Total synthesis and structural revision of lucentamycin A

Article abstract of DOI:10.1021/jo301723y

Lucentamycin A is a marine-derived peptide natural product harboring a unique 4-ethylidene-3-methylproline (Emp) subunit. The proposed structure of lucentamycin A and the core Emp residue have recently been called into question through synthesis. Here, we report the first total synthesis of lucentamycin A, which confirms that the ethylidene substituent in Emp bears an E geometry, in contrast to the originally assigned Z configuration. Synthesis of the desired (E)-Emp subunit required the implementation of a novel strategy starting from Garner's aldehyde.

Full text of DOI:10.1021/jo301723y

Note
pubs.acs.org/joc
Total Synthesis and Structural Revision of Lucentamycin A
Sujeewa Ranatunga,† Chih-Hang Anthony Tang,‡ Chih-Chi Andrew Hu,‡ and Juan R. Del Valle*,†
‡
†Drug Discovery Department and Department of Immunology, H. Lee Moffitt Cancer Center and Research Institute, Tampa,
Florida 33612, United States
S
* Supporting Information
ABSTRACT: Lucentamycin A is a marine-derived peptide natural product
harboring a unique 4-ethylidene-3-methylproline (Emp) subunit. The proposed
structure of lucentamycin A and the core Emp residue have recently been called
into question through synthesis. Here, we report the first total synthesis of
lucentamycin A, which confirms that the ethylidene substituent in Emp bears an
E geometry, in contrast to the originally assigned Z configuration. Synthesis of
the desired (E)-Emp subunit required the implementation of a novel strategy
starting from Garner’s aldehyde.
he lucentamycins are a family of tripeptides produced by
conspicuous absence of correlation between H-12 and H-9 (or
H-12 and H-14) left open the possibility of an (E)-alkene
geometry. During review of this paper, Fenical and co-workers
isolated an additional family member, lucentamycin E, and
observed ROESY correlations indicative of an (E)-Emp
configuration.3 Rexamination of the spectra for 1 supported
the same (E)-Emp structure for the other lucentamycin
peptides. Here, we report the first total synthesis of
lucentamycin A and confirmation of the (E)-Emp configuration
of the natural product. Our synthetic strategy is based on a
stereoselective enoate conjugate addition reaction followed by
ester α-ethylenation to provide a linear precursor to the desired
isomer. A detailed structural analysis of synthetic lucentamycin
A resolves recent conflicting reports on the true structure of the
natural product. We also prepared the simplified proline
analogue for comparative biological studies and found,
unexpectedly, that neither compound significantly inhibited
the growth of HCT-116 colon cancer cells by XTT assay.
Synthesis of the originally proposed structure of lucentamy-
cin A (1) has been reported using conceptually distinct
strategies to establish the desired 8S,9R,10Z configuration. Our
group had initially explored the utility of a stereoselective ester
enolate-Claisen rearrangement for the preparation of highly
substituted allylglycine derivatives, as shown in Scheme 1.2b
These linear precursors were subsequently cyclized to afford
various 3-alkyl-4-alkylideneprolines with moderate to high
stereoselectivities. One drawback to this strategy is the inability
to access the (E)-alkene due to the preferred equatorial
disposition of the R1 group in the chairlike transition state.4
Thus, our first attempts to synthesize the desired compound
relied on alkene cross-metathesis in the presence of 2-methyl-2-
butene5 with previously described tripeptides 5 and 6.2b
Despite exploration of various reaction conditions, no E
isomers were detected by NMR. When compound 7 was
T
Nocardiopsis lucentensis that harbor 4-ethylidene-3-meth-
ylproline (Emp) as a central residue.1 The reported activity of
lucentamycin A against HCT-116 human colon cancer cells
coupled with the structural novelty of its proline core has
prompted several synthetic efforts.2 Our group recently
completed the synthesis of the putative structure of
lucentmycin A (1, Figure 1), establishing the need for structural
Figure 1. Originally proposed structure of lucentamycin A (1) and
diastereomers.
revision on the basis of various discrepancies in the NMR
spectral regions corresponding to the Emp residue.2a Lindsley
and co-workers concurrently reported the synthesis of
diastereomer 2, which also showed key spectroscopic differ-
ences with the natural product.2c On the basis of NMR spectral
data from synthetic isomers and the natural isolate, we
hypothesized that the absolute stereochemistry of the proline
core may be in need of revision. However, synthesis of all four
diastereomers of Emp and their corresponding tripeptides
revealed that these stereocenters were not the source of
misassignment.2b
Two-dimensional NMR experiments originally carried out
with natural lucentamycin A indicated a ROESY correlation
between H-13 and H-11, indicative of a Z configuration.1
However, a weak signal from this interaction combined with the
Received: August 15, 2012
Published: October 5, 2012
© 2012 American Chemical Society
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presence of PTSA to give alcohol 14 in high yield. Oxidation to
the carboxylic acid and condensation with leucine tert-butyl
ester provided dipeptide 15 in 77% yield. After hydrolysis of the
benzoyl group in 15, the minor amount of Z isomer carried
through from the α-ethylenation reaction could be separated by
column chromatography. Chlorination of the now isomerically
pure alcohol with MsCl and NEt3 afforded allylic chloride 16,
which readily cyclized to the prolyl dipeptide upon acidolysis of
the Boc group and neutralization with base. DEPBT-mediated
condensation of 17 with Fmoc-Har(Boc)2-OH2a and con-
version to the N-benzoylated tripeptide provided 18. Finally,
global deprotection and purification by RP-HPLC on a
semipreparative C18 column afforded 19 as a white solid
upon lyophilization (Scheme 3).
Scheme 1. Attempted Synthesis of Emp Isomers via Ester
Enolate-Claisen Rearrangement and Alkene Cross-
Metathesis
Scheme 3. Synthesis of Lucentamycin A (19)
used as the substrate, we did isolate the alkene inversion
product, albeit in very low yield.
The difficulties encountered with producing the E isomer by
cross-metathesis prompted us to explore an alternative
synthetic strategy employing D-serine as a chiral progenitor.
As shown in Scheme 2, enoate 106 was subjected to a
Scheme 2. Synthesis of Allylic Alcohol 13
To prove that the structure of our synthetic material matched
that of natural lucentamycin A, we closely analyzed the 2D
NMR spectra for 19. As shown in Figure 2A, we observed
diagnostic ROESY correlations between H-11 and H-12 as well
as between H-9 and H-13 for 19. These correlations were also
noted in the recent reexamination of the ROESY spectrum for
natural lucentamycin A.3 The optical rotation we obtained for
19 was slightly more pronounced than that of the natural
diastereoselective conjugate addition reaction to afford 11 as a
single syn isomer in 90% yield.7 α-Ethylenation of 11 was
carried out using a three-step sequence involving enolate
alkylation with acetaldehyde, mesylation of the resulting
secondary alcohol, and elimination with DBU. Enoate 12 was
obtained in as an inseparable 94:6 E:Z mixture in 65% overall
yield from 11. Reduction of the ester group with DIBAL then
provided primary allylic alcohol 13 in 86% yield.
Various attempts at activating the alcohol in 13 (TsCl/NEt3,
MsCl/NEt3, CCl4/PPh3) ahead of pyrrolidine ring closure led
to complex product mixtures. As an alternative, we prepared the
leucyl dipeptide derivative prior to pyrrolidine ring formation.
Thus, 13 was benzoylated and the acetonide removed in the
isolate ([α]24 = −12.5 vs −6.3°; c 0.175, MeOH). However,
D
the 1H, 13C, COSY, HMQC, and HMBC NMR spectra
obtained for 19 were all in agreement with those reported for
the original sample (Figure 2B).1
Support for the configuration of the Emp residue in synthetic
lucentamycin A (19) initially relied on our use of a highly syn-
selective conjugate addition reaction onto a D-serine-derived
synthon.8 Sim and co-workers recently reported the synthesis
of the same tripeptide employing an asymmetric Rh-catalyzed
reductive cyclization strategy.9 Notably, the spectral data
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residues lead to a disruption of the head-to-tail salt bridge
observed in the solid-state structure of 1. The absence of this
H-bond constraint may explain the notable differences in the
NMR spectra of the two compounds. The signals for H-12 in
19 and natural lucentamycin A also exhibit a characteristic
1
downfield shift of ∼0.1−0.2 ppm in the H NMR spectrum
relative to each of the (Z)-Emp analogues 1−4.2b
Previously synthesized isomers of lucentamycin A have failed
to show significant cytotoxicity against HCT-116 colon
carcinoma cells. The change in global conformation shown in
Figure 3 suggested that the presence of an (E)-alkene may be
critical for bioactivity. However, in our hands, neither 19 or its
simplified proline analogue (Bz-Har-Pro-Leu-OH)2c exhibited
significant cytotoxicity toward HCT-116 cells at 20 μM by
XTT assay (Figure 4A). Dose−response experiments at various
Figure 4. XTT assay with HCT-116 human colon carcinoma cells: (A)
cells treated with 19 (20 μM) or Bz-Har-Pro-Leu-OH (20 μM) for a
course of 5 days; (B) cells treated with 0, 25, 50, or 100 μM 19 for a
course of 4 days. Percentages of cell growth were determined by
comparing treated with untreated groups. Each data point derived
from four independent groups receiving exactly the same treatment
was plotted as mean SD.
Figure 2. (A) Selected ROESY correlations observed for 19 in
DMSO-d6. (B) Comparison of H NMR spectra (0.5−5.5 ppm) of
natural lucentamycin A (blue) and 19 (red) in DMSO-d6.
1
obtained for their material were not in agreement with that of
natural lucentamycin A (or 19), leading to the conclusion that
the alkene geometry is not the source of missasignment. In light
of this report, we sought an unequivocal configurational
assignment of 19 by X-ray diffraction. Suitable crystals obtained
from MeOH/H2O led to a confirmation of the expected
structure (Figure 3). Our data suggest that the compound
time points showed compound 19 to have negligible growth
inhibitory activity up to 100 μM, in stark contrast to the 0.2 μM
GI50 previously reported by MTS assay (Figure 4B).1
Compound 19 and Bz-Har-Pro-Leu-OH also failed to
significantly inhibit the growth of two chronic lymphocytic
leukemia cell lines, MEC2 and WaC3 (see the Supporting
Information). This raises the possibility that the potent
cytotoxicity observed with the natural isolate may be due to
unidentified compounds in the original sample. Lucentamycin
B is also reported to exhibit moderate growth inhibition of
HCT-116 cells (GI50 = 10 μM), and efforts to synthesize and
evaluate this compound by XTT are currently underway.
In conclusion, we have described the first total synthesis of
lucentamycin A using D-serine as a chiral progenitor. Stereo-
selective introduction of the 3-methyl group in Emp was
achieved by conjugate addition onto an enoate derived from
Garner’s aldehyde. The desired (E)-alkene geometry was
established through base-promoted α-ethylenation of a linear
Emp precursor. Importantly, a combination of compelling
NMR and X-ray diffraction data obtained for 19 serves to
resolve recent conflicting reports on the true structure of the
natural product. We thus confirm, through synthesis, the alkene
geometry as the sole source of lucentamycin A misassignment.
In addition to providing access to lucentamycins B−E, our
strategy should find application in the synthesis of other natural
products featuring substituted (E)-4-alkylideneproline deriva-
tives.10
Figure 3. X-ray crystal structures of 1 and 19.
recently described by Sim and co-workers as the (E)-Emp
isomer of lucentamycin A9 may instead be a structural (or
conformational) isomer of 19.
Comparison between the crystal structure of 19 and that of
originally proposed lucentamycin A (1)2b revealed a significant
conformational change resulting from distinct alkene geo-
metries. Specifically, the pyrrolidine ring in 19 adopts an Cγ-exo
puckered conformation, whereas the proline derivative in 1
exists as a Cγ-endo conformer in the solid state. Moreover,
major changes in the ψ torsions for both the Har and Leu
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Note
found 342.229 30; [M + Na]+ calcd for C18H31NO5Na 364.209 44,
found 364.210 63.
EXPERIMENTAL SECTION
■
General Techniques. Unless stated otherwise, reactions were
performed in flame-dried glassware under a positive pressure of argon
or nitrogen gas using dry solvents. Commercial grade reagents and
solvents were used without further purification, except where noted.
Diethyl ether, toluene, dimethylformamide, dichloromethane, and
tetrahydrofuran were purified by a Glass Contour column-based
solvent purification system. Other anhydrous solvents were purchased
directly from chemical suppliers. Thin-layer chromatography (TLC)
was performed using silica gel 60 F254 precoated plates (0.25 mm).
Flash chromatography was performed using silica gel (60 μm particle
size). The purity of all compounds was judged by TLC analysis
(single-spot/two-solvent systems) using a UV lamp, CAM (ceric
ammonium molybdate), ninhydrin, or basic KMnO4 stain(s) for
detection purposes. NMR spectra were recorded on a 400 MHz
spectrometer. 1H and 13C NMR chemical shifts are reported as δ using
residual solvent as an internal standard. Analytical (4 × 150 mm
column, 1 mL/min flow rate) RP-HPLC was performed on a C18
column with acetonitrile/water (0.1% formic acid) as eluent.
(S)-tert-Butyl-4-((R,E)-3-(hydroxymethyl)pent-3-en-2-yl)-2,2-
dimethyloxazolidine-3-carboxylate (13). A solution of 12 (354
mg, 1.04 mmol) in 10 mL of DCM at −78 °C under an Ar atmosphere
was treated with a 1 M solution of DIBAL in THF (2.60 mL, 2.60
mmol) and stirred for 30 min. The reaction mixture was quenched
with 50% Rochelle salt, diluted with Et2O, and stirred vigorously for 2
h at room temperature. The organic layer was separated, and the
aqueous layer was diluted with brine and extracted with DCM. The
combined organic layers were dried over Na2SO4 and concentrated to
1
give 13 (94:6 E:Z mixture) as a colorless oil (267 mg, 86% yield): H
NMR (400 MHz, CDCl3, mixture of rotamers) δ 5.61 (q, J = 6.9 Hz,
1H), 4.08 (m, 2H), 3.96 (m, 1H), 3.84−3.58 (m, 2H), 3.07 − 2.81 (m,
1H), 2.75 (bs, 0.4H), 2.58−2.07 (m, 0.6H), 1.64 (m, 3H), 1.55 (m,
3H), 1.45 (m, 12H), 1.03 (d, J = 6.4 Hz, 3H); 13C NMR (101 MHz,
CDCl3, mixture of rotamers) δ 153.4, 152.6, 141.0, 140.9, 125.1, 124.8,
94.1, 93.7, 80.1, 79.8, 66.7, 66.0, 64.5, 60.7, 60.2, 37.1, 36.7, 28.4, 27.6,
27.3, 24.6, 23.1, 16.1, 15.7, 13.4.; HRMS (ESI-TOF) (m/z) [M + Na]+
calcd for C16H29NO4Na 322.198 88, found 322.197 60.
(S)-tert-Butyl-4-((R)-4-ethoxy-4-oxobutan-2-yl)-2,2-dimethy-
loxazolidine-3-carboxylate (11). Compound 11 was synthesized
using a slight modification to the published procedure.7 A slurry of CuI
(6.11 g, 32.1 mmol) in 75 mL of THF under Ar was cooled to −15 °C
and then treated with an 1.5 M solution of MeLi·LiBr in Et2O (42.76
mL, 64.14 mmol). After it was stirred at −15 °C for 20 min, the
solution was cooled to −70 °C and treated dropwise with TMSCl
(12.2 mL, 96.2 mmol) followed by a solution of 10 (1.60 g, 5.34
mmol) in 8 mL of THF. The reaction mixture was stirred at −70 °C
until TLC indicated completion (∼30 min), and then quenched
dropwise with 5 mL of 9/1 saturated aqueous NH4Cl/2 M aqueous
NaOH until gas evolution subsided. The mixture was then carefully
diluted with 100 mL of the same aqueous solution, warmed to room
temperature, and extracted with EtOAc. The organic layers were
washed with saturated aqueous NH4Cl, dried over Na2SO4, and
concentrated. Purification by flash chromatography over silica gel (8%
EtOAc/hexanes) afforded 11 as an amber liquid (1.51 g, 90%).
Spectral data for 11 matched those previously reported.7
(3R,4S,E)-4-((tert-Butoxycarbonyl)amino)-2-ethylidene-5-hy-
droxy-3-methylpentyl Benzoate (14). A solution of 13 (249 mg,
832 μmol) in 8 mL of DCM at room temperature under Ar was
treated with triethylamine (232 μL, 1.67 mmol) and benzoyl chloride
(193 μL, 1.67 mmol). After being stirred at room temperature for 17
h, the reaction mixture was washed with saturated aqueous NaHCO3
and extracted with DCM. The combined organic layers were dried
over Na2SO4 and concentrated. Purification by flash chromatography
over silica gel (5−20% EtOAc/hexanes) afforded the benzoylated
1
intermediate as a colorless oil (329 mg, 98% yield): H NMR (400
MHz, CDCl3) δ 8.00 (m, 2H), 7.52 (m, 1H), 7.41 (m, 2H), 5.78 (q, J
= 6.8 Hz, 1H), 4.75 (m, 2H), 4.24−4.00 (m, 1H), 3.94−3.62 (m, 2H),
2.98 (m, 1H), 1.71 (m, 3H), 1.59 (m, 3H), 1.53−1.32 (m, 12H), 1.13
(d, J = 7.2 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 166.2, 153.3,
152.6, 136.5, 136.3, 133.1, 132.9, 130.6, 130.4, 130.2, 130.0, 129.6,
128.9, 128.6, 128.5, 94.2, 93.7, 79.9, 79.8, 66.8, 66.7, 66.0, 60.3, 60.0,
37.4, 28.4, 28.1, 27.6, 24.6, 23.2, 16.1, 15.9, 14.4, 14.3, 13.6; HRMS
(ESI-TOF) (m/z) [MH]+ calcd for C23H34NO5 404.243 15, found
404.243 78; [M + Na]+ calcd for C23H33NO5Na 426.225 09, found
426.225 06.
The above ester (194 mg, 481 μmol) in 2.5 mL of MeOH at 0 °C
was treated with p-TsOH·H2O (68.0 mg, 361 μmol) and stirred from
0 °C to room temperature over 4 h. The reaction mixture was
concentrated under reduced pressure and loaded onto silica gel.
Purification by flash chromatography over silica gel (20−35% EtOAc/
hexanes) afforded 14 (94:6 E:Z mixture) as a white foam (167 mg,
96% yield): 1H NMR (400 MHz, CDCl3, mixture of rotamers) δ 8.00
(m, 2H), 7.52 (m, 1H), 7.41 (m, 2H), 5.75 (m, 1H), 5.07−4.62 (m,
3H), 3.81 (m, 1H), 3.72−3.45 (m, 2H), 2.99 (bs, 1H), 2.84 (m, 1H),
1.70 (d, J = 6.9 Hz, 3H), 1.46−1.29 (m, 9H), 1.16 (d, J = 7.1 Hz, 3H);
13C NMR (101 MHz, CDCl3, mixture of rotamers) δ 166.5, 156.6,
(S)-tert-Butyl-4-((R,E)-3-(ethoxycarbonyl)pent-3-en-2-yl)-2,2-
dimethyloxazolidine-3-carboxylate (12). A solution of diisopro-
pylamine (348 μL, 2.46 mmol) in 4 mL of THF was cooled to 0 °C
and treated dropwise with a 1.6 M solution of n-BuLi in hexanes (1.49
mL, 2.38 mmol). After the mixture was stirred for 10 min at the same
temperature, a solution of 11 (250 mg, 0.793 mmol) in 2 mL of THF
was added dropwise. The reaction mixture was stirred at 0 °C for 15
min and then cooled to −78 °C and treated with acetaldehyde (445
μL, 7.93 mmol). After it was stirred for 20 min at −78 °C, the reaction
mixture was quenched by dropwise addition of saturated aqueous
NH4Cl, warmed to room temperature, and extracted with EtOAc. The
organic layers were washed with 1 M aqueous HCl, dried over
Na2SO4, and concentrated. The crude alcohol was then taken up in 10
mL of DCM, cooled to 0 °C, and treated with NEt3 (442 μL, 3.17
mmol) and methanesulfonyl chloride (184 μL, 2.38 mmol). After it
was stirred for 1 h at room temperature, the reaction mixture was
diluted with DCM, washed with 1 M aqueous HCl, dried over
Na2SO4, and concentrated. The crude mesylate was then dissolved in
2.5 mL of toluene and treated with DBU (237 μL, 1.59 mmol). The
reaction mixture was stirred at 60 °C for 4 h and then at room
temperature for 16 h. The mixture was diluted with EtOAc, washed
with 1 M aqueous HCl, dried over Na2SO4, and concentrated.
Purification by flash chromatography over silica gel (5% EtOAc/
hexanes) afforded 12 (94:6 E:Z mixture) as an amber oil (176 mg,
135.9, 133.1, 130.2, 129.7, 128.5, 127.8, 79.5, 67.1, 63.9, 55.4, 35.0,
28.5, 16.0, 13.6; HRMS (ESI-TOF) (m/z) [M + Na]+ calcd for
C20H29NO5Na 386.193 79, found 386.192 70.
(3R,4S,E)-5-(((S)-1-(tert-Butoxy)-4-methyl-1-oxopentan-2-yl)-
amino)-4-((tert-butoxycarbonyl)amino)-2-ethylidene-3-meth-
yl-5-oxopentyl Benzoate (15). A solution of 14 (119 mg, 327
μmol) in 3 mL of MeCN at room temperature was treated with 1.5
mL of 0.67 M NaH2PO4 buffer followed by 20 mol % of TEMPO
(15.0 mg). The mixture was placed at 40 °C and simultaneously
treated with 700 μL of a solution of 6% bleach diluted with 3 mL of
water and 900 μL of a 2 M solution of 80% NaClO2 in water. After
being stirred at 40 °C for 3 h, the reaction mixture was quenched with
saturated aqueous Na2SO3 and partitioned with EtOAc. The aqueous
layer was acidified with 1 M aqueous HCl (pH 4) and extracted with
EtOAc. The combined organic layer was dried over Na2SO4 and
concentrated. The crude carboxylic acid in 3.5 mL of MeCN at room
temperature was treated with triethylamine (137 μL, 425 μmol);
HBTU (161 mg, 425 μmol) and HOBt (8.8 mg, 65 μmol) were added,
and the mixture was stirred for 3 min. H-Leu-Ot-Bu-HCl (95.0 mg,
1
65%, three steps): H NMR (400 MHz, CDCl3, mixture of rotamers)
δ 6.87 (m, 1H), 4.37 (m, 1H), 4.16 (m, 2H), 3.80 (m, 0.5H), 3.71 (m,
1H), 3.61 (m, 0.5H), 3.12 (m, 0.4H), 2.97 (m, 0.6H), 1.81 (d, J = 7.2
Hz, 3H), 1.70−1.37 (m, 15H), 1.26 (t, J = 7.1 Hz, 3H), 1.19 (d, J = 7.1
Hz, 3H); 13C NMR (101 MHz, CDCl3, mixture of rotamers) δ 167.6,
167.2, 153.1, 152.8, 139.4, 135.4, 135.2, 94.0, 93.5, 79.8, 66.5, 65.6,
60.7, 60.3, 59.9, 36.5, 35.7, 28.5, 27.7, 24.6, 23.2, 16.1, 15.8, 14.4, 14.4;
HRMS (ESI-TOF) (m/z) [MH]+ calcd for C18H31NO5 342.228 05,
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Note
425 μmol) was then added into the mixture and stirred for 20 h. The
solution was concentrated, and the resulting crude product was
directly purified by flash chromatography over silica gel (10−20%
EtOAc/hexanes) afforded 15 (94:6 E:Z mixture) as a white foam (138
(S)-tert-Butyl 2-((2S,3R,4E)-1-((S)-2-Benzamido-6-(2,3-bis-
(tert-butoxycarbonyl)guanidino)hexanoyl)-4-ethylidene-3-
methylpyrrolidine-2-carboxamido)-4-methylpentanoate (18).
A solution of 17 (43.0 mg, 132 μmol) in 2 mL of THF at 0 °C was
treated with triethylamine (37.0 μL, 264 μmol), DEPBT (60.0 mg, 199
μmol), and Fmoc-Har(Boc)2-OH (97.0 mg, 158 μmol) and stirred for
5 h at 0 °C. The reaction mixture was quenched with saturated
aqueous NH4Cl and evaporated. The residue was dissolved in EtOAc
and washed with 1 M aqueous HCl followed by saturated aqueous
NaHCO3. The organic layer was dried over Na2SO4 and concentrated.
Purification by flash chromatography over silica gel (30−60% EtOAc/
hexanes then 100% EtOAc) afforded 18 as a white foam (76.0 mg,
1
mg, 77% yield over two steps): H NMR (400 MHz, CDCl3, mixture
of rotamers) δ 8.06 (m, 2H), 7.53 (m, 1H), 7.42 (m, 2H), 6.43 (m,
1H), 5.75 (q, J = 7.0 Hz, 1H), 5.23 (m, 1H), 5.12−4.80 (m, 1H),
4.79−4.49 (m, 1H), 4.49−4.19 (m, 2H), 3.07 (m, 1H), 1.76−1.53 (m,
3H), 1.51−1.30 (m, 21H), 1.18 (d, J = 7.2 Hz, 3H), 0.97−0.84 (m,
6H); 13C NMR (101 MHz, CDCl3, mixture of rotamers) δ 171.4,
171.0, 166.6, 155.8, 134.3, 133.0, 130.5, 129.7, 129.1, 128.5, 127.1,
81.7, 79.9, 67.3, 57.5, 51.5, 42.2, 36.5, 28.4, 27.9, 24.8, 22.8, 22.3, 15.0,
13.7; HRMS (ESI-TOF) (m/z) [MH]+ calcd for C30H47N2O7
547.338 50, found 547.339 00.
1
62% yield): H NMR (400 MHz, CDCl3) δ 11.49 (s, 1H), 8.32 (s,
1H), 7.74 (d, J = 7.5 Hz, 2H), 7.58 (d, J = 7.4 Hz, 2H), 7.47−7.34 (m,
2H), 7.28 (m, 2H), 6.02 (d, J = 8.4 Hz, 1H), 5.65 (d, J = 8.6 Hz, 1H),
5.40 (m, 1H), 4.51 (m, 3.5H), 4.43−3.99 (m, 4.5H), 3.43 (m, 2H),
3.24 (m, 1H), 2.02−1.78 (m, 1H), 1.77−1.53 (m, 7H), 1.46 (m, 31H),
1.09 (d, J = 7.2 Hz, 3H), 0.92 (m, 6H); 13C NMR (101 MHz, CDCl3)
δ 172.5, 171.2, 168.3, 163.7, 156.2, 156.2, 153.3, 144.0, 143.9, 141.3,
139.0, 135.4, 132.8, 129.1, 127.8, 127.2, 125.9, 125.3, 125.2, 120.0,
117.8, 83.1, 82.0, 79.3, 67.1, 66.5, 64.6, 52.4, 51.6, 51.2, 47.2, 42.2,
40.8, 35.5, 32.6, 28.9, 28.4, 28.2, 28.1, 24.9, 22.8, 22.5, 22.4, 16.2, 16.1,
15.1, 13.7; HRMS (ESI-TOF) (m/z) [MH]+ calcd for C50H73N6O10
917.538 27, found 917.540 34; [M + Na]+ calcd for C50H72N6O10Na
939.520 21, found 939.521 97.
(S)-tert-Butyl 2-((2S,3R,E)-2-((tert-Butoxycarbonyl)amino)-4-
(chloromethyl)-3-methylhex-4-enamido)-4-methylpentanoate
(16). A solution of 15 (137 mg, 252 μmol) in 2 mL of 2/1 MeOH/
THF at room temperature was treated with 2.0 mL of 1 M aqueous
NaOH and stirred for 45 min. The reaction mixture was concentrated,
neutralized with 1 M aqueous HCl, and extracted with EtOAc. The
combined organic layer was dried over Na2SO4 and concentrated.
Purification by flash chromatography over silica gel (10−15% then
40% EtOAc/hexanes) afforded the allylic alcohol as a white foam (93.0
1
mg, 84% yield): H NMR (400 MHz, CDCl3) δ 6.79 (d, J = 8.5 Hz,
1H), 5.54 (q, J = 6.8 Hz, 1H), 5.23 (d, J = 9.3 Hz, 1H), 4.46 (td, J =
9.0, 5.8 Hz, 1H), 4.39−4.10 (m, 2H), 3.96 (dd, J = 12.0, 2.7 Hz, 1H),
3.26 (m, 1H), 2.94 (m, 1H), 1.64 (m, 1H), 1.55 (d, J = 6.9 Hz, 3H),
1.51−1.37 (m, 21H), 1.12 (d, J = 7.0 Hz, 3H), 0.90 (m, 6H); 13C
NMR (101 MHz, CDCl3) δ 173.1, 171.9, 155.9, 138.9, 127.6, 82.2,
79.7, 65.3, 58.2, 51.0, 42.1, 37.1, 28.4, 28.1, 28.0, 24.8, 22.8, 22.0, 15.5,
13.3; HRMS (ESI-TOF) (m/z) [MH]+ calcd for C23H43N2O6
443.311 56, found 443.312 09; [M + Na]+ calcd for C23H42N2O6Na
465.293 51, found 465.293 84.
A solution of the above allylic alcohol (102 mg, 230 μmol) in 2.5
mL of DCM at room temperature was treated with triethylamine (224
μL, 1.61 mmol) and MsCl (107 μL, 1.38 mmol). After being stirred at
room temperature for 18 h, the reaction mixture was washed with 1 M
aqueous HCl. The organic layer was dried over Na2SO4 and
concentrated. Purification by flash chromatography over silica gel
(10−20% EtOAc/hexanes) afforded 16 as a white foam (93.0 mg, 87%
yield): 1H NMR (400 MHz, CDCl3, mixture of rotamers) δ 6.32 (d, J
= 8.5 Hz, 1H), 5.76 (q, J = 7.0 Hz, 1H), 5.27−5.15 (m, 1H), 4.51−
4.32 (m, 2H), 4.22 (m, 1H), 4.02 (m, 1H), 2.99 (m, 1H), 1.64 (d, J =
7.0 Hz, 3H), 1.62−1.35 (m, 21H), 1.21 (d, J = 7.1 Hz, 3H), 0.91 (m,
6H); 13C NMR (101 MHz, CDCl3, mixture of rotamers) δ 171.5,
170.9, 155.8, 135.6, 131.7, 81.9, 79.9, 57.6, 51.7, 48.8, 42.4, 37.0, 28.4,
28.1, 24.9, 22.8, 22.3, 15.1, 14.1; HRMS (ESI-TOF) (m/z) [MH]+
calcd for C23H42ClN2O5 461.277 68, found 461.277 67; [M + Na]+
calcd for C23H41ClN2O5Na 483.259 62, found 483.259 14.
(S)-tert-Butyl 2-((2S,3R,E)-4-Ethylidene-3-methylpyrrolidine-
2-carboxamido)-4-methylpentanoate (17). A solution of 16 (92.0
mg, 199 μmol) in 2.0 mL of 15% TFA/DCM at 0 °C was stirred for
2.5 h and warmed to room temperature. The reaction was diluted with
EtOAc and concentrated under reduced pressure. This dilution and
evaporation sequence was repeated two more times, and the crude
residue was dried under high vacuum for 1 h. The resulting
trifluoroacetate salt was dissolved in 3 mL of acetone and treated
with K2CO3 (275 mg, 1.99 mmol). After being stirred at room
temperature for 24 h, the reaction mixture was filtered through a Celite
pad with acetone rinsing. The organic filtrate was concentrated and
purified by flash chromatography over silica gel (90% EtOAc/hexanes
then 10−15% MeOH/EtOAc) to afford 17 as a yellow oil (54.0 mg,
84% yield over two steps): 1H NMR (400 MHz, CDCl3) δ 7.53 (d, J =
8.6 Hz, 1H), 5.27 (m, 1H), 4.49 (m, 1H), 4.01 (m, 1H), 3.76 (m, 1H),
3.62 (m, 1H), 3.17 (m, 1H), 1.73−1.48 (m, 6H), 1.44 (s, 9H), 1.00−
0.86 (m, 9H); 13C NMR (101 MHz, CDCl3) δ 172.3, 170.9, 142.6,
115.8, 81.8, 65.3, 51.0, 49.4, 41.6, 36.7, 28.1, 25.0, 22.9, 21.9, 14.5,
14.3; HRMS (ESI-TOF) (m/z) [MH]+ calcd for C18H33N2O3
325.248 57, found 325.248 48; [M + Na]+ calcd for C18H32N2O3Na
347.230 51, found 347.230 52.
The tripeptide above (50.0 mg, 55.0 μmol) was dissolved in 2 mL of
THF at room temperature and was treated with diethylamine (226 μL,
2.18 mmol) and stirred for 5 h. The reaction mixture was concentrated
under reduced pressure, and the resulting crude amine was treated
with benzoyl chloride (13.0 μL, 109 μmol) at room temperature and
stirred for 2 h. After the solvent was evaporated, the crude product was
adsorbed onto silica gel and purified by flash chromatography over
silica (50% EtOAc/hexanes and 100% EtOAc then 10−20% MeOH/
1
EtOAc) to afford 18 as a white foam (33.0 mg, 76% yield): H NMR
(400 MHz, CDCl3) δ 11.47 (bs, 1H), 8.33 (m, 1H), 7.78 (m, 2H),
7.54−7.32 (m, 3H), 7.04 (d, J = 8.1 Hz, 1H), 6.00 (d, J = 8.3 Hz, 1H),
5.46 (m, 1H), 4.98 (m, 1H), 4.62−4.41 (m, 2H), 4.36 (m, 2H), 3.41
(m, 2H), 3.27 (m, 1H), 1.93 (m, 1H), 1.82−1.35 (m, 37H), 1.31 (m,
1H), 1.10 (d, J = 7.2 Hz, 3H), 0.95 (m, 6H); 13C NMR (101 MHz,
CDCl3) δ 172.5, 171.3, 168.3, 167.2, 156.2, 153.3, 138.9, 134.1, 131.7,
128.6, 127.3, 117.9, 83.1, 82.1, 79.4, 64.7, 51.7, 51.3, 50.9, 42.3, 40.8,
35.6, 32.6, 28.9, 28.4, 28.2, 28.1, 25.0, 22.8, 22.6, 22.4, 15.2, 13.7;
HRMS (ESI-TOF) (m/z) [MH]+ calcd for C42H67N6O9 799.496 40,
found 799.496 76; [M + Na]+ calcd for C42H66N6O9Na 821.478 35,
found 821.478 80.
Synthetic Lucentamycin A (19). Tripeptide 18 (32 mg, 40
μmol) was treated with a 2 mL solution of TFA/TES/DCM (90/5/5)
at room temperature and stirred for 7 h. The mixture was diluted with
EtOAc and evaporated under reduced pressure. The dilution and
evaporation sequence was repeated two more times. A portion of the
crude residue (85% by weight) was purified by semipreparative RP-
HPLC (15−70% MeCN/H2O linear gradient over 20 min, retention
time 7.4 min) to afford lucentamycin A (19) as a white fluffy solid (15
1
mg, 81% yield, based on the amount injected): H NMR (400 MHz,
d6-DMSO) δ 10.36 (bs, 1H), 8.45 (d, J = 7.3 Hz, 1H), 8.27 (m, 1H),
7.86 (m, 2H), 7.52 (t, J = 7.3 Hz, 1H), 7.43 (dd, J = 11.4, 4.3 Hz, 2H),
7.18−6.88 (m, 3H), 5.39 (q, J = 6.4 Hz, 1H), 4.79 (dd, J = 11.3, 7.1
Hz, 1H), 4.49 (d, J = 13.8 Hz, 1H), 4.38 (d, J = 12.3 Hz, 1H), 4.24 (d,
J = 8.5 Hz, 1H), 3.90 (dd, J = 13.3, 6.7 Hz, 2H), 3.22 (m, 2H), 3.12
(m, 2H), 3.02 (m, 2H), 1.98 (d, J = 7.0 Hz, 1H), 1.71 (m, 3H), 1.62
(d, J = 6.7 Hz, 3H), 1.59−1.45 (m, 3H), 1.44−1.21 (m, 3H), 1.02 (d, J
= 7.2 Hz, 3H), 0.86 (d, J = 3.4 Hz, 3H), 0.85 (d, J = 3.5 Hz, 3H); 13C
NMR (101 MHz, d6-DMSO) δ 175.9, 171.3, 167.3, 166.0, 157.2,
139.5, 133.8, 131.4, 128.3, 127.6, 116.3, 65.6, 52.4, 51.4, 51.0, 43.1,
41.2, 35.1, 31.4, 28.5, 24.5, 23.1, 22.7, 14.9, 13.2; HRMS (ESI-TOF)
(m/z) [MH]+ calcd for C28H43N6O5 543.328 94, found 543.328 36;
[M + Na]+ calcd for C28H42N6O5Na 565.310 89, found 565.309 33;
[α]24 = −12.5° (c 0.175, MeOH).
D
Cell Proliferation Assays. Human colorectal carcinoma cell line
HCT-116 was seeded and cultured in 96-well cell culture plates
9863
dx.doi.org/10.1021/jo301723y | J. Org. Chem. 2012, 77, 9859−9864

The Journal of Organic Chemistry
Note
overnight in Dulbecco’s Modified Eagle Medium (DMEM;
Invitrogen), supplemented with 10% heat-inactivated fetal bovine
serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin G sodium, 100
μg/mL streptomycin sulfate, 1 mM sodium pyruvate, and 0.1 mM
nonessential amino acids. On the next day, adherent HCT-116 cells
were treated with fresh phenol red free DMEM complete media
containing various concentrations of 19 and Bz-Har-Pro-Leu-OH, as
indicated in the legends. Human MEC2 and WaC3 chronic
lymphocytic leukemia cells were cultured in the RPMI 1640 media
(Invitrogen) supplemented with 10% heat-inactivated FBS, 2 mM L-
glutamine, 100 U/mL penicillin G sodium, 100 μg/mL streptomycin
sulfate, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and
0.1 mM β-mercaptoethanol (β-ME). Every 24 h, proliferative
capabilities were assessed by XTT assays (Roche) according to the
manufacturer’s instructions. Briefly, 50 μL of XTT labeling reagent
(sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]bis(4-methoxy-
6-nitro)benzenesulfonic acid hydrate), 1 μL of electron-coupling
reagent (N-methyldibenzopyrazine methyl sulfate), and 100 μL of
phenol red free culture media were combined and applied to each well
of the 96-well plates. The assay was based on cleavage of the yellow
tetrazolium salt XTT by mitochondrial dehydrogenases of the
metabolic active cells to form the orange formazan compound,
which can be quantified spectrophotometrically at 492 nm using a
BioTek microplate reader.
(5) Chatterjee, A. K.; Sanders, D. P.; Grubbs, R. H. Org. Lett. 2002, 4,
1939.
(6) (a) Dondoni, A.; Merino, P.; Perrone, D. Tetrahedron 1993, 49,
2939. (b) Ribes, C.; Falomir, E.; Carda, M.; Marco, J. A. J. Org. Chem.
2008, 73, 7779.
(7) (a) Flamant-Robin, C.; Wang, Q.; Chiaroni, A.; Sasaki, N. A.
Tetrahedron 2002, 58, 10475. (b) Flamant-Robin, C.; Wang, Q.;
Sasaki, N. A. Tetrahedron Lett. 2001, 42, 8483. (c) Yoda, H.; Shirai, T.;
Katagiri, T.; Takabe, K.; Kimata, K.; Hosoya, K. Chem. Lett. 1990,
2037.
(8) (a) Hanessian, S.; Wang, W.; Gai, Y. Tetrahedron Lett. 1996, 37,
7477. (b) Hanessian, S.; Sumi, K. Synthesis 1991, 1083.
(9) Selim, K. B.; Lee, B.; Sim, T. Tetrahedron Lett. 2012, 53, 5895.
(10) (a) Tozuka, Z.; Takaya, T. J. Antibiot. (Tokyo) 1983, 36, 142.
(b) Mansoor, T. A.; Ramalho, R. M.; Mulhovo, S.; Rodrigues, C. M.;
Ferreira, M. J. Bioorg. Med. Chem. Lett. 2009, 19, 4255. (c) Lorente, A.;
Pla, D.; Canedo, L. M.; Albericio, F.; Alvarez, M. J. Org. Chem. 2010,
75, 8508.
ASSOCIATED CONTENT
■
S
* Supporting Information
NMR spectra for all new compounds as well as HMQC,
HMBC, and ROESY spectra for synthetic and natural
lucentamycin A, dose−response curves for cell viability assays
with HCT-116, MEC2, and WaC3 cell lines, and X-ray
diffraction data for 19. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: juan.delvalle@moffitt.org.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Prof. William Fenical (UCSD/SIO) for copies of
NMR spectra for natural lucentamycin A and Dr. Lukasz
Wojtas (USF) for carrying out X-ray diffraction experiments.
This work was supported by a grant to J.R.D. from the
Bankhead-Coley Biomedical Research Program, Florida
Department of Health (1BN03).
REFERENCES
■
(1) Cho, J. Y.; Williams, P. G.; Kwon, H. C.; Jensen, P. R.; Fenical,
W. J. Nat. Prod. 2007, 70, 1321.
(2) (a) Pal, U.; Ranatunga, S.; Ariyarathna, Y.; Del Valle, J. R. Org.
Lett. 2009, 11, 5298. (b) Ranatunga, S.; Kim, J. S.; Pal, U.; Del Valle, J.
R. J. Org. Chem. 2011, 76, 8962. (c) Daniels, R. N.; Melancon, B. J.;
Wang, E. A.; Crews, B. C.; Marnett, L. J.; Sulikowski, G. A.; Lindsley,
C. W. J. Org. Chem. 2009, 74, 8852. (d) Ham, Y. J.; Yu, H.; Kim, N. D.;
Hah, J. M.; Selim, K. B.; Choi, H. G.; Sim, T. Tetrahedron 2012, 68,
1918.
(3) Cha, J. W.; Park, J. S.; Sim, T.; Nam, S. J.; Kwon, H. C.; Del Valle,
J. R.; Fenical, W. J. Nat. Prod. 2012, 75, 1648.
(4) (a) Kazmaier, U. In The Claisen Rearrangement; Hiersemmann,
M., Nubbemeyer, U., Eds.; Wiley-VCH: Weinheim, Germany, 2007; p
233. (b) Bartlett, P. A.; Barstow, J. F. J. Org. Chem. 1982, 47, 3933.
(c) Piero, V.; Hans-Jurgen, H.; Hans, S. Helv. Chim. Acta 1975, 58,
̈
1293.
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