Syntheses of the cylindrospermopsin alkaloids

Article abstract of DOI:10.1016/j.tet.2006.02.044

An intramolecular 1,3-dipolar cycloaddition has efficiently constructed the A-ring portions of the cylindrospermopsin alkaloids. A nitro-aldol addition of an elaborated nitroalkane to a pyrimidine aldehyde followed by an intramolecular reductive guanidiny

Full text of DOI:10.1016/j.tet.2006.02.044

Tetrahedron 62 (2006) 4549–4562
Syntheses of the cylindrospermopsin alkaloids
Ryan E. Looper,a Maria T. C. Runnegarb and Robert M. Williamsa,
*
aDepartment of Chemistry, Colorado State University, Fort Collins, CO 80523, USA
bResearch Center for Liver Diseases, University of Southern California Medical Center, Los Angeles, CA 90033, USA
Received 14 December 2005; revised 14 February 2006; accepted 15 February 2006
Available online 13 March 2006
Abstract—An intramolecular 1,3-dipolar cycloaddition has efficiently constructed the A-ring portions of the cylindrospermopsin alkaloids.
A nitro-aldol addition of an elaborated nitroalkane to a pyrimidine aldehyde followed by an intramolecular reductive guanidinylation has
enabled the syntheses of all three alkaloids in this family in 18–19 steps. We report the first asymmetric synthesis of cylindrospermopsin,
unambiguously assigning its absolute configuration.
q 2006 Elsevier Ltd. All rights reserved.
1. Introduction
This family of toxins has been implicated in the elevated
occurrence of liver cancer in China, where surface water is
relied upon.3 They are also the only toxins implicated in
human fatalities, tragically in the death of 60 people who
received microcystin contaminated water at a hemodialysis
center in Carauru, Brazil.4 These peptides have been shown
to be highly liver specific due to their active uptake into
Among the many toxic metabolites produced by cyano-
bacteria, the hepatotoxins pose the greatest threat to human
health.1 The peptidal toxins, microcystin-LR (1, LD50Z
50 mg/kg) is an example of the cyclic hepta-peptides
first isolated from Microcytis aeruginosa (Fig. 1).2
Figure 1. Hepatotoxic cyanobacterial metabolites.
Keywords: Cycloaddition; Guanidine; Alkaloid; Cyanobacteria; Hepatotoxin.
*
Corresponding author. Tel.: C1 970 491 6747; fax: C1 970 491 3944; e-mail: rmw@chem.colostate.edu
0040–4020/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.02.044

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hepatocytes via members of the organic anion transporting
polypeptide family.5 More importantly they have been
shown to be potent inhibitors of the protein phosphatases
PP1 and PP2A.6 Nodularin (2) and motopurin (3) are related
cyclic pentapeptides, with 3 remaining one of the most
potent inhibitors of these phosphatases (IC50!1.0 nM).7
Inhibition of these enzymes is thought to cause hyper-
phosphorylation of cytoskeletal proteins leading to the
disruption of the hepatic architecture resulting in cell death
of hepatocytes and liver hemorrhage.
intense synthetic investigation.19 Snider and co-workers
completed the first racemic total synthesis of cylindros-
permopsin 8 years after its discovery.19h Their accomplish-
ments however, failed to illuminate the missasigned
stereocenter at C7, elegantly corrected by Weinreb in a
racemic but highly stereocontrolled synthesis of 5 validating
the illustrated structures.19j,k Shortly thereafter Hansen and
White were able to complete an asymmetric total synthesis
of 5, confirming the absolute stereochemistry as 7S, 8R,
10S, 12S, 13R, 14S.19l,n
Cylindrospermopsin (4) was isolated as the principal
hepatotoxin from Cylindrospermopsis raciborskii in
1992 after suspicion of its involvement in an outbreak
of hepatoenteritis that hospitalized 150 people on Palm
Island, Australia.8,9 It has since been isolated in Japan
from Umezakia natans10 and Israel from Aphanizomenon
ovalisporum.11 Following the discovery of the parent
compound, 7-epi-cylindrospermopsin (5) was isolated
from A. ovalisporum as a toxic min or metabolite.12
7-Deoxy-cylindrospermopsin (6) was initially isolated
from C. raciborskii and has recently been co-isolated with
4 in China from Raphidiopsis curvata.13 Cylindrospermop-
sin has been shown to be a potent hepatotoxin (LD50Z
0.2 mg/kg in mice), 4 is equipotent with 5 while 6 was
thought to be non-toxic.9,13a,14 Unlike 1–3, the cylindros-
permopsins do not inhibit PP1 or PP2A. Their toxicity
appears to result at least in part from the inhibition of protein
synthesis. The translation step of protein synthesis is
inhibited by the cylindrospermopsins, but the mechanism
of this inhibition is not yet known.15 Cylindrospermopsin
has also been shown, in vitro, to be a non-competitive
inhibitor (KIZ10 mM) of the uridine monophosphate
(UMP) synthase complex, although in vivo assays do not
support a general inhibition of UMP synthesis.16
2. Synthetic considerations
At the onset of this project little was known about the
mechanism of action of this family of hepatotoxins. This
encouraged us to develop an efficient and flexible synthesis
of 4. We were intrigued by the observations that while 4 and
5 are toxic and 6 is not. Cytochrome P450 oxidation
had been purported to mediate their toxicity.14c We thought
that an oxidation event at C7 or C8 may produce the enol-
guanidine 7 (Fig. 2), alternatively C15 oxidation may
generate the guanidinimine 8. Both of these intermediates
are potentially redundant through extensive tautomeriza-
tion, and both are electrophilic intermediates, perhaps
responsible for the observation that oxidized metabolites
of 4 may alkylate DNA.14d These considerations helped
guide our synthetic investigations. We envisaged a late
stage guanidine installation via a reductive guanidinylation
of the nitronol 9. This nitro-aldol disconnection might lead
to both the anti and syn C7 diastereomers required for the
synthesis of 4 and 5, respectively.20 Further, diastereomers
at C8 would allow us to test the possibility that a C7/C8
oxidation event generates an identical metabolite (i.e., 7).
The threat posed to global public health by these molecules
in drinking water and the isolation of C. raciborskii in
several regions of the United States has prompted the NIH’s
national toxicology program (NTP) and the EPA’s
unregulated contaminant monitoring rule (UCMR) to elect
4 for toxicological and environmental evaluation.17
The three contiguous stereocenters in the A-ring were to be
created through an intramolecular nitrone dipolar cyclo-
addition producing 10.21 The ultimate starting point of the
synthesis would then be either antipode of the simple crotyl
glycine derivative 11. This was desirable as the absolute
configurations of 4–6 were unknown and could not be
discerned from their biogenesis. Although the absolute
configuration of 4 has been inferred, it was confounding
that 4 isolated from C. raciborskii and that isolated from
The intriguing biogenesis18 and challenging structural
features of the cylindrospermopsin alkaloids have garnered
Figure 2. Synthetic strategy.

R. E. Looper et al. / Tetrahedron 62 (2006) 4549–4562
4551
A. ovalisporum were characterized with opposite signs of
optical rotation.
the aminoalcohol be distilled prior to use, trace amounts of
water effect the annulation dramatically, and the use of the
hygroscopic hydrochloride salt results in considerably lower
yields. By introducing the lactone, we were confident that
we could obviate dipole isomerization, which leads to
diminished selectivity.26 Oxidation of the secondary amine
was most conveniently effected by treatment with purified
mCPBA in dichloromethane to give an 84% yield of
the oxazinone-N-oxide 17.27 Pleasingly, exposure of 17 to
elevated temperatures gave the tricyclic isoxazolidine 18 in
78% isolated yield as a 10:1 mixture contaminated with 19,
arising from endo-approach of the alkene to the dipole.
While treatment of 17 with scandium triflate can produce
the tricycle as an improved 12:1 mixture, the reaction takes
up to 3 days to reach completion at ambient temperatures.
The relative stereochemistry of 18 was secured by X-ray
crystallography.19i
3. Results and discussion
3.1. Synthesis of a common precursor
We investigated two strategies to obtain 11 (Scheme 1). The
first began with rac- or (R)-3-buten-2-ol (12), which was
coupled to N-Boc-Gly to give the ester 13. Enolate-Claisen
rearrangement of 13 gave good yields of 11 and was used to
generate large quantities of racemic material for initial
synthetic explorations.22 Rearrangement of the optically
pure ester through the chelated Z-enolate gave (R)-11 in
92:8 er, with sodium being the most effective counterion.
Unfortunately attempts to generate a non-chelated E-enolate
and thus (S)-11, were ineffective merely eroding the
selectivity for the R-enantiomer. Alternatively, the oxazi-
none 14 could be alkylated with crotyl iodide to give 15 as a
single diastereomer.23 Removal of the auxiliary with lithium
in ammonia gave (R)-11 in O99:1 er.24 Similar results were
obtained for the synthesis of (S)-11. Able to deliver both
antipodes with higher optical purity, the oxazinone became
the preferred method for the preparation of 11.
Having established the stereochemistry in the A-ring, we
needed to install N16 (Scheme 3). The lactone in 18 could
be opened with benzylamine to give 20, however,
purification on silica gel returned 18. To obviate this
reactivity the intermediate amide and N,O-bond could be
reduced with lithium aluminum hydride to afford the
diaminodiol 21. While benzylamine proved a convenient
way to introduce a protected nitrogen, we were concerned
about its orthogonality with the nitro group. para-
Methoxybenzylamine cleanly underwent the addition to
18, but to our surprise we were unable to effect the reduction
of this more electron rich amide. We then examined a
preemptive oxidation state change for C15. Thus 18 was
reduced with diisobutylaluminum hydride to give the lactol
Removal of the t-Boc group in 11 with concomitant methyl
ester formation followed by reduction with lithium
aluminumhydride gave the optically pure crotylglycinol
(Scheme 2). This was then transformed into the free
morpholinone 16 in a one-pot procedure by treatment with
a-bromophenyl acetate.25 It was found imperative that
Scheme 1. Preparation of crotyl glycine.
Scheme 2. Construction of the A-ring.

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R. E. Looper et al. / Tetrahedron 62 (2006) 4549–4562
Scheme 3. N16 introduction.
22 in 87% yield. Reductive amination of 22 with either
BnNH2 or PMBNH2 proceeded smoothly to afford 21 or 23,
respectively. To aid purification, these diaminodiols were
immediately converted to the thiourea using 1,10-thiocarbo-
nyldiimidazole or the urea using bis-p-nitrophenylcarbonate
giving 24–26 in good yields.
many of the reported conditions resulted in epimerization of
the sensitive ureidoaldehyde.28 Using PhI(OAc)2 as the re-
oxidant proved promising as it produced no epimerization,
however, the reaction failed to surpass w30% conversion
by 1H NMR.29 We were able to show that the rate of
oxidation or conversion was independent of the concen-
tration of TEMPO, PhI(OAc)2, or substrate. This suggested
that disproportionation of the nitroxyl radical to the active
oxoammonium salt may be the problematic step. This
equilibrium should be affected by the addition of acid,30 and
indeed the addition of 1 mol% methanesulfonic acid
resulted in complete conversion of the primary alcohol by
1H NMR and w75% isolated yields.31 Our concerns that the
aldehyde would epimerize to the more stable axial
configuration, avoiding pseudo A1,3 strain with the urea,
were negated by NOE correlations in 28.
Attempts to selectively oxidize the primary alcohol in the
thiourea were unsuccessful. Interestingly, this system
suffers from similar reactivity, used productively, in both
Weinreb’s and White’s syntheses.19j,l Treatment of 24 with
oxalyl chloride and DMSO surprisingly returned the
chloromethyl urea 27, presumably from preferential
activation of the thiourea (Scheme 4, Eq. 1). This was
also observed when treating 27 with mercuric chloride and
the correspoding acetoxymethyl urea was observed when
treating the thiourea with Dess–Martin periodinane or
PhI(OAc)2/TEMPO. Efforts to introduce productive nucleo-
philes (i.e., a C1–N synthon) such as cyanide or
nitromethane enolates in the presence or mercuric salts
failed, prompting us to rely on the ureas. Attempts to
oxidize the hydroxymethyl group in 25 utilizing Swern,
Dess–Martin, or Ley oxidations actually showed selectivity
for the secondary alcohol. Initial experiments utilizing the
hindered nitroxyl oxidant, TEMPO, were promising but
Homologation of the aldehydes 28 or 29 by the addition of
lithiated nitromethane provided an inseparable (w1.7:1)
diastereomeric mixture of nitroalcohols (Scheme 5).
Treatment of this mixture with acetic anhydride served
both to protect the secondary alcohol and dehydrate the
nitroalcohol. Fortunately this provided a single diastereo-
mer of the nitroalkene, assuring us that epimerization of the
aldehyde had not occurred. This nitroalkene was reduced
Scheme 4. Oxidation of the ureas.

R. E. Looper et al. / Tetrahedron 62 (2006) 4549–4562
4553
3.2. Synthesis of 7-epi-cylindrospermopsin
Having a substrate that successfully participated in the
reductive guandinylation we were poised to construct the
C7–C8 bond. Initial attempts to effect the nitro-aldol led to
disappointing selectivities, yielding equimolar amounts of
all four C7–C8 diastereomers. It was found imperative that
the nitro-aldol reaction be quenched with AcOH and
reduced. Treatment of 33 and 2,6-dimethoxypyrimidine-4-
carbaldehyde (36)34 with 2 equiv of tetra-n-butylammo-
nium fluoride for short reaction times gave the best
selectivities after reductive guanidinylation giving a 1:0.8
(37:38) mixture favoring the diastereomer required for the
synthesis of 7-epi-cylindrospermopsin (Scheme 6). If the
nitro-aldol products are purified without an acid quench, a
w1:1:1:1 mixture of diastereomers is formed, indicating
that the reaction is indeed highly reversible. Thus, all
reactions were quenched with 20% AcOH in THF and
immediately subjected to reductive guanidinylation. At this
stage the diastereomeric dimethoxypyrimidines were inse-
parable. Acidic hydrolysis of the pyrimidines gave a
separable mixture of 39 (32% yield from 33) and 40
(29%), isolated as their trifluoroacetate slats after purifica-
tion.19m The use of sulfultrioxide–pyridine complex in DMF
Scheme 5. Successful reductive guanidinylation.
in situ with sodium borohydride to give the homologated
nitroalkanes 30 and 31 in reasonable yield for the two steps.
Reduction of the nitro group in 30 provided an amine that
failed to cyclize to the guanidine 32. Attempts to force this
guanidinylation with heat, Lewis acids or protic acids were
unsuccessful. This forced us to pursue the deprotection of
the p-methoxybenzyl group in 31, anticipating the acti-
vation of the urea as an O-alkylisourea. Refluxing 31 in neat
trifluoroacetic acid32 cleanly provided the free urea that
could be O-alkylated with methyl or ethyl Meerwein’s salts
in the presence of an inorganic base. The O-Me isourea
could be synthesized, however, this proved to be unstable,
returning the urea after nucleophilic displacement of the
methyl group.33 A slightly more sterically hindered O-ethyl
isourea was superior and stable to subsequent reaction
conditions. Hydrogenolysis of 33 cleanly gave the tricyclic
guanidine 35 in 96% isolated yield. Interestingly, the
intermediate N-hydroxy guanidine 34 could be isolated if
the reduction was interrupted after 0.5 h and conducted
without the addition of protic acid. Conducting the
reduction in the presence of acetic acid accelerated the
reduction of 34, rendering it virtually undetectable.
˚
with 3 A molecular sieves reproducibly gave 5 in 59%
yield also as previously obtained as a w2:1 mixture with its
bis-sulfate.19j–l Synthetic 5 had spectroscopic properties
identical to those reported. The optical rotation also
agreed well: [a]D25 K12.5 (c 0.04, H2O); lit. [a]2D4 K20.5
(c 0.04, H2O).12
Attempts to control this nitro-aldol process through the use
of chiral Lewis acids that have been employed in the
asymmetric additions of nitromethane or silylnitronates to
aldehydes proved futile.35 This is in part due to the extreme
electrophilicity of the pyrimidine aldehyde 36, which
commonly underwent rapid disproportionation, returning
the corresponding pyrimidinemethanol.36 Cinchonidinium
fluoride catalysts also provided equimolar mixtures and
typically !10% conversion.37
Scheme 6. Synthesis of 7-epi-cylindrospermopsin.

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R. E. Looper et al. / Tetrahedron 62 (2006) 4549–4562
Scheme 7. Synthesis of cylindrospermopsin.
3.3. Synthesis of cylindrospermopsin
isolated from C. raciborskii it is now clear that cylindros-
permopsin does indeed carry the 7R, 8R, 10S, 12S, 13R,
14S configuration from both organisms (C. raciborskii and
A. ovalisporum).
At this juncture we were intrigued by the possibility of
conducting our reductive guanidinylation sequence while
simultaneous unmasking the uracil. The di-benzyloxypyr-
imidine aldehyde 41 was synthesized (Scheme 7).38
Treatment of 33 with 41 and 1.0 equiv TBAF for 0.5 h
followed by reductive guanidinylation gave an extremely
clean mixture of diastereomers by 1H NMR, indicating that
the benzyl groups are efficiently cleaved under the reducing
conditions. Although we had experienced partial cleavage
of the acetate group under hydrogenolysis conditions at
higher hydrogen pressures, we were unable to drive this
cleavage to completion. Thus it remained necessary to
expose the mixture to concd HCl briefly (0.5 h). At this
stage we could correlate all the diastereomers, with 42 and
43 being identical to the racemic diastereomers synthesized
by Snider and Xie. Although this 3-step reaction sequence
produces a w1:1:1:0.5 mixture of 42:43:39:40 the overall
chemical yield is excellent with 42 isolated in 20% yield
after HPLC purification. Sulfonation, again with sulfur
trioxide–pyridine complex, gives cylindrospermopsin in
60% yield, representing the first asymmetric synthesis of 4.
Figure 3. CD spectra of natural and synthetic 4.
3.4. Synthesis of 7-deoxycylindrospermopsin
Having completed the syntheses of the two oxygenated
cylindrospermopsin alkaloids we next focused on the
synthesis of 6 (Scheme 8).19o We were intrigued that 6
was thought to exist as a mixture of unconjugated uracil
Interestingly, synthetic cylindrospermopsin carrying the 7R,
8R, 10S, 12S, 13R, 14S configuration exhibits an [a]2D5 C7.7
(c 0.05, H2O). The natural material first isolated from C.
raciborskii displays an opposite rotation; [a]2D5 K30.1 (c
0.1, H2O).9 From A. ovalisporum, however, the optical
rotation is consistent with synthetic 4; [a]2D5 C12.5 (c 0.6,
H2O).12 It would seem unlikely that the two metabolites
would carry opposite absolute configurations as the
polyketide synthetases involved in their biogenesis are
highly conserved.39 To reconcile these differences in optical
rotation, Circular dichroism (CD) spectra were obtained in
water of natural 4 obtained from C. raciborskii and
compared to that of synthetic 4 at w44 mg/mL (Fig. 3).
Natural cylindrospermopsin displayed a Cotton effect
at 264 nm (D3ZK6.949) and 228 nm (D3ZK4.243).
Synthetic 4 showed identical Cotton effects at 264 nm
(D3ZK8.797) and 229 nm (D3ZK4.432). Although it is
unclear what caused the erroneous optical rotation for 4
1
tautomers, as the H NMR spectrum lacked the vinyllic
uracil proton, yet it displayed a lmaxZ263 nm, consistent
with the presence of a fully conjugated uracil.13
Treatment of the racemic isourea (rac-33) with the aldehyde
41, acetic anhydride, and cesium fluoride affords the
nitroalkene 44 in 67% yield. Although fluoride promoted
coupling and subsequent acetic anhydride mediated elim-
inations of nitroalcohols are known, they generally require
two distinct steps and require a molar excess of the
nitroalkane partner.40 This sequence generates 44 in a
single operation with only 1 equiv of both the aldehyde and
the nitroalkane, making this protocol amenable to complex
molecule synthesis. The nitoalkene is thought to carry the E
geometry around the tri-substituted double bond. It was

R. E. Looper et al. / Tetrahedron 62 (2006) 4549–4562
4555
Scheme 8. Synthesis of 7-deoxycylindrospermopin.
hoped that 44 could be directly reduced to 45 [via the
intermediate ene-guanidine]. However, subjection of 44 to
the reductive guanidinylation conditions returns a complex
mixture, containing products arising from hydrolysis of the
intermediate enamine prior to ring closure. To circumvent
this hydrolysis, 44 was subjected to a one-pot conjugate
reduction/reductive guanidinylation sequence giving a 1:1
mixture of diastereomers. Again the acetates could be
cleaved by brief heating in HCl to give 45 and 46. The
relative stereochemistry of these uracils was secured by
X-ray analysis of 46.41 Again, the reductive guanidinylation
sequence was clean enough that sulfonation could be
executed immediately, also uncomplicated by the need to
selectively sulfonate the C12 hydroxyl group. Thus racemic
6 and 47 were obtained in 66% combined yield over
the three steps. Co-HPLC-injection of synthetic 6 and
natural 7-deoxycylindrospermopsin produced a single peak,
corroborating both the structure of 6 and its natural
to achieve the same level of inhibition.14 Two orders of
magnitude less toxic, this suggests that they are not
processed through a common metabolic intermediate.
Most significantly, our synthetic 6 also proved to be a
potent inhibitor of protein synthesis, contrary to previous
results.19o Protein synthesis was completely inhibited at
12 mM, in vitro, and at 10 mM in whole cells; displaying
potency within an order of magnitude of 4. Resembling
intoxication by 4, synthetic 6 also inhibits the synthesis of
glutathione (GSH).15c The deoxygenated C8 diastereomer
47, also required a 100-fold increase in concentration to
elicit these effects. These results suggest that substitution at
C7 is not requisite for the toxicity of these alkaloids, and
that a common oxidized metabolite at C8 is not involved. In
agreement with previous studies, intermediates lacking the
uracil (i.e., 35) showed greatly diminished toxicity.14
However, the N-hydroxyguanidine 34 was shown to inhibit
protein synthesis on a dose dependent manner at millimolar
concentrations, whereas, 35 did not.
1
occurrence. Further the H NMR spectrum of 6 (Fig. 4)
clearly shows the vinyllic uracil proton at 5.72 ppm. To
reconcile these differences, we compared the spectrum that
led to the elucidation of 50s structure.13a However, it is clear
that the natural material is a mixture of compounds, and we
could not conclude whether 16 was a minor component of
that mixture.
4. Conclusion
The synthetic approach detailed herein has provided an
efficient and flexible route to these natural products. This
strategy has enabled the first enantioselective synthesis of
cylindrospermopsin and corroborated the absolute configur-
ation of this natural product. It also permitted the first
synthesis of 7-deoxycylindrospermopsin and corrected both
structural and toxicological misconceptions. We are further
exploiting this synthetic strategy, guided by the preliminary
toxicological data, to investigate alternative N18 or C15
oxidation events and their manifestation in the toxicity of
the cylindrospermopsin alkaloids.
3.5. Inhibition of protein synthesis
Having completed the total syntheses of all the cylidros-
permosin alkaloids, we were able to examine the feasibility
of our biomechanistic hypothesis for the intermediacy of 7
or 8. While synthetic 4 was a potent inhibitor of protein
synthesis in hepatocytes (4% of control at 3.3 mM), the C8
diastereomer (38) required a concentration of 320 mM

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R. E. Looper et al. / Tetrahedron 62 (2006) 4549–4562
Figure 4. 1H NMR of the synthetic cylindrospermopsins in D2O (CH3OH used as internal reference); (1) 4, (2) 5, (3) 6.
5. Experimental
relative to CHCl3, DMSO-d5, HOD, or HD2COD, and
J-values reported in Hertz.42 13C NMR spectra were
recorded at 75, 100, or 125 MHz. The chemical shifts of
carbon resonances are reported relative to the deuterated
solvent peak, except those in D2O, which are referenced to
methanol. IR spectra were recorded on a Nicolet Avatar
320-FT IR spectrometer (DepZdeposited). Mass spectra
were obtained on a Fisons VG Autospec. Optical rotations
were obtained with a 2 mL, 1 dm cell on a Rudolf Research
Autopol III polarimeter operating at 589 nm. CHCl3 was
distilled from CaCl2 for optical rotations where indicated.
HPLC data was obtained on a Waters 600 HPLC system
Interfaced with Varian Dynamax Integration software
using the indicated column and eluent conditions. Melting
points are uncorrected.
5.1. General
Dichloromethane, diisopropylamine, triethylamine, and
N,N-diisopropylethylamine were distilled from CaH2
immediately prior to use. Tetrahydrofuran, diethylether,
toluene, and dimethylformamide were degassed with argon
and passed through a solvent purification system (Meyer of
Glass Contour) containing either alumina or molecular
sieves. Flash chromatography was performed on Merk
silica gel Kieselgel 60 (230–400 mesh) from EM science
1
with the indicated solvent. H NMR spectra were recorded
on Varian 300, 400, or 500 MHz spectrometers. The
chemical shifts (d) of proton resonances are reported

R. E. Looper et al. / Tetrahedron 62 (2006) 4549–4562
4557
5.1.1. 3-(R)-But-2-enyl-2-oxo-5-(R),6-(S)-diphenyl-
morpholine-4-carboxylic acid tert-butyl ester (15). To a
solution of NaI (6.00 g, 40.0 mmol) in MeCN (30 mL)
under an argon atmosphere was added TMSCl (5.08 mL,
40.0 mmol) dropwise over 10 min. H2O (0.36 mL,
20.0 mmol) was then added followed by crotyl alcohol
(3.40 mL, 40.0 mmol). After 30 min the reaction was
diluted with H2O (100 mL) and extracted 3!50 mL
hexanes. The combined organics were washed with satd
Na2S2O3, brine, and dried (MgSO4). The organics were then
concentrated under aspirator pressure to w1/4 volume. To
this solution, under an argon atmosphere, was added the
oxazinone 14 (5.66 g, 16.0 mmol) and THF (100 mL). The
mixture was cooled to K78 8C and a 0.5 M solution of
KHMDS in PhMe (32.0 mL, 16.0 mmol) was added
dropwise over 10 min. After 0.5 h the reaction was
quenched with satd NH4Cl and diluted with Et2O. The
organics were washed with satd Na2S2O3, brine, and dried
(Na2SO4). Concentration of the organics afforded a white
solid, which was recrystallized from EtOH/H2O. The white
solid was dried at 60 8C to constant mass giving the
crotyloxazinone (5.97 g, 92%, mp 138–141 8C). [a]D25
C13.2 (c 1.00, CHCl3). Optical purity was determined by
HPLC, Chiracel OD-H column eluting with 97:3 hexanes/
iPrOH at 1 mL/min; (* indicates minor rotamer): 3(S), 5(S),
6(R) tRZ5.78*, 6.26 min; 3(R), 5(R), 6(S) tRZ7.66*,
9.35 min.43 1H NMR (CDCl3, 400 MHz, 273 K): (mixture
of rotamers, * indicates minor rotamer where discernable) d
7.28–7.10 (m, 6H), 7.05 (t, JZ7 Hz, 2H), 6.94 (d, JZ7 Hz,
2H), 6.55 (t, JZ8 Hz, 2H), 6.00* (br d, JZ2 Hz, 1H), 5.92
(br d, JZ3 Hz, 1H), 5.7–5.5 (m, 2H), 5.19* (d, JZ2 Hz,
1H), 5.05 (app t, JZ7 Hz, 1H), 4.96 (d, JZ3 Hz, 1H), 4.88*
(dd, JZ6, 8 Hz, 1H), 2.80 (br t, JZ6 Hz, 2H), 1.70
(overlapping d, JZ5 Hz, 3H), 1.43* (s, 9H), 1.08 (s, 9H).
13C NMR (CDCl3, 100 MHz, 273 K): (major rotamer) d
169.5, 153.9, 136.8, 134.7, 130.7, 128.7, 128.3, 127.9,
127.8, 127.7, 126.7, 125.2, 81.3, 79.1, 61.5, 57.2, 37.7, 28.0,
18.2. IR (Dep. CDCl3): 2975 (w), 1752, 1700 (both s), 1388,
1166, 700 (all m). HRMS (FABC): Calcd for C25H29NO4
(m/z) 407.2097; Found (m/z) 407.2094.
HClO4 (pH 1) at 0.8 mL/min: 2(R) tRZ3.95 min.; 2(S)
tRZ5.71 min. H NMR (CDCl3, 300 MHz): d 10.25 (br s,
1
1H), 5.60 (dq, JZ15.0, 6.3 Hz, 1H), 5.40–5.24 (m, 1H),
5.00 (d, JZ7.7 Hz, 1H), 4.34 (br m, 1H), 2.58–2.40 (m, 2H),
1.66 (dd, JZ6.3, 0.9 Hz, 3H), 1.44 (s, 9H). 13C NMR
(CDCl3, 75 MHz): d 177.2, 155.7, 130.5, 124.5, 80.5, 52.2,
35.4, 28.6, 18.3. IR (Dep. CDCl3): 3330 (m, br); 2978 (m);
1716 (s, br); 1508 (m); 1165 (s). HRMS (FABC): Calcd
for C11H20NO4 [MCH]: (m/z) 230.1392; Found (m/z)
230.1393.
5.1.3. tert-Butoxycarbonylamino-acetic acid 1-methyl-
allyl ester (13). To a solution of 3-buten-2-ol (12, 2.00 g,
27.7 mmol), 4-dimethylamino pyridine (10 mol%, 346 mg,
2.77 mmol), and N-tert-butoxycarbonyl glycine (5.35 g,
30.5 mmol) in CH2Cl2 (50 mL) was added diisopropylcar-
bodiimide (4.78 mL, 30.5 mmol) in CH2Cl2 (10 mL) at
0 8C. The mixture was stirred for 2 h and filtered through
Celite with CH2Cl2 (100 mL). The combined organics were
washed with 10% HCl, satd NaHCO3, brine, and dried
(Na2SO4). The concentrated organics were purified by flash
chromatography (6:1 hexanes/EtOAc) to give the ester as a
colourless oil (6.12 g, 96%). If the ester was derived from
(R)-(K)-3-buten-2-ol [a]2D5 C17.9 (c 1.50, CHCl3). 1H
NMR (CDCl3, 300 MHz): d 5.83 (ddd, JZ17.3, 10.5,
6.6 Hz, 1H), 5.40 (qd (app quintet), JZ6.6, 6.6 Hz, 1H),
5.25 (dd, JZ17.2, 1.2 Hz, 1H), 5.15 (dd, JZ10.5, 1.2 Hz,
1H), 5.00 (br s, 1H), 3.90 (app d, JZ3.9 Hz, 2H), 1.45 (s,
9H), 1.33 (d, JZ6.6 Hz, 3H). 13C NMR (CDCl3, 75 MHz):
d 169.7, 155.8, 137.2, 116.2, 80.1, 72.4, 42.8, 28.5, 20.1. IR
(Dep. CDCl3): 3381 (m); 2980 (m); 1751 (s, sh); 1719 (s);
1520 (m); 1368 (m); 1168 (s). HRMS (FABC): Calcd for
C11H20NO4 [MCH]: (m/z) 230.1393; Found (m/z)
230.1392.
5.1.4. rac-2-tert-Butoxycarbonylamino-hex-4-(E)-enoic
acid (11). To a solution of ester 13 (2.72 g, 11.9 mmol) in
THF (30 mL) under an Ar atmosphere was added a 1 M
solution of sodium bis(trimethylsilyl)amide in THF
(2.2 equiv, 26.1 mL, 26.1 mmol) at 0 8C. The mixture was
allowed to warm to rt. After 2 h the reaction was quenched
with satd NH4Cl (5 mL) and brought to pH 2 by the addition
of 10% HCl. The mixture was extracted with Et2O (3!
50 mL), the combined organics were washed with brine and
dried (Na2SO4). Concentration gave 11 as a light yellow
oil (2.69 g, 99%). All spectral characteristics agreed with
(R)-11.
5.1.2. 2-(R)-tert-Butoxycarbonylamino-hex-4-(E)-enoic
acid ((R)-11). A flame dried flask fitted with a CO2
condenser was charged with flattened lithium metal
(660 mg, 95.7 mmol) under argon. Ammonia (50 mL) was
condensed into the flask at K78 8C and the blue slurry
stirred for 15 min. A solution of the oxazinone 15 (3.00 g,
7.36 mmol) in THF (10 mL) and EtOH (1.29 mL,
22.08 mmol) was added dropwise over 5 min. The cooling
bath was removed and the mixture allowed to reflux at
K33 8C for 0.5 h. The reaction was quenched by the careful
addition of NH4Cl and the ammonia allowed to evaporate.
The resulting residue was taken up in satd NaHCO3
(100 mL) and extracted Et2O (2!50 mL). The aqueous
layer was acidified to pH 2 with NaHSO4 and extracted 3!
CH2Cl2 (50 mL). The combined organics were washed with
brine and dried (Na2SO4). Concentration gave the acid as a
light yellow oil (1.12 g, 67%), which was used without
further purification. Note: smaller reaction scale
(w1 mmol) resulted in increased w80% yields. [a]D25
K4.30 (c 1.0, CHCl3). Optical purity can be determined
by HPLC on the free amino acid after hydrolysis with concd
aqueous HCl, Crownpak CR column eluting with aqueous
5.1.5. 5-(R)-But-2-enyl-morpholin-2-one (16). Acetyl
chloride (1.39 mL, 19.5 mmol) was added dropwise to
MeOH (40 mL) at 0 8C and the solution stirred for 15 min.
A solution of the acid 11 (1.49 g, 6.49 mmol) in MeOH
(3 mL) was added and the mixture allowed to reach rt and
stirred an additional 12 h. The mixture was concentrated in
vacuo and further concentrated after the addition of Et2O
(2!20 mL) and PhMe (1!50 mL). The crude solid was
slurried in THF (50 mL) and LiAlH4 (500 mg, 13.2 mmol)
added in portions over 0.5 h at 0 8C. After stirring at rt for an
additional 3 h the reaction was quenched by the sequential
addition of H2O (0.5 mL), 15% NaOH (0.5 mL), and H2O
(1.5 mL). The mixture was filtered through Celite with THF
and concentrated. The crude oil was purified by Kugelhror
distillation, collecting material between 80 and 100 8C

4558
R. E. Looper et al. / Tetrahedron 62 (2006) 4549–4562
(0.5 mmHg) to give the amino alcohol as a clear oil
(487 mg, 65%). [a]D22 K14.3 (c 1.00, CHCl3). H NMR
(d, JZ6.9 Hz, 1H), 4.45 (dd, JZ12.3, 1.2 Hz, 1H), 3.58
(burried m, 1H), 3.58 (d, JZ3.6 Hz, 1H), 2.30 (ddd, JZ
11.7, 10.8, 5.4 Hz, 1H), 2.08 (qd, JZ6.9, 3.7 Hz, 1H), 1.56
(dd, JZ12.0, 6.0 Hz, 1H), 1.22 (d, JZ7.0 Hz, 3H). 13C
NMR (CDCl3, 75 MHz): d 169.9, 85.1, 70.4, 65.1, 57.7,
51.7, 34.7, 19.7. IR (Dep. CDCl3): 2966 (w), 1746 (vs),
14548, 1404 (both w), 1227 (m), 1117 (w), 988 (m). HRMS
(FABC): Calcd for C8H12NO3 [MCH]: 170.0817; Found
170.0812.
1
(CDCl3, 300 MHz): d 5.45 (dq, JZ15, 6 Hz, 1H), 5.31
(dddq, JZ15, 6, 6, 1.5 Hz, 1H), 3.59 (dd, JZ11, 4 Hz, 1H),
3.24 (dd, JZ11, 8 Hz, 1H), 2.78 (dddd, JZ8, 6, 6, 4 Hz,
1H), 2.60 (br s, 3H), 2.06 (ddd, JZ13, 6, 6 Hz, 1H), 1.86
(ddd, JZ13, 6, 6 Hz, 1H), 1.61 (dd, JZ6, 1.5 Hz, 3H). 13C
NMR (CDCl3, 75 MHz): d 128.3, 127.4, 66.2, 52.6, 37.5,
18.2. IR (Dep. CDCl3): 3335 (s), 1573, 1435, 1051, 968 (all
m). HRMS (FABC): Calcd for C6H13NO [MCH]: (m/z)
116.1075; Found (m/z) 116.1080.
Compound 19. 1H NMR (CDCl3, 400 MHz): d 4.49 (buried
dd, JZ10.8, 1.6 Hz, 1H), 4.00 (dd, JZ10.8, 2 Hz, 3.89 (br s,
1H), 3.80 (q, JZ6 Hz), 3.82–3.78 (buried m, 1H), 2.98 (d,
JZ4.8 Hz), 1.87 (ddd, JZ12.4, 4.8, 3.2 Hz, 1H), 1.58 (dd,
JZ12.4, 1.6 Hz, 1H), 1.15 (d, JZ6 Hz, 3H). 13C NMR
(CDCl3, 100 MHz, 2:1 mixture): d 168.4, 81.0, 70.9, 67.9,
61.9, 50.4, 29.5, 20.4.
A solution of the amino alcohol (395 mg, 3.43 mmol) and
iPr2NEt (745 mg, 3.46 mmol, 1.01 equiv) in MeCN (40 mL)
was added dropwise over 1 h to a solution of bromophenyl
acetate in MeCN (131 mL, final concd to be 0.02 M). The
mixture was stirred for an additional 4 h and concentrated.
Purification on silica with a Na2CO3 pre-pad eluting with
5% iPrOH/EtOAc gave the morpholinone 16 as a colourless
oil (335 mg, 63%). [a]2D2 K49.6 (c 1.00, CHCl3). 1H NMR
(CD3OD, 300 MHz): d 5.58 (dq, JZ15.0, 6.3 Hz, 1H), 5.43
(ddd, JZ15.0, 6.6, 1.5 Hz, 1H), 4.38 (dd, JZ10.9, 3.7 Hz,
1H), 4.07 (dd, JZ10.9, 10.9 Hz, 1H), 3.62 (ABq, dd, JZ
18.1, 18.1 Hz, 2H), 3.04 (m, 1H), 2.14 (dd, JZ6.6, 6.6 Hz,
2H), 1.68 (dd, JZ6.3, 1.2 Hz, 3H). 13C NMR (CD3OD,
75 MHz): d 170.8, 130.1, 126.9, 75.0, 52.2, 48.2, 35.6, 18.3.
IR (Dep. CD3OD): 3400 (br s), 2964 (s), 1636, 1404 (both
m), 1063 (vs). HRMS (FABC): Calcd for C8H14NO2 [MC
H]: 156.1025; Found 156.1025.
5.1.8. 2-(S)-Methyl-5(S),9-(R)-dioxa-8-aza-tricyclo-
[5.2.1.0.3,8]decan-4-ol (22) To a solution of the isoxazoli-
dine (167 mg, 0.99 mmol) in CH2Cl2 (20 mL) at K78 8C
under argon was added DIBAL-H (1 M/toluene, 0.99 mL,
0.99 mmol) over 0.5 h. The mixture was stirred for an
additional 1 h, quenched with water (0.2 mL), allowed to
warm to rt, and stirred for 2 h. The mixture was filtered
through Celite and concentrated. The resulting solid was
recrystallized from CHCl3/pentane to give the lactol as
white prisms (147 mg, 87%). 1H NMR (CDCl3, 400 MHz):
[w2:1 mixture of anomers] d 5.28 (s), 4.93 (d, JZ2.4 Hz),
4.39 (app d, JZ5.2 Hz), 4.34 (dd, JZ12.4, 2.0 Hz), 3.88
(dd, JZ12.8, 1.2 Hz), 3.69 (dd, JZ12.4, 1.2 Hz), 3.64 (dd,
JZ12.4, 0.8 Hz), 3.35 (ddd, JZ10.8, 4.4, 2.0 Hz), 3.25
(ddd, JZ10.4, 4.4, 2.4 Hz), 3.04 (dd, JZ4.4, 2.4 Hz), 2.96
(d, JZ4.4 Hz), 2.14–2.01 (m), 1.99 (qd, JZ6.8, 4.4 Hz),
1.79 (qd, JZ6.8, 4.4 Hz), 1.58 (dd, JZ11.2, 4.8 Hz), 1.51
(dd, JZ11.2, 4.8 Hz), 1.07 (d, JZ7.2 Hz), 1.05 (buried d,
JZ7.2 Hz). 13C NMR (CDCl3, 100 MHz): [w2:1 mixture
of anomers] d 92.2, 86.9, 86.9, 73.9, 73.1, 62.1, 59.9, 59.2,
58.2, 44.5, 40.3, 36.1, 35.9, 19.9, 19.0. IR (Dep. CDCl3):
3406, 3131 (br, s), 2965, 2930 (both s), 1452, 1124, 1092,
985, 710 (all m). HRMS (FABC): Calcd for C8H14NO3
[MCH]: 172.0974; Found 172.0976.
5.1.6. 5-(R)-But-2-enyl-4-oxy-5,6-dihydro-[1,4]oxazin-2-
one (17). A solution of the oxazinone 16 (260 mg,
1.67 mmol) in CH2Cl2 (1 mL) was added dropwise over
5 min to a solution of purified mCPBA (636 mg, 3.69 mmol)
and Na2HPO4 (1.18 g) in CH2Cl2 at K78 8C. The reaction
was allowed to proceed for 0.5 h and quenched with satd
Na2S2O3. The mixture was partitioned between H2O and
Et2O and the organics further washed with 9% Na2CO3,
brine, and dried (Na2SO4). The crude oil was purified on
silica eluting with 1:1 hexanes/EtOAc to afford the nitrone
as a colorless oil (236 mg, 84%). [a]2D5 C4.00 (c 4.00,
1
CHCl3). H NMR (CDCl3, 300 MHz): d 7.14 (s, 1H), 5.66
(dq, JZ15.0, 6.5 Hz, 1H), 5.46–5.30 (m, 1H), 4.58 (dd, JZ
12.3, 3.9 Hz, 1H), 4.43 (dd, JZ12.3, 3.9 Hz, 1H), 3.92
(dddd, JZ9.3, 3.9, 3.9, 3.9 Hz, 1H), 2.82–2.70 (m, 1H),
2.61–2.49 (m, 1H), 1.69 (d, JZ6.3 Hz, 3H). 13C NMR
(CDCl3, 75 MHz): d 158.2, 132.3, 124.7, 123.3, 68.1, 65.6,
32.8, 18.3. IR (Dep. CDCl3): 1715, 1556 (both s), 1209 (m),
1061, 968 (both w). HRMS (FABC): Calcd for C8H12NO3
[MCH]: 170.0818; Found 170.0817.
5.1.9. 7(S)-Hydroxy-5(R)-hydroxymethyl-2(S)-(4-meth-
oxy-benzyl)-8(S)-methyl-hexahydro-imidazo[1,5-a]-
pyridin-3-one (26). To a solution of the lactol (15 mg,
0.88 mmol) in EtOAc (3 mL) was added p-methoxybenzyl
amine (17 mg, 0.12 mmol). The solution was degassed with
argon and then 10% Pd/C (15 mg) was added. The solution
was then purged with H2 and stirred under a hydrogen
atmosphere for 12 h. The mixture was filtered and
concentrated. The crude oil was dissolved in MeCN
(5 mL) and cooled to 0 8C. A solution of bis-p-nitrophenyl
carbonate (32 mg, 0.11 mmol) in MeCN (5 mL) was added
dropwise over 15 min. After stirring an additional 0.5 h the
mixture was concentrated, taken up in EtOAc (20 mL) and
the organics washed 3!9% Na2CO3, 1!brine and dried
(Na2SO4). The crude material was purified on silica gel
eluting with EtOAc/5% iPrOH to give the urea 26 as a
5.1.7. 2-(S)-Methyl-5-(S),9-(R)-dioxa-8-(S)-aza-tri-
cyclo[5.2.1.0.3,8]decan-4-one (18) The nitrone 17 (60 mg,
0.35 mmol) was dissolved in dry toluene (7 mL) to be
0.05 M. This solution was heated in a sealed tube at 200 8C
(sand bath temperature) for 2.5 h. The mixture was then
cooled and the solvent removed in vacuo. The crude
organics were purified on silica eluting with 1:1 hexanes/
EtOAc to afford the tricyclic isoxazolidine 18 (47 mg, 78%)
as a colourless oil, which solidified upon standing. An
analytical sample was recrystallized from pet. ether/CH2Cl2
(mp 78–80 8C). [a]D25 C3.6 (c 0.52, CHCl3). 1H NMR
(CDCl3, 300 MHz): d 4.56 (dd, JZ12.3, 2.7 Hz, 1H), 4.53
1
clear oil (19 mg, 67%). [a]2D5 C37.7 (c 1.00, CHCl3). H
NMR (CDCl3, 300 MHz): d 7.18 (d, JZ8.4 Hz, 2H), 6.86
(d, JZ8.4 Hz, 2H), 5.80 (dd, JZ9, 5 Hz, 1H), 4.4 (1/2ABq,
JZ15 Hz, 1H), 4.19 (1/2ABq, JZ15 Hz, 1H), 3.94 (br dd,

R. E. Looper et al. / Tetrahedron 62 (2006) 4549–4562
4559
JZ2.4, 2.4 Hz, 1H), 3.90–3.72 (buried m, 3H), 3.80 (s, 3H),
3.51 (dddd, JZ9, 5, 3, 3 Hz, 1H), 3.45 (ddd, JZ10, 9, 9 Hz,
1H), 3.28 (dd, JZ9, 9 Hz, 1H), 2.76 (dd, JZ9, 9 Hz, 1H),
1.82 (d, JZ3 Hz, 1H), 1.72 (ddd, JZ14, 3, 3 Hz, 1H), 1.62
(ddd, JZ12, 12, 2 Hz, 1H), 1.48 (ddd, JZ14, 6, 3 Hz, 1H),
0.89 (d, JZ6 Hz, 3H). 13C NMR (CDCl3, 75 MHz): d 160.8,
159.0, 129.4, 114.0, 68.2, 64.8, 55.4, 54.4, 53.3, 47.9, 47.6,
40.0, 36.4. IR (Dep. CDCl3): 3385, 2933 (both m), 1664,
1513, 1246 (all s). HRMS (FABC): Calcd for C17H24N2O4
[MCH]: 322.1814; Found: 321.1811.
as a colorless oil (40 mg, 87%). [a]2D5 C15.2 (c 1.00,
CHCl3). H NMR (CDCl3, 400 MHz): d 7.12 (d, JZ8 Hz,
1
2H), 6.87 (d, JZ8 Hz, 2H), 5.12 (br d, JZ6.8, 3 Hz, 1H),
4.72 (ddd, JZ13.6, 8.4, 5.6 Hz, 1H), 4.61 (ddd, JZ13.6,
5.6, 5.6 Hz), 4.23 (s, 2H), 3.78 (s, 3H), 3.43 (dddd, JZ10.8,
10.8, 3, 3 Hz, 1H), 3.28 (ddd, JZ9, 8, 5.6 Hz, 1H), 3.18 (dd,
JZ8, 8 Hz, 1H), 2.78 (dd, JZ8, 5 Hz, 1H), 2.41 (dd, JZ
13.6, 8, 5, 5 Hz, 1H), 2.05 (s, 3H), 1.83 (ddd, JZ12, 3, 3 Hz,
1H), 1.70–1.60 (m, 2H), 0.78 (d, JZ6.8 Hz, 3H). 13C NMR
(CDCl3, 100 MHz): d 170.4, 159.7, 159.1, 129.5, 129.0,
114.2, 73.7, 71.5, 56.1, 55.5, 48.8, 47.3, 46.6, 36.8, 36.4,
29.6, 21.3, 13.3. IR (Dep. CDCl3): 2937 (m), 1737, 1693,
1550, 1513 (all s), 1442, 1374, 1351 (all m), 1242 (s).
HRMS (FABC): Calcd for C20H28N3O6 [MCH]:
406.1978; Found: 406.1969.
5.1.10. 7(S)-Hydroxy-2(S)-(4-methoxy-benzyl)-8(S)-
methyl-3-oxo-octahydro-imidazo[1,5-a]pyridine-5(R)-
carbaldehyde (29). To a solution of the diol 26 (211 mg,
0.66 mmol) in CDCl3 (3 mL) was added PhI(OAc)2
(318 mg, 0.99 mmol) and TEMPO (41 mg, 0.26 mmol).
Methanesulfonic acid (0.63 mg, 7 mmol, 1 mol%) was then
added as a solution in CDCl3. The mixture was stirred for
3 h, diluted with EtOAc (30 mL) and the organics washed
with satd Na2S2O3, satd NaHCO3, brine, and dried
(Na2SO4). The resulting oil was purified on silica gel
eluting with EtOAc/5% iPrOH) to give the aldehyde as a
white foam (156 mg, 75%). [a]2D5 C84.8 (c 1.13, CHCl3).
1H NMR (CDCl3, 300 MHz): d 9.81 (d, JZ2.1 Hz, 1H),
7.16 (d, JZ8.1 Hz, 2H), 6.86 (d, JZ8.1 Hz, 2H), 4.36
(1/2ABq, JZ15 Hz, 1H), 4.20 (1/2ABq, JZ15 Hz, 1H),
4.00 (br s, 1H), 3.82 (buried m, 1H), 3.79 (s, 3H), 3.40 (ddd,
JZ10.5, 9, 9 Hz, 1H), 3.28 (dd, JZ9, 9 Hz, 1H), 2.86 (dd,
JZ9, 9 Hz, 1H), 1.90 (br d, JZ13.5 Hz, 1H), 1.64 (dd, JZ
12, 12 Hz, 1H), 1.54 (br dd, JZ9, 9 Hz, 1H), 0.90 (d, JZ
6.6 Hz). 13C NMR (CDCl3, 75 MHz): d 198.3, 160.4, 158.9,
129.4, 128.7, 114.0, 67.9, 57.4, 55.4, 53.3, 47.9, 47.3, 38.4,
32.9, 13.4. IR (Dep. CDCl3): 3431, 2878 (both m), 1727,
1682, 1513, 1448, 1246 (all s). HRMS (FABC): Calcd for
C17H22N2O4 [MCH]: 319.1657; Found: 319.1664.
5.1.12. (5S,7S,8R,8aS)-3-Ethoxy-8-methyl-5-(2-nitro-
ethyl)-1,5,6,7,8,8a-hexahydroimidazo[1,5-a]pyridin-7-yl
acetate (33). The protected urea 31 (25 mg, 0.062 mmol)
was dissolved in trifluoroacetic acid (1.5 mL). The mixture
was refluxed for 1 h and concentrated under reduced
pressure. The purple residue was taken up in EtOAc
(10 mL) and washed H2O, satd NaHCO3, brine, and dried
(Na2SO4). The crude residue was purified on silica gel
eluting with EtOAc/5% iPrOH to give the urea (14 mg,
1
80%) as a white solid. [a]2D5 C17.3 (c 1.00, CHCl3). H
NMR (CDCl3, 300 MHz): d 5.14 (dd, JZ6, 3 Hz, 1H), 4.80
(br s, 1H), 4.76–4.54 (m, 2H), 3.52–3.38 (m, 3H), 3.1–2.9
(m, 2H), 2.37 (dddd, JZ15, 6, 6, 3 Hz, 1H), 2.09 (s, 3H),
1.86 (ddd, JZ12, 3, 3 Hz, 1H), 1.84–1.78 (buried m, 1H),
1.66 (ddd, JZ12, 3, 3 Hz, 1H), 0.87 (d, JZ6 Hz, 3H). 13C
NMR (CDCl3, 100 MHz): d 170.5, 161.6, 73.6, 71.5, 58.8,
48.5, 42.5, 36.5, 36.6, 29.4, 21.2, 13.2. IR (Dep. CDCl3):
3269, 2939 (both w), 1736, 1698, 1550 (all s), 1436, 1374
(both m), 1242 (s). HRMS (FABC): Calcd for C12H20N3O5
[MCH]: 286.1403; Found: 286.1409.
5.1.11. (5S,7S,8R,8aS)-2-(4-Methoxybenzyl)-8-methyl-5-
(2-nitroethyl)-3-oxo-octahydroimidazo[1,5-a]pyridin-7-
yl acetate (31). A solution of nitromethane in THF (10:1,
20 mL) under argon was cooled to 0 8C. A 1.6 M solution of
nBuLi (3.5 mL, 5.66 mmol) was added slowly (caution!
highly exothermic) over 20 min. The mixture was stirred an
additional 15 min and a solution of the aldehyde 29
(180 mg, 0.57 mmol) in THF added. The reaction was
allowed to proceed for 12 h, quenched with satd NH4Cl and
extracted with EtOAc (3!10 mL). The combined organics
were washed brine and dried (Na2SO4). The crude oil was
purified on silica eluting with 1:1 hexanes/EtOAc then
EtOAc/5% iPrOH to give the diastereomeric nitro alcohol
(183 mg, 84%). To a solution of the nitroalcohol (41 mg,
0.11 mmol) and N,N-dimethylaminopyridine (3 mg,
0.025 mmol, 20 mol%) in CH2Cl2 under an argon atmos-
phere was added acetic anhydride (0.10 mL, 1.1 mmol).
After stirring for 12 h the mixture was concentrated, taken
up in EtOH (3 mL) and added dropwise to a slurry of
NaBH4 (101 mg, 2.67 mmol) in EtOH (5 mL). The mixture
was stirred for 2 h and quenched by the addition of 50%
AcOH/H2O (0.4 mL). The mixture was concentrated under
reduced pressure and partitioned between H2O and EtOAc.
The aqueous phase was extracted again with EtOAc and the
combined organics washed with satd NaHCO3, brine, and
dried (Na2SO4). The crude oil was purified on silical gel
eluting with 1:1 hexanes/EtOAc to give the nitroalkane 31
To a solution of the urea (58 mg, 0.20 mmol) under argon in
CH2Cl2 (10 mL) was added Cs2CO3 (650 mg, 2.0 mmol)
and triethyloxonium tetrafluoroborate (386 mg, 2.0 mmol).
The reaction was stirred at rt for 15 h and quenched by the
addition of aqueous 9% Na2CO3 (5 mL). The aqueous layer
was extracted with CH2Cl2 (3!10 mL). The combined
organics were washed with brine and dried (Na2SO4). After
concentration the crude mixture was purified on silica gel
with 10% MeOH/CH2Cl2 to give the isourea 33 as a clear oil
(49 mg, 78%). [a]D25 C6.2 (c 1.00, CHCl3). 1H NMR
(CD3OD, 400 MHz): d 5.22 (app br dd, JZ8.0, 2.8 Hz, 1H),
4.61 (ddd, JZ7.6, 7.6, 2.4 Hz, 1H), 4.21 (q, JZ7.2 Hz, 2H),
3.59–3.46 (m, 2H), 3.55 (buried dd, JZ11.6, 4.0 Hz, 1H),
3.25 (dd, JZ11.6, 4.8 Hz, 1H), 2.66 (dddd, JZ18, 8, 8,
8 Hz, 1H), 2.37 (dddd, JZ18, 8, 8, 6 Hz, 1H), 2.08 (s, 3H),
1.94–1.79 (m, 2H), 1.65 (ddd, JZ14, 12, 2 Hz, 1H), 1.32 (q,
JZ7.2 Hz, 3H), 0.85 (d, JZ6.8 Hz, 3H). 13C NMR (CDCl3,
100 MHz): d 170.6, 163.3, 73.0, 71.9, 65.4, 63.8, 52.5, 48.6,
36.2, 35.5, 30.3, 21.2, 14.6, 13.0. IR (Dep. CDCl3): 2963
(m), 1735 (s), 1622, 1550, 1436, 1372, 1334 (all m), 1228
(s). HRMS (FABC): Calcd for C14H24N3O5 [MCH]:
314.1715; Found: 314.1710.
5.1.13. 7-epi-Cylindrospermopsin diol (37). To a solution
of 33 (8.0 mg, 26 mmol) and pyrimidine aldehyde 36
(5.2 mg, 31 mmol) in THF at K15 8C was added a 1 M

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R. E. Looper et al. / Tetrahedron 62 (2006) 4549–4562
solution of tetra-n-butylammonium fluoride (51 mL,
51 mmol). The reaction was allowed to proceed for 0.5 h
and quenched with Twenty percentage AcOH/THF
(0.5 mL). The mixture was concentrated and the crude oil
dissolved in 5% AcOH/MeOH (5.1 mL, to be 5 mM) and
the solution purged with argon. 20% Pd(OH)2 on carbon
(32 mg) was added and the solution purged with hydrogen.
After stirring for 12 h under an H2 atmosphere the mixture
was filtered through a 0.45 mm Acrodiscw and concentrated.
Purification (to remove 6-hydroxymethyl pyrimidine and
TBAF) by PTLC eluting with 20% MeOH/CH2Cl2 with 1%
HCO2H afforded an inseparable mixture (1:0.8) of the two
C-7 diastereomers after stripping the silica with 20% abs
EtOH/CH2Cl2. This mixture was then refluxed in concd HCl
for 8 h and concentrated. Purification of the uracils was
achieved by HPLC using a Waters Symmetryw C-18 colum
(4.6!250 mm) eluting with 4% MeOH/H2O with 1% TFA
at 1.5 mL/min, monitoring at 263 nm to give 7-epi-
cylindrospermopsin diol as a white solid (3.0 mg, 32%,
tRZ19.05 min) and the other C8 diastereomer also as a
white crystalline solid (2.7 mg, 29%, tRZ23.53 min).
(3!10 mL) and the combined organics washed with brine
and dried (Na2SO4). The crude oil was purified on silica gel
eluting with 15:1 hexanes/EtOAc to give the dibenzylox-
ypyrimidine as a clear oil (137 mg, 80%). 1H NMR (CDCl3,
300 MHz): d 7.47–7.32 (m, 10H), 6.66 (s, 1H), 5.43 (s, 2H),
5.40 (s, 2H). 13C NMR (CDCl3, 75 MHz): d 171.1, 163.8,
152.3, 135.9, 135.6, 128.7, 128.6, 128.5, 128.4, 128.3,
128.3, 105.5, 70.1, 69.1. IR (Dep. CDCl3): 2952 (w), 1549,
1404, 1323 (all s), 1130, 1003 (both m). HRMS (FABC):
Calcd for C18H16N2O281Br1 [MCH]: 373.0375; Found
373.0363. Calcd for C18H16N2O2Br1 [MCH]: 371.0395;
Found 371.0383.
5.1.16. Cylindrospermopsin (4). To a solution of 33
(4.5 mg, 14 mmol) and 41 (5.5 mg, 17 mmol) in THF
(120 mL) at K15 8C was added a 1 M solution of TBAF
(14 mL, 14 mmol). The solution was stirred for 0.5 h and
quenched with 20% AcOH/THF (0.2 mL). The mixture was
concentrated and taken up in 5% AcOH/THF (3 mL) and
Pd(OH)2 (20%/C, 5 mg) added. The solution was purged
with H2 and stirred under an H2 atmosphere for 12 h. The
mixture was taken up in MeOH and filtered through a
0.45 mm Acrodiscw. Purificataion by HPLC on a Waters
Symmetryw C-18 colum (4.6!250 mm) eluting with 8%
MeOH/H2O with 1% TFA at 1.5 mL/min, monitoring at
263 nm gave cylindrospermopsin diol (42) (tRZ9.47 min)
as a white solid after lyophilization (1.3 mg, 20%). [a]D25
C7.7 (c 0.13, H2O). Compound 42 (1.3 mg, 2.89 mmol) was
co-concentrated with MeCN (2!5 mL) and PhMe (2!
5 mL). The resulting solid was dried under vacuum for 0.5 h
and placed under argon. DMF (0.3 mL) and activated,
Compound 37. 1H and 13C NMR agreed with those
previously reported.19m [a]D25 K11.7 (c 0.06, H2O); (lit.
[a]2D4 K8.3 (c 0.06, H2O));12 38: [a]D25 C70.0 (c 0.20, H2O).
1H NMR (D2O, 400 MHz): d 5.80 (s, 1H), 4.62 (d, JZ
4.4 Hz, 1H), 4.04 (br s, 1H), 3.88–3.74 (m, 3H), 3.28 (app t,
JZ8.4 Hz, 1H), 2.26 (ddd, JZ14, 4, 3 Hz, 1H), 2.07 (ddd,
JZ14, 4, 4 Hz, 1H), 1.87 (ddd, JZ15, 10, 6 Hz, 1H), 1.78–
1.68 (m, 1H), 1.52 (app t, JZ13 Hz, 1H), 0.97 (d, JZ7 Hz,
3H). HRMS (FABC): Calcd for C15H22N5O4 [MCH]:
336.1672; Found: 336.1672.
˚
powdered 3 A molecular sieves (6 mg) were added and the
mixture stirred for 15 min. To this solution was added solid
SO3$pyr (4.6 mg, 29 mmol) and the mixture stirred for 1 h.
MeOH (0.1 mL) was added and the solvents removed in
vacuo. The mixture was taken up in MeOH and filtered
through a 0.45 mm Acrodiscw. Purification by HPLC on a
Waters Symmetryw C-18 colum (4.6!250 mm) eluting
with 4% MeOH/H2O with 1% TFA at 1.5 mL/min,
monitoring at 263 nm gave cylindrospermopsin 4 (tRZ
8.14 min) as a white solid after lyophilization (0.7 mg,
60%). This was preceded by its bis sulfate (tRZ5.32 min) as
a w6:1 mixture. 1H and 13C NMR agreed with those
previously reported.9,12,19h [a]D25 C8.0 (c 0.05, H2O).
5.1.14. 7-epi-Cylindrospermopsin (5). 7-epi-Cyclindros-
permopsin diol 37 (2.6 mg, 7.0 mmol) was co-concentrated
with MeCN (2!5 mL) and PhMe (2!5 mL). The resulting
solid was dried under vacuum for 0.5 h and placed under
˚
argon. DMF (0.4 mL) and activated, powdered 3 A
molecular sieves (6 mg) were added and the mixture stirred
for 15 min. To this solution was added solid SO3$pyr
(11 mg, 70 mmol) and the mixture was stirred for 1 h.
MeOH (0.1 mL) was added and the solvents removed in
vacuo. The mixture was taken up in MeOH and filtered
through a 0.45 mm Acrodiscw. Purificataion by HPLC on a
Waters Symmetryw C-18 colum (4.6!250 mm) eluting
with 2% MeOH/H2O with 1% TFA at 1.5 mL/min,
monitoring at 263 nm gave 7-epi-cylindrospermopsin 5
(tRZ9.22 min) as a white solid after lyophilization (1.7 mg,
59%). This was preceded by its bis sulfate (tRZ6.54 min) as
a w2:1 mixture. [a]D25 K12.5 (c 0.04, H2O); (lit. [a]D24
K20.5 (c 0.04, H2O),3 1H and 13C NMR spectra agree with
those reported.12 HRMS (FABC): Calcd for C15H22N5O7S
[MCH]: 416.1240; Found: 416.1247.
5.1.17. 5-((E)-3-(2,6-Bis(benzyloxy)pyrimidin-4-yl)-2-
nitroallyl)-3-ethoxy-8-methyl-1,5,6,7,8,8a-hexahydro-
imidazo[1,5-a]pyridin-7-yl acetate (44). To a solution of
the isourea 33 (23 mg, 73 mmol) and the pyrimidine
aldehyde (26 mg, 81 mmol, 1.1 equiv) in CH2Cl2 (1 mL)
under argon was added Ac2O (34 mL, 0.35 mmol, 5 equiv).
CsF (110 mg, 0.73 mmol) was then added as a solid in one
portion. The reaction was diluted with MeCN (3 mL) and
the mixture stirred for 4 h. The reaction was concentrated
under reduced pressure, taken up in CH2Cl2 and filtered to
remove the cesium salts. This mixture was again concen-
trated and purified on silica gel eluting with 10% MeOH/
CH2Cl2 to give the nitroalkene as a yellow oil (30 mg, 67%)
as a single geometric isomer. This compound is unstable,
decomposing overnight at rt. 1H NMR (CDCl3, 400 MHz): d
7.68 (s, 1H), 7.48–7.30 (m, 10H), 6.58 (s, 1H), 5.52–5.40
(m, 4H), 4.98 (br 2, JZ3.2 Hz), 4.28–4.18 (m, 3H), 4.00
(dd, JZ14, 5 Hz, 1H), 3.66 (ddd, JZ15, 10, 5 Hz, 1H), 3.55
5.1.15. 2,4-Bis(benzyloxy)-6-bromopyrimidine (41). To a
solution of benzyl alcohol (0.11 mL, 1.03 mmol) in THF
(0.5 mL) under an argon atmosphere at 0 8C was added a
1.6 M solution of nBuLi in hexanes (0.62 mL, 0.99 mmol).
The mixture was stirred 10 min and DMF (5 mL) added.
A solution of the tribromopyrimidine in DMF (1 mL) was
added and the mixture stirred at 0 8C for 3 h. The reaction
was quenched with satd NH4Cl and diluted with H2O
(10 mL). The aqueous phase was extracted with Et2O

R. E. Looper et al. / Tetrahedron 62 (2006) 4549–4562
4561
(dd, JZ10, 8 Hz, 1H), 3.40–3.30 (m, 1H), 3.12 (dd, JZ10,
8 Hz, 1H), 1.98 (s, 3H), 1.78–1.64 (m, 1H), 1.62–1.60 (m,
2H), 1.25 (t, JZ7.2 Hz, 3H), 0.76 (d, JZ6.8 Hz, 3H). 13C
NMR (CDCl3, 100 MHz): d 172.3, 170.6, 165.0, 164.3,
159.8, 155.2, 136.2, 135.7, 130.4, 128.9, 128.7, 128.5,
128.4, 127.8, 106.9, 71.6, 69.8, 69.1, 65.3, 64.1, 52.9, 50.2,
36.7, 35.4, 31.1, 21.2, 14.7, 13.0. HRMS (FABC): Calcd
for C33H38N5O7 [MCH]: (m/z) 616.2771; Found: (m/z)
616.2795.
3.8 Hz, 1H), 3.26 (dd, JZ10.8, 8.9 Hz, 1H), 2.76 (app d, JZ
6.8 Hz, 2H), 2.48 (ddd, JZ14.3, 3.8, 3.8 Hz, 1H), 2.32 (ddd,
JZ13.2, 3.6, 3.6 Hz, 1H), 1.87 (ddd, JZ8.9, 6.8, 2 Hz, 1H),
1.55 (app dd, JZ13.2, 11.3 Hz, 1H), 1.01 (d, JZ6.8 Hz,
3H). Calcd for C15H22N5O6S [MCH]: (m/z) 400.1296;
Found: 400.1282.
Acknowledgements
5.1.18. 7-Deoxycylindrospermopsin diol (45). A solution
of the nitroalkene 44 (18 mg, 29.2 mmol) in EtOH (0.5 mL)
was added dropwise to a slurry of NaBH4 (5 mg, 146 mmol)
in EtOH (0.5 mL) over 20 min. After stirring for 1.5 h the
reaction was quenched by the addition of 1:1 H2O/AcOH
(0.1 mL) and concentrated. The concentrate was diluted
with 5% AcOH:MeOH (5.8 mL, to be 5 mM) and purged
with argon. Pd(OH)2 (20%/C, 6 mg) was added and the
mixture stirred under a hydrogen atmosphere for 12 h,
filtered through a 0.45 mm Acrodiscw and concentrated. The
residue was dissolved in concd HCl and refluxed for 1 h and
concentrated. Purification of the uracils was achieved by
HPLC using a Waters Symmetryw C-18 colum (4.6!
250 mm) eluting with 8% MeOH/H2O with 1% TFA at
1.5 mL/min, monitoring at 263 nm to give 7-deoxy-
cylindrospermopsin diol 45 as a white solid (3.7 mg, 38%,
tRZ22.1 min) preceded by the C8 diastereomer 46 also
obtained as a white crystalline solid (4 mg, 38%, tRZ
12.6 min). A small sample of 45 (w1 mg) was recrystal-
lized from methanol (layered with pentane) to give X-ray
quality crystals. Compound 45 (8S*): 1H NMR (D2O,
500 MHz): d 5.68 (s, 1H), 4.03 (br s, 1H), 3.92 (m, 1H), 3.82
(dd, JZ9, 9 Hz, 1H), 3.78 (dd, JZ9, 9 Hz, 1H), 3.72 (dddd,
JZ11, 11, 4, 4 Hz, 1H), 3.25 (m, 1H), 2.71 (dd, JZ14,
5.5 Hz, 1H), 2.67 (dd, JZ14, 9 Hz, 1H), 2.16 (dt, JZ14, 4,
4 Hz, 1H), 2.06 (dt, JZ15, 3 Hz, 1H), 1.83 (ddd, JZ15, 11,
5 Hz, 1H), 1.72 (ddq, JZ14, 7, 3 Hz, 1H), 1.55 (ddd, JZ14,
14, 1.5 Hz, 1H), 0.95 (d, JZ7 Hz, 3H). HRMS (FABC):
Calcd for C15H22N5O3 [MCH]: (m/z) 320.1723; Found:
(m/z) 320.1723. Compound 46 (8R*): 1H NMR (D2O,
500 MHz): d 5.72 (s, 1H), 4.00 (br s, 1H), 3.86 (buried m,
1H), 3.82 (dd, JZ9.0, 9.0 Hz, 1H), 3.74 (dd, JZ10, 10 Hz,
1H), 3.61 (ddt, JZ11, 11, 3.5 Hz, 1H), 3.23 (dd, JZ10,
10 Hz, 1H), 2.73 (app d, JZ5 Hz, 1H), 2.26 (dt, JZ15, 5,
5 Hz, 1H), 2.07 (dt, JZ15, 3, 3, Hz, 1H), 1.70 (ddq, JZ9,
6.5, 2.5 Hz, 1H), 1.50 (app q, JZ11 Hz, 2H), 0.95 (d, JZ
6.5 Hz, 3H). HRMS (FABC): Calcd for C15H22N5O3 [MC
H]: (m/z) 320.1723; Found: 320.1712.
This work was supported by the National Institutes of
Health Grant #GM068011 (to R.M.W) and Grant
#DK51788 (to M.T.C.R) and the National Science
Foundation #CHE0202827 (to R.M.W). We are grateful to
Array Biopharma for fellowship support to R.E.L. We thank
Dr. Andrew Humpage for providing a sample of natural
7-deoxycylidrospermopsin and Dr. Glenn Shaw for provid-
ing us with spectra of 6. We thank Dr. Chris Rithner for
helpful NMR discussions and are indebted to Prof. Alan
Kennan and Dr. Nathan Schnarr for assistance with CD
measurements.
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