz), 4.17 (2H,
m), 4.05 (3H, m), 3.85 (1H, dd, J = 10 Hz, 6.0 Hz), 2.23 (5H, m),
1.74 (9H, m), 1.41 (9H, m), 1.20 (9H, s), 1.17 (3H, d, J = 6.4 Hz),
1.10 (18H, m), and 0.90 (30H, m); 13C NMR (CDCl3, 101 MHz) δ
172.33, 172.32, 172.14, 172.12, 171.81, 171.80, 171.13, 171.12, 170.35,
170.27, 170.19, 169.88, 74.76, 73.88, 73.37, 73.11, 70.91, 70.84, 70.64,
66.54, 60.82, 59.81, 59.12, 59.08, 58.53, 57.72, 40.96, 40.77, 40.71,
34.77, 29.64, 29.23, 28.78, 28.54, 28.53, 28.46, 25.38, 24.58, 24.55,
23.51, 23.50, 23.45, 21.47, 21.32, 21.20, 19.92, 19.86, 19.54, 19.53,
19.41, 19.38, 19.32, 19.04, 18.72, 18.45, 17.54, 17.13, and 17.05;
HRMS (APCI) m/z 1211.723 [M + H]+ (calcd for C60H103N6O19,
1211.728).
Cyclo-{Tyr(tBu)-D-Hica-D-Val-Lac-[Val-D-Hica-D-Val-Lac]2} (7c).
Using the strategy followed for the preparation and purification of
7a, depsipeptide 6c (700 mg, 0.44 mmol) was deprotected using 20%
palladium hydroxide-on-carbon (300 mg) in 10 mL of 4:1 EtOAc/
MeOH and then cyclized with PyBroP (373 mg, 0.80 mmol) and TEA
(111 μL, 81 mg, 0.80 mmol) in 200 mL of anhydrous DCM. After
separation by column chromatography 7c was obtained as a colorless
solid: yield 11% (66 mg, 52 μmol); TLC Rf 0.20 (1:4 EtOAc/
1
hexanes); mp 64−67 °C; [α]24 +25.7 (c 0.14, EtOH); H NMR
D
(CDCl3, 400 MHz) δ 7.94 (1H, d, J = 7.2 Hz), 7.85 (2H, m), 7.76
(1H, d, J = 8.0 Hz), 7.68 (1H, d, J = 7.2 Hz), 7.65 (1H, d, J = 6.0 Hz),
7.09 (2H, d, J = 8.8 Hz), 6.86 (2H, d, J = 8.4 Hz), 5.20 (4H, m), 5.05
(2H, m), 4.53 (q, J = 7.6 Hz, 1H), 4.13 (1H, t, J = 8.8 Hz), 4.02 (1H,
I
DOI: 10.1021/np501004h
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
26.6, 26.4, 26.2, 19.1, 17.7, and 17.1; HRMS (APCI), m/z 434.2542
[M + H]+ (calcd for C24H36NO6, 434.2543).
(3H, m), 4.42 (2H, m), 4.33 (1H, m), 4.22 (2H, m), 4.11 (1H, t, J =
7.6 Hz), 4.02 (1H, t, J = 7.6 Hz), 2.27 (3H, m), 2.15 (1H, m), 1.74
(14H, m), 1.41 (8H, m), 1.16 (6H, m), and 0.95 (28H, m); 13C NMR
(CDCl3, 101 MHz) δ 172.17, 171.33, 171.06, 171.01, 170.66, 170.54,
170.52, 170.35, 157.04, 144.01, 143.75, 141.41, 135.44, 128.60, 128.36,
128.25, 127.85, 127.19, 125.16, 120.10, 72.65, 72.58, 70.52, 69.24,
67.61, 67.04, 60.58, 59.18, 57.98, 47.16, 39.32, 39.13, 34.77, 33.94,
33.83, 33.81, 32.18, 32.11, 31.69, 30.49, 30.22, 29.85, 29.49, 26.47,
26.42, 26.39, 26.34, 26.10, 26.05, 25.39, 19.21, 19.16, 19.03, 18.94,
18.74, 18.47, 17.56, and 16.88; HRMS (APCI) m/z 1179.645 [M +
H]+ (calcd for C66H91N4O15, 1179.648).
Fmoc-[Val-D-Hcha-D-Val-Lac]3-OBn (13a). Again using the proce-
dure followed for the preparation and purification of 5, unit 12 (320
mg, 0.27 mmol) was deprotected with 2,2′,2″-triaminotriethylamine
(281 μL, 273 mg, 2.16 mmol) in 6 mL of DCM and then coupled to
unit 11a (179 mg, 0.27 mmol) using HBTU (205 mg, 0.54 mmol) and
TEA (37 μL, 27 mg, 0.27 mmol) in 5 mL of anhydrous DMF. After
separation by column chromatography 13a was obtained as a colorless
Fmoc-Val-D-Hcha-D-Val-Lac-OBn (10a). By the procedure fol-
lowed for the preparation and purification of 3a, unit 9 (1.80 g, 4.15
mmol) was coupled to Fmoc-Val (1.55 g, 4.57 mmol) using DCC
(943 mg, 4.57 mmol) and DMAP (152 mg, 1.25 mmol) in 50 mL of
anhydrous DCM. After separation by column chromatography 10a
was obtained as a colorless solid: yield 82% (2.56 g, 3.39 mmol); TLC
Rf 0.25 (1:4 EtOAc/hexanes); mp 49−53 °C; [α]24 +0.45 (c 0.22,
D
CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.74 (2H, d, J = 7.6 Hz), 7.57
(2H, d, J = 7.6 Hz), 7.39 (2H, m) 7.33 (7H, m), 6.74 (1H, d, J = 8.8
Hz), 5.34 (1H, d, J = 8.4 Hz), 5.28 (1H, m), 5.09 (3H, m), 4.55 (1H,
m), 4.41 (1H, m), 4.25 (2H, m), 4.18 (1H, m), 2.28 (1H, m), 2.15
(1H, m), 1.74 (7H, m), 1.41 (4H, m), 1.16 (3H, m), and 0.95 (14H,
m); 13C NMR (CDCl3, 101 MHz) δ 171.63, 170.59, 170.13, 170.08,
156.50, 144.02, 143.81, 141.40, 135.29, 128.67, 128.49, 128.26, 127.84,
127.17, 125.20, 120.09, 73.02, 69.32, 67.44, 67.16, 59.84, 57.38, 47.21,
39.43, 33.94, 33.89, 32.16, 30.87, 30.82, 26.41, 26.34, 26.07, 19.22,
19.11, 18.09, 18.06, and 16.89; HRMS (APCI) m/z 755.3901 [M +
H]+ (calcd for C44H55N2O9, 755.3907).
solid: yield 70% (304 mg, 0.19 mmol); TLC Rf 0.30 (1:4 EtOAc/
1
hexanes); mp 60−64 °C; [α]24 +20.9 (c 0.11, CHCl3); H NMR
D
Fmoc-Thr(tBu)-D-Hcha-D-Val-Lac-OBn (10b). With the method
followed for the preparation and purification of 3a, unit 9 (1.80 g, 4.15
mmol) was coupled to Fmoc-Thr(OtBu) (381 mg, 0.96 mmol) using
DCC (198 mg, 0.96 mmol) and DMAP (32 mg, 0.26 mmol) in 10 mL
of anhydrous DCM. After separation by column chromatography 10b
was obtained as a colorless solid: yield 79% (561 mg, 0.69 mmol);
(CDCl3, 400 MHz) δ 7.73 (2H, d, J = 7.2 Hz), 7.66 (7.6 Hz, 2H, J =
4.4 Hz, 2H), 7.58 (3H, d, J = 15.6 Hz), 7.51 (1H, d, J = 6.0 Hz), 7.36
(3H, m), 7.29 (8H, m), 5.79 (1H, d, J = 7.2 Hz), 5.24 (5H, m), 5.10
(3H, m), 4.38 (3H, m), 4.21 (1H, t, J = 7.2 Hz), 4.13 (2H, m), 4.02
(1H, m), 3.93 (2H, m), 2.27 (5H, m), 1.99 (1H, m), 1.74 (21H, m),
1.41 (12H, m), and 1.02 (51H, m); 13C NMR (CDCl3, 101 MHz) δ
172.44, 171.71, 171.39, 171.38, 171.29, 170.88, 170.68, 170.52, 170.46,
170.42, 170.26, 170.18, 157.07, 144.03, 143.63, 141.27, 135.35, 128.43,
128.17, 128.13, 127.69, 127.02, 124.99, 119.94, 72.57, 72.29, 71.86,
70.35, 69.94, 69.04, 67.49, 66.86, 60.67, 59.95, 59.66, 59.64, 58.92,
58.08, 47.03, 39.31, 38.96, 34.63, 33.85, 33.81, 33.69, 33.66, 33.61,
32.15, 32.03, 31.94, 31.86, 30.20, 29.92, 29.54, 29.26, 29.15, 29.02,
28.97, 26.33, 26.31, 26.26, 26.21, 25.95, 25.92, 25.24, 20.67, 19.35,
19.19, 19.15, 19.14, 19.11, 19.06, 19.02, 18.96, 18.91, 18.79, 18.54,
17.42, 17.15, and 16.74; HRFTMS (ESI) m/z 1625.885 [M + Na]+
(calcd for C88H126N6O21Na, 1625.887).
TLC Rf 0.27 (1:4 EtOAc/hexanes); mp 50−54 °C; [α]24 +2.97 (c
D
1
0.37, CHCl3); H NMR (CDCl3, 400 MHz) δ 7.74 (2H, d, J = 7.6
Hz), 7.57 (2H, t, J = 7.6 Hz), 7.39 (2H, t, J = 7.2 Hz), 7.33 (7H, m),
6.79 (1H, d, J = 8.8 Hz), 5.70 (1H, d, J = 8.0 Hz), 5.22 (1H, t, J = 6.8
Hz), 5.09 (3H, m), 4.55 (1H, dd, J = 8.4 Hz, 5.2 Hz), 4.35 (3H, m),
4.20 (3H, m), 2.28 (1H, m), 1.74 (7H, m), 1.41 (4H, m), 1.16 (15H,
m), and 0.95 (8H, m); 13C NMR (CDCl3, 101 MHz) δ 170.50,
170.48, 169.95, 169.78, 156.51, 143.99, 143.71, 141.28, 135.19, 128.56,
128.38, 128.13, 127.72, 127.06, 125.20, 119.97, 74.40, 73.37, 69.25,
67.39, 67.10, 67.02, 60.05, 57.21, 47.12, 39.43, 33.70, 33.49, 32.53,
30.91, 28.54, 26.33, 26.14, 25.96, 20.52, 18.94, 17.91, and 16.82;
HRMS (APCI) m/z 813.4324 [M + H]+ (calcd for C47H61N2O10,
813.4326).
Fmoc-Thr(tBu)-D-Hcha-D-Val-Lac-[Val-D-Hcha-D-Val-Lac]2-OBn
(13b). By the procedure followed for the preparation and purification
of 5, unit 12 (500 mg, 0.42 mmol) was deprotected with 2,2′,2″-
triaminotriethylamine (633 μL, 614 mg, 4.2 mmol) in 15 mL of DCM
and then coupled to unit 11b (306 mg, 0.42 mmol) using HBTU (319
mg, 0.84 mmol) and TEA (58 μL, 42 mg, 0.42 mmol) in 7 mL of
anhydrous DMF. After separation by column chromatography 13b was
obtained as a colorless solid: yield 64% (448 mg, 0.23 mmol); TLC Rf
Fmoc-Tyr(tBu)-D-Hcha-D-Val-Lac-OBn (10c). Using the strategy
followed for the preparation and purification of 3a, unit 9 (376 mg,
0.87 mmol) was coupled to Fmoc-Tyr(OtBu) (441 mg, 0.96 mmol)
using DCC (198 mg, 0.96 mmol) and DMAP (32 mg, 0.26 mmol) in
10 mL of anhydrous DCM. After separation by column chromatog-
raphy 10b was obtained as a colorless solid: yield 90% (688 mg, 0.79
0.40 (1:2 EtOAc/hexanes); mp 62−66 °C; [α]24 +10.6 (c 0.18,
D
mmol); TLC Rf 0.25 (1:4 EtOAc/hexanes); mp 52−56 °C; [α]24
CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.73 (2H, d, J = 7.2 Hz), 7.66
(4H, m), 7.36 (3H, m), 7.29 (8H, m), 7.14 (1H, d, J = 6.4 Hz), 5.66
(1H, d, J = 7.2 Hz), 5.24 (5H, m), 5.10 (3H, m), 4.38 (3H, m), 4.22
(1H, t, J = 7.2 Hz), 4.13 (4H, m), 4.02 (1H, m), 3.93 (1H, m), 2.27
(4H, m), 2.08 (1H, m), 1.74 (21H, m), 1.41 (12H, m), 1.21 (21H, m)
and 1.02 (36H, m); 13C NMR (CDCl3, 101 MHz) δ 171.45, 171.44,
171.41, 171.01, 170.98, 170.84, 170.79, 170.56, 170.54, 170.42, 170.41,
156.89, 143.98, 143.73, 141.39, 135.51, 130.57, 128.59, 128.33, 127.86,
127.18, 125.20, 120.11, 74.76, 72.86, 72.48, 72.01, 70.30, 70.14, 69.19,
67.64, 66.99, 60.36, 60.08, 59.98, 59.69, 59.39, 58.25, 47.17, 39.42,
39.05, 39.00, 34.76, 33.97, 33.80, 33.77, 33.71, 33.55, 32.50, 32.16,
32.08, 31.68, 30.30, 29.79, 29.75, 29.69, 29.48, 29.43, 28.63, 26.47,
26.45, 26.40, 26.38, 26.35, 26.23, 26.07, 26.03, 26.00, 25.38, 20.73,
19.47, 19.37, 19.27, 19.24, 19.18, 19.08, 19.05, 18.69, 17.33, 17.23, and
16.87; HRFTMS (ESI) m/z 1683.927 [M + Na]+ (calcd for
C91H132N6O22Na, 1683.929).
D
+11.2 (c 0.17, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.74 (2H, d, J
= 7.6 Hz), 7.57 (2H, t, J = 7.6 Hz), 7.39 (2H, t, J = 7.2 Hz), 7.33 (7H,
m), 7.06 (2H, d, J = 8.4 Hz), 6.90 (2H, d, J = 8.4 Hz), 6.83 (1H, d, J =
8.4 Hz), 5.45 (1H, d, J = 7.6 Hz), 5.23 (1H, t, J = 6.4 Hz), 5.09 (3H,
m), 4.55 (2H, m), 4.35 (1H, m), 4.26 (1H, m), 4.15 (1H, m), 3.17
(1H, m), 3.02 (1H, m), 2.28 (1H, m), 1.74 (7H, m), 1.42 (4H, m),
1.31 (12H, m), 1.15 (3H, m), and 0.95 (8H, m); 13C NMR (CDCl3,
101 MHz) δ 171.1, 170.6, 170.2, 170.1, 156.0, 154.7, 143.7, 141.3,
135.2, 130.3, 129.7, 128.6, 128.4, 128.2, 127.8, 127.1, 125.1, 124.2,
120.0, 78.5, 73.3, 69.3, 67.3, 67.1, 57.3, 55.5, 47.1, 39.4, 36.9, 33.8,
33.7, 32.4, 30.8, 28.9, 26.4, 26.1, 26.0, 19.1, 18.0 and 16.9; HRMS
(APCI) m/z 875.4487 [M + H]+ (calcd for C52H63N2O10, 875.4482).
Fmoc-[Val-D-Hcha-D-Val-Lac]2-OBn (12). Using the experimental
strategy followed for the preparation and purification of 5, unit 10a
(300 mg, 0.40 mmol) was deprotected with 2,2′,2″-triaminotriethyl-
amine (605 μL, 588 mg, 4.00 mmol) in 10 mL of DCM and then
coupled to unit 11a (266 mg, 0.40 mmol) using HBTU (303 mg, 0.80
mmol) and TEA (55 μL, 40 mg, 0.40 mmol) in 5 mL of anhydrous
DMF. After separation by column chromatography 12 was obtained as
Fmoc-Tyr(tBu)-D-Hcha-D-Val-Lac-[Val-D-Hcha-D-Val-Lac]2-OBn
(13c). By employing the procedure followed for the preparation and
purification of 5, unit 12 (500 mg, 0.42 mmol) was deprotected with
2,2′,2″-triaminotriethylamine (633 μL, 614 mg, 4.2 mmol) in 15 mL of
DCM and then condensed to unit 11c (329 mg, 0.42 mmol) using
HBTU (319 mg, 0.84 mmol) and TEA (58 μL, 42 mg, 0.42 mmol) in
7 mL of anhydrous DMF. Following separation by column
chromatography 13c was obtained as a colorless solid: yield 74%
(534 mg, 0.31 mmol); TLC Rf 0.40 (1:2 EtOAc/hexanes); mp 61−65
a colorless solid: yield 76% (360 mg, 0.31 mmol); TLC Rf 0.28 (1:4
1
EtOAc/hexanes); mp 60−65 °C; [α]24 +17.1 (c 0.25, CHCl3); H
D
NMR (CDCl3, 400 MHz) δ 7.74 (2H, d, J = 7.2 Hz), 7.58 (2H, dd, J =
14 Hz, 7.6 Hz), 7.39 (3H, m) 7.33 (7H, m), 7.21 (1H, d, J = 7.2 Hz),
7.16 (1H, d, J = 8.4 Hz), 5.50 (1H, d, J = 7.6 Hz), 5.26 (3H, m), 5.09
J
DOI: 10.1021/np501004h
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
°C; [α]24 +14.3 (c 0.30, CHCl3); H NMR (CDCl3, 400 MHz) δ
Hz), 6.88 (2H, d, J = 8.0 Hz), 5.23 (3H, m), 5.13 (3H, m), 4.57 (q, J =
7.2 Hz, 1H, J = 9.2 Hz, 4.0 Hz, dd), 4.07 (3H, dd, J = 9.2 Hz), 4.16
(1H, t, J = 8.8 Hz), 3.99 (1H, dd, J = 9.6 Hz, 7.2 Hz), 3.88 (3H, m),
3.11 (2H, m), 2.23 (5H, m), and 1.90−0.80 (87H, m); 13C NMR
(CDCl3, 101 MHz) δ 172.79, 172.76, 172.42, 172.15, 171.92, 171.75,
171.62, 171.55, 170.77, 170.45, 170.34, 170.10, 154.34, 131.42, 129.84,
124.13, 78.28, 73.37, 73.20, 72.54, 70.98, 70.71, 70.03, 60.76, 60.63,
59.69, 59.64, 58.33, 54.29, 39.50, 39.44, 39.25, 39.24, 35.29, 34.06,
33.89, 33.85, 33.69, 33.46, 32.88, 31.95, 31.85, 29.80, 29.02, 28.96,
28.76, 28.69, 28.67, 28.64, 28.61, 28.50, 26.52, 26.45, 26.39, 26.10,
26.07, 26.02, 19.96, 19.64, 19.49, 19.47, 19.40, 19.35, 19.32, 19.25,
18.92, 17.41, 17.27, 16.86, and 14.22; HRFTMS (ESI) m/z 1415.822
[M + Na]+ (calcd for C74H116N6O19Na, 1415.819).
D
7.73 (2H, d, J = 7.6 Hz), 7.69 (1H, d, J = 6.0 Hz), 7.65 (1H, d, J = 6.0
Hz), 7.53 (3H, m), 7.38 (4H, m), 7.29 (7H, m), 7.06 (2H, d, J = 8.4
Hz), 6.90 (2H, d, J = 8.4 Hz), 5.74 (1H, d, J = 6.4 Hz), 5.26 (5H, m),
5.09 (3H, m), 4.38 (4H, m), 4.20 (1H, m), 4.11 (2H, m), 4.00 (1H,
m), 3.94 (1H, m), 3.09 (2H, m), 2.28 (5H, m), 1.65 (21H, m), 1.41
(12H, m), 1.21 (18H, m), and 1.02 (36H, m); 13C NMR (CDCl3, 101
MHz) δ 171.67, 171.54, 171.63, 171.52, 171.45, 171.02, 170.93,
170.82, 170.59, 170.47, 170.42, 170.40, 156.64, 154.77, 143.92, 143.69,
141.37, 135.47, 130.19, 129.69, 128.54, 128.25, 127.82, 127.17, 125.15,
124.22, 120.06, 78.42, 72.95, 72.45, 72.03, 70.36, 70.15, 69.20, 67.58,
66.98, 59.95, 59.93, 59.73, 59.21, 58.26, 56.03, 47.09, 39.39, 39.08,
39.04, 39.03, 36.46, 34.74, 33.96, 33.95, 33.75, 33.70, 33.67, 33.63,
32.46, 32.18, 32.05, 31.66, 30.27, 29.69, 29.36, 29.27, 29.13, 28.94,
26.45, 26.39, 26.36, 26.06, 25.92, 25.35, 20.78, 19.41, 19.33, 19.30,
19.25, 19.24, 19.18, 19.05, 18.69, 17.38, 17.34, 16.83, 14.19, and 11.51;
Cyclo-{Thr-D-Hcha-D-Val-Lac-[Val-D-Hcha-D-Val-Lac]2} (15a, sil-
statin 5). By the experimental route followed for the preparation
and isolation of 8a, cyclodepsipeptide 14b (80 mg, 60 μmol) was
deprotected with TFA (250 μL) in 2 mL of anhydrous DCM. Isolation
by column chromatography yielded cyclodepsipeptide 15a (silstatin 5)
HRFTMS (ESI) m/z 1745.941 [M
+
Na]+ (calcd for
C96H134N6O22Na, 1745.944).
as a colorless solid: yield 91% (70 mg, 55 μmol); TLC Rf 0.27 (1:3
Cyclo-[Val-D-Hcha-D-Val-Lac]3 (14a, silstatin 4). Using the strategy
followed for the preparation and purification of 7a, depsipeptide 13a
(193 mg, 0.12 mmol) was deprotected using 20% palladium
hydroxide-on-carbon (100 mg) in 8 mL of 4:1 EtOAc/MeOH and
then cyclized with PyBroP (224 mg, 0.48 mmol) and TEA (35 μL, 24
mg, 0.24 mmol) in 120 mL of anhydrous DCM. After separation by
column chromatography silstatin 4 (14a) was obtained as a colorless
1
EtOAc/hexanes); mp 58−63 °C; [α]24 +32.0 (c 0.10, CHCl3); H
D
NMR (CDCl3, 400 MHz) δ 7.96 (1H, d, J = 6.8 Hz), 7.88 (1H, d, J =
7.2 Hz), 7.76 (3H, m), 7.57 (1H, d, J = 6.0 Hz), 5.20 (5H, m), 5.05
(1H, dd, J = 9.2 Hz, 4.4 Hz), 4.12 (1H, t, J = 9.2 Hz), 4.03 (4H, m),
3.88 (2H, m), 2.23 (4H, m), 2.11 (1H, m), 1.70 (21H, m), 1.41 (12H,
m), and 1.21−0.90 (48H, m); 13C NMR (CDCl3, 101 MHz) δ 173.36,
172.42, 172.26, 172.01, 171.96, 171.56, 171.36, 170.91, 170.61, 170.60,
170.28, 169.84, 73.80, 72.45, 72.34, 70.65, 70.60, 70.23, 66.01, 60.45,
60.36, 60.11, 59.60, 59.24, 58.37, 39.33, 39.07, 39.02, 34.62, 33.86,
33.75, 33.70, 33.65, 32.02, 31.96, 31.79, 28.61, 28.56, 28.47, 28.35,
28.11, 26.37, 26.35, 26.27, 26.22, 26.15, 25.97, 25.95, 25.93, 25.23,
19.62, 19.41, 19.40, 19.36, 19.34, 19.31, 19.30, 19.28, 19.27, 19.17,
19.15, 18.83, 17.35, 16.92, and 16.85; HRMS (APCI) m/z 1275.757
[M + H]+ (calcd for C65H107N6O19, 1275.759).
Cyclo-{Tyr-D-Hcha-D-Val-Lac-[Val-D-Hcha-D-Val-Lac]2} (15b, sil-
statin 6). Again by the procedure followed for the preparation and
purification of 8a, cyclodepsipeptide 14c (130 mg, 93 μmol) was
deprotected with TFA (250 μL) in 2 mL of anhydrous DCM. After
separation by column chromatography cyclodepsipeptide 15b
(silstatin 6) was obtained as a colorless solid: yield 96% (120 mg,
solid: yield 23% (35 mg, 27 μmol); TLC Rf 0.5 (1:4 EtOAc/hexanes);
1
mp 52−56 °C; [α]24 +35.0 (c 0.06, CHCl3); H NMR (CDCl3, 400
D
MHz) δ 7.79 (3H, d, J = 8.0 Hz), 7.73 (3H, d, J = 6.4 Hz), 5.23 (3H,
m), 5.17 (8.0 Hz, dd), 3.93 (3H, dd, J = 9.6 Hz, 6.0 Hz), 2.23 (6H, m),
1.74 (21H, m), 1.46 (12H, m), and 1.02 (51H, m); 13C NMR (CDCl3,
101 MHz) δ 172.4, 172.1, 171.5, 170.4, 72.9, 70.6, 60.3, 59.1, 39.4,
34.0, 33.9, 32.1, 28.7, 28.6, 26.6, 26.4, 26.1, 19.8, 19.6, 19.4, 19.2, and
17.3; HRMS (APCI) m/z 1273.779 [M + H]+ (calcd for
C66H109N6O18, 1273.780).
Cyclo-{Thr(tBu)-D-Hcha-D-Val-Lac-[Val-D-Hcha-D-Val-Lac]2} (14b).
Using the strategy followed for the preparation and purification of 7a,
depsipeptide 13b (530 mg, 0.26 mmol) was deprotected using 20%
palladium hydroxide-on-carbon (200 mg) in 10 mL of 4:1 EtOAc/
MeOH and then cyclized with PyBroP (485 mg, 1.04 mmol) and TEA
(144 μL, 105 mg, 1.04 mmol) in 260 mL of anhydrous DCM. After
separation by column chromatography 14b was obtained as a colorless
90 μmol); TLC Rf 0.28 (1:3 EtOAc/hexanes); mp 76−80 °C; [α]24
D
+26.2 (c 0.21, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.84 (1H, d, J
= 7.2 Hz), 7.81 (1H, d, J = 5.6 Hz), 7.77 (1H, d, J = 8.4 Hz), 7.69 (1H,
d, J = 6.4 Hz), 7.63 (1H, d, J = 6.0 Hz), 7.59 (1H, d, J = 7.2 Hz), 7.02
(2H, d, J = 8.8 Hz), 6.73 (2H, d, J = 8.4 Hz), 5.23 (3H, m), 5.10 (3H,
m), 4.50 (q, J = 8.0 Hz, 1H), 4.15 (1H, t, J = 8.8 Hz), 3.99 (2H, m),
3.88 (2H, m), 3.11 (2H, m), 2.23 (5H, m), and 1.90−0.80 (78H, m);
13C NMR (CDCl3, 101 MHz) δ 172.65, 172.62, 172.32, 172.15,
solid: yield 29% (100 mg, 75 μmol); TLC Rf 0.4 (1:4 EtOAc/
1
hexanes); mp 61−65 °C; [α]24 +27.1 (c 0.09, CHCl3); H NMR
D
(CDCl3, 400 MHz) δ 7.77 (1H, d, J = 8.4 Hz), 7.69 (1H, d, J = 7.2
Hz), 7.62 (1H, d, J = 8.4 Hz), 7.42 (1H, d, J = 8.0 Hz), 7.40 (1H, d, J
= 6.0 Hz), 7.29 (1H, d, J = 5.2 Hz), 5.23 (5H, m), 5.13 (1H, dd, J =
9.2 Hz, 3.6 Hz), 4.29 (1H, t, J = 8.0 Hz), 4.23 (1H, t, J = 8.0 Hz), 4.05
(4H, m), 3.85 (1H, dd, J = 10 Hz, 5.6 Hz), 2.23 (5H, m), 1.74 (21H,
m), 1.41 (12H, m), 1.21 (21H, m), and 1.02 (36H, m); 13C NMR
(CDCl3, 101 MHz) δ 172.29, 172.16, 172.08, 172.01, 171.78, 171.65,
171.17, 171.02, 170.19, 170.13, 169.94, 169.93, 74.84, 73.29, 72.77,
72.47, 71.04, 70.81, 70.70, 66.64, 60.82, 59.76, 59.10, 58.94, 58.23,
57.81, 39.50, 39.47, 39.43, 39.21, 36.77, 34.79, 34.13, 34.09, 34.03,
33.90, 33.83, 33.80, 32.22, 31.93, 31.83, 29.91, 29.35, 28.98, 28.68,
28.64, 28.59, 28.57, 28.52, 26.52, 26.42, 26.35, 26.09, 24.82, 20.15,
19.91, 19.53, 19.51, 19.42, 19.36, 19.29, 18.95, 18.58, 18.46, 17.50,
17.21, and 17.13; HRFTMS (ESI) m/z 1353.805 [M + Na]+ (calcd for
C69H114N6O19Na, 1353.803).
171.92, 171.79, 171.76, 171.43, 170.84, 170.53, 170.35, 170.09, 155.46,
130.45, 127.60, 115.75, 73.49, 73.10, 72.57, 70.90, 70.76, 70.28, 60.75,
60.36, 59.17, 59.15, 58.56, 54.59, 39.45, 39.42, 39.40, 39.25, 39.24,
35.30, 34.79, 34.07, 33.88, 33.63, 33.57, 32.57, 31.92, 31.84, 31.70,
28.95, 28.91, 28.75, 28.74, 28.69, 26.51, 26.41, 26.12, 26.07, 25.96,
25.40, 22.77, 19.91, 19.79, 19.60, 19.46, 19.40, 19.30, 19.28, 19.27,
19.11, 18.97, 18.94, 17.39, 17.06, and 16.99; HRFTMS (ESI) m/z
1359.755 [M + Na]+ (calcd for C70H108N6O19Na, 1359.756).
Fmoc-Thr(tBu)-D-Hcha-D-Val-Lac-[Val-D-Hica-D-Val-Lac]2-OBn
(16a). Applying the method followed for the preparation and isolation
of 5, unit 5 (264 mg, 0.24 mmol) was deprotected with 2,2′,2″-
triaminotriethylamine (362 μL, 351 mg, 2.4 mmol) in 15 mL of DCM
and then coupled to unit 11b (170 mg, 0.24 mmol) using HBTU (182
mg, 0.48 mmol) and TEA (33 μL, 24 mg, 0.24 mmol) in 5 mL of
anhydrous DMF. Separation by column chromatography provided
16a, which was obtained as a colorless solid: yield 74% (281 mg, 0.18
Cyclo-{Tyr(tBu)-D-Hcha-D-Val-Lac-[Val-D-Hcha-D-Val-Lac]2} (14c).
By means of the strategy followed for the preparation and purification
of 7a, depsipeptide 13c (502 mg, 0.29 mmol) was deprotected using
20% palladium hydroxide-on-carbon (200 mg) in 8 mL of 4:1 EtOAc/
MeOH and then cyclized with PyBroP (541 mg, 1.16 mmol) and TEA
(160 μL, 118 mg, 1.17 mmol) in 290 mL of anhydrous DCM. After
separation by column chromatography cyclodepsipeptide 14c was
obtained as a colorless solid: yield 34% (145 mg, 0.10 mmol); TLC Rf
mmol); TLC Rf 0.50 (1:2 EtOAc/hexanes); mp 56−60 °C; [α]24
D
+7.62 (c 0.11, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.74 (2H, d, J
= 7.2 Hz), 7.70 (1H, d, J = 6.0 Hz), 7.55 (4H, m), 7.39 (2H, m) 7.29
(8H, m), 7.14 (1H, d, J = 6.4 Hz), 5.65 (1H, d, J = 7.2 Hz), 5.24 (5H,
m), 5.10 (3H, m), 4.38 (3H, m), 4.22 (1H, t, J = 7.2 Hz), 4.13 (4H,
m), 3.98 (1H, m), 3.92 (1H, m), 2.27 (5H, m), 1.74 (13H, m), 1.41
(10H, m), 1.09 (15H, m), and 0.98 (44H, m); 13C NMR (CDCl3, 101
0.4 (1:4 EtOAc/hexanes); mp 68−72 °C; [α]24 +31.3 (c 0.12,
D
CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.97 (1H, d, J = 7.2 Hz), 7.93
(1H, d, J = 6.0 Hz), 7.85 (1H, d, J = 6.8 Hz), 7.75 (1H, d, J = 8.4 Hz),
7.63 (1H, d, J = 6.8 Hz), 7.58 (1H, d, J = 5.6 Hz), 7.11 (2H, d, J = 8.4
K
DOI: 10.1021/np501004h
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
MHz) δ 171.45, 171.38, 171.34, 171.03, 170.96, 170.91, 170.76,
170.69, 170.53, 170.48, 170.37, 170.36, 156.88, 143.95, 143.71, 141.38,
135.48, 128.58, 128.32, 128.26, 127.84, 127.16, 125.17, 120.09, 74.75,
73.10, 72.82, 72.62, 70.27, 70.18, 69.17, 67.63, 66.98, 66.94, 64.43,
60.35, 60.07, 59.97, 59.61, 59.42, 58.17, 47.15, 40.90, 40.43, 39.04,
33.80, 33.53, 32.47, 30.34, 29.76, 29.64, 29.44, 29.43, 28.61, 26.38,
26.21, 25.98, 24.48, 24.44, 23.33, 21.51, 21.44, 20.71, 19.47, 19.37,
19.31, 19.26, 19.23, 19.20, 19.15, 19.08, 19.06, 18.63, 17.36, 17.23, and
16.85; HRFTMS (ESI) m/z 1603.867 [M + Na]+ (calcd for
C85H124N6O22Na, 1603.866).
dd, J = 10 Hz, 7.6 Hz), 3.88 (3H, m), 3.11 (2H, m), 2.23 (5H, m), and
1.90−0.80 (73H, m); 13C NMR (CDCl3, 101 MHz) δ 172.64, 172.53,
172.29, 172.02, 171.78, 171.60, 171.52, 171.31, 170.57, 170.35, 170.10,
169.92, 154.19, 131.28, 129.68, 123.97, 78.15, 73.78, 73.18, 73.08,
70.81, 70.58, 69.86, 60.55, 59.58, 59.47, 58.13, 54.12, 40.73, 40.58,
39.29, 35.10, 35.09, 33.51, 33.33, 32.73, 29.66, 28.82, 28.62, 28.56,
28.49, 28.45, 28.33, 26.35, 25.91, 25.86, 24.46, 24.45, 23.35, 23.32,
21.15, 21.03, 19.82, 19.80, 19.50, 19.35, 19.31, 19.27, 19.18, 19.16,
18.75, 17.27, 17.15, and 16.68; HRMS (APCI) m/z 1313.777 [M +
H]+ (calcd for C68H109N6O19, 1313.775).
Fmoc-Tyr(tBu)-D-Hcha-D-Val-Lac-[Val-D-Hica-D-Val-Lac]2-OBn
(16b). By following the experimental procedures used for the synthesis
and purification of 5, that unit (5) (221 mg, 0.20 mmol) was
deprotected with 2,2′,2″-triaminotriethylamine (302 μL, 293 mg, 2.0
mmol) in 15 mL of DCM and then coupled to unit 11c (158 mg, 0.20
mmol) using HBTU (152 mg, 0.40 mmol) and TEA (28 μL, 20 mg,
0.20 mmol) in 5 mL of anhydrous DMF. Isolation by column
chromatography afforded 16b as a colorless solid in 71% yield (232
mg, 0.14 mmol): TLC Rf 0.45 (1:2 EtOAc/hexanes); mp 58−62 °C;
[α]24D +15 (c 0.09, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.74 (3H,
m), 7.65 (1H, d, J = 7.2 Hz), 7.55 (3H, m), 7.30 (11H, m), 7.06 (2H,
d, J = 8.0 Hz), 6.89 (2H, d, J = 8.4 Hz), 5.79 (1H, d, J = 6.8 Hz), 5.24
(5H, m), 5.08 (3H, m), 4.38 (3H, m), 4.05 (5H, m), 3.03 (2H, m),
2.25 (5H, m), 1.70 (13H, m), 1.41 (10H, m), 1.32 (9H, s), 1.09 (3H,
m), and 0.98 (44H, m); 13C NMR (CDCl3, 101 MHz) δ 171.68,
171.57, 171.54, 171.42, 171.38, 170.93, 170.72, 170.58, 170.45, 170.43,
170.42, 170.38, 156.64, 154.73, 143.89, 143.67, 141.35, 135.42, 129.67,
128.54, 128.29, 128.23, 127.81, 127.15, 125.13, 124.21, 120.05, 78.43,
73.08, 72.92, 72.66, 70.22, 69.21, 67.55, 66.99, 59.91, 59.89, 59.87,
59.86, 59.60, 59.21, 58.18, 56.01, 47.07, 40.86, 40.47, 39.07, 36.44,
34.72, 33.66, 33.61, 32.43, 31.64, 30.33, 29.73, 29.40, 29.36, 29.28,
29.11, 28.92, 26.37, 26.04, 25.90, 25.34, 24.46, 24.44, 23.32, 23.31,
21.52, 21.41, 19.39, 19.30, 19.26, 19.23, 19.19, 19.14, 19.05, 18.62,
17.37, and 16.83; HRFTMS (ESI) m/z 1665.877 [M + Na]+ (calcd for
C90H126N6O22Na, 1665.882).
Cyclo-{Thr-D-Hcha-D-Val-Lac-[Val-D-Hica-D-Val-Lac]2} (18a, silsta-
tin 7). Using the procedure used for the synthesis and isolation of 8a,
cyclodepsipeptide 17a (80 mg, 64 μmol) was deprotected with TFA
(250 μL) in 1 mL of anhydrous DCM. After separation by column
chromatography 18a (silstatin 7) was obtained as a colorless solid:
yield 92% (70 mg, 59 μmol); TLC Rf 0.27 (1:3 EtOAc/hexanes); mp
75−79 °C; [α]24D +19 (c 0.09, CHCl3); 1H NMR (CDCl3, 400 MHz)
δ 7.97 (1H, d, J = 6.8 Hz), 7.88 (1H, d, J = 7.2 Hz), 7.76 (3H, m), 7.57
(1H, d, J = 6.0 Hz), 5.20 (5H, m), 5.05 (1H, dd, J = 8.8 Hz, 4.0 Hz),
4.75 (1H, br s), 4.12 (1H, t, J = 8.8 Hz), 3.95 (6H, m), 2.23 (4H, m),
2.11 (1H, m), 1.74 (13H, m), 1.43 (10H, m), and 1.25−0.9 (50H, m);
13C NMR (CDCl3, 101 MHz) δ 173.4, 172.65, 172.30, 172.23, 172.13,
171.75, 171.57, 171.07, 170.94, 170.80, 170.40, 170.00, 73.91, 73.31,
73.19, 70.79, 70.72, 70.43, 66.16, 60.69, 60.48, 60.21, 59.86, 59.44,
58.59, 40.82, 40.73, 39.16, 34.00, 33.87, 32.12, 28.73, 28.68, 28.62,
28.52, 28.26, 26.50, 26.30, 26.10, 24.59, 24.58, 23.46, 23.34, 21.54,
21.34, 19.76, 19.58, 19.53, 19.49, 19.48, 19.47, 19.46, 19.45, 19.42,
19.32, 19.02, 17.50, and 17.05; HRMS (APCI) m/z 1195.696 [M +
H]+ (calcd for C59H99N6O19, 1195.697).
Cyclo-{Tyr-D-Hcha-D-Val-Lac-[Val-D-Hica-D-Val-Lac]2} (18b, silsta-
tin 8). By using the general procedure for the preparation and isolation
of 8a, cyclodepsipeptide 17b (30 mg, 23 μmol) was deprotected with
TFA (250 μL) in 1 mL of anhydrous DCM. Separation by column
chromatography led to 18b (silstatin 8) as a colorless solid: yield 87%
(25 mg, 20 μmol); TLC Rf 0.27 (1:3 EtOAc/hexanes); mp 78−82 °C;
1
[α]24 +21.3 (c 0.08, CHCl3); H NMR (CDCl3, 400 MHz) δ 7.80
Cyclo-{Thr(tBu)-D-Hcha-D-Val-Lac-[Val-D-Hica-D-Val-Lac]2} (17a).
By employing the route followed for obtaining 7a, depsipeptide 16a
(269 mg, 0.17 mmol) was deprotected using 20% palladium
hydroxide-on-carbon (200 mg) in 8 mL of 4:1 EtOAc/MeOH and
then cyclized employing PyBroP (317 mg, 0.68 mmol) and TEA (94
μL, 69 mg, 0.68 mmol) in 170 mL of anhydrous DCM. Separation by
column chromatography provided 17a as a colorless solid in 48% yield
D
(3H, m), 7.67 (2H, m), 7.61 (1H, d, J = 7.2 Hz), 7.05 (2H, d, J = 8.0
Hz), 6.74 (2H, d, J = 8.0 Hz), 6.03 (1H, br s), 5.23 (3H, m), 5.09 (3H,
m), 4.48 (q, J = 7.4 Hz, 1H), 4.14 (1H, t, J = 8.8 Hz), 4.05 (2H, m),
3.93 (2H, m), 3.05 (2H, m), 2.23 (5H, m), 1.77 (7H, m), 1.60 (7H,
m), 1.45 (6H, d, J = 6.8 Hz), 1.36 (3H, d, J = 6.8 Hz), and 1.11−0.80
(47H, m); 13C NMR (CDCl3, 101 MHz) δ 172.60, 172.44, 172.34,
172.22, 171.88, 171.78, 171.69, 171.49, 170.99, 170.45, 170.36, 170.14,
155.25, 130.52, 127.80, 115.81, 73.75, 73.54, 73.26, 70.86, 70.72,
70.38, 60.75, 60.31, 59.04, 59.01, 58.69, 54.69, 40.91, 40.74, 39.41,
35.32, 33.64, 33.62, 32.54, 29.85, 29.04, 28.91, 28.75, 28.72, 28.67,
26.53, 26.09, 25.97, 24.66, 24.63, 23.50, 23.49, 21.33, 21.21, 19.91,
19.76, 19.62, 19.50, 19.42, 19.36, 19.35, 19.07, 18.99, 18.89, 17.44, and
17.06; HRMS (APCI) m/z 1257.713 [M + H]+ (calcd for
C64H101N6O19, 1257.712).
(102 mg, 81 μmol); TLC Rf 0.3 (1:4 EtOAc/hexanes); mp 58−62 °C;
1
[α]24 +17.3 (c 0.08, CHCl3); H NMR (CDCl3, 400 MHz) δ 7.78
D
(1H, d, J = 8.0 Hz), 7.71 (1H, d, J = 7.2 Hz), 7.62 (1H, d, J = 8.4 Hz),
7.43 (2H, m), 7.33 (1H, d, J = 4.8 Hz), 5.18 (6H, m), 4.28 (1H, t, J =
8.0 Hz), 4.22 (1H, t, J = 8.0 Hz), 4.05 (4H, m), 3.85 (1H, dd, J = 10
Hz, 5.6 Hz), 2.23 (5H, m), 1.74 (13H, m), 1.43 (10H, m), and 1.25−
0.9 (59H, m); 13C NMR (CDCl3, 101 MHz) δ 172.31, 172.21, 172.06,
171.97, 171.69, 171.20, 171.09, 170.24, 170.22, 170.14, 169.99, 169.94,
74.80, 73.41, 73.28, 73.12, 70.99, 70.79, 70.68, 66.61, 60.79, 59.77,
59.12, 58.97, 58.27, 57.84, 40.94, 40.69, 39.46, 34.03, 33.77, 32.20,
29.83, 29.80, 29.29, 28.91, 28.65, 28.56, 28.49, 26.49, 26.33, 26.07,
24.60, 24.55, 23.50, 21.29, 21.18, 20.12, 19.87, 19.53, 19.51, 19.40,
19.37, 19.33, 19.29, 18.96, 18.60, 18.47, 17.48, 17.19, and 17.12;
HRMS (APCI) m/z 1251.760 [M + H]+ (calcd for C63H107N6O19,
1251.759).
Cyclo-{Tyr(tBu)-D-Hcha-D-Val-Lac-[Val-D-Hica-D-Val-Lac]2} (17b).
Using the preceding method followed for the synthesis and isolation
of 7a, depsipeptide 16b (220 mg, 0.13 mmol) was deprotected using
20% palladium hydroxide-on-carbon (200 mg) in 8 mL of 4:1 EtOAc/
MeOH and then cyclized with PyBroP (250 mg, 0.54 mmol) and TEA
(74 μL, 54 mg, 0.54 mmol) in 134 mL of anhydrous DCM. Isolation
by column chromatography yielded 17b as a colorless solid: yield 22%
(38 mg, 29 μmol); TLC Rf 0.3 (1:4 EtOAc/hexanes); mp 55−59 °C;
[α]24D +24 (c 0.07, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.98 (1H,
d, J = 7.2 Hz), 7.94 (1H, d, J = 5.6 Hz), 7.85 (1H, d, J = 7.2 Hz), 7.74
(1H, d, J = 8.8 Hz), 7.63 (1H, d, J = 6.8 Hz), 7.59 (1H, d, J = 5.6 Hz),
7.12 (2H, d, J = 8.4 Hz), 6.88 (2H, d, J = 8.4 Hz), 5.23 (5H, m), 5.13
(1H, m), 4.58 (q, J = 7.6 Hz, 1H), 4.18 (1H, t, J = 8.8 Hz), 4.01 (1H,
Methyl 1-(4-formyl-2-nitrophenyl)-2,3,4-tri-O-acetyl-β-D-glucuro-
nate (19). Employing the strategy presented by Duimstra et al.,11 1-
bromo-2,3,4-tri-O-acetyl-α-D-glucuronate (2.32 g, 5.84 mmol) was
coupled to 4-hydroxy-3-nitrobenzaldehyde (1.66 g, 9.93 mmol) using
Ag2O (6.14 g, 26.5 mmol) in 20 mL of anhydrous CH3CN. Yield 93%
1
(2.62 g, 5.42 mmol); H NMR (CDCl3, 400 MHz) δ 9.98 (1H, s),
8.32 (1H, d, J = 1.6 Hz), 8.10 (1H, dd, J = 8.0 Hz, 1.6 Hz), 7.52 (1H,
d, J = 8.8 Hz), 5.43 (2H, m), 5.31 (2H, m), 4.34 (1H, d, J = 8.8 Hz),
3.71 (3H, s), 2.13 (3H, s), 2.09 (3H, s), and 2.08 (3H, s); 13C NMR
(CDCl3, 101 MHz) δ 188.7, 170.0, 169.4, 169.2, 166.8, 153.4, 141.2,
134.4, 131.6, 126.8, 118.9, 98.7, 72.8, 70.3, 69.9, 68.2, 53.2, 20.7, and
20.7.
Methyl 1-(4-(tert-butyldimethylsilyloxy)methyl-2-nitrophenyl)-
2,3,4-tri-O-acetyl-β-D-glucuronate (21). By means of the synthesis
presented by Duimstra et al.,11 glucuronate 19 (2.60 g, 5.38 mmol)
was reduced with sodium borohydride (305 mg, 8.07 mmol) in the
presence of silica (1.08 g) in 65 mL of 1:5 2-propanol/CHCl3 and
then silyl protected using TBDMS-Cl (1.22 g, 8.07 mmol) and
imidazole (549 mg, 8.07 mmol) in 30 mL of anhydrous DCM. After
separation by column chromatography 21 was obtained as a colorless
L
DOI: 10.1021/np501004h
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
solid: yield 80% (2.58 g, 4.30 mmol); TLC Rf 0.25 (1:2 EtOAc/
hexanes); H NMR (CDCl3, 400 MHz) δ 7.75 (1H, d, J = 2.4 Hz),
of anhydrous DMF was added potassium carbonate (88 mg, 0.64
mmol) followed by compound 25 (256 mg, 0.32 mmol). The reaction
mixture was stirred at 23 °C for 30 min, diluted with 60 mL of EtOAc,
washed with 20 mL of 6% aqueous citric acid and 25 mL of brine,
dried over MgSO4, and concentrated under reduced pressure. The
residue was partially separated using a short silica gel plug to remove
the 4-nitrophenol and afford intermediate 26 as a colorless oil: crude
yield 80% (200 mg). Next, intermediate 26 (130 mg, 0.17 mmol) was
dissolved in 3 mL of anhydrous DCM, and compound 18a (167 mg,
0.14 mmol) was added followed by DMAP (5 mg, 0.04 mmol) and
DCC (31 mg, 0.15 mmol). The reaction mixture was stirred at 23 °C
for 16 h. The solvent was separated by filtration, and the filtrate was
concentrated under reduced pressure. The crude product was
separated by chromatography on a silica gel column. Elution with
3:2 hexanes/EtOAc gave the allyl-protected prodrug 27 as a colorless
1
7.45 (1H, dd, J = 8.8 Hz, 2.4 Hz), 7.32 (1H, d, J = 8.4 Hz), 5.31 (3H,
m), 5.17 (1H, d, J = 7.2 Hz), 4.72 (2H, s), 4.19 (1H, d, J = 9.2 Hz),
3.75 (3H, s), 2.13 (3H, s), 2.06 (3H, s), 2.05 (3H, s), 0.94 (9H, s), and
0.11 (6H, s); 13C NMR (CDCl3, 101 MHz) δ 170.16, 169.44, 169.43,
166.87, 147.92, 141.45, 138.24, 131.19, 122.56, 120.35, 100.23, 72.75,
71.37, 70.39, 68.97, 63.53, 53.18, 26.01, 20.74, 20.71, 20.65, and −5.18.
Allyl 1-(4-(tert-butyldimethylsilyloxy)methyl-2-nitrophenyl)-β-D-
glucuronate (22). By employing the procedure recorded by Grinda
et al.,12 glucuronate 21 (1.58 g, 2.63 mmol) was deacetylated with 0.5
N sodium methoxide in MeOH (5.26 mL, 2.63 mmol) in 50 mL of
anhydrous MeOH and then transesterified with a sodium allylate
solution (prepared by dissolving sodium (12 mg, 0.53 mmol) in 5 mL
of allyl alcohol). After separation by column chromatography 22 was
obtained as a colorless solid: yield 51% (671 mg, 1.34 mmol); TLC Rf
0.25 (1:1 acetone/hexanes); 1H NMR (CDCl3, 400 MHz) δ 7.71 (1H,
d, J = 2.4 Hz), 7.42 (1H, dd, J = 8.4 Hz, 2.0 Hz), 7.29 (1H, d, J = 8.4
Hz), 5.87 (1H, m), 5.32 (1H, m), 5.20 (1H, m), 5.02 (1H, d, J = 7.2
Hz), 4.81 (1H, br s), 4.66 (4H, m), 4.39 (1H, br s), 4.28 (1H, br s),
4.11 (1H, d, J = 9.6 Hz), 3.92 (1H, m), 3.78 (1H, m), 0.91 (9H, s),
and 0.07 (6H, s); 13C NMR (CDCl3, 101 MHz) δ 168.4, 149.1, 140.3,
137.0, 131.8, 131.4, 122.9, 119.1, 118.8, 102.3, 75.1, 74.8, 72.9, 71.0,
66.6, 63.5, 26.0, 18.5, and −5.2.
solid: yield 51% over two steps (176 mg, 90 μmol); TLC Rf 0.25 (2:3
1
EtOAc/hexanes); mp 76−79 °C; [α]24 +12.0 (c 0.03, CHCl3); H
D
NMR (CDCl3, 400 MHz) δ 7.93 (1H, m), 7.77 (6H, m), 7.53 (1H,
dd, J = 8.8 Hz, 2.0 Hz), 7.33 (1H, d, J = 8.8 Hz), 5.84 (4H, m), 5.25
(16H, m), 5.09 (4H, m), 4.65 (8H, m), 4.69 (8H, m), 4.48 (1H, dd, J
= 8.4 Hz, 6.4 Hz), 4.33 (1H, d, J = 8.8 Hz), 4.08 (5H, m), 3.90 (1H,
m), 3.47 (2H, m), 3.32 (3H, m), 2.92 (3H, s), 2.26 (5H, m), 1.77
(15H, m), 1.36 (13H, m), 1.08 (20H, m), and 0.94 (27H, m); 13C
NMR (CDCl3, 101 MHz) δ 172.32, 172.26, 172.06, 172.03, 172.01,
171.84, 171.69, 171.57, 171.49, 170.46, 170.28, 170.08, 165.54, 156.71,
153.89, 153.41, 153.40, 148.68, 140.87, 133.38, 131.12, 131.00, 130.88,
130.79, 124.74, 119.89, 119.34, 119.26, 119.24, 119.21, 118.97, 99.54,
74.87, 73.83, 73.40, 73.27, 73.15, 72.27, 72.22, 70.47, 70.41, 70.37,
69.45, 69.22, 69.11, 68.50, 66.86, 65.17, 60.52, 59.88, 58.74, 58.63,
57.62, 49.10, 40.63, 40.57, 39.15, 34.62, 33.92, 33.73, 33.69, 32.12,
31.54, 28.62, 28.53, 28.49, 26.30, 26.16, 25.92, 25.58, 25.23, 24.90,
24.45, 24.41, 23.45, 23.23, 22.62, 22.61, 21.24, 20.95, 20.66, 19.71,
19.43, 19.39, 19.29, 19.24, 19.03, 18.92, 18.87, 17.21, 16.98, 16.88, and
14.07; HRFTMS (ESI), m/z 1979.8054 [M + Na]+ (calcd for
C93H136N8O37Na, 1979.8899).
Allyl 1-(4-(tert-butyldimethylsilyloxy)methyl-2-nitrophenyl)-2,3,4-
tri-O-allyloxycarbonyl-β-D-glucuronate (23). Following the exper-
imental procedure recommended by Grinda et al.,12 the free hydroxy
groups of glucuronate 22 (200 mg, 0.40 mmol) were protected using
allyl chloroformate (1.28 mL, 1.45 g, 12.0 mmol) in 2 mL of
anhydrous pyridine, and following separation by column chromatog-
raphy 23 was obtained as a colorless oil: yield 72% (216 mg, 0.29
1
mmol); TLC Rf 0.4 (1:3 EtOAc/hexanes); H NMR (CDCl3, 400
MHz) δ 7.72 (1H, d, J = 2.4 Hz), 7.45 (1H, dd, J = 8.8 Hz, 2.4 Hz),
7.29 (1H, d, J = 8.4 Hz), 5.87 (4H, m), 5.33 (3H, m), 5.20 (7H, m),
4.68 (4H, m), 4.59 (6H, m), 4.31 (1H, m), 0.91 (9H, s), and 0.07 (6H,
s); 13C NMR (CDCl3, 101 MHz) δ 165.73, 154.03, 153.55, 153.54,
147.96, 140.99, 137.99, 131.31, 131.22, 131.15, 131.04, 130.97, 122.68,
119.38, 119.35, 119.32, 119.27, 119.05, 99.83, 75.15, 74.07, 72.49,
72.26, 69.54, 69.33, 69.18, 66.94, 63.42, 25.94, 18.41, and −5.24.
Allyl 1-(4-hydroxymethyl-2-nitrophenyl)-2,3,4-tri-O-allyloxycar-
bonyl-β-D-glucuronate (24). Again following the procedure presented
by Grinda et al.,12 compound 23 (409 mg, 0.54 mmol) was
deprotected using a 7:3 HF/pyridine solution (2.05 mL) in 10 mL
of anhydrous tetrahydrofuran (THF). Column chromatography using
EtOAc/hexanes gave 24 as a colorless oil: yield 92% (316 mg, 0.50
Glucuronide Prodrug 28. To a stirred solution containing
protected prodrug 27 (24 mg, 12 μmol) in 1 mL of anhydrous
THF was added a solution containing formic acid (1.4 μL, 1.7 mg, 36
μmol) and TEA (8.3 μL, 6.1 mg, 60 μmol) in 100 μL of anhydrous
THF. The reaction mixture was stirred at 23 °C for 10 min; then
Pd(PPh3)4 (1.4 mg, 1.2 μmol) was added, and the mixture was stirred
at 23 °C for 2.5 h and then concentrated under reduced pressure. The
crude product was separated by reversed-phase HPLC. Column:
Phenomenex Luna C8(2), 250 × 10 mm, 5 μm. Flow rate: 3.5 mL/
min. Solvents: (A) 50 mM NH4OAc (pH = 3.5); (B) CH3CN.
Isocratic elution with 20% A in B from 0 to 10 min; next, gradient
elution from 20% A in B to 1% A in B from min 10 to min 12; finally,
isocratic elution with 1% A in B from min 12 to min 18. Retention
time: 15.5 min. This provided a colorless solid: yield 50% (10 mg, 6
1
mmol); TLC Rf 0.20 (1:3 EtOAc/hexanes); H NMR (CDCl3, 400
MHz) δ 7.76 (1H, d, J = 2.0 Hz), 7.45 (1H, dd, J = 8.4 Hz, 2.0 Hz),
7.29 (1H, d, J = 8.4 Hz), 5.87 (4H, m), 5.33 (4H, m), 5.20 (8H, m),
4.67 (10H, m), and 4.35 (1H, d, J = 8.8 Hz); 13C NMR (CDCl3, 101
MHz) δ 165.80, 154.04, 153.58, 153.56, 148.16, 140.75, 137.52,
132.11, 131.24, 131.10, 131.00, 130.92, 123.33, 119.44, 119.37, 119.34,
119.12, 119.09, 99.54, 75.15, 74.07, 72.45, 72.07, 69.61, 69.36, 69.22,
67.01, and 63.25.
μmol); mp 133−136 °C; [α]24 +8.6 (c 0.04, CHCl3); 1H NMR
D
(CD3OD, 400 MHz) δ 7.84 (1H, m), 7.61 (1H, dd, J = 8.4 Hz, 1.6
Hz), 7.48 (1H, d, J = 8.8 Hz), 5.44 (1H, m), 5.12 (9H, m), 4.99 (1H,
m), 4.39 (2H, d, J = 6.8 Hz), 4.25 (3H, m), 3.85 (1H, d, J = 9.6 Hz),
3.52 (3H, m), 3.36 (2H, m), 2.94 (3H, s), 2.30 (7H, m), 1.74 (15H,
m), 1.41 (10H, m) 1.29 (6H, m), and 0.95 (44H, m); 13C NMR
(CD3OD, 101 MHz) δ 175.10, 173.42, 173.40, 173.29, 173.16, 172.82,
172.77, 172.74, 172.12, 172.10, 172.01, 171.99, 171.97, 170.56, 157.75,
151.12, 141.73, 134.57, 132.65, 125.51, 119.06, 102.39, 77.66, 76.57,
74.87, 74.49, 74.44, 73.18, 71.95, 71.86, 71.79, 66.75, 60.00, 59.86,
59.80, 59.72, 57.24, 49.28, 41.97, 40.85, 40.83, 35.67, 34.99, 34.94,
34.90, 34.46, 33.37, 32.15, 32.12, 31.50, 31.40, 31.22, 31.07, 31.02,
27.43, 27.24, 27.04, 26.19, 25.70, 24.20, 23.75, 23.68, 23.61, 21.71,
21.60, 19.68, 19.63, 19.62, 19.61, 19.60, 19.28, 19.16, 18.93, 18.85,
18.78, 18.08, 18.03, and 17.19; HRFTMS (ESI) m/z 1687.7934 [M +
Na]+ (calcd for C78H120N8O31Na, 1687.7952).
Allyl 1-(4-(O-4-nitrophenyloxycarbonyl)methyl-2-nitrophenyl)-
2,3,4-tri-O-allyloxycarbonyl-β-D-glucuronate (25). Following the
procedure presented by Grinda et al.,12 compound 24 (316 mg,
0.50 mmol) was activated using 4-nitrophenyl chloroformate (202 mg,
1.00 mmol) and pyridine (101 μL, 99 mg, 1.25 mmol) in 10 mL of
anhydrous dichloromethane. After separation by column chromatog-
raphy 25 was obtained as a colorless oil: yield 89% (356 mg, 0.44
1
mmol); TLC Rf 0.4 (2:3 EtOAc/hexanes); H NMR (CDCl3, 400
MHz) δ 8.27 (2H, d, J = 8.8 Hz), 7.87 (1H, d, J = 2.0 Hz), 7.58 (1H,
dd, J = 8.4 Hz, 2.0 Hz), 7.29 (3H, m), 5.84 (4H, m), 5.33 (14H, m),
4.65 (8H, m), and 4.35 (1H, d, J = 9.2 Hz); 13C NMR (CDCl3, 101
MHz) δ 165.70, 155.36, 154.04, 153.55, 153.51, 152.39, 149.50,
145.60, 140.87, 134.20, 131.20, 131.11, 130.99, 130.90, 130.36, 125.68,
125.44, 121.86, 119.52, 119.45, 119.42, 119.22, 119.13, 99.35, 74.89,
73.93, 72.31, 72.30, 69.65, 69.40, 69.27, 68.93, and 67.05.
Cancer Cell Line Procedures. Inhibition of human cancer cell
growth was assessed using the National Cancer Institute’s standard
sulforhodamine B assay as previously described.19 In summary, cells in
a 5% fetal bovine serum/RPMI1640 medium were inoculated in 96-
well plates and incubated for 24 h. Next, serial dilutions of the
Allyl-Protected Glucuronide Prodrug 27. To a stirred solution
containing 4-(methylamino)butyric acid (38 mg, 0.32 mmol) in 1 mL
M
DOI: 10.1021/np501004h
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
compounds were added. After 48 h, the plates were fixed with
trichloroacetic acid, stained with sulforhodamine B, and read with an
automated microplate reader. A growth inhibition of 50% (GI50, or the
drug concentration causing a 50% reduction in the net protein
increase) was calcd from optical density data with Immunosoft
software. Normal cells were treated in identical conditions. Normal
human prostate CRL-2221 (PZ-HPV-7) was grown in MEM 10%
FBS, and normal human breast MCF-10A in an MEGM kit.
2152. (e) Wu, C.; Tan, Y.; Gan, M.; Wang, Y.; Guan, Y.; Hu, X.; Zhou,
H.; Shang, X.; You, X.; Yang, Z.; Xiao, C. J. Nat. Prod. 2013, 76, 2153−
2157. (f) Felder, S.; Kehraus, S.; Neu, E.; Bierbaum, G.; Schaberle, T.
̈
F.; Koning, G. M. ChemBioChem 2013, 14, 1363−1371.
̈
(5) (a) Xu, D.-X.; Sun, P.; Kutan, T.; Mandi, A.; Tang, H.; Liu, B.;
Gerwick, W. H.; Wang, Z.-W.; Zhang, W. J. Nat. Prod. 2014, 77,
1179−1184. (b) Du, F.-Y.; Zhang, P.; Li, X.-M.; Li, C.-S.; Cui, C.-M.;
Wang, B.-G. J. Nat. Prod. 2014, 77, 1164−1169. (c) Wu, G.; Sun, X.;
Yu, G.; Wang, W.; Zhu, T.; Gu, Q.; Li, D. J. Nat. Prod. 2014, 77, 270−
275. (d) Haga, A.; Tamoto, H.; Ishino, M.; Kimura, E.; Sugita, T.;
Kinoshita, K.; Takahashi, K.; Shiro, M.; Koyama, K. J. Nat. Prod. 2013,
76, 750−754. (e) Bao, J.; Sun, Y.-L.; Zhang, X.-Y.; Han, Z.; Gao, H.-
C.; He, F.; Qian, P.-Y.; Qi, S.-H. J. Antibiot. 2013, 66, 219−223. (f) He,
F.; Bao, J.; Zhang, X.-Y.; Tu, Z.-C.; Shi, Y.-M.; Qi, S.-H. J. Nat. Prod.
2013, 76, 1182−1186.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of compounds 1−3c, 5−8b, 9−10c,
12−15b, 16a−18b, 19, 21−25, 27, and 28. This material is
available free of charge via the Internet at http://pubs.acs.org.
(6) (a) Pavlik, C. M.; Wong, C. Y. B.; Ononye, S.; Lopez, D. D.;
Engene, N.; McPhail, K. L.; Gerwick, W. H.; Balunas, M. J. J. Nat. Prod.
2013, 76, 2026−2033. (b) Thornburg, C. C.; Cowley, E. S.; Sikorska,
J.; Shaala, L. A.; Ishmael, J. E.; Youssef, D. T. A.; McPhail, K. L. J. Nat.
Prod. 2013, 76, 1781−1788. (c) Tan, L. T. Drug Discovery Today 2013,
18, 863−871.
AUTHOR INFORMATION
Corresponding Author
*Tel: (480) 965-3351. Fax: (480) 965-2747. E-Mail: bpettit@
asu.edu.
■
Notes
(7) (a) Guo, W.; Peng, J.; Zhu, T.; Gu, Q.; Keyzers, R. A.; Li, D. J.
Nat. Prod. 2013, 76, 2106−2112. (b) Um, S.; Kim, Y.-J.; Kwon, H.;
Wen, H.; Kim, S.-H.; Kwon, H.-C.; Park, S.; Shin, J.; Oh, D.-C. J. Nat.
Prod. 2013, 76, 873−879. (c) Peng, J.; Zhang, X.-Y.; Tu, Z.-C.; Xu, X.-
Y.; Qi, S.-H. J. Nat. Prod. 2013, 76, 983−987. (d) Pettit, R. K. Mar.
Biotechnol. 2011, 13, 1−11.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are pleased to acknowledge the financial support provided
by grants R01CA90441-01-05, 2R56CA090441-06A1, and 5-
RO1CA90441-07-08 from the Division of Cancer Treatment
and Diagnosis, NCI, DHHS; the Arizona Biomedical Research
Commission; J. W. Kieckhefer Foundation; Margaret T. Morris
Foundation; and the Robert B. Dalton Endowment Fund. For
other assistance we wish to thank Dr. N. Melody.
(8) Suwan, S.; Isobe, M.; Ohtani, I.; Agata, N.; Mori, M.; Ohta, M. J.
Chem. Soc., Perkin Trans. 1 1995, 765−775.
(9) Glueck, S. M.; Pirker, M.; Nestl, B. M.; Ueberbacher, B. T.;
Larissegger-Schnell, B.; Csar, K.; Hauer, B.; Stuermer, R.; Kroutil, W.;
Faber, K. J. Org. Chem. 2005, 70, 4028−4032.
(10) Bollenback, G. N.; Long, J. W.; Benjamin, D. G.; Lindquist, J. A.
J. Am. Chem. Soc. 1955, 77, 3310−3315.
(11) Duimstra, J. A.; Femia, F. J.; Meade, T. J. J. Am. Chem. Soc. 2005,
127, 12847−12855.
DEDICATION
■
Dedicated to Dr. William Fenical of Scripps Institution of
Oceanography, University of California−San Diego, for his
pioneering work on bioactive natural products.
(12) Grinda, M.; Clarhaut, J.; Tranoy-Opalinski, I.; Renoux, B.;
Monvoisin, A.; Cronier, L.; Papot, S. ChemMedChem 2011, 6, 2137−
2141.
(13) Alaoui, A. E.; Schmidt, F.; Monneret, C.; Florent, J.-C. J. Org.
Chem. 2006, 71, 9628−9636.
REFERENCES
■
(14) (a) Bouvier, E.; Thirot, S.; Schmedt, F.; Monneret, C. Org.
Biomol. Chem. 2003, 1, 3343−3352. (b) de Bond, D. B. A.; Leenders,
R. G. G.; Haisma, H. J.; Van Der Meulen-Muileman, I. H.; Scheeren,
H. W. Bioorg. Med. Chem. 1997, 5, 405−414. (c) DeWit, M. A.; Gillies,
E. R. Org. Biomol. Chem. 2011, 9, 1846−1854.
(1) Pettit, G. R.; Knight, J. C.; Herald, D. L.; Pettit, R. K.; Hogan, F.;
Mukku, V. J. R. V.; Hamblin, J. S.; Dodson, M. J., II; Chapuis, J.-C. J.
Nat. Prod. 2009, 72, 366−371.
(2) Pettit, G. R.; Hu, S.; Knight, J. C.; Chapuis, J.-C. J. Nat. Prod.
2009, 72, 372−379.
(15) Iacobazzi, R. M.; Annese, C.; Azzariti, A.; D’Accolti, L.; Franco,
M.; Fusco, C.; Gianluigi, L. P.; Laquintana, V.; Denora, N. ACS Med.
Chem. Lett. 2013, 4, 1189−1192.
(3) (a) Fu, P.; Johnson, M.; Chen, H.; Posner, B. A.; MacMillan, J. B.
J. Nat. Prod. 2014, 77, 1245−1248. (b) Bouhired, S. M.; Crusemann,
M.; Almeida, C.; Weber, T.; Piel, J.; Schaberle, T. F.; Koning, G. M.
̈
̈
̈
(16) Halsey, C. M.; Benham, D. A.; JiJi, R. D.; Cooley, J. W.
Spectrochim. Acta Mol. Biomol. Spectrosc. 2012, 96, 200−206.
(17) (a) Houba, P. H. J.; Boven, E.; Van Der Meulen-Muileman, I.
H.; Lenders, R. G. G.; Scheeren, J. W.; Pinedo, H. M.; Haisima, H. J.
Br. J. Cancer 2001, 84, 550−557. (b) Bosslet, K.; Straub, R.; Blumrich,
M.; Czech, J.; Gerken, M.; Sperker, B.; Kroemer, H. K.; Gesson, J.-P.;
Koch, M.; Monneret, C. Cancer Res. 1998, 58, 1195−1201.
(c) Woessner, R.; An, Z.; Li, X.; Hoffman, R. M.; Dix, R.; Bitonti,
A. Anticancer Res. 2000, 20, 2289−2296. (d) Legigan, T.; Clarhaut, J.;
Renoux, B.; Tranoy-Opalinski, I.; Monvoisin, A.; Jayle, C.; Alsarraf, J.;
Thomas, M.; Papot, S. Eur. J. Med. Chem. 2013, 67, 75−80.
(18) Legigan, T.; Clarhaut, J.; Renoux, B.; Tranoy-Opalinski, I.;
Monvoisin, A.; Berjeaud, J.-M.; Guilhot, F.; Papot, S. J. Med. Chem.
2012, 55, 4516−4520.
ChemBioChem 2014, 15, 757−765. (c) Myobatake, Y.; Takemoto, K.;
Kamisuki, S.; Inoue, N.; Takasaki, A.; Takeuchi, T.; Mizushina, Y.;
Sugawara, F. J. Nat. Prod. 2014, 77, 1236−1240. (d) Kong, F.; Wang,
Y.; Liu, P.; Dong, T.; Zhu, W. J. Nat. Prod. 2014, 77, 132−137.
(e) Sun, Y.-L.; Bao, J.; Liu, K.-S.; Zhang, X.-Y.; He, F.; Wang, Y.-F.;
Nong, X.-H.; Qi, S.-H. Planta Med. 2013, 79, 1474−1479. (f) Um, S.;
Choi, T. J.; Kim, H.; Kim, B. Y.; Kim, S.-H.; Lee, S. K.; Oh, K.-B.; Shin,
J.; Oh, D.-C. J. Org. Chem. 2013, 78, 12321−12329. (g) Meng, L.-H.;
Li, X.-M.; Lv, C.-T.; Li, C.-S.; Xu, G.-M.; Huang, C.-G.; Wang, B.-G. J.
Nat. Prod. 2013, 76, 2145−2149. (h) Kuramochi, K.; Tsubaki, K.;
Kuriyama, I.; Mizuchina, Y.; Yoshida, H.; Takeuchi, T.; Kamisuki, S.;
Sugawara, F.; Kobayashi, S. J. Nat. Prod. 2013, 76, 1737−1745.
(4) (a) Skellam, E. J.; Steward, A. K.; Strangman, W. K.; Wright, J. L.
C. J. Antibiot. 2013, 66, 431−441. (b) Lin, Z.; Koch, M.; Pond, C. D.;
Mabeza, G.; Seronay, R. A.; Concepcion, G. P.; Barrows, L. R.;
Oliveira, B. M.; Schmidt, E. W. J. Antibiot. 2013, 67, 121−126.
(c) Jang, K. H.; Nam, S.-J.; Locke, J. B.; Kauffman, C. A.; Beatty, D. S.;
Paul, L. A.; Fenical, W. Angew. Chem., Int. Ed. 2013, 52, 7822−7824.
(d) Lu, Z.; Koch, M.; Harper, M. K.; Matainaho, T. K.; Barrows, L. R.;
Van Wagoner, R. M.; Ireland, C. M. J. Nat. Prod. 2013, 76, 2150−
(19) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.;
Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Viagro-Wolff, A.; Gray-
Goodrich, M.; Campbell, H.; Mayo, J.; Boyd, M. J. Natl. Cancer Inst.
1991, 83, 757−766.
N
DOI: 10.1021/np501004h
J. Nat. Prod. XXXX, XXX, XXX−XXX