Synthesis and bioactivity of (±)-tetrahydrohaliclonacyclamine A

Article abstract of DOI:10.1016/j.tet.2010.03.117

The total synthesis of tetrahydrohaliclonacyclamine A (5) is described. A key step involves the hydrogenation of an unsaturated bis-piperidine incorporated into a 17-membered macrocycle to provide the cis-syn-cis stereochemistry common to haliclonacyclamines A-D. The hydrogenation product is advanced to the title compound following a five-step reaction sequence. Tetrahydrohaliclonacyclamine A is shown to bind to a variety of ion channels/GPCRs and act as a muscarinic M₁ antagonist.

Full text of DOI:10.1016/j.tet.2010.03.117

Tetrahedron 66 (2010) 4805–4810
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and bioactivity of (ꢀ)-tetrahydrohaliclonacyclamine A
,
Brian J. Smith a, Tao Qu b d, Matthew Mulder b, Meredith J. Noetzel b,
,
,c,
*
Craig W. Lindsley a b, Gary A. Sulikowski a
a Departments of Chemistry, Institute of Chemical Biology, Vanderbilt University, Nashville, TN 77842–3012, USA
b Departments of Pharmacology, Institute of Chemical Biology, Vanderbilt University, Nashville, TN 77842–3012, USA
c Departments of Biochemistry, Institute of Chemical Biology, Vanderbilt University, Nashville, TN 77842–3012, USA
d Departments of Chemistry, Texas A&M University, College Station, Texas 77482, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The total synthesis of tetrahydrohaliclonacyclamine A (5) is described. A key step involves the hydro-
genation of an unsaturated bis-piperidine incorporated into a 17-membered macrocycle to provide the
cis–syn–cis stereochemistry common to haliclonacyclamines A–D. The hydrogenation product is ad-
vanced to the title compound following a five-step reaction sequence. Tetrahydrohaliclonacyclamine A is
shown to bind to a variety of ion channels/GPCRs and act as a muscarinic M1 antagonist.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 28 January 2010
Received in revised form 29 March 2010
Accepted 30 March 2010
Available online 3 April 2010
Keywords:
Marine alkaloid
Ring-closing metathesis
Stereoselective hydrogenation
Stille cross-coupling
Muscarinic M1 antagonist
1. Introduction
The alkylpiperidine alkaloids are a group of natural products
isolated primarily from marine sponges and biosynthetically derived
from nicotinic acid.1,2 The simplest family members are monomeric
and dimeric 3-alkylpyridines. Architecturally unique 3-alkylpiper-
idine alkaloids incorporate an array of ring systems including tri-
cyclic, tetracyclic, and pentacyclic structures. Pentacyclic members
have attracted attention from synthetic chemists culminating in the
total syntheses of the manzamines and (ꢁ)-sarain A.3,4 Little prog-
ress has been reported toward the total synthesis of tetracyclic class
of alkylpiperidines such as haliclonacyclamines A–D (Fig. 1) despite
their unique molecular structure and reported biological activity.5
Tetracyclic alkaloids possess the general structure of a 3,4-linked
bis-piperidine core appended to two aliphatic macrocycles as rep-
resented by the halicyclamines,6 haliclonacyclamines,7 areno-
sclerins,8 and halichondramine.9 Over sixteen tetracyclic class
members have been isolated from a variety of marine sponges with
the haliclonacyclamines constituting the largest sub-group.
Haliclonacyclamines A–D (1–4) share a common molecular consti-
tution incorporating a core 3,4-bis-piperidine fused to ten (N1 to C7, 1)
and twelve (N2 to C2, 1) carbon bridges. Haliclonacyclamines A–D
Figure 1.
differ in the number and position of double bonds located within
bridging carbons. The bis-piperidine core of haliclonacyclamines
A–D possess identical relative stereochemistry that we describe as
cis–syn–cis configuration; cis denoting relative stereochemistry
* Corresponding author. E-mail address: gary.a.sulikowski@vanderbilt.edu
(G.A. Sulikowski).
0040-4020/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.03.117

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B.J. Smith et al. / Tetrahedron 66 (2010) 4805–4810
within the piperidine rings and syn across C9 and C3 stereocenters
interconnecting the piperidine rings (haliclonacyclamine number-
ing). Recently the absolute stereochemistry of haliclonacyclamines A
and B has been assigned by Garson and co-workers as shown in
Figure 1 based on single-crystal X-ray analysis.7c The Garson group
has demonstrated hydrogenation of haliclonacyclamines A–C lead
Our first approach to macrocycle
7 started from known
b
-ketoester 12,14 which was converted to stannane 14 by way of
intermediate enol triflate 13. To this end, the potassium enolate
derived from 12 was reacted with Comins’ triflating agent15 and the
resulting unsaturated ester reduced with DIBAL-H to afford the
corresponding allyl alcohol that was protected as a TBS ether (13).
Stille coupling of enol triflate 13 with hexamethyldistannane pro-
vided 14 in 43–65% yield.16 The latter was coupled with iodoenamide
1510 to provide bis-piperidine 16 in 67% yield (Scheme 1).17 The TBS
ether was then exchanged for an acetate (TBAF, THF, 0 ꢂC then Ac2O,
TEA, 86%) to set the stage for a third Stille cross-coupling this time
between 17 and vinyl stannane 1818 to provide 19 in 84% yield.
a
common reduction product, tetrahydrohaliclonacyclamine
A (5, Fig. 2), thereby confirming a common bis-piperidine core.7a,b
Herein we provide a full account of our synthesis of the tetrahy-
drohaliclonacyclamine A (5) and preliminary studies into its activity
against select ion channels and GPCR’s.10
Figure 2.
2. Results and discussion
Our synthetic strategy to tetrahydrohaliclonacyclamine A (5) is
shown retrosynthetically in Figure 2. We anticipated tetrahy-
drohaliclonacyclamine A (5) to be derived from bis-alkene 6 by
employing a ring-closing metathesis reaction followed by alkene
reduction.11 In order to set the cis–syn–cis relative stereochemistry of
the bis-piperidine core we elected to examine the hydrogenation of
a 17-membered macrocycle (7). While the stereoselective hydroge-
nation of a macrocyclic diene represented by 7 was unprecedented,
this approach was selected based in part upon the observed stereo-
selective hydrogenation of diene 10 that led to 11 as a single isomer
(C9 stereochemistry not assigned).12 Furthermore, we anticipated
incorporation of an equivalent 1,3-diene into a 17-membered mac-
rocycle (7) would enforce peripheral hydrogenation leading to the
desired cis–syn–cis relationship C2/C3 and C7/C9.13 Assembly of the
macrocyclic 7 was envisioned to be initiated by Stille cross-coupling
of a vinyl halide (8) and stannane (9) (Fig. 2).
Scheme 1. First approach to 17-membered macrocycle.
Our initial approach to fashioning the 17-memberd macrocycle
was to alkylate the enolate derived from 19 with (E,E)-dibromide
2019 followed by Boc removal and intramolecular N-alkylation.
Unfortunately, while enolate alkylation proceeded in 41% yield we
were unable to remove the Boc protecting group under a variety of
acidic and thermal conditions observing instead decomposition.
Rather then examining other nitrogen protecting groups we turned
our attention to a ring-closing metathesis (RCM) strategy to form
the key 17-membered macrocycle (Scheme 2).
To this end, bis-piperidine 27, the substrate required for the RCM
reaction leading to 17-membered ring formation, was prepared
starting from iodoenamide 22 and vinyl stannane 23.10 The four-
step reaction sequence leading to 27 utilized two Stille cross-
coupling reactions in a manner identical to the coupling series
described earlier in Scheme 1. With all carbons now in place we
turned our attention to formation of the 17-membered macrocycle
spanning C7 and the southern piperidine nitrogen. Initially, we
found the RCM reaction failed to proceed using the free amine,
leading primarily to unreacted starting material. Fortunately, when
the derived hydrochloride salt of 27 was treated with Fu¨rstner’s

B.J. Smith et al. / Tetrahedron 66 (2010) 4805–4810
4807
Scheme 2.
olefin, leading to a tentative assignment of the reduction products as
cis–syn–cis (29a) and cis–anti–cis (29b). The non-stereoselective re-
duction of the C2–C3 olefin of the southern piperidine is attributed
to the incorporation of a stereochemically dynamic tertiary amine
that due to rapid inversion equally exposes the diastereotopic faces
of the C2–C3 to the periphery of the macrocyclic structure and hy-
drogen addition (Scheme 2).
The mixture of diols (29) was oxidized with Dess–Martin peri-
odinane to give a moderately stable bis-aldehyde and without
purification was treated with 10 equiv of methylene-
triphenylphosphorane to provide bis-alkene 31 (Scheme 3). The
tetracyclic ring system common to the haliclonacyclamines was
completed upon treatment of the TFA salt derived from 31 with
Grubbs’ first-generation catalyst to provide 32 (6:1 trans/cis) and 33
(>95:5 trans/cis), separated by flash chromatography. Each isomer
was subjected to alkene hydrogenation (100 psi, Pd(OH)2, EtOH)
followed by lactam reduction (Red-Al, PhMe, reflux). Hydrogena-
tion of 32 provided lactam 34, which was subjected to a single-
crystal X-ray analysis revealed the cis–syn–cis stereochemistry of
the bis-piperidine core common to haliclonacyclamines A–D
(Fig. 1).10 Indeed Red-Al reduction of 34 followed by passing the
reaction product thru and SCX column provided tetrahy-
drohaliclonacyclamine A (5), identical to an authentic sample by 1H
and 13C NMR comparison.22 The cis–anti–cis isomer (33) was con-
verted to amine 36 by way of the same two-step reduction process
(Scheme 3). The stereochemistry of amine 36 was assigned based
on NMR analysis. Examination of the NOSY spectrum of 36 revealed
significant NOE between H7–H9 and H2–H3 (i.e., cis stereochem-
istry). This observation supported the stereochemical assignment
of bis-piperidine core of 36 as cis–anti–cis; partly by the process of
elimination as isomer 5 possessed the cis–syn–cis relationship.
Haliclonacylamines A and B have been reported to display anti-
biotic, antifungal, and cytotoxic properties.7a Berlinck and co-workers
have also reported on the antibiotic and cytotoxic properties of hal-
iclonacyclamine E and structurally related arenosclerins A–C.23 We
submitted the bis-TFA salt of tetrahydrohaliclonacyclamine A (5) to
a panel of ion channel and GPCR receptors for evaluation in radio-
Scheme 3.
ruthenium indenylidene catalyst in refluxing dichloromethane
macrocyle 28 was obtained in 80% yield (>90% trans).20 The RCM
reaction proceeded with equal efficiency when employing Grubbs
first-generation catalyst.21
With the key 17-membered macrocycle in hand, we next exam-
ined a range of reductive conditions to effect benzyl ether hydro-
genolysis and alkene saturation of macrocycle 28. Homogeneous
hydrogenation of 28 employing Wilkinson catalysis primarily pro-
duced starting material accompanied by hydrogenation of one al-
kene as determined by LC/MS analysis. Hydrogenation of the TFA salt
of 28 under one atmosphere hydrogen in methanol over Pearlman’s
catalyst led to variable results. However, when the same reaction was
repeated in ethanol at 500 psi at a temperature of 70 ꢂC for 8 days
a reproducible 1.3:1 mixture of two isomers tentatively assigned the
structures of 29a and 29b was produced in 79% yield. It was further
determined if the reduction was prematurely stopped enamide 30
was isolated as an approximate 1:1 mixture of 30a and 30b. These
observations led us to conclude hydrogenation of the C2–C3 carbon–
carbon double bond occurred first in a non-stereoselective fashion
followed by a stereoselective reduction of the C9–C10 enamide
ligand biochemical assays.24 At a concentration of 10
mM tetrahy-
drohaliclonacyclamine A (5) inhibited radioligand bindingof avariety
of receptors including muscarinic M1 (82%), opiate k (83%), and

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B.J. Smith et al. / Tetrahedron 66 (2010) 4805–4810
potassium channel hERG (98%). Since ligand binding assays provide
no information on functional activity (i.e., agonist or antagonist ef-
fects), we then performed a functional assay on the hM1 mAChR with
tetrahydrohydrohaliclonacyclamine A and the unnatural cis–anti–cis
isomer (36). Interestingly, tetrahydrohaliclonacyclamine A (5) was
found to be a full antagonist (ACh EC80 reduced to baseline) of hM1
extracts were dried over MgSO4, filtered, and concentrated. The
residue was purified by flash column chromatography on silica gel
(gradient elution: 2:1 to 1:1 to 1:1:0.01 hexanes/ethyl acetate/
triethylamine) to yield bis-piperidine 24 (1.90 g, 67%) as a yellow
oil: IR (neat) 2930, 2855, 1670, 1403 cmꢁ1
;
1H NMR (CDCl3,
300 MHz)
d
7.32–7.26 (m, 5H), 5.91 (s, 1H), 5.79 (m, 2H), 4.94
(albeit with modest potency, EC50¼5
m
M). The isomeric bis-
(m, 4H), 4.47 (s, 2H), 4.13 (s, 2H), 3.43 (m, 4H), 3.04 (m, 2H), 2.54
(m, 2H), 2.40–2.36, (m, 2H), 2.20 (m, 2H), 2.10–2.02 (m, 5H), 1.82,
(m, 2H), 1.62–1.50 (m, 6H), 1.43–1.31 (m, 8H), 0.88 (s, 9H), 0.02
piperidine (36) was a partial antagonist (ca. 60% of an ACh EC80
)
with similar potency.
(s, 6H); 13C NMR (CDCl3, 100 MHz)
d 171.1, 138.8, 138.6, 138.5, 131.0,
3. Conclusion
130.3, 128.3, 127.5, 127.4, 126.8, 116.7, 114.4, 114.3, 72.8, 70.1, 62.5,
58.4, 54.8, 50.2, 46.2, 40.4, 33.6, 33.5, 29.8, 29.3, 29.1, 28.9, 28.8,
28.5, 26.8, 26.6, 26.5, 25.8, 23.3, 18.2, ꢁ5.8. HRMS calculated for
C41H67N2O3Si (MþH)þ m/z: 663.4954, measured: 663.4921.
The synthesis of the tetrahydrohydrohaliclonacyclamine A (5)
and the unnatural cis–anti–cis isomer (36) have been completed in
16 steps (longest linear sequence). The former was determined to
bind to a range of ion channels and receptors, likely related to its
4.3. Stille cross-coupling of 25 and 26
reported cytotoxic properties. In
a functional assay tetrahy-
drohaliclonacyclamine A was shown to act as an antagonist against
the muscarinic M1 receptor.
To a solution of allylic acetate 25 (2.50 g, 4.23 mmol) in dime-
thylformamide (35 mL) at room temperature was added (E)-6-(trib-
utylstannyl)hex-5-en-1-ol (26) (2.47 g, 6.34 mmol), lithium chloride
(888 mg, 21.2 mmol), and bis(dibenzylidieneacetone)palladium
(245 mg, 0.42 mmol). The mixture was heated to 65 ꢂC and stirred for
22 h total with 0.01 equiv of bis(dibenzylidieneacetone)palladium
(24.5 mg, 0.04 mmol) added after 14 h. The reaction was filtered
through Celite and the filtrate was washed with brine (20 mL). The
aqueous layer was extracted with ethyl acetate (3ꢃ40 mL) and the
combined organic extracts were washed with brine (2ꢃ20 mL), dried
over MgSO4, filtered, and concentrated. The residue was purified by
flash column chromatography on silica gel (gradient elution with 2:1
to 1:1:0.01 hexanes/ethyl acetate/triethylamine) to yield tetraene 27
(2.13 g, 80%) as a yellow oil. IR (neat) 3625, 2928, 2856, 1666,
4. Experimental
4.1. General
All non-aqueous reactions were performed in flame-dried or
oven dried round-bottomed flasks under an atmosphere of argon.
Reaction temperatures were controlled using a thermocouple ther-
mometer and analog hotplate stirrer. Reactions were conducted at
room temperature (rt, approximately 23 ꢂC) unless otherwise noted.
Flash column chromatography was conducted using silica gel 230–
400 mesh. Where necessary, silica gel was neutralized by treatment
of the silica gel prior to chromatography with the eluent containing
1% triethylamine. Where indicated ammonium salts were converted
to free-amines using Strong Cation Exchange (SCX) cartridges pur-
chased from Varian. Analytical thin-layer chromatography (TLC) was
performed on E. Merck silica gel 60 F254 plates and visualized using
UV, ceric ammonium molybdate, potassium permanganate, and
anisaldehyde stains. Yields were reported as isolated, spectroscopi-
cally pure compounds. 1H NMR spectra were recorded on Bruker
300, 400, 500, or 600 MHz spectrometers and are reported relative
to deuterated solvent signals. 13C NMR spectra were recorded on
Bruker 75, 100, 125, or 150 MHz spectrometers and are reported
relative to deuterated solvent signals. LC/MS was conducted and
recorded on an Agilent Technologies 6130 Quadrupole instrument.
High-resolution mass spectra were obtained from the Department of
Chemistry and Biochemistry, University of Notre Dame using either
a JEOL AX505HA or JEOL LMS-GCmate mass spectrometer or from
Vanderbilt Institute of Chemical Biology Drug Discovery Program
laboratory using a Waters Acquity UPLC and Micromass Q-Tof Ultima
API.
1404 cmꢁ1 1H NMR (CDCl3, 300 MHz)
; d 7.30–7.24 (m, 5H), 5.85
(s, 1H), 5.78 (m, 2H), 5.00–4.92 (m, 4H), 4.46 (s, 2H), 3.57 (m, 2H),
3.43–3.32 (m, 4H), 2.88 (m, 2H), 2.73 (m, 2H), 2.52, (m, 2H), 2.39–2.35
(m, 3H), 2.18 (m, 2H), 2.18, (m, 2H), 2.07–2.02 (m, 6H), 1.62–1.50
(m,10H),1.41–1.33 (m,12)H; 13C NMR (CDCl3,100 MHz)
d 171.1, 138.8,
138.6, 138.5, 134.4, 129.3, 129.0, 128.5, 128.3, 127.6, 127.5, 125.8, 117.6,
114.4,114.3, 72.8, 70.1, 62.5, 58.3, 56.0, 50.4, 46.2, 40.5, 35.5, 33.5, 33.5,
32.2, 29.8, 29.6, 29.4, 29.2, 28.8, 28.5, 27.8, 26.9, 26.8, 26.5, 25.6, 23.3,
17.5, 13.5. HRMS calculated for C41H63N2O3 (MþH)þ m/z: 631.4839,
measured: 631.4850.
4.4. Ring-closing metathesis of 27
A hydrochloric acid solution (5 mL of 2 M solution in ether) was
added to a solution of tetraene 27 (220 mg, 0.35 mmol) in
dichloromethane (5 mL), and the resulting solution was stirred for
30 min at ambient temperature. The solution was then concen-
trated and dried in vacuo. The viscous, bright yellow hydrochloride
salt was dissolved in dichloromethane (1 L) and bis(tricyclohex-
ylphosphine)-3-phenyl-1H-inden-1-ylidene ruthenium(II) dichlor-
ide (32 mg, 0.035 mmol) was quickly added in one portion. The
solution was brought to reflux for 2 h, at which point an additional
(32 mg, 10 mol %) of the catalyst was added. The solution was
maintained at reflux for an additional 20 h, cooled to room tem-
perature and concentrated. The resulting residue was dissolved in
methanol and passed through a Varian SCX ion exchange column to
remove ruthenium byproducts, followed by eluting diene 28 with
2 N ammonia in methanol to release the free amine. Additionally,
the residue was purified by Biotage (KP-C18-HS) reverse phase
chromatography eluting with H2O (0.1% TFA)/Acetonitrile (20–60%
acetonitrile over 10 column volumes). The aqueous fractions were
concentrated by Genevac and the resulting material analyzed by LC/
MS to yield 134 mg, 64% of diene 28. The material was typically kept
as the trifluoroacetic acid salt for use in the next step. The salt was
converted to the free amine passage through a Varian SCX ion
4.2. Stille cross-coupling of 22 and 23
A solution of vinyl stannane 22 (2.47 g, 5.23 mmol) and vinyl
iodide 23 (2.05 g, 4.26 mmol) in dimethyl sulfoxide (38 mL) was
treated with copper chloride (2.26 g, 22.8 mmol), lithium
chloride (1.15 g, 27.3 mmol, flame dried under argon), and tetra-
kis(triphenylphosphine)palladium (526 mg, 0.46 mmol) at room
temperature. The mixture was immediately degassed (3ꢃ) under
high vacuum with an argon purge. The mixture was then warmed
to room temperature and stirred for 2 h, followed by heating to
60 ꢂC for 14 h. The black mixture was quenched with brine, and the
resulting solution was filtered over Celite. The filtrate was washed
with brine (100 mL) and 5% ammounium hydroxide (5 mL),
extracted with ethyl acetate (4ꢃ50 mL), and the combined organic
extracts were washed again with brine (25 mL). The combined

B.J. Smith et al. / Tetrahedron 66 (2010) 4805–4810
4809
exchange column for calculation of yield and analytical data. IR
(neat) 3530, 2932, 2846, 1666 cmꢁ1 1H NMR (CDCl3, 400 MHz)
7.30–7.24 (m, 5H), 5.87 (s, 1H), 5.44–5.30 (m, 4H), 4.46 (s, 2H),
(m, 1H), 2.71–2.58 (m, 4H), 2.37 (m, 1H), 2.25 (m, 1H), 2.07–1.95
(m, 7H), 1.69 (m, 1H), 1.71 (m, 2H), 1.54 (m, 4H), 1.42–1.24 (m, 29H);
;
d
13C NMR (CDCl3, 150 MHz)
d 171.8, 131.0, 130.8, 57.0, 54.7, 52.3, 47.7,
3.62–3.57 (m, 2H), 3.44 (app. t, J¼6.4 Hz, 2H), 3.32 (m, 2H), 3.06
(m, 2H), 2.73 (m, 2H), 2.60 (m, 2H), 2.50, (m,1H), 2.37 (m, 2H), 2.07–
2.02 (m, 6H), 1.61–1.52 (m, 10H), 1.42–1.35 (m, 7H), 1.23 (m, 6H),
46.3, 42.7, 42.0, 41.4, 36.0, 35.2, 34.2, 33.1, 32.4, 31.6, 31.3, 29.6, 28.4,
28.2, 27.4, 27.3, 27.2, 27.1, 27.1, 26.8, 26.5, 26.3, 26.2, 25.6, 21.6.
HRMS calculated for C32H57N2O (MþH)þ m/z: 485.4471, measured:
485.4471.
0.88–0.83 (m, 2H); 13C NMR (CDCl3, 100 MHz)
d 172.2, 133.1, 132.7,
132.0, 131.9, 130.8, 130.7, 130.6 (2C), 128.7, 128.2, 128.0, 127.9, 73.3,
70.5, 62.8, 51.8, 46.4, 41.1, 36.2, 32.8, 32.7, 31.9, 31.8, 31.0, 30.1 (2C),
29.8, 28.9 (2C), 27.6, 27.5, 26.2, 26.1 (2C), 25.1, 23.8. HRMS calculated
for C39H59N2O3 (MþH)þ m/z: 603.4526, measured: 603.4528.
Compound 33: light yellow oil: IR (neat) 2923, 2851, 1644 cmꢁ1
;
1H NMR (CDCl3, 600 MHz)
d 5.31–5.25 (m, 2H), 4.14 (m, 1H), 3.18,
(m, 1H), 2.99 (app. t, J¼11.4 Hz, 1H) 2.77 (m, 1H), 2.68 (m, 3H), 2.55
(m, 1H), 2.37 (app t, J¼11.4 Hz, 1H), 2.29 (m, 2H), 2.17 (m, 1H), 2.05–
1.97 (m, 5H), 1.84 (m, 3H), 1.51 (m, 3H),1.33–1.29 (m, 32H); 13C NMR
4.5. Hydrogenation of 28
(CDCl3, 150 MHz) d 171.7, 131.1, 130.1, 56.7, 55.3, 52.4, 47.4, 47.1, 41.0,
40.8, 32.3, 32.3, 31.7, 31.6, 31.4, 30.5, 29.7, 29.3, 28.3, 28.0, 27.6, 27.4,
27.3, 27.2, 27.2, 27.0, 26.9, 26.8, 26.5, 26.4, 26.0. HRMS calculated for
C32H57N2O (MþH)þ m/z: 485.4471, measured: 485.4469.
The trifluoroacetic acid salt of tetraene 28 (340.0 mg, 0.56 mmol,
free amine weight) was dissolved in ethanol (30 mL), treated with
palladium hydroxide (79.0 mg, 0.11 mmol), and transferred to a Parr
hydrogenator. Once the vessel was tightly secured, the solution was
purged with hydrogen, evacuated, and back-filled a total of five
times. The pressure was set to 500 psi and the mixturewas heated to
70 ꢂC with vigorous stirring. The progress of the reaction mixture
was monitored by LC/MS. After 8 days the reaction was filtered
through Celite and concentrated. The resulting residue was purified
by reverse phase HPLC chromatography eluting with H2O (0.1%
TFA)/Acetonitrile (12–40% acetonitrile) to afford a non-separable
mixture of 29a and 29b. Fractions were analyzed using LC/MS and
concentrated by Genevac. The products were converted to their free
amines by passing through a Varian SCX ion exchange column by
eluting first with methanol then 2 N ammonia in methanol to re-
lease 230 mg [79% yield of 29a and 29b (1.3:1 determined by 13C
NMR), characterized as a mixture]: IR (neat) 3400, 2920,1622,1494,
4.7. Tetrahydrohaliclonacyclamine A
A solution of lactam 34 (20.0 mg, 0.041 mmol) in toluene (4 mL)
was cooled to 0 ꢂC and sodium bis(2-methoxyethoxy)aluminium
hydride (130 mL, 0.410 mmol of a 65 wt.% solution in toluene) was
added dropwise. The solution was then placed into a pre-heated oil
bath at 130 ꢂC and stirred for 6 h. The resulting solution was cooled
to 0 ꢂC and slowly quenched with a saturated aqueous solution of
potassium sodium tartarate (4 mL) and stirred for 5 min. The mix-
ture was then diluted with ethyl acetate, warmed to room temper-
ature, and stirred for 30 min. The aqueous layer was extracted with
ethyl acetate (4ꢃ10 mL) and the combined extracts were dried over
MgSO4, filtered, and concentrated. The residue was purified by
passing through a Varian SCX ion exchange column by eluting first
with methanol then 2 N ammonia in methanol. After concentration,
the residue was subjected to flash column chromatography on silica
gel eluting with 3:6.5:0.5 (hexanes/ethyl acetate/triethylamine) to
yield tetrahydrohaliclonacyclamine A (4) (15.8 mg, 90%). 1H NMR
1461 cmꢁ1; 1H NMR (CDCl3, 400 MHz)
d
3.61 (app. t, J¼6.4 Hz, 4H),
3.37–3.27 (m, 2H), 3.23–3.17 (m, 1H), 3.03 (m, 1H), 2.85–2.77
(m, 3H), 2.65–2.53 (m, 5H), 2.26 (m, 1H), 2.11 (m, 2H), 1.93–1.86
(m, 3H), 1.67 (m, 1H), 1.63–1.55 (9H), 1.38–1.26 (28H); 13C NMR
(CDCl3, 100 MHz) d 172.1,171.9 (two diastereomers); 62.6 (2C); 62.3,
(CDCl3, 600 MHz)
d
2.96 (app. t, J¼11.1 Hz, 1H), 2.80–2.79 (m, 2H),
56.9, 56.8, 54.5, 53.9, 52.9, 52.2, 47.2, 47.0, 46.0, 42.0, 41.6, 41.0, 40.1,
39.9, 36.0, 34.7, 34.6, 33.7, 33.3, 32.9, 32.6, 32.5, 32.2, 31.4, 30.9, 30.6,
29.7, 29.3 (2C), 28.1, 27.9, 27.5, 27.4, 27.2, 27.1 (2C), 26.9 (2C), 26.6,
26.5, 26.3, 26.1, 25.9, 25.7 (2C), 25.3, 22.8, 21.8, 21.6. HRMS calcu-
lated for C32H61N2O3 (MþH)þ m/z: 521.4682, measured: 521.4682.
2.75–2.64 (m, 3H), 2.59 (m, 1H), 2.55–2.51 (m, 1H), 2.46–2.39
(m, 3H), 2.15 (app. t, J¼11.2 Hz, 1H), 1.95 (m, 1H), 1.85–1.76 (m, 4H),
1.72–1.69 (m, 2H), 1.53–1.50 (m, 5H), 1.39–1.20 (m, 34H), 0.92–0.88
(m, 2H); 13C NMR (CDCl3, 150 MHz)
d 60.7, 60.3, 58.4, 57.1, 53.2, 47.0,
45.5, 41.4, 38.3, 37.8, 36.4, 35.6, 34.1, 33.5, 29.3, 27.9, 27.8 (2C), 27.7,
27.6, 27.2, 27.1, 26.8 (2C), 26.5, 26.3, 26.1, 25.7 (2C), 25.6, 22.0, 21.5.
HRMS calculated for C32H61N2 (MþH)þ m/z: 473.4835, measured:
473.4833.
4.6. Ring-closing metathesis of 31
To a solution of dienes 31a/31b (20.0 mg, 0.039 mmol, free amine
weight) in dichloromethane (2.0 mL) was added trifluoroacetic acid
(two drops). The solution was stirred for 30 min and concentrated.
The residue was then dissolved in dichloromethane (250 mL) and
bis(tricyclohexylphosphine)benzylidine ruthenium(IV) chloride
(3.3 mg, 0.004 mmol) was added. The solution was refluxed for 2 h,
cooled to room temperature, and treated with additional catalyst
(3.3 mg, 0.004 mmol). This solution was heated at reflux for 16 h
and concentrated. The resulting residue was purified by a SCX ion
exchange column; eluting with methanol, then 2 N ammonia in
methanol. This residue was further purified by reverse phase HPLC
chromatography eluting with H2O (0.1% TFA)/Acetonitrile (30–60%
acetonitrile). The resulting fractions were concentrated by Genevac.
The purified TFA salt was converted to the free amine by running the
residue through an SCX ion exchange column. The fractions were
concentrated and the two pure diastereomers were separated by
flash column chromatography on silica gel (eluent 3:6.5:0.25 hex-
anes/ethyl acetate/triethylamine) to yield 9.5 mg of 26 and 5.6 mg of
27 (80% combined yield).
Acknowledgements
We thank the National Institutes of Health (GM067726) and the
Vanderbilt Institute of Chemical Biology for their support of this
research. We also thank Joseph Reibenspies (Center for Chemical
Characterization and Analysis, Texas A&M University) for de-
termining the X-ray crystal structure of 32. We gratefully ac-
knowledge Professor Mary Garson (University of Queensland) for
providing a sample of tetrahydrohaliclonacyclamine A and spectral
data.
Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2010.03.117.
References and notes
Compound 32: light yellow oil: IR (neat) 2925, 2853, 1639 cmꢁ1
1H NMR (CDCl3, 600 MHz)
5.31–5.23 (m, 2H), 4.34 (m, 1H), 3.53
(app t, J¼12.0 Hz, 1H), 2.99 (m, 1H), 2.87 (app. t, J¼10.8 Hz, 1H), 2.73
.
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