Amaranzoles B?F, imidazole-2-carboxy steroids from the marine sponge phorbas amaranthus. C24- N - And C24- O -analogues from a divergent oxidative biosynthesis

Article abstract of DOI:10.1021/jo1000324

Five new steroidal imidazoles, amaranzoles B?F, were isolated from extracts of the marine sponge Phorbas amaranthus along with the known amaranzole A. The C24-N-(4-p-hydroxyphenyl)imidazol-5-yl constitution found in amaranzoles A, C, and D is replaced by a C24-O-(4-p-hydroxyphenyl)imidazole-2-carboxylate motif in amaranzoles B, E, and F. The structures were elucidated by interpretation of spectroscopic data. The C24 side chain configuration was assigned by synthesis of a model ester followed by chiroptical comparisons of its CD spectrum with that of an amaranzole B derivative.

Full text of DOI:10.1021/jo1000324

pubs.acs.org/joc
Amaranzoles B-F, Imidazole-2-carboxy Steroids from the Marine
Sponge Phorbas amaranthus. C24-N- and C24-O-Analogues from a
Divergent Oxidative Biosynthesis
Brandon I. Morinaka,† Joseph R. Pawlik,§ and Tadeusz F. Molinski*,†,‡
†Department of Chemistry and Biochemistry, and ‡Skaggs School of Pharmacy and Pharmaceutical Sciences,
University of California, San Diego, La Jolla, California 92093-0358, and Department of Biology and
§
Marine Biology, Center for Marine Science, University of North Carolina Wilmington, 5600 Marvin K. Moss
Lane, North Carolina 28403-3297
tmolinski@ucsd.edu
Received January 13, 2010
Five new steroidal imidazoles, amaranzoles B-F, were isolated from extracts of the marine sponge
Phorbas amaranthus along with the known amaranzole A. The C24-N-(4-p-hydroxyphenyl)imidazol-5-yl
constitution found in amaranzoles A, C, and D is replaced by a C24-O-(4-p-hydroxyphenyl)imidazole-2-
carboxylate motif in amaranzoles B, E, and F. The structures were elucidated by interpretation of
spectroscopic data. The C24 side chain configuration was assigned by synthesis of a model ester followed
by chiroptical comparisons of its CD spectrum with that of an amaranzole B derivative.
Introduction
sponge that, after more extensive fractionation of the polar
extracts, led to pure compounds and characterization of
five minor steroidal alkaloids, amaranzoles B-F (2-6) in
addition to 1. Amaranzoles C (3) and D (4) were simple
double bond isomers of dehydro-1, but to our surprise,
amaranzoles B (2), E (5), and F (6) differed from 1 by
replacement of the unique C24-N imidazolyl structure
with 24-O-steroidal 2-carboxyimidazolecarboxylate esters.
Because nonfused imidazole rings, excluding simple hist-
amines, are less common among natural products,4 the
discovery of the latter two constitutional variants in the
same organism is intriguing from the perspective of biogen-
esis. A biosynthetic hypothesis is proposed that sheds light
Extracts of the marine sponge Phorbas amaranthus were
highly deterrent in feeding assays using the bluehead wrasse,
Thalassoma bifasciatum.1 In the course of investigations to
identify the antifeedant constituents from P. amaranthus,
we previously reported the ring-A contracted steroids, phor-
basterones A-D,2 and an unusual N-imidazolyl sterol,
amaranzole A (1).3 Partial fractionation of extracts of P.
amaranthus and interpretation of results of bioassay-guided
separations, using laboratory and field fish feeding assays,
pointed to the aqueous MeOH extracts of P. amaranthus as
the location of the feeding deterrent components. Scarcity of
material necessitated recollection of larger amounts of the
(1) Pawlik, J. R.; Chanas, B.; Toonen, R. J.; Fenical, W. Mar. Ecol.: Prog.
Ser. 1995, 127, 183–194.
(2) Masuno, M. N.; Pawlik, J. R.; Molinski, T. F. J. Nat. Prod. 2004, 67,
731–733.
(3) Morinaka, B. I.; Masuno, M. N.; Pawlik, J. R.; Molinski, T. F. Org.
Lett. 2007, 9, 5219–5222.
(4) For example, isobromotopsentin: (a) Murray, L. M.; Lim, T. K.;
Hooper, J. N. A.; Capon, R. J. Aust. J. Chem. 1995, 48, 2053. For
comprehensive reviews of imidazole-containing natural products, see: (b)
Jin, Z. Nat. Prod. Rep. 2009, 26, 382-445 and earlier reviews in the series,
including (c) Lewis, J. R. Nat. Prod. Rep. 1996, 13, 435-467 and references
cited within.
DOI: 10.1021/jo1000324
r
Published on Web 03/17/2010
J. Org. Chem. 2010, 75, 2453–2460 2453
2010 American Chemical Society

Morinaka et al.
JOCArticle
TABLE 1. 1H NMR Data for Amaranzoles B-F (2-5) [CD3OD, 600 MHz δ (mult, J (Hz))]
no.
2
3
4
5
6
1
1.17 (m) ax;
2.47 (dd, 14.8, 3.2) eq.
4.90 (brq, 2.8) eq.
4.26 (dt, 12.3, 4.0) ax.
1.78 (q, 12.3) ax;
2.29 (brd, 13.4) eq.
1.31 (m) ax.
4.20 (td, 11.0, 4.5) ax.
1.00 (m) ax.
2.34 (dt, 12.4, 4.5) eq.
1.53 (m)
1.36 (m)
2.54 (dd, 14.6, 3.3)
4.91 (m)a
4.28 (dt, 12.1, 3.9)
1.83 (q, 12.6)
2.36 (brd, 13.2)
1.63 (m)
4.51 (m)
2.10 (m)
2.73 (dd, 17.3, 6.5)
1.32 (dd, 14.6, 3.0)
2.47 (dd, 14.6, 3.0)
4.92 (brq, 3.0)
4.30 (ddd, 12.5, 4.3, 3.6)
1.80 (q, 12.5)
2.32 (m)
1.45 (ddd, 12.5, 10.9, 3.0)
4.12 (td, 10.9, 5.4)
1.82 (brq, 12.5)
1.31 (m)
2.48 (14.6, 3.3)
4.93 (brq, 3.1)
4.30 (ddd, 12.5, 4.5, 3.4)
1.79 (brq, 12.5)
2.31 (m)
1.46 (ddd, 12.5, 10.8, 3.0)
4.12 (td, 10.8, 5.4)
1.83 (brq, 12.6)
1.32 (m)
2.56 (dd, 14.7, 3.2)
4.92 (brq, 2.9)
4.30 (dt, 12.0, 4.0)
1.84 (q, 12.7)
2.37 (brd, 12.8)
1.63 (m)
4.51 (m)
2.10 (m)
2.74 (dd, 17.8, 6.7)
2
3
4
5
6
7
3.12 (dd, 13.8, 5.4)
3.13 (dd, 13.6, 5.4)
8
9
11
0.68 (m)
1.71 (m)
1.55 (m)
1.59 (m)
1.11 (m)
1.91 (brd, 12.6)
1.72 (m)
1.60 (m)
1.34 (m) ax.
1.53 (m) eq.
1.13 (m) ax.
2.00 (m) eq.
1.30 (m)
1.08 (m)
1.59 (m)
1.22 (m)
1.84 (m)
1.13 (m)
0.68 (s)
1.10 (s)
1.46 (m)
0.96 (d, 6.0)
1.15 (m)
1.50 (m)
1.70 (m)
1.92 (m)
5.39 (dd, 8.0, 5.6)
2.12 (m)
2.15 (m)
12
1.36 (m)
1.97 (m)
2.07 (m)
1.30 (m)
1.62 (m)
1.06 (m)
1.71 (m)
1.06 (m)
0.54 (s)
1.15 (m)
1.97 (brd, 12.6)
1.41 (m)
2.02 (m)
2.12 (m)
1.32 (m)
1.62 (m)
1.33 (m)
1.93 (m)
1.21 (m)
0.65 (s)
14
15
2.22 (m)
2.29 (m)
1.22 (m)
1.62 (m)
1.05 (m)
0.78 (s)
1.97 (m)
2.27 (m)
1.38 (m)
1.86 (m)
1.15 (m)
0.87 (s)
16
17
18
19
20
21
22
1.27 (s)
0.96 (s)
0.97 (s)
1.28 (s)
1.28 (m)
0.90 (d, 6.2)
0.86 (m)
1.29 (m)
2.00 (m)
1.39 (m)
0.91 (d, 6.6)
0.90 (m)
1.31 (m)
1.94 (m)
2.00 (m)
4.50 (m)
1.56 (m)
1.00 (d, 6.6)
1.24 (m)
1.55 (m)
1.71 (m)
1.92 (m)
5.41 (dd, 7.8, 5.4)
1.50 (m)
1.00 (d, 6.5)
1.16 (m)
1.50 (m)
1.71 (m)
1.93 (m)
5.41 (dd, 7.8, 5.4)
23
24
25
26
4.51 (m)
4.94 (s)
5.07 (s)
1.82 (s)
4.67 (s)
4.97 (s)
1.73 (s)
7.68 (brs)
6.83 (brs)
4.69 (s)
4.96 (s)
1.71 (s)
7.79 (brs)
6.92 (brs
4.95 (s)
5.09 (s)
1.83 (s)
4.95 (s)
5.08 (s)
1.83 (s)
27
28
29
30
31
32
33
7.41 (s)
7.43 (brs)
7.46 (brs)
7.57 (d, 8.4)
6.80 (d, 8.4)
7.14 (d, 8.4)
6.85 (d, 8.4)
7.08 (d, 8.4)
6.70 (d, 8.4)
7.59 (d, 8.4)
6.81 (d, 8.4)
7.62 (d, 8.4)
6.83 (d, 8.4)
aObscured by solvent peak.
on a possible origin of the two families of amaranzoles that
unifies their C24-N and C24-O constitutional differences
through a common intermediate.
Results and Discussion
Two specimens of P. amaranthus were collected in
November 2006 from the Florida Keys. The first specimen
(06-04-004a) was extracted sequentially with water, MeOH,
and CH2Cl2. The aqueous MeOH partition was filtered
through C18 reversed-phase column followed by repeated
reversed-phase HPLC to give 1 (6.8 ꢀ 10-4 % dry wt)
and a new analogue, amaranzole B (2, 2.0 ꢀ 10-4
%
dry wt). Similar treatment of the second sponge specimen
(06-04-004b) gave amaranzoles C (3, 1.4 ꢀ 10-4 % dry
wt), D (4, 2.3 ꢀ 10-4 %), E (5, 6.3 ꢀ 10-5 %), and F (6, 3.3 ꢀ
10-5 %) in addition to 1 and 2. Characterization of the new
compounds on submilligram samples was carried out by FTIR,
MS, and 600 MHz NMR facilitated by a state-of-the-art
1.7 mm microcryoprobe with exceptionally high mass
sensitivity.5 Amaranzole B (2) was assigned the molecular
formula C37H49N2Na3O15S3 (m/z 903.2108, [M - Na]-,
FIGURE 1. Selected gHMBC correlations and 13C NMR chemical
shifts for amaranzole B (2).
Δ = þ1.8 mmu) based on negative-ion HRESIMS that corres-
ponded the addition of the elements of CO2 to 1, con-
sistent with a carboxy derivative of amaranzole A. 1H NMR,
1
COSY, and HSQC data of 2 revealed H and 13C chemical
(5) Molinski, T. F. Nat. Prod. Rep. 2010, 27, 321-329.
2454 J. Org. Chem. Vol. 75, No. 8, 2010
shifts for the sterol core that were essentially identical with

Morinaka et al.
JOCArticle
those of 1; however, significant differences were observed for
the side chain in the vicinity of the C24 substituent compared to
1. The H24 methine signal (δ 5.39, dd, J = 8.0, 5.6 Hz) of 2 was
significantly deshielded from that of 1 (δ 4.48, m) indicating
a change in the electronic environment.
FIGURE 2. Two constitutional isomers of steroidal imidazole-2-
carboxylate, 2.
Two isomeric constitutional formulas (Figure 2) are
possible for the proposed 4-(p-hydroxyphenyl)imidazole-
2-carboxyl-substituted sterol: a, a carboxylic acid with a
C24-N linkage in which the imidazole is bonded to the side
chain through a nitrogen atom and carboxylic ester b with a
C24-O linkage. Both chemical and spectroscopic evidence
pointed to b as follows.
The deshielded 1H NMR signal for H24 in 2 (δ 5.39, dd,
J = 8.0, 5.6 Hz, 1H) is more consistent with an esterified C24
carbinol than the CH-N bond of H24 in 1 (δ 4.48, m, 1H).3
Imidazole-2-carboxylic acids are prone to spontaneous dec-
arboxylation under neutral or acidic conditions,7 even at
room temperature, however, prolonged heating of 2 in
pyridine or DMSO (>120 °C) failed to expel CO2 and
returned only starting material or small amounts of desul-
fated analogues of 2 (1H NMR). This evidence suggested that
the structure of amaranzole B could not contain substructure
a but was an ester including substructure b. Evidence for the
latter was provided by a HMBC (Figure 2) which showed
a weak cross peak between H24 and the carbonyl signal
(δ 158.8, s, (CdO)O). The latter is consistent with the
carboxyl chemical shifts of known 2-carboxyimidazoles
(e.g, ethyl 1-methylimidazole-2-carboxylate, δ 159.3 ppm).8
The structure 2 was confirmed in the present work by direct
comparisons of the natural product with model compounds
of defined constitution and absolute configuration prepared
by asymmetric synthesis (see below).
The molecular formula of amaranzole C (3), C36H47-
N2Na3O13S3, derived from negative-ion HRESIMS data
(m/z 857.2036 [M - Na]- Δmmu þ0.6 amu), is two hydrogen
atoms less than the molecular formula of 1 suggesting a
dehydro analogue. This was confirmed by analysis of the 1H
and 2D NMR of 3 which revealed a tetrasubstituted C8,9
CdC double bond in the sterol core. 1H chemical shifts and
coupling constants of 3 showed the substitution as well as
relative stereochemistry were identical to 1 at positions
H1-H6; however, the 1H NMR signals for H7 (δ 2.10 and
2.73), H11 (δ 2.12), and H14 (δ 2.07 ppm) in amaranzole C
(3) were shifted downfield with respect to those of 1 (H7; δ
1.01 and 2.36 ppm; H11: δ 1.31 and 1.51 ppm; H14: δ 1.06
ppm). The location of the double bond at C8, C9 in 3 was
secured by a gHMBC correlation from Me-19 to C9.9
Compound 2 showed only one sp2 heteroaromatic 1H
signal (δ 7.41 ppm, Table 1) suggesting substitution or
oxidation at C28 or C29 of the imidazole ring. In addition,
the H24 methine signal in 2 was shifted downfield compared
to that of 1 (δ 4.48, m). Coupled HSQC of 2 showed the 1JCH
1
of the single heteroaromatic H-13C couplet (δ 7.41 ppm,
1J = 187.8 Hz) was smaller than that characteristically
associated with H2-C2 of five-membered ring azole hetero-
aromatics (1J ∼200 Hz). For example, N-methylimidazole
shows coupling constants of 1J = 205.7 Hz and 1J = 187.9
Hz for the H2-C2 and H4-C4 spin pairs, respectively.6
Thus, the heteroaromatic singlet (δ 7.41, s) was assigned
to C29, and the (CdO)O group was placed at C28 (the
depicted imidazole tautomer is arbitrary). Although direct
observation of the CdO signal (δ 158.8, s) by 13C NMR
was not possible due to insufficient signal-to-noise in the
mass-limited sample of 2, the signal was observed in HMBC
(Figure 1).
1
Comparison of the H NMR signals for allylic protons of
the Δ8 sterol pentasulfate ester, hamigerol B,10 gave a good
(7) Krowicki, K.; Lown, J. W. J. Org. Chem. 1987, 52, 3493–3501.
(8) Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1996, 118, 6141–6146.
(9) Me-19 gave a strong gHMBC correlation to a C9 (δ 137.3, s).
(10) Chang, J.-F.; Lee, J. -S.; Sun, F.; Jares-Erijman, E. A.; Cross, S.;
Rinehart, K. L. J. Nat. Prod. 2007, 70, 1195–1199.
(6) Pachler, K. G. R.; Pachter, R.; Wessels, P. L. Org. Magn. Reson. 1981,
17, 278–284.
J. Org. Chem. Vol. 75, No. 8, 2010 2455

Morinaka et al.
JOCArticle
match with 3. The side-chain linkage of 3 was shown to be of
the C24-N type found in 1 by similarity of 1H and 13C NMR
signals for H24 and C24.
SCHEME 1
Amaranzole D (4), C36H47N2N3O13S3, was isomeric with
1
amaranzole C (3). H and 2D NMR evidence showed that
the CdC double bond in 4 was isomerized to C8,C14. Allylic
proton signals H9 (δ 1.71, m, 1H) and H15 (δ 2.22, m; 2.29,
m) were cross-correlated (gHMBC) to sp2 carbons C8
(δ 125.0, d), and C14 (δ 146.3, d). Additional gHMBC cross
peaks were observed from H9 to C8 and from C18 methyl
group to C14. Amaranzole E (5) was the 8,14-dehydro
derivative of amaranzole B (2) based on HRESITOFMS
(m/z 901.1934, [M - Na]- Δmmu = 0) and nearly identical
1H and 13C chemical shifts for the sterol core of 4. Similarly,
amaranzole F (6) (m/z 901.1948 [M - Na]- Δmmu = þ1.4)
was assigned as the 8,9-dehydro derivative of amaranzole
1
B (2) and comparison of H, 13C NMR data with amaran-
zole C (3).
Since there were no precedents for the C24-N and C24-O
variants that constitute the two subfamilies of amaranzoles,
we elected to determine the stereochemistry of 2 and ascer-
tain the relationship to 1. The absolute configuration of
stereocenters within the sterol cores in 1-6 are known with
certainty from firm principles of sterol biosynthesis, but the
C24 stereocenter in 1 was more difficult to assign because it is
located three bonds removed from the nearest stereogenic
center, C21. To solve the C24 stereocenter in 1,3 we exploited
Cotton effects observable in the CD spectrum that arise from
exciton coupling of π-π* transitions of the 4-(p-
hydroxyphenyl)imidazolyl group and the terminal vinyl-
idene group.11 However, the significant bond reorganiza-
tions in the side-chains of 2, 5, and 6 with respect to 1
required an independent solution to the configuration of
C24 in the new compounds. We turned to chiroptical com-
parisons of 2 with a different synthetic analog of defined
configuration.
In order to simplify comparative CD analyses, 2 was
converted to the N,O-dimethyl compound 7 with excess
CH2N2. Synthetic N,O-dimethyl analogues (()-8 and (-)-
R-9 were prepared by coupling (EDCI, DMAP) of the
acid-sensitive imidazole-2-carboxylic acid 10 with the
corresponding allylic alcohols (()-11 and (þ)-12 which
were made available as follows. Methylation of ethyl thio-
oxamate12,13 with Meerwein’s salt14 (Scheme 1) followed by
condensation with 2-amino-1-(4-methoxyphenyl)ethanone
(13)15 gave imidazolecarboxylate ester 14. Methylation of
14 (CH2N2, Et2O-MeOH) proceeded with concomitant
transesterification to afford a mixture of methyl esters. The
major product 15a (64%) was separated from the minor
regioisomer 15b (32%) by silica chromatography. Base
hydrolysis of 15a provided imidazolecarboxylic acid 10.
The latter compound was prone to spontaneous decarboxy-
lation at acidic pHs, even at room temperature, and required
careful handling.
Addition of n-propylmagnesium bromide to methacrolein
gave racemic allylic alcohol (()-11, while the optically
enriched homologue, (þ)-R-12, was obtained by asymmetric
addition of diethylzinc to methacrolein in the presence of
(2R)-(þ)-3-exo-N-morpholinoisoborneol (16, (þ)-MIB).16
The optical purity and assignment of configuration of (þ)-
R-12 (93% ee) were secured by the modified Mosher’s
method17 after conversion of (þ)-R-12 to the corresponding
(R)- and (S)-MTPA esters.
Racemic (()-8 and (-)-R-9 displayed NMR chemical
shifts (Figure 3), close to those of 2 and 7, particularly H3
(H24 steroid numbering, δ 5.45, dd, J = 8.0, 5.6 Hz, 1H and
5.36, t, J = 6.8 Hz, 1H, respectively), but significantly
different from those of 1 (δ 4.48, m).3
(11) Morinaka, B. I.; Molinski, T. F. Chirality 2008, 20, 1066–1070.
(12) Scheibye, S.; El-Barbary, A. A.; Lawesson, S.-O. Tetrahedron 1982,
38, 3753–3760.
(13) (a) Oliver, J. E.; Sonnet, P. E. J. Org. Chem. 1973, 38, 1437–1438. (b)
Yamanaka, H.; Mizugaki, M.; Sakamoto, T.; Sagi, M.; Nakagawa, Y.;
Takayama, H.; Ishibashi, M.; Miyazaki, H. Chem. Pharm. Bull. 1983, 31,
4549–4553.
(14) (a) Meerwein, H.; Hinz, G.; Hofmann, P.; Kroning, E.; Pfeil, E. J.
Prakt. Chem. 1937, 147, 257–285. (b) Meerwein, H.; Bettenberg, E.; Gold,
H.; Pfeil, E.; Willfang, G. J. Prakt. Chem. 1940, 154, 83–156.
(15) Holub, J. M.; O’Toole-Colin, K.; Getzel, A.; Argenti, A.; Evans,
M. A.; Smith, D. C.; Dalglish, G. A.; Rifat, S.; Wilson, D. L.; Taylor, B. M.;
Miott, U.; Glersaye, J.; Lam, K. S.; McCranor, B. J.; Berkowitz, J. D.; Miller,
R. B.; Lukens, J. R.; Krumpe, K.; Gupton, J. T.; Burnham, B. S. Molecules
2004, 9, 135–157.
As reported previously,3,11 the dominant feature in the CD
spectrum of 1 and, by extension, 2 and its dimethyl homo-
logue 7, is associated with a π-π* transition of imidazole
chromophore; the sign and magnitude of this Cotton effect
are dependent upon the configuration at C24. The CD
spectra of 7 and (-)-R-9 (Figure 4) showed the same weak
(16) (a) Nugent, W. Chem. Commun. 1999, 1369–1370. (b) Kelly, A. R.;
Lurain, A. E.; Walsh, P. J. J. Am. Chem. Soc. 2005, 127, 14668–14674.
(17) Kusumi, T.; Hamada, T.; Ishitsuka, M. O.; Ohtani, I.; Kakisawa, H.
J. Org. Chem. 1992, 57, 1033–1035.
2456 J. Org. Chem. Vol. 75, No. 8, 2010

Morinaka et al.
JOCArticle
FIGURE 3. 1H NMR (CD3OD, 600 MHz) of (a) 7, (b) (()-8, and (c) (-)-9.
Cotton effect at λ 221 nm (Δε -2.8 and Δε -2.9, respectively);
therefore, 2 and 7 have 24R configuration.
The new compounds, together with 1, derive from a
polar fraction that exhibits significant feeding deterrence
in assays using the blue head wrasse, Thalasoma bifascia-
tum, a common predatory reef fish. We chose to test
whether amaranzoles were responsible for this acti-
vity. Two mixtures of amaranzoles, one containing 1, 3,
and 4, and the other containing 2, 5, and 6, were tested
in fish feeding assays using a protocol we reported
previously.1 The mixture containing 1, 3, and 4 elicited
deterrent activity when tested at 16x natural concentra-
tion. We conclude that the amaranzoles are minor con-
tributors to the chemical defense of P. amaranthus against
T. bifasciatum.18
The structures of 1-6 suggest the involvement of a
common intermediate in their biosynthesis. Formation of
the p-hydroxyphenylimidazole side chain may commence
by coupling of an allylic alcohol (steroid side chain) and a
suitable imidazole precursor, for example, hamigeramine
isolated from the marine sponge Hamigera hamigera.19
Oxidative modification at C24 of the side chain is evident
in the two structural subfamilies represented by amaran-
zoles A-F; however, the biosynthesis of 1, 3, and 4
clearly diverges from that of 2, 5, and 6. Because all six
natural products occur together in the sponge consis-
tently, it is most likely their biosynthesis links the C24-N
and C24-O subfamilies through a common pathway. One
FIGURE 4. CD spectra of N,O-dimethylamaranzole B (7) and
model compound (-)-R-9 (MeOH, 23 °C).
possibility (Figure 5) invokes two allylic rearrangements of
imidazole 2: to give the primary allylic ester i that further
rearranges to the transient imidazolinium-2-carboxylic
acid ii with formation of a C-N bond. Facile loss of CO2
from ii would give a relatively stable carbene iii (4-sub-
stituted imidazole-2-ylidene) which undergoes 1,2-hydride
(18) The deterrent compounds, including amaroxocanes A and B segre-
gated into a different polar extract prepared from P. amaranthus and are the
subjects of an earlier report. Morinaka, B. I.; Pawlik, J. R.; Molinski, T. F. J.
Nat. Prod. 2009, 72, 259–264.
(19) Hassan, W.; Edrada, R.; Ebel, R.; Wray, V.; Proksch, P. Mar. Drugs.
2004, 2, 88–100.
J. Org. Chem. Vol. 75, No. 8, 2010 2457

Morinaka et al.
JOCArticle
Conclusions
We have isolated and structurally characterized five new
amaranzoles B-F (2-6) that belong to two related families
with different side-chain bond constitutions incorporating
p-hydroxyphenylimidazole. Structure elucidation of 2, 5,
and 6 revealed an unexpected C-O bond at C24 instead of
the C-N imidazole found in the other amaranzoles A (1), C
(3), and D (4). The two amaranzole families may be linked
through allylic rearrangements that interchange C24-N and
C24-O bonds with concomitant loss of CO2. The polar
sulfated 1 showed no activity against HCT-116 tumor cells;
however, the more liphophilic derivative 17 was cytotoxic
(IC50 = 4.4 μg/mL).
Experimental Section
General Experimental Procedures. 1H and 2D NMR spectra
were acquired using a 600 MHz NMR spectrometer equipped
with a {13C}1H 1.7 mm microcryoprobe or 500 MHz spectro-
meters {13C}1H and {1H}13C probes. All 1H spectra were
acquired in MeOH-d4 and referenced to δ 3.31 ppm. UV spectra
were recorded on a double-beam spectrophotometer. Optical
rotations were measured using a digital polarimeter. Circular
dichroism (CD) measurements were acquired on a grating
spectropolarimeter using dilute solutions in spectroscopic-grade
solvent and quartz cells (1 mm path length). IR spectra were
obtained using an FTIR spectrometer equipped with a ZnSe
ATR plate. HPLC was carried out using a dual-pump prepara-
tive instrument equipped with a high-dynamic range UV-vis
detector set to λ 260 nm. For semipreparative HPLC, an RP
column (5 μm C18-bonded silica, 10 ꢀ 250 mm) was used.
Preparative HPLC was carried on radial compression cartridges
(6 μm C18-bonded silica 25 ꢀ 100 mm) and commercial HPLC
grade solvents were used for liquid chromatography. THF,
CH2Cl2, and DMF were dried by passage through dual-alumina
cartridges under an atmosphere of Ar, and reagent-grade che-
micals were used as purchased.
Animal Material. Extraction and Isolation. P. amaranthus
Duchassaing & Michelotti 1864 was collected in November
2006 using scuba from Dry Rocks Reef, Key Largo, Florida
(25°7.8500 N, 80°17.5210 W), at a depth of 20-250. Specimens
were identified by J.R.P and stored at -20 °C until required.
Vouchers samples are archived at UCSD. Two specimens (06-
04-004a and 06-04-004b) of P. amaranthus collected at the same
site were analyzed separately. The first sample (06-04-004a,
88.3 g) was lyophilized, extracted with water (1 L), followed
by MeOH (1 L), and finally CH2Cl2 (1 L). The organic extracts
were combined, dried, and partitioned between hexane and
H2O/MeOH 1:9. The hexane layer (“A”) was separated, and
the MeOH layer was adjusted to 1:1 by addition of water and
then partitioned against CH2Cl2. The CH2Cl2 layer (“B”) was
separated from the aqueous MeOH layer (“C”), and the solvents
were removed under reduced pressure. The crude MeOH extract
C (33.5 g) was subjected to filtration through reversed-phase
silica (C18 cartridge, conditioned with H2O/MeOH 19:1) eluting
with water and then MeOH/CH3CN 1:1 to give two fractions.
The second fraction (7.03 g, 1.46 g further purified) was then
subject to reversed-phase chromatography (C18 cartridge) using
a stepwise gradient (H2O/MeOH 4:1, 7:3, 3:2, 1:1, 2:3, 3:7, 1:4,
1:9, then MeOH) to attain nine fractions. A portion (42.5 mg) of
the fraction (127.3 mg) eluting with H2O/MeOH (3:2) was
subject to gradient semipreparative reversed-phase HPLC
(flow rate: 2.5 mL/min; mobile phase: H2O/CH3CN containing
0.5 M NaClO4; gradient: 7:3 isocratic 15 min to 2:3 over 30 min;
λ = 254 nm) to give five fractions. The third fraction contained
amaranzole A (4.2 mg, 1). The fourth fraction was subjected to
FIGURE 5. Possible biosynthetic pathway linking amaranzoles
A (1) and B (2).
migration20 to restore the aromatic imidazole ring of 1.21
The enthalpic cost of replacing the stronger C-O bond
with a C-N bond would be paid in part by the energeti-
cally favorable loss of CO2.
Finally, it is noteworthy that the 24R configuration is
retained in all amaranzoles. If the allylic alcohol precursor to
5 (Figure 5) is formed with high stereochemical fidelity,
the subsequent interconversion of the two subfamilies of
amaranzoles must proceed through a tightly orchestrated,
enantiospecific mechanism with retention of configuration.
Although insufficient amounts of amaranzoles B-F (2-6)
were available for assay of biological activity, a brief evalu-
ation cytotoxicity of amaranzole A (1) and the less-polar
analogue 17 (obtained by acid hydrolysis of 1; 3 M
HCl-MeOH, 75 °C) toward human colon tumor cells
(HCT-116) was carried out. Compound 17 exhibited signi-
ficant cytotoxicity (IC50 = 4.4 μg/mL); however, the natural
product 1 was essentially inactive (IC50 > 32 μg/mL).
Presumably, cell permeability of 1 is restricted by the highly
charged sulfate groups, which is relaxed in the more lipo-
philic parent molecule 17.
(20) The carbene, 2,3-dihydroimidazole-2-ylidene, is calculated to be 26.3
kcal mol-1 higher in energy than its isomer, imidazole. (a) Maier, G.;
3
Endres, J. Eur. J. Chem. 1998, 1571–1520. (b) Freeman, F.; Lau, D. J.; Patel,
A. R.; Pavia, P. R.; Willey, J. D. J. Phys. Chem. A 2008, 112, 8775–8784.
(21) (a) Arduengo, A. J. III. Acc. Chem. Res. 1999, 32, 913–921. (b)
Boehme, C.; Frenking, G. J. Am. Chem. Soc. 1996, 118, 2039–2046. (c)
Heinemann, C.; Muller, T.; Apeloig, Y.; Schwartz, H. J. Am. Chem. Soc.
1996, 118, 2023–2038.
2458 J. Org. Chem. Vol. 75, No. 8, 2010

Morinaka et al.
JOCArticle
gradient semipreparative reversed-phase HPLC (same condi-
tions as above, except mobile phase: H2O/CH3CN containing
0.75 M NaClO4) to give amaranzole B (1.2 mg, 2).
HRESITOFMS m/z 901.1948 [M - Na]- calcd for C37H47-
N2Na2O15S3, 901.1934).
N,O-Dimethylamaranzole B (7). Excess ethereal diazo-
methane was added dropwise to a mixture of amaranzoles B,
E, and F (∼1:1:1, 1.5 mg) in 0.5 mL of MeOH. The mixture was
stirred at room temperature for 1.5 h. The solvent was evapo-
rated under a stream of N2. The mixture was subject to three
rounds of reversed-phase HPLC; first with a gradient of
10-100% (CH3CN/H2O þ 1 M NaClO4, over 40 min) followed
by two passages with 50-100% (CH3CN/H20 þ 1 M NaClO4
over 40 min) to give 7 (ca. 30 μg, based on comparison of UV to
(-)-9): UV (MeOH) λmax 261 nm (ε 16200), 286 nm (ε 15400),
A second lyophilized sample of P. amaranthus (06-04-004b,
131.2 g) was extracted with 1 L of H2O/MeOH 1:1 (23 °C,
overnight). The aqueous-MeOH phase was removed, and
extraction was repeated twice, first with 1 L H2O/MeOH 1:1
and then 1 L MeOH. The sponge tissue was then blended at high
speed and extracted once more with 1 L of MeOH (23 °C,
overnight), and the combined extracts were concentrated under
reduced pressure. The remaining sponge tissue was extracted
with CH2Cl2 (2 ꢀ 1 L). The CH2Cl2 extract was dried and
partitioned between hexane and H2O/MeOH 1:9. The aqueous
MeOH layer was dried and combined with the previous MeOH
extracts to give a crude aqueous MeOH extract (48.6 g). A
portion (33.9 g) of the aqueous MeOH crude extract was filtered
through a reversed-phase cartridge (C18, conditioned with H2O/
MeOH 19:1) eluting with H2O/MeOH 9:1, H2O/MeOH 1:9, and
i-PrOH to give three fractions. A portion (1.16 g) of the second
eluting fraction (5.82 g) was subjected to gradient preparative
reversed-phase HPLC (flow rate: 25 mL/min; mobile phase:
H2O/CH3CN containing 1.5 M NaClO4; gradient: 73:27 iso-
cratic 10 min to 23:77 over 30 min; λ=240 nm) to give seven
fractions. A portion (4.6 mg) of the third fraction (61.6 mg) was
subjected to a repeated gradient reversed-phase HPLC (flow
rate: 2.5 mL/min; mobile phase: H2O/CH3CN containing 0.5 M
NaClO4; gradient: 7:3 isocratic 15 min to 2:3 over 30 min; λ =
254 nm) to give amaranzole C (0.19 mg, 3), amaranzole D (0.32
mg, 4), and amaranzole A (0.35 mg, 1). A portion (1.4 mg) of the
fourth fraction (8.4 mg) was subjected to repeated gradient
reversed phase C18 HPLC (H2O/CH3CN containing 1.5 M
NaClO4, flow rate: 2.5 mL/min; gradient: 61:39 isocratic
15 min to 31:69 over 30 min; λ = 260 nm) to give amaranzole
F (0.10 mg, 6), amaranzole E (0.19 mg, 5), and amaranzole B
(0.15 mg, 2).
1
309 nm (ε 8600); CD (MeOH) 221 nm (Δε -2.8); H and 13C
NMR chemical shifts for the steroidal ABCD ring system are
nearly identical to those of amaranzole B (1); 1H NMR
(600 MHz, CD3OD) δ 7.73 (d, 2H, 8.8 Hz), 7.61 (s, 1H), 6.95
(d, J = 8.8 Hz, 2H), 5.41 (dd, J = 8.0, 5.4 Hz, 1H), 5.08 (m, 1H),
4.90 (m, 1H), 4.05 (s, 3H), 3.82 (s, 3H), 1.84 (s, 3H); 13C NMR
(150 MHz, CD3OD, partial data) δ 160.2 (C34), 144.2
(C25), 142.6 (C30), 137.2 (C28), 127.4 (C32), 126.6 (C31),
123.4 (C29), 114.7 (C33), 113.6 (C26), 79.9 (C24), 55.4 (OMe),
36.5 (NMe).
Ethyl 4-(4-Methoxyphenyl)-1H-imidazole-2-carboxylate (14).
Trimethyloxonium tetrafluoroborate (3.31 g, 22.4 mmol) was
added to a stirred solution of ethyl thiooxamate12 (2.59 g,
19.4 mmol) in CH2Cl2 (60 mL) in three portions over 1 h at
room temperature. After the mixture was stirred for an addi-
tional 30 min, the solvent was evaporated and the crude product
used immediately in the next step. Sodium acetate (3.18 g,
38.8 mmol), 2-amino-1-(4-methoxyphenyl)ethanone (13, 6.5 g,
19.3 mmol), and acetic acid (60 mL) were added, and the mixture
was stirred for 3 h at 100 °C. The mixture was allowed to cool to
room temperature, and the solvent was removed under reduced
pressure. Water (400 mL) was added, and the mixture was
extracted with ethyl acetate (3 ꢀ 100 mL). The combined
organic extracts were dried (MgSO4), and the solvent was
evaporated. The crude product was recrystallized from
MeOH/H2O to give pure 14 (4.18 g, 87% yield): mp 132-
134 °C; FTIR (ATR, ZnSe) νmax 2981, 2938, 2837, 1715, 1615,
1479, 1458, 1441, 1379, 1288, 1248, 1178, 1150, 1131, 1027,
795 cm-1; 1H NMR (400 MHz, CDCl3 þ 0.1% TFA-d) δ 7.67
(d, J = 8.4 Hz, 2H), 7.42 (s, 1H), 6.92 (d, J = 8.4 Hz, 2H), 4.42
(q, J = 7.2 Hz, 2H), 1.38 (t, J = 7.2 Hz, 3H); 13C NMR (100
MHz, CDCl3þ0.1% TFA-d) δ 159.6 (C), 158.6 (C), 139.5 (C),
136.8 (C), 126.9 (CH), 122.9 (C), 120.4 (CH), 114.2 (CH), 62.2
(CH2), 55.2 (CH3), 14.0 (CH3); HRESITOFMS m/z 247.1079
[M þ H]þ (calcd for C13H15N2O3, 247.1077)
Amaranzole B (2): colorless glass; [R]21 þ21.8 (c 0.52,
D
MeOH); FTIR (ATR, ZnSe) νmax 3487, 2945, 1652, 1449,
1232, 1065, 1001, 915, 839 cm-1; UV (H2O-CH3CN 7:3) λmax
256 nm (7800), 289 (9300), 310 (8000); CD (H2O-CH3CN 7:3)
218 nm (Δε -1.0); 1H NMR, see Table 1; 13C NMR (from 600
MHz, HSQC/gHMBC data, CD3OD) δ 160.2 (C34), 158.8
(C35), 144.3 (C25), 142.2 (C30), 138.4 (C28), 127.5 (C32),
122.5 (C31), 120.6 (C29), 116.9 (C33), 113.3 (C26), 79.6 (C24),
78.5 (C3), 77.9 (C6), 76.2 (C2), 56.9 (C17), 55.2 (C9), 51.6 (C5),
43.4 (C13), 42.2 (C1), 40.6 (C12), 39.6 (C7), 37.8 (C10), 36.1
(C20), 34.8 (C8), 32.3 (C22), 29.9 (C14), 29.9 (C23), 28.5 (C16),
25.4 (C4), 24.3 (C15), 21.8 (C11), 18.6 (C21), 17.9 (C27), 15.6
(C19), 11.8 (C18); HRESITOFMS m/z 903.2108 [M - Na]-
calcd for C37H49N2Na2O15S3, 903.2090.
Methyl 4-(4-Methoxyphenyl)-1-methyl-1H-imidazole-2-car-
boxylate (15a). A solution of ethereal diazomethane (∼0.2 M)
was added dropwise to a stirred solution of 5 (100 mg, 0.406
mmol) in MeOH/Et2O (8:3, 5.5 mL) at room temperature. When
a yellow color persisted, excess diazomethane (∼10 equiv) was
added, and the reaction was stirred for 24 h. The solvent was
evaporated under a stream of nitrogen, and the mixture was
subjected to silica flash chromatography (1:4 EtOAc/hexanes)
to give 15a (64 mg, 64% yield) and 15b (34 mg, 34% yield).
Methyl 4-(4-Methoxyphenyl)-1-methyl-1H-imidazole-2-car-
boxylate (15a): FTIR (ATR, ZnSe) νmax 2952, 2838, 1709,
1613, 1443, 1419, 1248, 1179, 1151, 1116, 1047, 1024, 930,
Amaranzole C (3): colorless glass; FTIR (ATR, ZnSe) νmax
3454, 2947, 1631, 1230, 1110, 1002, 949 cm-1; UV (H2O/
CH3CN 7:3) λmax 250 nm (ε 8300); CD (H2O-CH3CN 7:3)
201 nm (Δε þ3.5), 217 (Δε -8.0); 1H NMR, see Table 1;
HRESITOFMS m/z 857.2036 [M - Na]- calcd for C36H47-
N2Na2O13S3, 857.2030.
Amaranzole D (4): colorless glass; FTIR (ATR, ZnSe) νmax
3487, 2951, 1651, 1450, 1327, 1230, 1072, 1001, 964, 916, 840
cm-1; UV (H2O-CH3CN 7:3) λmax 250 nm (ε 8300); CD
(H2O-CH3CN 7:3) λ 201 nm (Δε þ4.7), 215 (Δε -1.9); 1H
NMR, see Table 1; HRESITOFMS m/z 857.2051 [M - Na]-
calcd for C36H47N2Na2O13S3, 857.2030.
832 cm-1 1H NMR (400 MHz, CDCl3) δ 7.72 (d, 2H, 8.8
;
Hz), 7.23 (s, 1H), 6.91 (d, 2H, 8.8 Hz), 4.04 (s, 3H), 3.96 (s, 3H),
3.82 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 161.6 (C), 161.2 (C),
143.9 (C), 138.0 (C), 128.5 (CH), 127.7 (C), 123.4 (CH), 115.9
(CH), 57.2 (CH3), 54.3 (CH3), 38.0 (CH3); HRESITOFMS m/z
247.1080 [M þ H]þ (calcd for C13H15N2O3, 247.1077).
Amaranzole E (5): colorless glass; FTIR (ATR, ZnSe) νmax
3559, 2952, 1651, 1453, 1386, 1232, 1113, 1001, 918, 843 cm-1
;
1H NMR, see Table 1; HRESITOFMS m/z 901.1941 [M - Na]-
calcd for C37H47N2Na2O15S3, 901.1934.
Methyl 5-(4-Methoxyphenyl)-1-methyl-1H-imidazole-2-car-
boxylate 15b: FTIR (ATR, ZnSe) νmax 2952, 1711, 1453, 1290,
1252, 1201, 1129, 1029, 949, 839, 789 cm-1; 1H NMR (400 MHz,
Amaranzole F (6): colorless glass; FTIR (ATR, ZnSe) νmax
3523, 2948, 1651, 1451, 1233, 1069, 1002, 915, 841 cm-1
;
J. Org. Chem. Vol. 75, No. 8, 2010 2459

Morinaka et al.
JOCArticle
CDCl3) δ 7.30 (d, J = 8.0 Hz, 2H), 7.14 (s, 1H), 6.98 (d, J = 8.0
Hz, 2H), 3.94 (s, 3H), 3.91 (s, 3H), 3.85 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 160.1 (C), 159.9 (C), 138.5 (C), 136.7 (C), 130.6
(CH), 128.5 (CH), 120.8 (C), 114.3 (CH), 55.3 (CH3), 52.1
(CH3), 33.7 (CH3); HRESITOFMS m/z 247.1076 [M þ H]þ
(calcd for C13H15N2O3, 247.1077).
1H), 4.03 (s, 3H), 3.81 (s, 3H), 1.95-1.80 (m, 2H), 1.83 (s, 3H),
0.98 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 160.8
(C), 159.4 (C), 144.3 (C), 143.0 (C), 137.7 (C), 127.7 (CH), 127.0
(C), 123.7 (CH), 115.0 (CH), 114.0 (CH2), 81.5 (CH), 55.7
(CH3), 36.7 (CH3), 26.7 (CH2), 18.4 (CH3), 10.4 (CH3); HRE-
SITOFMS m/z 315.1703 [MþH]þ (calcd for C18H23N2O3,
315.1703)
4-(4-Methoxyphenyl)-1-methyl-1H-imidazole-2-carboxylic Acid
(10). A solution of LiOH (10.4 mg, 0.248 mmol) in THF/H2O (8:3,
3.2 mL) was added to ester 15a (43 mg, 0.165 mmol), and the
mixture was stirred overnight at room temperature. The THF was
evaporated under reduced pressure, and the solution was cooled
to0 °C. Aqueous HCl (0.1 M) was added dropwise until pH ∼4, at
which point the free acid precipitated as a white solid. The mixture
was centrifuged, the supernatant removed, and the solid washed
with H2O (2 mL) and dried under high vacuum to give the free acid
10 (30.3mg, 79%) as a colorless solid. 10: FTIR (ATR, ZnSe) νmax
2909, 2834, 1659, 1327, 1270, 1254, 1023, 910, 811, 796 cm-1; 1H
NMR (400 MHz, DMSO-d6) δ 7.81 (s), 7.72 (d, J = 8.8 Hz, 2H),
6.97 (d, J = 8.8 Hz, 2H), 3.95 (s, 3H), 3.77 (s, 3H); 13C NMR (100
MHz, DMSO-d6) δ 159.3 (C), 158.7 (C), 138.3 (C), 137.5 (C),
126.0 (CH), 125.1 (C), 121.8 (CH), 114.1 (CH), 55.1 (CH3), 35.7
(CH3); HRESITOFMS m/z 233.0923 [M þ H]þ (calcd for
C12H13N2O3, 233.0921).
(R)-2-Methylpent-1-en-3-ol (þ)-12. Diethylzinc (8 mmol, 1 M
in hexanes) was added to a stirred solution of (þ)-MIB (48.5 mg,
0.2 mmol) in hexanes (20 mL) at 0 °C. Freshly distilled metha-
crolein (280 mg, 4 mmol) was added dropwise to the reaction and
stirred for 8 h at 0 °C. The reaction was quenched with saturated
ammonium chloride (40 mL) and extracted with pentane (3 ꢀ
100 mL). The combined organic layers were dried by filtration
through MgSO4, and filtered, and the solvent evaporated under
reduced pressure at 0 °C. The mixture was subjected to silica flash
chromatography (1:9 diethyl ether/pentane) to give (þ)-12
(220 mg, 55% yield, 93% ee): [R]24 = þ4.2 (c 1.0, CHCl3)
D
[lit.22b þ4.1 (CH2Cl2, 90% ee)]; (-)-12, [R]24 = -5.6 (c 1.0,
D
1
CHCl3, >98% ee).22a The % ee was determined by H NMR
analysis of the both (þ) and (-)-MTPA esters. 1H and 13C NMR
data were identical to literature values.22
Desulfation of Amaranzole A (1). A solution of amaranzole A
(1, 1 mg) was heated in 3 M HCl (MeOH-H2O, 2 mL) at 75 °C
for 45 min. The cooled solution was concentrated and passed
through a SiO2 cartridge, which was which was eluted with
MeOH-CH2Cl2 to give the desulfated compound 17 (quant):
1H NMR (500 MHz, CD3OD) (selected) δ 3.93 (brq, J = 4.0 Hz,
H2), 3.50 (dt, J = 10.9, 4.0 Hz, H3), 3.39 (td, J = 10.9, 4.6 Hz,
H6), 1.72 (s, H27), 1.00 (s, H19), 0.92 (d, J = 6.9 Hz, H21), 0.63
(s, H18). The remainder of the 1H NMR signals were essentially
identical to those of 1. HRESITOFMS: m/z 577.3996 [M þ H]þ,
calcd for C36H53N2O4 577.4005.
Cytotoxicity Assay. Amaranzole A (1) and the des-sulfato
derivative 17 were evaluated for in vivo cytotoxicity against
cultured human colon tumor cells (HTC-116) using a cell
viability assayed based on a colorimetric end point of the soluble
formazan dye from coincubated MTS [(3-(4,5-dimethylthiazol-
2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-
zolium, inner salt] as described elsewhere.23 Amaranzole
A (1) was inactive (IC50 >32 μg/mL), and 17 gave an IC50 of
4.4 μg/mL.
2-Methylhex-1-en-3-yl 4-(4-Methoxyphenyl)-1-methyl-1H-
imidazole-2-carboxylate (8). A solution of EDCI (30.0 mg,
0.156 mmol) in CH2Cl2 (3 mL) was added dropwise to a stirred
solution of the 10 (21.1 mg, 0.091 mmol), (()-2-methylhex-1-en-
3-ol22 (26 mg, 0.228 mmol), and DMAP (2.4 mg, 0.020 mmol) in
CH2Cl2 (2 mL) at 0 °C. The mixture was stirred for an additional
2 h and then at room temperature for 18 h before removal of
solvent under reduced pressure and purification of the residue
by silica flash chromatography (15:85 EtOAc/hexanes) to give
the ester (()-8 (22.1 mg, 74% yield): FTIR (ATR, ZnSe) νmax
1
2958, 1707, 1450, 1281, 1262, 1123, 1105, 836, 786 cm-1; H
NMR (400 MHz, CDCl3) δ 7.73 (d, J = 8.8 Hz, 2H), 7.20 (s,
1H), 6.91 (d, J = 8.8 Hz, 2H), 5.43 (t, 1H, 6.8 Hz), 5.09 (m, 1H),
4.95 (m, 1H), 4.00 (s, 3H), 3.82 (s, 3H), 1.93-1.85 (m, 1H), 1.83
(s, 3H), 1.79-1.70 (m, 1H), 1.50-1.34 (m, 2H), 0.96 (t, J = 7.2
Hz, 3H); 1H NMR (400 MHz, CD3OD) δ 7.70 (d, J = 8.8 Hz,
2H), 7.55 (s, 1H), 6.93 (d, J = 8.8 Hz, 2H), 5.45 (dd, J = 8.0, 5.6
Hz, 1H), 5.08 (m, 1H), 4.95 (m, 1H), 4.00 (s, 3H), 3.80 (s, 3H),
1.93-1.85 (m, 1H), 1.83 (s, 3H), 1.80-1.71 (m, 1H), 1.50-1.32
(m, 2H), 0.98 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CD3OD)
δ 160.8 (C), 159.4 (C), 144.7 (C), 143.0 (C), 137.7 (C), 127.7
(CH), 127.0 (C), 123.7 (CH), 115.0 (CH), 113.8 (CH2), 79.8
(CH), 55.7 (CH3), 36.7 (CH3), 35.9 (CH2), 19.9 (CH2), 18.4
(CH3), 14.1 (CH3); HRESITOFMS m/z 329.1866 [MþH]þ
(calcd for C19H25N2O3, 329.1860).
Acknowledgment. We thank T. Hong and M. Masuno for
preliminary work on Phorbas amaranthus, E. Rogers and A.
Jansma for assistance with NMR experiments, C. Skepper
for preparation of (þ)-16, J. Cowart, T.-L. Loh, and W.
Leong for running antifeedant assays, and UC Riverside
Mass Spectrometry Facility for HRMS measurements.
The 500 MHz NMR spectrometers were purchased with
a grant from the NSF (CRIF program CHE0741968).
This investigation was supported by grants from NIH
(CA122256 to T.F.M), a Ruth L. Kirschstein National
Research Service Award NIH/NCI (T32 CA009523 to B. I.
M), the National Undersea Research Program at UNCW,
and the Coral Reef Conservation Program (NOAA
NA96RU-0260 to J.R.P.). We are grateful to the captain
and crew of the RV Seward Johnson for logistical support
during collecting expeditions and in-field assays.
(R)-2-Methylpent-1-en-3-yl 4-(4-Methoxyphenyl)-1-methyl-
1H-imidazole-2-carboxylate (-)-9. A solution of EDCI (32.0
mg, 0.166 mmol) in CH2Cl2 (4 mL) was added dropwise to a
mixture of 7 (32.0 mg, 0.138 mmol), (R)-2-methylpent-1-en-3-ol
(27.6 mg, 0.276 mmol), and DMAP (1.7 mg, 0.014 mmol) in
CH2Cl2 (1 mL) at 0 °C with stirring. The mixture was stirred at
0 °C for 2 h and then at room temperature for 18 h. The solvent
was removed under reduced pressure and the residue subjected
to silica flash chromatography (15:85 EtOAc/hexanes) then
reversed-phase HPLC (C18, 3:1 CH3CN/H2O) to give the ester
(-)-9 (32.5 mg, 75% yield): [R]24 -61.9 (c 1.14, CHCl3); FTIR
(ATR, ZnSe) νmax 2966, 1705, 1505, 1448, 1400, 1246, 1120,
1089, 1030, 949, 903, 835, 794 cm-1; UV (MeOH) λmax 261 nm (ε
16200), 286 nm (ε 15200), 309 nm (ε 8600); CD (MeOH) λ 222
Supporting Information Available: 1H, 13C, and 2D NMR
spectra of 2-6 and synthetic compounds 8-10, 14, 15a,b, and
17. This material is available free of charge via the Internet at
http://pubs.acs.org.
1
nm (Δε -2.9), 287 (Δε -1.9), 306 (Δε -1.6); H NMR (400
MHz, CD3OD) δ 7.71 (d, J = 8.8 Hz, 2H), 7.57 (s, 1H), 6.94 (d,
J = 8.8 Hz, 2H), 5.36 (t, J = 6.8 Hz, 1H), 5.09 (m, 1H), 4.97 (m,
(22) (a) Paterson, I.; Perkins, M. V. Tetrahedron 1996, 52, 1811–1834. (b)
Cossy, J.; Bauer, D.; Bellosta, V. Tetrahedron 2002, 58, 5909–5922.
(23) Zhou, G.-X.; Molinski, T. F. Mar. Drugs 2003, 1, 46–53.
2460 J. Org. Chem. Vol. 75, No. 8, 2010