Total synthesis and configurational assignment of the marine natural product haliclamide

Article abstract of DOI:10.1021/jo3027643

The marine natural product haliclamide has been synthesized based on macrocyclization by ring-closing olefin metathesis. Using either enantiomer of two of the four building blocks that were employed to assemble the diene precursor for the metathesis reaction, three non-natural isomers of haliclamide were also prepared. On the basis of the comparison of the ¹H and ¹³C NMR spectra of the individual stereoisomers with literature data for the natural product, the configuration of the previously unassigned stereocenters at C9 and C20 of haliclamide could be determined to be S for both carbons. The absolute configuration of haliclamide thus is 2S, 9S, 14R, 20S. The antiproliferative activity of synthetic haliclamide against several human cancer cell lines was found to be in the high μM range. The compound showed no antifungal or antibiotic activity.

Full text of DOI:10.1021/jo3027643

Article
pubs.acs.org/joc
Total Synthesis and Configurational Assignment of the Marine
Natural Product Haliclamide
Bernhard Pfeiffer,† Sandra Speck-Gisler,† Luzi Barandun,† Ursula Senft,† Claire de Groot,†
Irene Lehmann,‡ Walter Ganci,‡ Jurg Gertsch,† and Karl-Heinz Altmann†,*
̀
̈
†Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, HCI H405, Wolfgang-Pauli
̈
Str. 10, CH-8093 Zurich, Switzerland
̈
‡Institute of Organic Chemistry, Laboratory for Process Research, University of Zurich, Wintherthurerstr. 190, CH-8057 Zurich,
̈
̈
Switzerland
S
* Supporting Information
ABSTRACT: The marine natural product haliclamide has been synthesized based on
macrocyclization by ring-closing olefin metathesis. Using either enantiomer of two of the four
building blocks that were employed to assemble the diene precursor for the metathesis
reaction, three non-natural isomers of haliclamide were also prepared. On the basis of the
comparison of the 1H and 13C NMR spectra of the individual stereoisomers with literature data
for the natural product, the configuration of the previously unassigned stereocenters at C9 and
C20 of haliclamide could be determined to be S for both carbons. The absolute configuration
of haliclamide thus is 2S, 9S, 14R, 20S. The antiproliferative activity of synthetic haliclamide
against several human cancer cell lines was found to be in the high μM range. The compound
showed no antifungal or antibiotic activity.
phenylalanine (N-Me-L-Phe), 5-hydroxy octanoic acid (HOA),
and 6-amino-7-hydroxy-2-methylheptanoic acid (AHMA). The
two latter have not been found in any other natural product so
far.
INTRODUCTION
■
Marine organisms are a highly prolific source of biologically
active secondary metabolites, many of which are potential lead
structures for drug discovery in different areas.1 At the same
time, the amount of material available from natural sources is
often insufficient for in-depth biological profiling; thus, the total
synthesis of marine natural products is of crucial importance
not only as a test ground for the assessment of the utility and
practicality of synthetic methods, but also as an enabling means
for natural product-based drug discovery and chemical biology.
Haliclamide (1; Figure 1) is a 16-membered cyclic
depsipeptide which was isolated from the marine sponge
Haliclona sp. by Randazzo et al.2 and reported to exhibit in vitro
antitumor activity against the human bronchopulmonary non-
small cell lung carcinoma cell line NSCLC-N6 (IC50 = 4 μg/mL
(8.7 μM)). Structurally, haliclamide (1) is composed of three
different hydroxy or amino acid units, including N-methyl-L-
Given its considerable bioactivity and in light of its structural
similarity with other depsipeptides of even higher potency, such
as, for example, the actin assembly inhibitors doliculide,3,4
jasplakinolide,5−7 and chondramide C,8,9 we felt that
haliclamide (1) should be an interesting and relevant target
for total synthesis and SAR studies.10 Unfortunately, however,
the stereochemistry of haliclamide (1) had not been fully
resolved by Randazzo et al. in their original structural work.2
While the configuration at C2 and C14 was firmly established
as S and R, respectively, the configuration of the stereocenters
at C9 and C20 remained unassigned. No attempts have been
reported subsequent to the original isolation work to determine
the configuration of these stereocenters, either through total
synthesis or by other means.
In this paper we now report the ring-closing metathesis
(RCM)-based stereoselective synthesis of natural haliclamide
and the concurrent elucidation of its absolute configuration as
that of 1 (Figure 1) by spectral comparison with published data
for the natural product. Three other isomers of haliclamide with
a 2S,14R configuration (atom numbering according to
Randazzo et al.; Figure 1) were also prepared as part of this
work following the same strategy. Although not all of these
compounds were ultimately obtained (or even attempted to be
obtained) as pure stereoisomers, their availability allowed the
Figure 1. Structure of haliclamide (1). The configuration at C9 and
C20 was only elucidated in the course of this work. Atom numbering is
according to Randazzo et al.2
Received: December 20, 2012
© XXXX American Chemical Society
A
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Scheme 1. Retrosynthesis of Haliclamide (1)
Chart 1
unambiguous identification of the diastereoisomer correspond-
ing to natural haliclamide. The latter was prepared in 92%
purity, with its 20R epimer as the only, but inseparable
impurity. With 1 in hand, we have assessed its cytotoxicity
against a broader range of human cancer cell lines than
previously reported.
ester 312 (er 93:7); silylation of 3 followed by ester
saponification provided the desired acid 4 in 58% overall
yield (Scheme 2).
Scheme 2. Synthesis of Carboxylic Acids 4 and ent-4
RESULTS AND DISCUSSION
■
As illustrated in Scheme 1 the retrosynthesis of diastereoisomer
1, which was eventually shown to correspond with natural
haliclamide, in the first major disconnection led to diene 14 as
the substrate for macrocyclization through RCM.11 This was to
be followed by simultaneous reduction of the double bond and
removal of the benzyl protecting group. Diene 14 would be
obtained by amide coupling of acid 6 with secondary amine 13;
the latter could be disconnected further into alcohol 10 and N-
Boc protected N-Me-L-Phe (11). Intermediate 10 was
envisaged to result from condensation of acid 4 with amine
9, which in turn was to be derived from L-serine as the source of
chirality at C14 (haliclamide numbering). Finally, the chiral
center in acid 4 was to be established by asymmetric Brown
allylation,12 while acid 6 would be accessible through
stereoselective allylation of (4R)-4-benzyl-3-propionyl-1,3-
oxazolidin-2-one.13,14
The three other haliclamide isomers with a 2S,14R
configuration, that is, 1-SR (9S,20R configuration), 1-RS
(9R,20S), and 1-RR (9R,20R) (Chart 1) were to be obtained
in a completely analogous fashion, employing the enantiomers
of building blocks 4 and 6, that is, ent-4 and ent-6, respectively.
Synthesis of Building Blocks. Carboxylic acid 4 was
obtained from δ-valerolactone via known aldehyde 215 and
The enantiomeric acid ent-4, which has not been described in
the literature previously, was obtained from 2 in complete
analogy to 4. Thus, Brown allylation of 2 with (+)-Ipc2B-
(CH2CHCH2) at −100 °C and subsequent hydrogenation of
the ensuing (crude) homoallylic alcohol with Ra−Ni gave ester
ent-3 in 39% yield (based on 2) with an er of 9:1. Silylation and
ester saponification then furnished ent-4.
Carboxylic acid 6 was prepared from oxazolidinone 513 by
hydrolytic removal of the auxiliary with LiOOH in 76% yield
B
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(Scheme 3). Likewise, saponification of ent-516 gave ent-6 in a
Scheme 5. Building Block Assembly and Synthesis of 1
yield of 73% (not shown).
Scheme 3. Synthesis of Carboxylic Acid 6
The synthesis of olefin 9 proceeded through primary alcohol
7 (Scheme 4), which was obtained from commercially available
Scheme 4. Synthesis of Amine 9
followed by Boc-removal with 20% TFA/DCM then gave the
free amino ester 13 in excellent yield (97% for two steps).
However, although homogeneous by TLC, 13C NMR analysis
showed 13 to be a mixture of at least two diastereoisomers; in
contrast, no clear indication for the presence of diaster-
eoisomers was evident from the corresponding 1H NMR
spectra, thus precluding even a rough determination of the
N-Boc-L-Ser(OBn)−OH by LAH reduction.17 Subsequent
oxidation with SO3•pyridine complex followed by Wittig
olefination of the crude aldehyde using Schlosser’s “instant
ylide”18 then gave the corresponding olefin 8 in 48% overall
yield (from 7). Boc-cleavage with 5% TFA in DCM in the
presence of triisopropylsilane (TIPS)19 finally delivered the
desired allylic amine 9 in 79% yield. The efficiency of the
olefination step critically depended on the method employed
for the oxidation of 7, with the use of the crude aldehyde
obtained through Swern oxidation resulting in significantly
lower yields of olefination product. Initial attempts at preparing
the intermediate aldehyde by DIBAL-H reduction of Boc-L-
Ser(OBn)-OMe20 only yielded the corresponding primary
alcohol 7.
As α-amino aldehydes generally undergo Wittig reactions
with little epimerization,21 we felt that the approach followed
for the synthesis of 9 should have delivered the compound in
good optical purity. On the other hand, the optical rotatory
power of this intermediate was somewhat lower than literature
values (+7.2° vs. +10.2°;22 −11.3°23 and −16.5°24 for ent-9),
which indicated that some erosion of stereochemical integrity
might have occurred in the elaboration of N-Boc-L-Ser(OBn)−
OH into olefin 9; this supposition was in fact corroborated
later.
Assembly of Building Blocks and Ring Closure. The
elaboration of building blocks 4, 6, and 9 into diene 14
commenced with the O-benzotriazolyl-N,N,N′,N′-tetramethyl-
uronium hexafluorophosphate (HBTU)25-mediated coupling of
acid 4 with amine 9, which gave the desired amide in excellent
chemical yield (89%, Scheme 5). Removal of the TBS group
from the coupling product with 20% TFA in DCM proceeded
smoothly to furnish the free alcohol 10 in high yield. While
routine analysis of the coupling product by 1H NMR
spectroscopy indicated the presence of a single compound
(before as well as after TBS deprotection), careful inspection of
the corresponding 13C NMR spectra showed signal splitting for
several carbon atoms for the immediate (TBS-protected)
coupling product and 10, respectively. At the same time,
amide 10 appeared to be homogeneous by TLC. DCC/DMAP-
mediated coupling of 10 with Boc-N-Me-L-Phe-OH (11)
1
isomer ratio by the integration of specific H NMR signals.
As for intermediates 10, 12, and 13, chromatographically
(TLC, FC) inseparable mixtures were also observed for the
corresponding intermediates derived from ent-4 or/and ent-6,
based on the presence of two sets of signals in their 13C NMR
spectra (for some carbons). However, while the presence of
mixtures of isomers was clearly unsatisfactory, the materials
obtained at this point were still processed further, assuming that
the separation of diastereoisomers would be feasible by HPLC
after macrocyclization. Thus, O-(7-azabenzotriazol-1-yl)-
N,N,N′,N′-tetramethyl-uronium hexafluoro-phosphate
(HATU)26-mediated coupling of acid 6 with amino ester 13
gave diene 14 as the substrate for the key cyclization step
(Scheme 5). HATU was preferred over other coupling reagents
in this step, as it has been shown to provide for optimal
coupling efficiency in reactions involving secondary amino
groups.26 Subsequent RCM of 14 was accomplished with
second generation Grubbs catalyst27 in refluxing toluene, to
provide a mixture of isomeric macrocycles in 49% total yield.
1
Not unexpectedly at this stage, H NMR analysis indicated the
presence of at least 3 isomers (at least partly due to the
(inconsequential) formation of double bond isomers in the
RCM step) that were inseparable either by TLC or FC. The
mixture of RCM-products (after filtration over silica gel) was
then submitted to catalytic hydrogenation over Pd/C in
MeOH, leading to simultaneous double bond reduction and
cleavage of the benzyl ether moiety. Purification of the
hydrogenation products by FC gave 38% of an inseparable
1
(by FC) 92:8 mixture of two isomers (based on H NMR;
isomer I and isomer II, respectively) and 32% of a ca. 2:3
mixture of the I/II mixture and a third isomer III (based on
reversed-phase (RP)-HPLC analysis; isomers I and II were
inseparable also by HPLC).28 Isomer I was eventually shown to
be haliclamide (1) (vide infra).
C
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Isomers 1-SR (9S, 20R configuration), 1-RS (9R, 20S), and
1-RR (9R, 20R) (Chart 1) were obtained from 13 (1-RS) and
20R-13 (haliclamide numbering; Figure 1) (1-SR and 1-RR) in
full analogy to the preparation of 1 from 13. As for the
synthesis of 1, mixtures of stereoisomers were obtained after
the final hydrogenation step that could not be simply accounted
for by the incomplete stereochemical purity of the starting
building blocks 4/ent-4. While samples of isomers 1-RS and 1-
SR with >90% purity could be isolated after FC, 1-RR was
obtained as a 3/1 mixture with its putative 14S-isomer (vide
infra); however, the spectral data collected for these materials
still allowed the unambiguous assignment of the C19 and C20
stereocenters in haliclamide. Thus, the 1H and 13C NMR
spectra of the major isomer I that was derived from starting
acids 4 and 6 matched perfectly with the spectral data published
for the natural product by Randazzo et al. (Table 1).2 In
contrast, clear deviations from the published NMR data were
apparent for isomers II and III (best visible in the 13C NMR
spectra, see Supporting Information); likewise, the NMR data
collected for either 1-SR, 1-RS, or 1-RR were clearly different
from those published for haliclamide (Table 2). On the basis of
these findings, the absolute configuration of haliclamide could
be deduced as 2S,9S,14R,20S (corresponding to structure 1 in
Figure 1). Isomer II was eventually shown to be 1-SR, with the
92/8 ratio of 1/1-SR in the final product reflecting the
imperfect optical purity of acid 4 (S/R ratio of ca. 93/7).
While the presence of isomer II could be readily traced to the
er for acid 4, the detection of substantial amounts of a third
haliclamide isomer in the final mixture of hydrogenation
products in the synthesis of 1 (and also the isolation of major
isomeric impurities in the syntheses of 1-SR, 1-RS, and 1-RR)
was somewhat puzzling. However, as briefly discussed above,
the optical rotatory power of amine 9 had indicated that this
building block might not to be a single enantiomer, which
would have been a plausible cause for the formation of
(additional) diastereomeric products after its coupling with acid
4 and at all subsequent stages of the synthesis. In order to
clarify this issue, the optical purity of 9 was analyzed in a more
rigorous way, based on its derivatization with Marfey’s reagent
(1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (FDAA)).29,30
HPLC-MS analysis of the resulting product mixture revealed
two peaks with the expected mass for the FDAA derivative of 9
in a ratio of ca. 5/1 (ΔRt ca. 0.8 min; see Supporting
Information). Comparison with the FDAA derivative of ent-9
(prepared from Boc-D-Ser(OBn)−OH according to Scheme 4)
clearly established the two peaks to be the FDAA derivatives of
R- and S-9, respectively. While these numbers do not fully
match with the ratio of isomers I and III in the final product
mixture (which was ca. 5/2), the data strongly suggest that the
difference between these isomers was indeed the configuration
at C14;31 isomer III was thus tentatively assigned as the 14S
analog of natural haliclamide.32 By inference, the major
isomeric side products obtained in the syntheses of 1-SR, 1-
RS, and 1-RR are assumed to be the respective 14S epimers;
independent of this assumption, it is clear that none of these
compounds corresponds to natural haliclamide. Interestingly,
the ratio of 14R/14S isomers for the total amount of material
isolated after FC in all three cases proved to be lower than the
5/1 R/S ratio in the starting building block 9 (1-SR: 14R/14S =
1.44, for a total combined yield of 21% for the cyclization and
hydrogenation steps; 1-RS: 1.39, 48%; 1-RR: 1.95, 50%). The
reasons for this apparent discrepancy are unclear at this point; a
Table 1. NMR Spectroscopic Data for Synthetic 1 and
Natural Haliclamide (CDCl3, 500 MHz)
a
1
haliclamide
atom
number
δC
[ppm]
δH [ppm]
(J in Hz)
δC
[ppm]
δH [ppm]
(J in Hz)
b
1
2
3
170.7
56.3
34.2
170.7
5.90, dd (11.9, 5.3) 56.2
5.90, dd (12.4, 5.6)
2.94, dd (14.6, 12.4)
2.95,
dd (15.1, 12.0)
34.1
3.51, dd (14.7, 4.7)
3.51, dd (14.6, 5.6)
4
136.6
128.5
128.4
126.8
31.2
136.5
128.4
128.4
126.7
31.2
5/5′
6/6′
7
7.27, m
7.18, m
7.20, m
2.84, s
7.27, t (7.7)
7.18, t (7.7)
7.20, t (7.7)
2.83, s
NMe
8
178.2
36.4
178.2
36.4
9
2.65−2.57, m
0.65, d (7.0)
1.74, m
2.60, m
10
11
18.0
18.0
0.63, d (6.8)
1.72, m
31.4
31.3
1.19, m
1.20, m
12
13
23.2
27.8
1.46, m
23.2
27.7
1.45, m
1.07, m
1.02, m
2.15, m
2.14, m
1.26, m
1.25, m
14
15
53.6
65.7
3.45−3.37, m
3.73−3.66, m
53.6
65.7
3.40, br m
3.68, br dd (11.6,
4.3)
3.87, dd (12.1, 1.8)
5.16, br s
3.87, d (11.6)
OH
NH
16
6.83, br d (5.7)
6.82, br d (5.6)
174.4
36.4
174.5
36.6
17
2.47−2.40, m
2.14, m
2.43, m
2.18, m
1.77, m
1.60, m
1.62, m
1.45, m
4.98, m
1.55, m
1.50, m
18
19
22.9
32.0
1.79, m
22.9
31.9
1.61, m
1.62, m
1.47, m
20
21
76.7
37.1
5.02−4.95, m
1.56, m
77.0
37.1
1.48, m
22
23
18.5
14.0
1.46, m
18.5
13.9
1.32, m
1.32, m
0.93, t (7.3)
0.92, t (7.3)
a
Data for natural haliclamide are from ref.2 Data for synthetic 1 were
obtained at 298 K; the measurement temperature for natural
b
haliclamide is not indicated in ref 2. For atom numbering see Figure
1.
detailed investigation of this finding, however, was outside of
the scope of the current study.
With regard to the loss of optical purity during the
elaboration of Boc-L-Ser(OBn)−OH into 9, we speculated
that partial racemization may have occurred at the stage of the
aldehyde obtained from 7, either during its formation/isolation
or in the ensuing Wittig reaction.33 In order to avoid this
problem, we have developed an alternative synthesis of amine 9
that does not proceed through a potentially epimerization-
prone α-amino aldehyde as an intermediate (Scheme 6).
The synthesis departed from acetonide 15,34,35 which was
converted into mono-PMB-protected triol 1634,35 by cleavage
with copper(II) chloride dihydrate36 in 92% yield. Conversion
of 16 into the corresponding cyclic dibutyltin acetal37,38
followed by in situ benzylation gave the primary benzyl ether
D
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a
1
Table 2. Comparison of Characteristic H-NMR Signals of Haliclamide (1), 1-SR, 1-RS, and 1-RR
b
haliclamide
1
1-SR
1-RS
1-RR
c
atom number
δH [ppm]
δH [ppm]
δH [ppm]
δH [ppm]
δH [ppm]
2
5.90, dd
2.83, s
0.63, d
4.98, m
0.92, t
5.90, dd
2.84, s
5.66, dd
2.93, s
3.69−3.64, m
2.68, s
3.65−3.58, m
2.62, s
NMe
10
0.65, d
0.81, d
1.05, d
0.90, d
20
5.02−4.92, m
0.93, t
4.95−4.89, m
0.83, t
5.06−5.00, m
0.93, t
4.90−4.82, m
0.82, t
23
a
b
c
CDCl3 at 500 MHz and 298K, except for 1-RR (400 MHz). Data from ref 2; the measurement temperature is not indicated in ref 2. For atom
numbering see Figure 1.
Scheme 6. Alternative Synthesis of Amine 9
Table 3. Antiproliferative Activity of Haliclamide (1) against
Human Cancer Cell Lines
a
cell line
IC50 [μM]
A549 (lung)
26 3.2
31 2.9
28 2.5
52 4.7
47 3.2
MCF-7 (breast)
HCT-116 (colon)
HL60 (leukemia)
Jurkat (leukemia)
a
Cells were exposed to 1 for 72 h. Cell numbers were determined by
an MTT assay. Numbers are average values of three experiments
standard deviations.
EXPERIMENTAL SECTION
■
(S)-5-((tert-Butyldimethylsilyl)oxy)octanoic Acid (4). To a
solution of ester 312 (4.63 g, 26.6 mmol) and imidazole (4.53 g,
66.5 mmol) in 50 mL of dry DCM was added TBSCl (6.03 g, 40
mmol) at 0 °C. The solution was allowed to warm to rt, stirred
overnight and poured into a mixture of 300 mL Et2O/300 mL of brine.
The aq. phase was extracted twice with 250 mL Et2O, the organic
extracts were dried (MgSO4) and the solvent evaporated. FC (hexane/
EtOAc 4:1) gave 5.06 g of (S)-methyl 5-((tert-butyldimethylsilyl)-
oxy)octanoate (66%) as a clear viscous liquid. [α]D20 = +0.01° (c 0.60,
CHCl3).43 1H NMR (400 MHz, CDCl3): δ 3.68−3.63 (m, 4H), 2.31
(t, J = 7.80 Hz, 2H), 1.75−1.57 (m, 2H), 1.49−1.25 (m, 6H), 0.90 (t, J
= 7.31 Hz, 3H), 0.89 (s, 9H), 0.04 (s, 6H). 13C NMR (100 MHz,
CDCl3): δ 174.1, 71.7, 51.4, 39.3, 36.4, 34.3, 25.93, 25.90, 20.8, 18.5,
18.1, 14.3, −4.5. IR: ν 2978, 2955, 2860, 1742, 1462, 1361, 1252, 1142
cm−1. HRMS (ESI) calcd for [C15H32O3Si + Na+] 311.2013, found
311.2013.
A solution of (S)-methyl 5-((tert-butyldimethylsilyl)oxy)octanoate
(5.79 g, 20 mmol) in 100 mL THF/water 4:1 and LiOH (2.49 g, 100
mmol) was stirred overnight at rt. The solvent was removed, 25 mL
water added, and the pH adjusted to 4 with 1M HCl followed by
extraction with DCM (2 × 200 mL). The organic extracts were dried
(MgSO4) and the solvent was evaporated to give 4 (4.86 g, 88%) as a
clear viscous liquid. [α]D20 = +0.01° (c 1.31, CHCl3).43 1H NMR (400
MHz, CDCl3): δ 9.75 (br s, 1H), 3.71−3.64 (m, 1H), 2.37 (t, J = 7.43
Hz, 2H), 1.76−1.26 (m, 8H), 0.91−0.87 (m, 12H), 0.04 (s, 6H). 13C
NMR (100 MHz, CDCl3): δ 179.4, 71.6, 39.3, 36.3, 34.1, 25.9, 20.5,
18.5, 18.1, 14.3, −4.45, −4.49. IR: ν 2956, 2860, 2360, 1708, 1254,
1140 cm−1. HRMS (ESI) calcd for [C14H30O3Si+Na+] 297.1856,
found 297.1855.
17 in excellent yield (87%);39 subsequent Mitsunobu reaction
with phthalimide as a nucleophile then furnished the N-
substituted phthalimide 18 (95%).24 DDQ-mediated cleavage
of the PMB protecting group followed by Grieco-Sharpless
olefination40,41 and final phthalimide cleavage with methyl-
amine42 yielded the desired amine 922 in 39% yield for the
three-step sequence from 18. (For syntheses of ent-9 see refs
23, 24). This material was elaborated into 1 through the same
sequence of reactions as depicted in Scheme 5, with the
chemical yields for the individual steps generally being
comparable with those obtained in the synthesis with partially
racemic 9. A notable exception is the final hydrogenation step,
which provided a 92% yield of a 92/8 mixture of 1 and 1-SR
(after FC) compared to a combined yield of 70% for the 92/8
mixture of 1/1-SR and the ca. 19/12/1 mixture of 14S
haliclamide/1/1-SR that had been obtained previously. None
of the (purported) 14S isomer of 1 was observed in this
product and the analytical data of the material were identical
with those of purified 1 derived from Boc-L-Ser(OBn)−OH
(vide supra).32 Unfortunately, we were not able to separate 1
and 1-SR either by FC or HPLC;28 given the moderate
biological activity of 1 (vide infra), no attempts were made to
further improve the synthesis by elaborating more selective
access to acid 4.
Synthetic haliclamide (1; 92% purity) was assessed for its
antiproliferative activity against an array of human cancer cell
lines, as only a single IC50 value had been reported for the
natural material in the context of the isolation work (8.7 μM;
against the NSCLC-N6 cell line).2 The compound was found
to inhibit human cancer cell growth in vitro with IC50 values
between 26 and 52 μM after a 72 h exposure period and thus to
be a moderately potent antiproliferative agent (Table 3). It
remains to be investigated whether modified analogs of 1 with
improved in vitro activity can be identified.
(S)-2-Methylpent-4-enoic Acid (6). To a solution of imide 513
(7.75 g, 29.2 mmol) in 90 mL THF/water 4:1 were added LiOH (2.79
g, 116.4 mmol) and 50% H2O2 (3.36 mL, 58.2 mmol). The mixture
was stirred overnight at rt, the pH adjusted to 1−2 with 90 mL 1 M
HCl, and the mixture extracted 3× with 300 mL Et2O. The organic
extracts were dried (MgSO4), the solvent was evaporated and the
residue distilled under reduced pressure (110 °C, 1 mbar) to give acid
6 (2.53 g, 76%) as a colorless liquid. [α]D20 = +10.5° (c 1.92, CHCl3)
(lit.:44 [α]D22 = +10.1° (c 1.0, CHCl3)). 1H NMR (400 MHz, CDCl3):
δ 5.82−5.72 (m, 1H), 5.12−5.04 (m, 2H), 2.59−2.50 (m, 1H), 2.50−
2.43 (m, 1H), 2.25−2.19 (m, 1H), 1.19 (d, J = 6.7 Hz, 3H). 13C NMR
E
dx.doi.org/10.1021/jo3027643 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
(100 MHz, CDCl3): δ 182.2, 135.1, 117.2, 39.1, 37.4, 16.3. IR: ν 2979,
2666, 1703, 1643, 1417, 1244, 916 cm−1. HRMS (ESI) calcd for
[C6H9O2 + 2Na+] 159.0392, found 159.0393.
The slurry was stirred for 24 h at rt, 120 mL of a 5% aq. KHSO4
solution were added, the phases were separated, and the aqueous
solution was extracted thrice with 100 mL Et2O. The organic extracts
were washed with water and brine and dried (MgSO4). Solvent
evaporation and FC (hexane/EtOAc 3:1) gave 950 mg (76%) of (R)-
2-(1-(benzyloxy)but-3-en-2-yl)isoindoline-1,3-dione as a yellow oil.
[α]D = +4.69° (c 0.50, CHCl3). H NMR (400 MHz, CDCl3): δ
7.86−7.80 (m, 2H), 7.74−7.68 (m, 2H), 7.29−7.18 (m, 5H), 6.16
(ddd, J = 17.4, 10.3, 7.2 Hz, 1H), 5.31 (td, J = 17.2, 1.2 Hz, 1H), 5.25
(td, J = 10.4, 1.1 Hz, 1H), 5.13−5.03 (m, 1H), 4.56 (d, J = 12.1 Hz,
1H), 4.49 (d, J = 12.1 Hz, 1H), 4.11 (t, J = 9.9 Hz, 1H), 3.75 (dd, J =
10.0, 5.7 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 168.1, 137.9,
133.9, 132.3, 132.0, 128.3, 127.6, 123.2, 119.0, 72.8, 68.9, 53.2. IR: ν
2866, 2364, 2339, 1773, 1707, 1517, 1498, 1468, 1454, 1384, 1357,
1333, 1304, 1173, 1099, 1029, 993, 958, 936, 883, 869, 718, 699, 679,
672, 531 cm−1. HRMS (ESI) calcd for [C19H17NO3+Na+] 330.1101,
found 330.1114.
(R)-tert-Butyl (1-(Benzyloxy)but-3-en-2-yl)carbamate (8). To
a solution of alcohol 717 (5.5 g, 19.5 mmol) and Et3N (8.0 mL, 58.4
mmol) in 140 mL DCM was added a solution of SO3•py-complex (9.3
g, 58.4 mmol) in 40 mL DMSO/30 mL DCM. After 30 min, 320 mL
water were added at 0 °C and the mixture extracted 4x with 400 mL
Et2O. Each individual organic extract was washed twice with 100 mL
10% citric acid, 100 mL water, 100 mL sat. NaHCO3, and again 100
mL water. The combined extracts were dried (MgSO4) and the solvent
was evaporated to give 4.85 g (89%) of crude aldehyde.
20
1
A suspension of MePPh3Br/NaH (12.2 g, 29.2 mmol) in 170 mL
THF was stirred for 1 h at 0 °C. A solution of the above crude
aldehyde (4.85 g, 17.4 mmol) in 160 mL THF was then slowly added
over a period of 1 h. After additional 2 h at 0 °C, 315 mL sat. NH4Cl,
400 mL water, and 700 mL Et2O were added at rt, the phases were
separated and the aq. phase was extracted thrice with 600 mL Et2O.
The combined extracts were dried (MgSO4) and the solvent
evaporated. FC (hexane/EtOAc 5:1) gave 2.60 g of olefin 8 (48%
A solution of (R)-2-(1-(benzyloxy)but-3-en-2-yl)isoindoline-1,3-
dione (790 mg, 2.6 mmol) in 80 mL of 8 M MeNH2 in EtOH was
stirred for 2 h at rt. After solvent evaporation FC (hexane/EtOAc 2:1
20
24
over two steps) as a yellowish oil. [α]D = +25.9° (c 1.40, CHCl3)
+ 2% Et3N) gave 270 mg (59%) of amine 9 as a yellow oil. [α]D
=
(lit.45 (ent-8): [α]D = −31.6° (c 1.0, CH2Cl2)). H NMR (400 MHz,
1
+10.2° (c 1.00, CHCl3). NMR data of this material were identical with
those of 9 derived from Boc-L-Ser(OBn)−OH.
CDCl3): δ 7.42−7.20 (m, 5H), 5.86 (ddd, J = 17.3, 10.4, 5.4 Hz, 1H),
5.32−5.10 (m, 2H), 4.90 (s, 1H), 4.63−4.45 (m, 2H), 4.32 (s, 1H),
3.53 (qd, J = 9.5, 4.6 Hz, 2H), 1.45 (s, 9H). 13C NMR (100 MHz,
CDCl3): δ 125.4, 138.0, 136.4, 128.4, 127.8, 127.7, 115.7, 80.0, 73.3,
72.1, 52.4, 28.4. IR: ν 3444, 3350, 3086, 3064, 3031, 2979, 2929, 2863,
2358, 1708, 1644, 1496, 1454, 1365, 1247, 1164, 1103, 1070, 1027,
991 cm−1. MS (ESI): m/z (%) 178.0 [M-Boc + 2H]+ (55), 222.1 [M-
t-Bu + 2H]+ (100), 278.3 [M + H]+ (35), 300.2 [M + Na]+ (10).
(R)-1-(Benzyloxy)but-3-en-2-amine (9). To a solution of 8 (1.2
g, 4.37 mmol) in 200 mL DCM were added triisopropylsilane (1.8 mL,
8.73 mmol) and 10 mL TFA and the mixture was stirred at rt for 2.5 h.
It was then slowly poured into 400 mL sat. NaHCO3 at 0 °C, 400 mL
DCM were added, the phases were separated and the aq. solution
extracted twice with 400 mL DCM. The combined extracts were dried
(S)-N-((R)-1-(Benzyloxy)but-3-en-2-yl)-5-hydroxyoctanamide
(10). To a solution of HBTU (1.95 g, 5.15 mmol) and 1-
hydroxybenzotriazole (HOBt) (695 mg, 5.15 mmol) in 17 mL DMF
were added acid 4 (1.41 g, 5.15 mmol) and N,N-diisopropylethylamine
(DIEA) (2.04 mL, 11.7 mmol). After 5 min amine 9 (830 mg, 4.68
mmol; obtained from Boc-L-Ser(OBn)−OH) was added and the
mixture was stirred at rt for 3.5 h. DMF was removed in vacuo, 20 mL
water and 20 mL DCM were added, the phases were separated and the
aq. solution was extracted twice with 20 mL DCM. The organic
extracts were dried (MgSO4) and the solvent evaporated. FC (hexane/
EtOAc 2:1) gave 1.8 g (89%) of (S)-N-((R)-1-(benzyloxy)but-3-en-2-
yl)-5-((tert-butyldimethylsilyl)oxy)octanamide as a slightly yellow oil.
20
1
[α]D = +2.7° (c 2.26, CHCl3). H NMR (400 MHz, CDCl3): δ
7.40−7.27 (m, 5H), 5.87 (ddd, J = 17.3, 10.4, 5.3 Hz, 1H), 5.78 (d, J =
8.5 Hz, 1H), 5.27−5.13 (m, 2H), 4.73−4.64 (m, 1H), 4.55 (d, J = 11.9
Hz, 1H), 4.51 (d, J = 11.3 Hz, 1H), 3.70−3.60 (m, 1H), 3.56 (d, J =
4.1 Hz, 2H), 2.19 (t, J = 7.6 Hz, 2H), 1.77−1.55 (m, 2H), 1.54−1.21
(m, 6H), 0.93−0.86 (m, 12H), 0.04 (s, 6H). 13C NMR (100 MHz,
CDCl3): 172.3, 137.8, 136.0, 128.4, 127.8, 127.7, 115.9, 73.3, 71.79,
71.76, 50.8, 39.3, 37.0, 36.58, 36.55, 25.9, 21.64, 21.62, 18.4, 18.1, 14.3,
−4.42, −4.45. IR: ν 3275, 3063, 2955, 2929, 2857, 2360, 2341, 1638,
1541, 1456, 1361, 1254, 1094, 1069, 1039, 920, 834, 773, 733, 696
cm−1. HRMS (ESI) calcd for [C25H43NO3Si+H+] 434.3085, found
434.3084.
(MgSO4) and the solvent evaporated. FC (hexane/EtOAc 2:1 + 2%
20
Et3N) gave 607 mg of amine 9 (79%) as a dark yellow oil. [α]D
=
+7.2° (c 1.35, CHCl3) (lit.:22 [α]D = +10.2° (c 1.35, CHCl3)). H
NMR (400 MHz, CDCl3): δ 7.42−7.20 (m, 5H), 5.83 (dddd, J = 16.9,
10.4, 6.1, 1.5 Hz, 1H), 5.24 (dq, J = 17.2, 1.5 Hz, 1H), 5.11 (dq, J =
10.4, 1.4 Hz, 1H), 4.54 (d, J = 1.7 Hz, 2H), 3.61 (dddd, J = 7.2, 5.1,
3.5, 1.8 Hz, 1H), 3.50 (ddd, J = 9.2, 4.1, 1.8 Hz, 1H), 3.31 (ddd, J =
9.4, 7.9, 1.7 Hz, 1H), 1.64 (s, 2H). 13C NMR (100 MHz, CDCl3): δ
139.1, 138.2, 128.4, 127.7, 115.2, 75.0, 73.3, 53.9. IR: ν 3375, 3063,
3030, 2980, 2858, 2360, 2341, 1645, 1586, 1496, 1454, 1362, 1092,
1028, 993, 920, 736, 697 cm−1. HRMS (ESI) calcd for [C11H15NO +
H+] 178.1226, found 178.1227.
24
1
A solution of the above (S)-N-((R)-1-(benzyloxy)but-3-en-2-yl)-5-
((tert-butyldimethylsilyl)oxy)octanamide (1.70 g, 3.92 mmol) in
DCM/TFA 4:1 (17 mL) was stirred for 2.5 h at 0 °C. Water (68
mL) was then added and the pH adjusted to 7 with sat. NaHCO3.
More water (30 mL) and DCM (120 mL) were added, the phases
were separated and the aq. solution was extracted 4x with 120 mL
DCM. The organic extracts were dried (MgSO4) and the solvent was
evaporated. FC (Et22O0 /MeOH 98:2) gave 1.02 g (82%) of amide 10 as
From Phthalimide 18. To a solution of 18 (1.03 g, 2.3 mmol) in
100 mL DCM/water 9:1 was added DDQ (630 mg, 2.8 mmol) at 0
°C. The mixture was stirred overnight at rt, diluted with DCM and
washed with NaHCO3. The organic phase was dried (MgSO4) and the
solvent evaporated. FC (hexane/EtOAc 1:1) furnished 657 mg (87%)
of (R)-2-(1-(benzyloxy)-4-hydroxybutan-2-yl)isoindoline-1,3-dione.
[α]D = −0.75° (c 1.0, CHCl3).43 1H NMR (400 MHz, CDCl3): δ
25
1
7.85−7.78 (m, 2H), 7.74−7.68 (m, 2H), 7.32−7.19 (m, 5H), 4.71
(dddd, J = 10.2, 9.0, 5.6, 4.7 Hz, 1H), 4.54 (d, J = 12.1 Hz, 1H), 4.48
(d, J = 12.1 Hz, 1H), 4.08 (dd, J = 9.9, 9.1 Hz, 1H), 3.76 (dd, J = 9.9,
5.8 Hz, 1H), 3.73−3.66 (m, 1H), 3.62−3.54 (m, 1H), 2.22−2.12 (m,
1H), 2.05−1.93 (m, 1H), 1.90 (br s, 1H). 13C NMR (100 MHz,
CDCl3): δ 168.9, 137.9, 134.0, 131.9, 128.3, 127.6, 123.3, 72.9, 69.5,
59.4, 48.3, 31.9. IR: ν 3463, 2871, 1771, 1702, 1375, 1089, 1040, 882,
721, 699, 531 cm−1. HRMS (ESI) calcd for [C19H19NO4 + H+]
326.1387, found 326.1384.
a colorless oil. [α]D = +7.8° (c 0.81, CHCl3). H NMR (400 MHz,
CDCl3): δ 7.41−7.27 (m, 5H), 6.00−5.77 (m, 2H), 5.28−5.13 (m,
2H), 4.74−4.63 (m, 1H), 4.54 (d, J = 12.0 Hz, 1H), 4.51 (d, J = 12.0
Hz, 1H), 3.61−3.52 (m, 3H), 2.31−2.15 (m, 2H), 1.81−1.69 (m, 3H),
1.55−1.24 (m, 6H), 0.96−0.86 (m, 3H). 13C NMR (100 MHz,
CDCl3): δ 172.5, 137.8, 135.8, 128.5, 127.8, 127.7, 116.0, 73.3, 71.8,
71.0, 70.9, 50.9, 39.7, 39.6 36.9, 36.4, 36.3, 21.6, 21.4, 18.8, 14.1. IR: ν
3291, 3064, 2956, 2930, 2870, 2360, 2342, 1637, 1541, 1455, 1361,
1124, 1028, 991, 921, 736, 697 cm−1. HRMS (ESI) calcd for
[C19H29NO3 + H+] 320.2220, found 320.2218.
To a solution of (R)-2-(1-(benzyloxy)-4-hydroxybutan-2-yl)-
isoindoline-1,3-dione (1.23 g, 3.8 mmol) and 2-nitrophenyl
selenocyanate (2.94 g, 12.9 mmol) in 30 mL THF was added (n-
Bu)3P (3.23 mL, 12.9 mmol) dropwise below 35 °C over a period of
15 min. The mixture was stirred for 1 h at rt, NaHCO3 (9.9 g, 0.12
mol) and 30% H2O2 (13.2 mL, 0.12 mol) were added at T < 35 °C.
10 (obtained from amine 9 as derived from acetonide 15): NMR,
IR, and TLC data were identical with those reported above (major
20
isomer). [α]D = +39.6° (c 1.00, CHCl3).
(S)-(S)-8-(((R)-1-(Benzyloxy)but-3-en-2-yl)amino)-8-oxooc-
tan-4-yl-2-((tert-butoxycarbonyl)-(methyl)-amino)-3-phenyl-
F
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The Journal of Organic Chemistry
Article
propanoate (12). To a solution of amide 10 (860 mg, 2.69 mmol) in
50 mL DCM were added Boc-N-Me-L-Phe-OH (11) (1.13 g, 4.04
mmol), DMAP (493 mg, 4.04 mmol), and DCC (1.67 g, 8.08 mmol)
and the mixture was stirred at rt for 40 min. Silica gel (10 g) was then
added and the solvent was evaporated. FC (hexane/EtOAc 1:1) gave
14 (obtained from amine 9 as derived from acetonide 15): NMR,
20
IR, and TLC data were identical with those reported above. [α]D
+12.9° (c 1.00, CHCl3).
=
Haliclamide (1). A solution of diene 14 (300 mg, 0.52 mmol) in
150 mL toluene was refluxed with Grubbs second generation catalyst
(66 mg, 0.08 mmol) for 22 h. After solvent removal the residue was
submitted to FC in DCM/acetone 5:1 (column diameter 2 cm,
column length 15 cm, 15 fractions of 12 mL each). Fractions 9−41
were repurified by FC with DCM/acetone 5:1 + 0.5% MeOH to give
20
1.55 g of ester 12 (99%) as a colorless oil. [α]D = −24.1° (c 0.50,
1
CHCl3). H NMR (400 MHz, CDCl3): Mixture of rotamers. δ 7.40−
7.10 (m, 10H), 6.08−5.75 (m, 2H), 5.29−5.10 (m, 2H), 5.00−4.60
(m, 3H), 4.59−4.45 (m, 2H), 3.60−3.47 (m, 2H), 3.34−3.19 (m, 1H),
3.09−2.90 (m, 1H), 2.81−2.62 (m, 3H), 2.26−2.09 (m, 2H), 1.75−
1.01 (m, 17H), 0.97−0.83 (m, 3H). 13C NMR (100 MHz, CDCl3): δ
172.0, 171.8, 171.2, 170.9, 155.2, 153.3, 137.9, 137.6, 135.9, 128.9,
128.9, 128.49, 128.45, 128.3, 127.8, 127.7, 116.0, 80.2, 79.9, 77.4, 77.3,
77.1, 76.7, 74.6, 73.2, 71.7, 60.8, 60.0, 50.9, 36.1, 36.0, 35.2, 34.9, 34.0,
33.2, 32.3, 28.3, 28.2, 26.1, 25.7, 25.0, 24.7, 21.2, 18.5, 13.9. IR: ν 3308,
2978, 2932, 2871, 2360, 2342, 1733, 1698, 1653, 1541, 1456, 1393,
1365, 1142, 748, 697, 669 cm−1. HRMS (ESI) calcd for [C34H48N2O6
+ Na+] 603.3405, found 603.3405.
1
130 mg (46%) of a multicomponent mixture as a brown resin: H
NMR (400 MHz, CDCl3): δ 7.40−7.10 (m, 10H), 6.02−5.85 (m,
1H), 5.59−5.37 (m, 1H), 5.04−4.84 (m, 1H), 4.73−4.60 (m, 1H),
4.56−4.44 (m, 2H), 3.52−3.37 (m, 2H), 2.95−2.76 (m, 4H), 2.65−
2.23 (m, 3H), 2.16−1.98 (m, 2H), 1.84−1.39 (m, 6H), 1.39−1.19 (m,
3H), 0.97−0.80 (m, 3H), 0.76−0.58 (m, 3H). HRMS (ESI) calcd for
[C33H44N2O5 + H+] 549.3323, found 549.3314.
The above material (70 mg, 0.13 mmol (based on the molecular
weight of the structure of the RCM product from diene 14)) was
hydrogenated over Pd−C (27 mg) in 12 mL MeOH at 3 bar of
hydrogen pressure for 18 h. The suspension was filtered and the
solvent evaporated. FC on silica gel 60 (20−45 μm) (DCM/acetone
7:3 + 3% MeOH) gave 22.5 mg (38%) of an inseparable 92/8 mixture
of haliclamide (1) and 1-SR, and 19 mg (32%) of a 9/1/15 mixture of
haliclamide (1), 1-SR, and 14S haliclamide, respectively, as amorphous
white solids. Haliclamide (1): [α]D24: −4.1° (c 0.02, CHCl3) (lit.:2
[α]D24: −4.8° (c 0.006 g/mL, CHCl3). 1H NMR (500 MHz, CDCl3):
δ 7.32−7.12 (m, 5H), 6.83 (d, J = 5.7 Hz, 1H), 5.90 (dd, J = 11.9, 5.3
Hz, 1H), 5.16 (br s, 1H), 5.02−4.95 (m, 1H), 3.87 (dd, J = 12.1, 1.8
Hz, 1H), 3.73−3.66 (m, 1H), 3.51 (dd, J = 14.7, 4.7 Hz, 1H), 3.45−
3.37 (m, 1H), 2.95 (dd, J = 15.1, 12.0 Hz, 1H), 2.84 (s, 3H), 2.65−
2.57 (m, 1H), 2.47−2.40 (m, 1H), 2.20−2.10 (m, 2H), 1.84−1.68 (m,
2H), 1.67−1.39 (m, 6H), 1.38−1.14 (m, 4H), 1.11−1.01 (m, 1H),
0.93 (t, J = 7.4 Hz, 3H), 0.65 (d, J = 7.0 Hz, 3H). 13C NMR (125
MHz, CDCl3): 178.2, 174.4, 170.7, 136.6, 128.5, 128.4, 126.8, 76.7,
65.7, 56.3, 53.6, 37.1, 36.41, 36.37, 34.2, 32.0, 31.4, 31.2, 27.8, 23.2,
22.9, 18.5, 18.0, 14.0. HRMS (ESI) calcd for [C26H40N2O5 + H+]
461.3010, found 461.3016.
12 (obtained from amine 9 as derived from acetonide 15): NMR,
IR, and TLC data were identical with those reported above (major
20
isomer). [α]D = −13.5° (c 1.00, CHCl3).
(S)-(S)-8-(((R)-1-(benzyloxy)but-3-en-2-yl)amino)-8-oxooc-
tan-4-yl-2-(methylamino)-3-phenyl-propanoate (13). To a
solution of 12 (1.55 g, 2.67 mmol) in 100 mL DCM were added
triisopropylsilane (0.6 mL, 2.93 mmol) and 25 mL TFA. The mixture
was stirred at rt for 30 min, cooled to 0 °C, and poured into 300 mL
sat. NaHCO3. DCM (300 mL) was added, the phases were separated
and the aq. solution was extracted twice with 300 mL DCM. The
extracts were dried (MgSO4) and the solvent was evaporated. FC
(hexane/EtOAc 1:1 + 1−2% Et3N) to furnish 1.25 g (98%) of amine
20
1
13 as a yellow oil: [α]D = +13.8° (c 1.00, CHCl3). H NMR (400
MHz, CDCl3): δ 7.40−7.14 (m, 10H), 5.93−5.77 (m, 2H), 5.25−5.13
(m, 2H), 4.93−4.82 (m, 1H), 4.71−4.62 (m, 1H), 4.55 (d, J = 11.7
Hz, 1H), 4.50 (d, J = 11.7 Hz, 1H), 3.55 (d, J = 5.2 Hz, 2H), 3.41 (t, J
= 6.6 Hz, 1H), 2.98−2.86 (m, 2H), 2.36 (s, 3H), 2.12−2.05 (m, 2H),
1.59−1.18 (m, 8H), 0.87 (t, J = 8.4 Hz, 3H). 13C NMR (100 MHz,
CDCl3): δ 174.4, 171.88, 171.86, 137.8, 137.3, 135.95, 135.91, 129.3,
128.5, 128.4, 127.72, 127.70, 126.6, 116.0, 115.97, 74.21, 74.16, 73.29,
73.26, 71.77, 71.75, 65.0, 64.9, 50.9, 39.6, 36.2, 35.94, 35.92, 34.7,
33.19, 33.15, 21.1, 21.0, 18.6, 13.9. IR: ν 3291, 2957, 2933, 2871, 2360,
2341, 1725, 1647, 1541, 1455, 1362, 1178, 1130, 921, 739, 698 cm−1.
HRMS (ESI) calcd for [C29H39N2O4 + H+] 481.3061, found 481.3067.
13 (obtained from amine 9 as derived from acetonide 15): NMR,
IR, and TLC data were identical with those reported above (major
(S)-4-((4-Methoxybenzyl)oxy)butane-1,2-diol (16). To a sol-
ution of PMB ether 1534,35 (217 mg, 0.82 mmol) in 15 mL MeOH
was added CuCl2•2H2O (750 mg, 4.5 mmol) and the mixture was
refluxed for 2 h. After cooling to 0 °C, NaHCO3 was added and the
solvent was evaporated. The residue was treated with EtOAc and
filtered and the filtrate was concentrated. FC (EtOAc) gave 170 mg
25
(92%) of monoprotected triol 16 as a clear oil. [α]D = +5.4° (c 1.0,
CHCl3) (lit.:34 [α]D = +4.4° (c 0.8, CHCl3)). H NMR (400 MHz,
CDCl3): δ 7.23−7.11 (m, 2H), 6.89−6.75 (m, 2H), 4.39 (s, 2H),
3.89−3.78 (m, 1H), 3.73 (s, 3H), 3.65−3.49 (m, 3H), 3.47−3.36 (m,
1H), 3.05 (br s, 1H), 2.25 (br s, 1H), 1.83−1.58 (m, 2H). 13C NMR
(100 MHz, CDCl3): δ 159.4, 129.8, 129.4, 113.9, 73.0, 71.4, 68.0, 66.6,
55.3, 32.8. HRMS (ESI) calcd for [C12H18O4 + Na+] 249.1097, found
249.1098.
25
1
20
isomer). [α]D = +42.9° (c 1.00, CHCl3).
(S)-(S)-8-(((R)-1-(Benzyloxy)but-3-en-2-yl)amino)-8-oxooc-
tan-4-yl-2-((S)-N,2-dimethylpent-4-enamido)-3-phenylpropa-
noate (14). To a solution of acid 6 (164 mg, 1.44 mmol) and HATU
(546 mg, 1.44 mmol) in 10 mL DMF was added DIEA (0.5 mL, 2.87
mmol). After 5 min at rt amine 13 (460 mg, 0.96 mmol) was added
and the solution was stirred at rt for 3 h. After removal of DMF, 20 mL
DCM and 20 mL 10% MeCN/water were added. The phases were
separated, the aq. solution was extracted twice with 20 mL DCM and
the organic extracts were dried (MgSO4) and the solvent evaporated.
FC (hexane/EtOAc 1:1 + 2% Et3N) gave 510 mg (92%) of amide 14
as a yellow oil. [α]D20 = −19.9° (c 1.28, CHCl3). 1H NMR (400 MHz,
CDCl3): δ 7.42−7.08 (m, 10H), 6.24−6.07 (m, 1H), 5.94−5.63 (m,
2H), 5.31−4.61 (m, 5H), 4.60−4.45 (m, 2H), 3.55 (d, J = 5.0 Hz,
2H), 3.42−3.21 (m, 1H), 3.07−2.75 (m, 4H), 2.66−2.52 (m, 1H),
2.45−2.32 (m, 1H), 2.28−2.10 (m, 2H), 2.07−1.79 (m, 2H), 1.71−
0.98 (m, 10H), 0.94−0.72 (m, 6H). 13C NMR (100 MHz, CDCl3): δ
176.3, 172.4, 170.7, 137.9, 137.2, 136.2, 135.9, 129.1, 128.9, 128.8,
128.5, 128.4, 128.3, 127.8, 127.7, 126.8, 116.3, 116.0, 77.4, 77.1, 76.7,
74.8, 74.7, 73.2, 71.8, 58.7, 50.9, 37.8, 36.14, 36.10, 35.7, 34.8, 33.3,
33.1, 21.33, 21.28, 18.6, 16.8, 13.9. IR: ν 3308, 3063, 2959, 2932, 2871,
2360, 2341, 1733, 1636, 1541, 1456, 1129, 1088, 917, 740, 698 cm−1.
HRMS (ESI) calcd for [C35H48N2O5 + Na+] 599.3455, found
599.3464.
(S)-1-(Benzyloxy)-4-((4-methoxybenzyl)oxy)butan-2-ol (17).
A suspension of monoprotected triol 16 (2.0 g, 8.8 mmol) and
Bu2SnO (2.41 g, 9.7 mmol) in 20 mL benzene was refluxed for 24 h
(Dean−Stark trap). TBAI (3.58 g, 9.7 mmol) and BnCl (1.11 mL, 9.7
mmol) were then added and refluxing was continued for 3 h. After
cooling to rt EtOAc was added (30 mL), the phases were separated
and the EtOAc solution was washed twice with each 15 mL NH4Cl
and 15 mL brine. The solvent was evaporated. FC (hexane/Et2O 1:1)
gave 2.42 g (87%) of 17 as a clear oil. [α]D25 = −12.0° (c 1.0, CHCl3).
1H NMR (400 MHz, CDCl3): δ 7.41−7.24 (m, 7H), 6.93−6.87 (m,
2H), 4.58 (s, 2H), 4.46 (s, 2H), 4.09−3.99 (m, 1H), 3.82 (s, 3H),
3.72−3.59 (m, 2H), 3.53−3.40 (m, 2H), 2.99 (d, J = 3.3 Hz, 1H),
1.84−1.78 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 159.1, 138.1,
130.2, 129.2, 128.3, 127.59, 127.56, 113.7, 74.3, 73.2, 72.7, 69.1, 67.5,
55.1, 33.1. HRMS (ESI) calcd for [C19H24O4 + H+] 317.1747, found
317.1744.
(R)-2-(1-(Benzyloxy)-4-((4-methoxybenzyl)oxy)butan-2-yl)-
isoindoline-1,3-dione (18). To a solution of 17 (91 mg, 0.28
mmol), phthalimide (84 mg, 0.57 mmol), and PPh3 (150 mg, 0.57
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mmol) in 5 mL THF was added diisopropyl azodicarboxylate (DIAD)
(112 μL, 0.57 mmol) at 0 °C. The mixture was stirred overnight at rt
and the solvent evaporated. FC (hexane/EtOAc 3:1) gave 121.6 mg
continued for 3.5 h. THF was then removed by evaporation, 15 mL
water were added, and the pH was adjusted to 4 with 1 M HCl. The
solution was then extracted twice with 100 mL DCM, the combined
20
(95%) of phthalimide 18 as a clear oil. [α]D = +0.47° (c 1.00,
organic extracts were dried over MgSO4 and the solvent was
CHCl3).43 1H NMR (400 MHz, CDCl3): δ 7.83−7.77 (m, 2H), 7.73−
7.67 (m, 2H), 7.31−7.21 (m, 5H), 7.18−7.12 (m, 2H), 6.80−6.75 (m,
2H), 4.75 (ddt, J = 9.8, 5.4, 4.5 Hz, 1H), 4.54 (d, J = 11.9 Hz, 1H),
4.45 (d, J = 12.1 Hz, 1H), 4.30 (s, 2H), 4.00 (dd, J = 9.9, 9.3 Hz, 1H),
3.78 (s, 3H), 3.72 (dd, J = 10.1, 5.6 Hz, 1H), 3.57−3.40 (m, 2H), 2.36
(dddd, J = 14.5, 10.3, 6.0, 4.5 Hz, 1H), 2.00 (tdd, J = 14.5, 7.8, 4.8 Hz,
1H). 13C NMR (100 MHz, CDCl3): δ 168.6, 159.0, 138.0, 133.7,
132.0, 130.2, 129.2, 128.3, 127.6, 127.5, 123.1, 113.6, 72.8, 72.7, 69.7,
67.0, 55.2, 48.9, 28.8. IR: ν 2928, 2863, 1773, 1751, 1734, 1708, 1611,
1513, 1468, 1455, 1376, 1337, 1303, 1289, 1248, 1173, 1146, 1098,
1036, 820, 741, 721, 699 cm−1. HRMS (ESI) calcd for [C27H27NO5 +
H+] 446.1962, found 446.1961.
20
evaporated to yield 1.65 g (97%) ent-4 as a clear oil: [α]D
=
−0.67° (c 1.16, CHCl3).43 1H NMR (400 MHz, CDCl3): δ 10.10 (br s,
1H), 3.70−3.63 (m, 1H), 2.36 (t, J = 7.4 Hz, 2H), 1.76−1.59 (m, 2H),
1.51−1.24 (m, 6H), 0.92−0.86 (m, 12H), 0.05 (s, 6H). 13C NMR
(100 MHz, CDCl3): δ 179.7, 71.7, 39.3, 36.3, 34.3, 25.9, 20.5, 18.5,
18.1, 14.3, −4.46, −4.49. IR: ν 2956, 2860, 2360, 1708, 1254, 1140
cm−1. HRMS (ESI) calcd for [C14H30O3Si + Na+] 297.1856, found
297.1855.
(R)-N-((R)-1-(Benzyloxy)but-3-en-2-yl)-5-((tert-butyl-
dimethylsilyl)oxy)octanamide. To a solution of HBTU (924 mg,
2.44 mmol) in 8 mL dry DMF were added ent-4 (668 mg, 2.51 mmol)
and DIEA (0.96 mL, 5.5 mmol) and the mixture stirred for 5 min.
Then 9 (388 mg, 2.19 mmol) was added and stirring was continued
for 3 h at rt. The DMF was removed by evaporation, 60 mL water 10%
MeCN and 60 mL DCM were added and the phases were separated.
The aq. phase was extracted twice with 60 mL DCM. The combined
organic extracts were dried over MgSO4 and the solvent was
evaporated. FC (hexane/EtOAc 2:1) gave 811 mg (85%) of (R)-N-
((R)-1-(benzyloxy)but-3-en-2-yl)-5-((tert-butyldimethylsilyl)-oxy)-
(R)-Methyl 5-hydroxyoctanoate (ent-3). To a solution of
(+)-(Ipc)2BCl (7.49 g, 23.3 mmol) in 50 mL Et2O was added a 1
M solution of allylmagnesium bromide (17.0 mL, 17 mmol) in Et2O
dropwise at 0 °C. The mixture was allowed to warm to rt and was then
cooled to −100 °C. A solution of methyl 5-oxopentanoate (2) (2.09 g,
16.1 mmol) in 20 mL Et2O was added dropwise strictly at −100 °C.
The mixture was then stirred at −100 °C for another hour; 3 mL dry
MeOH were added and the mixture was allowed to warm to rt. Three
M NaOH (8.5 mL) and 30% H2O2 (10 mL) were added and the
mixture was stirred for one hour at rt. It was then extracted twice with
70 mL Et2O, the combined organic extracts were washed with 100 mL
of brine and dried over MgSO4, and the solvent was evaporated. FC of
the residue (DCM/acetone/MeOH 97:2:1) gave 1.40 g (53%) of (S)-
methyl 5-hydroxyoct-7-enoate (er 9:1) as a clear oil. [α]D20 = −6.3° (c
1.03, CHCl3) (lit.12 ((+)-enantiomer). [α]D = +4.55° (c 1.67,
CHCl3)). 1H NMR (400 MHz, CDCl3): δ 5.88−5.77 (m, 1H),
5.18−5.12 (m, 2H), 3.70−3.62 (m, 4H), 2.39−2.27 (m, 3H), 2.21−
2.12 (m, 1H), 1.91−1.39 (m, 5H). 13C NMR (100 MHz, CDCl3): δ
174.1, 134.6, 118.2, 70.2, 51.5, 41.9, 36.0, 33.8, 21.0. IR: ν 3425, 3079,
2952, 2928, 2358, 1723, 1641, 1436, 1160 cm−1. HRMS (ESI) calcd
for [C9H16O3+Na+] 195.0992, found 195.0993.
20
octanamide as an amorphous white solid. [α]D = +14.0° (c 0.91,
1
CHCl3). H NMR (400 MHz, CDCl3): δ 7.39−7.29 (m, 5H), 5.88
(ddd, J = 17.3, 10.5, 5.3 Hz, 1H), 5.79 (d, J = 8.2 Hz, 1H), 5.26−5.16
(m, 2H), 4.72−4.66 (m, 1H), 4.56 (d, J = 12.0 Hz, 1H), 4.52 (d, J =
12.1 Hz, 1H), 3.69−3.62 (m, 1H), 3.56 (d, J = 4.2 Hz, 2H), 2.19 (t, J =
7.6 Hz, 2H), 1.76−1.60 (m, 2H), 1.49−1.31 (m, 6H), 0.90−0.86 (m,
12H), 0.05 (s, 6H). 13C NMR (100 MHz, CDCl3): 172.3, 137.9,
136.0, 128.5, 127.8, 127.7, 115.9, 73.3, 71.8, 50.8, 39.3, 37.0, 36.58,
36.56, 25.9, 21.64, 21.62, 18.4, 18.1, 14.3, −4.42, −4.46. IR: ν 3285,
3070, 2955, 2929, 2857, 1639, 1540, 1361, 1092 cm−1. HRMS (ESI)
calcd for [C25H43NO3Si + H+] 434.3085, found 434.3088.
(R)-N-((R)-1-(Benzyloxy)but-3-en-2-yl)-5-hydroxyoctana-
mide. A solution of (R)-N-((R)-1-(benzyloxy)but-3-en-2-yl)-5-((tert-
butyldimethylsilyl)-oxy)octanamide (838 mg, 1.93 mmol) in 1.7 mL
TFA and 6.7 mL dry DCM was stirred at 0 °C for 3.5 h. A total of 35
mL of water was added and the pH was adjusted to 7 with sat.
NaHCO3. Then 30 mL DCM were added, the phases were separated
and the aq. phase was extracted 4× with 30 mL DCM. The combined
organic phases were then dried over MgSO4 and the solvent was
evaporated. FC (Et2O/MeOH 98:2) gave 508 mg (82%) of (R)-N-
((R)-1-(benzyloxy)but-3-en-2-yl)-5-hydroxyoctanamide as a colorless
oil. [α]D20 = +17.3° (c 0.70, CHCl3). 1H NMR (400 MHz, CDCl3): δ
7.39−7.28 (m, 5H), 5.99−5.80 (m, 2H), 5.27−5.16 (m, 2H), 4.73−
4.66 (m, 1H), 4.56 (d, J = 12.0 Hz, 1H), 4.52 (d, J = 12.1 Hz, 1H),
3.64−3.54 (m, 3H), 2.31−2.16 (m, 2H), 1.83−1.71 (m, 3H), 1.52−
1.18 (m, 7H), 0.96−0.86 (m, 3H). 13C NMR (100 MHz, CDCl3): δ
172.6, 137.7, 135.8, 128.4, 127.8, 127.7, 116.0, 73.2, 71.7, 70.9, 70.8,
50.8, 39.7, 39.6, 36.6, 36.31, 36.26, 21.5, 21.4, 18.8, 14.0. IR: ν 3293,
2957, 2931, 2869, 2362, 1638, 1541, 1455, 1362, 1104 cm−1. HRMS
(ESI) calcd for [C19H30NO3 + Na+] 342.2040, found 342.2037.
(S)-(R)-8-(((R)-1-(Benzyloxy)but-3-en-2-yl)amino)-8-oxooc-
tan-4-yl-2-((tert-butoxycarbonyl)-(methyl)-amino)-3-phenyl-
propanoate. To a solution of (R)-N-((R)-1-(benzyloxy)but-3-en-2-
yl)-5-hydroxyoctanamide (462 mg, 1.45 mmol) in 25 mL DCM were
added Boc-N-Me-Phe-OH (11) (603 mg, 2.16 mmol), DMAP (272
mg, 2.23 mmol), and DCC (0.90 g, 4.37 mmol) and the mixture was
stirred at rt for 3 h. Then 4 g silica gel were added and the solvent was
removed by evaporation. FC (hexane/EtOAc 1:1) yielded 831 mg
(99%) of (S)-(R)-8-(((R)-1-(benzyloxy)but-3-en-2-yl)amino)-8-ox-
ooctan-4-yl-2-((tert-butoxycarbonyl)-(meth-yl)-amino)-3-phenyl-
(S)-Methyl 5-hydroxyoct-7-enoate (3.57 g, 21 mmol) was hydro-
genated over 1 g of Ra−Ni in 100 mL EtOAc/MeOH 1:1 for 3 h at rt
at atmospheric pressure. The catalyst was removed by filtration and
the filtrate was evaporated. Water (50 mL) was then added and the
mixture extracted 3× with 50 mL of EtOAc. The combined organic
phases were dried over MgSO4 and the solvent removed by
20
evaporation to yield 2.70 g (75%) of ent-3 as a clear oil. [α]D
=
−0.14° (c 1.03, CHCl3) (lit.12 (3): [α]D = +1.29° (c 1.25, CHCl3)).
1H NMR (400 MHz, CDCl3): δ 3.68 (s, 3H), 3.65−3.56 (m, 1H),
2.36 (t, J = 7.3 Hz, 2H), 1.84−1.64 (m, 2H), 1.56−1.31 (m, 7H), 0.93
(m, 3H). 13C NMR (100 MHz, CDCl3): δ 174.2, 70.9, 51.4, 39.6, 36.6,
33.8, 20.9, 18.7, 14.0. IR: ν 2922, 2852, 2337, 1731, 1247, 1050 cm−1.
HRMS (ESI) calcd for [C9H18O3 + Na+] 197.1148, found 197.1155.
(R)-5-((tert-Butyldimethylsilyl)oxy)octanoic acid (ent-4). To a
solution of ent-3 (2.38 g, 13.7 mmol) and imidazole (2.26 g, 34.2
mmol) in 20 mL dry DCM was added TBSCl (3.18 g, 20.5 mmol) 0
°C and the mixture was stirred at rt overnight. It was then poured into
a mixture of 300 mL Et2O and 300 mL of brine. The phases were
separated and the aq. phase was twice extracted with 250 mL Et2O.
The combined organic extracts were dried over MgSO4, the solvent
evaporated, and the residue was purified by FC (hexane/EtOAc 9:1)
to give 3.17 g (81%) of (R)-methyl 5-((tert-butyldimethylsilyl)oxy)-
octanoate as clear oil. [α]D = −1.21° (c 0.88, CHCl3).43 1H NMR
20
(400 MHz, CDCl3): δ 3.70−3.63 (m, 4H), 2.30 (t, J = 7.4 Hz, 2H),
1.76−1.59 (m, 2H), 1.49−1.24 (m, 6H), 0.91−0.88 (m, 12H), 0.05 (s,
6H). 13C NMR (100 MHz, CDCl3): δ 174.1, 71.7, 51.4, 39.3, 36.4,
34.3, 25.90, 20.8, 18.5, 18.1, 14.3, −4.46, −4.48. IR: ν 2955, 2930,
2857, 2360, 1742, 1252, 1069 cm−1. HRMS (ESI) calcd for
[C15H32O3Si + Na+] 311.2013, found 311.2011.
24
1
propanoate as a colorless oil. [α]D = −10.4° (c 1.57, CHCl3). H
NMR (400 MHz, CDCl3): δ 7.39−7.15 (m, 10H), 6.05−5.82 (m,
2H), 5.29−5.15 (m, 2H), 4.98−4.84 (m, 2H), 4.73−4.64 (m, 1H),
4.56 (d, J = 12.1 Hz, 1H), 4.52 (d, J = 12.1 Hz, 1H), 3.55 (d, J = 4.4
Hz, 2H), 3.34−3.23 (m, 1H), 3.08−2.90 (m, 1H), 2.81−2.62 (d, J =
16.3 Hz, 3H), 2.27−2.13 (m, 2H), 1.74−1.43 (m, 6H), 1.42−1.12 (m,
11H), 0.95−0.84 (m, 3H). 13C NMR (100 MHz, CDCl3): δ 172.1,
A solution of the above (R)-methyl 5-((tert-butyldimethylsilyl)oxy)
octanoate (1.78 g, 6.2 mmol) in 50 mL THF/water 4:1 was stirred
together with LiOH (711 mg, 9.7 mmol) at rt for 17 h. At this point
additional LiOH (727 mg, 9.9 mmol) was added and stirring was
H
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171.8, 171.4, 170.9, 155.8, 137.9, 137.6, 135.9, 128.9, 128.5, 128.4,
127.8, 127.7, 116.0, 80.2, 79.9, 75.0, 74.7, 73.2, 71.7, 60.8, 59.8, 50.9,
36.2, 36.1, 35.9, 35.1, 34.9, 33.4, 33.2, 32.1, 28.3, 28.2, 25.0, 21.3, 18.5,
13.9. IR: ν 3304, 2960, 2933, 2870, 2361, 2336, 1732, 1652, 1454,
1365, 1141 cm−1. HRMS (ESI) calcd for [C34H48N2O6 + Na+]
603.3405, found 603.3394.
0.64 (m, 6H). HRMS (ESI) calcd for [C33H44N2O5 + H+] 549.3323,
found 549.3316.
A solution of the above material (30 mg, 0.055 mmol) in 6 mL
MeOH was hydrogenated over Pd−C (15 mg, 0.014 mmol) at 3 bar
for 21 h. Additional Pd−C (7.8 mg, 0.007 mmol) was added and
hydrogenation was continued for 40 h. The suspension was filtered,
the filtrate was evaporated, and the residue was purified by FC (DCM/
acetone 7:3 + 3% MeOH) on silica gel 60 (20−45 μm) to yield 4 mg
(16%) of 1-SR as an amorphous white solid and 18 mg (72%) of a 2:1
mixture of 1-SR and 14S-1-SR. 1-SR: [α]D24 = +49.9° (c 0.16, CHCl3).
1H NMR (500 MHz, CDCl3): δ 7.24−7.10 (m, 5H), 6.00 (d, J = 6.8
Hz, 1H), 5.66 (dd, J = 11.7, 5.7 Hz, 1H), 4.95−4.89 (m, 1H), 3.72−
3.55 (m, 3H), 3.55−3.46 (m, 1H), 3.29 (dd, J = 15.1, 5.6 Hz, 1H),
2.95 (dd, J= 15.3, 11.7 Hz, 1H), 2.94 (s, 3H), 2.73−2.63 (m, 1H), 2.33
(td, J = 12.7, 3.8 Hz, 1H), 2.09−1.99 (m, 1H), 1.77−1.11 (m, 14H),
0.85−0.78 (m, 6H). 13C NMR (125 MHz, CDCl3): 177.7, 174.8,
173.5, 136.1, 128.6, 128.3, 126.9, 73.7, 66.8, 55.7, 52.4, 36.8, 36.4, 35.1,
34.7, 34.2, 32.8, 31.0, 29.6, 23.1, 22.8, 18.8, 18.1, 13.8. (Single isomer).
IR: ν 3308, 2929, 1730, 1649, 1541, 1458, 670 cm−1. HRMS (ESI)
calcd for [C26H40N2O5 + H+] 461.3010, found 461.3015.
(S)-(R)-8-(((R)-1-(Benzyloxy)but-3-en-2-yl)amino)-8-oxooc-
tan-4-yl-2-(methylamino)-3-phenyl-propanoate. To a solution
of (S)-(R)-8-(((R)-1-(benzyloxy)but-3-en-2-yl)amino)-8-oxooctan-4-
yl 2-((tert-butoxycarbonyl)-(methyl)-amino)-3-phenyl-propanoate
(735 mg, 1.23 mmol) in 40 mL DCM were added TIPS (290 μL,
1.42 mmol) and 10 mL TFA and the mixture was stirred at rt for 30
min. After cooling to 0 °C it was poured into 150 mL sat. NaHCO3,
150 mL DCM were added and the phases were separated. The aq.
phase was extracted twice with 150 mL DCM, the combined organic
phases were dried over MgSO4, and the solvent was evaporated. FC
(hexane/EtOAc 1:1 + 2% Et3N) gave 602 mg (99%) of (S)-(R)-8-
(((R)-1-(benzyloxy)but-3-en-2-yl)amino)-8-oxooctan-4-yl-2-(methyla-
24
mino)-3-phenyl-propanoate as clear viscous oil. [α]D = +23.4° (c
1
0.48, CHCl3). H NMR (400 MHz, CDCl3): δ 7.39−7.18 (m, 10H),
5.92−5.80 (m, 2H), 5.32−5.14 (m, 3H), 4.93−4.83 (m, 1H), 4.71−
4.63 (m, 1H), 4.55 (d, J = 12.1 Hz, 1H), 4.51 (d, J = 12.1 Hz, 1H),
3.55 (d, J = 4.2 Hz, 2H), 3.42 (t, J = 7.1 Hz, 1H), 2.92 (d, J = 7.0 Hz,
2H), 2.36 (s, 3H), 2.23−2.10 (m, 2H), 1.71−1.29 (m, 6H), 1.18−1.02
(m, 2H), 0.82 (t, J = 7.5 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ
174.4, 171.89, 171.84, 137.8, 137.3, 135.91, 129.3, 129.2, 128.5, 128.4,
127.9, 127.7, 126.7, 116.0, 74.2, 73.3, 71.8, 64.9, 64.8, 50.9, 39.6, 39.5,
36.2, 35.9, 34.7, 33.3, 33.2, 21.4, 21.0, 18.6, 18.3, 13.94, 13.91. IR: ν
3312, 2935, 2870, 2358, 1725, 1642, 1540, 1455, 1362, 1254, 1179,
1114 cm−1. HRMS (ESI) calcd for [C29H40N2O4+Na+] 503.2880,
found 503.2874.
(R)-2-Methylpent-4-enoic acid (ent-6). To a solution of (S)-4-
benzyl-3-((R)-2-methylpent-4-enoyl)-oxazolidin-2-one (6.24 g, 22.8
mmol) in 60 mL THF/water 4:1 was added LiOH (2.18 g, 91.2
mmol) and 50% H2O2 (2.90 mL, 50.2 mmol) and the mixture was
stirred at rt overnight. The pH was then adjusted to 1−2 with 90 mL 1
M HCl and the mixture was extracted thrice with 300 mL Et2O. The
combined organic extracts were dried over MgSO4 and the solvent was
removed. The product was distilled under reduced pressure to yield
20
ent-6 (1.91 g, 73%) as a colorless liquid. [α]D = −10.3° (c 1.92,
CHCl3). 1H NMR (400 MHz, CDCl3): δ 11.17 (s br, 1H), 5.77−5.63
(m, 1H), 5.07−4.94 (m, 2H), 2.56−2.43 (m, 1H), 2.43−2.31 (m, 1H),
2.20−2.09 (m, 1H), 1.12 (d, J = 7.0 Hz, 3H). 13C NMR (100 MHz,
CDCl3): δ 182.6, 135.1, 117.2, 39.1, 37.4, 16.3. IR: ν 2979, 2666, 1703,
1643, 1417, 1244, 916 cm−1. HRMS (ESI) calcd for [C6H9O2 − H+]
113.0608, found 113.0608.
(S)-(R)-8-(((R)-1-(Benzyloxy)but-3-en-2-yl)amino)-8-oxooc-
tan-4-yl-2-((S)-N,2-dimethylpent-4-enamido)-3-phenylpropa-
noate. To a solution of 6 (72 mg, 0.63 mmol) and HATU (239 mg,
0.63 mmol) in 3.7 mL dry DMF was added DIEA (0.2 mL, 1.15
mmol) and the mixture was stirred for 5 min at rt. Then (S)-(R)-8-
(((R)-1-(benzyloxy)but-3-en-2-yl)amino)-8-oxooctan-4-yl-2-(methyla-
mino)-3-phenyl-propanoate (196 mg, 0.41 mmol) was added and the
resulting solution was stirred at rt for 4 h. After removal of DMF 7.5
mL DCM and 7.5 mL 10% MeCN in water were added and the phases
were separated. The aq. phase was extracted twice with 7.5 mL DCM,
the combined organic extracts were dried over MgSO4, and the solvent
was evaporated. FC (hexane/EtOAc 1:1 + 2% Et3N) yielded 180 mg
(77%) of (S)-(R)-8-(((R)-1-(benzyloxy)but-3-en-2-yl)amino)-8-ox-
ooctan-4-yl 2-((S)-N,2-dimethylpent-4-enamido)-3-phenylpropanoate
(S)-(S)-8-(((R)-1-(Benzyloxy)But-3-en-2-yl)amino)-8-oxooc-
tan-4-yl-2-((R)-N,2-dimethylpent-4-enamido)-3-phenylpropa-
noate. To a solution of ent-6 (191 mg, 1.67 mmol) and HATU (635
mg, 1.67 mmol) in 10 mL dry DMF was added DIEA (0.6 mL, 3.34
mmol) and the mixture stirred for 5 min at rt. Then 13 (535 mg, 1.11
mmol) was added and the resulting solution was stirred at rt for 5 h.
After removal of DMF 20 mL DCM and 20 mL 10% MeCN in water
were added. The phases were separated and the aq. solution was
extracted twice with 20 mL DCM. The combined organic extracts
were dried over MgSO4 and the solvent was evaporated. FC (hexane/
EtOAc 1:1 + 2% Et3N) gave 630 mg (98%) of (S)-(S)-8-(((R)-1-
24
1
as yellowish resin. [α]D = −9.6° (c 0.48, CHCl3). H NMR (400
MHz, CDCl3): δ 7.41−7.10 (m, 10H), 6.20−5.65 (m, 3H), 5.35−5.15
(m, 3H), 5.05−4.60 (m, 4H), 4.59−4.49 (m, 2H), 3.62−3.52 (m, 2H),
3.44−3.25 (m, 1H), 3.09−2.94 (m, 1H), 2.93−2.81 (m, 3H), 2.70−
2.55 (m, 1H), 2.45−2.34 (m, 1H), 2.32−2.13 (m, 2H), 2.09−1.80 (m,
2H), 1.75−1.40 (m, 6H), 1.39−1.11 (m, 2H), 1.08−0.74 (m, 6H). 13C
NMR (100 MHz, CDCl3): δ 176.4, 172.1, 171.0, 137.9, 137.1, 136.2,
135.97, 135.92, 128.9, 128.44, 128.37, 127.7, 116.4, 116.0, 74.7, 73.20,
73.16, 71.8, 58.4, 51.0, 50.93, 50.88, 37.8, 36.2, 35.8, 35.7, 34.9, 33.3,
33.1, 21.4, 18.5, 16.8, 14.0, 13.9. IR: ν 3311, 3069, 3029, 2954, 2868,
2330, 1732, 1637, 1090 cm−1. HRMS (ESI) calcd for [C35H48N2O5 +
Na+] 599.3455, found 599.3454.
(benzyloxy)but-3-en-2-yl)amino)-8-oxooctan-4-yl-2-((R)-N,2-dime-
20
thylpent-4-enamido)-3-phenylpropanoate as dark yellow oil. [α]D
=
1
−41.6° (c 1.01, CHCl3). H NMR (400 MHz, CDCl3): δ 7.38−7.12
(m, 10H), 6.30−6.19 (m, 1H), 5.93−5.81 (m, 1H), 5.45−5.33 (m,
1H), 5.32−5.14 (m, 3H), 5.02−4.80 (m, 3H), 4.74−4.62 (m, 1H),
4.58−4.48 (m, 2H), 3.56 (d, J = 5.0 Hz, 2H), 3.38 (dd, J = 14.7, 5.7
Hz, 1H), 3.09−3.00 (m, 1H), 2.88−2.76 (m, 3H), 2.63−2.53 (m, 1H),
2.27−2.11 (m, 3H), 1.94−1.77 (m, 1H), 1.74−1.16 (m, 8H), 1.05 (d, J
= 7.0 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3):
δ 176.4, 172.3, 172.2, 170.7, 137.9, 137.2, 136.96, 136.92, 128.9, 128.4,
127.70, 127.67, 116.2, 116.0, 115.9, 74.79, 74.75, 73.2, 71.8, 71.7, 61.8,
58.7, 50.9, 38.6, 37.8, 36.10, 35.8, 34.6, 33.1, 29.7, 21.4, 21.3, 18.6,
18.5, 16.9, 16.6, 14.0. IR: ν 3310, 3064, 2974, 2933, 2871, 2360, 2341,
1732, 1636, 1541, 1456, 1129, 1089, 917, 738, 698 cm−1. HRMS (ESI)
calcd for [C35H48N2O5+Na+] 599.3455, found 599.3445.
(3S,6S,10R,16R)-3-Benzyl-10-(hydroxymethyl)-4,6-dimethyl-
16-propyl-1-oxa-4,11-diazacyclo-hexadecane-2,5,12-trione (1-
SR). A solution of (S)-(R)-8-(((R)-1-(benzyloxy)but-3-en-2-yl)-
amino)-8-oxooctan-4-yl 2-((S)-N,2-dimethylpent-4-enamido)-3-phe-
nylpropanoate (132 mg, 0.23 mmol) in 66 mL toluene was refluxed
with Grubbs II catalyst (28.6 mg, 0.03 mmol) for 16 h. After solvent
removal the residue was submitted to FC in hexane/EtOAc 1:2 to
yield 30 mg (24%) of (3S,6S,10R,16R)-3-benzyl-10-((benzyloxy)-
methyl)-4,6-dimethyl-16-propyl-1-oxa-4,11-diazacyclohexadec-8-ene-
2,5,12-trione as a brown resin. 1H NMR (400 MHz, CDCl3): δ 7.34−
7.02 (m, 10H), 5.77−5.54 (m, 1H), 5.37−5.20 (m, 1H), 5.00−4.77
(m, 1H), 4.51−4.34 (m, 2H), 4.28−3.74 (m, 2H), 3.63−3.14 (m, 2H),
3.06−2.55 (m, 5H), 2.34−2.03 (m, 3H), 1.99−0.96 (m, 11H), 0.90−
(3S,6R,10R,16S)-3-Benzyl-10-(hydroxymethyl)-4,6-dimethyl-
16-propyl-1-oxa-4,11-diazacyclohexa-decane-2,5,12-trione (1-
RS). To a solution of (S)-(S)-8-(((R)-1-(benzyloxy)but-3-en-2-yl)-
amino)-8-oxooctan-4-yl 2-((R)-N,2-dimethylpent-4-enamido)-3-phe-
nylpropanoate (300 mg, 0.52 mmol) in 150 mL toluene was added
Grubbs II catalyst (66 mg, 0.08 mmol) and the reaction mixture was
refluxed for 20 h. The solvent was evaporated and the residue was
filtered through silica gel (hexane/EtOAc 2:3) to yield 180 mg (63%)
of crude (3S,6R,10R,16S)-3-benzyl-10-((benzyloxy)methyl)-4,6-di-
I
dx.doi.org/10.1021/jo3027643 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
methyl-16-propyl-1-oxa-4,11-diazacyclohexadec-8-ene-2,5,12-trione as
a brown resin. H NMR (400 MHz, CDCl3): δ 7.32−7.06 (m, 10H),
5.88−5.27 (m, 2H), 5.02−4.38 (m, 3H), 4.28−3.74 (m, 2H), 3.62−
3.28 (m, 4H), 2.94−2.44 (m, 3H), 2.43−2.01 (m, 3H), 1.98−1.15 (m,
11H), 1.14−0.76 (m, 6H). HRMS (ESI) calcd for [C33H44N2O5 + H+]
549.3323, found 549.3319.
hydrogenation was continued for 8 h (3 bar). A third portion of Pd−C
(17.5 mg, 0.016 mmol) was added and hydrogenation was further
continued for 15 h. The catalyst was removed by filtration and the
solvent was evaporated. FC (DCM/acetone 7:3 + 3% MeOH) on
silica gel 60 (20−45 μm) gave 31 mg (44%) of a 3:1 mixture of 1-RR
and (the putative) 14S-1-RR and 4 mg (6%) of 14S-1-RR as
1
24
1
amorphous white solids. 1-RR: [α]D = −57.0° (c 0.31, CHCl3). H
NMR (400 MHz, CDCl3): δ 7.26−7.06 (m, 5H), 6.62 (d, J = 8.0 Hz,
1H), 4.91−4.82 (m, 1H), 3.87−3.78 (m, 1H), 3.65−3.55 (m, 2H),
3.54−3.43 (m, 2H), 3.37−3.18 (m, 2H), 2.69 (s, 3H), 2.40−2.32 (m,
1H), 2.29−2.20 (m, 1H), 2.08−1.99 (m, 1H), 1.76−1.00 (m, 15H),
0.97 (d, J = 7.0 Hz, 3H), 0.82 (t, J = 7.6 Hz, 3H). 13C NMR (125
MHz, CDCl3): 175.8, 174.4, 170.0, 138.3, 129.3, 128.4, 126.6, 74.9,
66.5, 66.4, 52.2, 39.4, 36.9, 36.8, 34.8, 34.0, 33.2, 31.2, 29.6, 25.1, 21.9,
18.9, 18.2, 14.0. IR: ν 3299, 2933, 2866, 1733, 1637, 1547, 1459, 1276,
1083, 752 cm−1. HRMS (ESI) calcd for [C26H40N2O5 + H+] 461.3010,
found 461.3011.
A solution of the above material (100 mg, 0.18 mmol) in 15 mL
MeOH was hydrogenated over Pd−C (39 mg, 0.04 mmol) at 3 bar for
8 h. The catalyst was then removed by filtration and the solvent was
evaporated. FC (DCM/acetone 7:3 + 3% MeOH) on silica gel 60
(20−45 μm) gave 18.3 mg (22%) of 1-RS (purity 94%), and 37.5 mg
(45%) of a 1:1 mixture of 1-RS and (the putative) 14S-1-RS and 7.5
24
mg (9%) of 14S-1-RS as amorphous white solids. 1-RS: [α]D
=
1
−89.9° (c 0.52, CHCl3). H NMR (500 MHz, CDCl3): δ 7.34−7.10
(m, 5H), 6.14 (d, J = 6.2 Hz, 1H), 5.08−5.00 (m, 1H), 5.04 (br s, 1H),
3.87−3.78 (m, 1H), 3.70−3.62 (m, 2H), 3.55 (dd, J = 11.2, 6.6 Hz,
1H), 3.47 (dd, J = 13.7, 10.8 Hz, 1H), 3.27 (dd, J = 13.7, 4.9 Hz, 1H),
2.68 (s, 3H), 2.65−2.47 (m, 2H), 2.15−2.06 (m, 1H), 1.91−1.74 (m,
4H), 1.70−1.17 (m, 11H), 1.05 (d, J = 6.9 Hz, 3H), 0.93 (t, J = 7.4 Hz,
3H). 13C NMR (125 MHz, CDCl3): 176.0, 175.3, 171.1, 138.4, 129.3,
128.5, 126.6, 73.7, 67.4, 67.1, 51.8, 39.3, 37.0, 36.1, 35.5, 34.6, 34.2,
32.3, 29.2, 22.3, 21.9, 18.9, 18.1, 13.9. (Single isomer). IR: ν 3308,
2935, 2867, 1722, 1638, 1538, 1455, 1243, 1067, 752 cm−1. HRMS
(ESI) calcd for [C26H40N2O5 + H+] 461.3010, found 461.3012.
(S)-(R)-8-(((R)-1-(Benzyloxy)But-3-en-2-yl)amino)-8-oxooc-
tan-4-yl-2-((R)-N,2-dimethylpent-4-enamido)-3-phenylpropa-
noate. To a solution of ent-6 (70 mg, 0.61 mmol) and HATU (240
mg, 0.63 mmol) in 3.7 mL dry DMF was added DIEA (0.2 mL, 1.15
mmol) and the mixture was stirred for 5 min at rt. Then (S)-(R)-8-
(((R)-1-(benzyloxy)but-3-en-2-yl)amino)-8-oxooctan-4-yl-2-(methyla-
mino)-3-phenyl-propanoate (198 mg, 0.41 mmol) was added and the
resulting solution was stirred at rt for 4.5 h. After removal of DMF 7.5
mL DCM and 7.5 mL 10% MeCN in water were added and the phases
were separated. The aq. solution was extracted twice with 7.5 mL
DCM, the combined organic extracts were dried over MgSO4, and the
solvent was evaporated. FC (hexane/EtOAc 1:1 + 2% Et3N) gave 156
mg (66%) of (S)-(R)-8-(((R)-1-(benzyloxy)but-3-en-2-yl)amino)-8-
oxooctan-4-yl-2-((R)-N,2-dimethylpent-4-enamido)-3-phenylpropa-
ASSOCIATED CONTENT
■
S
* Supporting Information
HPLC data for 1; HPLC data for FDAA derivatives of 9 and
1
ent-9; H and 13C NMR spectra for intermediates and final
products. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: karl-heinz.altmann@pharma.ethz.ch.
■
Present Address
J.G.: University of Bern, Institute of Biochemistry and
Molecular Medicine, Buhlstrasse 28, CH-3012 Bern, Switzer-
̈
land.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
24
1
noate as yellowish resin. [α]D = −27.9° (c 0.36, CHCl3). H NMR
(400 MHz, CDCl3): δ 7.38−7.14 (m, 10H), 6.14 (d, J = 8.0 Hz, 1H),
5.93−5.82 (m, 1H), 5.46−5.33 (m, 2H), 5.28−5.15 (m, 2H), 5.05−
4.79 (m, 3H), 4.74−4.63 (m, 1H), 4.58−4.48 (m, 2H), 3.59−3.52 (m,
2H), 3.38 (dd, J = 14.7, 5.4 Hz, 1H), 3.07−2.79 (m, 2H), 2.88 (s, 3H),
2.65−2.55 (m, 1H), 2.30−2.11 (m, 2H), 1.95−1.84 (m, 1H), 1.73−
1.42 (m, 6H), 1.37−1.19 (m, 2H), 1.05 (d, J = 6.7 Hz, 3H), 0.89 (d, J
= 7.3 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 176.5, 172.1, 172.1,
137.9, 137.0, 136.0, 135.9, 135.86, 128.8, 128.4, 127.7, 116.5, 116.4,
116.1, 116.0, 74.8, 74.7, 73.3, 73.20, 73.17, 71.8, 71.7, 58.1, 51.0, 50.92,
50.89, 37.9, 36.18, 36.10, 35.15, 35.8, 34.7, 33.3, 21.43, 21.36, 18.5,
16.9, 13.94, 13.86. IR: ν 3312, 3065, 3031, 2959, 2932, 2871, 1732,
1627, 1092 cm−1. HRMS (ESI) calcd for [C35H48N2O5 + Na+]
599.3455, found 599.3455.
■
We are indebted to Kurt Hauenstein for help with the HPLC
purification of 1 and to Louis Bertschi for HRMS spectra
acquisition. This work was conducted within the framework of
COST Action CM804 “Chemical Biology with Natural
Products”.
REFERENCES
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one as a brown resin. H NMR (400 MHz, CDCl3): δ 7.42−7.08 (m,
10H), 6.14−5.28 (m, 2H), 5.02−4.38 (m, 3H), 4.28−4.01 (m, 1H),
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dx.doi.org/10.1021/jo3027643 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
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μm). Different mobile phases were employed at different temperatures
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(31) It was suggested by one of the reviewers that the observed
enrichment of the 14S isomers in the final macrocycles could be the
result of kinetic resolution in one or several of the transformations that
led from (partially racemic) 9 to 1 and its different C9/C20 epimers.
(32) As pointed out by one of the reviewers, the formation of an
unexpected third isomer might also be due to partial racemization of
11 in the coupling reaction with 10. However, in model experiments,
no detectable racemization of 11 was observed upon reaction with (S)-
butan-2-ol under conditions identical with those employed for the
esterification of 11 with 10. In addition, the complete absence of
isomer III from the product obtained from enantiomerically pure 9
further confirms that isomer III is not the result of epimerization at
C2.
(33) The partial epimerization of a Boc-protected α-amino aldehyde
in a Wittig reaction has been reported: Thompson, W. J.; Ball, R. G.;
K
dx.doi.org/10.1021/jo3027643 | J. Org. Chem. XXXX, XXX, XXX−XXX