Xestoproxamines A-C from Neopetrosia proxima. assignment of absolute stereostructure of bis-piperidine alkaloids by integrated degradation-CD analysis

Article abstract of DOI:10.1021/np1008637

The complete stereostructures of xestoproxamines A-C, from the Bahamian sponge Neopetrosia proxima, were assigned from spectroscopic analysis, including MS, 2D NMR, and integrated degradation-CD analysis. Two new CD application protocols are described for defining absolute configuration: one for allylic methyl groups in branched chains and a second for the heterocyclic core bis-piperidine with specific applicability to other members of this class alkaloids - known for their stereoheterogeneity - and tertiary cyclic amines in general.

Full text of DOI:10.1021/np1008637

ARTICLE
pubs.acs.org/jnp
Xestoproxamines A-C from Neopetrosia proxima. Assignment
of Absolute Stereostructure of Bis-piperidine Alkaloids by Integrated
Degradation-CD Analysis
Brandon I. Morinaka‡ 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, United States
S Supporting Information
b
ABSTRACT: The complete stereostructures of xestoproxamines
A-C, from the Bahamian sponge Neopetrosia proxima, were
assigned from spectroscopic analysis, including MS, 2D NMR,
and integrated degradation-CD analysis. Two new CD application
protocols are described for defining absolute configuration: one for
allylic methyl groups in branched chains and a second for the
heterocyclic core bis-piperidine with specific applicability to other
members of this class alkaloids—known for their stereohetero-
geneity—and tertiary cyclic amines in general.
olycyclic amines comprise a family of biologically active
macrocycles reported from sponges of the genera Haliclona,
bonds. Because all sp2 carbons were accounted for and no CdN
bonds were present, the remaining N atoms must be sp3
hybridized; therefore, 1 is a ditertiary amine related to the known
alkaloids halicyclamines A (4)4 and B (5),5 haliclonacyclamines
A (6) and B (7),6 arenosclerin A (8), and haliclonacyclamine E
(9).7 The remaining four degrees of unsaturation were com-
pleted by four rings. Interpretation of 13C, DEPT, and HSQC
data showed six N-substituted CH2 groups (δ 58.5, 56.9, 56.4,
55.3, 48.9, and 47.4) that were linked to the remaining ring and
chain elements (substructures a-c, Figure 1), including two
substituted piperidine rings, from additional 2D NMR evidence
(DQF-COSY, TOCSY, gHSQC, and gHMBC), in particular,
HMBC cross-peaks of H3/C9, H4/C9, and H10/C3. Contig-
uous spin systems identified by TOCSY cross-peaks of H13b/
H16b and H11b/H14b secured two long hydrocarbon linking
chains between the CH2-N and CH-N groups. Substructure c
was joined to a by HMBC cross-peaks from H2 to both C29 and
C30. The final ring was closed by connecting substructure b to c
through HMBC correlations from H22 to C24.
P
Xestospongia, Petrosia, Neopetrosia, and Reniera, among others.1
Their structures contain two or more nitrogen atoms woven into
networks of fused heterocyclic and carbocyclic rings, mainly
based on piperidine or cyclohexane. From the standpoint of
structural characterization, polycyclic amines present difficulties
in their purification due to their amphiphilic physical properties,
while structure elucidation is hampered by both limited proton
chemical shift dispersion presented in their 1H NMR spectra and
stereochemical heterogeneity. In fact, the structures of several
alkaloids in this class have succumbed only to X-ray crystal-
lography. We report here three new bis-piperidine alkaloids,
xestoproxamines A-C (1-3), from Neopetrosia proxima2 and
assignment of their complete absolute stereostructures by an
integrated approach based on NMR and circular dichroism
(CD).3 A notable advancement is chiroptical analysis by CD,
following double quaternization of the bis-piperidine, to give N,
N0-bis-bromophenacyl derivatives that display characteristic split
Cotton effects that reliably reflect the absolute configuration of
the bis-piperidine core.
The relative configuration of the conjoined bis-piperidine
rings in 1 (designated rings A and B, Figure 2) could be established
from Jcoupling constants and NOESY data. The relative orientation
of H2ax and H3ax was established by a 1D-TOCSY experiment;
irradiation of H5eq (δ 3.48, brd) showed that H1ax (dd, J = 12.0,
12.0 Hz) revealed two large couplings to H1eq (δ 3.18, m) and
The molecular formula for 1 was established as C30H52N2
based on HRESIMS data (m/z 441.4207, [M þ H]þ). The
presence of two 1,2-disubstituted carbon-carbon double bonds
was supported by two coupled pairs of CHdCH spin systems in
the 1H-1H COSY spectrum (Table 1, δ 5.49 m; 5.37 m; 5.31, m,
and 5.28, m) and four CH sp2 carbons in the 13C DEPT spectrum
(δ 131.74, d; 131.69, d; 130.1, d, and 129.1, d). NOESY correla-
tions observed between allylic methylene groups (H16b/H19b
and H26b/H29b) confirmed the Z-configuration of both double
Special Issue: Special Issue in Honor of Koji Nakanishi
Received: November 26, 2010
Published: February 22, 2011
r
Copyright
2011 American Chemical Society and
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American Society of Pharmacognosy
|

Journal of Natural Products
ARTICLE
and H10eq and H3ax, led to the depicted relative configuration
and a conformer that places the averaged planes of ring A and B
orthogonal to each other (Figure 2).
Xestoproxamine B (2) showed a molecular formula of
C30H54N2, which has one degree of unsaturation less than
1
xestoproxamine A (1). The H NMR signals for 2 (Table 2)
were similar to those of 1 except for the presence of two vinyl
protons (δ 5.48, m and 5.33, m), indicating only one CdC double
bond. 2D NMR data confirmed both 1 and 2 share the same bis-
piperidine ring system, including relative configuration (JHH and
NOESY). The double bond was positioned between C17/C18 by
COSY and TOCSY correlations to H19. Therefore xestoproxamine
B (2) is a dihydro derivative of xestoproxamine A (1).
The third new bis-piperidine alkaloid, xestoproxamine C (3),
C31H56N2, showed a characteristic signal for a secondary methyl
branch in the 1H NMR spectrum (Table 3, δ 0.87, d, J = 6.7 Hz)
that was absent in 1 and 2. 2D NMR and NOESY data indicated
the configuration of 3 in the core bis-piperidine heterocycle
differed from 1 and 2 but corresponded to the relative config-
uration found in haliclonacyclamine A (6).5 While ring B
remained in the same conformation as 1 and 2, ring A in 3 had
undergone ring-flip to the opposite chair conformation, appar-
ently without inversion at the ring A sp3 nitrogen (NOESY,
Figure 2), a consequence of the inverted C2 configuration; yet in
the new torsional arrangement a dihedral angle of ∼90° for H3-
C3-C9-H9 (3JH3-H9 ≈ 0 Hz) was retained.
Compound 3 failed to produce suitable X-ray quality crystals
under a variety of conditions. Stereoanalysis by spectroscopic
means would require separate assignments of the two stereoele-
ments in 3: the C23 stereocenter and the bis-piperidine core. We
devised the following degradative approach for assignment of the
lone C23 stereocenter exo to the heterocyclic core. Hofmann
elimination of suitably quaternized nitrogen atoms in 3 would
provide a terminal olefin (Δ21), which could then be subjected to
cross metathesis with a styrenyl derivative to insert a chromo-
phore next to the secondary Me branch (first sphere of
asymmetry), rendering the molecule amenable to analysis by
CD. Due to the scarcity of 3, the method was first piloted with
more abundant xestoproxamine A (1) (Scheme 1). Catalytic
hydrogenation of 1 (Pd-C, MeOH, H2), purification and
conversion to the free base 10 by HPLC with buffered solvent
(Lux-cellulose; CH3CN/i-PrOH/Et2NH), and immediate ex-
haustive alkylation with CH3I cleanly provided the bis-methio-
dide salt 11 . The iodide counterion was exchanged with
hydroxide by treatment of 11 with either Ag2O or strong anion
exchange resin (Amberlite-IRA 400, HO- form). Two possible
major products were anticipated from cleavage of C21-N and
cleavage of either C5-N or C11-N, based on the Hofmann rule
that leads to the least substituted alkenes. In the event, Hofmann
elimination (10 min, 300 W, 140 °C) of the neat quaternary
ammonium hydroxide double salt afforded only one major
alkene product, 12. The structure of 12 was confirmed by 2D
NMR (COSY, TOCSY, HSQC, and HMBC), which showed
piperidine ring B was intact: exo cleavage of the C5-N bond was
supported by the multiplicity of H4 (δ 5.55, dt, J = 17.0, 10.3 Hz).
In contrast, ring A had undergone endo cleavage of the C21-N
bond, and the vinyl methine signal exhibited the more complex
pattern for vinyl proton H22 associated with a terminal allyl
group (δ 5.80, dddd, J = 17.0, 10.3, 6.8, 6.8 Hz, 1H). Alkene 12
was subjected to cross metathesis with excess 2-methoxy-6-
vinylnaphthalene 138 (∼10 equiv) in the presence of Grubbs’
second-generation catalyst,9 which selectively engaged only the
Figure 1. Substructures a-c of xestoproxamine A (1). Points of
attachment of linking chains are indicated by •.
to H2ax (δ 2.29, m) respectively. In addition, H3ax was vicinally
coupled by two large scalar couplings (J = 11.0 Hz) to H2ax and
H4ax. Additional large couplings (J = 12.0 and 12.0 Hz,
respectively) from H7ax and H9ax to H8ax (δ 1.06, ddd, J =
12.0, 12.0, 12.0 Hz) showed the three protons to be axially
disposed. The relative configuration of the remaining stereo-
centers in 1 was assigned by interpretation of NOESY correla-
tions. The dihedral angle of ∼90° between H3 and H9 was
supported by lack of vicinal coupling (3J ≈ 0 Hz). Conforma-
tional constraints placed on the rings by NOE and J data, in
particular, mutual correlations between the pairs H2ax and H8ax
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1
Table 1. H (600 MHz) and 13C NMR (125 MHz) for 1
(CD3OD)
δH, mult
DQF-
COSY
no. δC, mult.a
(J in Hz, ax/eq)
HMBCb
1
55.3, CH2 3.32, dd (12.0, ax)e
3.18, m (eq)
2
2, 3, 11, 30
2, 3, 5, 11, 30
2
3
4
29.7, CH
36.0, CH
2.29, m (ax)
1.91, m (ax)
1, 3, 30 3, 29, 30
2, 4, 4 4, 5, 8, 9, 10, 30
25.2, CH2 1.80, m (eq)
1.69, m (ax)
3
4
7
3, 5, 9,
2, 3
5
6
47.4, CH2 3.48, brd (13.2, eq)
3.19, m (ax)
1, 3, 4, 11
1, 3, 4
58.5, CH2 3.21, brd (12.0, eq)
7, 8, 9, 10, 20, 21
7, 8, 9, 10
2.84, dd (12.0, 12.0, 12.0, ax)
1.90, m (ax)
27.5, CH2 1.84, m (eq)
1.06, ddd (12.0, 12.0, 12.0ax)
1.74, m (ax) 8, 10 2, 3, 4, 7, 8
7
8
34.3, CH
6, 8, 20 6, 8, 9, 10, 20
7, 9
3,6, 7
6, 7, 20
9
42.1, CH
10 56.9, CH2 3.69, m (eq)
2.84, dd (12.0, 12.0 ax)
11 48.9c, CH2 3.32, m
3.02, ddd (12.8, 12.8, 3.7)
9
3, 6, 8, 9, 21
6, 8, 9
12
1, 5, 12, 13
5, 12, 13
Figure 2. Selected NOESY correlations between the piperidine rings of
(a) xestoproxamine A (1) and (b) xestoproxamine C (3). Points of
attachment of linking chains are indicated by •. Note, ring A has
undergone ring-flip in 3 with respect to 1.
12 25.2, CH2 1.79, m
1.66, m
11, 13 11, 13, 14
11, 14
13 21.7, CH2 1.79, m
1.65, m
12, 14 11, 12, 14, 15
11,12, 14
less hindered vinyl group derived from ring B to give the
conjugated naphthalene 14. The UV spectrum of 14 in CH3CN
showed characteristic λmax (246 and 292 nm) for the conjugated
naphthalene chromophore; however, as expected, this styrenyl
system was CD silent due to the remoteness of the chromophore
from the distal sphere of asymmetry near C2 (xestoproxamine
numbering, Figure 1).
14 27.3, CH2 1.52, m
1.44, m
13, 15 12, 13, 15, 16
12, 13, 15, 16
15 29.0, CH2 1.43, m
1.25, m
14, 16 14, 16, 17
14, 16, 17
16 29.4, CH2 2.25, m
2.00, m
15, 17 15, 17, 18
14, 17, 18
In order to assign the observed Cotton effect to the config-
uration of the C23 center in 3, an appropriate methyl-branched
model was required. Because the Cotton effect anticipated for the
degradation/cross metathesis product of 3 would arise from
asymmetric perturbation of the vinyl-conjugated 6-methoxy-
naphthalene, the model could consist of a chromophore adjacent
to an allylic methyl branch of known configuration, appended to
a short chain. Preparation of the model was achieved as depicted
in Scheme 2. The R-branched propionamide 15, obtained by
diastereoselective Myers alkylation of the enolate of (R,R)-(-)-
N-propionylpseudoephedrine10 (16) (LDA, LiCl),11 with 1-io-
dosilyloxy ether 17 was selectively reduced (LiBH3N(CH2)4, rt)
to the alcohol 1812 (% ee >94% by modified Mosher method).13
Swern oxidation of 18 to the corresponding aldehyde followed by
Wittig olefination (Ph3PCdCH2) gave terminal olefin 19.
Finally, cross metathesis of 19 (Grubbs’ II, MeO-NpV) provided
model alkene (S)-20.
17 131.69d, CH 5.49, m
18 129.1, CH 5.31, m
19 23.4, CH2 2.50, m
16, 18 15, 16, 18, 19
17, 19 16, 19, 20,
18, 20
2.12, brd (15.4)
7, 17, 18, 20
20 31.5, CH2 1.74, m
1.27, m
7
6, 7, 8, 18, 19
6, 7, 8
21 56.4, CH2 3.20, m
22 22.6, CH2 1.81, m
23 28.1, CH2 1.62, m
1.42, m
22
6, 22, 23
21, 23 21,23, 24, 25
22, 24 21, 22, 24, 25
21, 22, 24
24 27.0, CH2 1.42, m
25 29.0, CH2 1.53, m
1.45, m
23, 25 22, 23, 25, 26
24, 26 23, 24, 26, 27
23, 24, 26, 27
26 27.1, CH2 2.35, m
25, 27 28
1.91, m
24, 25, 27, 28
27 131.74d, CH 5.37, m
28 130.1, CH 5.28, m
29 21.5, CH2 2.24, m
1.82, m
26, 28 25, 26, 28, 29
27, 29 26, 27, 29, 30
28, 30 28, 30
Xestoproxamine C (3) was converted to 21, via the inter-
mediates 22-24 (Scheme 3), by a similar sequence of reactions
used to convert 1 to 14. The CD spectra (Figure 3 and Table 4)
of synthetic (S)-20 [λ 256 nm (Δε þ3.9)] and 21 [λ 255 nm (Δε
þ3.8)] were of the same sign and magnitude; therefore, 21 and
xestoproxamine C (3) have the 23S configuration.
27, 28, 30
30 31.9, CH2 1.72, m
29
1, 3, 28, 29
1.57, m
1, 3, 28, 29
a Determined from DEPT and HSQC. b HMBC correlations, optimized
for 8 Hz, are from proton(s) stated to the indicated carbon. c Observed
by HSQC. d May be interchanged. e Coupling constant assigned by 1D
TOCSY (irradiation of H5eq).
No simple spectroscopic method was available for defining the
absolute configuration of the bis-piperidine ring system in 1-3.
Since this is an outstanding problem of configurational analysis in
this class of alkaloids, we turned to refinement of a general
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1
Table 2. H (600 MHz) and 13C NMR (125 MHz) for Xestoproxamine B (2) (CD3OD)
no.
δC, mult.a
δH, mult (J in Hz, ax/eq)
COSY
3
HMBCb
1
55.7, CH2
3.23, dd (12.0, 12.0, ax)
3.16, m (eq)
2.24, m (ax)
1.87, m (ax)
1.81, m (eq)
1.68, m (ax)
3.45, brd (13.7, eq)
3.17, m (ax)
3.23, brd (12.0, eq)
2.85, dd (12.0, 12.0, ax)
1.91, m (ax)
1.87, m (eq)
1.08, ddd (12.0, 12.0, 12.0, ax)
1.78, m (ax)
3.53, m (eq)
2.98, dd (12.0, 12.0, ax)
3.30, m
2, 3, 5, 11, 30
2, 3, 5, 11, 30
1, 3, 29, 30
1, 2, 4, 5 8, 10, 29 30
2, 3, 9 11
2
3
4
29.9, CH
36.1, CH
25.3, CH2
1, 3, 30
2, 4
3, 5
2, 3, 9
5
6
47.5, CH2
57.4, CH2
4
7
1, 3, 4
1, 3, 4
7, 8, 10, 20, 21
7, 8, 10, 21
6, 8, 19, 20
3, 6, 7, 9, 10, 20
3, 6, 7, 9, 10, 20
4, 7,
7
8
34.5, CH
6, 8, 20
7, 9
27.9, CH2
9
42.1, CH
8, 10
9
10
56.7, CH2
6, 8 9, 21
6, 8, 9
11
12
13
49.3, CH2
21.7, CH2
25.4, CH2
12
1, 5, 12, 13
1. 5, 12, 13
11, 13, 14
11, 13
3.02, m
1.77,m
11, 13
12, 14
1.65, m
1.68, m
11, 12, 14, 15
11, 12, 15
12, 13, 15
13, 16, 17
13, 16, 17
14, 15, 17, 18
14, 15, 17, 18
16, 18, 19
17, 18, 19
1.60, m
14
15
27.5, CH2
28.9, CH2
1.44, m
13, 15
14, 16
1.44, m
1.26, m
16
29.5, CH2
2.22, m
15, 17
2.00, m
17
18
19
131.7, CH
129.2, CH
23.5, CH2
5.48, m
16, 18
17, 19
18, 20
5.33, m
2.45, m
2.15, m
17, 20
20
21
31.7, CH2
56.1, CH2
1.75, m
19, 7
22
6, 7, 18,
6, 7, 8, 18, 19
6, 10, 22, 23
6, 10, 22, 23
21, 23
1.28, m
3.30, m
3.07, m
22
23
20.0, CH2
26.2, CH2
1.77, m
21, 23
22, 24
1.50, m
22
1.37, m
21, 22
24-28
29.0-26.4, CH2
1.53-1.25, m
1.25, m
29
21.8, CH2
30
1.21, m
30
32.1, CH2
1.69, m
2, 29
1, 2, 3
1, 2, 3
1.44, m
a Determined from DEPT and HSQC. b HMBC correlations, optimized for 8 Hz, are from proton(s) stated to the indicated carbon.
method for assignment of cyclic tertiary amines involving
derivatization and CD first published by Nakanishi and co-
workers.14 Nakanishi’s assignment of the absolute configuration
of quinuclidinols14—bicyclic tertiary amines—followed simultaneo-
us quaternization of the tertiary nitrogen and esterification of the
pendant secondary OH group with excess p-phenylbenzylchloride
and interpretation of the resultant split CD spectrum arising
from exciton coupling of the two arene chromophores. Although
the xestoproxamines differ considerably from the quinuclidinols,
the same principle could be applied: quaternization of both N
atoms with a suitable chromophore and interpretation of exciton
coupling CD (ECCD) would be informative of the absolute
configuration of the heterocyclic bis-piperidine core. However,
three possible problems were anticipated. The distance between
chromophores attached to the N atoms in 1-3 is considerably
larger than those in derivatized quinuclidinols with an expectedly
weaker ECCD. This could be compensated for by substitution of
the p-phenylbenzyl group with a more polarized chromophore: a
phenacyl group. It was anticipated that possible differences in
conformations of the natural products and quaternized derivatives
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1
Table 3. H (600 MHz) and 13C NMR (125 MHz) NMR Data
configuration and conformation of the derivatives would be
revealed by CD spectra and could be interpreted as “fingerprint”
spectra that relate the handedness of the heterocyclic core. This
would confer an important advantage to chiroptical analysis of
antipodal bis-piperidines by CD over comparisons using [R]D.
Optical rotations of alkanamines are typically weak in magnitude,
and comparisons of specific rotation alone are notoriously unreli-
able due to changes in sign resulting from even slight structural
variants (unsaturation). By comparing a fingerprint Cotton effect
with that of a bis-piperidine phenacyl derivative of known absolute
configuration, the configuration of any member of the series could
be assigned.
for xestoproxamine C (3) (CDCl3)
δH, mult
no.
δC, mult.a
(J in Hz, ax/eq)
DQF-COSY
2
HMBCb
1
53.1, CH2 2.93, dd (12.0,
12.0, ax)
2.55, brd (12.0, eq)
2, 5
1, 2, 4, 8, 9, 10
2
3
4
40.1, CH 2.03, m (ax)
34.0, CH 1.96, m (eq)
34.7, CH2 2.12, m (ax)
1.80, m (eq)
1, 3, 30
2, 4
3, 5
2
To ensure a reasonably strong CD signal in the final product,
an N-(4-bromophenacyl) chromophore (λmax 265 nm, ε
∼12 000) was introduced by exhaustive quaternization of the
alkaloids. The bis-TFA salt of tetrahydroxestoproxamine (10)
was converted to the free base (KOH/MeOH) followed by
alkylation with p-bromophenacyl bromide (toluene, 90 °C) to
give 25 after HPLC purification (Scheme 4). The CD spectrum
(Table 4) of 25 showed a characteristic negative split Cotton
effect (λ 273 nm, Δε -6.0; 253 (þ2.6)) arising from exciton
coupling of equatorial and axial N-p-bromophenacyl chromo-
phores disposed with a negative helicity (see Figure 4). The
relative conformations of the conjoined bis-piperidine ring
system in 25 and 1 maintain the same conformations as starting
material (analysis of J and NOESY data); however, the exact
orientation of the transition dipole vectors that align along the
average axes of the N-p-Br-phenacyl groups is highly dependent
upon the conjoining C3-C9 bond, which is subject to subtle
torsional angle changes from nonbonding effects of the side
chains and not easily predictable. Instead, we chose to make
empirical chiroptical comparisons with p-Br-phenacyl derivatives
with a compound of known configuration.
The relative configuration of (-)-“perhaliclonacyclamine”
(26),15 the hydrogenation product of (þ)-tetradehydrohaliclo-
nacyclamine A (31) obtained from an Indonesian sponge,
Halichondria sp., differs from the tetrahydroxestoproxamine A
(10) in the relative configuration at C2 and the presence of two
additional CH2 groups in the lower linking chain.16 A sample of
(-)-2617 was alkylated in the same manner (see above) to give
derivative 27. The CD spectrum of 27 (λ 273 nm (Δε -6.1);
254, (þ3.2); Figure 4, Table 4) is almost identical to that of 25.
Therefore, despite differences in relative configuration in the
two piperidine rings in 25 and 27, both conform to the same
chromophore alignments (a negative N-(CdO)-(CdO)-N0
angle)—imposed largely by the constraint of theC3-C9 torsional
angle—and CD reveals they share the same absolute stereo-
structure form with the exception of the epimeric C2 position. A
sample of haliclonacyclamine E (9), isolated from a Brazilian
sample of Arenosclera braziliensis,18 was hydrogenated (H2, Pd-
C) to the tetrahydro derivative (28) and exhaustively alkylated (p-
bromophenacyl bromide, 90 °C) as before to the bis-p-Br-
phenacylated compound 29, which showed a CD spectrum (λ
273 nm, Δε -6.0; 255 (þ2.6), Table 4) almost identical with
those of 25 and 27. Finally, the CD spectrum of the bis-p-
bromophenylacyl derivative (30, λ 273 nm, Δε -7.1; 253
(þ3.5)), obtained from tetrahydroxestoproxamine C (22), was
identical to those of 25, 27, and 30.19 Therefore the absolute
configuration of the heterocyclic cores in xestoproxamine A (1),
haliclonacyclamine E (9), (-)-perhaliclonacyclamine (26), and
xestoproxamine C (3) are the same, with the exception of C2 as
noted above. Since the absolute configuration of (-)-26 was
5
6
46.7, CH2 3.14, dd (12.0,
12.0, ax)
4
7
2.80, m (eq)
60.0, CH2 2.76, m (eq)
1.84, dd (12.0,
12.0, ax)
7
8
37.4, CH 1.60, m (ax)
37.9, CH2 2.43, m (eq)
1.00, ddd (12.0,
12.0,
6, 8, 20
7, 9
6, 7, 9, 10, 20
12.0, ax)
9
44.9, CH 1.98, m (ax)
59.9, CH2 2.98, brd (9.8, eq)
2.17, m (ax)
8, 10
9
8
10
6, 8
11
12
56.5, CH2 2.91, m
22.0, CH2 1.65, m
1.56, m
12
1, 5, 12, 13
11, 13
11, 13
11, 12
11, 13
13
14
15
16
26.8, CH2 1.40, m
26.8-27.7 1.50-1.20, m
27.3, CH2 1.37, m
26.6, CH2 2.03, m
1.96, m
12
15, 17
15, 17, 18
15, 17, 18
16
17
18
19
129.5, CH 5.44, m
129.5, CH 5.41, m
24.8, CH2 2.37, m
1.81, m
16, 18
17, 19
18, 20
16
17, 18, 20
7, 17, 18, 20
8, 18, 19
6, 7, 8, 18, 19
10
v20
21
34.1, CH2 1.51, m
0.93, m
7, 19
22
55.0, CH2 2.70, m
2.48, m
6, 10
22
27.9, CH2 1.58, m
1.48, m
21, 23
21, 23, 24
21, 24, 31
23
24
30.5, CH 1.51, m
35.9, CH2 1.39, m
1.09, m
22, 24, 31
23, 25
22, 23, 31
25-30 26.8-27.7 1.50-1.20, m
31 21.2, CH3 0.87, d (6.7)
a Assigned from HSQC/HMBC. b HMBC correlations, optimized for 8
Hz, are from proton(s) stated to the indicated carbon.
23
22, 23, 24
and the introduction of two new stereocenters at N would
complicate nonempirical interpretation of ECCD effects. On the
other hand, the stereochemical outcome of N-quaternization
should be predictable. The alkylating reagent should approach
both N atoms in 1-3 along a trajectory in line with the lone pair
without changing the N-configuration. In any case, the resultant
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Scheme 1. Hofmann Degradation of Xestoproxamine A (1) and Cross Metathesis with 6-MeO-2-vinylnaphthalene (13)
Scheme 2. Synthesis of Model Compound 20
Scheme 3. Hofmann Degradation of Xestoproxamine C (3) and Cross Metathesis with 6-MeO-2-vinylnaphthalene (13)
established earlier by X-ray crystallography,15 the stereostructures of
1-3 and 9 are now completely assigned. Bis-piperidine 8 co-
occurs with 9,7 and it is likely both of these natural products share
the same heterocyclic core configuration.
A summary of the configurational analysis of 1-3 and the CIP
descriptors for the core stereocenters of related bis-piperidine
alkaloids is given in Table 5, along with a comparison of reported
[R]D values. No clear trend can be seen except that the compounds
have high stereochemical heterogeneity that may reflect their
geographic origins. The specific rotations vary in sign and magnitude
with a strong dependence upon the presence and position of
unsaturation in the top and bottom linking chains and possibly
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Table 4. CD Data (23 °C) for Vinylnaphthalene and Bis-p-
bromophenacyl Derivatives
no.
solvent
λ/nm
Δεb
λ/nm
Δεb
a
14c
20d
21e
25f
27f
29f
30f
CH3CN
CH3CN
CH3CN
CH3OH
CH3OH
CH3OH
CH3OH
255
256
254
254
254
254
þ3.8
þ3.9
þ2.6
þ3.2
þ2.6
þ3.5
276
272
272
276
-6.2
-6.9
-6.0
-7.1
a Only noise. b mol-1 dm3 cm-1. Scheme 1. d Scheme 2. Scheme 3.
c
e
f Scheme 4.
Figure 3. CD spectra (CH3CN, 23 °C) of (a) 20, (b) 21, and (c) 14.
Scheme 4. Preparation of N,N0-p-Br-phenacyl Derivatives of
Bis-piperidine Alkaloids
Figure 4. CD spectra (MeOH, 23 °C) of (a) 25, (b) 27, (c) 29, and
(d) 30.
quaternization of the tertiary amines, reliably reflects the absolute
configurations of the heterocyclic cores in 1-3, independent of
the relative configuration at C2. This observation was exploited
to show the two groups of structures, 1, 2, 8, 9, and 3, 26, have the
same absolute configuration in their core heterocyclic rings and
should be applicable to other bis-piperidine alkaloids in this class.
the chain lengths. Haliclonacyclamines A (6) and B (7), from
Haliclona sp. from the Great Barrier Reef, differ in the linking chains,
but the cores are antipodal to those of (-)-26 and (þ)-31 from
Halichondria sp. collected in Indonesia. We note that the recently
reported (-)-neopetrosiamine (32)20 from the sponge Neopetrosia
proxima,2 collected in the Caribbean sea south of the Bahamas, is
very similar to 1-3, but the configuration was not defined.
Bis-piperidine alkaloids have shown modest activity against
various cancer cell lines. Compounds 1-3 were assayed in vitro
against human colon tumor cells (HCT-116) and showed IC50
values of 21.2, 6.3, and 5.4 μM, respectively.
In conclusion, three new bis-piperidine alkaloids, xestoprox-
amines A-C (1-3), are reported and their complete structures
assigned by integrated MS, NMR, and chiroptical analysis. We
assigned the absolute configuration of the remote methyl group
in xestoproxamine C (3) by CD following a Hoffman degrada-
tion/cross metathesis protocol, a sensitive technique that can be
extended to other natural products containing acyclic allylic
methyl branches. Finally, we have shown that ECCD of
N,N0-di-p-bromophenacyl bis-piperidine alkaloids, obtained by
’ EXPERIMENTAL SECTION
General Experimental Procedures. General procedures are
described elsewhere.21 Yields were determined gravimetrically except
for those of masses of less than ∼100 μg, which are estimated from UV
extinction coefficients or by quantitative solvent 13C satellite (QSCS)22
analysis of cryomicroprobe-measured 1H NMR spectra. HPLC was
carried out using either a Gilson model 302 pump equipped with tandem
detectors—UV-visible (ISCO model UA-5, λ 254 nm) and refractive
index (Waters R401)—or a Rainin HPXL dual-pump with split flow
(7:1) between two detectors—a Jasco CD-2095 UV-CD and an ESA
model 301 evaporative light scattering detector (ELSD).
Animal Material. The sponge Neopetrosia proxima2 was collected
in June 2008 at Stirrup Cay, the Bahamas (25°49.511 N 77°53.9240 W)
at a depth of 8.5 m and identified by Sven Zea (Universidad Nacional de
Colombia, InveMar). The surface of the tissue was dark brown and free
of epiphytic zoanthids. A voucher sample of the sponge (08-13-073) is
stored at UC San Diego.
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Table 5. Specific Rotations and Configurational Assignment
and the residue subjected to HPLC (Phenomenex Lux-cellulose, 1.5 mL.
min-1, mobile phase: 9:1:0.02 CH3CN/i-PrOH/Et2NH) to give tetra-
hydroxestoproxamine A free base (10, 0.55 mg), which was used
of Bis-piperidine Alkaloidsa
immediately after characterization. Colorless solid; [R]23 -12.1 (c
linker chain Cn
D
0.7, CHCl3); 1H NMR (500 MHz, CDCl3) δ 2.90 (dt, 1H, J = 12.0, 5.5
Hz), 2.80 (dd, 1H, J = 12.5, 4.0 Hz), 2.73 (brd, 1H, J = 10.0 Hz), 2.70-
2.61 (m, 5H), 2.54 (brp, 1H, J = 5.5 Hz), 2.41 (brt, 1H, J = 9.8 Hz), 2.24
(t, 1H, 11 Hz), 2.04 (d, 1H, J = 12.5 Hz), 1.87 (t, 1H, J = 11 Hz), 1.82 (m,
1H), 1.64-1.21 (m, 40H), 0.97 (m, 1H), 0.72 (q, 1H, J = 12 Hz);
HRESIMS m/z 445.4518 [M þ H]þ (calcd for C30H57N2, 445.4516).
Tetrahydroxestoproxamine A Bis-methiodide Salt (11). Free base
10 (0.55 mg) was dissolved in CH2Cl2 (0.8 mL), excess CH3I (0.2 mL)
was added, and the mixture was stirred overnight in the dark. Volatiles were
removed under a stream of N2 to give the bis-methiodide salt (11, 0.9 mg)
as an off-white solid: 1H NMR (500 MHz, CD3OD) δ 3.84 (d, J = 10.5 Hz,
1H), 3.54-3.49 (m, 3H), 3.41-3.15 (m, 8H), 3.17 (s, 3H), 3.12 (s, 3H),
2.20 - 1.22 (m, 44H), 1.19 (q, J = 12.5 Hz, 1H); 13C NMR (125 MHz,
CD3OD) δ 69.4 (CH2), 66.5 (CH2), 64.6 (CH2), 63.6 (CH2), 60.4
(CH2), 57.9 (CH2), 53.3 (CH3), 47.6 (CH3), 37.0 (CH), 35.2 (CH), 32.2
(CH), 32.1 (CH2), 31.8 (CH2), 31.7 (CH), 29.2 (CH2), 29.1(CH2), 29.0
(CH2), 28.8 (CH2), 28.6 (2 ꢀ CH2), 28.3 (CH2), 28.2 (CH2), 28.1
(CH2), 27.4 (CH2), 27.3 (CH2), 26.8 (CH2), 26.6 (CH2), 25.7 (CH2),
25.3 (CH2), 22.3 (CH2), 20.7 (CH2), 20.2 (CH2); HRESIMS m/z
237.2453 [M]2þ (calcd for C32H62N2, 474.4908)
conc, g/
absolute
configna
cmpd [R]D 100 mL solvent
top
bottom
1b
2b
3b
4c
5d
6e
7e
8f
9f
þ4.4 c 2.0
þ2.7 c 2.4
-18.5 c 0.67 CHCl3
-7.3 c 0.73 CH2Cl2
CH3OH C10
CH3OH C10
C10
C10
C10
C12
C8
2R,3S,7S,9S
2R,3S,7S,9S
C10
C8
2S,3S,7S,9S,23S
2R*,3S*,9R* h
2R*,3S*,7S*h
2R,3R,7R,9R
2R,3R,7R,9R
2R,3S,7S,9S b
2R,3S,7S,9S b
2S,3S,7S,9S
-143.5 c 0.65
?
C10
C10
C10
C10
C10
C10
C10
C10
-3.4 c 1.21 CH2Cl2
þ3.4 c 0.55 CH2Cl2
C12
C12
C12
C12
C12
C12
C10
-3
c 0.015 MeOH
c 0.02 MeOH
þ14
26g
31g
32g
-20.9 c 0.205 CHCl3
þ19.4 c 0.515 CHCl3
2S,3S,7S,9S
2R*,3R*,7R*,9R*h
-10
c 1.0
CHCl3
a Core locants follow the numbering used for 1-3. Unless indicated by *,
depicted CIP assignments are absolute. Configuration of the N atoms
not given. b This work. c Ref 4. d X-ray, ref 5. e X-ray, ref 6a. f ref 7. g X-ray,
ref 15. h Relative configuration only. g Ref 20.
Hofmann Degradation of Tetrahydroxestoproxamine A Bis-
methiodide Salt. A solution of the bis-methiodide (11, 0.9 mg) was
eluted with MeOH through a short column of strong anion exchange
resin (Amberlite IRA-400, HO- form, prepared immediately before
from Cl- form by elution with aqueous 1 N NaOH, followed by washing
with distilled H2O until the eluate was neutral, then MeOH). The eluate
was concentrated under reduced pressure to remove the volatiles, and
solvent-free methohydroxide was irradiated (microwave, 300 W, 140 °C,
10 min). The crude product was taken up in MeOH and passed through
a reversed-phase silica (C18) cartridge (200 mg) by elution with MeOH
þ 0.1% TFA. The solvent was removed under a stream of N2, and the
residue subjected to HPLC (reversed-phase, C18 Phenomenex Luna, 5
μm, 10 ꢀ 250 mm, gradient: 40-100% CH3CN/H2O þ 0.1% TFA
over 40 min) to give a single major elimination product, 12 (0.45 mg).
12: 1H NMR (600 MHz, CD3OD, representative signals) δ 5.80 (dddd,
J = 17.0, 10.3, 6.8, 6.8 Hz, 1H), 5.55 (dt, J = 17.0, 10.3 Hz, 1H), 5.31 (dd, J =
10.3, 1.4 Hz, 1H), 5.18 (dd, J = 17.0, 1.4 Hz), 4.98 (dq, J = 17.0, 1.9 HZ,
1H), 4.92 (1H, under solvent), 3.44 (brd, J = 12.5 Hz, 1H), 3.40
(brd, J = 12.5 Hz, 1H), 3.20 (td, J = 12.5, 4.7 Hz, 1H), 3.10 (td, J = 12.5,
5.2 Hz, 1H), 2.92 (s, 3H), 2.85 (s, 3H), 2.54 (t, J = 12 Hz, 1H), 2.53 (t, J =
12 Hz, 1H), 2.18 (t, J = 10.4 Hz, 1H), 2.05 (q, 7.3 Hz, 2H), 0.88 (q, J = 12
Hz, 1H); HRESIMS m/z 473.4828 [M þ H]þ (calcd for C32H61N2,
473.4829).
Extraction and Isolation. A sample of N. proxima (252.6 g wet wt.) was
extracted with MeOH (3 ꢀ 2.5 L, 25 °C, overnight) and then CH2Cl2/
MeOH (2 ꢀ 2.5 L, 25 °C, overnight). The resulting extract was partitioned
between hexanes (3 ꢀ 500 mL) and 9:1 MeOH/H2O (1 L). The aqueous
MeOH layer was removed and the H2O content adjusted to 1:1 MeOH/
H2O before extraction with CH2Cl2 (3 ꢀ 1 L). The aqueous MeOH layer
was concentrated under reduced pressure and then applied directly onto a
reversed-phase (C18) silica column (∼400 g) successively eluting with 1:9,
3:7, 1:1, and 9:1 CH3CN/H2O þ 0.2% TFA, and i-PrOH þ 0.2% TFA to
give five fractions. A portion (305 mg) of the third fraction (1.22 g) was
subjected to preparative HPLC (reversed-phase, C18, 10 mL min-1, gradient
elution with 3:7 to 2:5 CH3CN/H2O þ 0.3% TFA over 40 min) to give 12
fractions. The third preparative HPLC fraction (27.2 mg) was subjected to
semipreparative HPLC (reversed-phase Synergi-HydroRP, 2.5 mL min-1
,
mobile phase: 2:2:6:0.05 CH3CN/i-PrOH/H2O/TFA) to give xestoprox-
amine A (1, 14.0 mg). A portion (4 mg) of the fourth preparative HPLC
fraction (24.0 mg) was subjected to semipreparative HPLC over the same
column (2.5 mL min-1, 22.5:22.5:55:0.5 CH3CN/i-PrOH/H2O/TFA) to
give xestoproxamine B (2, 2.9 mg). The entire sixth HPLC fraction was subj-
ected to HPLC (Phenomenex Lux-cellulose, 1.5 mL.min-1, mobile phase:
9:1:0.02 CH3CN/i-PrOH/Et2NH) to give xestoproxamine C (3, 1.5 mg).
Xestoproxamine A (1):. colorless glass; [R]24D þ4.4 (c 2.0, MeOH);
Cross Metathesis of 12 with 2-Methoxy-6-vinylnaphthalene
(13). To a solution of alkene 12 (450 μg, 0.642 μmol) and 6-meth-
oxy-2-vinylnaphthalene8 (13, 1.2 mg, 6.42 μmol) in CH2Cl2 (0.5 mL)
was added Grubbs’ second-generation catalyst9 (226 μg, 0.265 μmol) in
CH2Cl2 (1 mL) in three portions over a period of 11 h at 80 °C. The
solvent was evaporated under a stream of N2. The crude reaction
mixture was passed through a reversed-phase C18 cartridge by elution
with MeOH þ 0.1% TFA, then subjected to HPLC (Luna, C18, 250 ꢀ
10 mm, 40-100% CH3CN/H2O þ 0.1% TFA over 40 min) to give the
6-methoxy-2-naphthylethenyl derivative 14 (240 μg): UV (CH3CN)
FTIR (ATR) ν 1676, 1437, 1203, 1133, 841, 801, 723 cm-1; 1H and 13
C
NMR, see Table 1; HRESIMS m/z 441.4207 [M þ H]þ (calcd for
C30H53N2, 441.4203).
Xestoproxamine B (2):. colorless glass; [R]24D þ2.7 (c 2.4, MeOH);
FTIR (ATR) ν 2934, 2862, 1674, 1463, 1199, 1131, 833, 799, 721 cm-1
;
1H and 13C NMR, see Table 2; HRESIMS m/z 443.4362 [M þ H]þ
(calcd for C30H55N2, 443.4358).
Xestoproxamine C (3):. colorless solid; [R]24 -18.5 (c 0.67,
D
CHCl3); FTIR (ATR) ν 2933, 2862, 1673, 1470, 1199, 1128, 829,
797, 721 cm-1; 1H and 13C NMR, see Table 3; HRESIMS m/z 457.4513
[M þ H]þ (calcd for C31H57N2, 457.4516).
1
λmax 246, 292 nm; CD see Table 4; H NMR (600 MHz, CD3OD,
representative signals) δ 7.685 (d, J = 9.0 Hz, 1H), 7.681 (d, J = 8.6 Hz,
1H), 7.60 (brs, 1H), 7.55 (dd, J = 8.6, 2.0 Hz, 1H), 7.18 (d, J = 2.0 Hz,
1H), 7.09 (dd, J = 9.0, 2.0 Hz, 1H), 6.51 (d, J = 16.0 Hz, 1H), 6.32 (dt, J =
16.0, 7.0 Hz, 1H), 5.55 (dt, J = 17.0, 10.3 Hz, 1H), 5.31 (dd, J = 10.3, 1.4
Hz, 1H), 5.18 (dd, J = 17.0, 1.4 Hz), 3.90 (s, 3H), 2.91 (s, 3H), 2.83 (s,
3H), 2.50 (t, J = 12 Hz, 1H), 2.49 (t, J = 12 Hz, 1H), 2.26 (q, J = 7.0 Hz,
Tetrahydroxestoproxamine A Free Base (10). A solution of xesto-
proxamine A (1) TFA salt (1.0 mg) and Pd/C (10%, 0.2 mg) in MeOH
þ 2% AcOH (0.5 mL) was vigorously stirred under 1 atm of H2
overnight. The mixture was passed through a membrane filter (0.45
μm). The solvent was removed from the filtrate under reduced pressure,
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Journal of Natural Products
ARTICLE
2H), 0.86 (q, J = 12 Hz, 1H); HRESIMS m/z 629.5403 [M]þ (calcd for
C43H69N2O, 629.5404).
eluting with MeOH þ 0.1% TFA, and finally purified by HPLC (reversed-
phase, C18 Phenomenex Luna, 5 μm, 10 ꢀ 250 mm, gradient: 40-100%
CH3CN/H2O þ 0.1% TFA over 40 min) to give the 6-methoxy-2-
naphthylethenyl derivative 21 (6 μg): UV (CH3CN) λ 246, 292 nm; CD
(CH3CN) λ 256 (Δε þ3.9); 1H NMR (600 MHz, CD3OD, representa-
tive signals) δ 7.69 (d, J = 8.6 Hz, 2H), 7.62 (brs, 1H), 7.55 (dd, J = 8.6, 2.0
Hz, 1H), 7.19 (brs, J = 2.0 Hz, 1H), 7.10 (dd, J = 9.0, 2.0 Hz, 1H), 6.48 (d,
J = 16.2 Hz, 1H), 6.16 (dd, J = 16.2, 8.0 Hz, 1H), 5.58 (dt, J = 17.2, 10.0 Hz,
1H), 5.32 (d, J = 10.0 Hz, 1H), 5.27 (d, J = 17.2 Hz, 1H), 3.90 (s, 3H), 2.89
(s, 3H), 2.81 (s, 3H), 1.12 (d, J = 7 Hz, 3H); HRESIMS m/z 643.5563
[M]þ, calcd 643.5561 for C44H71N2O.
Preparation of p-Bromophenacyl Derivatives. Bis-p-bro-
mophenacyl Tetrahydroxestoproxamine A (25). The acetate salt of
tetrahydroxestoproxamine A (0.6 mg, 1.06 μmol) was converted to the free
base 10 by addition of KOH (238 μg, 4.25 μmol) in MeOH (0.5 mL). After
a few minutes the solvent was evaporated under a stream of N2 then on high
vacuum. Toluene (0.5 mL) and p-bromophenacyl bromide (5.9 mg, 21.2
μmol) were added, and the mixture was stirred at 90 °C for 12 h. The
solvent was evaporated under a stream of N2, and the mixture was subjected
to HPLC (reversed-phase Phenomenx, Luna, C18, 10 ꢀ 250 mm; 2 mL.
min-1 40-100% CH3CN/H2O þ 0.1% TFA over 40 min) to give the bis-
p-bromophenacyl TFA salt 25 (0.7 mg): UV (MeOH) λmax 265 (log ε
4.38) nm; CD (MeOH) λ 254 (Δε þ2.6), 276 (Δε -6.0) nm; 1H NMR
(500 MHz, CD3OD, representative signals) δ 7.99 (d, J = 8.8 Hz, 2H), 7.94
(d, J = 8.8 Hz, 2H), 7.78 (d, J = 8.8 Hz, 2H), 7.77 (d, J = 8.8 Hz, 2H), 5.32
(d, J = 18.2 Hz, 1H), 5.17 (d, J = 18.2 Hz, 1H), 5.17 (d, J = 18.2 Hz, 1H),
5.14 (s, 2H), 4.18 (brd, J = 11.3 Hz, 1H), 4.11 (brd, J = 12.7 Hz, 1H); 3.99
(m, 1H), 3.98 (m, 1H), 3.81 (m, 2H), 3.77 (m, 1H), 3.58 (td, J = 13.1, 3.3
Hz, 1H), 3.56 (m, 1H), 3.54 (t, J = 12.8 Hz, 1H), 3.31 (under solvent), 3.24
(t, J = 12.8 Hz, 1H), 1.21 (q, J = 12.6 Hz, 1H); HRESIMS m/z 838.3632
[M]2þ (calcd for C46H68Br2N2O2, 838.3636).
Degradation of Xestoproxamine C. Dihydroxestoproxamine
C TFA Salt (22). A solution of xestoproxamine C (3) (0.8 mg) and Pd/
C (10%, 0.2 mg) in MeOH þ 2% AcOH (0.5 mL) was vigorously stirred
overnight under H2 (1 atm). The solution was passed through a
membrane filter (0.45 m), and the volatiles were removed under a
stream of N2. The residue was purified by HPLC (reversed-phase,
Phenomenex, Synergi-HydroRP, 4 μm, 10 ꢀ 250 mm; 3 mL min-1
;
mobile phase: 25:25:50:0.5 CH3CN/i-PrOH/H2O/TFA) to give dihy-
droxestoproxamine C (22): colorless glass; 1H NMR (500 MHz,
CD3OD, representative signals) δ 3.68 (brd, J = 11.2 Hz, 1H), 3.48
(td, J = 13.0, 4.0 Hz, 1H), 2.91 (t, J = 12.0 Hz, 1H), 2.84 (t, J = 12.0 Hz,
1H), 2.33 (brd, J = 12.0 Hz, 1H), 0.95 (d, J = 7.0 Hz, 1H); 13C NMR
(125 MHz, CD3OD) δ 58.0 (CH2), 57.5 (CH2), 56.6 (CH2), 54.9
(CH2), 53.1 (CH2), 43.3 (CH), 40.3 (CH), 37.1 (CH), 35.74 (CH2),
35.67 (CH2), 33.7 (CH2), 33.6 (CH2), 33.3 (CH), 32.9 (CH2), 31.5
(CH), 28.8 (CH2), 25.56 (CH2), 28.53 (CH2), 27.8 (3 ꢀ CH2), 27.7
(CH2), 26.8 (CH2), 26.6 (CH2), 26.3 (CH2), 26.2 (CH2), 26.0 (CH2),
25.6 (CH2), 21.9 (CH2), 21.0 (CH3); HRESIMS m/z 460.4748 [M þ
H]þ (calcd for C31H60N2, 460.4751).
Tetrahydroxestoproxamine C Bis-methiodide Salt (23). The TFA
salt 22 was subjected to HPLC (Lux-cellulose, 1.5 mL min-1, mobile
phase: 9:1:0.02 CH3CN/i-PrOH/DEA) to give the free base (0.5 mg).
Immediately after removal of the volatiles, the free base (0.5 mg) was
dissolved in CH2Cl2 (0.8 mL) and treated with excess CH3I (0.2 mL),
and the mixture was stirred overnight in the dark. The solvent was
evaporated under a stream of N2 to give the methiodide (23, 0.8 mg) as
an off-white solid: 1H NMR (500 MHz, CD3OD, representative signals)
δ 3.87 (brd, J = 11.4 Hz, 1H), 3.77 (td, J = 13.8, 3.0 Hz, 1H), 3.21 (s,
3H), 3.12 (s, 3H), 3.04 (t, J = 12.0 Hz, 1H), 1.03 (d, J = 7.0 Hz, 3H); 13
C
NMR (125 MHz, CD3OD) δ 69.5 (CH2), 69.0 (CH2), 67.4 (CH2),
65.3 (CH2), 61.7 (CH2), 57.2 (CH2) [obscured by solvent] (CH3), 45.9
(CH3), 39.3 (CH), 36.4 (CH2), 36.2 (CH), 35.2 (CH2), 34.0 (CH2),
33.3 (CH), 33.1 (CH), 32.6 (CH2), 31.2 (CH), 30.2 (CH2), 29.5
(CH2), 29.3 (CH2), 28.7 (CH2), 28.4 (CH2), 28.2 (CH2), 27.94 (CH2),
27.86 (2 ꢀ CH2), 27.5 (CH2), 26.9 (CH2), 26.1 (CH2), 25.3 (CH2),
25.2 (CH2), 22.1 (CH3), 22.0 (CH2); HRESIMS m/z 244.2531 [M]2þ
(calcd for C33H64N2, 488.5064).
Bis-p-bromophenacyl Perhaliclonacyclamine (27). (-)-Perhali-
clonacyclamine15 (26, 150 μg, 0.317 μmol) was stirred with KOH (71
μg, 1.27 μmol) in MeOH (0.5 mL). After a few minutes the solvent was
evaporated under a stream of N2, then on high vacuum. Toluene (0.5
mL) and p-bromophenacyl bromide (1.8 mg, 6.34 μmol) were added,
and the mixture was stirred at 90 °C for 12 h. The solvent was evaporated
under a stream of N2, and the mixture was subjected to HPLC (reversed-
phase Phenomenex, Luna, C18, 10 ꢀ 250 mm; 2 mL min-1; mobile
phase/gradient: 40-100% CH3CN/H2O þ 0.1% TFA over 40 min) to
give the bis-p-bromophenacyl TFA salt 27 (ca. 44 μg): UV (MeOH) λ
265 nm; CD (MeOH) λ 254 (Δε þ3.2), 272 (-6.1) nm; HRESIMS m/
z 866.3946 [M]2þ (calcd for C48H72Br2N2O2, 866.3950).
Hofmann Degradation of Tetrahydroxestoproxamine C Bis-
methiodide Salt (23). A solution of the methiodide 23 in MeOH was
passed through a short column of strong anion exchange resin (Amberlite
IRA-400 (Cl-), convertedtotheHO- form with 1 N NaOH). After removal
of solvent under reduced pressure, the methohydroxide was transferred to a 10
mL microwave vessel, dried, and irradiated (microwave, 300 W, 140 °C over
7.5 min, then an additional 4.5 min). The crude product obtained was taken
up in MeOH passed through a reversed-phase silica cartridge (C18, 200 mg)
eluting with MeOH þ 0.1% TFA. Solvent was removed under a stream of N2,
and the residue purified by HPLC (reversed-phase, C18 Phenomenex Luna, 5
μm, 10 ꢀ 250 mm, gradient: 40-100% CH3CN/H2O þ 0.1% TFA over 40
min) to give the double-elimination product 24 (∼140 μg).
Bis-p-bromophenacyl Perhydrohaliclonacyclamine E (29). A solu-
tion of (þ)-haliclonacyclamine E7 (9, TFA salt, 0.8 mg, 1.15 μmol) in
MeOH/AcOH (49:1, 0.25 mL) was stirred with Pd/C (10%, 0.1 mg) at
room temperature under H2 (1 atm) for 48 h. The mixture was filtered
through a nylon syringe filter (0.45 μm), and the solvent removed from
the filtrate under a stream of N2. The residue was subjected to HPLC
(Phenomenex, Synergi Hydro-RP, 4 μm, 10 ꢀ 250 mm; 2.5 mL min-1
,
25:25:50:0.5 CH3CN/i-PrOH/H2O/TFA) to give perhydrohaliclona-
cyclamine E TFA salt (28, 0.3 mg). The TFA salt was subjected to HPLC
(Phenomenex Lux-cellulose, 1.5 mL min-1, 9:1:0.02 CH3CN/i-PrOH/
Et2NH), and the solvent removed under a stream of N2 and further dried
under high vacuum. Toluene (0.5 mL) and p-bromophenacyl bromide
(1.2 mg, 4.28 μmol) were added, and the mixture was stirred at 90 °C for
12 h. The solvent was removed under a stream of N2, and the mixture was
subjected to HPLC (reversed-phase, Phenomenex, Luna, C18, 10 ꢀ 250
mm; 2 mL min-1, 40-100% CH3CN/H2O þ 0.1% TFA over 40 min) to
give the bis-p-bromophenacyl TFA salt 29 (ca. 7.0 μg): UV (MeOH) λ 265
nm (log ε 4.38); CD (MeOH) λ 254 (Δε þ2.6), 272 (-6.0); HRESIMS
m/z 866.3940 [M]2þ (calcd for C48H72Br2N2O2, 866.3950).
1
24: H NMR (600 MHz, CD3OD, representative signals) δ 5.67
(ddd, J = 17.2, 10.2, 7.8 Hz, 1H), 5.62 (dt, J = 17.2, 10.0 Hz, 1H), 5.35 (d,
J = 10.0 Hz, 1H), 5.29 (d, J = 17.2 Hz, 1H), 4.94 (brd, J = 17.2 Hz, 1H),
2.93 (s, 3H), 2.86 (s, 3H), 2.58 (t, J = 12.2 Hz, 1H), 2.50 (t, J = 12.2 Hz,
1H), 0.98 (d, J = 7 Hz, 3H); HRESIMS m/z 487.4983 [M þ H]þ (calcd
for C33H63N2, 487.4886).
Cross Metathesis of 24 with 2-Methoxy-6-vinylnaphthalene
(13). Grubbs’ second-generation catalyst (95 μg, 0.112 μmol) in
CH2Cl2 (0.8 mL) was added in three portions over a period of 11 h
to alkene 24 (140 μg, 0.199 μmol) and 2-methoxy-6-vinylnaphthalene8
(0.8 mg, 4.20 μmol), and the mixture was stirred vigorously in a sealed
vial at 80 °C. The solvent was removed under a stream of N2, and the
residue was passed through a reversed-phase silica cartridge (C18, 200 mg)
Bis-p-bromophenacyl Dihydroxestoproxamine C (30). Xestoproxa-
mine C free base (22, 0.4 mg, 0.876 mmol) was stirred with Pd/C (10%,
438
dx.doi.org/10.1021/np1008637 |J. Nat. Prod. 2011, 74, 430–440

Journal of Natural Products
ARTICLE
0.1 mg) in 0.25 mL of MeOH/AcOH (49:1) at room temperature under
H2 (1 atm) for 48 h. The mixture was filtered through a membrane filter
(0.45 m), andthe solvent wasevaporatedunder astreamof N2 then by high
vacuum. The dihydroxestoproxamine C acetate salt was subjected to HPLC
(Phenomenex Lux-cellulose, 1.5 mL min-1, mobile phase: 9:1:0.02
CH3CN/i-PrOH/Et2NH), and the solvent was concentrated under a
stream of N2, then dried under reduced pressure. Dihydroxestoproxamine
Cfreebasewasstirredwithp-bromophenacyl bromide (3.0 mg, 10.8 mmol)
in toluene at 80 °C for 2 h in a microwave reactor (300 W). The solvent was
removed under a stream of N2, and the mixture subjected to RPHPLC
(column: Phenomenx, Luna, C18(2), 10 ꢀ 250 mm; 2 mL min-1; 40-
100% CH3CN/H2O þ 0.1% TFA over 40 min) to give the bis-p-
bromophenacyl TFA salt derivative 30 (137 μg): UV (MeOH) λmax 265
nm; CD (MeOH) λ 254 (Δε þ3.5), 276 (Δε -7.1) nm; HRESIMS m/z
852.3792 [M]2þ (calcd for C47H70Br2N2O2, 852.3799).
(m, 15H), 1.09 (m, 1H) 0.91 (d, J = 6.8 Hz, 3H), 0.89 (s, 9H), 0.04 (s, 6H);
13C NMR (100 MHz, CDCl3) δ 68.4 (CH2), 63.3 (CH2), 35.7 (CH), 33.2
(CH2), 32.9 (CH2), 29.9-29.4 (5 ꢀ CH2), 27.0 (CH2), 26.0 (3 ꢀ CH3),
25.8 (CH2), 18.4 (C), 16.6 (CH3), -5.3 (2 ꢀ CH3); HRESIMS m/z
353.2849 [M þ Na]þ (calcd for C19H42O2SiNa, 353.2846).
Aldehyde 19a. DMSO (64 mL, 0.91 mmol) was added dropwise to a
stirred solution of oxalyl chloride (51 mL, 0.60 mmol) in CH2Cl2
(3 mL) at -78 °C and stirred for 10 min. A solution of the alcohol 18
(100 mg, 0.302 mmol) in CH2Cl2 was added, and the mixture was stirred
for 30 min at -50 °C. Et3N (168 mL, 1.21 mmol) was added, and the
mixture was brought to -30 °C followed by stirring an additional 30
min. Saturated NH4Cl solution (5 mL) was added, and the layers were
separated. The aqueous layer was extracted with CH2Cl2 (2 ꢀ 3 mL),
the combined organic extracts were dried (MgSO4) and concentrated, and
the residue was purified by flash chromatography (SiO2, 3% EtOAc/
hexanes) to give the aldehyde 19a (91 mg, 92% yield): [R]24D þ12.8 (c
3.4, CHCl3); FTIR (ATR) ν 2926, 2854, 1730, 1463, 1254, 1099, 835, 774
cm-1; 1H NMR (400 MHz, CDCl3) δ 9.61 (d, J = 2.4 Hz, 1H), 3.59 (t, J =
6.4 Hz, 2H), 2.32 (sd, J= 7.2, 1.6 Hz, 1H), 1.69 (m, 1H), 1.50 (p, J= 7.2 Hz,
2H), 1.25(m, 15H), 1.08(d,J= 7.2 Hz, 3H), 0.89 (s, 9H), 0.04 (s, 6H); 13C
NMR (100 MHz, CDCl3) δ 205.5 (CH), 63.3 (CH2), 46.3 (CH), 32.9
(CH2), 30.5 (CH2), 29.6-29.4 (5 ꢀ CH2), 26.9 (CH2), 26.0 (3 ꢀ CH3),
25.8 (CH2), 18.4 (C), 13.3 (CH3), -5.3 (2 ꢀ CH3); HRESIMS m/z
351.2691 [M þ Na]þ (calcd for C19H40O2SiNa, 351.2690).
Synthesis of Model Compound 20. Amide 15. n-BuLi (2.21
M in hexanes, 3.0 mL, 6.64 mmol) was added to a stirred solution of
anhydrous lithium chloride (0.84 g, 19.8 mmol) and diisopropylamine
(0.94 mL, 6.64 mmol) in THF (20 mL) at -78 °C. The mixture was
kept at -78 °C for 20 min, then placed in an ice bath for 30 min, then
cooled back to -78 °C. Amide 16 (0.70 g, 3.16 mmol, prepared by
acylation of pseudoephedrine with propionyl chloride) was added over
10 min, and the solution was slowly warmed to room temperature over
1.5 h and stirred an additional 15 min, then cooled to -40 °C. The 1-O-
TBS-9-iododecane 17 (1.75 g, 4.39 mmol) was added dropwise, and the
mixture was allowed to warm to room temperature overnight with
stirring. The mixture was poured into saturated NH4Cl solution (75
mL), and the organic layer was separated. The aqueous layer was
extracted with EtOAc (4 ꢀ 50 mL), the combined organic layers were
dried (MgSO4), the solvent was removed under reduced pressure, and
the crude product was purified by flash chromatography (SiO2, 1:3
EtOAc/hexanes) to give the amide 15 (1.20 g, 77% yield): [R]22D -36.7
(c 3.1, CHCl3); FTIR (ATR) ν 3376, 2926, 2854, 1620, 1464, 1408,
1254, 1099, 1052, 835, 775, 701 cm-1; 1H NMR (400 MHz, CDCl3) δ
7.38-7.23 (m, 5H), 4.61 (t, J = 7.2 Hz, 1H, major), 4.59 (m, 1H, minor),
4.39 (brs, 1H, major), 4.08 (p, J = 8.0 Hz, 1H, minor), 3.59 (t, J = 6.4 Hz,
2H, major), 3.58 (t, J = 6.4 Hz, 2H, minor), 2.91 (s, 3H, minor), 2.84 (s,
3H, major), 2.78 (t, J = 6.4 Hz, 1H, minor), 2.58 (sex, J = 7.2 Hz, 1H,
major), 2.29 (brs, 1H, minor), 1.71 (brs, 1H, major), 1.60-1.46 (m),
1.31-1.19 (m), 1.14 (d, J = 6.8 Hz, 3H, major), 1.08 (d, J = 6.8 Hz, 3H,
Olefin 19. n-BuLi (1.2 M in hexanes, 143 uL, 0.171 mmol) was added
to a slurry of MePPh3Br (65 mg, 0.183 mmol) in THF (0.5 mL) at 0 °C;
then the mixture was stirred at room temperature for 5 min. The
aldehyde 19a (40 mg, 0.122 mmol) in THF (0.5 mL) was added, and
stirring continued for 2 h at room temperature. Saturated NH4Cl
solution (1 mL) was added followed by ether (2 mL), and the layers
were separated. The aqueous layer was extracted with ether (2 ꢀ 2 mL),
the combined organic extracts were dried (MgSO4) and concentrated,
and the residue was purified by flash chromatography (SiO2, hexanes) to
give the chiral olefin 19 (35 mg, 88% yield): [R]23D þ6.5 (c 2.8, CHCl3);
FTIR (ATR) ν 2925, 2854, 1463, 1254, 1099, 994, 909, 834, 773 cm-1; 1H
NMR (400 MHz, CDCl3) δ 5.69 (ddd, J = 17.2, 10.0, 7.2 Hz, 1H), 4.94
(ddd, J = 17.2, 2.0, 1.2 Hz, 1H), 4.89 (ddd, J = 10.0, 2.4, 1.2 Hz, 1H), 3.51 (t,
J = 6.8 Hz, 2H), 2.10 (m, 1H), 1.50 (p, J = 7.2 Hz, 1H), 1.25 (m, 16H), 0.97
(d, J = 6.8 Hz, 3H), 0.89 (s, 9H), 0.05 (s, 6H); 13C NMR (100 MHz,
CDCl3) δ 145.1 (CH), 112.2 (CH2), 63.3 (CH2), 37.8 (CH), 36.7 (CH2),
32.9 (CH2), 29.8-29.4 (5 ꢀ CH2), 27.2 (CH2), 26.0 (3 ꢀ CH3), 25.8
(CH2), 20.2 (CH3), 18.4 (C), -5.3 (2 ꢀ CH3); HRESIMS m/z 327.3081
[M þ H]þ (calcd for C20H43O2SiNa, 327.3078).
major), 1.02 (d, J = 6.8 Hz, 3H, minor), 0.89 (s, 9H), 0.04 (s, 6H); 13
C
NMR (75 MHz, CDCl3) δ 179.3 (C), 142.6 (C),128.3 (CH), 127.5
(CH), 126.3 (CH), 76.5 (CH), 63.3 (CH2), 36.6 (CH), 34.0 (CH2),
32.9 (CH2), 29.9-29.4 (5 ꢀ CH2), 27.4 (CH2), 26.0 (3 ꢀ CH3), 25.8
(CH2), 18.4 (C), 17.3 (CH3), 14.5 (CH3), -5.3 (2 ꢀ CH3); HRESIMS
m/z 514.3685 [M þ Na]þ (calcd for C29H53NO3SiNa, 514.3687).
Alkene 20. A mixture of alkene 19 (2 mg, 6.12 mmol) and
2-methoxy-6-vinylnaphthalene (11.3 mg, 61.2 mmol) was treated with
Grubbs’ second-generation catalyst (1 mg, 1.22 mmol) in CH2Cl2 (3
mL) at 70 °C in a sealed vial for 12 h. Additional catalyst (1 mg, 1.22
mmol) was added, and the mixture was stirred an additional 6 h at 80 °C.
The solvent was removed under a stream of N2, and the mixture was
passed through a reversed-phase (C18) silica cartridge (CH3CN then
MeOH), followed by HPLC (reversed-phase, C8, 85% CH3CN/H2O)
Alcohol 18. BH3 THF complex (1 M in THF, 3.0 mL, 3 mmol) was
3
added to pyrrolidine (246 mL, 3 mmol) at 0 °C, and the solution was
warmed to room temperature and stirred for 45 min. The solution was
cooled to 0 °C and treated, dropwise, with n-BuLi (2.21 M in hexanes,
1.36 mL, 3 mmol) with stirring for an additional 30 min. The amide 15
(500 mg, 1 mmol) in THF (10 mL) was added and stirred at room
temperature overnight. HCl (3 M, 5 mL) was added to quench the
excess hydride, and the layers were separated. Ether (5 mL) was added to
the aqueous layer, and the mixture cooled to 0 °C before being made
basic (pH 9-10) by addition of 2 N NaOH. The aqueous mixture was
extracted with ether (3 ꢀ 5 mL), and the combined organic extracts were
washed with 1:1 brine/1 N NaOH (2 ꢀ 10 mL) and dried (Na2SO4).
The solvent was evaporated and subjected to flash chromatography
(SiO2, 12.5% EtOAc/hexanes) to give the alcohol 18 (135 mg, 41%
yield): [R]23D -5.5 (c 2.1, CHCl3); FTIR (ATR) ν 3340, 2926, 2855,
1464, 1254, 1102, 1041, 835, 775 cm-1; 1H NMR (400 MHz, CDCl3) δ
3.59 (t, J = 6.4 Hz, 2H), 3.50 (dd, J = 10.0, 5.6 Hz, 1H), 3.41 (dd, J = 10.0,
6.8 Hz, 1H), 1.59 (m, 1H), 1.50 (p, J = 6.8 Hz, 2H), 1.41-1.26
to give alkene 20 (1.3 mg, 42%): [R]22 þ31.1 (c 1.0, CHCl3); UV
D
(CH3CN) 246 nm (log ε 4.62), 292 (4.20); CD (CH3CN) λ 255 nm
(Δε þ3.8); FTIR (ATR) ν 2925, 2853, 1256, 1100, 1035, 837, 777 cm-1
;
1H NMR (500 MHz, CDCl3) δ 7.68 (d, J = 9.0 Hz, 1H), 7.66 (d, J = 9.0,
1H), 7.62 (brs, 1H), 7.55 (dd, J = 9.0, 2.0 Hz, 1H), 7.11 (dd, J = 9.0. 2.0
Hz, 1H), 7.10 (brs, 1H), 6.46 (d, J = 16 Hz, 1H), 6.16 (dd, J = 16.0, 8.5
Hz, 1H), 3.91 (s, 3H), 3.59 (t, J = 7.0 Hz, 2H), 2.32 (sep, J = 6.5 Hz, 1H),
1.50 (m, 2H), 1.38 (m, 2H), 1.27 (m, 14H), 1.10 (d, J = 6.5 Hz, 3H), 0.88
(s, 9H), 0.04 (s, 6H); 13C NMR (125 MHz, CDCl3) δ 157.4 (C), 136.6
(CH), 133.7 (C), 133.4 (C), 129.3 (CH), 129.1 (C), 128.0 (CH), 126.9
(CH), 125.1 (CH), 124.2 (CH), 118.8 (CH), 105.8 (CH), 63.3 (CH2),
55.3 (CH3), 37.4 (CH), 37.2 (CH2), 32.9 (CH2), 29.8-29.4 (5 ꢀ CH2),
27.4 (CH2), 26.0 (3 ꢀ CH3), 25.8 (CH2), 20.8 (CH3), 18.4 (C), -5.3
439
dx.doi.org/10.1021/np1008637 |J. Nat. Prod. 2011, 74, 430–440

Journal of Natural Products
ARTICLE
(2 ꢀ CH3); HRESIMS m/z 482.3573 [M]þ (calcd for C31H50O2Si,
(10) Myers, A. G.; Yang, B. H.; Chen, H.; Gleason, J. L. J. Am. Chem.
Soc. 1994, 116, 9361–9362.
482.3575).
(11) Prepared from 1,10-decanediol. Tully, S. E.; Cravatt, B. F. J. Am.
Chem. Soc. 2010, 132, 3264–3265.
(12) Fisher, G. B.; Fuller, J. C.; Harrison, J.; Alvarez, S. G.; Burkhardt,
E. R.; Goralski, C. T.; Singaram, B. J. Org. Chem. 1994, 59, 6378–6385.
(13) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092–4096.
’ ASSOCIATED CONTENT
Supporting Information. 1Hand13CNMRand2DNMR
S
b
spectra of 1-3, 13C NMR spectra of 10, 11, 22, and 23, 1H NMR
spectra of 12, 14, 24, 25, and 30, and 1H and 13C NMR of all new
synthetic intermediates (15, 18, 19a, 19, and 20). This material is
available free of charge via the Internet at http://pubs.acs.org.
(14) Zhao, N.; Kumar, N.; Neuenschwander, K.; Nakanishi, K.;
Berova, N. J. Am. Chem. Soc. 1995, 117, 1844–1845.
(15) The absolute configuration of (-)-perhaliclonacyclamine was
established from X-ray single-crystal structure determination of the
parent compound, (þ)-tetradehydrohaliclonacyclamine A, obtained
with Cu KR radiation and interpretation of anomalous scattering
(Bijvoet method). Mudianta, I. W.; Katavic, P. L.; Lambert, L. K.; Hayes,
P. Y.; Banwell, M. G.; Munro, M. H. G.; Bernhardt, P. V.; Garson, M. J.
Tetrahedron 2010, 66, 2752–2760.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ1 (858) 534-7115. Fax: þ1 (858) 822-0386. E-mail:
tmolinski@ucsd.edu.
(16) It is worth recalling that it is not possible, strictly, to assign
absolute configuration to chiral compounds by comparison of signs of
[R]D, alone, except for enantiomers, even those as similar as 1-3 and
26. Two counter-examples are prescient and sufficient to illustrate this
often ignored tenet in stereochemistry. The paired compounds (-)-
perhydrohaliclonacyclamine (26) and the parent (þ)-tetradehydroha-
liclonacyclamine A (31), and (-)-haliclonacyclamine A (6) ([R]D = -
3.4) and (þ)-haliclonacyclamine B (7) ([R]D = þ3.4), differ only in the
position of a single double bond in the lower linking chain, yet the pairs
show opposite signs of specific rotation. Also, the absolute configuration
of (-)-26 ([R]D = -20.9])15 was shown to be antipodal to that of (-)-
6 despite the same sign of [R]D. Lastly, the signs of [R]D of alkaloid salts
and their parent free bases may also differ.
’ ACKNOWLEDGMENT
We thank S. Zea (INVEMAR, Universidad Nacional de
Colombia) for identification of the sponge, J. R. Pawlik (Univer-
sity of North Carolina, Wilmington), the captain and crew of the RV
Seward Johnson for logistical support during collecting expeditions,
and D. Dalisay for assistance with biological assays. The NSF
Biological Oceanography Program (OCE-0095724, 0550468
to J.R.P.) is acknowledged for ship time, and NSF (CRIF,
CHE0741968) for funds to acquire the 500 MHz NMR
spectrometer. This work was supported by grants from NIH
(CA122256 and AI039987 to T.F.M.) and a Ruth L. Kirschstein
National Research Service Award NIH/NCI (T32 CA009523 to
B.I.M.).
(17) We are grateful to Professor Mary Garson, University of
Queensland, for the generous gift of 26.
(18) We are grateful to Professor Roberto Berlinck, University of
S~ao Paulo, for the generous gift of 9.
(19) The chair-chair conformations and N-configurations adopted by
the bis-piperidine ring systems in the pairs 25 and 29 and 27 and 30 are
preserved with respect to their starting materials (see Supporting Informa-
tion for assignment of conformation of 25 using NOESY data). Although the
Br-phenacyl substitutents are disposed differently—equatorial/axial on rings
A and B of 25 and 29 and axial/axial for 27 and 30—examination of
molecular models shows the C-N bond vectors subtend similar angles (θ
∼90°) in all four derivatives. This explains why the diastereomeric variants
give the same CD spectra, because helicity and the consequent exciton
coupling is dictated largely by the orthogonality of the piperidine ring planes.
It is likely that the reactive conformers of each bis-piperidine ring require an
equatorial lone pair and, consequently, a ring flip for the B ring in all four
starting materials and an A ring flip in 22 then 26 (see Figure 2). Reaction of
22, with both lone pairs on N in the axial orientation, was particularly sluggish
and required more forcing conditions (microwave reactor, 140 °C) com-
pared to 10, 26, and 28 (90 °C), possibly a consequence of torsional
constraints imposed on 22 for the A ring flip (two less CH2 groups in the
“northern” linker chain) compared to the more flexible 26.
’ DEDICATION
Dedicated to Dr. Koji Nakanishi of Columbia University for his
pioneering work on bioactive natural products.
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