Straightforward access to spisulosine and 4,5-dehydrospisulosine stereoisomers: Probes for profiling ceramide synthase activities in intact cells

Article abstract of DOI:10.1021/jo400440z

A stereoselective synthesis of spisulosine (ES285) and 4,5- dehydrospisulosine stereoisomers is described. Hydrozirconation of 1-pentadecyne with Schwartz reagent, followed by diastereocontrolled addition to l- or d-alaninal afforded the required 2-amino-1,3-diol framework. The resulting sphingoid bases revealed as excellent probes for the profiling of ceramide synthase activity in intact cells. Among the sphingoid bases described in this work, spisulosine (ES285), RBM1-77, and RBM1-73 were the most suitable ones because of their highest acylation rates. These molecules should prove useful to study the role of the different ceramide synthases and the resulting N-acyl (dihydro)ceramides in cell fate.

Full text of DOI:10.1021/jo400440z

Article
pubs.acs.org/joc
Straightforward Access to Spisulosine and 4,5-Dehydrospisulosine
Stereoisomers: Probes for Profiling Ceramide Synthase Activities in
Intact Cells
Jose Luis Abad,*,† Ingrid Nieves,† Pedro Rayo,† Josefina Casas,† Gemma Fabrias,†
́
̀
and Antonio Delgado*,†,‡
†Consejo Superior de Investigaciones Científicas (CSIC), Institut de Química Avanca
̧
da de Catalunya (IQAC−CSIC), Research Unit
on Bioactive Molecules (RUBAM), Jordi Girona 18-26, 08034 Barcelona, Spain
‡Universidad de Barcelona (UB), Facultad de Farmacia, Unidad de Química Farmaceu
́
tica (Unidad Asociada al CSIC), Avda. Joan
XXIII s/n, 08028 Barcelona, Spain
S
* Supporting Information
ABSTRACT: A stereoselective synthesis of spisulosine
(ES285) and 4,5-dehydrospisulosine stereoisomers is de-
scribed. Hydrozirconation of 1-pentadecyne with Schwartz
reagent, followed by diastereocontrolled addition to L- or D-
alaninal afforded the required 2-amino-1,3-diol framework.
The resulting sphingoid bases revealed as excellent probes for
the profiling of ceramide synthase activity in intact cells.
Among the sphingoid bases described in this work, spisulosine
(ES285), RBM1-77, and RBM1-73 were the most suitable
ones because of their highest acylation rates. These molecules
should prove useful to study the role of the different ceramide synthases and the resulting N-acyl (dihydro)ceramides in cell fate.
reported,3,4 ES285 (Figure 1) resembles the natural SL in the
C18 sphingoid backbone and in the (2S,3R) configuration of
the amino and hydroxyl groups, respectively.
INTRODUCTION
■
Sphingolipids (SLs) constitute a group of natural products
characterized by the presence of a long chain 2-amino-1,3-diol
scaffold or sphingoid base. Structurally, this central scaffold can
accommodate a variety of functionalities that account for the
different families of SLs found in nature. In particular,
dihydrosphingosine shows a C18 2-amino-1,3-diol core with a
defined 2S,3R stereochemistry that is common to the most
representative group of mammal SLs (Figure 1). Acylation of
the amino group of sphingosine with acyl chains of different
lengths and unsaturations, followed by metabolic oxidation to
install an E unsaturation at C4 position of the sphingoid base,
affords ceramides. These are essential constituents of SL
metabolism in mammals, with crucial roles in cell fate and
homeostasis. Likewise, polar head groups, such as phosphate
derivatives or sugars, can be appended at the C1-hydroxy
position of ceramides to afford sphingomyelins and glyco-
sphingolipids, respectively (Scheme 1). Besides playing a
structural role and regulating the physical properties of cell
membranes, these SL metabolites also participate in cell
signaling and in the control of numerous cellular functions in
mammals.1
Interestingly, prior to its isolation and characterization as a
marine natural product, the structure of spisulosine had already
been described as a synthetic simplified analog of the ceramide
synthase (CerS) inhibitor Fumonisin B1 (FB1), together with
enigmol and other related 2-amino-3,5-octadecanediols with
different stereochemistries at the sphingoid backbone (Figure
1).5 Although spisulosine was initially developed as a promising
anticancer agent due to its ability to inhibit proliferation in the
prostate tumor PC-3 and LNCaP cell lines,6 it was discontinued
from phase I in 2008.7,8 Nevertheless, its close structural
relationship with other related 1-deoxysphingoids with
remarkable cytotoxic properties, such as obscuraminols,9
clavaminols,10,11 crucigasterins,12 and xestoaminols,13 (Figure
1) makes this type of compound an attractive lead for
anticancer-oriented drug discovery programs. In addition,
recent evidence of the presence of 1-deoxysphingolipids as
natural metabolites in mammalian cells have been postulated
from studies of a mutation in the SPTLC1 gene that is found in
human sensory neuropathy type 1 (HSN1).14 Interestingly, in a
recent study, the presence of several 1-deoxysphingolipids in
plasma has also been proposed as a novel class of biomarkers
for the metabolic syndrome.15
Several 1-deoxysphingolipids, derived from a central core
lacking the C1-OH group present in dihydrosphingosine, are
also found in nature. This is the case of spisulosines, a group of
antiproliferative compounds of marine origin isolated from the
clam Spisula polynyma (syn. Mactromeris polynima).2 Although
several spisulosine analogs of different chain lengths have been
Received: February 28, 2013
© XXXX American Chemical Society
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Figure 1. Spisulosine (ES285) and related 1-deoxysphingolipids.
a
Scheme 1. Main Metabolic Pathways in Sphingolipids
a
SPT, serine palmotyl transferase; CerS, ceramide synthase; Des1, dihydroceramide desaturase; SMS, sphingomyelin synthase; SMase,
sphingomyelinase; GCS, glucosylceramide synthase; GCase, glucocerebrosidase; CerPP, CerP phosphatase; CerK, ceramide kinase; CerS, ceramide
synthase; CDases, ceramide hydrolases; LCS, lactosyl ceramide synthase; SK, sphingosine kinase; S1PP, S1P phosphatase.
In light of the above considerations, we have recently
undertaken a work program aimed at the study of the cellular
properties of spisulosine and related analogs as SL modulators.
In this paper, we report on the design of efficient synthetic
protocols for this type of 1-desoxysphingolipids, with special
attention to the stereochemical control at C2 and C3 positions
and the presence of a C4−C5 unsaturation as part of the
sphingoid backbone. The usefulness of some of the compounds
as probes for ceramide synthase activity profiling in intact cells
is also reported.
ketimines,19 the regioselective azidolysis of epoxyalcohols,20 the
reaction of carbon nucleophiles with cyclic sulfamidates,21 or, in
its racemic form, the functional group transformations from a
Morita-Baylis-Hillman adduct.22 In addition, spisulosine has
also been obtained by multistep synthesis from Garner’s
aldehyde.23 In view of the above precedents, we reasoned that a
direct approach, based on the use of D-or L-alanine as starting
materials, would be more suitable for the straightforward
synthesis of spisulosine analogs with different chiralities and
degrees of unsaturation in the sphingoid backbone. Surpris-
ingly, despite its apparent simplicity, the use of D- or L-alanine
derivatives as starting materials for the synthesis of spisulosine
analogs has been scarcely reported in the literature. Thus, the
addition of Grignard reagents to N,N-dibenzylalaninal24,25 or to
the Weinreb amide of N-Boc alanine26 are the only reported
examples along this line.
RESULTS AND DISCUSSION
■
Aside from its interesting cytotoxic properties, spisulosine has
also been used as a target to illustrate the applicability of new
synthetic methodologies. In this context, several multistep
syntheses of enantiopure spisulosine, or that of advanced
precursors thereof, have been reported in the literature.
Examples are found in the reduction of enantiomerically pure
2-acylaziridines,16 the Cu-catalyzed Grignard addition to a
suitable N-Boc aminoepoxide,17 the organometallic addition to
N-tert-butylsulfinyl imines,18 the diastereoselective reduction of
In this work, we report on an alternative, straightforward
approach to spisulosines based on the diastereocontrolled
addition of a suitable alkenylzirconocene to N-Boc L- or D-
alaninal according to the general approach described in Scheme
2.27
B
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a
Scheme 2. General Approach to Spisulosine 1 (ES285) and the Analogs Described in This Work from N-Boc-L-alaninal
a
Conditions: (a) EtOH, aqueous 2 M NaOH, rfx (90-95%); (b) H2, 5% Rh in Al2O3 (quant); (c) EDC, HOBt, octanoic acid, CH2Cl2 (80−85%).
Hydrozirconation of 1-pentadecyne with Schwartz re-
agent,28,29 followed by reaction with N-Boc-L-alaninal, afforded
the required sphingoid base 5 as a mixture of diastereomers, the
syn adduct being predominant in all cases (see below). The
highest, albeit modest (4.5/1), diastereoselection in the
alkenylzirconocene addition was achieved in the presence of
AgClO4 (10 mol %) in CH2Cl2,30 while a modest 2.3/1 syn/anti
selectivity was observed in the presence of ZnBr2 as additive
(50 mol %, THF as solvent).31,32
Scheme 3. Formation of Oxazolidinones 6 by Retention or
Inversion Processes
a
Interestingly, transmetalation with Et2Zn of the initially
formed alkenylzirconocene to the corresponding organozinc
reagent,33,34 followed by addition to N-Boc alaninal in CH2Cl2,
increased the syn/anti ratio up to 7.3/1, in agreement with
reported results from Garner aldehyde.28,29 The major
formation of the syn adduct in all the conditions tested can
be explained either by operation of an apparent “chelation
controlled” transition state, following a classical Cram’s
model,35 or by a coordinated delivery from an alternative
nonchelated transition state.36,37
The resulting mixture of diastereomers syn/anti 5 was
difficult to separate at this stage. For preparative purposes, they
were converted into the corresponding oxazolidinones 6
(Scheme 2), which could be cleanly separated by flash
chromatography. Formation of 6 was initially carried out by
base-promoted (K2CO3 in MeOH) intramolecular cyclization
of the syn/anti mixture 5 (Scheme 3). Since this reaction
implied the formation of a transient alkoxide and the
nucleophilic intramolecular attack upon the N-Boc group,38
the ratio trans-6/cis-6 of the ensuing oxazolidinones matched
the syn-5/anti-5 ratio of the starting mixture, via an overall
“retention” process (Scheme 3).
a
Yields for cis-6: 70% (inversion); 10% (retention); trans-6: 12%
(inversion); 70% (retention).
6 through an “inversion” process via N-Boc promoted
intramolecular SN2 displacement (Scheme 3). As expected,
treatment of the diastereomeric mixture 5 (syn/anti 7.3/1) with
MsCl and Et3N followed by in situ intramolecular cyclization,
led to a mixture of oxazolidinones 6 in a roughly 7/1 cis/trans
ratio.40 Interestingly, this inversion process has been judiciously
used in our case to gain access to the corresponding anti
In light of the well established intramolecular reactivity of the
N-Boc group toward electrophiles,38,39 we reasoned that
conversion of the hydroxyl group of 5 into a suitable leaving
group would favor the formation of a mixture of oxazolidinones
C
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Scheme 4. General Approach to Enantiomeric Spisulosines and Dehydrospisulosines ent-1 to ent-4
spisulosines after hydrolysis of the oxazolidinone system (see
below and Scheme 2).
assignment of 8 was crucial for that of the remaining spisulosine
analogs described in this work.
Double bond reduction of oxazolidinones 6 afforded,
uneventfully, the saturated systems 7 present in natural
spisulosine. Finally, access to natural spisulosine 1 (ES285),
the unsaturated analog 3 (RBM1-74), and the diastereomeric
spisulosine analogs 2 (RBM1-77) and 4 (RBM1-73) required
the alkaline hydrolysis of oxazolidinones 6 or 7, a process that
took place in good overall yields. It is worthy of mention that
attempts to obtain spisulosines by direct addition of
pentadecylzirconocene to N-Boc-L-alaninal were of no synthetic
utility, both in terms of diastereoselectivities and overall yields.
Using N-Boc D-alaninal as starting material, the above process
(Scheme 2) allowed the access to the enantiomeric series ent-1
to ent-4, as summarized in Scheme 4.
Scheme 5. Derivatization of syn and anti-5 as (R)-(−)-MPA
Esters and Assignment of the Absolute Configuration at C3
(Sphingoid Numbering) based on ΔδRS between the C3(R)-
and C3(S)-(R)-(−)-OMPA Esters
Stereochemical Assignments. Initial attempts to assign
the stereochemistry of oxazolidinones 6 and 7 by NOE
experiments were not conclusive, since only a weak NOE
effects (around 2−3%) were observed for C(5)H in cis-6 cis-7
on irradiation at C(4)H (Figure 2).
Based on the above results, we can conclude that
hydrozirconation of 1-pentadecyne followed by diastereoselec-
tive addition of the intermediate zirconocene to D- or L-alaninal
represents a straightforward approach to stereochemically
defined 1-deoxysphingolipid analogs. Intramolecular, stereo-
contolled oxazolidinone formation from the mixture of the
initially formed syn/anti adducts 5 is crucial for the overall
efficiency of this synthetic approach. Interestingly, this
sequence can be easily extended to other related 1-
deoxysphingolipid analogs by a judicious choice of the starting
alkyne in the hydrozirconation step.
Enzymatic N-Acylation of Spisulosines. One of the steps
of de novo ceramide synthesis is the acylation of dihydros-
phingosine to form dihydroceramide. This reaction is catalyzed
by ceramide synthases (CerS), genetically encoded by longevity
assurance homologue of yeast lag1 (Lass) genes. Six
mammalian ceramide synthases (CerS1-CerS6) have been
identified. Each isoform has the ability to produce ceramides
with characteristic acyl-chain distributions, which may be, at
least in part, associated to specific enzyme compartmentaliza-
tion in a context-dependent manner. By participating in the
control of SL’s acyl-chain structure, CerS regulates multiple
aspects of sphingolipid-mediated cell biology.42−44 Recent
studies suggest that de novo-generated ceramides may have
different and opposing activities in tumor promotion/
suppression. In these different roles, the subcellular location
of the generated amide and its N-acyl moiety are probably of
importance by activating different downstream targets. For
example, CerS6/C16Cer is associated with increased tumor
growth in head and neck cancer cells, whereas CerS1/C18Cer
Figure 2. NOE effects from oxazolidionones cis-6 and cis-7.
Alternatively, we considered an indirect approach to
determine of the absolute configuration of the newly formed
stereogenic centers in syn/anti-5. Derivatization by means of
the α-methoxy-α-phenylacetic (MPA) esters is one of the most
frequently used auxiliary reagents for the assignment of the
41
1
absolute configuration of secondary alcohols by HNMR. In
our case, derivatization of the mixture syn/anti-5 with (R)-
(−)-MPA, followed by chromatographic separation of the
resulting diastereomeric (R)-(−)-OMPA esters 8 and 9,
allowed the configurational assignment of ester 8 as the 3R
isomer (sphingoid base numbering). Thus, positive Δδ for H4
to H7 and negative Δδ for Me and H2 are indicative of the
assigned configuration for ester 8. This was nicely corroborated
by its conversion into oxazolidinone cis-6 by “one-pot” ester
hydrolysis with in situ cyclization by treatment with methanolic
K2CO3 under the above “retention conditions”. Similar
reactivity was observed from the diastereomeric ester 9,
which was cleanly converted into trans-6 under identical
conditions (Scheme 5). The unambiguous configurational
D
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a
Table 1. Effect of Compounds (CC50, μM) on Viability of MDA MB 468 Cells
a
Cell viability was determined by MTT reduction. The CC50 values were obtained from dose−response curves. The % of viability numbers were
adjusted with the four parameter logistic equation using GraphPad Prism Software with top and bottom constraints at 100 and 0%, respectively. The
data were obtained from 2 or 3 experiments with triplicates. dhSo: dihydrosphingosine; So: sphingosine
has been shown to suppress tumor growth in several cancer
models.43 On the other hand, CerS1/C18Cer mediates lethal
mitophagy in human cancer cells,45 while CerS5/C14Cer are
required for cardiomyocyte autophagy in relation to lipotoxic
cardiomyopathy and hypertrophy.46
The increasing interest in CerS activity, compartmentaliza-
tion and function underscores the need for appropriate CerS
probes. Characterization of (dihydro)ceramidomes is not a
suitable means to profile CerS activities in intact cells, since
(dihydro)ceramide populations are the result of the overall
activities of ceramide metabolism enzymes. The compounds
described here were envisaged as useful tools in CerS activity
profiling, as they are inert toward enzymes of 1-O-
functionalization.
As a proof of concept, the metabolic N-acylation of
spisulosine 1 (ES285), the stereoisomeric 2 (RBM1-77), the
dehydroanalogs 3 (RBM1-74) and 4 (RBM1-73) and the
corresponding enantiomeric counterparts (ent-1: RBM1-76,
ent-2: RBM1-75, ent-3: RBM1-72, and ent-4: RBM1-71, see
Schemes 2 and 4) was investigated in the MDA MB 468 breast
cancer cell line. The effect of compounds on cell viability was
similar, as concluded from the CC50 values of the dose response
curves, which ranged from 8.3 to 13.2 μM in 24 h treatments
(one way ANOVA, P > 0.05, see Table 1).
Moreover, the natural bases sphingosine and dihydrosphin-
gosine (Scheme 1) had comparable CC50 values in the same
experimental conditions. Furthermore, similar viabilities were
observed for MCF-7 and MDA MB 231 cell lines in the
presence of the compounds (data not shown). The
cytotoxicities found here are lower than those reported for
ES285, RBM1-76 and RBM1-77 in other cell lines.24
Unfortunately, the lack of detailed experimental information,
including incubation time, precludes any comparison.
On the other hand, the eight 1-deoxyamines were N-acylated
in intact MDA MB 468 cells, although the total amount of
amides varied according to the probe stereochemistry (Figure
Figure 3. (A) Amounts (pmol) relative to C12Cer of N-acylated
derivatives of ES285 and RBM1-71 to RBM1-77 as measured by
3A). Not surprisingly, the 4 stereoisomers having the natural
configuration at the CNH2 center (RBM1-73, RBM1-74,
RBM1-77 and ES285) gave significantly higher quantities of N-
acylated derivatives than the corresponding analogs with
opposite CNH2 configuration (Figure 3A). Furthermore,
spisulosine (ES285) afforded the highest amounts of amide
metabolites, which were significantly higher than those
produced from the 4,5-dehydro analog (RBM1-74). These
results indicate that CerS are not only selective for the fatty acyl
donor, but also discriminate between acceptor bases, even
between the saturated and the unsaturated ones.
UPLC/TOF analyses of lipid extracts from cells incubated with the
compounds (5 μM, 3 h). (B) Percentage of endogenous ceramides
and (C) 1-deoxyceramides found in extracts from cells treated with
ES285, RBM1-77, RBM1-74, RBM1-73, and vehicle. (D) Total
amounts of recovered octanamides (C8) and their N-transacylated
derivatives (ΣC16−C24), as measured by UPLC/TOF analyses of
lipid extracts from cells incubated with the compounds (5 μM, 3 h).
(E) Percentage of 1-deoxyceramides formed in extracts from cells
treated with RBM1-35. Data, presented as mean SD, were obtained
from 2 separate experiments with duplicates. In panels A and D, means
are statistically different at the indicated P values (one way ANOVA).
Symbols on top of the SD bar indicate results from Bonferroni’s
Multiple Comparison post-test; (p < 0.05). See Table 1 and Scheme 2
for compounds stereochemistry.
Analysis of the different N-acylated species showed as the
most striking feature that, for compounds with the natural
E
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measured at the sodium D line (589 nm) and are reported as [α]D (c,
g/100 mL, solvent). UPLC-TOF analyses were carried out as
previously reported.47 Electrospray ionization was used for HRMS
analysis.
stereochemistry at CHNH2, which undergo the highest
acylation levels, the percentages of the N-C18 1-deoxyamides
(Figure 3C) were remarkably higher (30.2%, 39.7%, 15.2% and
25.3% for RBM1-73, RBM1-74, RBM1-77, ES285, respec-
tively) than those of the natural N-C18 ceramide (4.6%, 4.6%,
4.7% and 6.2% for RBM1-73, RBM1-74, RBM1-77 and
ES285, respectively) (compare Figure 3B and C). In contrast,
the percentages of the N-C24 species, mainly C24:0 were
significantly lower (Figure 3B and C), while other N-acyl
species remained essentially unchanged. Importantly, the
ceramides profile is not affected by the treatments, as similar
percentages of the different species are found in vehicle treated
controls (ethanol, Figure 3B). These data suggest that the levels
of endogenous CerS-generated-C18-ceramide are reduced by
metabolic interconversions that result in increased amounts of
the C24 species. In the case of the 1-deoxyamides, the amide
function is the only possible metabolic site, amenable to
transacylation by the sequential action of ceramidases and CerS.
To assess whether N-transacylation occurs, the formation of
different N-acyl species from the N-octanoyl derivatives of
amines RBM1-73, RBM1-74, RBM1-77 and ES285, namely
RBM1-39, RBM1-35, RBM1-43 and RBM1-33, respectively
(Scheme 2), was investigated using non toxic concentrations of
amides at 3 h incubation time.
As shown in Figure 3D, the compound undergoing the
highest transacylation (55.5%) was RBM1-35, (N-octanoyl-1-
deoxyceramide). The metabolization of RBM1-35 was
paralleled with a decrease in the amounts of the given probe
(Figure 3D). In contrast, the saturated counterpart, RBM1-33,
was only modestly transacylated (6.1%), while, the amounts of
the different amides formed from the other octanamides were
negligible (RBM1-39, 2.2%; RBM1-43, 3.0%). Regarding the
different N-transacylated species formed from RBM1-35, the
C16 and C18 species were the most abundant ones, with twice
as much C18 than C16 (Figure 3E). This profile resembles the
obtained from the parent amine, RBM1-74 (Figure 3C).
Likewise, not remarkably different N-acyldeoxy(dh)ceramide
profiles are produced from the other amine/octanamide pairs
(Figure S1, Supporting Information). These results suggest that
the different transacylation rates of the octanamides result from
the substrate specificity of ceramidase(s), which hydrolyze
RBM1-35 preferentially. Which of the different ceramidases are
involved in the hydrolysis will be investigated in the near future.
In summary, ES285, RBM1-77 and RBM1-73 are suitable
probes for CerS profiling in intact cells, as they are
metabolically stable at both C1 and the amide linkage and
thus the resulting amide composition reflects the overall CerS
activities in a given experimental condition. Among the three
compounds, ES285 gives the highest acylation rates, thus being
the compound of choice. These molecules should prove useful
to study the role of the different CerS and N-acyl (dihydro)-
ceramides in cell fate.
(2S,3RS,E)-tert-Butyl 3-hydroxyoctadec-4-en-2-yl)carbamate
(syn- and anti-5). a. Via Schwartz Reagent in the Presence of
ZnBr2. To an ice-cooled stirred suspension of Cp2Zr(H)Cl (520 mg,
2.0 mmol) in THF (1 mL) under Ar was added neat 1-pentadecyne
(530 μL, 2.0 mmol). After stirring at rt for 1h, the reaction mixture was
cooled to 0 °C and a solution of N-Boc L-alaninal48 (173 mg, 1.0
mmol) in THF (1.5 mL) was added dropwise, followed by ZnBr2 (112
mg, 0.5 mmol), previously dried by evaporation from an anh THF
solution. The resulting mixture was stirred for 24 h at rt, diluted with
EtOAc (10 mL) and aq. potassium sodium tartrate (10 mL) and
stirred for additional 10 min. The resulting suspension was filtered off
and washed with EtOAc (10 mL). The combined filtrate and washings
were washed with brine, dried, and evaporated. The residue was flash
chromatographed using a stepwise gradient from 0 to 20% hexanes−
EtOAc to afford a 2.3:1 mixture of syn/anti-5 in 50% yield on elution
with a 88:12 hexanes−EtOAc mixture.
b. Via Schwartz Reagent in the Presence of AgClO4. Neat 1-
pentadecyne (135 μL, 0.5 mmol) was added dropwise to Cp2Zr(H)Cl
(130 mg, 0.5 mmol) in CH2Cl2 (4 mL) at 0 °C under Ar. The reaction
mixture was allowed to warm to rt (around 20 min) to give a clear pale
yellow solution. A solution of N-Boc L-alaninal (87 mg, 0.5 mmol) in
CH2Cl2 (1 mL) was next added followed by AgClO4 (10 mg, 0.05
mmol). After 30 min, the dark brown reaction mixture was diluted
with Et2O (5 mL) and the organic phase was washed with sat
NaHCO3 aqueous solution and then filtered through a pad of Celite.
The organic extracts were washed with aqueous sat NaHCO3 and
brine. Usual workup afforded a 4.5:1 mixture of syn/anti-5 in 74%
yield.
c. Via Transmetalation with Et2Zn.28 To an ice-cooled stirred
suspension of Cp2Zr(H)Cl (260 mg, 1.0 mmol) in CH2Cl2 (4 mL)
under Ar was added neat 1-pentadecyne (265 μL, 1 mmol). After
stirring at rt for 1h, the reaction mixture was cooled to −40 °C and
next treated with a 1.0 M solution of Et2Zn in hexane (1 mL, 1.0
mmol), followed by N-Boc L-alaninal (173 mg, 1.0 mmol) in CH2Cl2
(1.5 mL). The resulting mixture was allowed to warm gradually and
stirred at rt for 1 h. The mixture was next diluted with EtOAc (10 mL)
and aq. potassium sodium tartrate (10 mL). A similar workup as
described in a) afforded a 7.3:1 mixture of syn/anti-5 in 82% yield.
1HNMR (CDCl3, 400 MHz); data for the major syn isomer: 5.71−
5.59 (dt, J = 13.4, 6.7, C(5)H), 5.46−5.35 (m, 1H, C(4)H), 3.93 (dd, J
= J′ = 6.2, 1H, C3H), 3.63 (m, 1H, C(2)H), 2.03 (m, 2H), 1.43 (s,
9H), 1.35 (broad, 2H), 1.25 (broad, 20H), 1.13 (d, J = 6.8, 3H,
C(1)H3), 0.87 (t, J = 6.9, 3H, C(18)H3). 13CNMR (CDCl3, 101
MHz): 134.2 (C5), 129.4 (C4), 79.6 (C(CH3)3), 76.1 (C3), 51.2
(C2), 32.5 (C6), 32.1 (C16), 29.8−29.2, 28.3 (C(CH3)3), 17.8 (C1),
14.3 (C18). HRMS (mixture of syn/anti-5): Calculated for
C23H46NO3 (M + 1)+: 384.3472; Found 348.3478. Application of
this protocol using N-Boc D-alaninal afforded ent-syn/anti-5 in
comparable yields.
(4S,5RS)-4-Methyl-5-[(E)-pentadec-1-en-1-yl]oxazolidin-2-
one (cis-6 and trans-6). a. Retention Conditions. A solution of 5
(syn/anti 7.3/1 mixture, 380 mg, 1 mmol) in MeOH (10 mL) is
treated with K2CO3 (600 mg, 4.3 mmol) and warmed gently in a water
bath at 50 °C until consumption of the starting material (TLC). The
reaction mixture is next evaporated to dryness, taken up in H2O (10
mL) and extracted with CH2Cl2 (3 × 15 mL). Usual workup affored a
crude that was flash chromatographed on a stepwise gradient of
hexanes/EtOAc from 1 to 40%. Carbamate trans-6 was isolated on
elution with a 72:28 gradient mixture in 70% yield and cis-6 was
obtained in 10% yield on elution with a 66:34 gradient mixture.
b. Inversion Conditions. A solution of mesyl chloride (170 mg, 1.5
mmol) in CH2Cl2 (4 mL) was added dropwise to a solution of
aminoalcohols 5 or ent-5 (syn/anti 7.3/1 mixture, 380 mg, 1 mmol) in
CH2Cl2 (6 mL) containing Et3N (415 μL,303 mg, 3.0 mmol). After
stirring for 1h at rt, the reaction mixture was quenched with H2O (5
mL) and the organic phase was separated, dried over Na2SO4 and
EXPERIMENTAL SECTION
■
General. Solvents were distilled prior to use and, if required, dried
by standard methods. 1H and 13CNMR spectra were obtained in
CDCl3 solutions at 400 MHz (for 1H) and 100 MHz (for 13C),
respectively, unless otherwise indicated. Chemical shifts (δ) are
reported in ppm relative to the solvent (CDCl3) signal. All reactions
were monitored by thin layer chromatography (TLC) on aluminum
foil precoated silica gel plates. Flash column chromatography was
performed with the indicated solvents using silica gel 60 (particle size
0.035−0.070). Yields refer to chromatographically and spectroscopi-
cally pure compounds, unless otherwise stated. Optical rotations were
F
dx.doi.org/10.1021/jo400440z | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
evaporated to dryness. The resulting crude was taken up in 1,2-DCE
(10 mL) and Et3N (690 μL, 505 mg, 5.0 mmol) was next added
dropwise. The resulting solution was refluxed until TLC indicated the
total formation carbamates 6 (typically around 4h). Evaporation to
dryness afforded a crude mixture that was flash chromatographed
under the above conditions to give trans-6 in 12% yield and cis-6 in
70% yield.
C18H40NO (M + 1)+: 286.3104; Found 286.3107. Mp: 76−77 °C; lit23
65−67 °C; lit17 64.5−66 °C. Base: [α]D +8.2 (c 1, CHCl3); lit3 +24.9
(c 1, CHCl3); lit18 +5.32 (c 0.36, MeOH); lit20 +21.4 (c 0.5 CHCl3);
lit17 +24.0 (c 1, CHCl3); lit23 +25.3 (c 0.95, CHCl3); lit25 +24.2 (c 1,
CHCl3). Hydrochloride: lit17 +3.2 (c 1, MeOH, HCl); lit21 +7.15 (c
0.42, MeOH).
2 (2S,3S, RBM1-77).24 Yield: 92% from carbamate trans-7. 1HNMR
(CD3OD, 400 MHz, sphingoid base numbering): 3.21 (m, 1H,
C(3)H), 2.69 (m, 1H, C(2)H), 1.50 (m, 2H), 1.29 (m, 26H), 1.04 (d,
J = 9.2, 3H, C(1)H3), 0.90 (t, 3H, C(18)H3). 13C NMR (CD3OD, 101
MHz): 77.2, 52.4, 34.6, 33.1, 30.9−30.8, 30.5, 26.9, 23.7, 19.4, 14.5.
HRMS: Calculated for C18H40NO (M + 1)+: 286.3104; Found
286.3100. Mp: 87−88 °C. Base: [α]D −7.6 (c 1, CHCl3); lit20 +3.4 (c
1.8, MeOH); no alfaD.24
1
trans-6: HNMR (CDCl3, 400 MHz, sphingoid base numbering):
5.84 (dt, J = 15.3, 6.8, 6.8, 1H, C(5)H), 5.49 (dd, J = 15.3, 7.9, 1H,
C(4)H), 4.42 (dd, J = J′ = 7.9, 1H, C(3)H), 3.62 (m, 1H, C(2)H),
2.08 (m, 2H, C(6)H2), 1.38 (m, 2H), 1.26 (m, broad, 23H), 0.87 (t,
3H). 13CNMR (CDCl3, 101 MHz, sphingoid base numbering): 159.3,
137.9, 125.5, 85.3, 54.5, 32.3, 32.1, 29.9−29.8 (3C), 29.7, 29.6, 29.5,
29.2, 28.8, 22.8, 19.4, 14.2. [α]D −33.0 (c 1, CHCl3). HRMS:
Calculated for C19H36NO2 (M + 1)+: 310.2741; Found 310.2747.
cis-6:24 1HNMR (CDCl3, 400 MHz, sphingoid base numbering):
5.86 (dd, J = 14.6, 7.6, 1H, C(5)H), 5.51 (dd, J = 14.6, 7.9, 1H,
C(4)H), 4.99 (dd, J = J′ = 7.9, 1H, C(3)H), 3.95 (m, 1H, C(2)H),
2.12 (m, 2H), 1.38 (m, 2H), 1.25 (m, broad, 20H), 1.14 (d, J = 6.5,
3H, C(1)H3), 0.88 (t, 3H). 13CNMR (CDCl3, 101 MHz, sphingoid
base numbering): 138.4, 122.6, 81.7, 51.8, 32.4, 32.0, 29.8−28.9, 22.8,
17.2, 14.3. [α]D +1.0 (c 1, CHCl3). HRMS: Calculated for C19H36NO2
(M + 1)+: 310.2741; Found 310.2736.
Starting from ent-5 (syn/anti mixture) carbamates ent-cis-6 and ent-
trans-6 were obtained in comparable yields; ent-trans-6: [α]D +29.6 (c
1, CHCl3). HRMS: Calculated for C19H36NO2 (M + 1)+: 310.2741;
Found 310.2750. ent-cis-6: [α]D −1.0 (c 1, CHCl3). HRMS:
Calculated for C19H36NO21 (M + 1)+: 310.2741; Found 310.2752.
Both compounds showed H and 13C spectra identical to those of
trans-6 and cis-6, respectively.
3 (2S,3R, RBM1-74).18,26 Yield: 91% from carbamate cis-6. 1HNMR
(CD3OD, 400 MHz, sphingoid base numbering): 5.71 (dt, 1H, J =
15.6, J′ = J″ = 6.4, C(5)H), 3.82 (m, 1H, C(3)H), 2.80 (m, 1H,
C(2)H), 2.07 (m, 2H, C(6)H2), 1.40 (m, 2H), 1.30 (m, 22H), 1.04 (d,
J = 6.6, 3H, C(1)H3),), 0.90 (t, 3H, C(18)H3). 13CNMR (CD3OD,
101 MHz, sphingoid base numbering): 135.1, 130.5, 78.1, 52.2, 33.5,
33.1, 30.8−30.7, 30.6, 30.5, 30.4, 30.3, 23.7, 18.2, 14.5. HRMS:
Calculated for C18H38NO (M + 1)+: 284.2948; Found 284.2949. Mp:
70−71 °C; Base: [α]D +7.5 (c 1, CHCl3).
4 (2S,3S, RBM1-73).49 Yield: 94% from carbamate trans-6. 1HNMR
(CD3OD, 400 MHz, sphingoid base numbering): 5.69 (dt, 1H, J =
15.4, J′ = 6.6, C(5)H), 5.43 (dd, 1H, J = 15.4, J′ = 7.44, C(4)H), 3.65
(t, 1H, C(3)H), 2.71 (m, 1H, C(2)H), 2.07 (m, 2H, C6(H)2), 1.39
(m, 2H), 1.30 (broad, 20H), 1.01 (d, J = 6.8, 3H, C(1)H3), 0.91 (t,
3H, C(18)H3). 13CNMR (CD3OD, 101 MHz): 134.7 (C5), 131.6
(C4), 79.0 (C3), 52.4 (C2), 33.4 (C6), 33.1, 30.8−30.7, 30.6, 30.5,
30.4, 30.3, 23.8, 18.9 (C1), 14.5 (C18). HRMS: Calculated for
C18H38NO (M + 1)+: 284.2948; Found 284.2946. Mp: 71−72 °C;
Base: [α]D +6.5 (c 1, CHCl3).
(4S,5R)-4-Methyl-5-pentadecyloxazolidin-2-one (cis-7) and
(4S,5S)-4-Methyl-5-pentadecyloxazolidin-2-one (trans-7). A
solution of the starting carbamate cis-6 or trans-6 (309 mg, 1.0
mmol) in EtOAc (20 mL) was hydrogenated at 1 atm and rt in the
presence of 5% (w/w) Rh on alumina. After stirring for 12 h, the
catalyst was removed by filtration over Celite and the resulting clear
solution was evaporated to dryness to afford the required carbamates 7
in quantitative yield.
The enantiomeric spisulosines (ent-1 and ent-2) and dehydrospi-
sulosines (ent-3 and ent-4) were obtained similarly from the
corresponding carbamates ent-6 or ent-7
ent-1 (2R,3S, RBM1-76):24 Yield: 93% from carbamate ent-cis-7. 1H
and 13C spectra identical to those of 1 (ES285), see above. Mp: 74−75
°C; Base: [α]D −8.5 (c 1, CHCl3). HRMS: Calculated for C18H40NO
(M + 1)+: 286.3104; Found 286.3103.
1
trans-7: HNMR (CDCl3, 400 MHz, sphingoid base numbering):
4.07 (m, 1H, C(3)H), 3.56 (m, 1H, C(2)H), 1.65 (m, 2H,C(4)H2),
1.25 (broad, 29H), 0.87 (t, 3H). 13CNMR (CDCl3, 101 MHz): 159.6,
84.4, 53.8, 34.3, 32.1, 29.8−29.4, 25.0, 22.8, 20.8, 14.2. [α]D −34.6 (c,
1.0 CHCl3). HRMS: Calculated for C19H38NO2 (M + 1)+: 312.2897;
Found 312.2892.
ent-2 (2R,3R, RBM1-75): Yield: 95% from carbamate ent-trans-7.
1H and 13C spectra identical to those of 2 (RBM1-77), see above. Mp:
84−85 °C; Base: [α]D +6.5 (c 1, CHCl3). HRMS: Calculated for
C18H40NO (M + 1)+: 286.3104; Found 286.3115.
24
1
cis-7 : HNMR (CDCl3, 400 MHz, sphingoid base numbering):
4.55 (m, 1H, C(3)H), 3.89 (m, 1H, C(2)H), 1.72 (m, 2H), 1.52 (m,
2H), 1.35−1.20 (broad, 24H), 1.15 (d, J = 6.4, 3H, C(1)H3), 0.87 (t,
3H, C(18)H3). 13CNMR (CDCl3, 101 MHz, sphingoid base
numbering): 159.7, 80.4 (C3), 51.3 (C2), 32.1, 29.8−29.3, 26.0,
22.83, 16.1 (C1), 14.3 (C18). [α]D +11.5 (c, 1.0 CHCl3). HRMS:
Calculated for C19H38NO2 (M + 1)+: 312.2897; Found 312.2903.
The enantiomeric series was obtained similarly from carbamates ent-
6: ent-cis-7: [α]D −15.2 (c, 1.0 CHCl3). HRMS: Calculated for
C19H38NO2 (M + 1)+: 312.2897; Found 312.2906. ent-trans-7: [α]D
+33.6 (c, 1.0 CHCl3). HRMS: Calculated for C19H38NO2 (M + 1)+:
312.2897; Found 312.2901. Both compounds showed 1H and 13C
spectra identical to those of trans-7 and cis-7, respectively.
1
ent-3 (2R,3S, RBM1-72): Yield: 94% from carbamate ent-cis-6. H
and 13C spectra identical to those of 3 (RBM1-74), see above. Mp:
67−68 °C; Base: [α]D −6.8 (c 1, CHCl3). HRMS: Calculated for
C18H38NO (M + 1)+: 284.2948; Found 284.2963.
ent-4 (2R,3R, RBM1-71): Yield: 92% from carbamate ent-trans-6.
1H and 13C spectra identical to those of 4 (RBM1-73), see above. Mp:
69−70 °C; Base: [α]D −7.0 (c 1, CHCl3). HRMS: Calculated for
C18H38NO (M + 1)+: 284.2948; Found 284.2945.
Synthesis of MPA Esters 8 and 9. EDC (120 mg, 0.65 mmol)
was added portionwise to a solution of 155 mg (0.4 mmol) of a
mixture syn/anti-5 in CH2Cl2 (10 mL) containing (R)(−)MPA (100
mg, 0.6 mmol), DMAP (73 mg, 0.6 mmol) and Et3N (111 μL, 0.8
mmol). After stirring for 2h at rt, the reaction mixture was washed with
aq. sat NaHCO3, the organic phase was separated, dried, evaporated to
dryness and the resulting residue was flash chromatographed
(hexanes/EtOAc from 0 to 10% gradient).
Spisulosines and Dehydrospisulosines by Hydrolysis of
Carbamates 6 and 7. A solution of the starting carbamate 6, ent-
6, 7 or ent-7 (0.5 mmol) in a 1:1 EtOH−aqueous 2N NaOH (20 mL)
was refluxed for 2 h or until disappearance of the starting carbamate.
The reaction mixture was next concentrated, diluted with H2O (5 mL)
and extracted with CH2Cl2 (3 × 5 mL). Usual workup afforded the
required spisulosines and dehydrospisulosines in optimal purities and
yields.
Compound 8 was obtained on elution with hexanes/EtOAc 8%, (Rf
1
0.66, hexanes/EtOAc 7/3) in 82% isolated yield. HNMR (CDCl3,
400 MHz, sphingoid base numbering): 7.45 (m, 2H), 7.38 (m, 3H),
5.72 (m, 1H, C(5)H), 5.34 (m, 1H, C(3)H), 5.32 (m, 1H, C(4)H),
4.77 (s, 1H), 3.78 (broad, 1H, NHBoc), 3.66 (m, 1H, C(2)H), 3.42 (s,
3H), 2.00 (m, 2H, C(6)H2), 1.62 (broad, 2H), 1.39 (s, 9H), 1.25
(broad, 20H), 0.88 (t, 3H, C(18)H3), 0.81 (d, J = 6.8, 1H, C(1)H3).
13CNMR (CDCl3, 101 MHz, sphingoid base numbering): 169.9 (CO),
154.9 (CO), 136.9, 136.3 (C5), 129.0, 128.9, 127.4, 124.5 (C4), 82.6,
1 (2S,3R, Spisulosine, ES285).24 Yield: 90% from carbamate cis-7.
1HNMR (CD3OD, 400 MHz, sphingoid base numbering): 3.40 (m,
1H), 2.78 (m, 1H), 1.51 (m, 2H), 1.39−1.23 (m, 26H), 1.04 (d, J =
6.4, 3H), 0.90 (t, 3H). 13CNMR (CD3OD, 101 MHz): 76.5, 52.1, 33.4,
33.1, 30.8−30.7, 30.5, 27.3, 23.7, 17.2, 14.5. HRMS: Calculated for
G
dx.doi.org/10.1021/jo400440z | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
79.4, 77.2 (C3), 57.4, 49.3 (C2), 32.4 (C6), 32.1, 29.8−29.5, 29.2,
28.9, 28.5, 22.8, 14.7 (C1), 14.2 (C18).
Cell Viability. Cells were seeded in 96-well plates at a density of
50,000 cells/mL (0.1 mL/well). Twenty four hours after seeding, the
medium was replaced with fresh complete medium containing vehicle
(0.3% ethanol, 100% viability) or the test compounds at
concentrations of 30, 20, 13.3, 8.9, 5.9, 4.0, 2.6, and 1.8 μM (2/3
serial dilution). The 30 μM solution was made by dilution of 0.9 μL of
a 10 mM solution in ethanol in 300 μL of complete medium. After 24
h of treatment, the medium was removed and cell viability was
determined by the MTT test.
Metabolization of Compounds in Intact Cells. Cells were
seeded in 6-well plates at a density of 500 000 cells/mL (2 mL/well).
Twenty four hours after seeding, the medium was removed and 1 mL
of complete fresh medium containing the test compounds at 5 μM
(0.5 μL of a 10 mM solution in ethanol) was added. After 3 h of
incubation at 37 °C, 5% CO2 cells were collected by trypsinization,
pelleted by centrifugation and lipids were extracted and analyzed as
reported.47
Compound 9 was obtained on elution with hexanes/EtOAc 10%,
(Rf 0.60, hexanes/EtOAc 7/3) in 86% isolated yield. 1HNMR (CDCl3,
400 MHz, sphingoid base numbering):): 7.43 (m, 2H), 7.33 (m, 3H),
5.42 (m, 1H, C(5)H), 5.21 (m, 1H, C(3)H), 5.20 (m, 1H, C(4)H),
4.76 (s, 1H), 4.48 (broad, 1H, NHBoc), 3.83 (m, 1H, C(2)H), 3.40 (s,
3H), 1.86 (m, 2H, C(6)H2), 1.42 (s, 9H), 1.29−1.15 (broad, 22H),
1.03 (d, J = 6.8, 1H, C(1)H3), 0.87 (t, 3H, C(18)H3). 13CNMR
(CDCl3, 101 MHz, sphingoid base numbering): 169.9 (CO), 155.3
(CO), 136.7 (C5), 136.2, 128.8, 128.6, 127.3, 124.1 (C4), 82.7, 79.4,
77.4 (C3), 57.4, 49.1 (C2), 32.2 (C6), 32.1, 29.8−29.7, 29.6, 29.5,
29.4, 29.1, 28.8, 28.5, 22.8, 17.8 (C1), 14.2 (C18).
One-pot Hydrolysis and Cyclization of MPA Esters 8 and 9.
A solution of the MPA ester 8 (50 mg, 94 μmol) in MeOH (1 mL) is
treated with K2CO3 (60 mg, 0.43 mmol) at rt until consumption of the
starting material (TLC). The reaction mixture is next evaporated to
dryness, taken up in H2O (1 mL) and extracted with CH2Cl2 (3 × 1.5
mL). Usual workup afforded 26 mg (89% yield) of oxazolidinone cis-6
(TLC, hexanes/EtOAc 70:30), whose spectroscopical data were
identical to those reported above. Following the same procedure,
MPA ester 9 afforded 24 mg (83% yield) of oxazolidinone trans-6.
General Method for the Synthesis of N-octanoyl Amines. To
a solution of EDC (1.62 mmol) and HOBt (1.19 mmol) in anhydrous
CH2Cl2 (5 mL) was added the corresponding carboxylic acid (1.08
mmol) under argon atmosphere. The resulting mixture was vigorously
stirred at rt for 10 min, and next added dropwise to a solution of the
corresponding amine (1.00 mmol) and Et3N (3.90 mmol) in
anhydrous CH2Cl2 (5 mL). The reaction mixture was stirred at rt
for 1 h under argon atmosphere. The mixture was next diluted by
addition of CH2Cl2 (10 mL), and washed successively with 1 M
NaHCO3 solution and brine (5 mL each). The organic layer was dried
over MgSO4, and filtered. Concentration under reduced pressure
afforded crude compounds, which were purified by flash chromatog-
raphy (hexanes/EtOAc, 7:3) to give the corresponding amides in 80−
85% yield.
ASSOCIATED CONTENT
* Supporting Information
■
S
Copies of NMR spectra for the compounds described in this
work, UPLC-TOF analyses of the percentage of 1-deoxycer-
amides formed in extracts from cells treated with RBM1-33,
RBM1-43, and RBM1-39. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: jlaqob@cid.csic.es (J.L.A.); delgado@rubam.net
(A.D.).
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank Dr. Yolanda Per
■
(2S,3R, RBM1-33): 1HNMR (CDCl3, 400 MHz): 5.81 (broad,
NH), 4.00 (m, 1H), 3.62 (m, 1H), 2.16 (t, 2H), 1.62 (m, 2H), 1.25
(broad, 36H), 1.09 (d, J = 8 Hz, 3H), 0.87 (2t, 6H). 13C NMR
(CDCl3, 101 MHz): 173.3, 74.5, 49.6, 37.0, 33.7, 32.0, 31.8, 29.8−
29.7, 29.5, 29.4, 29.1, 26.6, 25.9, 22.8, 22.7, 14.3, 14.2. HRMS:
Calculated for C26H54NO2 (M + 1)+: 412.4149; Found 412.4153.
́
ez for her technical
assistance in the NMR experiments and Mrs. Eva Dalmau for
her experimental contribution. This work was supported by
grants SAF2011-22444 (Ministry of Science and Innovation)
̀ ́
and 2009SGR1072 (Agencia de Gestio d’Ajuts Universitaris i
de Recerca de la Generalitat de Catalunya) to G.F. I.N. was
supported by a JAE-predoc fellowship (CSIC).
1
(2S,3R, RBM1-35): H NMR (CDCl3, 400 MHz): 5.69 (m, 2H,
C(5)H + NH), 5.41 (dd, 1H, J = 16 Hz, J′= 8 Hz, C(4)H), 4.10 (m,
2H, C(2)H, C(3)H), 2.17 (t, 2H, C(2′)H2), 2.04 (m, 2H, C(6)H2),
1.62 (m, 2H), 1.28 (m, 30H), 1.09 (d, J = 8 Hz, 3H, C1(H)3), 0.87
(2t, 6H, C8′(H)3 + C18(H)3). 13C NMR (CDCl3, 101 MHz): 173.9,
134.2 (C5), 128.3 (C4), 75.8 (C3), 50.2 (C2), 37.0 (C2′), 32.5 (C6),
32.1, 32.8, 29.8−29.7, 29.6, 29.5, 29.4, 29.2, 26.0, 22.8, 22.7, 15.4 (C1),
14.3, 14.2. HRMS: Calculated for C26H52NO2 (M + 1)+: 410.3993;
Found 410.4011.
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■
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(2S,3S, RBM1-39): H NMR (CDCl3, 400 MHz): 5.67 (m, 2H,
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(2S,3S, RBM1-43): H NMR (CDCl3, 400 MHz): 5.78 (broad,
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(t, 2H), 1.40 (m, 2H), 1.28 (broad, 36H), 1.18 (d, J = 8 Hz, 3H), 0.88
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37.2 (C2′), 34.6, 32.1, 31.9, 29.8−29.7, 29.5, 29.4, 29.2, 26.0, 25.8,
22.8, 22.7, 18.5 (C1), 14.3, 14.2. HRMS: Calculated for C26H54NO2
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