Isolation, structural assignment, and total synthesis of barmumycin

Article abstract of DOI:10.1021/jo101834c

Barmumycin was isolated from an extract of the marine actinomycete Streptomyces sp. BOSC-022A and found to be cytotoxic against various human tumor cell lines. On the basis of preliminary one- and two-dimensional ¹H and ¹³C NMR spect

Full text of DOI:10.1021/jo101834c

pubs.acs.org/joc
Isolation, Structural Assignment, and Total Synthesis of Barmumycin
‡
,†,§,
Adriana Lorente,† Daniel Pla,†,# Librada M. Canedo, Fernando Albericio,*
and
~
Mercedes Alvarez*
,†,§,^
ꢀ
†Institute for Research in Biomedicine, Barcelona Science Park, University of Barcelona, Baldiri Reixac 10,
E-08028 Barcelona, Spain, ‡Instituto Biomar, S.A. Parque Tecnoloꢀgico de Leoꢀn-M10.4, E-24009 Armunia,
§
Leon, Spain, CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine,
ꢀ
Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain, Department of Organic Chemistry,
University of Barcelona, 08028 Barcelona, Spain, and ^Laboratory of Organic Chemistry, Faculty of Pharmacy,
University of Barcelona, 08028 Barcelona, Spain. #Current address: Memorial Sloan-Kettering Cancer Center,
New York, NY 10065. E-mail: plaquerd@mskcc.org
fernando.albericio@irbbarcelona.org; mercedes.alvarez@irbbarcelona.org
Received September 23, 2010
Barmumycin was isolated from an extract of the marine actinomycete Streptomyces sp. BOSC-022A and
found to be cytotoxic against various human tumor cell lines. On the basis of preliminary one- and two-
1
dimensional H and 13C NMR spectra, the natural compound was initially assigned the structure of
macrolactone-type compound 1, which was later prepared by two different routes. However, major spectro-
scopic differences between isolated barmumycin and 1 led to revision of the proposed structure as E-16. On
the basis of the synthesis of this new compound, and subsequent spectroscopic comparison of it to an authen-
tic sample of barmumycin, the structure of the natural compound was indeed confirmed as that of E-16.
Introduction
the culture broth of the marine actinomycete Streptomyces
sp. BOSC-022A, isolated from a tunicate collected off the
Scottish coast. Barmumycin and its diacetate show antitu-
mor activity at micromolar concentrations in all 12 cancer
cell lines tested (see Table 1 in the Supporting Information).
Herein we report the isolation, total synthesis, and structure
elucidation of barmumycin.
Natural products from terrestrial plants and microorgan-
isms have long been a traditional source of drugs; however,
over the past few years, marine organisms have garnered
ever-increasing attention as a rich bank of new bioactive
compounds.1 Marine actinomycetes have also proven to be
an important source of biologically active compounds.2
Among the marine actinomycetes that our group has studied,
those of the genus Streptomyces have clearly shown the most
pharmacological potential; however, in many bioactive cultures
they have yielded only compounds that are already known.
During ongoing research efforts to explore the biosynthetic
potential of rare marine microorganisms, we isolated two known
compounds, pretomaymycin3 and oxotomaymycin4 (Figure 1),
plus the previously unknown compound barmumycin from
Results and Discussion
The molecular formula of barmumycin was determined to
be C15H19NO4 by HRMS MALDI-TOF; it gave an (M þ H)þ
ion at m/z 278.13840 (calcd m/z 278.13869 for C15H20NO4).
Reaction of barmumycin with acetic anhydride and pyridine
gave a diacetyl derivative, confirmed by MS, pointing the pres-
ence of two OH and/or NH protons (see Figure 2).
1H NMR shows three groups of protons (see Table 2 in the
Supporting Information). The first group is in the aromatic
region and contains three protons at 6.90 (d), 7.03 (d), and
7.08 (s) ppm; the chemical shifts indicated a 1,2,4-substituted
electron-rich benzene ring. The second group corresponds to
a single vinylic proton at 5.34 ppm (m). The third region
comprises an upfield CH shift at 4.67 ppm (m); two methyl
(1) Molinski, T. F.; Dalisay, D. S.; Lievens, S. L.; Saludes, J. P. Nat. Rev.
Drug Discov. 2009, 8, 69–85.
(2) (a) Olano, C.; Mendez, C.; Salas, J. A. Mar. Drugs 2009, 7, 210–248.
(b) Bull, A. T.; Stach, J. E. Trends Microbiol. 2007, 15, 491–499.
(3) Shimizu, K. I.; Kawamoto, I.; Tomita, F.; Morimoto, M.; Fujimoto,
K. J. Antibiot. 1982, 35, 972–978.
(4) Kariyone, K.; Yazawa, I.; Kohsaka, M. Chem. Pharm. Bull. 1971, 19,
2289–2293.
8508 J. Org. Chem. 2010, 75, 8508–8515
Published on Web 11/23/2010
DOI: 10.1021/jo101834c
r
2010 American Chemical Society

Lorente et al.
JOCArticle
Reductive amination of 4 with aniline 510 required special con-
ditions due to the poor nucleophilicity of the aniline (which is
deactivated by the methyl ester group in the ortho position): thus,
reaction of 4, 5, phenylsilane, and dibutyltin dichloride under
microwave irradiation for short reaction times gave the ami-
noalkene 6a in 67% yield.11,12 Attempts at protecting the aniline
NH in 6a as a tBu carbamate failed due to its poor reactivity;
therefore, 6a was treated with K2CO3 in MeOH to give the
deacetylated derivative 6b. However, all attempts at oxidizing 6b
to its ketone derivative resulted in decomposition of the starting
material.13 Thus, the aniline had to be protected, but this was not
possible in the presence of the unprotected alcohol. Exploiting
the lack of reactivity of the aromatic amine toward Boc protec-
tion, and using standard conditions, 6b was converted into its
tBu carbonate derivative 6c in 43% yield.14 The aniline group of
6c was then orthogonally protected using (CF3CO)2O in pyr-
idine to afford the trifluoroacetamide derivative 6d in quantita-
tive yield. Treatment of 6d with 10% TFA in CH2Cl2 to remove
the carbonate gave the free alcohol 6e in quantitative yield.
Compound 6e was thenoxidizedwith Dess-Martin periodinate
(DMP)15 to yield the ketone 7in 93% yield. Slow addition of 7to
N-methylmorpholine oxide (NMO) and a catalytic amount of
OsO4 in acetone-H2O to generate the corresponding diol 8
while preventing double-bond isomerization gave 8 in good
yield. Diol 8 was further protected by conversion into its 2,
2-dimethyl-1,3-dioxolane derivative 9 using 2,2-dimethoxypro-
pane plus pyridinium p-toluenesulfonate (PPTS) as catalyst
(quantitative yield). Deprotection of the amine in 9 via mild
basic hydrolysis gave the free amine 10 in 97% yield.
A faster and better yielding synthesis of 10 (Scheme 3) was
done in parallel to the route described above. The first step was
dihydroxylation of isobutyl but-3-enoate. The introduction of the
bromomethyl residue was planned for a later step. The oxidation
conditions described above afforded isobutyl 3,4-dihydroxybu-
tanoate (11), which was then further protected as the 2,2-dimeth-
yl-1,3-dioxolane derivative 12, in excellent overall yield for both
steps. The key step, transformation of 12 into the bromoketone 13
using bromomethyllithium, gave 13 in 49% yield. N-Alkylation
of 5 with 13 under microwave irradiation gave 10.
Wittig chemistry was employed to introduce the ethylidene
chain. Reaction of 10 with the Wittig ylide derived from
ethyltriphenylphosphonium bromide yielded 14 (43%) as a
mixture of Z/E diastereomers.16
Z/E-14 was transformed into 1 in three successive reactions:
hydrolysis of the methyl ester, acetonide deprotection under
acidic conditions, and macrocyclization. The acid Z-15 was ob-
tained by purification of the Z/E mixture of acids by semipre-
parative HPLC.17
FIGURE 1. Two known compounds isolated from Streptomyces
sp. BOSC-022A.
1
FIGURE 2. Structure of 1 showing H NMR (blue) and 13C NMR
(red) chemical shifts (left) and its HMBC and NOE correlations (right).
groups, seen at 3.90 ppm (s) and 1.62 ppm (d); and the signals
of three CH2 at 4.07 (bt), 4.18 (bd), 3.75 (m), 2.29 (m) and
2.71 (m) ppm. On the basis of these data, the MS findings,
and further data from one-dimensional and two-dimensional
1
(COSY, HMBC, and NOESY) H and 13C NMR experi-
ments, we initially proposed that the structure of the isolated
natural product was that of benzomacrolactone 1, derived
from 5-methoxy-2-aminobenzoic acid with an exocyclic (E)-
ethylidene and one alcohol function (Figure 2).
Decanolides are chemical entities abundant in terrestrial
organisms, though only a few (e.g., modiolides A and B5 and
xestodecalactones A-C6) have been isolated from marine
sources. To the best of our knowledge, the aniline moiety within
its 10-membered lactone had never been reported in naturally
occurring macrocycles.
We sought to synthesize 1 to compare it against an authentic
sample of barmumycin in order to assess its structural assign-
ment. Our retrosynthetic analysis of 1 entailed formation of the
exocyclic double bond via Wittig reaction; dihydroxylation of a
double bond to give the alcohol required for lactonization; and
finally, introduction of a functionalized five-carbon chain onto
the nitrogen of methyl 2-amino-5-methoxybenzoate (Scheme 1).
The functionalized five-carbon chain on the aniline nitro-
gen was introduced by two different ways: via reductive
amination (Scheme 2) and via N-alkylation (Scheme 3).
Homoallylic alcohol 2 was obtained by Barbier reaction of
2,2-dimethoxyacetaldehyde with allyl bromide and indium
powder in water (95% yield).7
Attempts at direct deprotection of the dimethyl acetal under
acidic conditions led to polymerization of 2;8 therefore, the
alcohol had to be protected. Acetylation of the alcohol to give
compound 3,7 followed by dimethyl acetal deprotection using
LiBF4 in MeCN-H2O,9 gave the aldehyde 4 in excellent yield.
Racemic Z-1 was obtained in 35% yield by acetal depro-
tection followed by macrocyclization using EDC HCl and
3
€
(11) Kangasmetsa, J. J.; Johnson, T. Org. Lett. 2005, 7, 5653–5655.
(12) Other reduction conditions proved unsuccessful. These included NaBH-
(OAc)3 in THF at room temperature for 16 h, NaBH(OAc)3 in CH2Cl2/AcOH at
room temperature for 5 h, and NaBH(OAc)3 in toluene at 110 ꢀC for 2 h.
(13) The aniline 5 was isolated from the oxidation degradation mixture.
Its formation could be rationalized through hydrolysis of the enamine
resulting from enolization of the keto compound.
(5) Tsuda, M.; Mugishima, T; Komatsu, K.; Sone, T.; Tanaka, M.;
Mikami, Y.; Kobayashi, J. J. Nat. Prod. 2003, 66, 412–415.
€
(6) Edrada, R. A.; Heubes, M.; Brauers, G.; Wray, V.; Berg, A.; Grafe, U.;
Wohlfarth, M.; Muhlbacher, J.; Schaumann, K.; Sudarsono; Bringmann, G.;
€
Proksch, P. J. Nat. Prod. 2002, 65, 1598–1604.
(7) Chenevert, R.; Gravil, S.; Bolte, J. Tetrahedron: Asymetry 2005, 16,
2081–2086.
(14) Under these conditions, methyl 2-(5-allyl-2-oxooxazolidin-3-yl)-
5-methoxybenzoate was isolated as a byproduct in 27% yield.
(15) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155–4156.
(16) The Z/E stereoisomers were in a ratio of 73:27 (based on NMR signal
areas).
(8) The instability of R-hydroxy aldehydes due to their partial isomerization
ꢀ
into R-hydroxy ketones has previously been reported; see: Crestia, D.; Guerard,
C.; Bolte, J.; Demuynck, C. J. Mol. Catal. B: Enzym. 2001, 11, 207–212.
(9) Lipshutz, B. H.; Harvey, D. F. Synth. Commun. 1982, 12, 267–277.
(10) Theeraladanon, C.; Arisawa, M.; Nishida, A.; Nakagawa, M. Tetra-
hedron 2004, 60, 3017–3035.
(17) The NOESY correlations between 4CH2 (2.27 and 2.41 ppm) and the
vinyl proton (5.58 ppm) confirmed the stereochemistry of (Z)-15 (see
NOESY interactions in the Supporting Information).
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SCHEME 1. Retrosynthetic Analysis of Compound 1
SCHEME 2. Route A for the Synthesis of 10
solid-supported DMAP in a 5 mM CH2Cl2 solution. The 1H
NMR spectrum of Z-1 showed two doublets for the CH3
linked to the double bond (1.42 ppm and 1.45 ppm) and two
quadruplets for the vinylic proton (5.51 and 5.53 ppm).18
These data could be explained by the presence of two highly
populated conformations of Z-1 at room temperature.
Therefore, we studied peak coalescence by 1H NMR run at
different temperatures. Spectra from the initial experiments,
run up to 55 ꢀC in CDCl3 as solvent, exhibited this trend,
but coalescence was not reached at this temperature limit.
Finally, coalescence was almost reached in DMSO-d6 as
solvent at 145 ꢀC (see Table 4 in the Supporting Informa-
tion). Moreover, comparison of spectroscopic data for bar-
mumycin with those for Z-1 revealed dramatic differences in
the chemical shifts (see Tables 2 and 3 in the Supporting Infor-
mation). This discrepancy, despite the conflicting stereochemis-
try of the two compounds (Z-1 and E-barmumycin), led us to
pursue a new structural assignment.
all three molecules could derive from the same biogenetic
pathway.
In order to confirm that the structure of barmumycin is
actually that of E-16, we synthesized the latter and subsequently
compared it to an authentic sample of the former. This began
with selective silyl protection of the primary alcohol in the com-
mercially available N-Boc-trans-4-hydroxy-L-prolinol followed
by oxidation of the secondary alcohol in the derivative 17, which
afforded ketone 18 in 62% yield over two steps (Scheme 4).
Wittig chemistry was again employed to introduce the ethylidene
chain: reaction of 18 with the Wittig ylide derived from ethyl-
triphenylphosphonium bromide yielded 19 as a 9:1 mixture of Z/
E-diastereomers. Z/E-19 was used directly without separation, as
a single purificationwas planned for the final step of the synthesis.
The TMS ether and the tert-butyl carbamate of Z/E-19 were
deprotected with 10% TFA in CH2Cl2 to give the pyrrolidine
derivative Z/E-20. Condensation of Z/E-20 to vanillic acid using
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluoro-
phosphate (PyBOP) and N,N-diisopropylethylamine (DIEA)
gave a 9:1 mixture of Z/E-16 diastereomers. The configuration
of the double bond was established by the NOESY correlations
Re-evaluation of all possible alternative structures led us
to systematic elucidation of E-16 as a novel structure for
barmumycin (Figure 3). Interestingly, the very close struc-
tural resemblance of 16 to the pretomaymycin and oxoto-
maymycin isolated from the extract (Figure 1) suggests that
5
in Z/E-16 between CH2 (4.00-4.20 ppm) and the CH3 (1.60
ppm) (see NOESY correlations in the Supporting Information).
None of the Horner-Wadsworth-Emmons reactions tested
(using the appropriate phosphonate and different bases19) gave
(18) The NOESY correlations between 2CH2 (4.35 and 4.82 ppm) and
CH3 (1.42 and 1.45 ppm), and between 4CH2 (2.09-2.20 ppm) and the vinyl
proton (5.51 and 5.53 ppm), confirmed the stereochemistry of (Z)-1 (see
NOESY interactions in the Supporting Information).
(19) The reaction was performed with (EtO)2P(O)CH2CH3 and either
LDA, KtBuO, or NaHMDS as base.
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SCHEME 3. Route B for the Synthesis of 10
which was subsequently prepared (in five steps and 18%
overall yield) for comparison with the natural compound.
The spectroscopic data for E-16 fully coincided with that
for barmumycin, thereby confirming that the two struc-
tures are equivalent. This work is a new example of the impor-
tance of total synthesis for structural characterization and
confirmation of natural products.21
FIGURE 3. Structure proposed for barmumycin upon re-evalua-
tion of NMR data.
Experimental Section
the desired E-19. However, the Kocienski variant of the Julia-
Lythgoe olefination20 afforded a 2:1 mixture of E/Z-19. This
process entails nucleophilic addition of 5-(ethylsulfonyl)-1-phe-
nyl-1H-tetrazole anion to the ketone followed by transposition
and elimination to give the double bond. Again, deprotection of
the hydroxyl group and the amine group was obtained using
10% TFA in CH2Cl2, and condensation of E/Z-20 to vanillic
acid using PyBOP and DIEA gave a 2:1 mixture of E/Z-16. This
mixture was purified by semipreparative HPLC to obtain E-16
as a single diastereomer. The configuration of the double bond
was established by the NOESY correlations in E-16 between
5CH2 (4.00-4.22 ppm) and the vinyl proton (5.30-5.38 ppm)
(see the NOESY correlations in the Supporting Information).
Comparison of spectroscopic data obtained for E-16 and
barmumycin confirmed that the revised structure is indeed
the structure of the natural product.
In summary, the previously unreported marine compound
barmumycin was isolated, and its chemical formula was deter-
mined via mass spectrometry. On the basis of preliminary NMR
data, barmumycin was initially assigned the structure of com-
pound 1. To confirm this assignment, compound 1 was syn-
thesized following two different strategies starting from an
o-aminobenzoic ester: one based on reductive amination, and
one based on N-alkylation, which was shorter and higher
yielding. However, comparison of the NMR spectra for 1
with those for isolated barmumycin showed dramatic
differences. The structure of barmumycin was reassessed,
and most probable option conceived was compound E-16,
See the Supporting Information for general procedures.
Extraction and Isolation of Barmumycin. The culture broth
(10 L) was separated by filtration into a mycelial cake and
cultured filtrate (9 L). A 500 mL aliquot of the absorber resin
XAD-1180 was added to the filtrate. Compound barmumycin
was eluted from the resin by double extraction with a 3:1:1
mixture of EtOAc-MeOH-H2O (1.8 L). The active fractions
were concentrated in the organic phase, which was concentrated
to dryness in vacuo to yield 950 mg of crude extract. This extract
purified by vacuum flash chromatography using a mixture of
n-hexane-EtOAc and EtOAc-MeOH, whereby the fractions
containing barmumycin (220 mg) were eluted with 9:1 EtOAc-
MeOH. The active fractions were purified by silica gel chroma-
tography using CHCl--MeOH mixtures. Cytotoxicity was
detected in the fractions eluted with 96:4 CHCl--MeOH
(20 mg). Further purification with a C18 column by HPLC
afforded 6 mg of pure barmumycin (elution with 54:46 H2O/
MeOH). This quantity of barmumycin was treated with 0.5 mL
of pyridine and 0.5 mL of Ac2O to afford 7 mg of the corresponding
diacetate. The molecular formula of barmumycin was determined to
be C15H19NO4 by HPLC-APESI MS, in which it gave an (M þ
Na)þ peak at 300 and (M - H)- 276. Barmumycin gave an (M þ
H)þ peak at 278 and (M - H)- 276 in HPLC-APCI MS and it gave
an (M þ H)þ ion at m/z 278.13840 (calcd m/z 278.13869 for
C15H20NO4) in HRMALDI-TOF MS.
The diacetyl derivative of barmumycin gave an (M þ H)þ
peak at 362 and an (M þ Na)þ peak at 384 by HPLC-APCI MS
and HPLC-ESI MS.
(21) (a) Nicolaou, K. C.; Snyder, S. A. Angew. Chem., Int. Ed. 2005, 44,
1012–1044. (b) Cornella, I; Kelly, T. R. J. Org. Chem. 2004, 69, 2191–2193.
(20) Blakemore, P. R. J. Chem. Soc., Perkin Trans. 1 2002, 2563–2585.
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SCHEME 4. Preparation of Z/E-16 Starting from N-Boc-trans-4-hydroxy-L-prolinol
2-Acetoxypent-4-enal (4). LiBF4 (6.05 g, 64.5 mmol) was
added to a solution of 3 (4.05 g, 21.5 mmol) in 98:2 MeCN/
H2O (110 mL), and the mixture was stirred for 72 h at room
temperature. The solvents were removed in vacuo. The crude
was dissolved in CH2Cl2, washed with water and brine, and then
concentrated in vacuo to yield 4 (2.82 g, 92%) as a yellowish oil.
J = 3.1 Hz, 1H). 13C NMR (100.6 MHz, CDCl3): δ 39.5 (t); 49.3
(t); 51.7 (q); 56.0 (q); 69.3 (d); 110.8 (s); 113.6 (d); 114.5 (d); 118.5
(t); 123.2 (d); 133.9 (d); 145.8 (s); 149.9 (s); 168.6 (s). MS (ESI-
TOF): 266 (M þ 1, 100). HRMS: m/z calcd for C14H20NO4
266.1392, found 266.1398.
Methyl 2-(2-(tert-Butoxycarbonyloxy)pent-4-enylamino)-
5-methoxybenzoate (6c). Boc2O (823 mg, 3.77 mmol) was added to
a solution of 6b (909.4 mg, 3.43 mmol) and DMAP (125.6 mg, 1.03
mmol) in dry CH2Cl2 (50 mL). The reaction mixture was stirred for
40 h at room temperature and then concentrated in vacuo. Purifica-
tion by silica gel column chromatography (100:0 to 95:5 hexane-
EtOAc) yielded 6c (543 mg, 43%) as a yellowish oil. IR (KBr film): ν
1
IR (KBr film): ν 3080, 2932, 1744, 1373, 1237, 1048 cm-1. H
NMR (400 MHz, CDCl3): δ 2.18 (s, 3H); 2.32-2.65 (m, 2H);
5.06-5.30 (m, 3H); 5.65-5.87 (m, 1H); 9.54 (s, 1H). 13C NMR
(100.6 MHz, CDCl3): δ 20.4 (q); 33.1 (t); 77.2 (d); 119.0 (t); 131.4
(d); 170.4 (s); 197.9 (d). MS (ESI-TOF): 143 (M þ 1, 100).
Methyl 2-(2-Acetoxypent-4-enylamino)-5-methoxybenzoate
(6a). PhSiH3 (2.01 g, 18.6 mmol) was added to a THF solution
(12 mL) of 5 (2.02 g, 9.3 mmol), 4 (1.32 g, 9.3 mmol), and
Bu2SnCl2 (282.3 mg, 0.9 mmol) in a sealed tube. The mixture
was heated to 100 ꢀC under MW irradiation for 15 min. The
solvents were removed in vacuo. Purification by silica gel
column chromatography (100:0 to 95:5 hexane-Et2O) yielded
6a (1.93 g, 67%) as a yellowish oil. IR (KBr film): ν 3479, 3370,
2951, 1739, 1691, 1520, 1223, 1042 cm-1. 1H NMR (400 MHz,
CDCl3): δ 2.06 (s, 3H, Me); 2.41-2.46 (m, 2H); 3.35-3.37 (m,
2H); 3.76 (s, 3H, OMe); 3.86 (s, 3H, OMe); 5.09-5.17 (m, 3H);
5.78 (ddt, J = 17.2, 10.1, and 7.1 Hz, 1H); 6.74 (d, J = 9.2 Hz,
1H); 7.04 (dd, J = 9.2, 3.1 Hz, 1H); 7.42 (d, J = 3.1 Hz, 1H); 7.55
(bs, 1H, NH). 13C NMR (100.6 MHz, CDCl3): δ 21.0 (q); 36.3
(t); 45.6 (t); 51.5 (q); 55.9 (q); 71.6 (d); 110.2 (s); 112.9 (d); 114.3
(d); 118.3 (t); 123.3 (d); 133.0 (d); 146.0 (s); 149.5 (s); 168.6 (s);
170.6 (s). MS (ESI-TOF): 308 (M þ 1, 82). HRMS: m/z calcd.
for C16H22NO5 308.1498, found 308.1504.
Methyl 2-(2-Hydroxypent-4-enylamino)-5-methoxybenzoate
(6b). K2CO3 (953.5 mg, 6.9 mmol) was added to a solution of
6a (1.92 g, 6.27 mmol) in MeOH (75 mL). The mixture was
stirred for 1 h at room temperature. The solvent was removed in
vacuo. The residue was dissolved in CH2Cl2 and then washed
with water and brine. The organic extracts were dried over
MgSO4 and then concentrated in vacuo. Purification by silica
gel column chromatography (85:15 to 75:25 hexane/EtOAc)
yielded 6b (1.55 g, 93%) as a yellow oil. IR (KBr film): ν 3370,
1688, 1518, 1437, 1222, 1073 cm-1. 1H NMR (400 MHz, CDCl3)
δ 2.27-2.43 (m, 2H); 3.20 (dd, J = 13.0 and 7.7 Hz, 1H); 3.33
(dd, J = 13.0 and 4.5 Hz, 1H); 3.77 (s, 3H, OMe); 3.87 (s, 3H,
OMe); 3.96 (m, 1H); 5.17 (m, 1H); 5.19 (m, 1H); 5.87 (m, 1H);
6.75 (d, J = 9.1 Hz, 1H); 7.04 (dd, J = 9.1, 3.1 Hz, 1H); 7.44 (d,
1
3369, 3078, 1730, 1709, 1500, 1368 cm-1. H NMR (400 MHz,
CDCl3): δ1.47 (s, 9H); 2.41-2.48 (m, 2H); 3.39 (d, J = 6.1 Hz, 2H);
3.76 (s, 3H, OMe); 3.85 (s, 3H, OMe); 4.91 (p, J = 6.1 Hz, 1H); 5.12
(m, 1H); 5.17 (m, 1H); 5.82 (m, 1H); 6.74 (d, J = 9.2 Hz, 1H); 7.04
(dd, J = 9.2, 3.1 Hz, 1H); 7.42 (d, J= 3.1 Hz, 1H). 13CNMR (100.6
MHz, CDCl3):δ27.7 (q); 36.5 (t); 45.9 (t); 51.6 (q); 56.0 (q); 74.4 (d);
82.2 (s); 110.4 (s); 112.9 (d); 114.4 (d); 118.4 (t); 123.3 (d); 132.9 (d);
145.8 (s); 149.6 (s); 153.2 (s); 168.5 (s). MS (ESI-TOF): 366 (M þ 1,
100). HRMS: m/z calcd for C19H28NO6 366.1917, found 366.1917.
Methyl 2-[N-(2-(tert-Butoxycarbonyloxy)pent-4-enyl)trifluoro-
acetamido]-5-methoxybenzoate (6d). TFAA (0.3 mL, 2.2 mmol) was
added to a cooled (0 ꢀC) solution of 6c (542.9 mg, 1.49 mmol) in
pyridine (20 mL), and the mixture was stirred at 0 ꢀC for 90 min. The
crude was concentrated in vacuo, dissolved in CH2Cl2, and then
washed with NH4Cl, water, and brine. The organic extracts were
dried over MgSO4 and then concentrated in vacuo to yield 6d as a
mixture of rotamers (685 mg, quant) as a yellowish oil. IR (KBr
film): ν 1736, 1707, 1502, 1282 cm-1. 1H NMR (400 MHz, CDCl3):
δ 1.42 and 1.47 (2s, 9H); 2.32-2.40 (m, 2H, CH2); 3.04 and 3.31
(2dd, J = 14.4, 9.3 and 14.8, 2.2 Hz, 1H, CH2); 3.86 (s, 3H, OMe);
3.87 (s, 3H, OMe); 4.41 and 4.51 (2dd, J = 14.4, 2.9 and 14.8, 8.9
Hz, 1H, CH2); 4.82-4.87 and 5.17-5.23 (2 m, 1H, CH); 5.05-5.17
(m, 2H); 5.65-5.81 (m, 1H); 7.04 and 7.10 (2dd, J = 8.8, 3.0 Hz,
1H); 7.24 and 7.51 (2d, J = 8.8 Hz, 1H); 7.55 and 7.57 (2d, J = 3.0
Hz, 1H). 13C NMR (100.6 MHz, CDCl3): δ 27.6 and 27.7 (3q); 36.7
and 37.1 (t); 52.7 (q); 54.0 and 55.8 (t); 55.7 (q); 73.3 and 73.7 (d);
82.3 (s); 116.4 and 116.8 (d); 118.5 and 118.7 (d); 118.6 (t); 132.2 and
132.3 (d); 132.7 (d); 152.7 (s); 153.1 (s); 159.6 and 159.7 (s); 164.8
and 164.9 (s); 171.1 (s). 19F NMR (376 MHz, CDCl3): δ -68.8
and -69.0 (2s). MS (ESI-TOF): 945 (2M þ Na, 15). HRMS: m/z
calcd for C42H52N2O14F6Na 945.3220, found 945.3208.
8512 J. Org. Chem. Vol. 75, No. 24, 2010

Lorente et al.
JOCArticle
Methyl 5-Methoxy-2-[N-(2-hydroxypent-4-enyl)trifluoroaceta-
mido]benzoate (6e). A 10% solution of TFA in CH2Cl2 (50 mL) was
added to 6d (494.9 mg, 1.07 mmol), and the mixture was stirred at
room temperature for 25 min. Elimination of the solvent gave 6e
(387 mg, quant) as a yellow oil. IR (KBr film): ν 3370, 2950, 1688,
3.85 (s, 3H, OMe); 3.87 (s, 3H, OMe); 4.12-4.18 (m, 1H);
4.40-4.48 (m, 1H); 5.08 (d, J = 17.9 Hz, 1H); 5.13 (d, J = 17.9
Hz, 1H); 7.048 and 7.051 (2dd, J = 8.8 and 3.0 Hz, 1H); 7.50 (d,
J = 3.0 Hz, 1H); 7.55 and 7.57 (2d, J = 8.8 Hz, 1H). 13C NMR
(100.6 MHz, CDCl3): δ 25.4 (q); 26.8 (q); 44.3 and 44.4 (t); 52.6 (q);
55.7 (q); 61.2 and 61.4 (t); 69.0 and 69.2 (t); 71.3 (d); 109.1 (s); 114.6
(s); 116.4 and 116.5 (d); 117.5 (s); 118.4 (d); 129.0 (s); 131.6 (s); 132.5
and 132.6 (d); 159.8 (s); 164.9 (s); 200.8 (s). 19F NMR (376 MHz,
CDCl3): δ -68.8 (s). MS (ESI-TOF): 434 (M þ 1, 100).
1
1519, 1437, 1222, 1074 cm-1. H NMR (400 MHz, CDCl3): δ
2.20-2.30 (m, 2H, CH2); 3.46-3.49 and 3.54-3.61 (m, 1H); 3.88 (s,
3H, OMe); 3.90 (s, 3H, OMe); 4.02-4.15 (m, 2H); 4.89 (bs, 1H,
OH); 5.10-5.14 (m, 2H); 5.72-5.81 (m, 1H); 7.09-7.14 (m, 1H);
7.31 and 7.39 (2d, J = 8.8 Hz, 1H); 7.55 (d, J = 2.5 Hz, 1H). 13C
NMR (100.6 MHz, CDCl3): δ 39.3 and 39.4 (t); 52.9 (q); 55.8 (q);
58.4 and 59.0 (t); 68.2 and 68.8 (d); 114.9 (s); 116.2 and 116.7 (d);
118.8 (t); 118.9 and 119.0 (d); 120.1 (s); 120.9 (s); 122.8 (s); 131.1 (s);
131.9 and 132.1 (d); 133.1 and 133.4 (d); 159.8 (s). 19F NMR (376
MHz, CDCl3): δ -68.7 (s). MS (ESI-TOF): 362 (M þ 1, 37).
HRMS: m/z calcd for C16H19NO5F3 362.1215, found 362.1211.
Methyl 5-Methoxy-2-[N-(2-oxopent-4-enyl)trifluoroacetamido]-
benzoate (7). Dess-Martin periodinane (606.4 mg, 1.43 mmol) was
added to a solution of 6e (469.6 mg, 1.30 mmol) in anhydrous
CH2Cl2 (35 mL). The mixture was stirred for 1 h and then diluted
with Et2O and hexane to a final concentration of 30:20:50 CH2Cl2/
Et2O/hexane. The solution was filtered through a silica gel pad.
The solvents were removed in vacuo to yield 7 (433 mg, 93%) as a
yellowish oil. IR (KBr film): ν 1707, 1704, 1502, 1291, 1204, 1153
cm-1. 1H NMR (400 MHz, CDCl3): δ 3.18 (dd, J = 16.8 and 6.7
Hz, 1H); 3.27 (dd, J = 16.8 and 7.2 Hz, 1H); 3.85 (m, 1H); 3.86 (s,
3H, OMe); 3.89 (s, 3H, OMe); 5.13-5.23 (m, 3H); 5.95 (m, 1H);
7.06 (dd, J = 8.8 and 3.0 Hz, 1H); 7.51 (d, J = 3.0 Hz, 1H); 7.59 (d,
J = 8.8 Hz, 1H). 13C NMR (100.6 MHz, CDCl3): δ 45.0 (t); 52.7
(q); 55.7 (q); 60.4 (t); 114.7 (s); 116.5 (d); 117.6 (s); 118.4 (d); 119.9
(t); 129.0 (s); 129.1 (d); 131.6 (s); 132.6 (d); 159.8 (s); 165.0 (s); 200.8
(s). 19F NMR (376 MHz, CDCl3):δ-68.8(s). MS(ESI-TOF):360
(M þ 1, 100). HRMS: m/z calcd for C16H17NO5F3 360.1059,
found 360.1072.
Methyl 5-Methoxy-2-[N-(2,4,5-trihydroxypent-2-enyl)trifluoro-
acetamido]benzoate (8). A solution of 7 (775 mg, 2.16 mmol) in
acetone (20 mL) was added dropwise over 10 h to a stirring
solution of N-methylmorpholine oxide (337 mg, 2.37 mmol) and
OsO4 (catalytic amount) in 60:40 acetone/H2O (64 mL). The
mixture was stirred for 20 h at room temperature, quenched with
40% aq NaHSO3 (3 mL), and subsequently concentrated in vacuo.
The residue was dissolved in EtOAc, dried over MgSO4, filtered,
and then reconcentrated in vacuo. Purification by silica gel column
chromatography (100:0 to 90:10 CH2Cl2-MeOH) yielded 8 (628
mg, 84%) as a brownish oil. IR (KBr film): ν 1725, 1605, 1503,
1293, 1204 cm-1. 1HNMR(400MHz, DMSO-d6) δ3.25-3.30 (m,
1H); 3.36-3.43 (m, 1H); 3.63-3.71 (m, 1H, CH); 3.84 (s, 6H,
2OMe); 4.12-4.13 (m, 1H); 4.16 and 4.33 (2d, J = 18.5 Hz, 1H);
5.14 and 5.33 (2d, J = 18.5 Hz, 1H); 7.27 and 7.29 (2dd, J = 3.0
Hz, 1H); 7.41-7.48 (m, 2H). 13C NMR (100.6 MHz, DMSO-d6):δ
52.6 (q); 55.7 (q); 59.4 and 60.2 (t); 61.3 (t); 72.2 and 72.9 (d); 76.0
and 76.1 (d); 116.0 (d); 118.5 (d); 128.6 (s); 128.7 (s); 131.2 (s); 131.9
(d); 159.2 (s); 164.4 (s); 206.2 (s); 207.5 (s). 19F NMR (376 MHz,
CDCl3): δ -68.8 (s). MS (ESI-TOF): 392 (M - 1, 100); 394 (M þ
1, 40). HRMS: m/z calcd for C16H17NO7F3 392.0957, found
392.0946.
Isobutyl 3,4-Dihydroxybutanoate (11). A solution of isobutyl
but-3-enoate (10.5 g, 73.6 mmol) in acetone (50 mL) was added
dropwise over 20 h to a solution of N-methylmorpholine oxide
(10.9 g, 80.9 mmol) and a catalytic amount OsO4 in 60:40 acetone/
H2O (250 mL). The reaction was quenched with NaHSO3 40%
aq solution (3 mL) and concentrated in vacuo. The crude was
dissolved in EtOAc and filtered through silica gel, and the eluent
was concentrated in vacuo to yield 11 (12.6 g, 97%) as a yellow oil.
IR (KBr film): ν 3402, 2962, 1729, 1470, 1381, 1170, 1043 cm-1. 1H
NMR (400 MHz, CDCl3): δ 0.94 (d, J = 6.8 Hz, 6H); 2.00-1.89
(m, 1H); 2.51 (dd, J = 16.4 and 4.0 Hz, 1H, CH2); 2.58 (dd, J =
16.4 and 8.4 Hz, 1H, CH2); 3.58 (dd, J = 11.2 and 6.4 Hz, 1H,
CH2);3.69(dd, J = 11.2 and 3.6 Hz, 1H, CH2);3.91(d, J= 6.8 Hz,
2H); 4.10-4.18 (m, 1H). 13C NMR (100.6 MHz, CDCl3): δ 19.2
(2q); 27.8 (d); 37.9 (t); 65.9 (t); 68.8 (d); 71.2 (t); 172.8 (s). MS (ESI-
TOF): 177 (M þ 1, 45); 199 (M þ Na, 100). HRMS (þESI): m/z
calcd for C8H17O4 (M þ 1) 177.1127, found 177.1127; calcd for
C8H16O4Na (M þ Na) 199.0946, found 199.0945.
Isobutyl 2,2-Dimethyl-1,3-dioxolan-4-yl acetate (12). Pyridinium
p-toluenesulfonate (120 mg, 0.48 mmol) was added to a solution of
11 (15.4 g, 87.26 mmol) in 50:50 2,2-dimethoxypropane/CH2Cl2
(300 mL). The reaction mixture was stirred at room temperature
for 16 h, and then the solvent was removed in vacuo. Purification
by silica gel column chromatography (50:50 hexane-EtOAc)
yielded 12 (17.4 g, 92%) as a yellowish oil. IR (KBr film): ν
1736, 1380, 1370 cm-1. 1H NMR (400 MHz, CDCl3): δ 0.93 (d,
J = 6.8 Hz, 6H); 1.35 (s, 3H); 1.41 (s, 3H); 1.98-1.87 (m, 1H); 2.52
(dd, J = 15.7 and 7.6 Hz, 1H, CH2); 2.72 (dd, J = 15.7 and 6.4 Hz,
1H, CH2); 3.65 (dd, J = 8.4 and 6.4 Hz, 1H, CH2); 3.88 (d, J = 6.4
Hz, 2H); 4.16 (dd, J = 8.4 and 6.0 Hz, 1H, CH2); 4.40-4.51 (m,
1H). 13C NMR (100.6 MHz, CDCl3):δ 19.0 (2q); 25.5 (q); 26.9 (q);
27.6 (d); 39.0 (t); 69.2 (t); 70.8 (t); 72.1 (d); 109.1 (s); 170.6 (s).
1-Bromo-3-(2,2-dimethyl-1,3-dioxolan-4-yl)propan-2-one (13). A
1.6 M solution of MeLi in Et2O (5 mL, 8 mmol) was added to
a solution of 12 (865 mg, 4 mmol) and dibromomethane (557 μL,
8 mmol) in THF (20 mL) at -116 ꢀC. The solution was stirred for
3 h and then quenched with satd NH4Cl (60 mL). The residue was
immediately extracted with CH2Cl2. The organic layers were dried
over MgSO4, filtered, and then concentrated in vacuo. Purification
by silica gel column chromatography (80:20 to 70:30 hexane-
EtOAc) yielded 13 (460 mg, 49%) as a yellow oil. IR (KBr film): ν
1719, 1370, 840 cm-1. 1H NMR (400 MHz, CDCl3): δ 1.35 (s, 3H);
1.42 (s, 3H); 2.82 (dd, J = 16.6 and 6.0 Hz, 1H, CH2); 3.07 (dd, J=
16.6 and 6.8 Hz, 1H, CH2); 3.60 (dd, J = 8.4 and 6.4 Hz, 1H, CH2);
3.94 (s, 2H); 4.18 (dd, J = 8.4 and 6.0 Hz, 1H, CH2); 4.44-4.51 (m,
1H). 13C NMR (100.6 MHz, CDCl3): δ 25.4 (q); 26.8 (q); 34.6 (t);
44.16 (t); 69.2 (t); 71.8 (d); 109.3 (s); 199.7 (s). MS (ESI-TOF): 259
(MBr79 þ Na, 100); 261 (MBr81 þ Na, 98). HRMS (þESI): m/z
calcd for C8H13O3NaBr (M þ Na) 258.9944, found 258.9946.
Methyl 2-[3-(2,2-Dimethyl-1,3-dioxolan-4-yl)-2-oxopropyl-
amino]-5-methoxybenzoate (10). Route A. K2CO3 (68 mg, 0.5 mmol)
was added to a solution of 9 (50 mg, 0.11 mmol) in MeOH, and the
mixture was stirred for 1 h. The solvent was removed in vacuo, and
the resulting residue was purified by silica gel column chroma-
tography (95:5 to 80:20 hexane/EtOAc) to yield 10 (38 mg, 97%)
as a yellowish oil.
Methyl 2-[N-(3-(2,2-Dimethyl-1,3-dioxolan-4-yl)-2-oxopropyl)-
trifluoroacetamido]-5-methoxybenzoate (9). Pyridinium p-toluene-
sulfonate (18.7 mg, 74 mmol) was added to a solution of 8 (584 mg,
1.49 mol) and 2,2-dimethoxypropane (1.82 mL, 14.8 mmol) in
CH2Cl2 (30 mL). The mixture was stirred at 40 ꢀC for 16 h and
then concentrated in vacuo. Purification by silica gel column
chromatography (90:10 to 40:60 hexane-EtOAc) yielded 9
(642 mg, quant) as a yellowish oil. IR (KBr film): ν 1775, 1730,
1381, 1172, 1087, 1047 cm-1. 1H NMR (400 MHz, CDCl3): δ 1.30
and 1.36 (2s, 6H); 2.59 and 2.69 (2dd, J = 16.3, 6.8 and 16.7, 6.0 Hz,
1H, CH2); 2.81 and 2.90 (2dd, J=16.7, 6.8 and 16.3, 6.0 Hz, 1H,
CH2); 3.54 and 3.60 (2dd, J = 8.4, 6.6 Hz, 1H); 3.77-3.83 (m, 1H);
Route B. 2,6-Lutidine (1.6 mL, 13.74 mmol) and tetrabuty-
lammonium iodide (3.08 g, 8.33 mmol) were added to a solution
of 5 (1.21 g, 6.66 mmol) and 13 (1.37 g, 5.76 mmol) in 1,4-dioxane
J. Org. Chem. Vol. 75, No. 24, 2010 8513

Lorente et al.
JOCArticle
(12 mL). The reaction mixture was stirred at 40ꢀC for 15 min under
microwave irradiation. Purification by silica gel column chroma-
tography (95:5 to 80:20 hexane-EtOAc) yielded 10 (1.44 g, 74%) as
a yellow oil. IR (KBr film): ν 3350, 1705, 1692, 1521, 1286, 1225,
1045 cm-1. 1H NMR (400 MHz, CDCl3): δ 1.34 (s, 3H); 1.40 (s,
3H); 2.64 (dd, J = 16.2, 6.5 Hz, 1H, CH2); 2.94 (dd, J = 16.2, 6.5
Hz, 1H, CH2); 3.58 (dd, J = 8.4, 6.7 Hz, 1H); 3.76 (s, 3H, OMe);
3.89 (s, 3H, OMe); 4.08 (bd, 2H, CH2);4.18(dd, J=8.4and6.0Hz,
1H); 4.46-4.52 (m, 1H); 6.46 (d, J = 9.1 Hz, 1H); 7.02 (dd, J = 9.1
(q); 69.2 (t); 75.1 (d); 109.1 (s); 109.8 (s); 113.9 (d); 114.6 (d); 124.5
(d); 125.8 (d); 133.3 (s); 146.3 (s); 150.0 (s); 172.5 (s). MS (ESI-
TOF): 336 (M þ 1, 100); 337 (M þ 2, 23).
(Z )-3-Ethylidene-5-hydroxy-10-methoxy-1,2,3,4,5,6-hexahydro-
benzo[c][1,5]oxazecin-8-one (1). Z-15 (30 mg, 0.09 mmol) was
stirred with 4 M HCl (4 mL) for 30 min and then concentrated in
vacuo to yield (Z)-2-(2-ethylidene-4,5-dihydroxypentylamino)-
5-methoxybenzoic acid hydrochloride. The resulting residue was
used in the following step without further purification. A mixture of
and 3.1 Hz, 1H); 7.45 (d, J = 3.1 Hz, 1H); 7.99 (bt, 1H, NH). 13
C
EDC HCl (69 mg, 0.36 mmol), (Z)-2-[2-ethylidene-4,5-dihy-
3
NMR (100.6 MHz, CDCl3): δ 25.4 (q); 26.8 (q); 44.2 (t); 51.7 (q);
54.2 (t); 55.9 (q); 69.3 (t); 71.8 (d); 109.1 (s); 110.8 (s); 112.7 (d);
114.8 (d); 123.1 (d); 144.7 (s); 149.9 (s); 168.4 (s); 204.7 (s). MS (ESI-
TOF): 338 (M þ 1, 47); 675 (2M þ 1, 100). HRMS: m/z calcd for
C17H24NO6 338.1598, found 338.1603.
droxypentylamino]-5-methoxybenzoic acid hydrochloride, solid-
supported DMAP (18 mg, 0.09 mmol), and molecular sieves 4 A
(254 mg) in dry CH2Cl2 (20 mL) was stirred at room temperature
for 3 h. The crude mixture was filtered, treated with satd NH4Cl,
and then extracted with CH2Cl2. The combined organic extracts
were dried over MgSO4 and then concentrated in vacuo. Purifica-
tion by silica gel column chromatography (100:0 to 80:20 CH2Cl2/
MeOH) yielded Z-1asabrownishoil(9mg, 35%). IR(KBrfilm):ν
(Z/E )-Methyl 2-[2-((2,2-Dimethyl-1,3-dioxolan-4-yl)methyl)-
but-2-enylamino]-5-methoxybenzoate (Z/E-14). A 2.5 M solution
of BuLi in hexane (1.27 mL, 3.17 mmol) was added to a mixture of
ethyltriphenylphosphonium bromide (1.18 g, 3.17 mmol) in anhy-
drous THF (13 mL). The reaction mixture was stirred at room
temperature for 1 h and then cooled to -78 ꢀC. A solution of 10
(337 mg, 1.58 mmol) in anhydrous THF (3 mL) was added
dropwise to the reaction mixture. The reaction mixture was stirred
at -78 ꢀC for 30 min and subsequently allowed to warm to room
temperature for an additional 30 min. The solvent was removed in
vacuo, and the residue was purified by silica gel column chroma-
tography with hexane-EtOAc (95:5 to 80:20) to yield Z-14 and
E-14 as a yellowish oil (239 mg, 43%; 73:27 Z/E, as determined by
1HNMR).IR(KBrfilm):ν3373, 1689, 1517, 1222, 1065 cm-1.Z-14.
1H NMR (400 MHz, CDCl3): δ 1.33 (s, 3H); 1.40 (s, 3H); 1.74 (d,
J=6.9 Hz, 3H); 2.23-2.44 (m, 2H, CH2); 3.53 (dd, J=7.8 and 7.4
Hz, 1H); 3.76 (s, 3H, OMe); 3.82 (bs, 2H, CH2); 3.85 (s, 3H, OMe);
4.00 (dd, J=7.8 and 6.0 Hz, 1H); 4.19-4.26 (m, 1H); 5.56 (q, J=
6.9 Hz, 1H); 6.63 (d, J = 9.2 Hz, 1H); 7.03 (dd, J = 9.2 and 3.2 Hz,
1H); 7.42 (d, J=3.2 Hz, 1H). E-14: 1H NMR (100.6 MHz, CDCl3):
δ 1.35 (s, 3H); 1.43 (s, 3H); 1.65 (d, J = 6.9 Hz, 3H); 2.23-2.44 (m,
2H, CH2); 3.56 (dd, J=7.6 and 7.6 Hz, 1H); 3.75 (s, 3H, OMe); 3.82
(bs, 2H, CH2); 3.86 (s, 3H, OMe); 4.02-4.05 (m, 1H); 4.19-4.26
(m, 1H); 5.62 (q, J=6.9 Hz, 1H); 6.63 (d, J=9.2 Hz, 1H); 7.00 (dd,
J=9.2 and 3.1 Hz, 1H); 7.41 (d, J=3.1 Hz, 1H). 13C NMR (400
MHz, CDCl3): δ 13.4 (q); 25.7 (q); 27.0 (q); 32.6 and 42.8 (t); 40.0
(t); 51.5 (q); 56.0 (q); 69.3 (t); 75.0 (d); 108.9 (s); 112.8 and 114.1 (d);
114.4 (d); 123.3 (d); 125.4 (d); 133.0 (s); 133.7 (s); 146.4 (s); 149.4 (s);
168.6 (s). MS (ESI-TOF): 350 (M þ 1, 35); 372 (M þ Na, 45); 721
(2M þ Na, 100). HRMS: m/z calcd for C19H28NO5 350.1962,
found 350.1961.
(Z )-2-[2-((2,2-Dimethyl-1,3-dioxolan-4-yl)methyl)but-2-enyl-
amino]-5-methoxybenzoic Acid (Z-15). LiOH (310 mg, 7.39 mmol)
was added to a solution of Z/E-14 (258 mg, 0.74 mmol) in 75:25
H2O/THF (20 mL). The reaction mixture was sonicated at room
temperature for 1 h and then stirred at 50 ꢀC for 24 h. The crude
mixture was washed with Et2O. The aqueous phase was cooled to
0 ꢀC, acidified to pH 7 with 4 M HCl (1.7 mL), and extracted with
CH2Cl2. The combined organic extracts were dried over MgSO4
and then concentrated in vacuo to yield Z/E-15 (250 mg, quant;
73:27 Z/E, as determined by 1H NMR) as a yellowish oil. Purifica-
tion by semipreparative HPLC performed on a 15.5 g Redisep Gold
C18 (20-40 μm) column, with UV detection at 254 nm, a flow rate
of 18 mL/min, and H2O-CH3CN as eluents (gradient: 80:20 to
60:40 in 50 min), yielded Z-15 (80 mg, 72% recovery). IR (KBr
film): ν 3375, 2985, 1668, 1577, 1516, 1371, 1216, 1041 cm-1. 1H
NMR (400 MHz, CDCl3): δ 1.34 (s, 3H); 1.42 (s, 3H); 1.74 (d, J=
6.9 Hz, 3H); 2.27 (dd, J=14.3 and 5.7 Hz, 1H, CH2); 2.41 (dd, J=
14.3 and 7.1 Hz, 1H, CH2); 3.55 (t, J=8.0 Hz, 1H); 3.78 (s, 3H,
OMe); 3.86 (bs, 2H); 4.02 (dd, J=8.0 and 6.0 Hz, 1H); 4.20-4.27
(m, 1H); 5.58 (q, J=6.9 Hz, 1H); 6.69 (d, J=9.1 Hz, 1H); 7.08 (dd,
J = 9.1 and 3.1 Hz, 1H); 7.49 (d, J = 3.1 Hz, 1H). 13C NMR (100.6
MHz, CDCl3): δ 13.5 (q); 25.7 (q); 27.0 (q); 39.8 (t); 43.3 (t); 56.0
1
3397, 1640, 1498, 1436, 1292, 1228, 1039 cm-1. H NMR (400
MHz, CDCl3): δ 1.42 and 1.45 (2d, J = 6.9 Hz, 3H); 2.09-2.20 (m,
2H, CH2); 3.44-3.51 and 3.59-3.69 (2 m, 2H, CH2); 3.73 (s, 3H,
OMe); 3.79-3.90 (m, 1H); 4.35 (dd, J = 14.3 and 6.1 Hz, 1H,
CH2); 4.82 (dd, J = 14.3, 3.1 Hz, 1H, CH2); 5.51 and 5.53 (2q, J =
6.9 Hz, 1H); 6.72-6.76 (m, 2H); 6.92 (2d, J = 8.5, 1H). 13C NMR
(100.6 MHz, CDCl3): δ 13.4 (q); 38.2 and 38.9 (t); 46.9 and 47.1 (t);
55.5 (q); 66.2 and 66.8 (t); 70.4 and 70.9 (d); 111.5 and 111.6 (d);
116.5 (d); 127.0 and 127.1 (d); 127.9 and 128.4 (d); 130.2 and 130.5
(s); 130.7 (s); 136.1 and 136.2 (s); 158.9 (s); 169.1 and 169.2 (s). MS
(ESI-TOF): 299 (M þ Na, 13); 555 (2M þ 1, 100); 577 (2M þ Na,
100). HRMS: m/z calcd. for C30H38N2O8Na 577.2520, found
577.2521.
(2S,4R)-1-tert-Butoxycarbonyl-4-hydroxy-2-(trimethylsilyloxy-
methyl)pyrrolidine (17). TMSCl (0.65 mL, 4.85 mmol) was added
to a solution of N-Boc-trans-4-hydroxy-L-prolinol (1.054 g, 4.85
mmol), Et3N (0.67 mL, 4.85 mmol) in CH2Cl2 (25 mL). The
reaction mixture was stirred at 0 ꢀC for 16 h. After this time the
reaction mixture was washed with water, dried over MgSO4,
filtered, and the solvent was removed under reduced pressure.
Purification by silica gel column chromatography with hexane-
EtOAc (70:30 to 50:50) yielded 17 (1.05 g, 75%) as a colorless oil.
1
IR (KBr film) ν 3434, 1692, 1678, 1408, 1119 cm-1. H NMR
(400 MHz, CD3OD) δ 0.12 and 0.13 (2s, 9H); 1.50 and 1.51 (2s,
9H); 1.90-2.04 (m, 1H, CH2); 2.18 (dt, J = 17.2 and 5.8 Hz, 1H,
CH2); 3.35-3.48 (m, 2H, CH2); 3.63 and 3.65 (2d, J = 10.2 Hz,
1H, CH2); 3.78 and 3.90 (2dd, J = 10.2 and 4.6 Hz, 1H, CH2);
3.94-4.02 (m, 1H, CH); 4.38-4.44 (m, 1H, CH). 13C NMR (100.6
MHz, CD3OD) δ-0.5 (q); 28.8 (q); 37.3 and 38.2 (t); 55.9 and 56.4
(t); 58.8 and 58.9 (d); 63.6 and 64.6 (t); 70.1 and 70.6 (d) 80.8 and
81.1 (s); 155.3 (s). HRMS m/z calcd. for C13H28NO4Si 290.1782,
found 290.1784.
(S )-1-tert-Butoxycarbonyl-2-(trimethylsilyloxymethyl)pyrroli-
din-4-one (18). DMP (369 mg, 0.87 mmol) was added to a solu-
tion of 17 (228 mg, 0.78 mmol) in CH2Cl2 (10 mL), and the reaction
mixture was stirred at room temperature for 15 min. After this
time, satd NaHCO3 and satd Na2S2O3 were added, and the
reaction mixture was stirred for additional 10 min. The residue
was extracted with CH2Cl2, the combined organic extracts were
dried over MgSO4 and filtered, and the solvent was removed under
reduced pressure. Purification by silica gel column chromatography
with hexane-EtOAc (80:20) yielded 18 (182 mg, 82%) as a color-
less oil. IR (KBr film): ν 1765, 1701, 1401, 1106 cm-1. 1H NMR
(400 MHz, CD3OD): δ 0.12 (s, 9H); 1.54 (s, 9H); 2.41 (d, J=
18.0 Hz, 1H, CH2); 2.77-2.93 (m, 1H, CH2); 3.57-3.68 (m, 2H,
CH2); 3.86 (d, J=18.0 Hz, 1H, CH2); 3.99 (dd, J=22.1, 10.0 Hz,
1H, CH2); 4.38 and 4.40 (2bs, 1H, CH). 13C NMR (100.6 MHz,
CD3OD): δ -0.9 (q); 28.7 (q); 41.1 and 41.7 (t); 54.4 and 54.9 (t);
56.8 and 57.4 (d); 65.3 and 66.1 (t), 81.6 (s). HRMS: m/z calcd for
C13H26NO4Si 288.1626, found 288.1628.
8514 J. Org. Chem. Vol. 75, No. 24, 2010

Lorente et al.
JOCArticle
(S,Z )-1-tert-Butoxycarbonyl-4-ethylidene-2-((trimethylsilyloxy)-
methyl)pyrrolidine (Z-19). tBuOK (213 mg, 1.9 mmol) was added to
a solution of ethyltriphenylphosphonium bromide (705 mg, 1.9
mmol) in THF (5 mL), and the mixture was stirred for 1 h. After this
time, 18 (180 mg, 0.62 mmol) was added, and the mixture was
stirred for additional 30 min. Water was added, the residue was
extracted with Et2O, dried over MgSO4, and filtered, and the
solvent was removed under reduced pressure. Purification by silica
gel column chromatography with hexane-EtOAc (95:5) yielded Z/
E-19 (136 mg, 73%) in a 9:1 ratio as a colorless oil. IR (KBr film): ν
1702, 1397, 1251, 1109 cm-1. 1H NMR (400 MHz, CDCl3): δ 0.09
(s, 9H,); 1.48 (s, 9H); 1.58 (d, J = 6.8 Hz, CH3); 2.45-2.54 (m, 1H,
CH2); 2.56-2.67 (m, 1H, CH2); 3.20-3.45 (m, 1H, CH2); 3.55-
3.67 (m, 1H, CH2); 3.78-4.05 (m, 3H, CH þ CH2); 5.32-5.40 (m,
1H, CH). 13C NMR (100.6 MHz, CDCl3): δ -0.5 (q); 14.5 (q); 28.5
(q); 34.0 and 34.5 (t); 47.5 (t) 57.5 and 57.7 (d); 62.7 and 63.1 (t); 79.4
(s); 115.9 and 116.2 (d); 136.7 (s); 154.2 (s). HRMS: m/z calcd for
C15H30NO3Si 300.1989, found 300.1990.
2H); 5.32-5.42 (m, 1H, CH). 13C NMR (100.6 MHz, CDCl3) (E
diastereomer): δ -0.5 (q); 14.4 (q); 28.5 (q); 29.8 and 30.4 (t); 50.8
and 51.4 (t); 57.5 and 57.7 (d); 62.7 and 63.1 (t); 79.4 (s); 115.5 and
116.1 (d); 136.7 (s); 154.2 (s). HRMS: m/z calcd for C15H30NO3Si
300.1989, found 300.1990.
(S,E )-4-Ethylidene-2-(hydroxymethyl)pyrrolidine (E-20). A solu-
tion of Z/E-19 in 1:2 ratio (95 mg, 0.31 mmol) in 10% TFA in
CH2Cl2 (5 mL) was stirred at room temperature for 1 h. The solvent
was removed under reduced pressure, and the residue was purified
by silica gel column chromatography with CH2Cl2-MeOH(98:2to
90:10) to obtain Z/E-20 in a 1:2 ratio in quantitative yield. IR (KBr
film): ν 3380, 1677, 1435, 1135 cm-1. 1H NMR (400 MHz, CDCl3)
(E diastereomer): δ 1.63 (d, J = 6.7 Hz, 3H, CH3); 2.21-2.30 (m,
1H, CH2); 2.58-2.68 (m, 1H, CH2); 3.65-3.95 (m, 5H); 5.50-5.58
(m, 1H, CH). 13C NMR (100.6 MHz, CDCl3) (E diastereomer): δ
14.7 (q); 28.2 (t); 48.7 (t); 60.8 (t); 61.6 (d); 120.6 (d); 130.9 (s). 1H
NMR (400 MHz, CD3OD) (E diastereomer): δ 1.68 (d, J = 6.9 Hz,
3H, CH3); 2.40 (dd, J = 16.4 and 8.4 Hz, 1H, CH2); 2.75 (dd, J =
16.4 and 7.0 Hz, 1H, CH2); 3.60-3.98 (m, 5H); 5.55-5.67 (m, 1H,
CH). 13CNMR(100.6MHz, CD3OD) (Ediastereomer): δ14.8 (q);
29.1 (t); 50.1 (t); 61.2 (t); 62.7 (d); 121.0 (d); 133.1 (s). HRMS: m/z
calcd for C7H14NO 128.1069, found 128.1070.
(S,E )-N-(4-Hydroxy-3-methoxybenzoyl)-4-ethylidene-2-(hydro-
xymethyl)pyrrolidine (E-16). PyBOP (93 mg, 0.18 mmol) was
added to a solution of DIEA (64 μL, 0.37 mmol) and vanillic acid
(30 mg, 0.18 mmol) in THF (5 mL), and the mixture was stirred for
10 min. After this time, a solution of Z/E-20 in a 1:2 ratio (19 mg,
0.15 mmol) in THF was added, and the mixture was stirred at
room temperature for 1 h. The solvent was removed under reduced
pressure, and the residue was dissolved in EtOAc and washed
with satd NaHCO3 and satd NH4Cl. Purification by silica gel
column chromatography with EtOAc yielded Z/E-16 (25 mg,
60%) in a 1:2 ratio. Purification by semipreparative HPLC using
a Waters XBridge C18 column (10ꢀ100 mm, 5 μm), UV detec-
tion at 254 nm, with a flow of 3 mL/min, and H2O-CH3CN 18:82
as solvent system in isocratic conditions yielded E-16 (5.7 mg).
The 1H NMR and 13C NMR are identical as described for
the natural product in Table 2 in the Supporting Information.
[R]D = -51.2 (c 0.25, CH2Cl2). IR (KBr film): ν 3288, 1600, 1585,
1431, 1277, 1207 cm-1. 1H NMR (400 MHz, CDCl3): δ 1.62 (d,
J = 6.8, 3H, CH3); 2.20-2.35 (m, 1H, CH2); 2.67-2.77 (m, 1H,
CH2); 3.75 (bs, 2H, CH2); 3.90 (s, 3H, OMe); 4.00-4.22 (m, 2H,
CH2); 4.67 (bs, 1H, CH); 5.34 (bs, 1H, CH); 6.91 (d, J = 7.8 Hz,
1H, Ar); 7.04 (d, J = 7.8, 1H, Ar); 7.09 (s, 1H, Ar). 13C NMR
(100.6 MHz, CDCl3): δ 14.5 (q); 30.1 (t); 55.1 (t); 56.3 (q); 60.6 (d);
67.1 (t); 110.6 (d); 114.1 (d); 117.6 (d); 121.0 (d); 128.3 (s) 134.7 (s);
146.7 (s); 147.8 (s); 172.0 (s). HRMS: m/z calcd for C15H20NO4
278.1387, found 278.1387.
(S,Z )-4-Ethylidene-2-(hydroxymethyl)pyrrolidine (Z-20). A
solution of Z/E-19 in a 9:1 ratio (136 mg, 0.45 mmol) in 10%
TFA in CH2Cl2 (5 mL) was stirred at room temperature for 1 h.
The solvent was removed under reduced pressure, and the residue
was purified by silica gel column chromatography with CH2Cl2-
MeOH (98:2 to 90:10) to obtain Z/E-20 (105 mg) in a 9:1 ratio in
quantitative yield. IR (KBr film): ν 3380, 1677, 1435, 1135 cm-1
.
1H NMR (400 MHz, CDCl3): δ 1.60 (d, J = 8.0 Hz, 3H, CH3);
2.34-2.45 (m, 1H, CH2); 2.58-2.68 (m, 1H, CH2); 3.65-3.95 (m,
5H); 5.50-5.58 (m, 1H, dCH). 13C NMR (100.6 MHz, CDCl3): δ
14.7 (q); 32.2 (t); 45.8 (t); 60.5 (t); 61.4 (d); 120.6 (d); 130.9 (s).
HRMS: m/z calcd for C7H14NO 128.1069, found 128.1070.
(S,Z )-N-(4-Hydroxy-3-methoxybenzoyl)-4-ethylidene-2-(hy-
droxymethyl)pyrrolidine (Z-16). PyBOP (135 mg, 0.26 mmol) was
added to a solution of DIEA (0.1 mL, 0.55 mmol) and vanillic acid
(44 mg, 0.26 mmol) in THF (5 mL), and the mixture was stirred for
10 min. Then, a solution of Z/E-20 in a 9:1 ratio (26 mg, 0.21 mmol)
in THF was added, and the mixture was stirred for 1 h. The solvent
was removed under reduced pressure, and the residue was dissolved
in EtOAc and washed with satd NaHCO3 and satd NH4Cl. Purifi-
cation by silica gel column chromatography with EtOAc yielded
Z-16 (45 mg, 73%) in a 9:1 ratio as a colorless oil. [R]D=-21.5
(c 0.75, CH2Cl2). IR (KBr film): ν 3288, 1600, 1585, 1431, 1277,
1207 cm-1. 1H NMR (400 MHz, CDCl3): δ 1.60 (bs, 3H, CH3);
2.27-2.40 (m, 1H, CH2); 2.66-2.78 (m, 1H, CH2); 3.65-3.75 (m,
2H, CH2); 3.90 (s, 3H, OMe); 4.00-4.20 (m, 2H, CH2); 4.53-4.69
(m, 1H, CH);5.30-5.45 (m, 1H); 6.91 (d, J= 8.1 Hz, 1H); 7.05 (dd,
J = 8.1 and 1.8 Hz, 1H); 7.09 (d, J = 1.8 Hz, 1H). 13C NMR (100.6
MHz, CDCl3):δ14.5 (q); 34.1 (t); 51.5 (t); 56.1 (q); 59.9 (d); 66.4 (t);
110.4 (d); 114.0 (d); 117.7 (d); 120.7 (d); 128.0 (s); 134.4 (s); 146.6
(s); 147.6 (s); 172.0 (s). HRMS: m/z calcd for C15H20NO4 278.1387,
found 278.1387.
Acknowledgment. This study was partially supported by
CICYT (Grant No. CTQ 2009-07758), Generalitat de Cata-
lunya, and the Barcelona Science Park. We express our thanks
ꢀ
to Dr. F. Romero and Dr. R. Fernandez Chimeno, leaders of
our microbiological effort, for their excellent microbiological
(S,E )-1-tert-Butoxycarbonyl-4-ethylidene-2-(trimethylsilyloxy-
methyl)pyrrolidine (E-19). A 2 M solution of LDA in THF
(0.25 mL, 0.5 mmol) was added to a solution of 5-(ethylsulfonyl)-
1-phenyl-1H-tetrazole (119 mg, 0.5 mmol) in THF (4 mL) at -78 ꢀC,
andthe mixturewas stirred for 10 min. After thistime, 18(116 mg,
0.4 mmol) was added, and the mixture was stirred for an addi-
tional 30 min. The reaction mixture was quenched with satd
NH4Cl and extracted with CH2Cl2. The organic fractions were
dried over MgSO4 and filtered, and the solvent was removed
under reduced pressure. Purification by silica gel column chro-
matography with hexane-EtOAc (97:3) yielded Z/E-19 (58 mg,
50%) in a 1:2 ratio as a colorless oil. IR (KBr film): ν 1702, 1397,
1251, 1109 cm-1. 1H NMR (400 MHz, CDCl3) (E diastereomer):
δ 0.09 (s, 9H); 1.46 (s, 9H); 1.62 (d, J = 6.7 Hz, CH3); 2.41-2.52
(m, 1H, CH2); 2.54-2.66 (m, 1H, CH2); 3.20-3.45 (m, 1H, CH2);
3.55-3.65 (m, 1H, CH2); 3.72-3.80 (m, 1H, CH2); 3.87-4.05 (m,
ꢀ
work. We thank to Mr. Jose Antonio Alonso Garcı
´
a for
helpful technical assistance.
Supporting Information Available: General procedures;
tables of the bioactivity of isolated barmumycin and of its
diacetyl derivative; NMR data Tables 2 and 3; 1H and 13C
NMR spectra of compounds 6a-e, 7-14, 17, 18, (Z/E)-19, and
1
(Z/E)-20; and the H and 13C NMR spectra with two-dimen-
sional NMR experiments for compounds (Z)-15, (Z)-1, (Z)-16,
and (E)-16. This information is available free of charge via the
Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 75, No. 24, 2010 8515