Total synthesis and biological evaluation of (+)-kalkitoxin, a cytotoxic metabolite of the cyanobacterium Lyngbya majuscula

Article abstract of DOI:10.1039/b404205k

(+)-Kalkitoxin, a metabolite of the marine cyanobacterium Lyngbya majuscula, was synthesized from (R)-2-methylbutyric acid, (R)-cysteine, and (3S, 4S, 6S)-3,4,6-trimethyl-8-(methylamino)octanoic acid. A key step in the synthesis was installation of the anti,anti methyl stereotriad by means of a tandem asymmetric conjugate addition of an organocopper species to an α,β-unsaturated N-acyl oxazolidin-2-one followed in situ by α-methylation of the resultant enolate. The thiazoline portion of kalkitoxin was assembled by titanium tetrachloride catalyzed cyclization of a vinyl substituted amido thiol.

Full text of DOI:10.1039/b404205k

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Total synthesis and biological evaluation of (؉)-kalkitoxin,
a cytotoxic metabolite of the cyanobacterium Lyngbya majuscula†
James D. White,*a Qing Xu,a Chang-Sun Leea and Frederick A. Valerioteb
a Department of Chemistry, Oregon State University, Corvallis, OR 97331-4003, USA.
E-mail: james.white@oregonstate.edu
b Henry Ford Health System, Detroit, MI 48202, USA
Received 19th March 2004, Accepted 30th April 2004
First published as an Advance Article on the web 28th June 2004
(ϩ)-Kalkitoxin, a metabolite of the marine cyanobacterium Lyngbya majuscula, was synthesized from (R)-2-
methylbutyric acid, (R)-cysteine, and (3S, 4S, 6S)-3,4,6-trimethyl-8-(methylamino)octanoic acid. A key step in the
synthesis was installation of the anti,anti methyl stereotriad by means of a tandem asymmetric conjugate addition
of an organocopper species to an α,β-unsaturated N-acyl oxazolidin-2-one followed in situ by α-methylation of the
resultant enolate. The thiazoline portion of kalkitoxin was assembled by titanium tetrachloride catalyzed cyclization
of a vinyl substituted amido thiol.
Introduction
Results and discussion
The cyanobacterium Lyngbya majuscula is known to be a source
of toxic metabolites such as lyngbyatoxin1 and debromo-
aplysiatoxin,2 but a wide variety of other biologically active
metabolites including the antiproliferative agent curacin A (1)3
have also been isolated from this prolific marine organism.
Curacin A, in particular, has captured interest as a result of its
potent cytotoxic activity.4 In screening extracts of L. majuscula
for additional substances with biological activity, Wu et al.
discovered the lipopeptide kalkitoxin (2) and deduced both
its gross structure and relative configuration by means of
NMR experiments.5 The absolute configuration of natural
(ϩ)-kalkitoxin was assigned as (3R, 7R, 8S, 10S, 2ЉR) by means
of synthesis of all possible stereoisomers and comparison of
their 13C spectra with that of the natural product.
Synthesis
A retrosynthetic analysis of kalkitoxin suggested that the
thiazoline portion could be constructed from the constituent
subunits 3 and 4 shown in Scheme 1, 4 being accessible in
principle from (R)-(ϩ)-cysteine (5). Amidocarboxylic acid 3
was foreseen as the coupling product of (R)-2-methylbutyric
acid (6) with amine 7, the latter bearing an anti,anti methyl triad
whose absolute configuration would be set through the agency
of a chiral reagent or auxiliary.
A striking similarity between kalkitoxin and curacin A
emerges from these structural studies. For example, both
structures contain a 2,4-disubstituted thiazoline with one of the
substituents being a lipophilic chain and the other an unsatur-
ated unit. However, a significant difference between 1 and 2 is
the presence in the latter of three methyl substituents arranged
in a 1,2,4-anti,anti sequence along the aliphatic chain. The
synthetic interest presented by this stereochemical array, as well
as intriguing reports of kalkitoxin’s biological properties,6–8
attracted our attention soon after the disclosure of its
structure.9 We now describe the full details of our work which
has led to the total synthesis of natural (ϩ)-kalkitoxin (2), and
we also report studies of its pharmacological properties.
Scheme 1
Our plan from the outset envisioned installation of the anti-
1,3-dimethyl array of 7 as the first operation, with insertion of
the third methyl group as a subsequent and separate step. This
strategy required an asymmetric method for positioning stereo-
genic methyl-bearing centers in a 1,3-relationship, for which
there are a limited number of solutions. One of these is a tactic
devised independently by Evans10 and Sonnet11 using prolinol
as a chiral auxiliary, and which was employed with good results
for the asymmetric assembly of an anti-1,3-dimethyl unit in our
synthesis of bourgeanic acid.12 Thus, the dianion of N-prop-
ionylprolinol (8) was reacted with the known iodide 913 to give
10 as the sole detectable diastereomer (Scheme 2). Amides of
prolinol, when exposed to hydrochloric acid, undergo transfer
of the acyl group from the nitrogen atom to the primary alcohol
† Electronic supplementary information (ESI) available: 1H NMR
spectrum of synthetic (ϩ)-kalkitoxin in C6D6. See http://www.rsc.org/
suppdata/ob/b4/b404205k/
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 2 0 9 2 – 2 1 0 2
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
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Scheme 2
in a process that is forced by generation of the pyrrolidinium
salt. Reduction of the resultant ester therefore leads to a
primary alcohol. When this protocol was applied to 10 using
lithium aluminium hydride as the reducing agent, the alcohol
11 was produced in excellent yield along with recovered (R)-
prolinol. Oxidation of 11 to the corresponding aldehyde with
Dess–Martin periodinane,14 followed by Wittig olefination with
the phosphorane 12, afforded trans α,β-unsaturated ester 13.
This substance now became the platform for a substrate-
directed conjugate addition intended to introduce the third
methyl substituent in the desired (S) configuration. Good
precedent for this outcome appeared to exist in published work
by Yamamoto,15 who has shown that conjugate addition to
γ-alkyl-α,β-unsaturated esters leads predominantly to an anti
relationship between the new and preexisting alkyl substituents.
However, it was found that the reaction of 13 with methyl-
lithium and cuprous bromide under a variety of conditions
resulted in a mixture of 14 and 15, a typical ratio of products
being 1.5 : 1 in favor of 14. These esters were inseparable by
chromatography, and the conjugate addition route was there-
fore abandoned as a means for installing the methyl stereotriad
of kalkitoxin. Instead, a new pathway was sought which
eschewed substrate stereocontrol for this purpose.
A tactical revision involving a reagent controlled reaction for
setting configuration of the C7 methyl group of kalkitoxin
appeared to be the logical alternative. Pfaltz has shown that
reduction of β,β-disubstituted α,β-unsaturated esters can be
accomplished in high yield and good enantiomeric excess16
using the cobalt complex of a semicorrin and sodium boro-
hydride, and this precedent became the basis for our next
approach to 7. Our route to the precursor required for this
approach diverged from the alkylation sequence of Scheme 2
in that we now used Myers’ pseudoephedrine auxiliary17 for
constructing our anti-1,3-dimethyl platform. The enolate of the
N-propionyl derivative 16 of (1R, 2R)-pseudoephedrine was
reacted with iodide 9 to give amide 17 as a single diastereomer
in excellent yield (Scheme 3). An advantage of 17 over its
prolinol analogue 10 is that the former can be converted directly
to a methyl ketone, and when 17 was treated with methyllithium
a clean reaction took place to yield 18. Wadsworth–Emmons
reaction of this ketone with the anion of phosphonate 19
furnished an (E,Z) mixture of α,β-unsaturated esters in which
the (E) isomer 20 predominated by a ratio of 11 : 1. Catalytic
hydrogenation of 20 over palladium-on-carbon not only
produced a 1 : 1 mixture of inseparable stereoisomers from
Scheme 3
saturation of the trisubstituted double bond but also cleaved
the benzyl ether. We nevertheless hoped that the reducing
system prepared from cobalt() chloride, sodium borohydride,
and (S,S)-semicorrin 21 would effect stereoselective saturation
of 20. In practice, reduction of 20 with this set of reagents was
very slow and resulted in only 30% conversion after three days.
Furthermore, it gave a disappointing ratio of only 1.6 : 1 in
favor of the desired product 22 over its isomer 23. The reason
for this unsatisfactory outcome probably lies in the steric
hindrance presented by the substrate 20 to the bulky cobalt–
semicorrin complex from 21 and suggests there is a practical
limit to the application of this asymmetric reduction method in
sterically unfavorable situations. In any event, we were forced
to search again for methodology which would allow us to
install the anti,anti-1,2,4-trimethyl array of 2 in an efficient
asymmetric fashion.
At this point, we were drawn to a report by Hruby18 in which
it was shown that conjugate addition of an organocopper
reagent to trans-3-crotonyl-4-phenyloxazolidin-2-one resulted
in a high level of stereoinduction. Williams has generalized this
reaction,19 and in an elegant synthesis of sambutoxin20 he made
use of Hruby’s finding for the asymmetric construction of an
anti-1,3-dimethyl assemblage. For this strategy to be of greatest
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 2 0 9 2 – 2 1 0 2
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practical value in our synthesis of kalkitoxin, the organocopper
species used for conjugate addition should be one derived from
halide 24, i.e. a homologue of 9. Since 24 was known only in
racemic form,21 its preparation as the (R) enantiomer was
carried out using the sequence shown in Scheme 4. The enolate
of N-propionylpseudoephedrine 2517 (enantiomeric with 16)
was alkylated with 2-iodoethyl benzyl ether,22 and the resultant
amide 26 was reduced with lithium amidotrihydroborate23 to
furnish (R)-27. This alcohol was shown to be enantiomerically
pure within the limits of detection, and it was converted to
bromide 24 with N-bromosuccinimide and triphenylphosphine
in an overall yield of 64% from 25.
Fig. 1 Transition state for the reaction of 28 with 29 leading to 30.
substituent in the desired (R) orientation, since both the oxazol-
idinone configuration and that adjacent to the enolate should
favor attack from the rear face of 30. In fact, addition of excess
methyl iodide to the reaction mixture from 28 and 29 gave two
methylated products in the ratio 3.6 : 1. These compounds were
separated chromatographically, after which the major isomer
crystallized. Its structure was established as 32 by X-ray crystal-
lographic analysis.9 For comparison, methylation of 30 using
potassium hexamethyldisilylazide as the base gave a 2.8 : 1
mixture of 32 and 33, respectively. As previously noted by
Williams,24 replacement of the phenyl substituent of the
oxazolidinone 29 by a benzyl group resulted in decreased
stereoselectivity in the conjugate addition step with 28.
With the methyl stereotriad of kalkitoxin in place, attention
next turned to elaboration of the benzyl ether terminus of 32.
To this end, the benzyl ether was cleaved by hydrogenolysis and
the resulting alcohol was oxidized under Swern conditions to
aldehyde 34 (Scheme 6). Reductive amination of this aldehyde
with methylamine and sodium cyanoborohydride25 furnished
the secondary amine 35 which then became the partner for
amide formation with (R)-2-methylbutyric acid (36). The reac-
tion of activated α-branched carboxylic acids with secondary
amines is typically difficult,26 and this case was no exception.
For example, the use of activating agents such as 1-hydroxy-
benzotriazole or diethyl cyanophosphate, as conventionally
employed in peptide coupling,27 resulted in sluggish reaction
rates and low yields (29–46%) of amide 37. It was discovered,
however, that activation of 36 with Carpino’s 1-hydroxy-7-
azabenzotriazole28 (HOAt) accomplished the desired coupling
very efficiently with no detectable epimerization at the stereo-
center α to the amide carbonyl. The product, as shown by
Scheme 4
The alkylcopper reagent 28 was prepared by first converting
bromide 24 to its Grignard reagent and then treating this
with cuprous bromide dimethyl sulfide complex (Scheme 5).
Addition of three equivalents of 28 to (S)-trans-3-crotonyl-4-
phenyloxazolidin-2-one (29) followed by quenching of the
intermediate enolate 30 with water, gave 31 as the sole detect-
able stereoisomer in high yield. The use of less than three
equivalents of the organocopper species 28 resulted in a signifi-
cant decrease in both stereoselectivity and yield of 31. For
example, the reaction of two equivalents of 28 with 29 afforded
a (1.2 : 1) mixture of conjugate addition products only slightly
favoring anti isomer 31. Although a transition state of the
reaction of 28 with 29 along lines proposed by Williams20
suggests that this is a stereochemically matched case, in which
both the methyl configuration in 28 and the phenyl configur-
ation in 29 contribute to attack by the former at the si face of
the latter (see Fig. 1), the need for an excess of 24 indicates that
this rationale may be too simple. The assumed formation of (Z)
enolate 30 in this process implies that in situ methylation of
this intermediate will lead to incorporation of the third methyl
Scheme 5
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Scheme 7
Scheme 6
NMR, was a 1 : 1 mixture of rotamers of 37 resulting from
restricted rotation around the amide bond.
A trivial but necessary task for advancing 37 towards
carboxylic acid 3 was a one-carbon homologation at the acyl
oxazolidinone terminus, and for this purpose the auxiliary was
cleaved from 37 by reduction with lithium borohydride. This
gave primary alcohol 38 which was oxidized to the corre-
sponding aldehyde under Swern conditions (Scheme 7). A
Wittig reaction of this aldehyde with methoxymethylenephos-
phorane prepared from phosphonium chloride 3929 afforded
the enol ether 40 as a 1 : 1 mixture of (E) and (Z) isomers, and
acidic hydrolysis of the mixture furnished an aldehyde which
was oxidized immediately with sodium chlorite to 3. The five-
step homologation sequence from 37 proceeded in an overall
51% yield.
The amine 4 required for coupling with 3 as a prelude to
assembling the thiazoline moiety of kalkitoxin30 was prepared
from (R)-cysteine (5, Scheme 8). Thus, S-benzylation of 5
followed by protection of the amine as its tert-butoxycarbonyl
(Boc) derivative gave carboxylic acid 4131 which was trans-
formed to Weinreb amide 42.32 Reduction of 42 with lithium
aluminium hydride yielded aldehyde 43, and Wittig olefination
of this substance with methylenetriphenylphosphorane gave 44.
Final cleavage of the Boc group from 44 led to benzyl (R)-2-
amino-3-butenyl sulfide (4). Coupling of 4 with carboxylic acid
3 was accomplished by activating the latter with Carpino’s
O-(7-azabenzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium
hexafluorophosphate (HATU)33 in the presence of Hunig’s
base and afforded bis amide 45 in excellent yield (Scheme 9).
Reductive cleavage of the benzyl group from 45 with sodium–
ammonia and treatment of the resulting thiol 46 with titanium
tetrachloride in dichloromethane34 led directly to (ϩ)-kalki-
toxin (2) with spectroscopic properties identical to those of
Scheme 8
the natural material (ESI†reports the 1H NMR spectrum of syn-
thetic (ϩ)-kalkitoxin in C6D6; for that of natural (ϩ)-kalkitoxin
see the supporting information of ref. 5).
Biological studies
The cytotoxicity of kalkitoxin (2), thiol 46 and benzylthio ether
45 was measured against the human colon cell line HCT-116.
The IC50 values for these three compounds are presented in
Table 1, and a comparison of the data indicates that kalkitoxin
is 200- to 400-fold more potent than either 45 or 46. The latter
are comparable in their toxicity. The data demonstrate that the
intact thiazoline moiety of kalkitoxin is required for full
cytotoxicity.
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Table 1 IC50 values for kalkitoxin and synthetic precursors
and 46 there was no cytotoxic effect for exposures up to 24 h at
10 µg mLϪ1. However, when exposure was extended for 168 h,
significant cytotoxicity appeared for kalkitoxin with 10%
survival at 2 ng mLϪ1. For 46 at this time interval there was less
cytotoxicity, with 10% survival at 1 µg mLϪ1, and for 45 there
was 10% survival at 3 µg mLϪ1. These results indicate that
tumour cells need to be exposed to these compounds for greater
than 24 h in order to produce cytotoxic effects. In the case of
kalkitoxin, the cytotoxic effect could probably be maintained
by daily administration of the drug in vivo since activity is at the
ng mLϪ1 level. For 45 and 46, which are ca. 1000-fold less
potent than kalkitoxin, it appears unlikely that long term levels
in the µg mLϪ1 range could be maintained.
Compound
IC50/µg mLϪ1
2
46
45
1.0 × 10Ϫ3
1.9 × 10Ϫ1
4.0 × 10Ϫ1
Table
2
Zone differential results for kalkitoxin and synthetic
precursors
a
a
a
Compound
µg/disk
C38∆sL1210
C38∆sCFU
H116∆sCEM
2
6
45
3
800
—
950
400
1000
—
450
450
350
300
—
46
Experimental
45
12
—
a See text for definition of ∆s.
Starting materials and reagents were obtained from commercial
sources and were used without further purification. Solvents
were dried by distillation from the appropriate drying agents
immediately prior to use. Tetrahydrofuran (THF), diethyl ether
(Et2O), and toluene were distilled from sodium benzophenone
under an argon atmosphere. Diisopropylamine, triethylamine,
benzene, acetonitrile and dichloromethane (CH2Cl2) were
distilled from calcium hydride under argon. All solvents used
for routine isolation of products and chromatography were
reagent grade. Moisture and air sensitive reactions were carried
out under an atmosphere of argon. Reaction flasks were flame
dried under a stream of argon gas, and glass syringes were oven
dried at 120 ЊC and cooled in a dessicator over anhydrous
calcium sulfate prior to use. Unless otherwise stated, concen-
tration under reduced pressure refers to a rotary evaporator at
water aspirator pressure.
Analytical thin layer chromatography (TLC) was performed
using precoated aluminium E. Merck TLC plates (0.2 mm layer
thickness of silica gel 60 F-254). Compounds were visualized by
ultraviolet light, and/or by heating the plate after dipping in a
solution of 14% ammonium molybdate tetrahydrate and 1.4%
cerium() sulfate in 1.6 M sulfuric acid in water or 1% solution
of vanillin in 0.1 M sulfuric acid in ethanol or 1% solution of
potassium permanganate in 2% 1 N sodium hydroxide in water.
Flash chromatography was carried out using E. Merck silica gel
60 (230–400 mesh ASTM). Radial chromatography was carried
out on individual rotors with layer thickness of 1, 2, or 4 mm
using a Chromatotron manufactured by Harrison Research,
Palo Alto, California.
Melting points were measured using a Büchi melting point
apparatus, and are uncorrected. Infrared (IR) spectra were
recorded with a Nicolet 5DXB FT-IR spectrometer using a thin
film supported between NaCl plates or KBr discs. Specific
optical rotations were measured at ambient temperature (23 ЊC)
from CHCl3 solutions on a Perkin-Elmer 243 polarimeter using
a 1 mL cell with 1 dm path length. Proton and carbon nuclear
magnetic resonance (NMR) spectra were obtained using
either a Bruker AC-300 or a Bruker AM-400 spectrometer. All
chemical shifts are reported in parts per million (ppm) down-
field from tetramethylsilane using the δ scale. 1H NMR spectra
data are reported in the order: chemical shift, multiplicity
(s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet
and br = broad), coupling constant (J) in Hertz (Hz) and
number of protons.
Chemical ionization (CI) high and low resolution mass
spectroscopy (HRMS and MS) were obtained using a Kratos
MS-50 spectrometer with a source temperature of 120 ЊC and
methane gas as the ionizing source. Perfluorokerosene was used
as a reference. Electron impact (EI) mass spectra (HRMS and
MS) were obtained using a Varian MAT311 or a Finnegan 4000
spectrometer. X-Ray crystallographic data were collected on
a Siemens P4 instrument and these data were interpreted
using the direct methods program contained in the SHELXTL
(Silicon Graphics/Unix) software package.
Scheme 9
An in vitro cell-based assay was employed to identify solid
tumour selectivity for kalkitoxin and its synthetic precursors
45 and 46. The differential cytotoxicity was determined by
observing a zone differential between a solid tumour cell
(Colon 38, Colon HCT-116, Lung H-125) and either leukemia
cells (L1210 or CEM) or normal cells (CFU-GM).35 A sample
is designated as “solid tumour selective” if the zone units for
the solid tumour (Colon 38, for example) minus the zone units
for a leukemia cell (L1210, for example), expressed as C38∆sL1210
,
is greater than 250 units. The results of this assay are presented
in Table 2 and show that kalkitoxin, 45 and 46 all show differen-
tial cytotoxicity for Colon 38 versus both L1210 leukemia and
normal CFU-GM cells. Furthermore, kalkitoxin and thiol 46
show differential cytotoxicity for HCT-116 versus human
leukemia CEM. While kalkitoxin is the most potent, 45 and 46
are only two- to eight-fold less potent by this assay.
Finally, a clonogenic assay was carried out in which con-
centration and time-survival measurements were made with
kalkitoxin, 45 and 46 against HCT-116 cells. The results are
shown in Fig. 2 and indicate that for either kalkitoxin or for 45
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 2 0 9 2 – 2 1 0 2
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Fig. 2 Clonogenic dose response with HCT-116 cells for kalkitoxin, 45 and 46.
(2R,2ЈS,4ЈS )-1-(5Ј-Benzyloxy-2Ј,4Ј-dimethylpentanoyl)-2-
hydroxymethylpyrrolidine (10)
735, 697; HRMS (FAB, M ϩ Hϩ) calcd for C14H23O2 223.1698,
found 223.1696.
To a solution of diisopropylamine (1.12 mL, 8.0 mmol) in THF
(2 mL) was added n-BuLi (2.5 M, 3.2 mL, 8 mmol) at 0 ЊC, and
the resulting solution was stirred at 0 ЊC for 30 min. A solution
of 8 (0.495 g, 3.15 mmol) in THF (3 mL) was added at 0 ЊC,
the mixture was stirred for 1 h at room temperature, HMPA
(1.4 mL, 8.0 mmol) was added, and the mixture was cooled to
Ϫ78 ЊC. Iodide 9 (1.2 g, 4.1 mmol) was added, and the mixture
was stirred for 1 h at Ϫ78 ЊC and allowed to warm to room
temperature during 3 h. After stirring for 1 h at room temper-
ature the reaction was quenched with saturated NH4Cl solution
and the mixture was extracted with EtOAc (100 mL). The
organic layer was washed with brine, dried over MgSO4, and
concentrated. Purification of the residual oil by flash column
chromatography (hexanes/ethyl acetate, 3 : 2) gave 10 as a color-
Methyl (4S,6S )-7-benzyloxy-4,6-dimethylhept-2-enoate (13)
To a stirred solution of Dess–Martin periodinane (0.541 g, 1.27
mmol) in CH2Cl2 (8 mL) at 0 ЊC was added a solution of 11
(0.218 g, 0.98 mmol) in CH2Cl2 (10 mL). The mixture was
stirred for 9 h, and the reaction was quenched by the addition
of 20% Na2S2O3 solution (8 mL) and saturated NaHCO3
solution (8 mL). The organic layer was separated and diluted
with ether (80 mL), and the ethereal solution was washed with
saturated NaHCO3 solution and brine. After drying over
MgSO4, the solution was concentrated to give an aldehyde
(0.217 g, 99%). To a solution of this aldehyde (16.0 mg, 0.073
mmol) in toluene (5 mL) was added 12 (69 mg, 0.20 mmol) and
the mixture was heated to 80 ЊC for 3 h. After cooling to room
temperature, the mixture was diluted with ether (20 mL) and
washed with water. The separated organic phase was dried over
MgSO4 and concentrated, and the residual oil was purified by
flash column chromatography (hexanes/ethyl acetate, 9 : 1) to
give 13 (18.6 mg, 93%) as a pale yellow oil: [α]2D0 ϩ31.7 (c 0.6,
CHCl3); 1H NMR (300 MHz, CDCl3) δ 7.33 (m, 5H), 6.87 (dd,
J = 15.7, 7.9 Hz, 1H), 5.77 (dd, J = 15.7, 1.1 Hz, 1H), 4.49 (s,
2H), 3.74 (s, 3H), 3.26 1.90 (m, 1H), 1.75 (m, 1H), 1.56 (br, 1H),
1.20 (m, 2H), 0.91 (d, J = 6.1 Hz, 3H), 0.89 (d, J = 6.5 Hz, 3H);
13C NMR (75 MHz, CDCl3) δ 138.6, 128.3, 127.6, 127.5, 76.6,
73.3, 68.9, 37.2, 33.0, 30.6, 17.0, 16.3; IR (film, cmϪ1) 3381,
2956, 2924, 2854, 1454, 1363, 1099, 1029, 735, 697; HRMS
(FAB, MϩHϩ) calcd for C14H23O2 223.1698, found 223.1696.
less oil (0.738 g, 74%): [α]2D0 ϩ50.4 (c 2.0, CH2Cl2); H NMR
1
(300 MHz, CDCl3) δ 7.29 (m, 5H), 5.23 (br, 1H), 4.47 (q, J = 8.0
Hz, 2H), 4.18 (m, 1H), 3.55–3.25 (m, 6H), 2.68 (m, 1H), 2.05–
1.43 (m, 7H), 1.09 (d, J = 6.8 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H);
13C NMR (75 MHz, CDCl3) δ 178.3, 138.5, 128.3, 127.6, 127.5,
76.2, 73.1, 67.6, 60.9, 47.5, 38.0, 35.7, 31.4, 28.1, 24.3, 17.8,
17.5; IR (film, cmϪ1) 2293, 2960, 2929, 2872, 1616, 1454, 1435,
1361, 1093; HRMS (FAB, M ϩ Hϩ) calcd for C19H30NO3
320.2226, found 320.2223.
(2S,4S )-5-Benzyloxy-2,4-dimethylpentanol (11)
A solution of 10 (0.342 g, 1.07 mmol) in 1 N HCl (10 mL) was
heated at reflux for 6 h, then was cooled to 0 ЊC, and treated
with 15% NaOH solution for 10 min. The mixture was acidified
to pH 3 and extracted with ether. The extract was dried over
MgSO4, and concentrated to give the crude carboxylic acid
(0.253 g, 100%). To a solution of this acid in dry ether (10 mL)
at 0 ЊC was added LiAlH4 (0.085 g, 2.24 mmol) slowly during 10
min. The mixture was stirred for 30 min at 0 ЊC and then for 3.5
h at room temperature. The reaction was quenched by careful
addition of water (2 mL) at 0 ЊC, and the mixture was treated
with 15% NaOH solution (2 mL) for 20 min and diluted with
ether (40 mL). The ethereal solution was washed with water and
brine, dried over MgSO4, and concentrated. Purification of the
residual oil by flash column chromatography (hexanes/ethyl
acetate, 4 : 1) yielded 11 as a colorless oil (0.232 g, 98%): [α]2D0
ϩ50.4 (c 2.0, CH2Cl2); 1H NMR (300 MHz, CDCl3) δ 7.32 (m,
5H), 4.51(s, 2H), 3.45 (m, 2H), 3.30 (m, 2H), 1.90 (m, 1H), 1.75
(m, 1H), 1.56 (br, 1H), 1.20 (m, 2H), 0.91 (d, J = 6.1 Hz, 3H),
0.89 (d, J = 6.5 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 138.6,
128.3, 127.6, 127.5, 76.6, 73.3, 68.9, 37.2, 33.0, 30.6, 17.0, 16.3;
IR (film, cmϪ1) 3381, 2956, 2924, 2854, 1454, 1363, 1099, 1029,
Methyl (3S,4S,6S )-7-benzyloxy-3,4,6-trimethylheptanoate (14)
and methyl (3R,4S,6S )-7-benzyloxy-3,4,6-trimethylheptanoate
(15)
To a suspension of CuBrؒDMS (0.042 g, 0.20 mmol) in THF
(1 mL) was added MeLi (1.0 M solution, 0.4 mL, 0.4 mmol) at
Ϫ40 ЊC, and the mixture was stirred for 20 min. To this mixture
at Ϫ40 ЊC were added chlorotrimethylsilane (0.032 mL, 0.25
mmol), HMPA (0.044 mL, 0.25 mmol) and 13 (0.014 g, 0.051
mmol), and the mixture was stirred for 1.5 h at 0 ЊC. The
reaction was quenched by the addition of saturated NH4Cl
solution, and the mixture was extracted with ether. The extract
was dried over MgSO4 and concentrated. Flash column
chromatography (hexanes/ethyl acetate, 9 : 1) of the residual oil
gave a mixture (0.004 g, 29%, 6 : 4 ratio) of 14 and 15: 1H NMR
(asterisk denotes signals due to minor diastereomer, 300 MHz,
CDCl3) δ 7.32 (m, 5H), 4.49 (s, 2H), 3.66 (s, 3H), 3.65* (s, 3H),
3.33* (m, 2H), 3.27 (m, 2H), 2.31* (m, 1H), 2.29 (m, 1H), 2.09
(m, 1H), 2.07* (m, 1H), 1.99 (m, 1H), 1.99* (m, 1H), 1.81* (m,
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1H), 1.81* (m, 1H), 1.65 (m, 1H), 1.65* (m, 1H), 0.90* (d,
J = 4.9 Hz, 3H), 0.88 (d, J = 4.4 Hz, 3H), 0.87 (d, J = 4.4 Hz,
3H), 0.85* (d, J = 4.7 Hz, 3H), 0.83 (d, J = 4.2 Hz, 3H), 0.79* (d,
J = 4.7 Hz, 3H).
concentrated under reduced pressure. Column chromatography
(hexanes/ethyl acetate, 93 : 7) of the residue gave 20 (0.078 g,
1
93%) as an oil (E : Z = 11 : 1): H NMR (300 MHz, CDCl3)
δ 7.33 (m, 5H), 5.67 (s, 1H), 4.48 (s, 2H), 4.13 (q, J = 7.2 Hz,
2H), 3.27 (m, 2H), 2.29 (m, 1H), 2.09 (s, 3H), 1.79 (m, 1H), 1.44
(m, 1H), 1.26 (t, J = 7.2 Hz, 3H), 1.01 (d, J = 6.8 Hz, 3H), 0.93
(d, J = 6.7 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 167.0, 164.6,
138.7, 128.3, 127.5, 127.4, 115.0, 75.5, 73.0, 59.4, 41.3, 38.8,
31.2, 19.0, 17.5, 15.5, 14.3. IR (film, cmϪ1) 2962, 2928, 2854,
1715, 1643, 1455, 1222, 1158, 1096.
(1ЈR,2ЈR,2S,4S )-5-Benzyloxy-2,4-dimethylpentanoic acid
(2Ј-hydroxy-1Ј-methyl-2Ј-phenylethyl)methylamide (17)
To a solution of lithium chloride (0.58 g, 13.7 mmol) and diiso-
propylamine (0.404 g, 4.0 mmol) in THF (2 mL) at Ϫ78 ЊC was
added n-BuLi (2.1 M solution in hexane, 1.80 mL, 3.80 mmol)
and the mixture was stirred for 10 min at Ϫ78 ЊC and then for
15 min at 0 ЊC. To this mixture at Ϫ78 ЊC was added a solution
of 16 (0.450 g, 2.04 mmol) in THF (2 mL), and the mixture was
stirred for 1 h at Ϫ78 ЊC, for 15 min at 0 ЊC and for 5 min
at room temperature. The solution was cooled to 0 ЊC and a
solution of 9 (0.298 g, 1.03 mmol) in THF (1 mL) was added.
The mixture was stirred for 10 min at 0 ЊC and for 15 h at room
temperature, and the reaction was quenched by the addition of
saturated NH4Cl solution. The mixture was extracted with
EtOAc (60 mL × 3) and the extract was washed with water,
dried over anhydrous MgSO4, and concentrated under reduced
pressure. Purification of the residue by flash column chromato-
graphy (hexanes/ethyl acetate, 3 : 2) gave 17 (0.451 g, 98%) as
Ethyl (3S,4S,6S )-7-benzyloxy-3,4,6-trimethylheptanoate (22)
and ethyl (3R,4S,6S )-7-benzyloxy-3,4,6-trimethylheptanoate
(23)
To a solution of 20 (0.036 g, 0.118 mmol) in EtOH (0.05 mL)
and diglyme (0.05 mL) were added 21 (1.3 mg, 2.8 µmol) and
CoCl2ؒ6H2O (0.85 mg, 3.5 µmol). The blue solution was
degassed by three freeze–thaw cycles, NaBH4 (9.8 mg, 0.26
mmol) was added, and the mixture was degassed again by three
freeze–thaw cycles. The mixture was stirred for 3 days at room
temperature, diluted with CH2Cl2 (10 mL) and washed with
water. The separated organic phase was washed with saturated
NaCl solution, dried over anhydrous MgSO4, and concentrated
under reduced pressure to give a 1.6 : 1 mixture of 22 and 23
a pale yellow oil: [α]2D0 Ϫ49.9 (c 1.1, CHCl3); H NMR (4 : 1
1
1
rotamer ratio, asterisk denotes signals due to minor rotamer,
300 MHz, CDCl3) δ 7.31 (m, 10H), 4.58 (m, 1H), 4.56* (m, 1H),
4.50 (m, 3H), 4.40* (m, 2H), 4.09* (m, 1H), 3.32* (m, 2H), 3.21
(m, 2H), 3.09* (m, 1H), 2.88* (s, 3H), 2.73 (s, 3H), 2.70 (m, 1H),
1.86* (m, 1H), 1.70 (m, 1H), 1.45* (m, 2H), 1.40 (m, 2H), 1.09
(d, J = 6.9 Hz, 3H), 1.05* (overlapped, 3H), 1.03 (d, J = 6.8 Hz,
3H), 0.96* (m, 6H), 0.90 (d, J = 6.7 Hz, 3H); 13C NMR (4 : 1
rotamer ratio, asterisk denotes signals due to minor rotamer, 75
MHz, CDCl3) δ 179.0, 177.5*, 142.5, 141.4*, 138.5, 128.6*,
128.2, 128.1, 127.5, 127.4, 126.8*, 126.2, 76.3, 76.2*, 75.8, 75.1,
73.0, 72.9*, 58.6, 57.7*, 38.2, 34.2, 33.4*, 32.8*, 31.3, 29.6,
26.8*, 18.1*, 17.7*, 17.5, 17.3, 15.5*, 14.3; IR (film, cmϪ1) 3384,
2956, 2929, 2871, 1617, 1455, 1097; HRMS (CI, MϩHϩ) calcd
for C24H34NO3 384.2539, found 384.2541.
(0.011 g, 30%). The H NMR spectrum of the mixture of di-
astereomers was essentially identical to that of the homologous
methyl esters 14 and 15.
(1ЈS,2ЈS,2R)-4-Benzyloxy-N-(2Ј-hydroxy-1Ј-methyl-2Ј-phenyl-
ethyl)-2,N-dimethylbutyramide (26)
In a 100 mL round-bottomed flask was placed LiCl (2.16 g, 54.8
mmol) and the flask was flame-dried under argon. To the flask
at Ϫ78 ЊC were added dry THF (8 mL) and diisopropylamine
(2.24 mL, 16 mmol), and the resulting suspension was stirred as
n-BuLi (7.6 mL, 15.2 mmol, 2.0 M hexane solution) was added.
The mixture was stirred for 10 min at Ϫ78 ЊC and for 15 min at
0 ЊC, and was then cooled again to Ϫ78 ЊC as a solution of 25 in
THF (8 mL) was added slowly by cannula. The mixture was
stirred for 1 h at Ϫ78 ЊC, for 15 min at 0 ЊC and for 5 min at
ambient temperature. To this mixture at 0 ЊC was added a solu-
tion of 2-benzyloxy-1-iodoethane (1.15 g, 4.4 mmol) in THF (4
mL), the mixture was stirred for 15 h at ambient temperature,
and the reaction was quenched by the addition of aqueous
NH4Cl. The solution was extracted with EtOAc (200 mL × 2)
and the extract was washed with NH4Cl solution and brine. The
extract was dried over MgSO4, the solvent was evaporated
under reduced pressure and the residue was purified by flash
column chromatography (hexanes/ethyl acetate, 3 : 2) to give 26
(1.392 g, 98%) as a colorless oil: [α]2D0 = ϩ52.8 (c 0.8, CHCl3); 1H
NMR (7 : 3 rotamer mixture, asterisk denotes signals due to
minor rotamer, 300 MHz, CDCl3) δ 7.29 (m, 10H), 4.78–4.40
(m, 4H), 4.12* (m, 1H), 3.68* (m, 1H), 3.55 (m, 1H), 3.45 (m,
1H), 3.29 (m, 1H), 3.21* (m, 1H), 2.88 (m, 1H), 2.83 (s, 3H),
2.81* (s, 3H), 2.25* (m, 1H), 1.93 (m, 1H), 1.68* (m, 1H), 1.65
(m, 1H), 1.1 (m, 6H).; 13C NMR (75 MHz, CDCl3) δ 178.1,
176.9*, 142.4, 141.4*, 138.1, 128.3*, 128.1, 128.0*, 127.8, 127.5,
127.4*, 127.3, 127.2*, 126.7, 126.1*, 75.8, 75.0*, 72.6, 68.2*,
67.7, 57.9, 33.8, 32.8, 32.3*, 26.6*, 18.1*, 17.1, 15.5*, 14.1.; IR
(film, cmϪ1) 3386, 2968, 2932, 2869, 1618, 1454, 1089; HRMS
(CI, Mϩ) calcd for C22H30O3N 356.2226, found 356.2224.
(3R,5R)-6-Benzyloxy-3,5-dimethylhexan-2-one (18)
To a solution of 17 (0.115 mg. 0.30 mmol) in THF (5 mL) at
Ϫ78 ЊC was slowly added MeLi (0.55 mL, 1.3 M in hexane),
and the mixture was stirred for 5 min at Ϫ78 ЊC and for 10 min
at 0 ЊC. To the mixture at 0 ЊC was added a solution of diiso-
propylamine (0.06 mL, 0.043 mmol) in THF (1 mL), and the
mixture was stirred for 15 min, diluted with EtOAc (20 mL) and
washed with NH4Cl solution. The separated organic phase was
dried over MgSO4 and concentrated under reduced pressure.
Flash column chromatography (hexanes/ethyl acetate, 4 : 1) of
the residue gave 18 (0.065 g, 93%) as a colorless oil: [α]2D0 ϩ1.1
1
(c 1.9, CHCl3); H NMR (300 MHz, CDCl3) δ 7.31 (m, 5H),
4.49 (s, 2H), 3.28 (dd, J = 6.1, 1.3 Hz, 2H), 2.61 (m, 1H), 2.12
(s, 3H), 1.79 (m, 1H), 1.47 (m, 2H), 1.06 (d, J = 7.0 Hz, 3H),
0.92 (d, J = 6.7 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 212.9,
138.5, 128.3, 127.5, 75.8, 73.0, 44.7, 36.7, 31.3, 27.9, 17.0, 16.2;
IR (film, cmϪ1) 2928, 2854, 1714, 1456, 1362, 1097; HRMS (CI,
Mϩ) calcd for C15H22O2, 234.1620, found 234.1617.
Ethyl (2E,4R,6R)-7-benzyloxy-3,4,6-trimethylhept-2-enoate
(20)
To a solution of NaH (60% dispersion in mineral oil, 0.06 g,
1.50 mmol) in toluene (2 mL) at room temperature was added a
solution of 19 (0.38 g, 1.50 mmol) in toluene (2 mL). The
mixture was stirred for 1 h, and a solution of 18 (0.065 g, 0.28
mmol) in toluene (2 mL) was added. The mixture was heated
for 8 h at 70 ЊC, cooled to room temperature, and diluted with
ether (30 mL). The mixture was washed with NH4Cl solution,
and the separated organic phase was dried over MgSO4 and
(2R)-4-Benzyloxy-2-methylbutanol (27)
To a solution of diisopropylamine (8.45 mL, 60.3 mmol) in
THF (80 mL) at 0 ЊC was added n-BuLi (2.1 M, 28.7 mL, 60.3
mmol) at 0 ЊC and the resulting solution was stirred for 20 min.
Borane–ammonia complex (90%, 1.90 g, 61.5 mmol) was added
in one portion, and the suspension was stirred at 0 ЊC for 20
min and then warmed to room temperature. After 15 min, the
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suspension was cooled to 0 ЊC and a solution of 26 (4.28 g,
12.06 mmol) in THF (12 mL) was added via cannula. The
mixture was warmed to room temperature after 10 min, held
at that temperature for 2.5 h, and then cooled to 0 ЊC while
3 N HCl was added slowly to quench excess hydride. The
biphasic solution was stirred for 30 min at 0 ЊC and for 15 min
at room temperature, and was then separated. The aqueous
layer was extracted with ether and the ethereal extract was
washed with brine, dried, and concentrated. Purification of the
residual oil by flash column chromatography (hexanes/ethyl
acetate, 2 : 1) afforded 27 as a colorless oil (2.11g, 90%): [α]2D0
ϩ10.4 (c 7.5, CHCl3); 1H NMR (300 MHz, CDCl3) δ 7.26–7.38
(m, 5H), 4.51 (s, 2H), 3.38–3.62 (m, 4H), 2.94 (bs, 1H),
1.66–1.83 (m, 2H), 1.49–1.59 (m, 1H), 0.92 (d, 3H, J = 6.6 Hz);
13C NMR (75 MHz, CDCl3) δ 137.9, 128.3, 127.6, 127.5, 72.9,
68.5, 67.8, 33.8, 17.0; IR (film, cmϪ1) 3404, 2926, 2869, 1454,
1097; HRMS (CI, Mϩ) calcd for C12H18O2 194.1307, found
194.1303.
(4S,2ЈS,3ЈS,5ЈS )-3-(7Ј-Benzyloxy-2Ј,3Ј,5Ј-trimethylheptanoyl)-
4-phenyloxazolidin-2-one (32)9 and (4S,2ЈR,3ЈS,5ЈS )-3-(7Ј-benz-
yloxy-2Ј,3Ј,5Ј-trimethylheptanoyl)-4-phenyloxazolidin-2-one
(33)
To a suspension of magnesium (0.10 g, 4.14 mmol) in THF (2
mL) was added 1,2-dibromoethane (0.0074 mL, 0.086 mmol),
and the mixture was heated at reflux for 10 min and then cooled
to room temperature. A solution of 24 (0.443 g, 1.72 mmol) in
THF (0.5 mL) was added via syringe, and the mixture was
heated to reflux for 20 min and then cooled to Ϫ78 ЊC. This
mixture was added to a solution of CuBrؒDMS (0.34 g, 1.72
mmol) in THF (2 mL), and the resulting suspension was
warmed to Ϫ20 ЊC, stirred for 25 min and cooled again to
Ϫ78 ЊC. To the suspension was added dropwise a solution of 29
(0.14 g, 0.57 mmol) in THF (1.5 mL), and the suspension was
stirred for 2.5 h at Ϫ78 ЊC and was then slowly warmed to
Ϫ30 ЊC during 45 min. The mixture was cooled again to
Ϫ78 ЊC, MeI (0.54 mL, 8.6 mmol) was added, and the mixture
was allowed to warm to room temperature and was stirred
overnight. Saturated NH4Cl was added to quench the reaction,
and the mixture was extracted with ether. The ether extract was
washed with brine, dried over MgSO4, and concentrated. Flash
column chromatography (hexanes/ethyl acetate, 5 : 1) of the
residue gave 32 (0.117 g, 50%) and 33 (0.038 g, 14%).
(2R)-4-Benzyloxy-1-bromo-2-methylbutane (24)
To a solution of 27 (0.25 g, 1.31 mmol) in CH2Cl2 (2 mL) was
added PPh3 (0.24g, 1.37 mmol) and N-bromosuccinimide (0.36
g, 1.37 mmol) at room temperature. The solution was stirred for
10 min and concentrated under vacuum. The crude solid was
triturated with hexane, the mixture was filtered, and the filtrate
was concentrated. The residue was purified by flash column
chromatography (hexanes/ethyl acetate, 20 : 1) to provide 24 as
Recrystallization of 32 from warm hexanes gave colorless
crystals: mp 75–76 ЊC; [α]2D0 ϩ49.7 (c 1.65, CHCl3); H NMR
1
(300 MHz, CDCl3) δ 7.27–7.41 (m, 10H), 5.40 (dd, J = 8.4, 3.3
Hz, 1H), 4.62 (t, J = 8.7 Hz, 1H), 4.50 (s, 2H), 4.22 (dd, J = 9.0,
3.3 Hz, 1H), 3.70 (quint, J = 7.2 Hz, 1H), 3.51 (t, J = 6.6 Hz,
2H), 1.84–1.92 (m, 1H), 1.46–1.71 (m, 3H), 1.15 (ddd, J = 10.5,
10.5, 3.6 Hz, 2H), 1.04 (d, J = 6.9 Hz, 3H), 0.94 (d, J = 6.6 Hz,
3H), 0.84 (d, J = 6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3)
δ 176.2, 153.4, 139.3, 138.6, 129.1, 128.5, 128.3, 127.6, 127.4,
125.6, 72.9, 69.6, 68.4, 57.7, 43.1, 39.6, 37.9, 32.7, 27.0, 18.9,
17.8, 13.6; IR (film, cmϪ1) 2925, 1780, 1704; HRMS (FAB, M ϩ
Hϩ) calcd for C26H34NO4 424.2488, found 424.2509.
a colorless oil (0.25 g, 74%): [α]2D0 Ϫ4.7 (c 0.32, CHCl3); H
1
NMR (300 MHz, CDCl3) δ 7.29–7.42 (m, 5H), 4.54 (s, 2H), 3.57
(dt, J = 3.3, 0.1 Hz, 2H), 3.47 (dd, J = 9.9, 4.8 Hz, 1H), 3.40 (dd,
J = 9.9, 5.7 Hz, 1H), 2.03–2.11 (m, 1H), 1.83 (ddd, J = 12.9,
12.9, 6.3 Hz, 1H), 1.59 (ddd, J = 13.8, 13.8, 6.3 Hz, 1H), 1.07
(d, J = 6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 138.9, 128.8,
128.0, 73.4, 68.3, 42.0, 35.1, 32.6, 19.2; IR (film, cmϪ1) 2961,
2858, 1454; HRMS (FAB, M ϩ Hϩ) calcd for C12H18OBr
259.0521, found 259.0532.
Data for 33: [α]2D0 Ϫ 20.6 (c 1.88, CHCl3); H NMR (300
1
(3ЈS,4S,5ЈS )-3-(7Ј-Benzyloxy-3Ј,5Ј-dimethylheptanoyl)-4-
phenyloxazolidin-2-one (31)
MHz, CDCl3) δ 7.26–7.41 (m, 10H), 5.46 (dd, J = 9.0, 4.5 Hz,
1H), 4.68 (t, J = 9.0 Hz, 1H), 4.51 (s, 2H), 4.28 (dd, J = 9.0, 4.5
Hz, 1H), 3.68 (quint, J = 6.6 Hz, 1H), 3.48 (t, J = 6.6 Hz, 2H),
1.89–1.97 (m, 1H), 1.46–1.62 (m, 2H), 1.36–1.42 (m, 1H), 1.16
(ddd, J = 13.5, 10.5, 3.6 Hz, 1H), 1.05 (d, J = 6.9 Hz, 3H), 0.83–
0.96 (m, 1H), 0.74 (d, J = 6.3 Hz, 3H), 0.62 (d, J = 6.6 Hz, 3H);
13C NMR (75 MHz, CDCl3) δ 177.0, 153.8, 139.6, 139.1, 129.4,
129.1, 128.8, 128.0, 127.9, 126.8, 73.3, 69.9, 69.0, 58.1, 43.2,
42.9, 38.1, 33.4, 27.6, 19.3, 14.7, 12.3; IR (film, cmϪ1) 2926,
1780, 1704; HRMS (FAB, M ϩ Hϩ) calcd for C26H34NO4
424.2488, found 424.2490.
To a suspension of magnesium (0.52 g, 21.4 mmol) in THF
(10 mL) was added 1,2-dibromoethane (0.0384 mL, 0.44 mmol)
and the mixture was heated at reflux for 10 min and then cooled
to room temperature. A solution of 24 (2.29 g, 8.91 mmol) in
THF (2 mL) was added via syringe, the mixture was heated at
reflux for 20 min, then was cooled and added to a solution of
CuBrؒDMS (1.83 g, 8.91 mmol) in THF (8 mL) at Ϫ78 ЊC. The
suspension was warmed to Ϫ20 ЊC, stirred for 25 min and
cooled again to Ϫ78 ЊC. To this suspension was added dropwise
a solution of 29 (0.68 g, 2.94 mmol) in THF (6 mL), the dark
brown suspension was stirred for 2.5 h at Ϫ78 ЊC, and then was
slowly warmed to Ϫ30 ЊC during 45 min. The reaction was
quenched with saturated NH4Cl, the mixture was extracted
with ether, and the extract was washed with brine, dried over
MgSO4, and concentrated. Purification of the residual oil by
flash column chromatography (hexanes/ethyl acetate, 3 : 1)
gave 29 (1.16 g, 96%) as a colorless oil: [α]2D0 ϩ20.0 (c 2.30,
(4S,2ЈS,3ЈS,5ЈS )-3-(7Ј-Oxo-2Ј,3Ј,5Ј-trimethylheptanoyl)-4-
phenyloxazolidin-2-one (34)
To a solution of 32 (0.753 g, 1.78 mmol) in EtOAc (12 mL) at
room temperature was added Pd(OH)2 on carbon (Pearlman’s
catalyst, 0.116 g), and the mixture was stirred for 2 h under an
atmosphere of hydrogen. The catalyst was filtered off and the
filtrate was concentrated. Flash column chromatography (hex-
anes/ethyl acetate, 1 : 1) of the residue gave (4S,2ЈS,3ЈS,5ЈS)-3-
(7Ј-hydroxy-2Ј,3Ј,5Ј-trimethylheptanoyl)-4-phenyloxazolidin-
2-one as a pale yellow oil (0.60 g, 100%): [α]2D0 ϩ68.6 (c 2.50,
CHCl3); 1H NMR (300 MHz, CDCl3) δ 7.26–7.41 (m, 5H), 5.42
(dd, J = 8.4, 3.3 Hz, 1H), 4.66 (t, J = 8.7 Hz, 1H), 4.24 (dd,
J = 9.0, 3.3 Hz, 1H), 3.61–3.73 (m, 3H), 1.83–1.92 (m, 1H),
1.62–1.64 (m, 1H), 1.34–1.55 (m, 3H), 1.09–1.20 (m, 2H), 1.03
(d, J = 6.9 Hz, 3H), 0.92 (d, J = 6.6 Hz, 3H), 0.83 (d, J = 6.6 Hz,
3H); 13C NMR (75 MHz, CDCl3) δ 176.3, 153.4, 139.2, 129.1,
128.5, 125.6, 69.6, 60.8, 57.7, 43.0, 41.0, 40.0, 32.6, 26.6, 18.8,
17.7, 13.7; IR (film, cmϪ1) 3385, 2926, 1779, 1702; HRMS
(FAB, M ϩ Hϩ) calcd for C19H28O4N 334.2018, found
334.2018.
1
CHCl3); H NMR (300 MHz, CDCl3) δ 7.29–7.45 (m, 10H),
5.47 (dd, J = 8.7, 3.6 Hz, 1H), 4.71 (t, J = 8.7 Hz, 1H), 4.52 (s,
2H), 4.30 (dd, J = 9.0, 3.9 Hz, 1H), 3.50 (dt, J = 7.5, 0.9 Hz, 2H),
2.96 (dd, J = 15.9, 5.4 Hz, 1H), 2.76 (dd, J = 15.9, 8.1 Hz, 1H),
2.09–2.22 (m, 1H), 1.66–1.75 (m, 1H), 1.58 (ddd, J = 12.6, 12.6,
6.6 Hz, 1H), 1.44 (ddd, J = 13.2, 13.2, 6.9 Hz, 1H), 1.22
(ddd, J = 13.5, 9.6, 4.5 Hz, 1H), 1.09 (ddd, J = 13.8, 9.3, 4.8 Hz,
1H), 0.88 (d, J = 6.6 Hz, 3H), 0.85 (d, J = 6.6 Hz, 3H); 13C
NMR (75 MHz, CDCl3) δ 172.1, 153.6, 139.2, 138.6, 129.1,
128.6, 128.3, 127.6, 127.4, 125.9, 72.8, 69.8, 68.4, 57.5, 44.2,
43.2, 37.4, 27.1, 19.1; IR (film, cmϪ1) 2925, 1782, 1705; HRMS
(FAB, M ϩ Hϩ) calcd for C25H32NO4 410.2331, found
410.2339.
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To a solution of oxalyl chloride (0.261 mL, 3.0 mmol) in
CH2Cl2 (3 mL) at Ϫ78 ЊC was added DMSO (0.284 mL, 4.0
mmol) and CH2Cl2 (3 mL). After stirring the mixture for 15
min, a solution of the alcohol prepared above (0.60 g, 1.78
mmol) in CH2Cl2 (8 mL) was added dropwise. The solution was
stirred at Ϫ78 ЊC for 15 min, a solution of NEt3 (1.09 mL, 8.0
mmol) in CH2Cl2 (4 mL) was added and the mixture was slowly
warmed to 0Њ C during 30 min. The reaction was quenched by
addition of saturated NH4Cl, the layers were separated and the
aqueous layer was extracted with ether. The extract was washed
with brine, dried over MgSO4, and concentrated. Flash column
chromatography (hexanes/ethyl acetate, 3 : 1) of the residue
yielded 34 as a pale yellow oil (0.51 g, 85%): [α]2D0 ϩ69.3 (c 1.0,
CHCl3); 1H NMR (300 MHz, CDCl3) δ 9.74 (t, J = 2.1 Hz, 1H),
7.26–7.41 (m, 5H), 5.42 (dd, J = 8.7, 3.3 Hz, 1H), 4.67 (t, J = 9.0
Hz, 1H), 4.25 (dd, J = 9.0, 3.6 Hz, 1H), 3.69 (quin, J = 6.9 Hz,
1H), 2.29–2.33 (m, 2H), 2.08–2.19 (m, 1H), 1.84–1.93 (m, 1H),
1.28 (ddd, J = 13.5, 10.8, 3.0, 1H), 1.10 (ddd, J = 14.1, 10.8, 3.6,
1H), 1.03 (d, J = 6.9 Hz, 3H), 0.96 (d, J = 6.9 Hz, 3H), 0.90 (d, J
= 6.3 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 203.0, 176.6,
153.8, 140.0, 129.6, 129.1, 126.1, 70.1, 58.2, 52.6, 43.5, 40.0,
33.0, 25.8, 19.4, 18.1, 14.2; IR (film, cmϪ1) 2964, 2930, 1777,
1721, 1702, 1383; HRMS (FAB, M ϩ Hϩ) calcd for C19H26NO4
332.1862, found 332.1872.
1.11 (m, 9H), 0.84–0.93 (m, 9H); 13C NMR (75 MHz, CDCl3)
δ 176.9, 176.6, 176.5, 153.9, 139.8, 139.6, 129.6, 129.1, 129.0,
126.1, 70.1, 58.2, 48.4, 46.5, 43.5, 40.2, 37.8, 37.7, 37.6, 35.9,
35.6, 34.1, 33.2, 33.0, 28.4, 27.8, 27.5, 19.2, 18.2, 18.0, 17.5,
14.4, 14.1, 12.6, 12.4; IR (film, cmϪ1) 2926, 1779, 1704, 1639,
1456; HRMS (FAB, M ϩ Hϩ) calcd for C25H39 N2O4 431.2910,
found 431.2912.
(2R,3ЈS,5ЈS,6ЈS )-2,N-Dimethyl-N-[7Ј-hydroxy-3Ј,5Ј,6Ј-tri-
methylheptyl]butyramide (38)
To a solution of 37 (0.084 g, 0.20 mmol) in THF (1.7 mL) at
0 ЊC was added LiBH4 (2 M solution in THF, 0.30 mL,
0.6 mmol), followed by MeOH (0.042 mL, 1.04 mmol). The
solution was stirred for 40 min at 0 ЊC, for 10 min at room
temperature, and the reaction was quenched with water. The
mixture was extracted with EtOAc, and the extract was washed
with brine, dried over MgSO4, and concentrated. Flash column
chromatography (hexanes/ethyl acetate, 3 : 2) of the residue
afforded a mixture of rotamers of 38 as a pale yellow oil (0.042
g, 80%): [α]2D0 Ϫ36.2 (c 0.50, CHCl3); 1H NMR (400 MHz,
CDCl3) δ 3.58–3.62 (m, 1H), 3.25–3.48 (m, 3H), 3.02 (s, 3H,
rotamer), 2.93 (s, 3H, rotamer), 2.58 (m, 1H), 1.92 (s, 1H), 1.33–
1.73 (m, 6H), 1.11 (d, J = 6.8 Hz, 3H), 1.09 (d, J = 6.8 Hz, 3H),
0.84–0.91 (m, 12H); 13C NMR (100 MHz, CDCl3) δ 177.0,
176.7, 66.3, 66.2, 48.5, 46.7, 41.5, 41.3, 40.5, 40.2, 37.8, 37.6,
35.7, 35.6, 34.1, 31.8, 31.7, 28.8, 28.6, 27.8, 27.4, 19.6, 19.4,
(4S,2ЈS,3ЈS,5ЈS )-3-(7Ј-Methylamino-2Ј,3Ј,5Ј-trimethylhepta-
noyl)-4-phenyloxazolidin-2-one (35)
18.1, 17.5, 17.4, 17.3, 13.8, 13.6, 12.6, 12.4; IR (film, cmϪ1
)
To a solution of 34 (0.50g, 1.51 mmol) in MeOH (10 mL) at
room temperature was added MeNH2ؒHCl (0.204 g, 3.02
mmol), MeNH2 (2 M solution in THF, 2.76 mL, 4.53 mmol)
and Na2SO4 (0.4 g). The mixture was stirred for 20 min and
then cooled to 0 ЊC as a solution of NaCNBH3 (0.142 g, 2.26
mmol) in MeOH (1.5 mL) was added. The mixture was warmed
to room temperature and stirred for 1 h, MeOH was removed
under vacuum and the residue was purified by flash column
chromatography (CH2Cl2/MeOH, 20 : 1) to give 35 (0.314 g,
61%) as a viscous oil: [α]2D0 ϩ52.8 (c 1.70, CHCl3); 1H NMR (400
MHz, CDCl3) δ 7.29–7.44 (m, 5H), 5.51 (dd, J = 8.4, 3.6 Hz,
1H), 4.76 (t, J = 8.8 Hz, 1H), 4.26 (dd, J = 8.8, 3.6 Hz, 1H), 3.74
(quint, J = 6.8 Hz, 1H), 2.98 (dd, J = 8.4, 6.0 Hz, 2H), 2.72 (s,
3H), 1.85–1.95 (m, 1H), 1.60–1.80 (m, 3H), 1.22–1.34 (m, 2H),
1.14–1.16 (m, 1H), 1.08 (d, J = 7.2 Hz, 3H), 0.96 (d, J = 6.4 Hz,
3H), 0.91 (d, J = 5.6 Hz, 3H); 13C NMR (100 MHz, CDCl3)
δ 176.7, 154.2, 139.7, 129.6, 129.0, 126.0, 70.4, 58.3, 48.6, 43.4,
3419, 2961, 1624, 1458; HRMS (FAB, M ϩ Hϩ) calcd for C16
H34NO2 272.2589, found 272.2605.
(2R,3ЈS,5ЈS,6ЈS )-2,N-Dimethyl-N-[8Ј-Methoxy-3Ј,5Ј,6Ј-tri-
methyloct-7-enyl]-butyramide (40)
To a solution of oxalyl chloride (0.073 mL, 0.84 mmol) in
CH2Cl2 (0.8 mL) at Ϫ78 ЊC was added a solution of DMSO
(0.079 mL, 1.12 mmol) in CH2Cl2 (0.8 mL). The mixture was
stirred for 15 min at Ϫ78 ЊC and a solution of 38 (0.151 g, 0.56
mmol) in CH2Cl2 (2 mL) was added dropwise. The mixture was
stirred for an additional 15 min at Ϫ78 ЊC, and a solution of
NEt3 (0.314 mL, 2.24 mmol) in CH2Cl2 (0.8 mL) was added.
The mixture was slowly warmed to Ϫ10 ЊC during 30 min and
the reaction was quenched with saturated NH4Cl. The mixture
was extracted with ether, and the extract was washed with brine,
dried over MgSO4, and concentrated to give the crude aldehyde
which was subjected immediately to the next reaction.
To a suspension of 39 (dried under vacuum at 50 ЊC, 0.955 g,
2.80 mmol) in THF (4.5 mL) at 0 ЊC was added n-BuLi (1.6 M
in hexane, 1.57 mL, 2.52 mmol), and the resulting deep red
solution was stirred for 30 min at room temperature. The mix-
ture was cooled to Ϫ20 ЊC and a solution of the crude aldehyde
prepared above in THF (2 mL) was added. The mixture was
kept at Ϫ20 ЊC for 30 min and then warmed to room temper-
ature. The reaction was quenched with MeOH and the mixture
was diluted with ether (100 mL). The separated ethereal layer
was washed with saturated NH4Cl and brine, dried over
MgSO4, and concentrated. Flash column chromatography
(hexanes/ethyl acetate, 5 : 1) of the residue gave 40 (1 : 1 mixture
of E and Z isomers, 0.086 g, 52% over two steps): [α]2D0 Ϫ20.4 (c
1.60, CHCl3); 1H NMR (400 MHz, CDCl3) δ 6.21 (dd, J = 12.4,
5.2 Hz, 1H), 5.85 (dd, J = 9.6, 6.4 Hz, 1H), 4.60 (dd, J = 12.8,
9.2 Hz, 1H), 4.18 (dd, J = 10.0, 6.4 Hz, 1H), 3.49–3.55 (s, 6H),
3.39 (dd, J = 7.6, 7.6 Hz, 2H), 3.26–3.30 (m, 2H), 3.02 (s, 6H,
rotamer), 2.92 (s, 6H, rotamer), 2.50–2.63 (m, 3H), 1.90–2.01
(m, 1H), 1.66–1.74 (m, 2H), 1.25–1.56 (m, 10H), 1.03–1.18 (m,
10H), 0.86–0.99 (m, 18H), 0.75–0.80 (m, 6H); 13C NMR (100
MHz, CDCl3) δ 176.8, 176.5, 147.1, 146.9, 146.0, 145.8, 111.2,
110.7, 106.8, 106.5, 59.8, 56.4, 56.3, 48.5, 48.4, 46.6, 42.5, 42.4,
38.2, 38.1, 37.8, 37.6, 37.5, 37.4, 36.0, 35.9, 35.7, 35.6, 34.05,
34.00, 33.8, 28.6, 28.4, 27.8, 27.4, 19.8, 19.6, 19.5, 19.2, 19.1,
39.8, 34.2, 33.9, 33.0, 28.3, 18.8, 17.9, 14.3; IR (film, cmϪ1
)
2965, 2930, 2333, 2173, 1777, 1703, 1384; HRMS (CI, Mϩ)
calcd for C20H30N2O3 346.2256, found 346.2249.
(2R,3ЈS,5ЈS,6ЈS,4ЉS )-2,N-Dimethyl-N-[3Ј,5Ј,6Ј-trimethyl-7Ј-
oxo-7Ј-(2Љ-oxo-4Љ-phenyloxazolidin-3-yl)heptyl]butyramide (37)
To a solution of 35 (0.314 g, 0.90 mmol) in DMF (4 mL) at 0 ЊC
was added a solution of (R)-2-methylbutyric acid (36, 0.183 g,
1.80 mmol) in DMF (5 mL), followed by 1-hydroxy-7-aza-
benzotriazole (HOAt, 0.245 g, 1.80 mmol), 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.345
g, 1.80 mmol) and diisopropylethylamine (DIPEA, 0.313 mL,
1.80 mmol). The mixture was kept at room temperature for 12 h
and then diluted with EtOAc. The separated organic phase was
washed with 1 N HCl and brine, and was dried over MgSO4 and
concentrated. Flash column chromatography (hexanes/ethyl
acetate, 3 : 2) of the residual oil gave 37 (0.326 g, 84%) as a pale
yellow oil: [α]2D0 ϩ40.0 (c 0.50, CHCl3); H NMR (300 MHz,
1
CDCl3) δ 7.26–7.40 (m, 5H), 5.43 (dd, J = 8.4, 3.0 Hz, 1H), 4.67
(t, J = 8.7 Hz, 1H), 4.25 (dd, J = 8.4, 4.2 Hz, 1H, rotamer), 4.24
(dd, J = 8.4, 3.3 Hz, 1H, rotamer), 3.66–3.72 (m, 1H), 3.37 (ddd,
J = 10.2, 6.0, 3.6 Hz, 1H), 3.28 (ddd, J = 10.4, 6.0, 4.0 Hz, 1H),
3.00 (s, 3H, rotamer), 2.91 (s, 3H, rotamer), 2.50–2.63 (m, 1H),
1.81–1.90 (m, 1H), 1.64–1.75 (m, 1H), 1.31–1.48 (m, 3H), 1.01–
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 2 0 9 2 – 2 1 0 2
2100

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18.2, 17.5, 15.8, 15.7, 15.6, 12.5, 12.4; IR (film, cmϪ1) 2929,
1647, 1463; HRMS (CI, Mϩ) calcd for C18H35NO2 297.2668,
found 297.2663.
(3R)-4-Benzylthio-3-(N-tert-butyloxycarbonyl)amino-1-butene
(44)
To
a stirred suspension of methyltriphenylphosphonium
bromide (1.27 g, 3.5 mmol) in THF (15 mL) was added
KHMDS (0.5 M solution in toluene, 6.48 mL, 3.24 mmol), and
the resulting yellow suspension was stirred at room temperature
for 1 h and then cooled to Ϫ78 ЊC. A solution of 43 (0.478 g,
1.62 mmol) in THF (10 mL) was added, the cooling bath was
removed, and the mixture was allowed to warm to room tem-
perature. The mixture was diluted with ether, and the separated
ethereal layer was washed with a half-saturated solution of
Rochelle’s salt, dried over MgSO4, and concentrated. Purifica-
tion of the residue by flash column chromatography (hexanes/
ethyl acetate, 8 : 1) gave 44 (0.263 g, 55%) as a colorless oil:
[α]2D0 ϩ12.7 (c 2.63, CHCl3); 1H NMR (300 MHz, CDCl3)
δ 7.20–7.32 (m, 5H), 5.78 (ddd, J = 17.1, 10.5, 5.4 Hz, 1H), 5.19
(d, J = 17.4 Hz, 1H), 5.13 (d, J = 10.2 Hz, 1H), 4.80 (bs, 1H),
4.33 (bs, 1H), 3.73 (s, 2H), 2.58 (d, J = 6.0 Hz, 2H), 1.46 (s, 9H);
13C NMR (75 MHz, CDCl3) δ 155.6, 138.4, 137.8, 129.4, 129.0,
127.5, 116.0, 80.0, 52.2, 37.0, 36.8, 28.8; IR (film, cmϪ1) 3344,
2977, 1701, 1495; HRMS (CI, Mϩ) calcd. for C16H23NO2S
293.1449, found 293.1448.
(2ЈR,3R,4S,6S )-3,4,6-Trimethyl-8-[methyl-2Ј-(methyl-
butyryl)amino]octanoic acid (3)
To a solution of 40 (0.086 g, 0.29 mmol) in CH3CN (2.1 mL) at
room temperature was added 1 N HCl (0.7 mL), and the
mixture was stirred for 1 h and then diluted with ether. The
ethereal layer was separated and was washed with brine, dried
over MgSO4, and concentrated to give the crude aldehyde.
To a solution of the aldehyde in MeOH (10 mL) was added
2-methyl-2-butene (1.02 g, 14.5 mmol) followed by a freshly
prepared solution of NaClO2 (1.25 M in 20% NaH2PO4, 1.16
mL, 1.45 mmol). The mixture was stirred for 2 h at room
temperature and then diluted with ether. The separated ethereal
layer was washed with brine, dried over MgSO4, and concen-
trated. Flash column chromatography (hexanes/ethyl acetate/
MeOH, 47.5 : 47.5 : 5) of the residue gave 3 as a colorless oil
(0.083 g, 96%): [α]2D0 Ϫ35.0 (c 0.40, CHCl3); 1H NMR (400
MHz, CDCl3) δ 3.24–3.41 (m, 2H), 3.02 (s, 3H, rotamer), 2.93
(s, 3H, rotamer), 2.53–2.62 (m, 1H), 2.35 (dd, J = 10.8, 4.8 Hz,
1H), 2.10 (dd, J = 9.6, 7.2 Hz, 1H, rotamer), 2.06 (dd, J = 9.2,
6.8 Hz, 1H, rotamer), 1.91–2.01 (m, 1H), 1.65–1.74 (m, 1H),
1.30–1.58 (m, 5H), 1.04–1.16 (m, 2H), 1.10 (d, J = 6.8 Hz, 3H,
rotamer), 1.08 (d, J = 6.8 Hz, 3H, rotamer), 0.80–0.94 (m, 12H);
13C NMR (100 MHz, CDCl3) δ 179.0, 178.8, 177.3, 177.0, 48.6,
46.7, 40.8, 40.6, 38.6, 38.5, 37.8, 37.6, 37.5, 35.8, 35.7, 35.6,
35.4, 34.7, 34.6, 34.3, 28.6, 27.7, 27.4, 19.5, 18.0, 17.4, 17.0,
16.9, 16.44, 16.39, 13.5, 12.4; IR (film, cmϪ1) 2963, 1728,
1611; HRMS (CI, Mϩ) calcd for C17H33NO3 299.2460, found
299.2466.
(1ЉR,2ЈR,3R,4S,6S )-3,4,6-Trimethyl-8-[methyl-(2Ј-methyl-
butyryl)amino]octanoic acid (1Љ-benzylsulfanylmethylallyl)amide
(45)
To a solution of 44 (0.244 g, 0.833 mmol) in CH2Cl2 (4 mL) at
0 ЊC was added trifluoroacetic acid (TFA, 3 mL), and the
mixture was stirred at 0 ЊC for 30 min. The solvent was
removed, and the residue was taken up into CH2Cl2. The solu-
tion was washed with 5% NaOH aqueous solution and brine,
dried over MgSO4, and concentrated to give (3R)-4-benzylthio-
3-amino-1-butene (4) which was used without further purifi-
cation. To 3 (0.083 g, 0.278 mmol) at room temperature was
added a solution of 4 in CH2Cl2 (2.5 mL), followed by O-(7-
azabenzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium-hexa-
fluorophosphate (HATU, 0.158 g, 0.417 mmol) and diisoprop-
ylethylamine (DIPEA, 0.073 mL, 0.417 mmol). The mixture
was kept for 12 h at room temperature and the solvent was re-
moved under vacuum. Flash column chromatography (hexanes/
ethyl acetate, 1 : 1) of the residue afforded 45 (0.122 g, 93%):
S-Benzyl-Nꢀ-(tert-butyloxycarbonyl)cysteine N-methoxy-N-
methylamide (42)
To a solution of 41 (0.59 g, 1.90 mmol) in THF (38 mL) at 0 ЊC
was added 1-hydroxybenzotriazole (HOBt, 0.282 g, 2.08 mmol)
and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (EDCI, 0.40 g, 2.08 mmol). The mixture was stirred at
room temperature for 15 min, and MeNHOMeؒHCl (0.208 g,
2.09 mmol) followed by diisopropylethylamine (0.362 mL, 2.08
mmol) were added. The solution was kept at room temperature
for 12 h and then concentrated. Flash column chromatography
(hexanes/ethyl acetate, 2 : 1) of the residue gave 42 (0.56 g, 84%)
[α]2D0 Ϫ15.0 (c 1.16, CHCl3); H NMR (400 MHz, CDCl3) δ
1
7.22–7.31 (m, 5H), 6.03 (d, J = 8.4 Hz, 1H, rotamer), 5.97 (d, J =
8.4 Hz, 1H, rotamer), 5.79 (ddd, J = 17.2, 10.4, 5.2 Hz, 1H),
5.18 (d, J = 17.2 Hz, 1H), 5.14 (d, J = 10.4 Hz, 1H), 4.71 (m,
1H), 3.73 (s, 2H), 3.26–3.41 (m, 2H), 3.00 (s, 3H, rotamer), 2.91
(s, 3H, rotamer), 2.53–2.66 (m, 3H), 2.22 (dt, J = 13.6, 3.6 Hz,
1H), 1.92–2.04 (m, 1H), 1.82–1.88 (m, 1H), 1.65–1.74 (m, 1H),
1.31–1.53 (m, 5H), 1.06–1.16 (m, 2H), 1.10 (d, J = 6.4 Hz, 3H,
rotamer, 1.07 (d, J = 6.8 Hz, 3H, rotamer), 0.80–0.89 (m, 12H);
13C NMR (100 MHz, CDCl3) δ 176.8, 176.5, 172.8, 172.7,
138.5, 138.4, 137.4, 129.4, 129.0, 127.5, 116.2, 50.3, 48.5, 46.5,
41.1, 41.0, 40.9, 40.6, 37.7, 37.6, 36.9, 36.4, 36.1, 35.8, 35.7,
35.6, 34.8, 34.7, 34.1, 28.6, 28.5, 27.8, 27.5, 19.64, 19.59, 18.2,
17.6, 16.9, 16.7, 16.6, 16.5, 12.6, 12.4; IR (film, cmϪ1) 3302,
2917, 1625; HRMS (CI, Mϩ) calcd for C28H46N2O2S 474.3280,
found 474.3283.
as an oil: [α]2D0 Ϫ 30.2 (c 1.04, CHCl3); H NMR (400 MHz,
1
CDCl3) δ 7.25–7.36 (m, 5H), 5.38 (d, J = 8.4 Hz, 1H), 4.92 (bs,
1H), 3.76 (s, 2H), 3.74 (s, 3H), 3.22 (s, 3H), 2.82 (dd, J = 14.0,
5.6 Hz, 1H), 2.65 (dd, J = 14.0, 7.2 Hz, 1H), 1.47 (s, 9H); 13C
NMR (100 MHz, CDCl3) δ 171.9, 155.7, 138.3, 129.4, 128.9,
127.5, 80.2, 62.0, 50.0, 36.7, 33.9, 32.6, 28.8; IR (film, cmϪ1
)
3321, 2976, 1711, 1662, 1169; HRMS (CI, Mϩ) calcd for
C17H26NO4N2S 354.1613, found 354.1616.
(3R)-4-Benzylthio-3-(N-tert-butyloxycarbonyl)amino-
propionaldehyde (43)
To a solution of 42 (0.632 g, 1.78 mmol) in ether (8 mL) at 0 ЊC
was added LiAlH4 (0.085 g, 2.23 mmol), and the mixture was
stirred for 30 min at 0 ЊC. The mixture was extracted with ether,
and the ethereal extract was washed with 1 N HCl and brine,
dried over MgSO4, and concentrated. Flash column chromato-
graphy (hexanes/ethyl acetate, 2 : 1) of the residue gave 43
(0.478 g, 91%) as a colorless oil: [α]2D0 ϩ3.47 (c 1.96, CHCl3); 1H
NMR (300 MHz, CDCl3) δ 9.51 (s, 1H), 7.21–7.34 (m, 5H), 5.39
(m, 1H), 4.24–4.32 (m, 1H), 3.72 (s, 2H), 2.86 (dd, J = 13.8, 5.7
Hz, 1H), 2.80 (dd, J = 14.1, 6.0 Hz, 1H), 1.45 (s, 9H); 13C NMR
(75 MHz, CDCl3) δ 199.2, 155.8, 138.0, 129.4, 129.1, 127.8,
80.8, 59.5, 37.3, 31.0, 28.7; IR (film, cmϪ1) 3354, 2977, 1709,
1495.
(1ЉR,2ЈR,3R,4S,6S )-3,4,6-Trimethyl-8-[methyl-(2Ј-methyl-
butyryl)amino]octanoic acid (1Љ-mercaptomethylallyl)amide (46)
To a solution of sodium (0.023 g, 1 mmol) in liquid NH3 (2 mL)
at Ϫ78 ЊC was added a solution of 45 (0.018 g, 0.042 mmol) in
THF (0.5 mL) and the deep blue solution was stirred for 30 min
at Ϫ78 ЊC. Solid NH4Cl was added to quench the reaction, and
ammonia was evaporated in a stream of argon. The residue was
triturated with CH2Cl2, and the separated organic phase was
washed with saturated NaHCO3, dried over Na2SO4, and
concentrated. Flash column chromatography (hexanes/ethyl
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 2 0 9 2 – 2 1 0 2
2101

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acetate/MeOH, 47.5 : 47.5 : 5) of the residue afforded 46 as a
Acknowledgements
colorless oil (0.0125 g, 86%): [α]2D0 Ϫ4.3 (c 0.65, CHCl3); H
1
We are grateful to Professor Alexandre F. T. Yokochi, Oregon
State University, for determining the X-ray crystal structure of
32. Financial support was provided by the National Institute
of General Medical Sciences (grant GM50574 to JDW).
NMR (300 MHz, CDCl3) δ 5.93 (d, J = 8.1 Hz, 1H, rotamer),
5.82 (d, J = 8.1 Hz, 1H, rotamer), 5.76 (ddd, J = 16.2, 11.1, 5.1
Hz, 1H), 5.20–5.26 (m, 2H), 4.72–4.83 (m, 1H), 3.21–3.45 (m,
2H), 3.01 (s, 3H, rotamer), 2.92 (s, 3H, rotamer), 2.51–2.85 (m,
3H), 2.25–2.30 (m, 1H), 1.88–2.04 (m, 2H), 1.61–1.75 (m, 2H),
1.28–1.57 (m, 6H), 1.12–1.16 (m, 1H), 1.10 (d, J = 7.8 Hz, 3H,
rotamer, 1.07 (d, J = 6.9 Hz, 3H, rotamer), 0.80–0.94 (m, 12H);
13C NMR (75 MHz, CDCl3) δ 176.9,176.6, 172.8, 172.5, 136.32,
136.26, 117.2, 51.9, 51.7, 48.5, 46.5, 41.2, 41.1, 41.0, 40.5, 37.8,
37.6, 36.1, 35.9, 35.7, 34.7, 34.6, 34.1, 30.0, 29.7, 28.6, 27.8,
27.5, 19.7, 19.6, 18.2, 17.5, 16.9, 16.8, 16.6, 12.6, 12.4; IR (film,
cmϪ1) 3297, 2961, 1640, 1624; HRMS (CI) calcd for C21H40
N2O2S 384.2810, found 384.2811.
References
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(؉)-Kalkitoxin (2)
To a solution of 46 (0.012 g, 0.031 mmol) in CH2Cl2 (1 mL) at
room temperature was added freshly distilled TiCl4 (0.034 mL,
0.31 mmol), and the orange coloured solution was stirred for
12 h at room temperature. The reaction was quenched by
addition of saturated NaHCO3, the mixture was extracted with
CH2Cl2, and the extract was dried over Na2SO4 and concen-
trated. Flash column chromatography (hexanes/ethyl acetate,
1 : 1) of the residue furnished (ϩ)-kalkitoxin (2) as a colorless oil
(0.0076 g, 67%): [α]2D0 ϩ11.5 (c 0.34, CHCl3) [lit.5 [α]2D5 ϩ16 (c
0.07, CHCl3)]; 1H NMR (400 MHz, C6D6) δ 5.85 (ddd, J = 16.8,
10.4, 6.0 Hz, 1H), 5.24 (d, J = 17.2 Hz, 1H), 5.01 (d, J = 10.4 Hz,
1H), 4.75 (m, 1H), 3.35 (m, 1H), 2.94 (m, 1H), 2.93 (dd, J =
10.8, 8.4 Hz, 1H), 2.80 (s, 3H, rotamer), 2.71 (dd, J = 10.8, 8.4
Hz, 1H), 2.46–2.55 (m, 1H), 2.42 (s, 3H, rotamer), 2.20–2.37
(m, 2H), 2.06 (m, 1H), 1.84–1.99 (m, 1H), 1.21–1.59 (m, 5H),
1.18 (s, 3H, rotamer), 1.10 (m, 1H), 1.09 (s, 3H, rotamer), 0.69–
0.96 (m, 13 H); 13C NMR (100 MHz, C6D6) δ 175.5, 175.2,
170.1, 170.0, 138.5, 138.3, 115.5, 115.4, 79.4, 47.9, 46.1, 40.5,
40.4, 39.0, 38.8, 38.6, 38.2, 37.7, 37.6, 37.4, 36.1, 34.7, 34.6,
34.5, 33.6, 28.5, 28.1, 27.7, 19.6, 19.4, 18.4, 17.8, 16.6, 16.5,
12.6, 12.4; IR (film, cmϪ1) 2961, 2874, 1643, 1463; HRMS (FAB
M ϩ Hϩ) calcd for C21H38 N2OS 367.2783, found 367.2793.
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IC50 Determination
HCT-116 cells were plated at 5 × 104 cells in T25 tissue culture
flasks (Falcon Plastics, New Jersey) with 5 mL media RPMI
1640 (Cellgro, Virginia) supplemented with 15% BCS (Hyclone,
Utah), 5% Pen. Strep. and 5% Glutamine (Cellgro). After three
days (cells in logarithmic growth phase; 5 × 105 cells/flask), test
compound was added to the flasks to achieve concentrations
ranging from 10 to 10Ϫ4 µg mLϪ1. At day 3, the flasks were
washed, trypsinized, spun down and the cells counted for
both viable and dead cells using 0.08% trypan blue (Gibco,
Maryland). Viable cell number as a function of concentration
was plotted and the IC50 values determined by interpolation.
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Clonogenic assay
HCT-116 cells were seeded at 200 to 20 000 cells in 60 mm
dishes. Drug was added to the medium (RPMI ϩ 10% FBS) at
concentrations of 1 mg mLϪ1 and 10-fold dilutions thereof. At
either 2 h or 24 h, the drug-containing media was removed and
fresh media without drug was added. The dishes were incubated
for 7 days, media was removed and the colonies were stained
with methylene blue. Colonies containing 50 cells or more were
counted and the results were normalized to an untreated
control. Plating efficiency for the untreated cells was ca. 90%.
Repeat experiments were carried out to define the cell survival
range between 100 and 10Ϫ3 survival. Values were determined
for three exposure times (2 h, 24 h, 168 h).
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O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 2 0 9 2 – 2 1 0 2
2102